Citation
Nitrate as an Endocrine Disrupting Contaminant in Captive Siberian Sturgeon, Acipenser baeri

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Title:
Nitrate as an Endocrine Disrupting Contaminant in Captive Siberian Sturgeon, Acipenser baeri
Copyright Date:
2008

Subjects

Subjects / Keywords:
Aquaculture ( jstor )
Fish ( jstor )
Hormones ( jstor )
Messenger RNA ( jstor )
Nitrates ( jstor )
Nitrites ( jstor )
Plasmas ( jstor )
Sex hormones ( jstor )
Species ( jstor )
Sturgeon ( jstor )

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University of Florida
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University of Florida
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All applicable rights reserved by the source institution and holding location.
Embargo Date:
7/12/2007

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NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN
STURGEON, Acipenser baeri














By

HEATHER J. HAMLIN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2007

































Copyright 2007

by

Heather J. Hamlin

































To my family









ACKNOWLEDGMENTS

First and foremost I thank Lou Guillette for his tremendous mentorship, for sharing with

me his wealth of knowledge and experience and for having more faith in me than I did at times.

I am a better person for having worked with him. I thank my advisor and other members of my

committee for their encouragement and support: Ruth Francis-Floyd was graciously supportive

and gave me the freedom to do what I love; Kevan Main gave me the opportunity and

encouragement to fulfill my dream; Daryl Parkyn read my manuscripts and gave me valuable

comments; Roy Yanong gave me valuable opinions and comments on my manuscripts and I'll

always appreciate his enthusiasm for disease. I'd also like to thank Jim Michaels for allowing

me access to my research animals and supporting my research.

This j ourney would not have been nearly as fulfilling were it not for the comradery of my

fellow lab mates: Thea Edwards, who taught me EIAs and introduced me to the lab experience.

I'll always treasure our late night conversations about everything from egg cups to egg

development; Brandon Moore, who took the time to mentor me and with whom I'll always enjoy

scientific discussions; Satomi Kohno, whose patience in teaching lab techniques deserves an

award; Iske Larkin, for teaching me the wonders of RIAs. Lori Albergotti, Ashley Boggs and

Nicole Botteri, who made me wish I could spend more time in the lab. I'd also like to thank all

the undergraduate students who assisted me with collections and lab analyses.

Finally, I' d like to thank my friends and family, without whom the j ourney wouldn't be

worth it: my Mother, Holly Paulsen, who let me have every creature known to man as a child,

and spent countless hours with me as an adult collecting data and analyzing samples in this work.

It wouldn't have been the same without her help; my Father, Greg Hamlin, who bought me my

first aquarium; my best friend Maria Piccioni, who supported me tremendously in this journey;









I'd love to be half the person she thinks I am; Dave Jenkins, who supported me more than he

knows; and Amy Leighton, who made my childhood something to treasure.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ................. ................. iv.............


TABLE OF CONTENTS............... ................vi


LI ST OF T ABLE S ................. ................. viii............


LI ST OF FIGURE S .............. .................... ix


AB STRAC T ................ .............. xi


1 INTRODUCTION ................. ...............1.......... ......


Background ............... ... .. .......... .. .......... ....... .........
Overview of Reproductive Endocrinology in Fishes ................. .......... ................1
Stress in Fish and Its Effects on Reproduction............... ..............
Endocrine Disruption in Aquatic Vertebrates .............. ...............6.....
Nitrate in Natural Water Systems............... ............ ........
Nitrate in Aquaculture and Its Implications as an EDC .............. .....................
Sturgeon as a Model Species ................. ...............12........... ...
Research Obj ectives and Hypotheses ...._ ......_____ .......___ ...........1


2 NITRATE TOXICITY IN SIBERIAN STURGEON .............. ...............18....


Introduction............... ..............1
M ethods ............... .. .. ............ .. ...........2

Study Animals and Pre-Testing Conditions .............. ...............20....
Range-Finding Studies .............. ...............20....
Test Procedures .............. ...............21....
Statistical Analyses............... ...............22
Re sults.........____....... ___ ...............22.....
Discussion............... ...............2


3 STRESS AND ITS RELATION TO ENDOCRINE FUNCTION IN CAPTIVE
FEMALE SIBERIAN STURGEON............... ...............30


Introduction............... ..............3
M ethods .............. .. ...............33...

Fish and Sampling .............. ...............33....
Surgical Sexing............... ...............34.
Treatm ents .............. ...............3 4....
Hormone Evaluations .............. ...............3 5....

Statistical Analyses............... ...............36
Re sults................ .. ....... .. ...............36.......

Morphology and Chemistry............... ...............3












Horm ones .............. ...............37....
Discussion............... ...............3


4 NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT INT CAPTIVE
S BERIAN S TURGEON. ............. ...... ...............46...


Introduction............... ..............4
M ethods .............. .. ... .... .... ..........4

Fish and Sampling Procedures .............. ...............49....

Surgical Sexing............... ...............50.
Experim ent 1 .............. ...............51....
Experim ent 2 .............. ...............52....
Hormone Evaluations .............. ...............53....

Statistical Analyses ............. ...... ._ ...............54...
Re sults............. .. ... ._ ...............54...

Experim ent 1 .............. ...............54....
Experim ent 2 .............. ...............56....
Discussion............... ...............5


5 EFFECTS OF NITRATE ON STEROIDOGENIC GENE EXPRESSION INT CAPTIVE
FEMALE SIBERIAN STURGEON............... ...............69


Introduction............... ..............6
M ethods .............. .... .... ........ .........7

Fish and Experimental Systems............... ...............73
Surgical Sexing and Tissue Collection............... ...............7
Treatments and Experimental Conditions .............. ...............74....
RNA Isolation and Primer Design ................. ...............74........... ...
Quantitative Real-Time PCR............... ...............75..
Sequence Data .............. ...............76....
Statistical Analyses............... ...............76
Re sults ................ ........... ...............77.......

W ater Chemistry ................. ............. ........... .. .. .........7
Steroidogenic Gene Expression and Hormone Regressions from Previous Studies.......78

Sequence Data .............. ...............77....
Discussion............... ...............7


6 SUMMARY AND FUTURE DIRECTIONS ...._ ......_____ .......___ ............0


Sum m ary ............. ...... ._ ...............103....
Future Directions .............. ...............107....
Conclusions............... ..............10


LIST OF REFERENCES ............. ......___ ...............110...


BIOGRAPHICAL SKETCH ............. ......___ ...............130...











LIST OF TABLES
Table page

1-1 LCso results and test conditions for three size classes of Siberian sturgeon exposed to
sodium nitrate............... ...............27

1-2 Representative acute toxicity data for nitrate .............. ...............28....

5-1 Forward and reverse primers used for quantitative real-time PCR ................. ................85

5-2 Regression data mRNA expression patterns for P450 side chain cleavage enzyme
(P450sec), estrogen receptor P (ERP), glucocorticoid receptor (GR), testosterone (T),
11-ketotestosterone (11KT), 17P-estradiol (E2) COrtisol and glucose in sturgeon
exposed to 1.5 and 57 mg/L NO3-N ................. ...............86........... .

5-3 Regression data for testosterone (T), 11-ketotestosterone (11KT), 17P-estradiol (E2)
cortisol and glucose in sturgeon exposed to 1.5 and 57 mg/L NO3-N from Chapter 4 .....87










LIST OF FIGURES
Figure page

1-1 Overview of the hypothalamic-pituitary-gonadal axis in sturgeon ................. ................15

1-2 A representative steroidogenic pathway of steroid hormones in gonadal cells..................16

1-3 A representative steroidogenic pathway of cortisol production in an interrenal cell .........17

2-1 Linear regression of loglo transformed nitrate-N (mg/L) lethal concentration values
versus log transformed fish weight (g). ............. ...............29.....

3-1 Blood sampling times for treatments 1-4 of fish held under confinement stress for 4-h ...43

3-2 Plasma cortisol (A) and plasma glucose (B) concentrations (mean f S.E.M.) during a
4-h capture and confinement period .............. ...............44....

3-3 Sex steroid data for treatment 2. Plasma 17P-Estradiol (A), testosterone (B), and 11-
ketotestosterone (C) taken from serial bleeds of cultured female Siberian sturgeon
throughout the 4-h period of confinement stress .............. ...............45....

4-1 Blood sampling times for treatments 1 and 2 of fish held under confinement stress for
6-h. ............. ...............62.....

4-2 Plasma cortisol (A) and glucose (B) concentrations (mean f 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
11.5 or 57 mg/L nitrate-N ................. ...............63........... ..

4-3 Plasma testosterone (A), 11-ketotestosterone (B) and estradiol (C) concentrations
(mean f 1 S.E.M.) in cultured female Siberian sturgeon (Acipenser baeri) exposed
for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N ............... ...................6

4-4 Plasma cortisol (A) and glucose (B) concentrations (mean f 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
11.5 or 57 mg/L nitrate-N ................. ...............65........... ..

4-5 Plasma cortisol (A), glucose (B) testosterone concentrations (mean f 1 S.E.M.) in
cultured female Siberian sturgeon (Acipenser baeri) exposed for 30 days to
concentrations of 1.5 or 57 mg/L nitrate-N .............. ...............66....

4-6 Plasma cortisol testosterone (A), 11-ketotestosterone (B) and estradiol-17P (C)
concentrations (mean f 1 S.E.M.) in cultured female Siberian sturgeon (Acipenser
baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N .......................67










4-7 Plasma cortisol (A) and glucose (B) concentrations (mean f 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
1.5 or 57 mg/L nitrate-N ................. ...............68........... ..

5-1 Nucleotide and deduced amino acid sequences of Siberian sturgeon ribosomal protein
L8 (RPL8) .............. ...............88....

5-2 Nucleotide and deduced amino acid sequences of Siberian sturgeon P450scec.................. 89

5-3 Nucleotide and deduced amino acid sequences of Siberian sturgeon ERP ......................90

5-4 Nucleotide and deduced amino acid sequences of Siberian sturgeon GR..........................91

5-5 Sequence comparison of deduced amino acid sequences for ribosomal protein L8
(RPL 8) ................ ...............92................

5-6 Sequence comparison of deduced amino acid sequences for P450scec............... .... ...........93

5-8 Sequence comparison of deduced amino acid sequences for ERP ........._._. ............_.....95

5-9 Mean (f SE) expression of P450sce mRNA in 4.5 year-old Siberian sturgeon. ................96

5-10 Mean (f SE) expression of glucocorticoid (GR) receptor mRNA in 4.5 year-old
Siberian sturgeon. ............. ...............97.....

5-11 Mean (f SE) expression of estrogen receptor-P (ERP) mRNA in 4.5 year-old
Siberian sturgeon. ............. ...............98.....

5-12 Linear regression of glucose (mmol/L) vs GR mRNA (normalized to L8 expression)
for fish exposed to 1.5 mg/L nitrate-N ................. ...............99........... .

5-13 Linear regression of ERP mRNA and GR mRNA (normalized to L8 expression) for
fish exposed to 1.5 mg/L nitrate-N. ............. ...............100....

5-14 Linear regression of P450sce mRNA (normalized to L8 expression) and T for fish
exposed to 57 mg/L nitrate-N ................. ...............101.......... ...

5-15 Linear regression of P450sce mRNA (normalized to L8 expression) and 1 1-KT for
fish exposed to 57 mg/L nitrate-N ................ ...............102..............

6-1 Possible alterations in nitrate induced elevations of sex steroid concentrations in
Siberian sturgeon .............. ...............109....










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN
STURGEON, Acipenser baeri

By

Heather J. Hamlin

May 2007

Chair: Ruth Francis-Floyd
Major: Fisheries and Aquatic Sciences

Numerous environmental contaminants have been shown to alter reproductive endocrine

function. Such compounds have been termed endocrine-disrupting contaminants (EDCs). EDCs

exert their effects through numerous physiological mechanisms, including alterations in

steroidogenesis. Although a global pollutant of most aquatic systems, nitrate has only recently

begun to receive attention for its ability to alter endocrine function in wildlife. We examined

nitrate-induced endocrine disruption using the Siberian sturgeon (Acipenser baeri) as a model

species. Comparisons of captive populations of sturgeon cultured in nitrate concentrations of

1.5, 11.5 and 57.5 mg/L nitrate-N revealed nitrate induced elevations in plasma concentrations of

sex steroids including testosterone, 11l-ketotestosterone and 17P-estradiol. Alterations in

circulating concentrations of sex steroids can be a response to several physiological mechanisms,

including an up-regulation of gonadal steroid synthesis, altered biotransformation and clearance

by the liver or alterations in plasma storage by steroid binding proteins.

To gain a better mechanistic understanding of the observed sex steroid elevations we

examined mRNA expression patterns of steroidogenic enzymes (P450sec) and receptor proteins

(ERP and GR). We found no significant differences in mRNA expression patterns, indicating










that the observed sex steroid increases were not likely due to an up-regulation of gonadal

synthesis.

Cortisol and glucose, commonly examined as indicators of perceived stress, were not

found to vary among groups exposed to any of the nitrate concentrations. Because responses to

stress can be cumulative, endocrine responses to stress events in fish residing in the various

nitrate concentrations were also investigated. Results showed that nitrate does alter the

associated stress response, defined by plasma glucose concentrations.

These data suggest that long-term exposure to nitrate is associated with altered endocrine

parameters (e.g., plasma hormone concentrations) in Siberian sturgeon. Future work must begin

to examine the underlying causes of these changes. Although the data of gene expression

suggest that mRNA concentrations of at least one steroidogenic enzyme were not altered, other

enzymes in the pathway need to be examined. These data indicated that nitrate concentrations

must now be considered in the effective management of sturgeon populations in both natural and

captive environments.









CHAPTER 1
INTRODUCTION

Background

Overview of Reproductive Endocrinology in Fishes

The production of circulating hormones is the result of numerous physiological

reactions spanning many levels of biological organization. The regulation of hormone

production is controlled by mechanisms that both create and destroy these chemical

messengers, and is Eine-tuned by various stimulatory and feedback mechanisms (Norris,

1997). Tropic hormones regulate many of the activities of the thyroid gland, adrenal gland

and the gonads (Norris, 1997). The endocrine regulation of reproduction is initiated in

resp on se to envi ronmental cue s, whi ch sti mulate the rel eas e of gonad otropi n-rel easi ng

hormone (GnRH) from the hypothalamus (Detlaff et al., 1993; Norris, 1997) (Figure 1-1).

In response to GnRH, the anterior pituitary releases gonadotropins, which circulate

throughout the body, targeting various organs, such as the gonads.

Two chemically distinct gonadotropins have been characterized in fish, GTH-I and

GTH-II, which are purportedly analogous to follicle stimulating hormone (FSH) and

luteinizing hormone (LH), respectively, in terrestrial animals (Norris, 1997). Because few

Hish species have defined chemical hormone structures to date, much of the research

literature employs heterologous hormones (Van Der Kraak et al., 1998). FSH stimulates

oogenesis and spermatogenesis, and LH stimulates Einal gamete maturation and release.

Like FSH and LH, GTH-I and GTH-II consist of an a and P subunit; the a subunit is the

same for both gonadotropins, with only the P subunit conferring biological specificity

(Norris, 1997; Vasudevan et al., 2002). The P subunits of both gonadotropins have been

cloned in Siberian sturgeon (A. baeri) and Russian sturgeon (A. gueldenstaedti), and based









on their function and position in the phylogenetic tree, it was suggested these compounds

be termed FSH and LH, respectively (Querat et al., 2000; Hurvitz et al., 2005).

FSH and LH stimulate gonadal steroidogenesis, and the three steroid hormones

relevant to this study are estradiol-17P (E2), testosterone (T) and 11l-ketotestosterone (11-

KT). In females, E2 Stimulates gonadal growth, sexual maturation, vitellogenesis by the

liver and oogenesis (Knobil and Neill, 1994; Norris, 1997; Denslow et al., 2001). In males,

T stimulates sexual maturation, spermatogenesis and spawning, and is implicated in sexual

behavior for both males and females (Norris, 1997; Toft et al., 2003). In addition to

inducing spermatogonial proliferation, 11-KT likely also participates in the former

processes (Schultz and Miura, 2002).

Circulating hormones can be detected by receptors at the periphery of the cell, and

through a cAMP mediated process ultimately leads to increased levels of intracellular

cholesterol (Stocco, 1999). This cholesterol is mobilized to the outer mitochondrial

membrane and is the precursor for steroid biosynthesis. A protein inserted in the

mitochondrial membrane, steroidogenic acute regulatory (StAR) protein, functions to

transport cholesterol from the outer mitochondrial membrane to the inner mitochondrial

membrane, and this process is now thought to be the rate limiting step in steroidogenesis

(Stocco, 1999). Its function has received considerable attention in recent studies of

vertebrates (Stocco, 2001), including fish (Goetz et al., 2004). The inner mitochondrial

membrane is the site of activity for the P450 side chain cleavage enzyme (P450sec) that

cleaves cholesterol to form the first steroid in the pathway, pregnenolone. Pregnenolone is

then converted to progesterone by 3P-hydroxysteroid dehydrogenase (3P-HSD). Both

P450sec and 3 P-HSD are often evaluated in studies of steroidogenesis and related










physiological mechanisms (Takase et al., 1999; Pozzi et al., 2002; Inai et al., 2003).

Further pathways of steroid production are shown in Figures 1-2 and 1-3. Quantifying

compounds in the biosynthetic pathways will assist in developing a mechanistic

understanding of which pathways can be disrupted.

Stress in Fish and Its Effects on Reproduction

Stress has been defined in the literature in a number of ways, encompassing such

definitions as diversions of metabolic energy, adaptive changes resulting in modifications

to normal physiological states, and any change that impacts long term survival (Selye,

1956; Esch and Hazen, 1978; Wedemeyer and McLeay, 1991; Bayne, 1985; Barton and

Schreck, 1987). Ultimately our interest in stress is attendant upon the causative factors

mitigating the deleterious response. Once these causative factors are determined, we can

then begin the process of remediation. In this sense, understanding stress is a means to an

end and becomes a useful tool to predict if negative outcomes are likely to ensue. We can

use this diagnostic tool to understand environmental impact and determine at what point

action is necessary to effectuate relief.

As in other vertebrates, concentrations of coricosteroid hormones are sensitive

indicators of acute stress in fish, and circulating concentrations generally reflect synthesis

rates since little hormone is stored in the adrenal (mammals) or interrenal tissue (fish)

(Norris, 1997). The production of corticosteroids is initiated by perceived stress events,

triggering the release of corticotropin releasing hormone (CRH) from the hypothalamus,

which then triggers the release of ACTH from the pituitary (Figure 1-2) (Flik et al., 2006).

Circulating ACTH triggers the release of corticosteroids from the interrenal cells of the

head kidney in most fish species; in sturgeon cortisol releasing adrenocortical cells are

present in small clusters throughout the kidney (Norris, 1997).









The principal corticosteroid in most Hish species is cortisol (Kime, 1997; Barton,

2002; Overli et al., 2005), which has been implicated as a causal factor in many of the

deleterious effects of stress (Barton and Iwama, 1991; Harris and Bird, 2000; Schreck,

2001; Bernier et al., 2004). Cortisol shows two primary actions in fish, regulation of water

and mineral balance and energy metabolism (Wendelaar Bonga, 1997). The effects of

corticosteroid hormones are mediated through intracellular receptors, which act as ligand

binding transcription factors (Norris, 1997). Fish possess both a glucocorticoid receptor

(GR) and a mineralcorticoid receptor (MR) with GR possessing various isoforms (Bury et

al., 2003). While cortisol is the predominant physiological ligand for GR, it is still unclear

what is the primary ligand for MR in fish, which shows a high affinity for both

deoxycorticosterone and aldosterone (Prunet et al., 2006). This is particularly interesting

since there is no reliable evidence for the presence of aldosterone in teleosts, and it is

becoming accepted that aldosterone is likely absent in most or potentially all Eish groups

(Prunet et al., 2006).

The molecular characterization of corticosteroid receptors (CR) in the last 10 years

has modified the initial consensus of a unique high affinity binding site for cortisol, and

now depicts a multiple CR family with two classes of receptors (GR and MR) with splicing

isoforms and duplicated genes (GR1 and GR2) (Prunet et al., 2006). Functional analyses in

trout show that GR2 has a higher sensitivity to cortisol when compared to GR1, and that

these isoforms show different patterns of expression sensitivity depending on the tissues

targeted (Greenwood et al., 2003). It has also been shown that GR can be less sensitive to

corticosteroids than MR, suggesting that the latter could serve as a high affinity cortisol

receptor in fishes, a condition already described in humans (Hellal-Levy et al., 2000).









The significance of cortisol in assessments of stress may be limited when examining

chronic stressors, due in part to the acclimation of the interrenal tissues during chronic

stre ss, whi ch i s miti gated by negative feedb ack m ech ani sm s on th e hyp othal amo-pituitary -

interrenal (HPI) axis (Rotllant et al., 2000). Other bio-markers, such as expression levels

of GR, have been shown to be more sensitive indicators of chronic stress. Quantification of

GR in seabass (Dicentrarchus labrax) showed significantly reduced GR concentrations

after a 3-month exposure to elevated stocking densities (Terova et al, 2005).

Environmental contaminants have been shown to alter the stress response by altering

GR activation. Organotins, compounds used as industrial stabilizers in paints now present

in aquatic environments, have been shown to block GR activation (Odermatt et al., 2006).

Other ubiquitous pollutants such as PCBs and arsenic have also been shown to alter GR

receptor functioning (Johansson et al., 1998; Bodwell et al., 2004).

The effects of stress can be manifest at multiple levels of the reproductive endocrine

axis (Guillette et al., 1995; Pankhurst and Van Der Kraak, 1997). Although there is limited

information on the effects of stress on the release of GnRH on aquatic inhabitants, several

studies have been conducted identifying stress impacts on circulating concentrations of

GTH-I and GTH-II. For some species of fish such as brown trout (Salmo trutta),

confinement stress results in an increase in circulating concentrations of GTHs (Pickering

et al., 1987; Sumpter et al., 1987). For other species, such as the white sucker (Catostonaus

conanersoni), capture and transport stress results in depression of GTHs to undetectable

concentrations within 24 h of capture (Stacey et al., 1984).

The effects of stress on concentrations of gonadal steroids in both terrestrial and

aquatic animals is well documented, resulting in a depression in plasma concentrations of









both androgens and estrogens in most species studied to date (Francis, 1981; Pickering et

al., 1987; Carragher and Pankhurst, 1991). These reductions can be attributed to altered

secretion of gonadotropins (Gray et al., 1978) as well as by direct inhibition of gonadal

steroid synthesis (Saez et al., 1977; Sapolsky, 1985).

Cortisol has also been implicated in altering endocrine function. Cortisol's negative

effects on reproduction includes depressed plasma concentrations of sex steroids

(Pankhurst and Dedual, 1994; Pottinger et al, 1999). However, this response is dependent

upon the hormones involved and the species investigated. Elevated plasma concentrations

of cortisol in Stellate sturgeon (A. stellatus) females have been shown to result in

correspondingly lower concentrations of circulating plasma T and 1 1-KT, however, E2 and

progesterone (P) remain constant (Semenkova et al., 2002). Similarly, Bayunova (2002)

observed an inverse relationship between cortisol and T after a 9-h period of confinement

stress for both male and female stellate sturgeon. Consten et al. (2002) investigated

whether the decrease in plasma 1 1-KT of male carp was caused by a direct effect of

cortisol, or by an indirect effect (such as a decrease in plasma LH). Experimental animals

were fed cortisol-containing food pellets over a prolonged period, and the results indicated

that cortisol had a direct inhibitory effect on testicular androgen secretion that was

independent of LH secretion. Reductions in reproductive hormones can lead to a myriad of

deleterious reproductive effects such as decreased gamete quality, embryo mortality, and

behavioral changes (Pankhurst and Van Der Kraak, 1997; Pankhurst, et al., 1995).

Endocrine Disruption in Aquatic Vertebrates

Xenobiotics, or man-made chemicals, have been shown to disrupt normal hormone

function, and have received considerable attention over the last decade (Colborn and

Clement, 1992; Guillette, 2000; McLachlan 2001). Compounds evaluated as endocrine









disrupting contaminants have generally included common environmental pollutants which

have demonstrated abilities to mimic hormones, alter hormone production, or act as anti-

hormones (Guillette, 2000). Molecularly, xenobiotics have the ability to bind directly to

steroid hormone receptors or other proteins that initiate or facilitate the transcription of

genes (Thomas, 1990; Rooney and Guillette, 2000). Compounds such as polychlorinated

hydrocarbon pesticides (e.g., DDT derivatives), polychlorinated biphenyls (PCBs) and

others have been shown to bind to estrogen receptors manifesting estrogenic or anti-

estrogenic actions in mammals and birds (Bulger and Kupfer, 1985; Rooney and Guillette,

2000). Extensive work has been conducted in fishes, and evidence indicates similar

mechanisms occur (Thomas, 1990; White et al., 1994; Tyler et al., 1998a; 1998b; 1999;

Jobling et al., 1995; 1996; 1998; 2002).

Numerous studies document a vast array of endocrine disruptive effects in fish

located in polluted aquatic systems and areas downstream of sewage or other industrial

treatment plants (Jobling et al., 2003; Toft et al., 2004). Male walleye (Stizostedion

vitreum) collected near a metropolitan sewage treatment plant exhibited depressed serum T

concentrations and elevated serum E2 COncentrations compared to reference males (Folmar

et al., 2001). Reduced plasma concentrations of T have also been documented in lake

whitefish (Coregonus clupeaformis) and white sucker (Catostomus commersonii) exposed

to bleached Kraft mill and pulp mill effluent respectively (Munkittrick et al., 1992; 1994).

Female mosquitofish downstream from Kraft paper-mill effluent in Florida demonstrated

masculinization of the anal fins, which is an androgen-dependent trait (Parks, et al., 2001).

Male mosquitofish from a Florida lake contaminated with known endocrine disruptors

displayed shorter gonopodium, significantly reduced whole body T concentrations, reduced









liver weights and had reduced sperm counts versus those of a reference population (Toft et

al., 2003).

Compounds such as the natural steroid E2 have been measured in both industrial

and municipal sewage treatment effluents, which represent the principle sources of natural

estrogens in the aquatic environment (Lai et al., 2002). Exposure to E2 CauSed disruptions

in sexual differentiation in young zebrafish and altered egg production patterns in adults

(Brion et al., 2004). Exposure of the riverine species the roach (Rutilus rutilus) to a host of

chemicals persistent in typical British waters, revealed significantly increased incidences of

intersexuality and plasma vitellogenin concentrations and attributed these alterations to

estrogenic constituents of sewage effluents (Jobling et al., 1998).

Considerable work also has been conducted on abnormalities of the reproductive

system of Florida' s alligators in relation to environmental contamination, notably in Lake

Apopka, located northwest of Orlando. These studies report reductions in circulating

concentrations of sex steroids, alterations in gonadal morphology, phallus size, enzyme

activity and steroidogenesis (Guillette, et al., 1999; 2000). These modifications were

attributed to both embryonic and post-hatching exposure to a complex mixture of

chemicals from agricultural activities and stormwater runoff, including PCBs, p,p'-DDE,

dieldrin, endrin, mirex, and oxychlordane. Excess nitrate has also been shown to alter

steroidogenesis and endocrine function in several aquatic species (Guillette and Edwards,

2005; Barbaeu, 2004). Detailed lists of known endocrine disrupting contaminants and

their documented effects are readily available (Edwards, 2006), and will be discussed in

further detail in Chapters 3, 4 and 5.









Nitrate in Natural Water Systems

In nature, organic and inorganic nitrogen is cycled through various environmental

processes such as nitrifieation, denitrifieation, Eixation and decay. Nitrifieation and

denitrifieation processes are essential to the health of aquatic ecosystems. These processes

generally begin with ammonia, which is broken down to nitrite by aerobic nitrifying

bacteria (usually Nitrosomona~s sp.), which is then converted by another group of bacteria

to nitrate (usually by Nitrobacter sp.). Nitrate is often then fixed by plants as a nutrient, or

undergoes denitrification (Sharma and Ahlert, 1977). Complete denitrification converts

nitrate to either nitrogen gas or organic nitrogen. Incomplete denitrification, resulting from

inadequate sources of carbon or environmental conditions, results in nitrate's conversion

back to nitrite, or even ammonia, by anaerobic denitrifying bacteria (Van Rijn et al. 2006).

Over the last several decades, concentrations of nitrate in natural water bodies from

anthropogenic impact has increased significantly (Pucket, 1995), which has resulted in

nitrate concentrations in many water sources far in excess of the EPA drinking standard of

10 mg/L nitrate-N (Kross et al., 1993; U.S. EPA, 1996). In northern Florida,

concentrations as high as 38 mg/L nitrate-N were recorded in the Suwannee River (Katz et

al., 1999). In addition to its direct effects, nitrate can encourage excessive algal and plant

growth, adversely impacting the ecology of the affected area (Attayde and Hansson, 1999;

Capriulo et al., 2002).

Nitrate in Aquaculture and Its Implications as an EDC

As discussed previously, elevated concentrations of stress hormones have been

shown to result in decreased concentrations of circulating sex steroids. Environmental

contaminants have been shown to elicit a stress response, thereby decreasing circulating

concentrations of sex steroids. In fact, some of the earliest reports of vertebrate stress










responses were induced by chemical exposure (Selye, 1936). While it is clear many man-

made chemicals have considerable impact on hormone function in aquatic animals, it is less

clear if naturally occurring compounds could also have the same effect. Contaminated

aquatic ecosystems such as Lake Apopka, Florida provide ample opportunity to observe

severe abnormalities of the reproductive system, and are decidedly "unhealthy" for aquatic

life. In aquaculture, aquatic animals are exposed to xenobiotic and natural compounds

often far in excess of those experienced in nature, but resultant abnormalities are often

overlooked since aquaculture fish are not necessarily expected to mimic wild fish. After

all, they are held at higher densities, eat dramatically different diets, and are often held

under unnatural temperature and light regimes. Additionally, definitions of acceptable

water quality standards of natural water environments (generally under EPA regulation)

versus those of intensive aquaculture systems (under the regulation of the farm manager)

are usually dramatically different. Commercial aquaculture operations have limited

budgets (if any) for in-depth research into the factors that are contributing to the success or

failure of husbandry practices and protocols. Therefore, water quality estimates of "safe"

operating levels in aquaculture are often the result of trial and error practices based on

growth or mortality events. For species such as sturgeon, which take many years to reach

reproductive maturity, and whose economic viability relies heavily on proper egg

production, it may be important to investigate more thoroughly the sublethal effects a

potential hazard may impose.

Nitrate has been overlooked as a material water quality hazard in both natural and

aquaculture settings. Emerging information implicates nitrate as a hazard at concentrations

once thought to be innocuous for both reptile and amphibian species (see Guillette and









Edwards, 2005). It has been shown that vertebrate mitochondria are capable of nitric oxide

(NO) synthesis via non nitric oxide synthase (NOS) activity (Zweier et al., 1999) using

nitrite as a precursor. Nitrate can be converted to nitrite in-vivo (Panesar and Chan, 2000),

and it is thought other enzymes can generate NO directly from nitrate (Meyer, 1995).

Nitric oxide is a gas that plays diverse roles in cellular signaling, vasodilation, immunity

and has been documented to inhibit steroid hormone synthesis (DelPunta et al., 1996;

Panesar and Chan, 2000; Weitzberg and Lundberg, 1998). As discussed previously in this

chapter, StAR and P450sce are key factors regulating steroidogenesis. NO has been shown

to alter the activity of StAR and may also alter P450sce by binding to the heme group

which is present in all enzymes of the P450 family (White et al., 1987). Bulls fed nitrate

showed reduced sperm motility and degenerative lesions of the germ layers of the testes

(Zraly et al., 1997). Medaka exposed for 2-months to no more than 75 mg/L NO3-N

showed reduced fertilization and hatching rates (Shimura et al., 2002). A study of female

mosquitofish (Gamnbusia holbrooki) in Florida showed reduced reproductive activity and

embryo number in fish exposed to 5.06 mg/L NO3-N (Edwards et al., 2006b).

Reproductive hormone concentrations have been shown to be especially vulnerable

to chemical and physical strain (Pickering, 1987), which as discussed can cause numerous

reproductive complications. Since nitrate has been shown to negatively impact the

reproductive physiology of a number of aquatic species (Edwards et al. 2006a; Edwards et

al., 2006b) and sturgeon have been shown to be unusually susceptible to environmental

impact (Akimova and Ruban; Dwyer et al., 2005), it stands to reason that nitrate could be

an endocrine disrupting contaminant for Siberian sturgeon, and is worthy of investigation.









In the United States and elsewhere, water is becoming a valuable and limited

commodity, and its use is tightly regulated. New aquaculture operations will not be

afforded the vast quantities of water established facilities have been permitted to use, and

will therefore need to use recirculating technologies which enable these facilities to reuse a

significant portion of their water. In most of these recirculating facilities, the limiting

factor for water exchange is nitrate concentration.

Sturgeon as a Model Species

Sturgeons belong to one of the most ancient groups of Owridukes ~ and are

naturally distributed above the 30th parallel. Although they can be found almost

everywhere along the Pacific and Atlantic coasts, the Mediterranean and Black Seas, as

well as rivers, lakes and inland seas, most sturgeon populations are sparse and occur in

significant numbers in only a few regions (Detlaff et al., 1993). The Caspian Sea

represents a unique reservoir, producing the bulk of the world' s sturgeon capture fisheries.

Sturgeon include anadromous, semi-anadromous and river-resident (freshwater) forms.

The Siberian sturgeon have both semi-anadromous and river resident populations (Detlaff

et al., 1993).

Sturgeon have preserved primitive structural features relating them to

chondrosteans, while at the same time the structure of their eggs is more similar to

amphibians than either chondrosteans or teleosts, since the inclusions of yolk are

distributed throughout the cytoplasm. Although sturgeon produce great numbers of large

eggs, affording them great ecological advantage in hostile environments, ironically this

production is at the nexus of their dwindling population. Sturgeon eggs, termed caviar

when processed, are a prized delicacy and commands very high prices. This has lead to

over fishing on a grand scale (Birstein, 1993; Williot et al., 2002). This over fishing, in









concert with other anthropogenic impacts, such as river damming and pollution, has

resulted in the reduction, or in some cases decimation, of sturgeon stocks worldwide

(Williot et al., 2002). Aquaculture has been proposed as a mechanism to help save wild

populations, either by reducing fishing pressures or by providing animals for stock

enhancement. Due to the high value of caviar, sturgeon aquaculture has great promise.

As discussed above, nitrate is the limiting factor for water exchange in recirculating

aquaculture systems. The less water a facility uses, the greater the possible concentrations

of nitrate, and although research is underway to develop technologies to reduce nitrate

concentrations, it is unclear what affects nitrate has on fish residing in these systems.

Additionally, environmental nitrate from anthropogenic sources is increasing at an

alarming rate worldwide (Rouse et al., 1999), and with pollution implicated in reductions in

wild sturgeon populations in the Caspian Sea, the world's largest sturgeon reservoir, the

need to understand the affects of nitrate on sturgeon is becoming more and more apparent.

That egg production is paramount to the viability of sturgeon as an aquaculture species, and

is of obvious ecological importance, necessitates an understanding of the affects of nitrate

on the reproductive system in particular.

Research Objectives and Hypotheses

The goal of this study was to gain a better mechanistic understanding of the

potential for nitrate-induced disruptions in reproductive function, using Siberian sturgeon

as a model. Based on previous studies reviewed in this Chapter, I hypothesize that given

nitrate's ability to alter steroidogenic activity, notably through NO induced alterations in

P450 enzyme activities, that the fish exposed to elevated nitrate will demonstrate reduced

concentrations of plasma sex steroid concentrations, and these reductions will be mirrored

in gonadal mRNA expression patterns of P450sc, ERP and GR. I theorize that these










alterations would not be caused by a generalized stress response, but by disruptions in

steroidogenic mechanisms directed at the production of sex steroids, notably T, 11-KT and

E2.

Compensatory mechanisms required to combat physiological challenges consumes

energy and physiological resources that could otherwise be used to carry out other essential

functions. Therefore, an animal experiencing simultaneous stressors, such as nitrate

exposure in combination with an induced stressor such as confinement, may not be as adept

at responding to the stress events as an animal experiencing a single stressor. I therefore

hypothesize that long-term exposure to elevated nitrate will alter the associated stress

response. In addition, given that GR has been shown to parallel chronic stress, I predict

GR mRNA expression will be significantly reduced in animals exposed for 30 days to

elevated nitrate.
























ACTIONt


a~i~P
~~CmlTC~


PBRODUTID


pR~oDUnrlDN


I I~D


Figure 1-1.


Overview of the hypothalamic-pituitary-gonadal axis in sturgeon. The
hypothalamic-pituitary-gonadal axis in sturgeon is similar to that of other
vertebrates. Gonadotropic releasing hormone (GnRH) from the hypothalamus
controls the release of gonadotropins (GTHs) from the pituitary that then enter
circulation. The gonad responds by producing various sex steroids including
17P-estradiol, which stimulates hepatic vitellogenin production. These
processes are essential for normal ovarian follicle development. Similar to
other fish species, the hypothalamic release of corticotropin-releasing
hormone (CRH) controls the release of adreno-corticotropin hormone (ACTH)
from the pituitary, which controls the release of glucocorticoids from the
interrenal cells of the head kidney.



















































Figure 1-2. Representative steroidogenic pathway of steroid hormones in gonadal cells. In
response to ligand binding of the receptor, the transfer of free cholesterol into
the mitochondria facilitated by steroidogenic acute regulatory (StAR) protein, is
considered the acute rate limiting step in steroidogenesis. The enzymatic
conversion of cholesterol to pregeneolone by P450sec is considered the chronic
regulatory step in steroidogenesis. Pregnenolone or progesterone is released
into the cytoplasm/smooth endoplasmic reticulum to be converted to
androstenedione, which is in turn converted into testosterone and 17P-estradiol
by 17P-HSD or aromatase respectively.

















































Figure 1-3. Representative steroidogenic pathway of cortisol production in an interrenal
cell. In response to ligand binding of the receptor, the transfer of free
cholesterol into the mitochondria facilitated by steroidogenic acute regulatory
(StAR) protein, is considered the acute rate limiting step in steroidogenesis.
The enzymatic conversion of cholesterol to pregeneolone by P450se is
considered the chronic regulatory step in steroidogenesis. 1700-
hydroxyprogesterone is released into the smooth endoplasmic reticulum for
further processing and eventual conversion 11l-deoxycortisol and cortisol.











CHAPTER 2
NITRATE TOXICITY INT SIBERIAN STURGEON

Introduction

Ammonia is a product of the biological degradation of proteins and nucleic acids.

Nitrifying bacteria convert ammonia to nitrite, which is in turn converted to nitrate, the end

product of nitrification (Sharma and Ahlert, 1977). Ammonia, and to a less extent nitrite,

are ecologically relevant compounds and the toxicity of these compounds, both in terms of

tolerable thresholds and physiologic mechanism to aquatic animal health, has been well

documented (Rubin and Elmaraghy, 1977; Meade, 1985; Huertas et al., 2002). Nitrate,

however, does not normally reach toxic concentrations in natural environments or in

recirculating systems with high water exchange, and has therefore received comparatively

less attention as a material water quality hazard (Knepp and Arkin, 1973; Russo, 1985;

Bromage et al., 1988; Meade and Watts, 1995). The absence of obvious patho-

physiological effects in most aquatic species at ecologically relevant concentrations of

nitrate, rationalizes the belief that nitrate is relatively non-toxic (Jensen, 1996). While

nitrate is indeed much less toxic than ammonia or nitrite on a mg/L basis, nitrate commonly

rises to levels far in excess of those of the other compounds in intensive aquaculture

environments with limited water exchange (Knepp and Arkin, 1973; Hrubec, 1996), and

warrants more detailed investigations into the effects these levels may have.

Excess nitrate in aquaculture has traditionally been reduced by water exchange or the

operation of denitrification filters (Timmons et al., 2001). Current trends in environmental

regulation are limiting the amount of water which may be consumed or discharged,

reducing the feasibility of using large influxes of water to remove excess nitrate.









Denitrification filters can be technically challenging and costly, and as aquaculture

operations become water limited, nitrate will become a considerable concern.

The levels of nitrate that are likely to cause concern are unknown for many aquatic

species, as are how susceptibilities to nitrate change ontogenetically. For large species

such as sturgeon, it is logistically difficult and costly to conduct acute toxicity evaluations

on broodstock size animals. However, evaluations using smaller animals may not mimic

responses of larger fish. New evidence implicates nitrate as a material water quality hazard

at levels much lower than previously suspected for other aquatic species (Guillette and

Edwards, 2005) and recommended levels of nitrate for warm-water fishes (90 mg NO3-N)

(U. S. E.P.A., 1986) has been shown to be highly toxic to amphibians (Marco et al., 1999).

Although a great deal of research needs to be conducted to elucidate the effects of

sublethal exposures, acute testing will assist researchers in understanding how sensitive a

particular species is to nitrate, and can be used as a tool to predict if susceptibilities may

change over time. The most common analytical method for evaluating acute toxicity in

fish is the LCso (Parish, 1985). An LCso describes a lethal concentration (LC) at which 50%

of the experimental population dies in a specified period of time. LCso data allows us to

determine if a substance is toxic, how toxic it is, and allows for multi-species comparisons

of sensitivity. The obj ectives of this study were to determine the acute toxicity of three

ontogenetic size classes of Siberian sturgeon (Acipenser baeri) to nitrate, using the LCso

criterion, to determine how life stage influences this response.









Methods

Study Animals and Pre-Testing Conditions

Siberian sturgeon were reared from eggs in 250 L troughs in a recirculating system

containing well water. Fish were initially fed Artemia and a soft moist formulated feed

(Silver CupTM, Nelson and Sons Inc., Murray, UT). When the fish reached 1.5 g they were

transferred to 1300 L tanks and were fed only formulated feeds by this time. Dissolved

oxygen was monitored daily and rarely went below 90% saturation (Oxyguard Handy Beta,

Point Four Systems Inc., Richmond, BC, Canada). Temperatures were slowly increased

throughout the fish's development, and ranged from 150C (at hatch) to 23.50C. Other water

quality parameters prior to the toxicity trials were evaluated weekly (ammonia-N and

nitrite-N, Lamotte Smart Colorimeter, Chestertown, MD; nitrate, lon 6 Acorn Series,

Oakton InstrumentsTM, Vernon Hills, IL; pH, Acorn 6 Series, Oakton InstrumentsTM, Vernon

Hills, IL). In addition to the above parameters, alkalinity, chloride, total hardness and

calcium hardness (Hach CompanyTM, Loveland, CO) were tested at the beginning and end

of each 96-h toxicity trial.

Range-Finding Studies

Small-scale range Einding studies using at least three nitrate concentrations with five

fish/concentration were conducted prior to each test until a suitable test range was

determined. Suitability was defined by total mortality in the highest concentration and no

mortality in the lowest concentration in 96 hours within a narrow test range. Tests

generally required 2-3 range finding studies per toxicity trial. Tanks were evaluated for

mortalities every 3-4 hours from 08:00 to 20:00, and dead fish were immediately removed

and inspected for condition.









Test Procedures

Three partial exchange 96-h toxicity tests were conducted in triplicate using three

weight classes of Siberian sturgeon spanning 3 orders of magnitude, with 10 Hish per test

container. Experiments were conducted over time using Eish from the same cohort to

eliminate cohort variability. New experimental animals were used for each trial. Water

for each of the evaluations consisted of degassed well water (nitrate-N 1.4 & 0.3 mg/L)

from which nitrate solutions were created from food-grade sodium nitrate (JLM Marketing,

Tampa, FL). Initial concentrations were confirmed with an Auto AnalyzerTM, and were

periodically spot-checked with an ion specific probe (lon 6 Acorn Series, Oakton

InstrumentsTM, Vernon Hills, IL) throughout the trials to ensure concentrations matched

initial target values. Each trial evaluated four geometrically constant concentrations of

nitrate, as well as triplicate well water and sodium controls. Sodium controls were

achieved with NaCl (Morton SaltTM, Chicago, 1L) with concentrations adjusted to match

the sodium in the highest nitrate concentration in the trial. Tanks were randomly assigned

to each treatment. Tanks were evaluated for mortalities every 3-4 hours from 08:00 to

20:00 and dead fish were immediately removed and inspected for condition.

The first trial evaluated concentrations of 555, 888, 1420, and 2273 mg/L nitrate-N

using 6.9 & 0.31Ig fish. This trial was conducted in glass aquaria filled with 32.4 L of test

solution, submersed in a water bath to maintain a temperature of 210C. A 50% water

exchange with the appropriate nitrate concentration was conducted half way through the

trial to eliminate collateral effects from elevated ammonia or nitrite. Fish were not fed two

days prior to and throughout the trial, and fecal debris was siphoned twice daily.









At least twice daily, observations were made of fish behavior (orientation, gill

ventilation rate, swimming speed) and appearance throughout the trial. The second trial

evaluated concentrations of 216, 323, 485, and 727 mg/L nitrate-N using 66.9 & 3.4 g fish.

This trial was conducted in fiberglass tanks filled to 670 L. The water was maintained at

23.50C. The third trial evaluated concentrations of 234, 421, 758 and 1364 mg/L using

673.8 & 18.6 g fish. This trial was conducted in fiberglass tanks filled to 587 L, and the

temperature was maintained at 23.50C.

Statistical Analyses

Data from replicates were pooled prior to calculating the median lethal concentration.

Median lethal concentrations and 95% confidence intervals were evaluated by the trimmed

Spearman-Karber method for 24, 48, 72, and 96-hr time periods. Testing ranges,

determined by range finding studies, were designed to evaluate a 96-hr time period.

Therefore, shorter time periods did not always result in enough mortality to compute the

LC5o values. Normal distribution was evaluated with the Shapiro-Wilk' s test. A linear

regression of loglo transformed data was conducted to predict susceptibilities of larger

sturgeon using StatView@ statistical software package (SAS@ Institute, Cary, NC).

Results

No animals died in either the well water or sodium controls for any of the size classes

tested, and appeared healthy throughout the trial. The 96-h LC5o of nitrate to 6.9 & 0.3 1 g

Siberian sturgeon was 1028 mg/L nitrate-N (Table 2-1). Moribund fish in this size class

tended to gill rapidly, but most showed few outward signs of toxicity except a stiffening of

the musculature and lethargy (decreased swimming speed, frequent resting periods). The

96-h LCso of nitrate to the 66.9 & 3.4 g and 673.8 & 18.6 g sturgeon was 601 mg/L and 397










mg/L nitrate-N respectively. Moribund fish in these treatments tended to exhibit additional

evidence of the toxicity such as reddening around the mouth, and red specks and/or patches

along the length of the body, most notably at the base of the pectoral fins. Log transformed

nitrate vs. log transformed LCso values are shown in Fig. 2-1. Water chemistry parameters

were as follows: unionized ammonia-N (NH3)<; 0.04 f 0.02 mg/L; nitrite-N
pH 7.9 f 0.2; alkalinity 208 f 12 mg/L; chloride 90 f 5 mg/L (exclusive of the NaCl

control); total hardness 260 f 10 mg/L; calcium hardness 160 f 10 mg/L. Dissolved

oxygen levels were maintained at >95% saturation throughout the trials. The Shapiro-

Wilk's test indicated normal distribution for all treatments. The 6.9 + 0.31 g sturgeon were

maintained at 21.00C while the latter two size classes were maintained at 23.50C, which are

typical temperatures for these size stages. Placing all three size classes at the same

temperature would not represent a realistic rearing condition, and previous toxicity tests

with this species has not demonstrated a significant difference in LC50 values for

temperatures ranging from 200C-250C for 6.0 g to 1 kg Siberian sturgeon (H. Hamlin,

unpublished data).

Discussion

The United States is now recognizing water as a valuable and limited commodity,

and its tight regulation is forcing aquaculture technology to shift toward more sustainable

and ecologically responsible practices. Therefore, as the land-based aquaculture industry

continues to grow, management strategies are shifting to recirculating systems with lower

water exchange. This trend is creating new husbandry concerns as less clean water is

available to flush out nitrate. In systems with limited water exchange, nitrate can build to

levels of 150 mg/L nitrate-N or more (personal observation), and it is unclear the impact









these elevated levels may have. Critical for the design of any aquaculture operation are the

water quality standards to be maintained, and it is important to know what levels of

substances are likely to cause concern (Bohl, 1977). The etiology and effects of nitrate

toxicity are relatively unknown in fishes, leaving open future opportunities for research in

this area. This information can then be used to understand toxicity thresholds and

physiologic impact, as well as appropriately engineer remediation systems and

technologies.

Results of this study demonstrated the 96-h LCso for fish of 7-700 g to range between

397-1028 mg/L nitrate-N. These numbers are appreciably lower than those reported for

most aquatic species tested to date. Comparative nitrate data from representative toxicity

studies suggests that the maj ority of test populations can handle nitrate-N levels of 1000

mg/L nitrate-N or more (4426 mg/L total nitrate) without reaching 50% mortality, when

sodium nitrate is used as the source of nitrate (Table 2). Some fish, such as the

beaugregory (Stegastes leucostictus), exhibit LCso values of over 3000 mg/L NO3-N

(13,280 mg/L total nitrate), substantially above the tolerance of most freshwater fish

including Siberian sturgeon (Peirce et al., 1993). Although diet may affect the relative

toxicity of nitrate (Chow and Hong, 2002), a pervasive theory in the etiology of nitrate

toxicity is that it is endogenously converted to nitrite (Hill, 1999), and it is in fact nitrite

that is the biotoxic agent. In terrestrial animals this theory has been the source of numerous

debates (Hartman, 1982), and the mechanism of nitrate toxicity in fishes is still unclear.

Anecdotal evidence at Mote Marine Laboratory's Aquaculture Park (Sturgeon

Commercial Demonstration Proj ect) has shown Siberian sturgeon to be especially sensitive

to nitrate, with larger animals exhibiting increased incidence of toxicity and mortality









starting at levels as low as 90 mg/L nitrate-N (398 mg/L total nitrate, see Guillette and

Edwards (2005) for an explanation of the reporting of nitrate concentrations) (H. Hamlin,

unpublished data). Susceptibilities have been strongly affected by cohort variability, with

certain cohorts being more sensitive to elevated nitrate than others. Although the results in

this study demonstrate a strong correlation between size and LCso values, caution must be

taken in predicting susceptibilities of varying cohorts of Eish, or even fish within the same

cohort, since LCso values have been shown to be highly variable (Buikema et al., 1982).

Regression analysis of the current data yield a predicted LCso of 247 mg/L nitrate-N (1093

mg/L total nitrate) for 6 kg fish (Fig. 2-1). Regardless of the high variability of

toxicological responses to nitrate, it is clear from this study that young Siberian sturgeon

are far more tolerant to elevated nitrate than their adult counterparts, and this is the first

study to demonstrate this finding.

Often, the dose-response relationship is a scaled association between the

concentration of chemical tested and the severity of the elicited response (Lloyd, 1979). In

general, younger or immature animals tend to be more susceptible to chemical insult or

perturbation than are adults (Macek et al., 1978; Sprague, 1985). In fact, a common

chronic toxicity test is the early life stage test, because although this test does not provide

total life cycle exposure, it is purported to include exposure during the most sensitive life

stages (McKim, 1985). This study found an increased tolerance of Siberian sturgeon to

nitrate at younger stages. Although this opposes general convention, this phenomenon has

been reported for other fish species with other toxic compounds (Rosenberger et al., 1978).

Acute toxicity tests are an effective tool to establish baseline toxicity thresholds in

terms of responses to nitrate over time, and to compare the toxicity of nitrate to other










species. Given the increased sensitivity of Siberian sturgeon to nitrate as compared to

other species, it is clear much more work is needed to elucidate the sublethal effects of

elevated nitrate exposure. The sensitive nature of sturgeon to nitrate renders them suitable

candidates for further investigation of the etiology and nature of nitrate exposure and

toxicosis.














Table 1-1. LCso results and test conditions for three size classes of Siberian sturgeon
exposed to sodium nitrate
Average weight 6.9f0.31 g 66.9f3.4 g 673.8f18.6 g
24-h LCso (mg/L NO3-N) 1510 n/a 803
95% confidence interval (1826-2631) (720-897)

48-h LCso (mg/L NO3-N) 1443 n/a 522
95% confidence interval (1309-1590) (486-562)

72-h LCso (mg/L NO3-N) 1195 n/a 438
95% confidence interval (1086-1316) (394-487)

96-h LCso (mg/L NO3-N) 1028 601 397
95% confidence interval (941-1124) (557-649) (3 57-441)
* Not enough partial kill responses to obtain a valid lethal concentration estimate.












Table 1-2. Representative acute toxicity data for nitrate


NO3
Source
NaNO3


NO3-N
mg/L
5081


Species
Cape sole
(Hr. capensisl
Common bluegill
(L. nzacrochirus)
Goldfish
(C. carassius)
Tiger shrimp

Catla
(C. catla)
Channel catfish
(I. punctatus)
Chinook salmon
(0. tshaw/tscha)
Fathead Minnows
(P. pronzela~s)
Guadalupe Bass
(M~ treculi)
African clawed frog
(X. laevis)
Aquatic Snail
(P. antipod arunt) d~~~dd~~~dd
Florida pompano
(T. carolinus)
Sao Paulo shrimp
(P. paulensis)
Pacific treefrog
(P. regilla)
Guppy fry
(P. reticulatus)
Caddi sflies
(C. pettiti)


LCso
24-h LCso


Reference
Brownell 1980


NaNO3 2909*

NaNO3 2761*


24-h LC5o Dowden and Bennett
1965
24-h LC5o Dowden and Bennett
1965
96-h LC5o Tsali and Chen 2002

96-h LC5o Tilalk et al. 2002

96-h LC5o Colt and
Tchobanoglous 1976
96-h LC5o Westin 1974

96-h LC5o Scott and Crunkilton
2000
96-h LCso Tomasso and
Carmichael 1986
240-h LCso Schuytema and
Nebeker 1999
96-h LCso Alonso and Camargo
2003
96-h LCso Pierce et al. 1993

96-h LCso Cavalli et al. 1996

240-h LCso Schuytema and
Nebeker 1999
72-h LCso Rubin and Elmarachy
1977
96-h LCso Comargo and Ward
1992


NaNO3

NaNO3

NaNO3

NaNO3

NaNO3

NaNO3

NaNO3

NaNO3

NaNO3

NaNO3


1575

1565

1409

1318

1349

1269

1236

1042

1006

494


NaNO3 266


KNO3

NaNO3


200

114


* Publication did not specify whether results were values for NO3 Or NO3-N














Y=3.177 .208*X; R^`2 = .994


2.5
0.5 1 1.5 2 2.5 3 3.5

Log fish weight (g)


Figure 2-1. Linear regression of loglo transformed nitrate-N (mg/L) lethal concentration
values versus log transformed fish weight (g).











CHAPTER 3
STRESS AND ITS RELATION TO ENDOCRINE FUNCTION IN CAPTIVE FEMALE
SIBERIAN STURGEON

Introduction

The central focus of comparative physiology and endocrinology involves understanding

how various organisms respond to environmental influences. Fish are affected by stress in both

their natural and captive environments. It is well recognized that common fishery and

aquaculture practices, including crowding, transport and confinement are stressful to Hish and can

negatively affect reproduction (Pankhurst and Van Der Kraak, 1997). The effects of stress can

be manifested at many levels of the reproductive endocrine axis, and measuring the

concentration of circulating hormones is a useful endpoint to understand if a stressor affects

endocrine function. Numerous environmental stressors, including capture and confinement

(Pankhurst and Dedual, 1994), time of day (Lankford et al., 2003), hypoxia (Maxime et al.,

1995), and environmental contaminants (Orlando et al., 2002; Guillette and Edwards, 2005) have

been shown to induce stress in fish. For most Hish, including the Siberian sturgeon and other

freshwater chondrosteans, cortisol is the predominant stress hormone (Maxime et al., 1995;

Barton et al., 1998; Mommsen et al., 1999). Plasma glucose concentration has also been shown

to be an indicator of secondary stress responses (Bayunova et al., 2002).

Sex steroids can have an inverse relationship with plasma concentrations of stress steroids,

an effect evident in fish and some other animals (Carragher and Sumpter, 1990; Cooke et al.,

2004). Negative effects of stress on reproduction have been attributed to the suppression of LH

and FSH secretion from the pituitary gland, disruptions in steroidogenesis pathways, or alteration

of hormone degradation by the liver and/or kidney (Krulich et al., 1974). Although plasma

concentrations of corticosteroids often parallel acute stress, there is evidence in teleosts that the









estrogenic inhibitory effects of stress are not necessarily mediated by cortisol, and that these

effects arise higher in the endocrine pathway than at the level of ovarian steroidogenesis

(Pankhurst et al., 1995).

Contradictory evidence has shown that the addition of cortisol to the culture medium

reduces the secretions of 17P-estradiol (E2) and testosterone (T) from cultured ovarian follicles

of rainbow trout (Oncorhynchus mykiss) (Carragher and Sumpter, 1989). Likewise, carp fed

with cortisol-containing food pellets showed reduced androgenic production, independent of LH

secretion (Consten et al., 2002). Acute confinement stress in male brown trout (Salmo trutta L.)

resulted in low concentrations of plasma T and 1 1-KT in sexually mature animals (Pickering et

al., 1987). White sturgeon (Acipenser transmontanus) injected with an ACTH analog exhibited a

dose-dependent increase in cortisol concentration more than the cortisol concentrations induced

by stress events such as transport and handling (Belanger et al., 2001). A few studies, including

one examining the effects of stress on serum cortisol concentration in cultured stellate sturgeon,

actually demonstrated significantly increased gamete quality in fish with elevated cortisol

concentration, speculating that cortisol could be a normal endocrine component of the

reproductive system, even though later studies of the same species showed reduced plasma

concentrations of sex steroids during stress (Semenkova et al., 1999; Bayunova et al., 2002). It

has also been shown that fish require prolonged periods to recover from an acute stress event

(Jardine et al., 1996). Other studies have shown that blood removal, a practice often necessary

for evaluating endocrine endpoints, can alter blood hemoglobin concentration (Hogasen, 1995).

Stress studies typically focus on the causative factors mitigating the deleterious response,

but defining these relationships often requires sampling and research measures that themselves

contribute to enhancing the stress response. Understanding the effects of potential stressors is









critical to properly manage wild fisheries or successfully culture endangered or economically

important fishes. It is important to know which stressors are naturally present in the fish's

environment, which are caused by typical aquaculture practices, and which are induced by the

testing procedures themselves (Conte, 2004).

Sturgeons (Acipenseriformes) are among the most ancient fishes on earth, originating

over 200 million years ago (see review by Birstein, 1993). Twenty-five extant sturgeon species

occupy the Northern Hemisphere; however, excessive fishing, loss of spawning grounds and

other environmental pressures have contributed to the reduction of sturgeon stocks worldwide,

particularly Caspian Sea varieties (Williot et al., 2002). Today, all 25 species of sturgeon are

listed as endangered or threatened in some regard (Birstein, 1993). Aquaculture has been

proposed as a means to conserve sturgeon, and generating commercial stocks has the dual benefit

of providing fish for stock enhancement, as well as for food production, thus conserving wild

populations (Beamesderfer and Farr, 1997; Waldman and Wirgin, 1997; Chebanov et al., 2002;

Stone, 2002). The Siberian sturgeon is rapidly becoming a species of great economic interest in

the United States, and is currently the most widespread sturgeon species utilized for commercial

aquaculture in Europe (Gisbert and Williot, 2002). Despite this, very few studies have been

conducted to clarify the physiological effects of stress on this species. Understanding the

endocrine disruptive effects of induced stress will serve as a baseline for understanding the

effects of other environmental stressors, such as contaminants commonly found in both natural

and constructed environments. Nitrate, for example, has recently been shown to be highly toxic

to Siberian sturgeon in aquaculture environments with limited water exchange (Hamlin, 2006),

and is predicted to be of considerable concern for commercial aquaculture operations, which are

already being forced to significantly reduce their water usage. Nitrates and other ions have also









been established as ecologically relevant endocrine disruptors in natural environments for

numerous other vertebrates (see review by Guillette and Edwards, 2005). For late maturing

species such as sturgeons, whose economic viability relies heavily on successful egg production

(caviar), it is of particular importance to understand the relationships between stress and

reproductive health.

The purpose of this study is to define the relationship between induced stress and

circulating concentrations of steroid hormones in cultured Siberian sturgeon, and to identify

mitigating stress factors in typical testing procedures, most notably the techniques of blood

withdrawal and surgical sexing, to understand what factors contribute significantly to the stress

response.

Methods

Fish and Sampling

Three-year-old Siberian sturgeon were collected from two 30,000 L tanks, each from

separate commercial recirculating aquaculture systems at Mote Marine Laboratory's Aquaculture

Park (Commercial Sturgeon Demonstration Project) in Sarasota, Florida. Experiments were

started at approximately 10:30 a.m. in May of 2004. Water chemistry in each of these systems

was analyzed weekly for the levels of ammonia-N, nitrite-N, nitrate, and pH prior to the start of

experiments. Dissolved oxygen and temperature were monitored continuously using stationary

probes, which were spot-checked biweekly for calibration using portable probes. Hardness,

alkalinity, and chloride concentration was analyzed the day prior to the start of experiments.

The sturgeon were pulled from the water by hand at the side of the tank and immediately

held down on a padded V-shaped surgical table. Pulling the sturgeon from the tank by hand

(versus netting) decreased the likelihood of stressing the remaining fish in the tank and allowed

immediate access to the fish for blood sampling. Blood was extracted from the caudal vein (5










ml) using a 10 ml syringe (20 gauge needle) within 1 min of capture; most captures took 30 sec

for the full blood sample to be drawn. The blood sample was placed into lithium heparin

VacutainerTM tubes, and stored on ice for less than 30 minutes before centrifugation. Plasma was

separated via centrifugation (5-10 min at 2000 g), placed into cryovials, rapidly frozen in liquid

nitrogen and stored at -80 oC for 2-3 weeks prior to analysis.

Surgical Sexing

For surgical sexing, the sturgeon were anesthetized in a 5 OC water bath containing carbon

dioxide. Carbon dioxide was used because it is a low regulatory priority anesthetic for fish that

are grown for food production and requires no withdrawal period; the sturgeon used in this study

were part of a commercial food production program. Pure oxygen gas administered through a

fine air stone was used to maintain dissolved oxygen concentrations in the range of 8.0 12.0

mg/L in the anesthetic bath, and sodium bicarbonate was added to maintain pH in the range of

6.8 7.5 throughout the procedure. The sturgeon generally took 3 5 min for full

anesthetization. A 2.5 3.8 cm incision was made on the ventral side of each fish, approximately

7.5 cm anterior to the vent, along the median axis to allow inspection of the gonads on either side

of the fish for sex determination. The incision in each fish was closed by suturing with coated

vicryl absorbable suture (Ethicon IncTM., Somerville, New Jersey), and the fish was allowed to

recover in a confinement tank. Once anesthetized, the surgical procedure took approximately 1

min/fish, and the fish recovered fully from the anesthesia in 5 10 min.

Treatments

Six fish (3 fish/tank) were used for each treatment. All fish were sexed immediately after

initial bleedings/sham bleedings; if the fish was male, the sample was discarded, and another fish

was extracted until 3 females had been sampled from each tank for each treatment. In this study,









we focused on female sturgeon because they are part of a larger set of studies examining various

environmental factors and ovarian development leading to commercial caviar production. The

female sturgeon were then weighed and measured just after sexing while they were still under

anesthesia. The fish were then placed into a square 0.64 m3 inSulated plastic tote filled with 530

liters of system water for a 4-h period of confinement stress. A numbered cable tie placed

around the caudle peduncle identified individual Eish. The time at which the fish was removed

from the tank for initial bleeding/sham bleeding was considered 0-h.

In all treatments, fish were sexed immediately after initial blood drawing/sham drawing

prior to placement in the confinement tank. In treatment 1, fish were bled at 0-h only and placed

into an insulated tote as described previously. In treatment 2, fish were bled at 0-h, 1-h and 4-h.

In treatment 3, fish were bled at time 1-h and 4-h only, and in treatment 4, fish were bled at 4-h

only. For treatments 3 and 4, during the sampling periods when the fish were not bled, the fish

were held down on the surgical table momentarily to mimic the bleeding procedure but were not

pricked with the needle. Blood sampling times for all treatments during the 4-h period of

confinement stress are shown in Fig. 3-1.

Hormone Evaluations

Plasma samples for steroid evaluations were thawed on ice, and the steroid fraction was

extracted with diethyl ether. Extraction was repeated twice to enhance extraction efficiency.

Plasma cortisol, E2, T and 11-KT concentrations were analyzed according to the instructions

provided with the commercial competitive enzyme immunoassay kits (Cayman Chemical Co.,

Ann Arbor, MI), specific to each hormone. Each hormone was previously validated for Siberian

sturgeon plasma by verifying that serial dilutions were parallel to the standard curve. Samples

were run in duplicate and each plate contained duplicate wells for interassay variance and a

blank. Individual hormones were all run with plates from the same kit lot # and were completed










in the same testing session to reduce testing variance. Sample plates were analyzed using a

microplate reader (BioRad, Hercules, CA). Intra-assay and interassay variances, respectively,

were as follows: estradiol, 3.5% and 7.0%; cortisol, 2.0% and 9.1%; testosterone, 3.7% and

12.8%; 11-KT, 4.9% and 11.9%.

Plasma samples for glucose concentration determination were thawed on ice and

evaluated according to the instructions provided with the commercial glucose oxidase assay kit

(Invitrogen, Amplex@ Red, Eugene OR). The sample plate was analyzed using a microplate

reader (BioRad, Hercules, CA).

Statistical Analyses

Statistical analyses were performed using StatView for Windows (SAS Institute, Cary,

NC, USA). Initial comparisons were made to determine if there was a significant tank effect

within treatments. F-tests were conducted to test variances among treatment groups for

homogeneity. If variance was heterogenous, data were loglo transformed to achieve

homogeneity of variance; however, all reported means (+ 1 SE) are from nontransformed data.

Analyses of variance (ANOVA) of weight, length and hormone concentration was used to

compare differences among treatment groups. If significance was determined (P < 0.05),

Fisher' s protected least-significant difference was used to determine differences among treatment

means.

Results

Morphology and Chemistry

The average fish weights in this experiment ranged from 4.13 to 4.55 kg, and the average

fish length ranged from 88.8 to 92.2 cm. Neither weight nor length was significantly different

among treatments, and there was no significant tank effect for any tested parameter. Water









chemistry parameters were tested on the day of the experiment and were as follows: un-ionized

ammonia (NH3), < 4.55 Clg/1; nitrite, < 0.2 mg/L; pH, 7.5; alkalinity, 200 mg/L; chloride

concentration, 85 mg/L; total hardness, 230 mg/L; and calcium hardness, 130 mg/L. Dissolved

oxygen concentrations were maintained at > 95% saturation throughout the trial and the

temperature was 240C.

Hormones

The 0-h plasma cortisol concentrations for treatments 1 and 2 averaged 6.65 f 3.58 and

4.63 f 1.02 ng/ml, respectively (Fig. 3-2A), and were statistically similar. The 0-h plasma

glucose concentrations were statistically similar and averaged 2. 13 f 0. 12 and 2.21 f 0. 11

mmol/L for treatments 1 and 2, respectively (Fig. 3-2B). The plasma concentrations of T, 1 1-

KT, and E2 WeTO Statistically similar at 0-h for treatments 1 and 2 and averaged 25.53 f 2.9, and

10.2 f 0.8 ng/ml and 672.4 f 45.9 pg/ml, respectively.

Plasma cortisol concentrations increased significantly (P < 0.05) in the Siberian sturgeon

from 0-h to the 1-h sampling period averaging 70.9 f 18.7 ng/ml at 1-h, and were not

significantly different between treatments 2 and 3 (Fig. 3-2A). Plasma glucose concentrations

increased significantly from 0-h to the 1-h sampling period and averaged 4.67 f 0.40 mmol/L at

1-h, and there were no significant differences among treatments 2 and 3 (Fig. 3-2B). At 4-h,

plasma cortisol concentrations were similar for Hish in treatments 2 (46.2 f 15.4 ng/ml) and 3

(36.27 f 14.0 ng/ml), but were significantly elevated compared with those observed for fish in

the treatment 4 group (10.44 f 2.53 pg/ml) (Fig. 3-2A). Plasma glucose concentrations at the 4-h

sampling period were similar for treatment 2 (4.70 f 0.27 mmol/L) and treatment 4 (4.14 f 0.38










mmol/L), but were significantly lower than plasma glucose concentration in treatment 3 (5.65 f

0.41 mmol/L) (Fig. 3-2B).

The evaluation of treatment 2, in which the same group of Eish at 0-h, 1-h and 4-h were

sampled, demonstrated that plasma T concentrations increased significantly from time 0 to 1-h

(20.3 f 1.76 and 31.45 f 4. 19 ng/ml respectively), with a subsequent decrease at 4-h to a

concentration similar to that observed at 0-h (Fig. 3-3A). In the same fish, we observed no

differences between bleeding times for E2 Or 11-KT (Fig. 3-3 B,C).

Discussion

The Siberian sturgeon that were exposed to capture and confinement stress exhibited

significantly elevated plasma cortisol concentrations 1-h after the initiation of stress, which

persisted throughout the 4-h sampling period. This response is similar to the reactions of other

fish species exposed to acute stressors (Thomas et. al., 1990). Cortisol and glucose have been

shown to be more sensitive to stress than most other plasma constituents except catecholamines,

and respond rapidly to a wide range of environmental stressors. Stress in Hish and the

concomitant increase in cortisol have been implicated in numerous physiological conditions

including impaired immune function (Tort et al., 1996), altered feeding behavior (Kentouri et al.,

1994), oxygen radical production (Ruane et al., 2002), and reproductive impairment (Pankhurst

and Van Der Kraak, 1997). Responses to stress are largely dependent on the severity and type of

environmental stressor. Previous studies with Siberian sturgeon exposed to acute and severe

hypoxia have shown significantly elevated plasma cortisol concentrations, with a peak

concentration of 35,000 pg/ml (Maxime et al., 1995). The basal cortisol concentration in that

study was approximately 5000 pg/ml, which is comparable to the basal cortisol concentration

obtained in this study. However, the peak concentration of cortisol in our study increased to









nearly 75,000 pg/ml, demonstrating the plasticity of the physiological stress response in this

species. In some species, plasma cortisol concentrations can persist for days if the stressor is

chronic or severe (Sumpter, 1997).

This study is distinct from other studies in several regards. This is the first study to

define the relationship between stress and potential reproductive function, as indicated by the

plasma concentrations of various sex steroids, in Caspian Sea sturgeon, habituated to a warm

environment and reared under commercial culture conditions from the egg stage. This is also the

first study to show the endocrine effects of surgical sexing, a procedure often necessary for

sturgeon and other species that do not exhibit sexually dimorphic characteristics. The induced

stressors in this study, caused by capture and confinement, bleeding, and surgical sexing are

common stressors in a laboratory or fishery environment, and it is important to understand what

effects these stressors can have on mitigating experimental responses.

In this experiment, fish underwent capture and confinement stress, with multiple

disturbances at 1-h and 4-h. It has been shown that serial stressors evoke cumulative

physiological stress responses in other fish species (Waring et al., 1997; Di Marco et al., 1999)

and multiple stress events cause fish to be more sensitive to additional acute stress (Ruane et al.,

2002). The multiple disturbances in this study likely mitigated the expected decreases in plasma

cortisol concentrations after 4-h, because in treatment 4, where fish were captured but not bled

until the fourth hour of capture, fish exhibited lower plasma cortisol concentrations than fish in

treatment 2 or 3. These lower concentrations could result from a more rapid return toward basal

concentrations, due to the lack of repeated stressors, or a reduced stress effect as they were not

bled initially, adding additional handling and blood loss to the stress. Our data indicate that

serial bleedings intensify the associated stress response, as evidenced by significantly lower









concentrations ofF in fish in which a blood sample was not drawn at 0-h or 1-h. This is an

important consideration for future studies of this species involving multiple blood samples.

Whether elevations in cortisol concentration for the serially bled fish are due to blood volume

loss or its associated stressors such as pricking of the fish with a needle, or longer handling times

to ensure that a fish is still for actual blood drawing versus sham drawing, is uncertain. It is

likely, however, that it is a combination of events, and not solely blood loss that leads to elevated

stress in serially bled fish. Note that surgical sexing, an invasive procedure that is often

necessary in aquaculture or fishery practice, did not induce a prolonged stress reaction, because

fish in treatment 4, which were similarly sexed at 0-h, exhibited plasma cortisol concentrations

similar to basal concentrations less than 4-h after the procedure.

The 0-h blood sampling period was started in the morning and the experiment was

concluded in the early afternoon. Cortisol concentrations in sturgeon (Belanger et al., 2001;

Lankford et al., 2003) and other animals (Young et al., 2004) have been shown to be highly

sensitive to diurnal variation, so care was taken in this study to ensure that all samples were

collected within a relatively short period to reduce the possibility of daily hormone fluctuations

as confounding variables. In addition to the concentrations of sex steroids, it has been shown

that plasma cortisol concentration can be altered depending on the reproductive stage in sturgeon

(Barannikova et al., 2000) and other species (Pickering and Pottinger, 1985). The female

sturgeon in this study were 3 years old, and although all female sturgeon had formed clearly

visible ovigerous lamellae or ovarian folds, none of them exhibited vitellogenic oocytes, and

they appeared to be in a similar reproductive stage. However, the plasma concentrations of sex

steroids in this study were similar to those of fish possessing fully vitellogenic oocytes in

subsequent studies.









Interestingly, the concentrations of sex steroids evaluated in this study did not demonstrate

an inverse relationship with stress as defined by plasma cortisol concentrations; in fact, plasma T

concentration was significantly elevated during periods of peak plasma cortisol concentration

(Fig. 3-3A). Although there have been no studies of this kind, in which stress and reproductive

function in Siberian sturgeon reared in commercial culture conditions are evaluated, this

response is distinct from that in published data with other fish species, including other sturgeon

species. Of the reproductive hormones, testosterone has been shown to be highly responsive to

stress-induced alterations in sturgeons and other species (Pickering et al., 1987; Bayunova et al.,

2002). Plasma E2 and 11-KT concentrations were not significantly affected by stress within the

timeframe of this study. In American alligators, certain environmental toxicants were found to

increase plasma T concentrations in juveniles, but did not affect the plasma concentrations of

other circulating hormones (Milnes et al., 2004). Our findings do not necessarily indicate,

however, that stress is not detrimental to the reproduction of this species. Circulating

concentrations of sex steroids are only one endpoint in the reproductive endocrine axis, and

stress can manifest itself at many levels of the steroidogenic pathway. For example, sex steroids

are generally removed from circulation via clearance by the liver. Reductions in sex steroid

production would not necessarily be reflected in circulating concentrations if clearance is

concomitantly affected. Other possible mechanisms that would result in the alteration of the

reproductive biology of this species include alterations in hypothalamic-pituitary stimulation or

alterations in transport mechanics (i.e., transport proteins).

Finally, the elevation in plasma T concentrations described here could be due to a technical

problem; that is, although commercial antibodies are screened for cross reactivity and specificity

to a wide range of steroids, little is known about the steroid milieu released during stress in










sturgeon. Although unlikely, it is possible that a unique androgen of adrenal origin is released

during stress in this species that cross reacts with the antibody used in the testosterone but not in

the 11-KT kits. Studies using advanced analytical chemistry could determine the steroids

released from stressed Siberian sturgeon. The data presented here indicate that the

concentrations of sex steroids in Siberian sturgeon do not show an inverse relationship with

elevated plasma cortisol concentration following acute stress, as has been observed for most fish.

This altered response needs further study, as this study differed from previous studies of sturgeon

in that it coupled sturgeon habituated to warm temperature with a specific stress response. This

is the first study to define the relationship between stress and endocrine function in cultured

Siberian sturgeon, a threatened and commercially important species. Future studies need to

address various aspects of the aquaculture environment (e.g., temperature and water quality),

reproductive stage (e.g., juvenile versus adult) and seasonality to determine which variables

modify the stress response and thus potentially alter growth and reproductive potential. This

work will also serve as a baseline to evaluate the effects of material water quality hazards, such

as nitrate, present in both natural and constructed environments.














~ Treatment 1


Treatment 2

Treatment 3

Treatment 4


To T1-h


Figure 3-1. Blood sampling times for Treatments 1 to 4 of fish held under confinement stress for
4 hours. Six female Siberian sturgeon were used for each treatment.











100-
A. 90bb

80
70 b
S 60
50
40
O 30
20 aa

10 -

0-hr 1-hr 4-hr
B.



bb b
5b




3 a a






0-hr 1-hr 4-hr

Blood sampling time



Figure 3-2. Plasma cortisol (A) and plasma glucose (B) concentrations (mean + S.E.M.) during a
4-h capture and confinement period. Means with the same superscript are not
significantly different (P > 0.05).














40b
S35
S30
25
S20
59 15



0-hr 1-hr 4-hr

B.

14
S12
10





0
n 0-hr 1-hr 4-hr

C.
900
800
700
~~600




100

0-hr 1-hr 4-hr

Blood sampling time


Figure. 3-3. Sex steroid data for treatment 2. Plasma 17P-Estradiol (A), testosterone (B), and
11l-ketotestosterone (C) taken from serial bleeds of cultured female Siberian sturgeon
throughout the 4-h period of confinement stress (mean + 1 S.E.M.). Fish were
serially bled at 0-h, 1-h and 4-h (see Fig. 3-1 legend for a description of treatment 2
bleeding times). Means with the same superscript or no superscript are not
significantly different (P > 0.05).









CHAPTER 4
NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN
STURGEON

Introduction

The endocrine disrupting actions of various chemical contaminants have become a

significant concern for comparative endocrinologists (Colborn et al., 1993; Guillette and Crain,

2000). A growing literature describes the effects of endocrine disrupting contaminants (EDCs)

for both terrestrial (Iguchi and Sato, 2000) and aquatic (Sumpter, 2005; Milnes et al., 2006)

species. These effects include altered reproductive morphology, endocrine physiology and

behavior, and involves such endpoints as reduced phallus size, decreased sperm count, depressed

reproductive behaviors and altered circulating concentrations of sex steroids (e.g., Guillette et al.,

1999; Orlando et al., 2002; Toft and Guillette, 2005). EDCs exert their effects by mimicking

hormones, acting as hormone antagonists, altering the function or concentration of serum-

binding proteins, or altering the synthesis or degradation of hormones. Aquatic organisms can

receive continuous exposure to environmental contaminants throughout their lives, as the aquatic

environment receives most of the intentionally released environmental pollutants. Thus, the

effects of EDC exposure on aquatic life have received considerable attention (Kime, 1999;

McMaster, 2001; Sumpter, 2005; Milnes et al., 2006).

Although nitrate is a ubiquitous component of aquatic environments, and has become a

global pollutant in a variety of aquatic systems (Sampat, 2000), it has only recently begun to

receive attention for its ability to alter endocrine function (Guillette and Edwards, 2005). The

toxicological effects of nitrate have long been known. As early as 1945, nitrate induced

methemoglobinemia (Blue Baby Syndrome) in humans was associated with drinking well water

contaminated with nitrate (Comly, 1945). Fish are also vulnerable to methemoglobinemia

(Brown Blood Disease), and in Siberian sturgeon methemoglobinemia has been associated with a










significant chloride imbalance (Gisbert et al., 2004). Toxicity studies with fish (LCso) have

shown lethal concentrations of nitrate to range an order of magnitude or more (Brownell, 1980;

Pierce et al., 1993; Hamlin, 2006), demonstrating significant plasticity in response to elevated

nitrate among fish species.

Sublethal effects of nitrate include endocrine alterations which have been shown to alter

metabolism, reproductive function and development. Frogs (Rana ca~scadae) exposed to 3.5

mg/L nitrate-N metamorphosed more slowly, and emerged from the water in a less developed

state than control animals (Marco and Blaustein, 1999). Rodents exposed to nitrate (50 mg/L

NaNO3) in their drinking water had significantly lower circulating testosterone (T)

concentrations than control animals (Panesar and Chan, 2000). Bulls given oral administration

of nitrate (100 250 g/day/animal) showed reduced sperm motility, depressed Leydig cell

function, and degenerative lesions in the germ layers of the testes (Zraly et al., 1997). Studies in

Southern toad tadpoles showed nitrate induced alterations in growth and thyroxine

concentrations were mitigated by the source of culture water used, indicating that environmental

context plays a significant role in mitigating the effects of nitrate (Edwards et al., 2006a).

Mosquitofish (Gamnbusia holbrooki) experienced significant reproductive alterations, such as

reduced gonopodium length and fecundity (number of females per unit of female size), in nitrate

concentrations as low as 5 mg/L NO3-N (Toft et al., 2004; Edwards et al., 2006b). Proposed

mechanisms for nitrate induced steroidogenic disturbances include mitochondrial conversion to

nitric oxide (NO), altered chloride ion concentrations and altered enzymatic action by binding to

the heme region of P450 enzymes associated with steroidogenesis (Guillette and Edwards, 2005).

Stress effects on reproduction can be manifest at various levels of the reproductive

endocrine axis, and stress has been shown to have inhibitory effects on reproduction for most










aquatic species studied to date (Pickering et al., 1987; Carragher and Sumpter, 1990; Pankhurst

and Van Der Kraak, 1997; Consten et al., 2002). For many species of fish, including sturgeon

and other chondrosteans, cortisol is the primary stress hormone (Idler and Sangalang, 1970;

Barton et al., 1998) and cortisol has been implicated in mediating the inhibitory reproductive

effects induced by stress (Pankhurst and Van Der Kraak, 1997; Semenkova et al., 1999;

Bayunova et al., 2002). There is evidence in teleosts, however, that the estrogenic inhibitory

effects of stress are not mediated by cortisol and that the effects arise higher in the reproductive

endocrine pathway (Pankhurst et al., 1995). Tilapia (Oreochromis mossamnbicus) fed pellets

containing cortisol to achieve plasma cortisol concentrations typical of acutely stress fish,

resulted in decreased plasma concentrations of T and 17P-estradiol (E2), reduced oocyte diameter

and gonad size in females, and reduced plasma T concentrations in males (Foo and Lam,

1993a,b). Female brown trout (Salmo trutta) exposed to 2 weeks of confinement stress had

significantly reduced plasma T concentrations compared to unstressed fish (Campbell et al.,

1994). Plasma glucose concentrations have also been shown to be reliable indicators of

secondary stress responses. An animal under chronic stress can demonstrate a reduced capacity

to handle subsequent stress events, and studies have shown responses of fish to multiple stressors

are cumulative (Barton et al., 1986). Fish residing in laboratories or fish farms are often

subj ected to chronic stress (sub-optimal water chemistry, crowding, confinement) followed by

acute stress events (sampling, netting), which can lead to dramatic and prolonged stress

responses (Rotllant and Tort, 1997; Heugens et al., 2001).

Sturgeon are among the most ancient groups of Osteichthyes, and twenty-five extant

species occupy the Northern Hemisphere (Birstein, 1993). The dramatic decline in sturgeon

populations due to overfishing, pollution, and habitat degradation have led to the necessity of









commercial aquaculture as a means to provide animals for stock enhancement, as well as food

production, reducing pressures on wild populations (Beamesderfer and Farr, 1997; Waldman and

Wirgin, 1997; Williot et al., 2002; Chebanov et al., 2002). The Siberian sturgeon is one of the

leading species of sturgeon adapted to aquaculture (reviewed by Gisbert and Williot, 2002). It

was recently discovered that Siberian sturgeon are more sensitive to nitrate toxicosis than most

fish species reported to date (Hamlin, 2006). Further, Siberian sturgeon juveniles become less

tolerant to nitrate as they grow, a finding of considerable importance for the commercial culture

of this species, since adult populations reared in recirculation systems often experience higher

nitrate concentrations than their juvenile counterparts. Although understanding what

concentrations of nitrate are necessary to avert mortality is generally understood in commercial

aquaculture, mortality is not an effective endpoint for producers interested in optimizing growth

and reproductive function. Understanding nitrate's effects on reproductive function is especially

critical to sturgeon, whose economic viability relies heavily on proper endocrine function,

notably the production of eggs (caviar).

The purpose of this study is to begin to determine the potential effects of elevated

environmental nitrate on endocrine function, and investigate whether elevated nitrate alters the

stress response in captive female Siberian sturgeon.

Methods

Fish and Sampling Procedures

Siberian sturgeon were collected from four 30,000 liter tanks, from separate commercial

recirculating aquaculture systems at Mote Marine Laboratory's Aquaculture Park (Commercial

Sturgeon Demonstration Project) in Sarasota, FL. Water chemistry in each of these systems was

analyzed weekly for ammonia, nitrite, nitrate, and pH prior to commencement of the

experiments. Dissolved oxygen and temperature were monitored continuously with stationary










probes, which were spot-checked bi-weekly for calibration with portable probes. Hardness,

alkalinity and chloride were analyzed the day prior to commencement of the experiment.

The sturgeon were pulled by hand at the side of the tank and immediately held down on a

padded V-shaped surgical table. Pulling the fish from the tank by hand (versus netting)

decreased the likelihood of stressing Eish remaining in the tank and allowed for more immediate

access to the fish for blood sampling. Blood was extracted from the caudal vein (5 ml) with a 10

ml syringe (20 gauge needle) within 1 minute of capture; most captures took 30 seconds for the

full sample to be drawn. The blood was placed into lithium heparin VacutainerTM tubes, and

stored on ice for no more than 30 minutes before centrifugation. The plasma was separated via

centrifugation (5 10 min at 2000 g), transferred to cryovials, flash frozen in liquid nitrogen and

stored at -800 C for 1 3 weeks prior to analysis.

Surgical Sexing

For surgical sexing, the fish were anesthetized in a 5 80 C water bath containing carbon

dioxide (CO2) gaS; CO2 WAS used because it is a low regulatory priority anesthetic for fish that

are grown for food production and requires no withdrawal period; the sturgeon used in this study

were part of a commercial food production program. Pure oxygen gas administered through a

fine air stone was used to maintain a dissolved oxygen concentration of 9.0 13.0 mg/L, and

sodium bicarbonate was added to maintain a pH of 6.8 7.6 in the bath throughout the procedure.

Fish generally took 3 5 minutes for full anesthetization. A 2.5 3.5 cm incision was made on

the ventral side of the fish, approximately 8 cm anterior to the vent, along the median axis to

allow inspection of the gonads on either side of the fish for sex determination. The fish was

sutured closed with coated vicryl absorbable suture (Ethicon Inc., Somerville, New Jersey).












Experiment 1

Experiment 1 was conducted in July of 2004 and consisted of two treatments, which

sampled fish from each of four commercial culture tanks (30,000 1 each) located in separate

recirculating systems at Mote Marine Laboratory' s Aquaculture Park. Two of the culture tanks

were held at a nitrate concentration of 11.5 mg/L nitrate-N (50 mg/L total nitrate) for one month,

and the other two tanks were held at 57 mg/L nitrate-N (250 mg/L total nitrate) for the same time

period (two replicates each). Nitrate concentrations were achieved by adjusting the freshwater

input to each system, typical of commercial culture practices. Prior to the 1-month exposure,

nitrate concentrations in the four study tanks oscillated between 20 60 mg/L nitrate-N routinely.

A nitrate concentration of 57 mg/L nitrate-N was chosen as the upper limit in this study, as this is

the maximum concentration deemed safe, defined by feeding behavior and mortality, at Mote' s

Commercial Sturgeon Demonstration Project. The lower concentration of 11.5 mg/L nitrate-N~

was chosen as this was considered extremely safe, yet realistically achievable under normal

aquaculture practices. Although these concentrations may be typical of commercial recirculating

aquaculture facilities, these levels are elevated relative to environmental levels or approved

drinking water limits of 10 mg/L nitrate-N (U.S EPA, 1996).

Treatment 1 sampled 15 Eish from each of the four commercial recirculating culture tanks

(two tanks/nitrate concentration; N = 30 per nitrate treatment). Each Hish was sampled at time 0

and was surgically sexed immediately after the blood sample was drawn. Only blood samples

from female fish were used in the analyses for this study. Each Hish was weighed and placed

into a holding tank until treatment 2 Eish were removed, to avoid stressing fish remaining in the

tank.









Treatment 2 sampled 18 Eish from each of the four commercial recirculating culture tanks

(N = 36 per nitrate treatment). Fish were sampled at time 0, and were then placed into square

0.64 m3 inSulated plastic totes (one tote per nitrate concentration) Eilled with 530 L of system

water for a 6-h period of confinement stress. A numbered tag (DuffexTM, St. Paul, MN) was

placed on the pectoral fin of each Hish for identification. Fish were bled at 1 and 6 h during the

confinement period (Fig. 4-1). After the 6-h sampling period, the fish were surgically sexed as

previously described.

Experiment 2

Experiment 2 was conducted in May of 2005 and was procedurally identical to

experiment 1 with the following exceptions. Two of the culture tanks were held at a nitrate

concentration of 1.5 mg/L nitrate-N (6.5 mg/L total nitrate) for one month, and two tanks were

held at 57 mg/L (250 mg/L total nitrate) for the same time period. It should be noted that

although the same tanks and population (different individuals) of animals was used in this second

experiment, the tanks that previously held the low nitrate concentrations in experiment 1, now

held the elevated nitrate concentration and vice versa, to reduce the possibility of tank affect

among treatment groups. The exposure in the first experiment should not affect the fish in either

nitrate group in the second experiment, since nitrate concentrations typically oscillate in the

range of the upper limit (57 mg/L nitrate-N) and the lower limit (1 1.5 mg/L nitrate-N) routinely

in recirculating aquaculture settings, including our facility. Although 11.5 mg/L nitrate-N is

considered low in commercial aquaculture, this concentration exceeds that which would occur in

unpolluted natural environments. Therefore 1.5 mg/L nitrate-N was chosen in this experiment as

it would be more reflective of ecologically relevant exposures. Treatment 1 sampled 15 fish

from each of the four commercial recirculating culture tanks (N = 30 per nitrate treatment) and

treatment 2 sampled 25 fish from each of the four tanks (N = 50 per nitrate treatment).









Hormone Evaluations

Plasma samples were thawed on ice, and the steroid fraction was extracted twice with

diethyl ether. Plasma cortisol (F), E2 (experiment 1), T and 11-KT were analyzed according to

instructions provided with the commercial competitive enzyme immunoassay kits (Cayman

Chemical, Ann Arbor, MI), specific to each hormone. Each hormone was previously validated

for Siberian sturgeon by verifying that serial dilutions were parallel to the standard curve.

Samples were run in duplicate and each plate contained duplicate wells for interassay variance

and a blank. Individual hormones were all run with plates from the same kit lot number and

were completed in the same testing session to reduce testing variance. Sample plates were

analyzed with a plate reader (BioRadTM, Hercules CA). Glucose was evaluated with an

AmplexTM Red glucose/glucose oxidation kit (InvitrogenTM, CaTISbad, CA).

Radioimmunoassays for E2 (validated for Siberian sturgeon) in experiment 2 were

conducted as described previously by this lab (Milnes et al., 2004). Briefly, extracted samples

were reconstituted in Borate Buffer (50 ul, 0.05 M, pH 8.0). Antibody (Endocrine Sciences,

Tarazana, CA, USA) and radiolabeled steroid (2, 4,6,7, 16,17-3H) were added at 12,000 cpm per

100 C1l. Interassay variance tubes were similarly prepared from pooled Siberian sturgeon plasma.

Standards were prepared in duplicate at 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 and 800

pg per tube. Assay tubes were incubated at 40C overnight. Bound free separation was

performed by adding charcoal and centrifuging for 30-min. The supernatant was then drawn off

and diluted with scintillation cocktail and counted on a Beckman LS 5801 scintillation counter.










Statistical Analyses

Statistical analyses were performed using StatView for Windows (SAS Institute, Cary,

NC, USA). Initial comparisons were made to determine significance within treatments. F-tests

were conducted to test variances among treatment groups for homogeneity. If variance was

heterogenous, data were loglo transformed to achieve homogeneity of variance, however, all

reported mean (f 1 SE) values are from non-transformed data. Analyses of variance (ANOVA)

of weights and hormone concentrations were used to compare differences among treatment

groups. If significance was determined (p < 0.05), Fisher' s protected least-signifieant difference

was used to determine differences among treatment means.

Results

Experiment 1

In treatment 1, of the 30 fish sampled and sexed in each nitrate concentration, 19 were

females in the 11.5 mg/L nitrate-N group, and 18 were females in the 57 mg/L nitrate-N group.

Of the 36 Eish sampled and sexed in each nitrate concentration for treatment 2, 16 were females

in the low nitrate group, whereas 13 were females in the high nitrate group. The average weight

for females in treatment 1 was 4.16 f 0.53 kg whereas females sampled in treatment 2 was 4.29

f 0.36 kg. There were no significant differences among the tanks within each nitrate group for

any tested parameter.

Water chemistry parameters were tested the day of experimentation and were as follows:

unionized ammonia (NH3) < 4.35 Clg/L, nitrite <; 0. 15 mg/L; pH 7.4, alkalinity 230 mg/L,

chloride 94 mg/L, total hardness 240 mg/L and calcium hardness 140 mg/L. Dissolved oxygen

concentrations were maintained at > 95% saturation throughout the trial and temperature was

23.3 oC.









Time 0 females in treatment 1 were combined with time 0 females from treatment 2 to

evaluate the effects of nitrate exposure for each experiment. Fig. 4-2 and 4-3 illustrates time 0

data for each hormone for experiment 1. Initial concentrations of plasma F or glucose were not

different between females in the 11.5 and the 57 mg/L nitrate-N groups, averaging 5.95 f 1.08

ng/ml and 255.9 f 6.8 pg/ml respectively. Plasma T, 11-KT and E2 COncentrations were

significantly elevated in the 57 mg/L nitrate-N group when compared to concentrations observed

in females exposed to 11.5 mg/L nitrate-N (p < 0.05).

Data for plasma F and glucose concentrations in treatment 2 are shown in Fig. 4-4. There

was no significant difference in the stress response, defined by plasma F concentrations, when

the females exposed to the two nitrate concentrations were compared. The females in both the

11.5 mg/L and 57 mg/L nitrate-N concentration groups demonstrated a dramatic increase in

plasma F concentrations at the 1-h sampling period averaging 42.0 f 5.7 ng/ml, followed by a

significant decrease at the 6-h sampling period. The 6-h plasma F concentrations were still

significantly elevated when compared to time 0 concentrations (11.5 f 1.7 ng/ml). Plasma

glucose concentrations were similar for both nitrate groups at time 0 and 1-h, averaging 227.5 f

12.2 pg/ml at time 0, and rising significantly to an average of 428 f 17.5 pg/ml by 1-h. The 1 1.5

mg/L nitrate-N concentration group females demonstrated a significant increase in plasma

glucose from time 1-h to 6-h (517.6 f 19 pg/ml at 6-h), whereas the 57 mg/L nitrate-N

concentration group females exhibited no increase in plasma glucose between the 1-h and 6-h

sampling period (427.9 f 25.1 pg/ml). During the six hour captive stress period, we observed no

significant changes in plasma T, 11-KT or E2 COncentrations with plasma concentrations within

each respective nitrate concentration averaging 10.9 f 0.8 ng/ml, 4.4 f 0.4 ng/ml and 784 f 16.6

pg/ml respectively.










Experiment 2

In treatment 1, of the 30 fish sampled and sexed in each nitrate concentration, 14 were

females in the 1.5 mg/L nitrate-N group, and 12 were females in the 57 mg/L nitrate-N group.

Of the 50 fish sampled and sexed in each nitrate concentration for treatment 2, 22 were females

in the 1.5 mg/L nitrate-N group, and 24 were females in the 57 mg/L nitrate-N group. The

average weight for females in treatment 1 was 5.84 f 0.89 kg and the average weight for females

sampled in treatment two was 6.14 f 1.10 kg. There were no significant differences among the

tanks within each nitrate group for any tested water parameter.

Water chemistry parameters were tested the day of experimentation and were as follows:

unionized ammonia (NH3) < 5.35 Clg/L, nitrite <; 0.20 mg/L; pH 7.6, alkalinity 240 mg/L,

chloride 90 mg/L, total hardness 240 mg/L and calcium hardness 135 mg/L. Dissolved oxygen

concentrations were maintained at > 95% saturation throughout the trial and temperature was

23.5 oC.

Time 0 females in treatment 1 were combined with time 0 females from treatment 2 to

evaluate the effects of nitrate exposure for each experiment. Fig. 4-5 and 4-6 illustrates time 0

data for each hormone for experiment 2. Plasma F concentrations were not significantly

different among females when the 1.5 mg/L or the 57 mg/L nitrate-N groups were compared at

time 0. Plasma T concentrations were significantly elevated in the 57 mg/L nitrate-N

concentration group (p = 0.010), with an average of 17.28 f 4.57 ng/ml for the 1.5 mg/L nitrate-

N group, and 31.17 f 4.57 for the 57 mg/L nitrate-N group. Plasma 11-KT concentrations were

not significantly different for either nitrate group at time 0 (p = 0.091) with an average of 8.5 f

2.1 ng/ml for the 1.5 mg/L nitrate-N group, and 13.3 f 2.9 ng/ml for the 57 mg/L nitrate-N

group.









Data for treatment 2 is shown in Fig. 4-7. There was no significant difference in plasma F

concentrations between nitrate groups. Initial plasma F concentrations averaged 6.9 f 1.1 ng/ml,

rose to an average of 68.1 f 6.2 ng/ml at the 1-h sampling period and dropped to an average of

26.8 f 2.6 ng/ml by 6-h. Plasma F concentrations were significantly different for each sampling

period. There was no significant difference in stress response for plasma T or 11-KT for

treatment 2 with plasma concentrations averaging 26.4 f 1.9 ng/ml and 11.7 f 1.4 ng/ml

respectively, across all sampling periods.

Discussion

Absent from most investigations assessing the endocrine disrupting effects of

environmental pollutants on aquatic inhabitants, have been studies examining the effects of ions,

such as nitrate and nitrite, which are ubiquitous components of most aquatic ecosystems.

Anthropogenic activities have dramatically impacted the amount of nitrogenous compounds

entering freshwater systems, and recent reports have identified agricultural non-point source

pollution, often caused by nitrate laden fertilizers, as the leading cause of water quality

deterioration to freshwater systems (Sampat, 2000).

This paper describes the effects of a chronic 30 day exposure of Siberian sturgeon to

elevated nitrate on circulating concentrations of plasma glucocorticoids (F and glucose) and sex

steroids (T, 11-KT, and E2). Results of the first experiment, in which animals were exposed to

concentrations of 11.5 and 57 mg/L nitrate-N (50 mg/L and 250 mg/L total nitrate respectively),

revealed significantly elevated concentrations of plasma T, 1 1-KT and E2 in animalS exposed to

the higher nitrate concentration. Experiment 2, which evaluated the effects of animals exposed

to 1.5 and 57 mg/L nitrate-N (6.6 and 250 mg/L total nitrate respectively), also demonstrated an

elevated concentration of plasma T and E2 in animalS exposed to the higher nitrate concentration.









Although the results of Experiment 2 did not demonstrate a significant elevation in plasma 1 1-

KT concentration (p = 0.09) as shown in Experiment 1 (p = 0.05), it should be noted that the

second experiment was conducted at a slightly different time of the year, and in animals which

were almost 1-yr older. Seasonal variation and stage of reproductive development can have

significant impacts on steroid profiles of most fish species (Stacey et al., 1984).

This is the first study to demonstrate a nitrate-induced elevation in concentrations of

plasma sex steroids, using a Caspian Sea sturgeon species habituated to a warm environment,

typical of commercial culture. Since small-scale trials do not always reflect the scale-up

challenges of commercial culture environments, or mimic similar effects on physiologic

response, this experiment is unique in that it was conducted at a commercial farm under typical

culture conditions. This study is also distinct in that it used naturally occurring nitrate produced

by nitrification, to achieve desired nitrate concentrations, versus altering the nitrate environment

by chemical addition (e.g. sodium nitrate).

It has been proposed that nitrates and nitrites disrupt endocrine function by entering

steroidogenic tissues, where they are metabolized to nitric oxide (NO). NO possesses the ability

to bind to the heme moiety of the cytochrome P450 enzymes, which are present at multiple

locations along the steroidogenic pathway. The mechanism by which nitrate has led to the

elevated concentrations of plasma sex steroids seen in this study is unclear, and more work is

necessary to understand the mechanisms involved. Nitrate induced elevations in plasma

concentrations of sex steroids does not necessarily imply that nitrate is not detrimental to the

reproductive health of this species. Concentrations of circulating plasma sex steroids are only

one endpoint in the reproductive-endocrine axis, and disruptions can occur which will not be

manifest at the level of circulating steroids. I offer three potential explanations for the elevations










in plasma concentrations of sex steroids seen in this study. First, nitrate triggered an up-

regulation of steroidogenic function resulting in increased gonadal synthesis of sex steroids.

Second, nitrate induced alterations to transport proteins hamper transport to the liver and

concomitantly affect clearance. And lastly, elevated nitrate may impair liver function, thereby

reducing its ability to clear these steroids from the blood.

The female fish in this study demonstrated increased plasma concentrations of androgens,

as well as E2. COnsiderable attention in the literature evaluating the effects of endocrine

disrupting contaminants on aquatic animals has been directed at the estrogenic effects of

compounds, because many effects reported in wildlife populations are a consequence of the

feminization of males (Stoker et al., 2003; Sumpter 2005; Milnes et al., 2006). However, a

growing literature recognizes that populations of female fish exposed to environmental

contaminants exhibit masculinized features (Parrott et al., 2004). Toft et al. (2004) found that

female mosquitofish (Gamnbusia holbrooki) exposed to paper mill effluent exhibited

masculinized anal fins, and exhibited lower fecundity (number of embryos per unit of female

size) than reference fish. 17P-trenbolone is an anabolic steroid used to promote growth in beef

cattle and has shown strong androgenic activity, and is thought to be the cause of reproductive

alterations in fish living downstream from animal feedlot operations (Jegou et al., 2001; Wilson

et al., 2002; Orlando et al., 2002). It is unclear what effects elevated androgens, or estrogens for

that matter, have on Siberian sturgeon reproduction, and this lab is currently investigating the

mechanisms involved.

In aquaculture systems, nitrate has been neglected as a material water quality hazard.

Commercial aquaculture operations have traditionally used large influxes of water to maintain

water chemistry, and it is not uncommon to have water exchanges of 100% or more per day.









Consequently, nitrate has not traditionally been a concern in commercial aquaculture since this

flush rate is sufficient to maintain relatively low nitrate concentrations. Water is rapidly

becoming recognized as a valuable and limited resource, and legislative mandate is becoming

more stringent in its limits of the amount of water which may be consumed or discharged. As

aquaculture attempts to keep pace with global demand, the growing number of aquaculture

operations will be forced to utilize recirculating aquaculture technology, and significantly reduce

the heavy water usage in current practice. Nitrification systems are well understood in

aquaculture, and are decidedly effective at reducing ammonia and nitrite to nitrate (Timmons,

2001). In recirculating aquaculture systems with limited water exchange, nitrate can rise to

concentrations far in excess of those of natural environments, and it is unclear what impact these

concentrations can have on species residing in these environments. Understanding the sublethal

effects of exposure to nitrate is especially critical to sturgeon, whose economic viability relies

heavily on proper egg production and reproductive performance.

Fish are highly sensitive to the chemical influences in their environment, and negative

influences are often reflected in an acute stress response, indicated by elevations in

concentrations of glucocorticoids (Guillette et al., 1997). Stress in fish, and the concomitant

increase in plasma F concentrations, has been implicated in numerous physiological maladies,

including reproductive impairment (Pankhurst and Van Der Kraak, 1997). Stress induced effects

on reproduction include decreased plasma concentrations of sex steroids, depressed vitellogenin

production and decreased gamete quality (Pankhurst and Van Der Kraak, 1997). Although

plasma concentrations of sex steroids were significantly elevated in the groups of fish exposed to

57 mg/L nitrate-N, time 0 plasma F and glucose concentrations were not affected by nitrate









concentration in this study, indicating that the alterations to concentrations of plasma sex steroids

were unlikely to be mediated by glucocorticoid action.

Induced stress in both experiments in this study, caused by confinement and associated

blood sampling stressors, caused a dramatic increase in plasma F concentrations after 1-h, with a

significant decrease by the 6-h sampling period; this response was not influenced by nitrate

concentration in this study. Previous studies with gilthead sea bream (Sparus aurata) have

shown a decreased acute stress response in chronically stressed fish, speculating that the reduced

plasma F response likely resulted from negative feedback of mild but chronically elevated F

caused by the confinement stressor on the hypothalamic-pituitary-interrenal axis (Barton et al.,

2005). Since the initial blood samples (time 0) were taken generally within 30 s of capture, it is

likely initial concentrations of plasma F seen in this study (= 6 ng/ml) are representative of basal

plasma F concentrations of captive sturgeon in our facility. Previous studies with Siberian

sturgeon exposed to severe hypoxic stress, demonstrated peak plasma F concentrations of 35

ng/ml (Maxime et al., 1995). Peak concentrations of plasma F in our study rose to over 40 ng/ml

in one experiment, and nearly 70 ng/ml in the second experiment, demonstrating the plasticity of

physiological response for this species. Nitrate in this study was shown to alter at least one

component of the stress response, defined by plasma glucose concentrations, during a 6-h period

of confinement stress.

In conclusion, elevated nitrate is capable of altering the steroid profiles of cultured female

Siberian sturgeon, and is able to alter the secondary stress response, defined by plasma glucose

concentrations. We also show that responses to nitrate can change over time, and more work is

necessary to uncover the mechanisms involved in steroid alterations seen in this study, as well as

understand the impact these effects may have on reproductive performance.












Y Treatment 1

Treatment 2
To T1-hr T6-hr


Figure 4-1. Blood sampling times for treatments 1 and 2 of fish held under confinement stress for
6-h.













8

6
-
-4
o

-,
05~


11.5 mg/1 57 mg/1


11.5 mg/L 57 mg/L


Figure 4-2. Plasma cortisol (A) and glucose (B) concentrations (mean + 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
11.5 or 57 mg/L nitrate-N (n = 35 and n = 31 respectively). Means with no
superscript are not significantly different (p > 0.05).














14 b 6-b
S12 a5 a




10 a,0
115m / 7 g11 m / 7m /

C.
900B -
800 I
a 700- -y
600 -




0
11.5 mg/1 57 mg/1 15m/ 7m/


Nirt-Ncnenrto


Figur 4-.Pam etseoe() 1kttsoteoe()adetail()cnetain
(ma ... ncutrdfml ieia tren(cpnerbei xoe o
30 0 dy ocnetain f1. r5 gLntaeN( 5adn=3
repcivl) Suesrpsdsgae infcnl ifretvle p<00)










601 b o Time 0
A. b I Time 1-hr
J- 50 m Time 6-hrs
~5 40
.w 30
0 20a
10


11.5 57 mg/L


B.


a 12 bb b
B 10
E8a

o 4
O3 2

11.5 mg/L 57
mm II
Nitrate-N concentration



Figure 4-4. Plasma cortisol (A) and glucose (B) concentrations (mean + 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
11.5 or 57 mg/L nitrate-N (n = 16 and n = 13 respectively). The fish were bled at
time 0, 1-h and 6-h during a 6-h period of confinement stress. Means with the same
superscript are not significantly different (p > 0.05).













10

E 8-

O
.w4

S2-


1.5 mg/1 57 mg/1

B.
7.2
S7.0
0 6.8 -
E~ 6.6
8 6.4
o 6.2
O3 6.0
5.8
1.5 mg/L 57 mg/L

Nitrate-N concentration




Figure 4-5. Plasma cortisol (A), glucose (B) testosterone concentrations (mean + 1 S.E.M.) in
cultured female Siberian sturgeon (Acipenser baeri) exposed for 30 days to
concentrations of 1.5 or 57 mg/L nitrate-N (n = 36 for both nitrate groups). Means
with no superscript are not significantly different (p > 0.05).




















1.5 mg/L 57 mg/L


1.5 mg/L 57 mg/L


600
500
400
300
200
100


1.5 57 mg/L
m es I
Nitrate-N concentration


Figure 4-6. Plasma cortisol testosterone (A), 11-ketotestosterone (B) and estradiol-17P (C)
concentrations (mean + 1 S.E.M.) in cultured female Siberian sturgeon (Acipenser
baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N (n = 36 for
both nitrate groups). Superscripts designate significantly different values (p < 0.05).










o Time 0
I Time 1-hr
90 b I Time 6-hrs
80 -1 b
70
j~ 60
r 50
S40 c

20 -
10 -
20


1.5 mg/1 57 mg/1


12- c c

10

28



04-




1.5 57
mg/L mg/L

Figure 4-7. Plasma cortisol (A) and glucose (B) concentrations (mean + 1 S.E.M.) in cultured
female Siberian sturgeon (Acipenser baeri) exposed for 30 days to concentrations of
1.5 or 57 mg/L nitrate-N (n = 22 and n = 24 respectively). The Hish were bled at time
0, 1-h and 6-h during a 6-h period of confinement stress. Means with the same
superscript are not significantly different (p > 0.05).










CHAPTER 5
EFFECTS OF NITRATE ON STEROIDOGENIC GENE EXPRESSION IN CAPTIVE
FEMALE SIBERIAN STURGEON

Introduction

Environmental contaminants capable of altering steroidogenic regulation and function are

well documented in the literature for both terrestrial and aquatic inhabitants (Guillette and

Gunderson, 2001; Mills and Chichester, 2005; Sumpter, 2005; Edwards et al., 2006c). These

endocrine disrupting contaminants (EDCs) can exert their effects through numerous

physiological mechanisms including mimicking naturally occurring steroids, altering hormone

synthesis and degradation and interacting directly with steroid receptors (vom Saal et al., 1995;

Rooney and Guillette, 2000). In the latter case, EDCs can either stimulate (Parks et al., 2001) or

inhibit (Kelce et al., 1995) the expression of the target genes for that receptor. The endocrine

system is responsible for numerous physiological processes, and as such, perturbations to this

system have the potential to deleteriously affect reproductive and developmental performance of

the affected organism.

Stress has also been shown to alter endocrine function, and is generally negatively

correlated with concentrations of sex steroids (Pankhurst and Van Der Kraak, 1997; Orlando et

al., 2002). Cortisol, a predominant glucocorticoid, is the most commonly accepted plasma

indicator of the degree to which an animal is stressed and has been associated with inhibitory

effects on reproduction (Pankhurst and Van Der Kraak, 1997). Commonly studied stressors in

fishes include capture and confinement or handling and alterations to various environmental

parameters such as temperature, pH or salinity (Pankhurst and Dedual, 1994). Certain

contaminants, however, have also been shown to increase plasma glucocorticoid concentrations,

further contributing to the suppression of circulating sex steroids (Schreck and Lorz, 1978).









In the United States, the input of nitrogen from terrestrial agriculture has increased 20-

fold in the past 50 years (Pucket, 1995). Aquatic nitrate concentrations of over 100 mg/L have

been reported in some locations (Kross et al., 1993; Rouse et al., 1999), a ten-fold increase over

the U.S. drinking water standards of 10 mg/L NO3-N (EPA, 1996). A growing body of literature

implicates agricultural non-point source pollution as the leading cause of these elevations in

freshwater systems, posing a direct health risk to both humans and wildlife (Sampat, 2000). A

global pollutant of aquatic habitats, the ubiquitous presence of nitrate has only recently begun to

receive attention for its ability to alter endocrine function, and now j oins the list of

environmental contaminants implicated in reproductive dysgenesis (see review by Guillette and

Edwards, 2005). Unlike most environmental endocrine disrupting contaminants, nitrate is

unique in that it exists naturally at low concentrations in the aquatic environment as the

degradative end product of nitrification. Therefore, the physiological disruptive actions of nitrate

stem from its relative concentration, as well as its interactions within the environment in which it

persists (Edwards et al., 2006a).

The seafood trade deficit in the United States is exceeding eight billion dollars annually,

a natural resource deficit second only to oil and natural gas in magnitude. With the oceans at or

exceeding their maximum sustainable yields for 75% of commercially relevant species,

aquaculture, or the culture of fish and other aquatic organisms, has been proposed as the only

viable alternative to keep pace with global demand (FAO, 2004). Like seafood, water is also

becoming a limited and increasingly valuable resource, and the necessary increase in aquaculture

operations will not be afforded the liberal quantities of water permitted to established facilities.

Although recirculating aquaculture facilities, which recycle and reuse a significant

portion of their water, are becoming increasingly common, the limiting factor for water exchange









for most of these facilities is nitrate. Work is ongoing to develop technologies to reduce nitrate

in commercial aquaculture, but it is still unclear what concentrations of nitrate are safe,

especially for sensitive physiological systems such as the endocrine system which have been

shown to be vulnerable to the effects of nitrate (Suzuki et al., 2003; van Rijn et al., 2006).

Sturgeon species are ideally suited to serve as models to study the endocrine disruptive

effects of elevated nitrate exposure. Many species are commercially viable, highly endangered

and have documented sensitivities to environmental contaminants, including nitrate (Akimova

and Ruban, 1995; Dwyer et al., 2005; Hamlin, 2006). The Caspian Sea, which houses some of

the most endangered sturgeon species, is becoming increasingly affected by contaminants

(Birstein, 1993; Stone, 2002) many of which are implicated in the disruption of reproduction in

sturgeon species (Akimova and Ruban, 1995).

It has been proposed that aquaculture, incorporating the development of captive broodstock

programs, could be the best solution to reduce Hishing pressures, facilitating recovery of wild

populations (Williot et al., 2002). The economic viability of sturgeon culture rests squarely with

the successful production of eggs, or caviar, the commercial hallmark of this family of Eishes.

Therefore, environmental contaminants, that have the potential to alter reproductive endpoints

such as egg production, are critical areas of investigation for threatened species whose promise

in aquaculture relies almost entirely on proper egg development.

In many aquatic animals, including most fish, nitrate enters the bloodstream by crossing

the gill epithelia, either by diffusion or against a concentration gradient by substituting for

chloride, and accumulating in extracellular fluid (Lee and Prichard, 1985; Jensen, 1995).

Ingested nitrate is readily absorbed by the proximal small intestine in mammals (Walker, 1996),

or can also be converted to nitrite, although the degree and mechanism of the latter has been a










significant point of debate (Hartman, 1982). Concentrations of excess nitrite can cause the

potentially fatal methemoglobinemia, or brown blood disease in fishes, caused by an inability to

reversibly carry oxygen in the blood (Scott and Crunkilton, 2000). Both nitrate and nitrite are

capable of generating nitric oxide (NO) (Meyer, 1995; Cadenas et al., 2000; Lepore, 2000).

Nitric oxide has been shown to inhibit steroidogenesis through its interactions with steroidogenic

acute regulatory protein (StAR) or the enzyme cytochrome P450 side chain cleavage (P450sec)

(White et al., 1987).

In the mitochondria of steroidogenic cells, free cholesterol, the precursor for

steroidogenesis, is transported across the mitochondrial membrane by StAR. This cholesterol is

then converted to pregnenolone by the P450sce enzyme (Stocco, 1999). Pregnenolone is

subsequently converted to progesterone by mitochondrial 3 P hydroxysteroid dehydrogenase (3 P-

HSD) (Stocco, 1999). Progesterone then exits the mitochondria and depending on the tissue,

will be converted to either mineralcorticoids, glucocorticoids, progestins, androgens or estrogens

in the smooth endoplasmic reticulum (Norris, 1997). Noticeably absent from nitrate studies

describing the mechanisms of altered steroid concentrations, are studies of enzymes and

receptors involved in regulating the earliest stages of steroidogenesis. In fact, the maj ority of

steroidogenic research has focused on enzymes and receptors further downstream from the

conversions of cholesterol to pregnenolone (Goto-Kazeto et al., 2004).

The goal of this study is to examine nitrate-induced alterations in endocrine function and

identify mechanisms through which environmental exposure to nitrate alters steroidogenesis at

the molecular level. These mechanisms will be investigated by comparing the mRNA expression

of a regulatory enzyme functioning at an early stage of steroidogenesis (P450sec) as well as

receptor proteins at the end of the steroidogenic cascade for both sex steroids and










glucocorticoids, estrogen receptor P (ERP) and glucocorticoid receptor (GR), the mRNA

expression patterns of which have not been previously characterized in sturgeon.

Methods

Fish and Experimental Systems

Siberian sturgeon were collected from four 30,000 liter tanks, from separate commercial

recirculating aquaculture systems at Mote Marine Laboratory's Aquaculture Park (Commercial

Sturgeon Demonstration Proj ect) in Sarasota, FL. The fish were 4.5 years old and weighed an

average of 6. 14 + 1.10 kg. Water chemistry in each of these systems was analyzed weekly for

ammonia, nitrite, nitrate, and pH prior to commencement of the experiments. Dissolved oxygen

and temperature were monitored continuously with stationary probes, which were spot-checked

bi-weekly for calibration with portable probes. Hardness, alkalinity and chloride were analyzed

the day prior to commencement of the experiment.

Surgical Sexing and Tissue Collection

The sturgeon were pulled by hand at the side of the tank and immediately held down on a

padded V-shaped surgical table. Pulling the fish from the tank by hand (versus netting)

decreased the likelihood of stressing Eish remaining in the tank and allowed for more immediate

access to the fish for sampling.

For surgical sexing, the fish were anesthetized in a 5 80 C water bath containing carbon

dioxide (CO2) gaS; CO2 WAS used because it is a low regulatory priority anesthetic for Hish that

are grown for food production and requires no withdrawal period; the sturgeon used in this study

were part of a commercial food production program. Pure oxygen gas administered through a

Eine air stone was used to maintain a dissolved oxygen concentration of 9.0 13.0 mg/L, and

sodium bicarbonate was added to maintain a pH of 6.8 7.6 in the bath throughout the procedure.









Fish generally took 3 5 minutes for full anesthetization. A 2.5 3.5 cm incision was made on

the ventral side of the fish, approximately 8 cm anterior to the vent, along the median axis to

allow inspection of the gonads on either side of the fish for sex determination and tissue

collection. A piece of gonad approximately 5 mm3 was removed with a biopsy force (Ethicon

Inc., Somerville, New Jersey), flash frozen in liquid nitrogen and stored at -800C. The fish was

sutured closed with coated vicryl absorbable suture (Ethicon Inc., Somerville, New Jersey).

Treatments and Experimental Conditions

Two treatments were established which sampled fish from each of four commercial

culture tanks (30,000 L each) located in separate recirculating systems at Mote Marine

Laboratory's Aquaculture Park in Sarasota, FL. Two of the culture tanks were held at a nitrate

concentration of 1.5 mg/L nitrate-N (6.5 mg/L total nitrate) for one month, and two tanks were

held at 57 mg/L (250 mg/L total nitrate). Nitrate concentrations were achieved by adjusting the

freshwater input to each system, typical of commercial aquaculture practices. A nitrate

concentration of 57 mg/L nitrate-N was chosen as the upper limit in this study, as this is the

maximum concentration deemed safe, defined by feeding behavior and mortality, at Mote's

Commercial Sturgeon Demonstration Project. The lower concentration of 1.5 mg/L nitrate-N

was chosen because it reflects ecologically relevant exposures. Eight fish were sampled from

each of the four commercial recirculating culture tanks (N = 16 per nitrate treatment).

RNA Isolation and Primer Design

Frozen gonadal tissues were weighed and immediately homogenized in TRIzol reagent

(Invitrogen, Carlsbad, CA) at a ratio of 1 ml TRIzol / 100 mg tissue. Total RNA was isolated by

collecting the aqueous phase of a chloroform/phenol extraction and precipitated in isopropanol.

The pellet was washed in 80% ethanol and then dissolved in DEPC treated water. An SV Total

RNA Isolation System kit (Promega, Madison, WI) was used to purify the samples and perform









a DNase treatment The quality and concentration of the total RNA was determined with

agarose gel electrophoresis and spectrophotometer, respectively. First strand cDNA was

synthesized with 2 Clg total RNA with Oligo (dT)12-18 Primer (Invitrogen) and SuperScript III

RNase H- Reverse Transcriptase.

Degenerate primers for L8 (a ribosomal protein used for normalization of mRNA levels),

glucocorticoid receptor (GR), P450sec, and ERP were designed from conserved regions of the

respective genes from other species. The PCR primers were used to amplify fragments of the

sturgeon cDNA. Amplified cDNA were purified by Wizard SV Gel and PCR Clean-up System

(Promega) and cloned by pGEM-T Vector System (Promega). Cloned plasmids were isolated by

Wizard Plus SV Miniprep DNA Purification System (Promega). We used the BigDye

Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) to sequence the

amplifed fragments which were analyzed with an ABI PRISM 3100. BLAST

(http://www.ncbi .nlm.nih.gov/BLAST/) was used to check for nucleotide and amino acid

homology. Primer Express (Applied Biosystems, Foster City, CA) was used to design the real-

time PCR primers (Table 5-1).

Quantitative Real-Time PCR

Quantitative real-time PCR (Q-PCR) was conduced using SYBR Green PCR Master Mix

using a MyiQ Single Color Real-Time PCR Detection System (Bio-Rad) in a reaction volume of

15C1l following the manufacturer' s protocol as previously described by this lab (Katsu et al.,

2004). Conditions for Q-PCR for all genes were 3 min at 950C and 40 cycles of 950C for 10

seconds and 1 min. at the best annealing temperature for each gene. The best annealing

temperature for P450se was 60.60C, with L8, ERP and GR running at an annealing temperature

of 650C. Starting quantities of cDNA (copies/ml) for each gene were calculated according to









(Yin, 2001), based on optical density and molecular weight values. The expression of mRNA of

the samples was calculated from a standard curve created from a serially diluted sample.

Samples were run in triplicate and were normalized for ribosomal L8 expression.

Sequence Data

The sequence data were analyzed using CLC Free Workbench (CLC Bio A/S, Cambridge,

MA), and homologous sequences of their deduced amino acid sequences were searched by

BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The amino acid sequences were aligned using

ClustalX (Thompson et al., 1997). Genebank accession numbers for the amino acid sequences of

RPL8 are Q6POV6 (zebrafish), P41116 (X. leaves), XP_416772 (Chicken), P62918 (Mouse) and

P62917 (Human); those of GR are BAE92737 (zebrafish), P49844 (X. laevis), XP_420437

(chicken), NP_032199 (mouse) and PO4150 (human); those of ER-beta are NP_85 1297

(zebrafish), NP_001035101 (X. tropicalis), NP_990125 (chicken), NP_034287 (mouse) and

NP_001428 (human); those ofP450 SCC are XP_691817 (zebrafish), NP_001001756 (chicken),

Q9QZ82 (mouse) and AAH32329 (human). The Conserved Domains in amino acid sequences

were searched by CD-search (http://www.ncbi_ nlm. nih.gov/Structure/cdd/wrpsb .cgi).

Statistical Analyses

Statistical analyses were performed using StatView for Windows (SAS Institute, Cary,

NC, USA). Initial comparisons were made to determine significance within treatments. F-tests

were conducted to test variances among treatment groups for homogeneity. If variance was

heterogenous, data were loglo transformed to achieve homogeneity of variance, however, all

reported mean (+ 1 SE) values are from non-transformed data. The relative expression of each

gene was computed as a ratio with L8 and then multiplied by a consistent multiplier of 10 to

ensure all values were greater than one prior to analyses of variance (ANOVA). Figures,










however, display original values. If significance was determined (p < 0.05). Fisher' s protected

least-significant difference was used to determine differences among treatment means.

Results

Sequence Data

Nucleotide and deduced amino acid sequences of RPL8 (L8), P450sec, ERP and GR are

shown in Figures 5-1 to 5-4. Cloned cDNAs are 309, 584, 698 and 845 base pairs encoding 102,

194, 232 and 281 amino acids, and are similar to L8, GR, ERP and P450sec, respectively (Figs.

5-1 to 5-8). These are partial cDNA sequences and it is 40, 26, 42 and 55% of the length of the

zebrafish coding region for L8, GR, ER-P and P450sec, respectively. Cloned L8 included a

partial conserved domain of ribosomal protein L2 C-terminal domain, and revealed higher than

97% of sequence identity among the vertebrates (Fig. 5-5). Cloned P450sce encoded a part of

conserved region for cytochrome P450s, and revealed 77, 67, 49 and 51% of sequence identity

compared with zebrafish, chicken, mouse and human, respectively (Fig. 5-6).

Partially cloned GR included a complete hinge region, and a partial DNA- and ligand-

binding domain (Fig. 5-7). Sturgeon GR showed 74, 67, 59, 67 and 65% of sequence identity

with GR cloned from zebrafish, Xenopus leavis, chicken, mouse and human, respectively (Fig. 5-

7). Cloned partial cDNA for ERP included a partial hinge region and ligand binding domain,

and revealed 71, 58, 57, 59 and 57% of sequence identity with ERP of zebrafish, Xenopus

tropicalis, chicken, mouse and human, respectively (Fig. 5-8).

Water Chemistry

Water chemistry parameters were tested the day of experimentation and were as follows:

unionized ammonia (NH3) < 5.35 Clg/L, nitrite <; 0.20 mg/L; pH 7.6, alkalinity 240 mg/L,

chloride 90 mg/L, total hardness 240 mg/L and calcium hardness 135 mg/L. Dissolved oxygen










concentrations were maintained at > 95% saturation throughout the trial and temperature was

23.5 oC.

Steroidogenic Gene Expression and Hormone Regressions from Previous Studies

Expression levels, normalized to L8 expression, of all genes evaluated were statistically

similar between the fish residing in the 1.5 or 57 mg/L nitrate-N (Figs. 5-9 to 5-11). As

expected, the expression of L8 was not significant for either treatment. Additionally, there was

no tank effect among treatments. Mean expression levels for P450se were 0.027 (f 0.007) and

0.026 (f 0.006) for the 1.5 and 57 mg/L nitrate-N treatments respectively (Fig. 5-8). Mean

expression levels for GR averaged 0.359 (f 0.056) and 0.341 (f 0.035) for the 1.5 and 57 mg/L

nitrate-N treatments respectively (Fig. 5-9). Mean expression level for ERP was 0.440 (f0. 109)

and 0.583 (f 0. 160) for the 1.5 and 57 mg/L nitrate-N concentrations respectively (Fig. 5-10).

Simple regression analyses of mRNA expression levels (normalized to L8) of P450sec,

ERP and GR, as well as sex steroid and stress hormone plasma concentrations from Chapter 4

are summarized in Tables 5-2 and 5-3 as well as Figs. 5-12 to 5-15. Fish exposed to 1.5 mg/L

NO3-N demonstrated significant regressions (p < 0.05) for the following comparisons: GR vs.

ERP; GR vs. glucose; and T vs. 11-KT. Fish exposed to 57 mg/L NO3-N demonstrated

significant regressions for the following comparisons: ERP vs. P450sec; ERP vs. 11-KT;

P450sce vs. T; P450sce vs. 11-KT.

Discussion

This is the first study to successfully clone and describe the mRNA expression patterns of

sturgeon P450sec, ERP and GR, key constituents in steroidogenic and stress receptor

functioning. These genes represent both early (P450sec) and late (ERP and GR) steroidogenic

endpoints, with their expressions offering insight into several steroidogenic pathways.









In mammals, two ERs have been identified, in contrast to teleosts in which there are three

known ERs, ERa and two isoforms of ERP (Filby and Tyler, 2005). Although both ERa and

ERP are found in the gonads of fish and mammals, there is currently no agreement regarding the

relative importance of one form over the other (Hall et al., 2001). ERP has been shown to

attenuate the ligand stimulated transcriptional activity of ERu, and has been shown to

heterodimerize with ERa in vitro, suggesting that relative expression levels of the receptors

could dictate cellular sensitivities to estrogens (Hall et al., 2001).

ERP is most strongly expressed in the gonad in most fishes. In a study of largemouth bass

(M~icropterus salmoides) the gonadal mRNA expression of ERP was many fold greater than

ERu, however its relative expression was strongly dependent upon time of the year (Sabo-

Attwood et al., 2004). This study also showed that ERa was more strongly expressed in the

liver, but only for certain periods of the year. In rivulus (Rivulus marmoratus) the greatest

expression of ERP is found in the gonad and it has been shown that environmental pollutants can

dramatically alter ER expression in this species (Seo et al., 2006). Rivulus has both

hermaphroditic and primary male forms, and it has been shown that expression levels of ERP can

vary dramatically depending on the form (Orlando et. al., 2006). ERP has been shown to be

preferentially sensitive to synthetic antiestrogens and phytoestrogens versus ERa (Bodo and

Rissman, 2006). Taken together, these data demonstrate the plasticity of ERP mRNA expression

and its capacity to be altered by environmental variables.

The fish in this study were part of a larger body of work examining several endocrine

endpoints associated with nitrate exposure. In Chapter 4, we documented a significant rise in

plasma concentrations of sex steroids under conditions of elevated nitrate. In that study, I

offered three possible explanations for the observed rise in plasma sex steroid concentrations,









which included increased steroidogenesis and a concomitant increase in gonadal synthesis of sex

steroid hormones, alterations in transport proteins or reductions in liver clearance. The enzyme

P450sce is regarded as the chronically regulated rate-limiting step in steroidogenesis (Miller,

2002) and functions at the early stages of steroidogenesis. The P450sce enzyme is expressed

very early in development; in mice expression begins at embryonic day 11 (Hsu et al., 2006).

During these early embryonic stages, mice with targeted disruption of the P450sce gene produce

no steroids and have severe adrenal defects, and die shortly after birth; zebrafish with blocked

P450sce function do not survive as well (Hsu et al., 2006).

In general, gonadotropins regulate P450sce expression, however, sex steroids have been

found to alter its expression in several tissues (Von Hofsten et al., 2002). In Arctic char

(Salvelinus alpinus) 11-KT has been shown to up-regulate P450sce expression in the gonads

(Von Hofsten et al., 2002). Although nitrate exposure did not appear to alter the mRNA

expression of P450sce in sturgeon in this study, there was a significantly positive correlation

with P450sce and both androgens (Chapter 4) in fish exposed to 57 mg/L NO3-N, that was not

apparent in fish exposed to 1.5 mg/L NO3-N. Given this difference, I hypothesize that the sex

steroids at the upper nitrate concentration, that were significantly elevated compared to the

population of fish exposed to low nitrate, approached a threshold for feed back; that is, the

binding of a critical number of receptors sufficient to trigger a response, and this elevated gene

expression. It is logical to suggest, that although the fish in this study possessed vitellogenic

oocytes, they were nonetheless early in their development, and it is possible that the fish in the

1.5 mg/L NO3-N concentration would experience an elevation in sex steroid hormones

concomitant with progressive egg development, and once these sex steroids reached a critical

concentration, they too would demonstrate similar correlations. It is also possible that nitrate is









affecting an unknown mechanism, that itself regulates both P450sce and sex steroid expression,

and that their correlation is not necessarily directly causative.

Interestingly, there was a positive correlation between ERP and 11-KT in the fish exposed

to 57 mg/L NO3-N that was not evident in fish exposed to 1.5 mg/L NO3-N. It has been shown

in female sturgeon that both T and 11-KT rise significantly during vitellogenesis, and often peak

just prior to Einal maturation (Barannikova et al., 2004). It is possible that under a normal

reproductive cycle, that during a key period of development in Siberian sturgeon, androgens of

ovarian origin rise, providing a precursor for estrogen synthesis, and thus, serving as a signal for

the production of aromatase to facilitate the conversion of androgens to estrogens.

The estrogen receptor protein expression examined in this study represents an endpoint

regulated far downstream, via negative feedback, in the steroidogenic pathway. That we did not

observe an increase in mRNA expression for a chronically regulated upstream enzyme, nor for

downstream estrogenic receptors, suggests that sex steroid elevations were not likely due to

increased gonadal output. It is more likely then, that the discord between plasma sex steroid

concentrations and mRNA expression patterns could be explained by altered hepatic metabolism,

either via alterations in transport proteins to the liver, or by direct action on the liver itself.

Although these results do not provide a mechanism for hepatic or transport protein failure,

they do support the need for future studies clarifying liver performance under high nitrate

conditions. Thibaut and Porte (2004) found significantly reduced metabolic liver clearance when

carp (C. carpio) were exposed to estrogenic nonylphenol and androgenic fenarimol at

concentrations as low as 10 CIM and 50 CIM, respectively. Several other studies have shown that

altered plasma sex steroid concentrations, induced by xenobiotics, could be caused by altered

hydroxylase enzyme activity in the liver (see review by Guillette and Gunderson, 2001).










NO, derived from nitrate or nitrite, has been shown to have inhibitory effects on

steroidogenesis via its actions on StAR or P450sce by binding to the heme groups of these

compounds (White et al., 1987). Heme groups characterize all enzymes of the P450 family, and

have been shown to be susceptible to chemical perturbation (White et al., 1987; Walsh and

Stocco, 2000; Danielson, 2002). These studies provide a possible mechanism for nitrate induced

hepatic alterations by inhibiting enzymatic action of the various P450s in the liver responsible for

clearance (Guillette and Edwards, 2005).

This study is unique in several regards. It is the first study to evaluate the steroidogenic

effects of nitrate exposure in a commercially viable and ecologically threatened species,

habituated to a warm environment under commercial culture conditions. Of significant

importance is the fact that this study used nitrate produced through nitrifieation as its source.

Most studies examining nitrate exposure use a purified aquatic medium dosed with various

nitrate salts (e.g. NaNO3, KNO3). Nitrate produced through nitrification brings with it a host of

metabolites and oxidative end products not present in a purified medium, and is more relevant to

ecological exposure. This is of particular importance because it has been shown that the nitrate

medium itself can significantly alter its toxic effects, even if the same source of nitrate (i.e.

NaNO3) is used. Edwards et al. (2006a), found that Southern Toad (Bufo terrestris) tadpoles

exposed to various concentrations of nitrate responded differently depending on the source of

freshwater used, and this difference could not be attributed to differential electrolyte balances

since both sources were equivalent.

Although we did not observe nitrate induced alterations in mRNA gene expression patterns

of P450sec, ERP or GR in this experiment, it is important to note that these animals were

exposed to the nitrate concentrations for 30 days, and it is probable that the fish were adapted to









the nitrate concentrations in terms of gene expression, since most alterations in gene expression

are observable hours or days after a disrupting event. However, a goal of this study was to

understand the implications of long-term exposure to elevated nitrate, and these adaptive and

persistent mRNA expression patterns are relevant to aquaculture environments.

It is now known that a maj or function of glucocorticoids (GCs), including cortisol, is to

protect against over stimulation by host defenses in a stress event (Li and Sanchez, 2005). GCs

regulate numerous biological processes and play diverse roles in growth, development and

maintenance of stress related homeostasis (Sapolsky et al., 2000). GCs effectuate their responses

by their association with glucocorticoid receptors (GRs), and altered GRs have been implicated

as a causative factor in several pathologic states (Barden, 2004; Marchetti et al., 2005). That

GR-deficient mice die within a few hours after birth clearly shows that proper GR function is

essential for survival (Cole et al., 1995).

Although nitrate did not alter the mRNA expression of GR in this study, there was a

positive correlation between GR and both ERP and glucose. There is no evidence in the

literature of an overt regulatory mechanism for GR induction of either ERP or glucose, or a

mechanism by which glucose alters GR or ERP expression, and it is possible this relationship is

the result of an unknown or unapparent factor that is co-regulating these genes. However, it has

been shown recently that glucose has the ability to regulate hepatic gene expression in a

transcriptional manner, through the carbohydrate responsive element binding protein (ChREBP)

(Dentin et al., 2006). In addition, glucose has been shown to directly up-regulate the mRNA

expression of P-defensin-1, an immune system peptide, in cultured human renal cells (Malik and

Al-Kafaji, 2006). Therefore, although the relationship between glucose and GR mRNA










expression is not yet clear, given that glucose has been shown to regulate gene expression in

other systems, it is possible that glucose could regulate the expression patterns of these receptors.

Cortisol bio-synthesis commences with the stimulation of interrenal tissues by

adrenocorticotropic hormone, resulting in an enzymatic conversion of cholesterol which

progresses through the steroidogenic cascade through a series of enzymatic steps, including the

cytochrome P450 family of proteins. It was recently shown in rainbow trout (0. mykiss) that

xenobiotic stressors that activate aryl hydrocarbon signaling, impair the corticosteroid response

to stress by inhibiting both StAR and P450sec (Aluru and Vijayan, 2006). Other studies have

also documented the impairment of the adaptive stress response by decreasing the capacity for

interrenal cortisol production (Wilson et al., 1998; Hontela, 2005). In Chapter 4 it was shown

that basal cortisol production was not increased in animals exposed to elevated nitrate for 30

days. Expectedly, we did not observe a change in mRNA expression for GR in animals exposed

to elevated nitrate, indicating nitrate may not alter the enzymes involved in the adaptive stress

response long term as these animals are likely adapted to the elevated nitrate at the tissue

(interrenal) level, although the question of hepatic alteration and clearance still remains a

concern.

This study contributes a better mechanistic understanding of the endocrine disruptive

effects of nitrate exposure. Future studies of the endocrinological effects of nitrate should focus

on mechanisms of hepatic alteration including examining enzymes involved in clearance,

expression of gonadal and hepatic StAR protein and vitellogenin production, as well as transport

protein kinetics.










Table 5-1. Forward and reverse primers used for quantitative real-time PCR

Forward Primer (5' 3') Product
Gene
Reverse Primer (3' 5') Size (bp)
CCGGTGACCGTGGTAAACTG
L8 67
TCAGGGTTGTGGGAGATGACA
AGCCTCAGCGTCTCCTTTAT
P450sc 159
ccCCCTGTTGTGGACCATGTT
TGGTCAGCTGGGCCAAA
ERP 69
CCAATAGGCATACCTGGTCATACA
CAAGCAACACCGCTACCAGAT
GR 66
CGTTAGCTGTGGCATCGATTT












Table 5-2. Regression data mRNA expression patterns for P450 side chain cleavage enzyme
(P450sec), estrogen receptor P (ERP), glucocorticoid receptor (GR), testosterone (T),
11-k~etotestosterone (11KT), 17P-estradiol (E2) COrtisol and glucose in sturgeon
exposed to 1.5 and 57 mg/L NO3-N. Bold numbers represent significant, positive
correlations.

1.5


mg/L
NO3-N

ERP

GR

T

11-KT


P450sce E
p = 0.4821
r2 = 0.064
p = 0.3927 p = 0.02
r2 = 0.093 r2 = 0.471
p = 0.2849 p = 0.3249
r2 = 0.161 r2 = 0.121
p = 0.3640 p = 0.9435
r2 = 0. 119 r2 = 0.001
p = 0.0704 p = 0.7351


GR





p = 0.3923
r2 = 0.093
p = 0.1477
r2 = 0.243
p = 0.6785
r2 = 0.031

rp = 0.5109= .4

p = 0.035
r2 = 0.444


r2 = 0.021
p = 0.8008
r2 = 0.007

p = 02263


r2 = 0.512
p = 0.7455
r2 = 0.014


Corti sol

Glucose


57.0
mg/L
NO3-N
P450scec
ERP p = 0.0278


GR

T

11-KT


ERP GR



,p = 0.48330.3

p = 0.0827 p = 0.9835
r2 = 0.214 r2 = 0.000
p = 0.0193 p: = 0.4818
r2 = 0.378 r2= 0.042
p = 0.0678 p = 0.2330
r2 = 0.324 r2 = 0.060
rp = 0.8467 ,p = 0.7247
r2= 0.004 r2= 0.013
p = 0.6392 p = 0.7834
r2 = 0.019 r2 = 0.007


r2 = 0.320
p = 0.3069
r2 = 0.080
p = 0.0002
r2 = 0.673
p = 0.0019
r2 = 0.567


p = 0.9510
r2 = 0.000
p = 0. 1735
r2 = 0.149


Cortisol

Glucose
























11-KT p =0.58

E2 p = 0.9919 p = 0.8984
r2 = 0.000 r2 = 0.003
Corti sol p: = 0.5528 p: = 7108 p: = 0.4648
r2= 0.046 r2= 0.018 r2= 0.092
Glucose p = 0.6245 p = 0.4601 p = 0.5398 p = 0.4326
r2 = 0.031 r2 = 0.070 r2 = 0.066 r2 = 0.079


57.0
mg/L
NO3-N
T 1 1-KT E2 COrti sol
11-KT p = 0.0001
r2 = 0.819
E2 p = 0.9221 p = 0.4658
r2 = 0.001 r2 = 0.061
Cortisol p = 0.5190 p = 0.4652 p = 0.1247
r2 = 0.043 r2 = 0.061 r2 = 0.347
Glucose p = 0.0563 p = 0. 1029 p = 0.3525 p = 0.3397
r2 = 0.271 r2 = 0.223 r2 = 0.109 r2 = 0.091


Table 5-3. Regression data for testosterone (T), 11l-ketotestosterone (11KT), 17P-estradiol (E2)
cortisol and glucose in sturgeon exposed to 1.5 and 57 mg/L NO3-N from Chapter 4.
Bold numbers represent significant, positive correlations.


1.5
mg/L
NO3-N


11-KT


Corti sol














CTCAGCTGAATATTGGCAATGTTCTCCCAGTTGGCACCATCTGATCATTT 60
QLNIGNVLPVGTMPEGTIIC 20
GCTGCCTGGAAGAGAAGCCCGGTGACCGTGGTAAAACTGGCCGGCCGGATC 120
CLEEKPGDRGKLARASGNYA 40
CCACTGTCATCTCCCACAACCCTGAA~ACTAAGAA~ATCCCGGGACGCACGG 180
TVISHNPETKKSRVKLPSGS 60
CCAAGAAAGTAATCTCCTCTGCCAACAGAGCCGTAGTCGGTTGGCGGTGC 240
KKVI SSANRAVVGVVAGGGR 80
GTATTGACAA~ACCAATCCTGAAGGCGGGTCGAGCCTATCACATCAGCAAA 300
IDKPILKAGRAYHKYKAKRN 100
ACTGCTGGC 309
C W 102




Figure 5-1. Nucleotide and deduced amino acid sequences of Siberian sturgeon ribosomal
protein L8 (RPL8). Partial cDNA of RPL8 was 309 base pairs encoding 102 amino
acids.




Full Text

PAGE 1

NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN STURGEON, Acipenser baeri By HEATHER J. HAMLIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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Copyright 2007 by Heather J. Hamlin

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To my family

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ACKNOWLEDGMENTS First and foremost I thank Lou Guillette for his tremendous mentorship, for sharing with me his wealth of knowledge and experience and for having more faith in me than I did at times. I am a better person for having worked with him. I thank my advisor and other members of my committee for their encouragement and support: Ruth Francis-Floyd wa s graciously supportive and gave me the freedom to do what I love ; Kevan Main gave me the opportunity and encouragement to fulfill my dream; Daryl Parkyn read my manuscripts and gave me valuable comments; Roy Yanong gave me valuable opinion s and comments on my manuscripts and Ill always appreciate his enthusiasm for disease. Id also like to thank Jim Michaels for allowing me access to my research animals and supporting my research. This journey would not have been nearly as fulfilling were it not fo r the comradery of my fellow lab mates: Thea Edwards, who taught me EI As and introduced me to the lab experience. Ill always treasure our late night conversations about ever ything from egg cups to egg development; Brandon Moore, who took the time to mentor me and with whom Ill always enjoy scientific discussions; Satomi Kohno, whose pa tience in teaching lab techniques deserves an award; Iske Larkin, for teaching me the wonders of RIAs. Lori Alberg otti, Ashley Boggs and Nicole Botteri, who made me wish I could spend mo re time in the lab. Id also like to thank all the undergraduate students who assisted me with collections and lab analyses. Finally, Id like to thank my friends and family, without whom the journey wouldnt be worth it: my Mother, Holly Paulsen, who let me have every creature known to man as a child, and spent countless hours with me as an adult coll ecting data and analyzing samples in this work. It wouldnt have been the same without her he lp; my Father, Greg Hamlin, who bought me my first aquarium; my best friend Ma ria Piccioni, who supported me tr emendously in this journey; iv

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Id love to be half the person she thinks I am; Dave Jenkins, who supported me more than he knows; and Amy Leighton, who made my childhood something to treasure. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .............................................................................................................iv TABLE OF CONTENTS ...............................................................................................................vi LIST OF TABLES .......................................................................................................................viii LIST OF FIGURES .......................................................................................................................ix ABSTRACT ...................................................................................................................................xi 1 INTRODUCTION................................................................................................................. ...1 Background ...............................................................................................................................1 Overview of Reproductive Endocrinology in Fishes ........................................................1 Stress in Fish and Its Effects on Reproduction ..................................................................3 Endocrine Disruption in Aquatic Vertebrates ...................................................................6 Nitrate in Natural Water Systems ......................................................................................9 Nitrate in Aquaculture and Its Implications as an EDC ....................................................9 Sturgeon as a Model Species ...........................................................................................12 Research Objectives and Hypotheses ..............................................................................13 2 NITRATE TOXICITY IN SIBERIAN STURGEON............................................................18 Introduction .............................................................................................................................18 Methods ..................................................................................................................................20 Study Animals and Pre-Testing Conditions ....................................................................20 Range-Finding Studies ....................................................................................................20 Test Procedures ...............................................................................................................21 Statistical Analyses ..........................................................................................................22 Results .....................................................................................................................................22 Discussion ...............................................................................................................................23 3 STRESS AND ITS RELATION TO ENDOCRINE FUNCTION IN CAPTIVE FEMALE SIBERIAN STURGEON.......................................................................................30 Introduction .............................................................................................................................30 Methods ..................................................................................................................................33 Fish and Sampling...........................................................................................................33 Surgical Sexing................................................................................................................34 Treatments .......................................................................................................................34 Hormone Evaluations ......................................................................................................35 Statistical Analyses ..........................................................................................................36 Results .....................................................................................................................................36 Morphology and Chemistry .............................................................................................36 vi

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Hormones ........................................................................................................................37 Discussion ...............................................................................................................................38 4 NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN STURGEON........................................................................................................46 Introduction .............................................................................................................................46 Methods ..................................................................................................................................49 Fish and Sampling Procedures ........................................................................................49 Surgical Sexing................................................................................................................50 Experiment 1 ...................................................................................................................51 Experiment 2 ...................................................................................................................52 Hormone Evaluations ......................................................................................................53 Statistical Analyses .................................................................................................................54 Results .....................................................................................................................................54 Experiment 1 ...................................................................................................................54 Experiment 2 ...................................................................................................................56 Discussion ...............................................................................................................................57 5 EFFECTS OF NITRATE ON STEROIDOGE NIC GENE EXPRESSION IN CAPTIVE FEMALE SIBERIAN STURGEON.......................................................................................69 Introduction .............................................................................................................................69 Methods ..................................................................................................................................73 Fish and Experimental Systems .......................................................................................73 Surgical Sexing and Tissue Collection............................................................................73 Treatments and Experimental Conditions .......................................................................74 RNA Isolation and Primer Design ...................................................................................74 Quantitative Real-Time PCR ...........................................................................................75 Sequence Data .................................................................................................................76 Statistical Analyses ..........................................................................................................76 Results .....................................................................................................................................77 Water Chemistry ..............................................................................................................77 Steroidogenic Gene Expression and Horm one Regressions from Previous Studies .......78 Sequence Data .................................................................................................................77 Discussion ...............................................................................................................................78 6 SUMMARY AND FUTURE DIRECTIONS.......................................................................103 Summary ...............................................................................................................................103 Future Directions ..................................................................................................................107 Conclusions ...........................................................................................................................108 LIST OF REFERENCES .............................................................................................................110 BIOGRAPHICAL SKETCH .......................................................................................................130 vii

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LIST OF TABLES Table page 1-1 LC50 results and test conditions for three size classes of Siberian sturgeon exposed to sodium nitrate .....................................................................................................................27 1-2 Representative ac ute toxicity data for nitrate ....................................................................28 5-1 Forward and reverse prim ers used for quantitative real-time PCR ...................................85 5-2 Regression data mRNA expressi on patterns for P450 side chain cleavage enzyme (P450SCC), estrogen receptor (ER ), glucocorticoid receptor (GR), testosterone (T), 11-ketotestosterone (11KT), 17 -estradiol (E2) cortisol and glucose in sturgeon exposed to 1.5 and 57 mg/L NO3-N. ..................................................................................86 5-3 Regression data for testoste rone (T), 11-ketotestosterone (11KT), 17 -estradiol (E2) cortisol and glucose in sturge on exposed to 1.5 and 57 mg/L NO3-N from Chapter 4 .....87 viii

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LIST OF FIGURES Figure page 1-1 Overview of the hypothalami c-pituitary-gonadal axis in sturgeon ....................................15 1-2 A representative steroidogenic pathway of st eroid hormones in gonadal cells ..................16 1-3 A representative st eroidogenic pathway of cortisol production in an interrenal cell .........17 2-1 Linear regression of log10 transformed nitrate-N (mg/L) lethal concentration values versus log transformed fish weight (g). .............................................................................29 3-1 Blood sampling times for treatments 1-4 of fish held under confinement stress for 4-h ...43 3-2 Plasma cortisol (A) and pl asma glucose (B) concentrations (mean S.E.M.) during a 4-h capture and confinement period ..................................................................................44 3-3 Sex steroid data for treatment 2. Plasma 17 -Estradiol (A), testosterone (B), and 11ketotestosterone (C) taken fr om serial bleeds of cultured female Siberian sturgeon throughout the 4-h period of confinement stress ...............................................................45 4-1 Blood sampling times for treatments 1 and 2 of fish held under confinement stress for 6-h. .....................................................................................................................................62 4-2 Plasma cortisol (A) and glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N ..................................................................................................63 4-3 Plasma testosterone (A), 11-ketote stosterone (B) and estrad iol (C) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N ..............................................64 4-4 Plasma cortisol (A) and glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N ..................................................................................................65 4-5 Plasma cortisol (A), glucos e (B) testosterone concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N .......................................................................66 4-6 Plasma cortisol testosterone (A), 11-ketotestosterone (B) and estradiol-17 (C) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N .......................67 ix

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4-7 Plasma cortisol (A) and glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N ....................................................................................................68 5-1 Nucleotide and deduced amino acid sequ ences of Siberian stur geon ribosomal protein L8 (RPL8) ..........................................................................................................................88 5-2 Nucleotide and deduced amino acid sequences of Siberian sturgeon P450SCC..................89 5-3 Nucleotide and deduced amino acid sequences of Siberian sturgeon ER ........................90 5-4 Nucleotide and deduced amino acid sequences of Siberian sturgeon GR ..........................91 5-5 Sequence comparison of deduced ami no acid sequences for ribosomal protein L8 (RPL8) ................................................................................................................................92 5-6 Sequence comparison of deduced amino acid sequences for P450SCC...............................93 5-8 Sequence comparison of deduced amino acid sequences for ER .....................................95 5-9 Mean ( SE) expression of P450SCC mRNA in 4.5 year-old Siberian sturgeon. ................96 5-10 Mean ( SE) expression of glucocorticoid (GR) receptor mRNA in 4.5 year-old Siberian sturgeon. ..............................................................................................................97 5-11 Mean ( SE) expression of estrogen receptor(ER ) mRNA in 4.5 year-old Siberian sturgeon. ..............................................................................................................98 5-12 Linear regression of glucose (mmol/L) vs GR mRNA (normalized to L8 expression) for fish exposed to 1.5 mg/L nitrate-N...............................................................................99 5-13 Linear regression of ER mRNA and GR mRNA (normali zed to L8 expression) for fish exposed to 1.5 mg/L nitrate-N. .................................................................................100 5-14 Linear regression of P450SCC mRNA (normalized to L8 expression) and T for fish exposed to 57 mg/L nitrate-N ..........................................................................................101 5-15 Linear regression of P450SCC mRNA (normalized to L8 expression) and 11-KT for fish exposed to 57 mg/L nitrate-N ...................................................................................102 6-1 Possible a lterations in nitrate indu ced elevations of sex st eroid concentrations in Siberian sturgeon .............................................................................................................109 x

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN STURGEON, Acipenser baeri By Heather J. Hamlin May 2007 Chair: Ruth Francis-Floyd Major: Fisheries and Aquatic Sciences Numerous environmental contaminants have been shown to alter reproductive endocrine function. Such compounds have been termed e ndocrine-disrupting contam inants (EDCs). EDCs exert their effects through numerous physiolo gical mechanisms, including alterations in steroidogenesis. Although a global pollutant of most aquatic systems, nitrate has only recently begun to receive attention for its ability to alter endocrine function in wildlife. We examined nitrate-induced endocrine disrupti on using the Siberian sturgeon (Acipenser baeri ) as a model species. Comparisons of captive populations of st urgeon cultured in nitr ate concentrations of 1.5, 11.5 and 57.5 mg/L nitrate-N revealed nitrate induced elevations in plasma concentrations of sex steroids including testoste rone, 11-ketotestosterone and 17 -estradiol. Alterations in circulating concentrations of sex steroids can be a response to several ph ysiological mechanisms, including an up-regulation of gona dal steroid synthesis, altered biotransformation and clearance by the liver or alterations in plasma storage by steroid bi nding proteins. To gain a better mechanistic understanding of the observed sex steroid elevations we examined mRNA expression patterns of steroidogenic enzymes (P450 SCC ) and receptor proteins (ER and GR). We found no significant differences in mRNA expression patterns, indicating xi

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that the observed sex steroid increases were not likely due to an up-regulation of gonadal synthesis. Cortisol and glucose, commonly examined as indicators of perceived stress, were not found to vary among groups exposed to any of the nitrate concentrations. Because responses to stress can be cumulative, endocrine responses to stress events in fish residing in the various nitrate concentrations were also investigate d. Results showed that nitrate does alter the associated stress response, defined by plasma glucose concentrations. These data suggest that long-term exposure to nitrate is associated with altered endocrine parameters (e.g., plasma hormone concentrations) in Siberian sturgeon. Future work must begin to examine the underlying causes of these cha nges. Although the data of gene expression suggest that mRNA concentrations of at least one steroidogenic enzyme were not altered, other enzymes in the pathway need to be examined. Th ese data indicated that nitrate concentrations must now be considered in the effective management of sturgeon populations in both natural and captive environments. xii

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CHAPTER 1 INTRODUCTION Background Overview of Reproductive Endocrinology in Fishes The production of circulating hormones is the result of numerous physiological reactions spanning many levels of biological organization. The regulation of hormone production is controlled by mechanisms that both create and destroy these chemical messengers, and is fine-tuned by various stimul atory and feedback mechanisms (Norris, 1997). Tropic hormones regulate many of the ac tivities of the thyroid gland, adrenal gland and the gonads (Norris, 1997). The endocrine regulation of reproduc tion is initiated in response to environmental cues, which stim ulate the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus (Detlaff et al., 1993; Norris, 1997) (Figure 1-1). In response to GnRH, the anterior pituita ry releases gonadotropins, which circulate throughout the body, targeting various or gans, such as the gonads. Two chemically distinct gonadotropins have been characterized in fish, GTH-I and GTH-II, which are purportedly analogous to follicle stimulating hormone (FSH) and luteinizing hormone (LH), respectively, in terrestrial animals (Norris, 1997). Because few fish species have defined chemical hormone structures to date, much of the research literature employs heterologous hormones (Van Der Kraak et al., 1998). FSH stimulates oogenesis and spermatogenesis, and LH stimulates final gamete maturation and release. Like FSH and LH, GTH-I and GTH-II consist of an and subunit; the subunit is the same for both gonadotropins, with only the subunit conferring bi ological specificity (Norris, 1997; Vasudevan et al., 2002). The subunits of both gonadotropins have been cloned in Siberian sturgeon ( A. baeri ) and Russian sturgeon ( A. gueldenstaedti ), and based 1

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on their function and position in the phylogenetic tree, it wa s suggested these compounds be termed FSH and LH, respectively (Que rat et al., 2000; Hurvitz et al., 2005). FSH and LH stimulate gonadal steroidoge nesis, and the three steroid hormones relevant to this study are estradiol-17 (E 2 ), testosterone (T) and 11-ketotestosterone (11KT). In females, E 2 stimulates gonadal growth, sexua l maturation, vite llogenesis by the liver and oogenesis (Knobil and Neill, 1994; Norris, 1997; Denslow et al., 2001). In males, T stimulates sexual maturation, spermatogenesi s and spawning, and is implicated in sexual behavior for both males and females (Norris, 1997; Toft et al., 2003). In addition to inducing spermatogonial prolifer ation, 11-KT likely also participates in the former processes (Schultz and Miura, 2002). Circulating hormones can be detected by recep tors at the periphery of the cell, and through a cAMP mediated proce ss ultimately leads to increa sed levels of intracellular cholesterol (Stocco, 1999). This cholestero l is mobilized to the outer mitochondrial membrane and is the precursor for steroid biosynthesis. A protein inserted in the mitochondrial membrane, steroidogenic acute re gulatory (StAR) protein, functions to transport cholesterol from the outer mitoc hondrial membrane to the inner mitochondrial membrane, and this process is now thought to be the rate limiting step in steroidogenesis (Stocco, 1999). Its function has received cons iderable attention in recent studies of vertebrates (Stocco, 2001), including fish (G oetz et al., 2004). The inner mitochondrial membrane is the site of activity for the P450 side chain cleavage enzyme (P450 SCC ) that cleaves cholesterol to form the first steroid in the pathway, pregnenolone. Pregnenolone is then converted to progesterone by 3 -hydroxysteroid dehydrogenase (3 -HSD). Both P450scc and 3 -HSD are often evaluated in studies of steroidogenesis and related 2

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physiological mechanisms (Takase et al., 1999; Pozzi et al., 2002; Inai et al., 2003). Further pathways of steroi d production are shown in Figur es 1-2 and 1-3. Quantifying compounds in the biosynthetic pathways will assist in developing a mechanistic understanding of which pathways can be disrupted. Stress in Fish and Its Effects on Reproduction Stress has been defined in the literature in a number of ways, encompassing such definitions as diversions of metabolic ener gy, adaptive changes resulting in modifications to normal physiological states, and any change that impacts long term survival (Selye, 1956; Esch and Hazen, 1978; Wedemeyer and McLeay, 1991; Bayne, 1985; Barton and Schreck, 1987). Ultimately our interest in stress is attendant upon the causative factors mitigating the deleterious response. Once these causative factors are determined, we can then begin the process of remediation. In this sense, understanding stress is a means to an end and becomes a useful tool to predict if negative outcomes are likely to ensue. We can use this diagnostic tool to unde rstand environmental impact and determine at what point action is necessary to effectuate relief. As in other vertebrates, concentrations of coricosteroid hormones are sensitive indicators of acute stress in fi sh, and circulating concentratio ns generally reflect synthesis rates since little hormone is stored in the ad renal (mammals) or interrenal tissue (fish) (Norris, 1997). The production of corticostero ids is initiated by perc eived stress events, triggering the release of cort icotropin releasing hormone (CRH) from the hypothalamus, which then triggers the release of ACTH from the pituitary (Figure 12) (Flik et al., 2006). Circulating ACTH triggers the release of corticosteroids from the interrenal cells of the head kidney in most fish species; in sturge on cortisol releasing adrenocortical cells are present in small clusters throughout the kidney (Norris, 1997). 3

PAGE 16

The principal corticosteroid in most fi sh species is cortisol (Kime, 1997; Barton, 2002; Overli et al., 2005), which has been imp licated as a causal f actor in many of the deleterious effects of stress (Barton and Iwama, 1991; Ha rris and Bird, 2000; Schreck, 2001; Bernier et al., 2004). Cor tisol shows two primary actions in fish, regulation of water and mineral balance and energy metabolism (Wendelaar Bonga, 1997). The effects of corticosteroid hormones are mediated through intracellular re ceptors, which act as ligand binding transcription factors (N orris, 1997). Fish possess bot h a glucocorticoid receptor (GR) and a mineralcorticoid receptor (MR) w ith GR possessing various isoforms (Bury et al., 2003). While cortisol is the predominant ph ysiological ligand for GR, it is still unclear what is the primary ligand for MR in fish, which shows a high affinity for both deoxycorticosterone and al dosterone (Prunet et al., 2006). Th is is particularly interesting since there is no reliable evidence for the pres ence of aldosterone in teleosts, and it is becoming accepted that aldosterone is likely absent in most or potentially all fish groups (Prunet et al., 2006). The molecular characterization of corticoste roid receptors (CR) in the last 10 years has modified the initial consensus of a unique high affinity binding site for cortisol, and now depicts a multiple CR family with two cla sses of receptors (GR and MR) with splicing isoforms and duplicated genes (GR1 and GR2) (Prunet et al., 2006). Functional analyses in trout show that GR2 has a higher sensitivity to cortisol when compared to GR1, and that these isoforms show different patterns of expression sensit ivity depending on the tissues targeted (Greenwood et al., 2003). It has also been shown that GR can be less sensitive to corticosteroids than MR, suggesting that the la tter could serve as a high affinity cortisol receptor in fishes, a condition already described in humans (Hellal-Levy et al., 2000). 4

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The significance of cortisol in assessments of stress may be limited when examining chronic stressors, due in part to the acclimation of the interrenal tissues during chronic stress, which is mitigated by negative feedb ack mechanisms on the hypothalamo-pituitaryinterrenal (HPI) axis (R otllant et al., 2000). Other bio-ma rkers, such as expression levels of GR, have been shown to be more sensitive indicators of chronic stre ss. Quantification of GR in seabass ( Dicentrarchus labrax ) showed significantly reduced GR concentrations after a 3-month exposure to elevated stoc king densities (Terova et al, 2005). Environmental contaminants have been s hown to alter the stre ss response by altering GR activation. Organotins, compounds used as industrial stabilizers in paints now present in aquatic environments, have been shown to block GR activation (Odermatt et al., 2006). Other ubiquitous pollutants such as PCBs and arsenic have also been shown to alter GR receptor functioning (Johansson et al., 1998; Bodwell et al., 2004). The effects of stress can be manifest at multiple levels of the reproductive endocrine axis (Guillette et al., 1995; Pankhurst and Van Der Kraak, 1997). Although there is limited information on the effects of stress on the rel ease of GnRH on aquati c inhabitants, several studies have been conducted identifying stre ss impacts on circulating concentrations of GTH-I and GTH-II. For some species of fish such as brown trout ( Salmo trutta ), confinement stress results in an increase in circulating concentrati ons of GTHs (Pickering et al., 1987; Sumpter et al., 1987). For othe r species, such as the white sucker ( Catostomus commersoni ), capture and transport st ress results in depression of GTHs to undetectable concentrations within 24 h of capture (Stacey et al., 1984). The effects of stress on concentrations of gonadal steroids in both terrestrial and aquatic animals is well documented, resulting in a depression in plasma concentrations of 5

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both androgens and estrogens in most species st udied to date (Francis, 1981; Pickering et al., 1987; Carragher and Pankhurst, 1991). These reductions can be attributed to altered secretion of gonadotropins (Gra y et al., 1978) as well as by direct inhibition of gonadal steroid synthesis (Saez et al., 1977; Sapolsky, 1985). Cortisol has also been implicated in alteri ng endocrine function. Cortisols negative effects on reproduction includes depressed pl asma concentrations of sex steroids (Pankhurst and Dedual, 1994; Pott inger et al, 1999). However, this response is dependent upon the hormones involved and the species investigated. Elev ated plasma concentrations of cortisol in Stellate sturgeon ( A. stellatus) females have been shown to result in correspondingly lower concentrations of ci rculating plasma T and 11-KT, however, E 2 and progesterone (P) remain constant (Semenkova et al., 2002). Similarly, Bayunova (2002) observed an inverse relationship between cortisol and T after a 9-h period of confinement stress for both male and female stellate st urgeon. Consten et al. (2002) investigated whether the decrease in plas ma 11-KT of male carp was ca used by a direct effect of cortisol, or by an indirect effect (such as a decrease in plasma LH). Experimental animals were fed cortisol-containing food pellets over a prolonged peri od, and the results indicated that cortisol had a direct i nhibitory effect on testicular androgen secretion that was independent of LH secretion. Reductions in reproductive hormones can lead to a myriad of deleterious reproductive effects such as d ecreased gamete quality, embryo mortality, and behavioral changes (Pankhurst and Va n Der Kraak, 1997; Pankhurst, et al., 1995). Endocrine Disruption in Aquatic Vertebrates Xenobiotics, or man-made chemicals, have been shown to disrupt normal hormone function, and have received considerable attention over the last decade (Colborn and Clement, 1992; Guillette, 2000; McLachlan 2001 ). Compounds evaluated as endocrine 6

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disrupting contaminants have generally in cluded common environmental pollutants which have demonstrated abilities to mimic hormone s, alter hormone production, or act as antihormones (Guillette, 2000). Molecularly, xenobiot ics have the ability to bind directly to steroid hormone receptors or other proteins that initiate or facilitate the transcription of genes (Thomas, 1990; Rooney and Guillette, 2000) Compounds such as polychlorinated hydrocarbon pesticides (e.g., DDT derivatives), polychlorinated biphenyls (PCBs) and others have been shown to bind to estroge n receptors manifesting estrogenic or antiestrogenic actions in mammals and birds (B ulger and Kupfer, 1985; Rooney and Guillette, 2000). Extensive work has been conducted in fishes, and evidence indicates similar mechanisms occur (Thomas, 1990; White et al., 1994; Tyler et al., 1998a; 1998b; 1999; Jobling et al., 1995; 1996; 1998; 2002). Numerous studies document a vast array of endocrine disruptive effects in fish located in polluted aquatic systems and areas downstream of sewage or other industrial treatment plants (Jobling et al., 2003; Toft et al., 2004). Male walleye ( Stizostedion vitreum ) collected near a metropolitan sewage treatment plant exhibite d depressed serum T concentrations and elevated serum E 2 concentrations compared to reference males (Folmar et al., 2001). Reduced plasma concentrations of T have also been documented in lake whitefish ( Coregonus clupeaformis) and white sucker (Catostomus commersonii ) exposed to bleached Kraft mill and pulp mill effluent respectively (Munkittrick et al., 1992; 1994). Female mosquitofish downstream from Kraft paper-mill effluent in Florida demonstrated masculinization of the anal fins, which is an androgen-dependent trait (Parks, et al., 2001). Male mosquitofish from a Florida lake cont aminated with known endocrine disruptors displayed shorter gonopodium, significantly redu ced whole body T concentrations, reduced 7

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liver weights and had reduced sperm counts versus those of a reference population (Toft et al., 2003). Compounds such as the natural steroid E 2 have been measured in both industrial and municipal sewage treatment effluents, whic h represent the principl e sources of natural estrogens in the aquatic environment (Lai et al., 2002). Exposure to E 2 caused disruptions in sexual differentiation in young zebrafish and altered egg production patterns in adults (Brion et al., 2004). Exposure of the riverine species the roach ( Rutilus rutilus ) to a host of chemicals persistent in typical British waters, revealed significantly in creased incidences of intersexuality and plasma vitellogenin concentrations and attributed these alterations to estrogenic constituents of sewage effluents (Jobling et al., 1998). Considerable work also has been conducted on abnormalities of the reproductive system of Floridas alligators in relation to environmental co ntamination, notably in Lake Apopka, located northwest of Orlando. These studies report reducti ons in circulating concentrations of sex steroids, alterati ons in gonadal morphology, phallus size, enzyme activity and steroidogenesis (G uillette, et al., 1999; 2000). These modifications were attributed to both embryonic and post-hatc hing exposure to a complex mixture of chemicals from agricultural activities and stormwater runoff, including PCBs, p,p -DDE, dieldrin, endrin, mirex, and oxychlordane. Ex cess nitrate has also been shown to alter steroidogenesis and endocrine function in seve ral aquatic species (G uillette and Edwards, 2005; Barbaeu, 2004). Detailed lists of know n endocrine disrupting contaminants and their documented effects are readily availabl e (Edwards, 2006), and wi ll be discussed in further detail in Chapters 3, 4 and 5. 8

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Nitrate in Natural Water Systems In nature, organic and inorganic nitrogen is cycled through various environmental processes such as nitrifica tion, denitrification, fixation a nd decay. Nitrification and denitrification processes are esse ntial to the health of aquatic ecosystems. These processes generally begin with ammonia, which is broken down to nitrite by aerobic nitrifying bacteria (usually Nitrosomonas sp.), which is then converted by another group of bacteria to nitrate (usually by Nitrobacter sp.). Nitrate is often then fi xed by plants as a nutrient, or undergoes denitrification (Sharma and Ahlert, 1977). Complete deni trification converts nitrate to either nitrogen gas or organic nitrogen. Incomplete denitrification, resulting from inadequate sources of carbon or environmenta l conditions, results in nitrates conversion back to nitrite, or even ammoni a, by anaerobic denitrif ying bacteria (Van Rijn et al. 2006). Over the last several decades, concentrati ons of nitrate in natural water bodies from anthropogenic impact has increased significantly (Pucket, 1995), which has resulted in nitrate concentrations in many water sources fa r in excess of the EPA drinking standard of 10 mg/L nitrate-N (Kross et al., 1993; U. S. EPA, 1996). In northern Florida, concentrations as high as 38 mg /L nitrate-N were recorded in the Suwannee River (Katz et al., 1999). In addition to its direct effects, nitrate can encourage ex cessive algal and plant growth, adversely impacting the ecology of th e affected area (Attayde and Hansson, 1999; Capriulo et al., 2002). Nitrate in Aquaculture and It s Implications as an EDC As discussed previously, elevated con centrations of stress hormones have been shown to result in decreased concentrations of circulating sex ster oids. Environmental contaminants have been shown to elicit a st ress response, thereby decreasing circulating concentrations of sex steroids. In fact, some of the earliest reports of vertebrate stress 9

PAGE 22

responses were induced by chemical exposur e (Selye, 1936). While it is clear many manmade chemicals have considerable impact on horm one function in aquatic animals, it is less clear if naturally occurring compounds could also have the same effect. Contaminated aquatic ecosystems such as Lake Apopka, Fl orida provide ample opportunity to observe severe abnormalities of the reproductive system and are decidedly unhealthy for aquatic life. In aquaculture, aquatic animals are exposed to xenobiotic and natural compounds often far in excess of those experienced in nature, but resultant abnormalities are often overlooked since aquaculture fish are not necess arily expected to mimic wild fish. After all, they are held at higher densities, eat dramatically diffe rent diets, and are often held under unnatural temperature and light regimes. Additionally, defin itions of acceptable water quality standards of natu ral water environments (generally under EPA regulation) versus those of intensive aquaculture system s (under the regulation of the farm manager) are usually dramatically diffe rent. Commercial aquaculture operations have limited budgets (if any) for in-depth research into the factors that are contri buting to the success or failure of husbandry practices and protocols. Therefore, water quality estimates of safe operating levels in aquaculture are often the result of trial and error practices based on growth or mortality events. For species such as sturgeon, which take many years to reach reproductive maturity, and whose economic vi ability relies heavily on proper egg production, it may be important to investigat e more thoroughly the sublethal effects a potential hazard may impose. Nitrate has been overlooked as a material water quality hazard in both natural and aquaculture settings. Emerging information implic ates nitrate as a hazard at concentrations once thought to be innocuous for both reptile and amphibian species (see Guillette and 10

PAGE 23

Edwards, 2005). It has been shown that vertebrate mitochondria are capable of nitric oxide (NO) synthesis via non nitric oxide synthase (NOS) activity (Zweier et al., 1999) using nitrite as a precursor. Nitrat e can be converted to nitrite in-vivo (Panesar and Chan, 2000), and it is thought other enzyme s can generate NO directly from nitrate (Meyer, 1995). Nitric oxide is a gas that pl ays diverse roles in cellular signaling, vasodilation, immunity and has been documented to inhibit steroi d hormone synthesis (DelPunta et al., 1996; Panesar and Chan, 2000; Weitzberg and Lundberg, 1998). As discussed previously in this chapter, StAR and P450 SCC are key factors regulating steroi dogenesis. NO has been shown to alter the activity of St AR and may also alter P450 SCC by binding to the heme group which is present in all enzymes of the P450 fa mily (White et al., 1987). Bulls fed nitrate showed reduced sperm motility a nd degenerative lesions of the germ layers of the testes (Zraly et al., 1997). Medaka exposed for 2-months to no more than 75 mg/L NO 3 -N showed reduced fertilization and hatching rates (Shimura et al., 2002). A study of female mosquitofish ( Gambusia holbrooki ) in Florida showed reduced reproductive activity and embryo number in fish exposed to 5.06 mg/L NO 3 -N (Edwards et al., 2006b). Reproductive hormone concentrations have b een shown to be especially vulnerable to chemical and physical strain (Pickering, 1987), which as discussed can cause numerous reproductive complications. Since nitrate has been shown to negatively impact the reproductive physiology of a number of aquatic species (Edwards et al. 2006a; Edwards et al., 2006b) and sturgeon have been shown to be unusually susceptible to environmental impact (Akimova and Ruban; Dwyer et al., 2005), it stands to reason that nitrate could be an endocrine disrupting contaminant for Siberian sturgeon, and is worthy of investigation. 11

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In the United States and elsewhere, water is becoming a valuable and limited commodity, and its use is tightly regulated. New aquaculture operations will not be afforded the vast quantities of water establishe d facilities have been permitted to use, and will therefore need to use recirculating technolog ies which enable these facilities to reuse a significant portion of their water. In most of these recirculating facilities, the limiting factor for water exchange is nitrate concentration. Sturgeon as a Model Species Sturgeons belong to one of th e most ancient groups of Osteichthyes and are naturally distributed above the 30 th parallel. Although they can be found almost everywhere along the Pacific and Atlantic coasts, the Medite rranean and Black Seas, as well as rivers, lakes and inland seas, most sturgeon populations are sparse and occur in significant numbers in only a few regions (Detlaff et al., 1993). The Caspian Sea represents a unique reservoir, producing the bulk of the world s sturgeon capture fisheries. Sturgeon include anadromous, semi-anadromous and river-resident (freshwater) forms. The Siberian sturgeon have both semi-anadrom ous and river resident populations (Detlaff et al., 1993). Sturgeon have preserved primitive structural features relating them to chondrosteans, while at the same time the structure of their eggs is more similar to amphibians than either chondrosteans or teleosts, since th e inclusions of yolk are distributed throughout the cytoplasm. Alt hough sturgeon produce great numbers of large eggs, affording them great ecological advant age in hostile environm ents, ironically this production is at the nexus of their dwind ling population. Sturgeon eggs, termed caviar when processed, are a prized delicacy and commands very high prices. This has lead to over fishing on a grand scale (Birstein, 1993; W illiot et al., 2002). This over fishing, in 12

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concert with other anthropogenic impacts, such as river damming and pollution, has resulted in the reduction, or in some cas es decimation, of stur geon stocks worldwide (Williot et al., 2002). Aquaculture has been pr oposed as a mechanism to help save wild populations, either by reducing fishing pre ssures or by providing animals for stock enhancement. Due to the high value of cavia r, sturgeon aquaculture has great promise. As discussed above, nitrate is the limiting f actor for water exchange in recirculating aquaculture systems. The less water a facility uses, the greater the possible concentrations of nitrate, and although resear ch is underway to develop technologies to reduce nitrate concentrations, it is unclear what affects nitr ate has on fish residing in these systems. Additionally, environmental nitrate from anthropogenic sources is increasing at an alarming rate worldwide (Rouse et al., 1999), and with pollution implicat ed in reductions in wild sturgeon populations in the Caspian Sea, the worlds largest stur geon reservoir, the need to understand the affects of nitrate on st urgeon is becoming more and more apparent. That egg production is paramount to the viabilit y of sturgeon as an aquaculture species, and is of obvious ecological importance, necessitate s an understanding of the affects of nitrate on the reproductive system in particular. Research Objectives and Hypotheses The goal of this study was to gain a better mechanistic understanding of the potential for nitrate-induced disruptions in reproductive function, using Siberian sturgeon as a model. Based on previous studies review ed in this Chapter, I hypothesize that given nitrates ability to alter steroidogenic activity, notably thr ough NO induced alterations in P450 enzyme activities, that the fish exposed to elevated nitrate will demonstrate reduced concentrations of plasma sex steroid concentr ations, and these reductions will be mirrored in gonadal mRNA expre ssion patterns of P450 SCC ER and GR. I theorize that these 13

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alterations would not be caused by a generali zed stress response, but by disruptions in steroidogenic mechanisms directed at the produ ction of sex steroids, notably T, 11-KT and E 2 Compensatory mechanisms required to combat physiological challenges consumes energy and physiological resources that could otherwise be used to carry out other essential functions. Therefore, an animal experien cing simultaneous stressors, such as nitrate exposure in combination with an induced stresso r such as confinement, may not be as adept at responding to the stress events as an anim al experiencing a single stressor. I therefore hypothesize that long-term exposur e to elevated nitrate will alter the associated stress response. In addition, given th at GR has been shown to parallel chronic stress, I predict GR mRNA expression will be significantly reduced in animals exposed for 30 days to elevated nitrate. 14

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Figure 1-1. Overview of the hypothalamicpituitary-gonadal axis in sturgeon. The hypothalamic-pituitary-gonadal axis in st urgeon is similar to that of other vertebrates. Gonadotropic releasing hormone (GnRH) from the hypothalamus controls the release of gona dotropins (GTHs) from the pituitary that then enter circulation. The gonad responds by produc ing various sex steroids including 17 -estradiol, which stimulates hepa tic vitellogenin production. These processes are essential for normal ovarian follicle development. Similar to other fish species, the hypothalamic release of corticotropin-releasing hormone (CRH) controls the release of adreno-corticotropin hormone (ACTH) from the pituitary, which controls the release of glucocorticoids from the interrenal cells of the head kidney. 15

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CHOL P450SCCPREG PROG 3 -HSDAndrostenedione Testosterone 17 -Estradiol 17 -HSDP450AROMATASECholesterol Pool MITOCHONDRION STAR LH LH-R P450 17 hydroxylase 17 hydroxypregnenolone dehydroepiandrosterone 17 -h y drox yp ro g esterone Figure 1-2. Representative steroi dogenic pathway of steroid hormones in gonadal cells. In response to ligand binding of the receptor, the transfer of free cholesterol into the mitochondria facilitate d by steroidogenic acute regul atory (StAR) protein, is considered the acute rate limiting step in steroidogenesis. The enzymatic conversion of cholesterol to pregeneolone by P450 SCC is considered the chronic regulatory step in steroidogenesis. Pr egnenolone or progesterone is released into the cytoplasm/smooth endoplasmi c reticulum to be converted to androstenedione, which is in turn converted into testosterone and 17 -estradiol by 17 -HSD or aromatase respectively. 16

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ACTH CHOL P450SCCPREG PROG 3 -HSD 17 -h y drox y pro g esterone 11-deoxycortisol Cortisol P450 21-hydroxylase P450 11 -hydroxylase Cholesterol Pool MITOCHONDRION STAR P450 17 -hydroxylase ACTH Figure 1-3. Representative steroidogenic pathway of cortisol production in an interrenal cell. In response to ligand binding of the receptor, the transfer of free cholesterol into the mitochondria fac ilitated by steroidogeni c acute regulatory (StAR) protein, is consider ed the acute rate limiting step in steroidogenesis. The enzymatic conversion of chol esterol to pregeneolone by P450 SCC is considered the chronic regulatory step in steroidogenesis. 17 hydroxyprogesterone is released into the smooth endoplasmic reticulum for further processing and eventual conv ersion 11-deoxycortisol and cortisol. 17

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CHAPTER 2 NITRATE TOXICITY IN SIBERIAN STURGEON Introduction Ammonia is a product of the biological degrad ation of proteins and nucleic acids. Nitrifying bacteria convert ammoni a to nitrite, which is in turn converted to nitrate, the end product of nitrification (Sharma and Ahlert, 1977). Ammonia, a nd to a less extent nitrite, are ecologically relevant compounds and the to xicity of these compounds, both in terms of tolerable thresholds and physiologic mechanis m to aquatic animal health, has been well documented (Rubin and Elmaraghy, 1977; Meade, 1985; Huertas et al., 2002). Nitrate, however, does not normally reach toxic concentr ations in natural environments or in recirculating systems with high water exchange and has therefore received comparatively less attention as a material water quality hazard (Knepp and Arkin, 1973; Russo, 1985; Bromage et al., 1988; Meade and Watts, 1995). The absence of obvious pathophysiological effects in most aquatic species at ecologically relevant concentrations of nitrate, rationalizes the belief that nitrate is relatively non-toxic (Jensen, 1996). While nitrate is indeed much less toxic than ammoni a or nitrite on a mg/L basis, nitrate commonly rises to levels far in excess of those of the other compounds in intensive aquaculture environments with limited water exchange (Knepp and Arkin, 1973; Hrubec, 1996), and warrants more detailed investigations in to the effects thes e levels may have. Excess nitrate in aquaculture has traditionally been reduced by water exchange or the operation of denitrification filters (Timmons et al., 2001). Current trends in environmental regulation are limiting the amount of water which may be consumed or discharged, reducing the feasibility of using large infl uxes of water to rem ove excess nitrate. 18

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Denitrification filters can be technically challenging and costly, and as aquaculture operations become water limited, nitrate wi ll become a considerable concern. The levels of nitrate that are likely to cause concern are unknown for many aquatic species, as are how susceptibili ties to nitrate change ontoge netically. For large species such as sturgeon, it is logistical ly difficult and costly to cond uct acute toxicity evaluations on broodstock size animals. However, evalua tions using smaller animals may not mimic responses of larger fish. New evidence implicat es nitrate as a material water quality hazard at levels much lower than previously suspect ed for other aquatic sp ecies (Guillette and Edwards, 2005) and recommended levels of nitrate for warm-water fishes (90 mg N0 3 -N) (U.S. E.P.A., 1986) has been shown to be highly toxic to amphibians (Marco et al., 1999). Although a great deal of research needs to be conducted to elucid ate the effects of sublethal exposures, acute testing will assist researchers in understa nding how sensitive a particular species is to nitrate, and can be used as a tool to predict if susceptibilities may change over time. The most common analytic al method for evaluating acute toxicity in fish is the LC 50 (Parish, 1985) An LC 50 describes a lethal concentr ation (LC) at which 50% of the experimental population dies in a specified period of time. LC 50 data allows us to determine if a substance is toxic, how toxic it is, and allows for multi-species comparisons of sensitivity. The objectives of this study were to determine the acute toxicity of three ontogenetic size classes of Siberian sturgeon ( Acipenser baeri ) to nitrate, using the LC 50 criterion, to determine how life stage influences this response. 19

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Methods Study Animals and Pre-Testing Conditions Siberian sturgeon were reared from eggs in 250 L troughs in a recirculating system containing well water. Fish were initially fed Artemia and a soft moist formulated feed (Silver Cup, Nelson and Sons Inc., Murray, UT). When the fish reached 1.5 g they were transferred to 1300 L tanks and were fed only fo rmulated feeds by this time. Dissolved oxygen was monitored daily and rarely went below 90% saturation (Oxyguard Handy Beta, Point Four Systems Inc., Richmond, BC, Canada ). Temperatures were slowly increased throughout the fishs devel opment, and ranged from 15 C (at hatch) to 23.5 C. Other water quality parameters prior to the toxicity tr ials were evaluated weekly (ammonia-N and nitrite-N, Lamotte Smart Colorimeter, Ches tertown, MD; nitrate, Ion 6 Acorn Series, Oakton Instruments Vernon Hills, IL; pH, Acorn 6 Series, Oakton Instruments Vernon Hills, IL). In addition to the above parameters, alkalinity, chloride, total hardness and calcium hardness (Hach Company, Loveland, CO) were tested at the beginning and end of each 96-h toxicity trial. Range-Finding Studies Small-scale range finding studies using at le ast three nitrate concentrations with five fish/concentration were conducted prior to each test until a suitable test range was determined. Suitability was defined by total mortality in the highest concentration and no mortality in the lowest concentration in 96 hours within a narrow test range. Tests generally required 2-3 range findi ng studies per toxicity trial. Tanks were evaluated for mortalities every 3-4 hours from 08:00 to 20: 00, and dead fish were immediately removed and inspected for condition. 20

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Test Procedures Three partial exchange 96-h toxicity tests were conducted in tr iplicate using three weight classes of Siberian sturgeon spanning 3 orders of magnitude, with 10 fish per test container. Experiments were conducted ove r time using fish from the same cohort to eliminate cohort variability. New experimental animals were used for each trial. Water for each of the evaluations consisted of degassed well wa ter (nitrate-N 1.4 0.3 mg/L) from which nitrate solutions were created from food-grade sodium nitrate (JLM Marketing, Tampa, FL) Initial concentrations were confirmed with an Auto Analyzer and were periodically spot-checked with an ion specific probe (Ion 6 Acorn Series, Oakton Instruments Vernon Hills, IL) throughout the trials to ensure concentrations matched initial target values. Each trial evaluated f our geometrically consta nt concentrations of nitrate, as well as triplicate well water a nd sodium controls. Sodium controls were achieved with NaCl (Morton Salt, Chicago, IL ) with concentrations adjusted to match the sodium in the highest nitrat e concentration in the trial. Tanks were randomly assigned to each treatment. Tanks were evaluated for mortalities every 3-4 hours from 08:00 to 20:00 and dead fish were immediately removed and inspected for condition. The first trial evaluated concentratio ns of 555, 888, 1420, and 2273 mg/L nitrate-N using 6.9 0.31g fish. This trial was conducted in glass aquaria filled with 32.4 L of test solution, submersed in a water bath to maintain a temperature of 21 C. A 50% water exchange with the appropriate nitrate con centration was conducted half way through the trial to eliminate collateral effects from elevated ammonia or nitrite. Fish were not fed two days prior to and throughout the trial, a nd fecal debris was siphoned twice daily. 21

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At least twice daily, observations were made of fish behavior (orientation, gill ventilation rate, swimming speed) and appearan ce throughout the trial. The second trial evaluated concentrations of 216, 323, 485, and 727 mg/L nitrate-N using 66.9 3.4 g fish. This trial was conducted in fiberglass tanks fill ed to 670 L. The water was maintained at 23.5 C. The third trial evaluated concentr ations of 234, 421, 758 and 1364 mg/L using 673.8 18.6 g fish. This trial was conducted in fiberglass tanks filled to 587 L and the temperature was maintained at 23.5 C. Statistical Analyses Data from replicates were pooled prior to ca lculating the median le thal concentration. Median lethal concentrations and 95% confidence intervals were evaluated by the trimmed Spearman-Karber method for 24, 48, 72, and 96-hr time periods. Testing ranges, determined by range finding studies, were de signed to evaluate a 96-hr time period. Therefore, shorter time periods did not always result in enough mortality to compute the LC 50 values. Normal distribution was evaluated with the Shapiro-Wilks test. A linear regression of log 10 transformed data was conducted to predict susceptibilities of larger sturgeon using StatView statistical software package (SAS Institute, Cary, NC). Results No animals died in either the well water or sodium controls for any of the size classes tested, and appeared healthy th roughout the trial. The 96-h LC 50 of nitrate to 6.9 0.31 g Siberian sturgeon was 1028 mg/L nitrate-N (Tab le 2-1). Moribund fish in this size class tended to gill rapidly, but most showed few outwa rd signs of toxicity except a stiffening of the musculature and lethargy (decreased swim ming speed, frequent resting periods). The 96-h LC 50 of nitrate to the 66.9 3.4 g and 673.8 18.6 g sturgeon was 601 mg/L and 397 22

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mg/L nitrate-N respectively. Moribund fish in these treatments tended to exhibit additional evidence of the toxicity such as reddening around the mouth, and red specks and/or patches along the length of the body, most notably at the base of the pectoral fins. Log transformed nitrate vs. log transformed LC 50 values are shown in Fig. 2-1. Water chemistry parameters were as follows: unionized ammonia-N (NH 3 ) 0.04 0.02 mg/L; nitrite-N 0.01 mg/L; pH 7.9 0.2; alkalinity 208 12 mg/L; chloride 90 5 mg/L (exclusive of the NaCl control); total hardness 260 10 mg/L; calcium hardness 160 10 mg/L. Dissolved oxygen levels were maintained at 95% saturation throughout the trials. The ShapiroWilks test indicated normal distribution for a ll treatments. The 6.9 0.31 g sturgeon were maintained at 21.0 C while the latter two size cl asses were maintained at 23.5 C, which are typical temperatures for these size stages. Placing all three size classes at the same temperature would not represen t a realistic rearing condition, and previous toxicity tests with this species has not demonstrated a significant difference in LC50 values for temperatures ranging from 20 C-25 C for 6.0 g to 1 kg Siberian sturgeon (H. Hamlin, unpublished data). Discussion The United States is now recognizing wate r as a valuable and limited commodity, and its tight regulation is forc ing aquaculture technology to sh ift toward more sustainable and ecologically responsible practices. Theref ore, as the land-based aquaculture industry continues to grow, management strategies are shifting to recirculati ng systems with lower water exchange. This trend is creating new husbandry concerns as less clean water is available to flush out nitrate. In systems with limited water exchange, nitrate can build to levels of 150 mg/L nitrate-N or more (perso nal observation), and it is unclear the impact 23

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these elevated levels may have. Critical for the design of any aquaculture operation are the water quality standards to be maintained, a nd it is important to know what levels of substances are likely to cause concern (Bohl 1977). The etiology and effects of nitrate toxicity are relatively unknown in fishes, l eaving open future opportunities for research in this area. This information can then be used to understand toxi city thresholds and physiologic impact, as well as appropriately engineer remediation systems and technologies. Results of this study demonstrated the 96-h LC 50 for fish of 7-700 g to range between 397-1028 mg/L nitrate-N. These numbers are appreciably lower than those reported for most aquatic species tested to date. Comparative nitrate data from representative toxicity studies suggests that the majo rity of test populations can handle nitrate-N levels of 1000 mg/L nitrate-N or more (4426 mg/L total n itrate) without reachi ng 50% mortality, when sodium nitrate is used as the source of n itrate (Table 2). Some fish, such as the beaugregory ( Stegastes leucostictus ), exhibit LC 50 values of over 3000 mg/L NO 3 -N (13,280 mg/L total nitrate), substantially abov e the tolerance of most freshwater fish including Siberian stur geon (Peirce et al., 1993). Although diet may affect the relative toxicity of nitrate (Chow a nd Hong, 2002), a pervasive theory in the etiology of nitrate toxicity is that it is endogenous ly converted to nitrite (Hill, 1999), and it is in fact nitrite that is the biotoxic agent. In terrestrial anim als this theory has been the source of numerous debates (Hartman, 1982), and the mechanism of nitr ate toxicity in fishes is still unclear. Anecdotal evidence at Mote Marine Laboratorys Aquaculture Park (Sturgeon Commercial Demonstration Projec t) has shown Siberian sturgeon to be especially sensitive to nitrate, with larger animals exhibiting in creased incidence of t oxicity and mortality 24

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starting at levels as low as 90 mg/L nitrate-N (398 mg/L total nitrate, see Guillette and Edwards (2005) for an explanation of the re porting of nitrate concentrations) (H. Hamlin, unpublished data). Susceptibilities have been strongly affected by c ohort variability, with certain cohorts being more sensitive to elevated nitrate than others. Although the results in this study demonstrate a strong correlation between size and LC 50 values, caution must be taken in predicting susceptibilities of varying co horts of fish, or even fish within the same cohort, since LC 50 values have been shown to be highly variable (Buikema et al., 1982). Regression analysis of the curr ent data yield a predicted LC 50 of 247 mg/L nitrate-N (1093 mg/L total nitrate) for 6 kg fish (Fig. 2-1). Regardless of the high variability of toxicological responses to nitr ate, it is clear from this study that young Siberian sturgeon are far more tolerant to elevated nitrate than their adult counterparts, and this is the first study to demonstrate this finding. Often, the dose-response relationship is a scaled association between the concentration of chemical tested and the severi ty of the elicited re sponse (Lloyd, 1979). In general, younger or immature animals tend to be more susceptible to chemical insult or perturbation than are adults (Macek et al., 1978; Sprague, 1985). In fact, a common chronic toxicity test is the ear ly life stage test, because althou gh this test does not provide total life cycle exposure, it is purported to include exposur e during the most sensitive life stages (McKim, 1985). This study found an incr eased tolerance of Si berian sturgeon to nitrate at younger stages. Although this opposes general co nvention, this phenomenon has been reported for other fish species with othe r toxic compounds (Rosenberger et al., 1978). Acute toxicity tests are an effective tool to establish baseline toxicity thresholds in terms of responses to nitrate over time, and to compare the toxicity of nitrate to other 25

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species. Given the increased sensitivity of Si berian sturgeon to nitrate as compared to other species, it is clear much more work is needed to elucidate the sublethal effects of elevated nitrate exposure. The sensitive natu re of sturgeon to nitrat e renders them suitable candidates for further investig ation of the etiology and natu re of nitrate exposure and toxicosis. 26

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Table 1-1. LC 50 results and test conditions for thr ee size classes of Siberian sturgeon exposed to sodium nitrate Average weight 6.9 0.31 g 66.9 3.4 g 673.8 18.6 g 24-h LC 50 (mg/L NO 3 -N) 1510 n/a 803 95% confidence interval (1826-2631) (720-897) 48-h LC 50 (mg/L NO 3 -N) 1443 n/a 522 95% confidence interval (1309-1590) (486-562) 72-h LC 50 (mg/L NO 3 -N) 1195 n/a 438 95% confidence interval (1086-1316) (394-487) 96-h LC 50 (mg/L NO 3 -N) 1028 601 397 95% confidence interval (941-1124) (557-649) (357-441) Not enough partial kill res ponses to obtain a valid lethal concentration estimate. 27

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Table 1-2. Representative acute toxicity data for nitrate NO 3 NO 3 -N Species Source mg/L LC 50 Reference Cape sole ( H. capensis ) NaNO 3 5081 24-h LC 50 Brownell 1980 Common bluegill ( L. macrochirus ) NaNO 3 2909* 24-h LC 50 Dowden and Bennett 1965 Goldfish (C. carassius ) NaNO 3 2761* 24-h LC 50 Dowden and Bennett 1965 Tiger shrimp ( P. monodon) NaNO 3 1575 96-h LC 50 Tsai and Chen 2002 Catla ( C. catla ) NaNO 3 1565 96-h LC 50 Tilak et al. 2002 Channel catfish ( I. punctatus ) NaNO 3 1409 96-h LC 50 Colt and Tchobanoglous 1976 Chinook salmon ( O. tshawtscha) NaNO 3 1318 96-h LC 50 Westin 1974 Fathead Minnows ( P. promelas ) NaNO 3 1349 96-h LC 50 Scott and Crunkilton 2000 Guadalupe Bass ( M. treculi ) NaNO 3 1269 96-h LC 50 Tomasso and Carmichael 1986 African clawed frog ( X. laevis ) NaNO 3 1236 240-h LC 50 Schuytema and Nebeker 1999 Aquatic Snail ( P. antipodarum ) NaNO 3 1042 96-h LC 50 Alonso and Camargo 2003 Florida pompano ( T. carolinus ) NaNO 3 1006 96-h LC 50 Pierce et al. 1993 Sao Paulo shrimp ( P. paulensis) NaNO 3 494 96-h LC 50 Cavalli et al. 1996 Pacific treefrog ( P. regilla ) NaNO 3 266 240-h LC 50 Schuytema and Nebeker 1999 Guppy fry ( P. reticulatus) KNO 3 200 72-h LC 50 Rubin and Elmarachy 1977 Caddisflies ( C. pettiti ) NaNO 3 114 96-h LC 50 Comargo and Ward 1992 Publication did not specify whet her results were values for NO 3 or NO 3 -N 28

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2.5 2.6 2.7 2.8 2.9 3 3.1 0.511.522.533.5 Log fish weight (g)Log nitrate-N (ppm) Y=3.177 .208*X; R^2 = .994 Figure 2-1. Linear regression of log 10 transformed nitrate-N (mg/ L) lethal concentration values versus log transformed fish weight (g). 29

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CHAPTER 3 STRESS AND ITS RELATION TO ENDOCRI NE FUNCTION IN CAPTIVE FEMALE SIBERIAN STURGEON Introduction The central focus of comparative physiol ogy and endocrinology i nvolves understanding how various organisms respond to environmental infl uences. Fish are affected by stress in both their natural and captive environments. It is well recognized that common fishery and aquaculture practices, including cr owding, transport and confinement are stressful to fish and can negatively affect reprod uction (Pankhurst and Van Der Kraak 1997). The effects of stress can be manifested at many levels of the reproductive endocrine axis, and measuring the concentration of circulating hormones is a usef ul endpoint to understand if a stressor affects endocrine function. Numerous environmental stressors, including capture and confinement (Pankhurst and Dedual, 1994), time of day (Lankford et al., 2003), hypoxia (Maxime et al., 1995), and environmental contaminants (Orlando et al., 2002; Guillette and Edwards, 2005) have been shown to induce stress in fish. For most fish, including the Sibe rian sturgeon and other freshwater chondrosteans, cort isol is the predominant stress hormone (Maxime et al., 1995; Barton et al., 1998; Mommsen et al., 1999). Plasma glucose con centration has also been shown to be an indicator of secondary st ress responses (Bayunova et al., 2002). Sex steroids can have an invers e relationship with plasma conc entrations of stress steroids, an effect evident in fish and some other anim als (Carragher and Sump ter, 1990; Cooke et al., 2004). Negative effects of stress on reproduction have been attribut ed to the suppression of LH and FSH secretion from the pituita ry gland, disruptions in steroi dogenesis pathways, or alteration of hormone degradation by the liver and/or kidney (Krulich et al., 1974). Although plasma concentrations of corticosteroids often parallel acute st ress, there is evidence in teleosts that the 30

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estrogenic inhibitory effects of stress are not necessarily mediat ed by cortisol, and that these effects arise higher in the endocrine pathway than at the le vel of ovarian steroidogenesis (Pankhurst et al., 1995). Contradictory evidence has shown that the a ddition of cortisol to the culture medium reduces the secretions of 17 -estradiol (E 2 ) and testosterone (T) from cultured ovarian follicles of rainbow trout ( Oncorhynchus mykiss) (Carragher and Sumpter, 1989 ). Likewise, carp fed with cortisol-containing food pellets showed reduced androgeni c production, independent of LH secretion (Consten et al., 2002). Acute c onfinement stress in male brown trout ( Salmo trutta L.) resulted in low concentrations of plasma T and 11-KT in sexually mature animals (Pickering et al., 1987). White sturgeon ( Acipenser transmontanus ) injected with an AC TH analog exhibited a dose-dependent increase in cortisol concentration more than the co rtisol concentrations induced by stress events such as transport and handling (Belanger et al., 2001). A few studies, including one examining the effects of stress on serum cortis ol concentration in cult ured stellate sturgeon, actually demonstrated significantly increased game te quality in fish with elevated cortisol concentration, speculating that cortisol could be a normal endocrine component of the reproductive system, even though later studies of the same species showed reduced plasma concentrations of sex steroids during stress (Semenkova et al ., 1999; Bayunova et al., 2002). It has also been shown that fish require prolonged periods to recover from an acute stress event (Jardine et al., 1996). Other studies have show n that blood removal, a practice often necessary for evaluating endocrine endpoint s, can alter blood hemoglobin concentration (Hogasen, 1995). Stress studies typically focus on the causative factors mitigating the deleterious response, but defining these relationships often requires sampling and research measures that themselves contribute to enhancing the stress response. Understanding the eff ects of potential stressors is 31

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critical to properly manage wild fisheries or successfully culture endangered or economically important fishes. It is importa nt to know which stressors are na turally present in the fishs environment, which are caused by typical aqu aculture practices, and which are induced by the testing procedures th emselves (Conte, 2004). Sturgeons ( Acipenseriformes ) are among the most ancient fishes on earth, originating over 200 million years ago (see review by Birstein, 1993). Twenty-five extant sturgeon species occupy the Northern Hemisphere; however, exce ssive fishing, loss of spawning grounds and other environmental pressures have contributed to the reduction of sturgeon stocks worldwide, particularly Caspian Sea varieties (Williot et al ., 2002). Today, all 25 species of sturgeon are listed as endangered or threatened in some regard (Birstein, 1993). Aquaculture has been proposed as a means to conserve sturgeon, and ge nerating commercial stocks has the dual benefit of providing fish for stock enhancement, as we ll as for food production, thus conserving wild populations (Beamesderfer and Farr, 1997; Wald man and Wirgin, 1997; Chebanov et al., 2002; Stone, 2002). The Siberian sturgeon is rapidly beco ming a species of great economic interest in the United States, and is currently the most wide spread sturgeon species utilized for commercial aquaculture in Europe (Gisbert and Williot, 2002) Despite this, very few studies have been conducted to clarify the physiolo gical effects of stress on this species. Understanding the endocrine disruptive effects of induced stress will serve as a baseline for understanding the effects of other environmental stressors, such as contaminants commonly found in both natural and constructed environments. Nitrate, for exam ple, has recently been shown to be highly toxic to Siberian sturgeon in aquaculture environm ents with limited water exchange (Hamlin, 2006), and is predicted to be of cons iderable concern for commercial aquaculture operations, which are already being forced to significantly reduce their water usage. Nitr ates and other ions have also 32

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been established as ecologically relevant endoc rine disruptors in natural environments for numerous other vertebrates (see review by Gu illette and Edwards, 2005). For late maturing species such as sturgeons, w hose economic viability relies heavily on successful egg production (caviar), it is of particular importance to understand the relationships between stress and reproductive health. The purpose of this study is to define the relationship between induced stress and circulating concentrations of steroid hormones in cultured Siberian sturgeon, and to identify mitigating stress factors in typical testing pr ocedures, most notably the techniques of blood withdrawal and surgical sexing, to understand what factors contribu te significantly to the stress response. Methods Fish and Sampling Three-year-old Siberian sturgeon were collected from two 30,000 L tanks, each from separate commercial recirculating aquaculture sy stems at Mote Marine Laboratorys Aquaculture Park (Commercial Sturgeon Demonstration Project) in Sarasota, Florida. Experiments were started at approximately 10:30 a.m. in May of 2004. Water chemistry in each of these systems was analyzed weekly for the levels of ammonia-N, nitrite-N, nitrate, and pH prior to the start of experiments. Dissolved oxygen a nd temperature were monitored continuously using stationary probes, which were spot-checked biweekly for calibration using portable probes. Hardness, alkalinity, and chloride concentr ation was analyzed the day prior to the start of experiments. The sturgeon were pulled from the water by ha nd at the side of the tank and immediately held down on a padded V-shaped surgical table. Pulling the sturgeon from the tank by hand (versus netting) decreased the likelihood of stre ssing the remaining fish in the tank and allowed immediate access to the fish for blood sampling. Blood was extracted from the caudal vein (5 33

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ml) using a 10 ml syringe (20 gauge needle) within 1 min of captur e; most captures took 30 sec for the full blood sample to be drawn. The blood sample was placed into lithium heparin Vacutainer tubes, and stored on ice for less than 30 minutes before centrifugation. Plasma was separated via centrifugation (5-10 min at 2000 g), placed into cryovials, rapidly frozen in liquid nitrogen and stored at -80 C for 2-3 weeks prior to analysis. Surgical Sexing For surgical sexing, the sturge on were anesthetized in a 5 C water bath containing carbon dioxide. Carbon dioxide was used be cause it is a low regulatory prio rity anesthetic for fish that are grown for food production and requires no withdr awal period; the sturgeon used in this study were part of a commercial food production pr ogram. Pure oxygen gas administered through a fine air stone was used to maintain dissolved oxygen concentrations in the range of 8.0 12.0 mg/L in the anesthetic bath, and sodium bicarbon ate was added to maintain pH in the range of 6.8 7.5 throughout the procedure. The stur geon generally took 3 5 min for full anesthetization. A 2.5 3.8 cm incision was made on the ventral side of each fish, approximately 7.5 cm anterior to the vent, along the median axis to allow inspection of the gonads on either side of the fish for sex determination. The incision in each fish was closed by suturing with coated vicryl absorbable suture (Ethicon Inc ., Somerville, New Jersey), and the fish was allowed to recover in a confinement tank. Once anesthetized, the surgical procedure took approximately 1 min/fish, and the fish recovered fully from the anesthesia in 5 10 min. Treatments Six fish (3 fish/tank) were used for each trea tment. All fish were sexed immediately after initial bleedings/sham bleedings; if the fish was male, the sample was discarded, and another fish was extracted until 3 females had been sampled from each tank for each treatment. In this study, 34

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we focused on female sturgeon because they are part of a larger set of studies examining various environmental factors and ovarian development leading to commercial ca viar production. The female sturgeon were then weighed and measured just after sexing while they were still under anesthesia. The fish were th en placed into a square 0.64 m 3 insulated plastic tote filled with 530 liters of system water for a 4-h period of confinement stress. A numbered cable tie placed around the caudle peduncle id entified individual fish. The time at which the fish was removed from the tank for initial bleeding/sh am bleeding was considered 0-h. In all treatments, fish were sexed immediat ely after initial blood drawing/sham drawing prior to placement in the confinement tank. In treat ment 1, fish were bled at 0-h only and placed into an insulated tote as described previously. In treatment 2, fi sh were bled at 0-h, 1-h and 4-h. In treatment 3, fish were bled at time 1-h and 4h only, and in treatment 4, fish were bled at 4-h only. For treatments 3 and 4, during the sampling periods when the fish were not bled, the fish were held down on the surgical table momentaril y to mimic the bleeding procedure but were not pricked with the needle. Blood sampling times for all treatments during the 4-h period of confinement stress are shown in Fig. 3-1. Hormone Evaluations Plasma samples for steroid evaluations were thawed on ice, and the steroid fraction was extracted with diethyl ether. Extraction was re peated twice to enhance extraction efficiency. Plasma cortisol, E 2 T and 11-KT concentrations were an alyzed according to the instructions provided with the commercial competitive enzyme immunoassay kits (Cayman Chemical Co., Ann Arbor, MI), specific to each hormone. Each hormone was previously validated for Siberian sturgeon plasma by verifying that serial dilutions were parallel to the standard curve. Samples were run in duplicate and each plate contained d uplicate wells for interassay variance and a blank. Individual hormones were all run with plates from the sa me kit lot # and were completed 35

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in the same testing session to reduce testing va riance. Sample plates were analyzed using a microplate reader (BioRad, Hercules, CA). Intr a-assay and interassay variances, respectively, were as follows: estradiol, 3.5% and 7.0%; cortisol, 2.0% and 9.1%; testosterone, 3.7% and 12.8%; 11-KT, 4.9% and 11.9%. Plasma samples for glucose concentrati on determination were thawed on ice and evaluated according to the inst ructions provided with the comme rcial glucose oxidase assay kit (Invitrogen, Amplex Red, Eugene OR). The sample plat e was analyzed using a microplate reader (BioRad, Hercules, CA). Statistical Analyses Statistical analyses were performed using StatView for Windows (SAS Institute, Cary, NC, USA). Initial comparisons we re made to determine if there was a significant tank effect within treatments. F-tests were conducted to test variances am ong treatment groups for homogeneity. If variance was heterogenous, data were log 10 transformed to achieve homogeneity of variance; how ever, all reported means ( 1 SE) are from nontransformed data. Analyses of variance (ANOVA) of weight, leng th and hormone concentration was used to compare differences among treatment groups. If significance was determined ( P 0.05), Fishers protected least-signifi cant difference was used to dete rmine differences among treatment means. Results Morphology and Chemistry The average fish weights in this experime nt ranged from 4.13 to 4.55 kg, and the average fish length ranged from 88.8 to 92.2 cm. Neither weight nor length was significantly different among treatments, and there was no significant tank effect for any tested parameter. Water 36

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chemistry parameters were tested on the day of the experiment and were as follows: un-ionized ammonia (NH 3 ), 4.55 g/l; nitrite, 0.2 mg/L; pH, 7.5; alkalinity, 200 mg/L; chloride concentration, 85 mg/L; total ha rdness, 230 mg/L; and calcium ha rdness, 130 mg/L. Dissolved oxygen concentrations were maintained at 95% saturation throughout the trial and the temperature was 24 C. Hormones The 0-h plasma cortisol concentrations for treatments 1 and 2 averaged 6.65 3.58 and 4.63 1.02 ng/ml, respectively (Fig. 3-2A), and were statistically similar. The 0-h plasma glucose concentrations were sta tistically similar and averaged 2.13 0.12 and 2.21 0.11 mmol/L for treatments 1 and 2, respectively (Fig. 3-2B). The plasma concentrations of T, 11KT, and E 2 were statistically similar at 0-h for treatments 1 and 2 and averaged 25.53 2.9, and 10.2 0.8 ng/ml and 672.4 45.9 pg/ml, respectively. Plasma cortisol concentrati ons increased significantly (P 0.05) in the Siberian sturgeon from 0-h to the 1-h sampling period averaging 70.9 18.7 ng/ml at 1-h, and were not significantly different between treat ments 2 and 3 (Fig. 3-2A). Plasma glucose concentrations increased significantly from 0-h to th e 1-h sampling period and averaged 4.67 0.40 mmol/L at 1-h, and there were no significant differences am ong treatments 2 and 3 (Fig. 3-2B). At 4-h, plasma cortisol concentrations were similar for fish in treatments 2 (46.2 15.4 ng/ml) and 3 (36.27 14.0 ng/ml), but were significantly elevated compared with those observed for fish in the treatment 4 group (10.44 2.53 pg/ml) (Fig. 3-2A). Plasma gl ucose concentrations at the 4-h sampling period were similar for treatment 2 (4.70 0.27 mmol/L) and treatment 4 (4.14 0.38 37

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mmol/L), but were significantly lower than plas ma glucose concentrati on in treatment 3 (5.65 0.41 mmol/L) (Fig. 3-2B). The evaluation of treatment 2, in which the same group of fish at 0-h, 1-h and 4-h were sampled, demonstrated that plasma T concentrations increased significantly from time 0 to 1-h (20.3 1.76 and 31.45 4.19 ng/ml respectively), with a su bsequent decrease at 4-h to a concentration similar to that observed at 0-h (F ig. 3-3A). In the same fish, we observed no differences between bleeding times for E 2 or 11-KT (Fig. 3-3 B,C). Discussion The Siberian sturgeon that were exposed to capture and confinement stress exhibited significantly elevated plasma cortisol concentrat ions 1-h after the ini tiation of stress, which persisted throughout the 4-h sampling period. This re sponse is similar to the reactions of other fish species exposed to acute stressors (Thomas et. al., 1990). Cortisol and glucose have been shown to be more sensitive to stress than most other plasma constituents except catecholamines, and respond rapidly to a wide range of environmental stresso rs. Stress in fish and the concomitant increase in cortisol have been implicated in numerous physiological conditions including impaired immune functi on (Tort et al., 1996), altered feed ing behavior (Kentouri et al., 1994), oxygen radical production (Ruane et al., 2 002), and reproductive impairment (Pankhurst and Van Der Kraak, 1997). Responses to stress are largely dependent on the severity and type of environmental stressor. Previous studies with Siberian sturgeon exposed to acute and severe hypoxia have shown significantly elevated plas ma cortisol concentrations, with a peak concentration of 35,000 pg/ml (Maxime et al., 1995). The basal cortisol concentration in that study was approximately 5000 pg/ml, which is comparable to the basal cortisol concentration obtained in this study. However, the peak concen tration of cortisol in our study increased to 38

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nearly 75,000 pg/ml, demonstrating the plasticity of the physiological stress response in this species. In some species, plasma cortisol concentr ations can persist for da ys if the stressor is chronic or severe (Sumpter, 1997). This study is distinct from other studies in several regards. This is the first study to define the relationship between stress and pot ential reproductive functi on, as indicated by the plasma concentrations of various sex steroids, in Caspian Sea sturgeon, habituated to a warm environment and reared under commercial culture condi tions from the egg stage. This is also the first study to show the endocrine effects of surgical sexing, a procedure often necessary for sturgeon and other species that do not exhibit se xually dimorphic characteristics. The induced stressors in this study, caused by capture and c onfinement, bleeding, and surgical sexing are common stressors in a laboratory or fishery envi ronment, and it is important to understand what effects these stressors can have on mitigating experimental responses. In this experiment, fish underwent captur e and confinement stress, with multiple disturbances at 1-h and 4-h. It has been shown that serial stressors evoke cumulative physiological stress responses in other fish spec ies (Waring et al., 1997; Di Marco et al., 1999) and multiple stress events cause fish to be more sensitive to additional ac ute stress (Ruane et al., 2002). The multiple disturbances in this study likel y mitigated the expected decreases in plasma cortisol concentrations after 4-h, because in treatment 4, where fish were captured but not bled until the fourth hour of capture, fish exhibited lowe r plasma cortisol concentrations than fish in treatment 2 or 3. These lower concentrations co uld result from a more rapid return toward basal concentrations, due to the lack of repeated stresso rs, or a reduced stress effect as they were not bled initially, adding additional handling and blood loss to the stress. Our data indicate that serial bleedings intensify the associated stre ss response, as evidenced by significantly lower 39

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concentrations of F in fish in which a blood samp le was not drawn at 0-h or 1-h. This is an important consideration for future studies of this species involving multiple blood samples. Whether elevations in cortisol concentration for the serially bl ed fish are due to blood volume loss or its associated stressors su ch as pricking of the fish with a needle, or longer handling times to ensure that a fish is still for actual blood draw ing versus sham drawing, is uncertain. It is likely, however, that it is a combination of events and not solely blood loss that leads to elevated stress in serially bled fish. Note that surg ical sexing, an invasive procedure that is often necessary in aquaculture or fishery practice, did not induce a prolonged stress reaction, because fish in treatment 4, which were similarly sexed at 0-h, exhibited plasma cortisol concentrations similar to basal concentrations less than 4-h after the procedure. The 0-h blood sampling period was started in the morning and the experiment was concluded in the early afternoon. Cortisol conc entrations in sturgeon (Belanger et al., 2001; Lankford et al., 2003) and other animals (Young et al., 2004) have been shown to be highly sensitive to diurnal variation, so care was taken in this study to ensure that all samples were collected within a relatively short period to reduc e the possibility of daily hormone fluctuations as confounding variables. In addition to the con centrations of sex steroids, it has been shown that plasma cortisol concentration can be alte red depending on the reproduc tive stage in sturgeon (Barannikova et al., 2000) and other species (Pickering and Pottinger, 1985). The female sturgeon in this study were 3 years old, and a lthough all female sturgeon had formed clearly visible ovigerous lamellae or ovarian folds, none of them exhibited vitellogenic oocytes, and they appeared to be in a similar reproductive stag e. However, the plasma concentrations of sex steroids in this study were sim ilar to those of fish possessing fully vitellogenic oocytes in subsequent studies. 40

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Interestingly, the concentrations of sex steroi ds evaluated in this study did not demonstrate an inverse relationship with stress as defined by plasma cortisol c oncentrations; in fact, plasma T concentration was significantly elevated during pe riods of peak plasma cortisol concentration (Fig. 3-3A). Although there have been no studies of this kind, in which stress and reproductive function in Siberian sturgeon re ared in commercial culture conditions are evaluated, this response is distinct from that in published data with other fi sh species, including other sturgeon species. Of the reproductive hormones, testoste rone has been shown to be highly responsive to stress-induced alterations in sturgeons and othe r species (Pickering et al., 1987; Bayunova et al., 2002). Plasma E 2 and 11-KT concentrations were not si gnificantly affected by stress within the timeframe of this study. In Amer ican alligators, certain enviro nmental toxicants were found to increase plasma T concentrations in juveniles, but did not affect the plasma concentrations of other circulating hormones (Milnes et al., 2004). Our findings do not necessarily indicate, however, that stress is not detr imental to the reproduction of this species. Circulating concentrations of sex steroids are only one e ndpoint in the reproductive endocrine axis, and stress can manifest itself at many levels of th e steroidogenic pathway. Fo r example, sex steroids are generally removed from circulation via cleara nce by the liver. Reduc tions in sex steroid production would not necessarily be reflected in circulating concentrations if clearance is concomitantly affected. Other possible mechanis ms that would result in the alteration of the reproductive biology of this species include alterations in hypotha lamic-pituitary stimulation or alterations in transport mechanic s (i.e., transport proteins). Finally, the elevation in plasma T concentrations described here could be due to a technical problem; that is, although commercial antibodies ar e screened for cross r eactivity and specificity to a wide range of steroids, little is known a bout the steroid milieu released during stress in 41

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sturgeon. Although unlikely, it is possible that a unique androgen of adrenal origin is released during stress in this species that cross reacts with the antibody used in the testosterone but not in the 11-KT kits. Studies using advanced analy tical chemistry could determine the steroids released from stressed Siberian sturgeon. The data presented here indicate that the concentrations of sex steroids in Siberian st urgeon do not show an inverse relationship with elevated plasma cortisol concentration following acu te stress, as has been observed for most fish. This altered response needs further study, as this study differed from previ ous studies of sturgeon in that it coupled sturgeon habituated to warm temperature with a specific stress response. This is the first study to define the relationship be tween stress and endocrine function in cultured Siberian sturgeon, a threatened and commercially important species. Future studies need to address various aspects of the aquaculture envi ronment (e.g., temperature and water quality), reproductive stage (e.g., juvenile versus adult) and seasonality to determine which variables modify the stress response and thus potentially alter growth and reproductive potential. This work will also serve as a baseline to evaluate th e effects of material water quality hazards, such as nitrate, present in both natu ral and constructed environments. 42

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T 0 T 1h T4h Treatment 1 Treatment 2 Treatment 3 Treatment 4 Figure 3-1. Blood sampling times for Treatments 1 to 4 of fish held under confinement stress for 4 hours. Six female Siberian sturgeon were used for each treatment. 43

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a b b b b a a 100 90 80 70 60 50 40 30 20 10 0 A. Cortisol (ng/ml) 1-hr 0-hr 4-hr 0 1 2 3 4 5 6 7 0-hr 1-hr 4-hr Glucose (mmol/l) c B. bb b b a a Blood sampling time Figure 3-2. Plasma cortisol (A) and plas ma glucose (B) concentrations (mean S.E.M.) during a 4-h capture and confinement period. Mean s with the same superscript are not significantly different (P 0.05). 44

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A. 0 5 10 15 20 25 30 35 40 0-hr 1-hr 4-hra b a Plasma Testosterone (ng/ml) B. 0 2 4 6 8 10 12 14 0-hr 1-hr 4-hrPlasma 11-ketotestosterone (ng/ml) 0 100 200 300 400 500 600 700 800 900 0-hr 1-hr 4-hr Plasma Estradiol (pg/ml) C. Blood sampling time Figure. 3-3. Sex steroid data for treatment 2. Plasma 17 -Estradiol (A), testosterone (B), and 11-ketotestosterone (C) taken from serial bleeds of cultured female Siberian sturgeon throughout the 4-h period of confinement stress (mean 1 S.E.M.). Fish were serially bled at 0-h, 1-h and 4-h (see Fig. 3-1 legend for a description of treatment 2 bleeding times). Means with the same superscript or no superscript are not significantly different (P 0.05). 45

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CHAPTER 4 NITRATE AS AN ENDOCRINE DISRUPTING CONTAMINANT IN CAPTIVE SIBERIAN STURGEON Introduction The endocrine disrupting actions of various chemical contaminants have become a significant concern for comparativ e endocrinologists (Col born et al., 1993; Guillette and Crain, 2000). A growing literature describes the effects of endocrine disrupting contaminants (EDCs) for both terrestrial (Iguchi a nd Sato, 2000) and aquatic (Sumpt er, 2005; Milnes et al., 2006) species. These effects include altered reproductive morphology, endocrine physiology and behavior, and involves such endpoi nts as reduced phallus size, decreased sperm count, depressed reproductive behaviors and altered ci rculating concentrations of sex steroids (e.g., Guillette et al., 1999; Orlando et al., 2002; Toft and Guillette, 2005). EDCs exert their effects by mimicking hormones, acting as hormone antagonists, altering the function or concentration of serumbinding proteins, or altering the synthesis or degradation of hormones. Aquatic organisms can receive continuous exposure to environmental co ntaminants throughout their lives, as the aquatic environment receives most of the intentionally released environmental pollutants. Thus, the effects of EDC exposure on aquatic life have re ceived considerable attention (Kime, 1999; McMaster, 2001; Sumpter, 2005; Milnes et al., 2006). Although nitrate is a ubiquitous component of aquatic environments, and has become a global pollutant in a variety of aquatic syst ems (Sampat, 2000), it has only recently begun to receive attention for its ability to alter endocrine function (G uillette and Edwards, 2005). The toxicological effects of nitr ate have long been known. As early as 1945, nitrate induced methemoglobinemia (Blue Baby Syndrome) in huma ns was associated with drinking well water contaminated with nitrate (Comly, 1945). Fi sh are also vulnerable to methemoglobinemia (Brown Blood Disease), and in Siberian sturgeon methemoglobinemia has been associated with a 46

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significant chloride imbalance (Gisbert et al ., 2004). Toxicity studies with fish (LC 50 ) have shown lethal concentrations of nitrate to range an order of magnitude or more (Brownell, 1980; Pierce et al., 1993; Hamlin, 2006), demonstrating significant plastici ty in response to elevated nitrate among fish species. Sublethal effects of nitrate include endocrine alterations which have been shown to alter metabolism, reproductive function and development. Frogs ( Rana cascadae) exposed to 3.5 mg/L nitrate-N metamorphosed more slowly, an d emerged from the water in a less developed state than control animals (Mar co and Blaustein, 1999). Rodents exposed to nitrate (50 mg/L NaNO 3 ) in their drinking water had significan tly lower circulating testosterone (T) concentrations than control anim als (Panesar and Chan, 2000). Bulls given oral administration of nitrate (100 250 g/day/animal) showed reduced sperm motility, depressed Leydig cell function, and degenerative lesions in the germ layers of the testes (Z raly et al., 1997). Studies in Southern toad tadpoles showed nitrate indu ced alterations in growth and thyroxine concentrations were mitigated by the source of cu lture water used, indicating that environmental context plays a significant role in mitigating th e effects of nitrate (Edwards et al., 2006a). Mosquitofish ( Gambusia holbrooki ) experienced significant reproductive alterations, such as reduced gonopodium length and fecundity (number of females per unit of female size), in nitrate concentrations as low as 5 mg/L NO 3 -N (Toft et al., 2004; Edward s et al., 2006b). Proposed mechanisms for nitrate induced steroidogenic di sturbances include mitochondrial conversion to nitric oxide (NO), altered chloride ion concentr ations and altered enzy matic action by binding to the heme region of P450 enzymes associated with steroidogenesis (Guille tte and Edwards, 2005). Stress effects on reproduction can be manife st at various levels of the reproductive endocrine axis, and stress has been shown to ha ve inhibitory effects on reproduction for most 47

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aquatic species studied to date (Pickering et al., 1987; Carragher and Sumpter, 1990; Pankhurst and Van Der Kraak, 1997; Consten et al., 2002). For many species of fish, including sturgeon and other chondrosteans, cortisol is the primary stre ss hormone (Idler and Sangalang, 1970; Barton et al., 1998) and cortisol has been impli cated in mediating the inhibitory reproductive effects induced by stress (P ankhurst and Van Der Kraak, 1997; Semenkova et al., 1999; Bayunova et al., 2002). There is evidence in tele osts, however, that the estrogenic inhibitory effects of stress are not mediated by cortisol and that the effects arise hi gher in the reproductive endocrine pathway (Pankhurst et al., 1995). Tilapia ( Oreochromis mossambicus) fed pellets containing cortisol to achieve pl asma cortisol concentrations t ypical of acutely stress fish, resulted in decreased plasma concentrations of T and 17 -estradiol (E 2 ), reduced oocyte diameter and gonad size in females, and reduced plasma T concentrations in males (Foo and Lam, 1993a,b). Female brown trout ( Salmo trutta ) exposed to 2 weeks of confinement stress had significantly reduced plasma T concentrations co mpared to unstressed fish (Campbell et al., 1994). Plasma glucose concentrations have also been shown to be reliable indicators of secondary stress responses. An animal under ch ronic stress can demonstrate a reduced capacity to handle subsequent stress events, and studies have shown responses of fish to multiple stressors are cumulative (Barton et al., 1986 ). Fish residing in laborato ries or fish farms are often subjected to chronic stress (sub-optimal water chemistry, cr owding, confinement) followed by acute stress events (sampling, netting), which can lead to dramatic and prolonged stress responses (Rotllant and Tort 1997; Heugens et al., 2001). Sturgeon are among the most ancient groups of Osteichthyes, and twenty-five extant species occupy the Northern Hemisphere (Birst ein, 1993). The dramatic decline in sturgeon populations due to overfishing, pollution, and habita t degradation have led to the necessity of 48

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commercial aquaculture as a means to provide an imals for stock enhancement, as well as food production, reducing pressures on wild populations (Beamesderfer and Farr, 1997; Waldman and Wirgin, 1997; Williot et al., 2002; Chebanov et al., 2002). The Siberian sturgeon is one of the leading species of sturgeon adapted to aquacult ure (reviewed by Gisbert and Williot, 2002). It was recently discovered that Siberi an sturgeon are more sensitive to nitrate toxicosis than most fish species reported to date (Hamlin, 2006). Fu rther, Siberian sturge on juveniles become less tolerant to nitrate as they grow a finding of considerable importance for the commercial culture of this species, since adult popul ations reared in recirculation systems often experience higher nitrate concentrations than their juvenile counterparts Although understanding what concentrations of nitrate are necessary to aver t mortality is generall y understood in commercial aquaculture, mortality is not an effective endpoi nt for producers interested in optimizing growth and reproductive function. Unde rstanding nitrates effects on re productive function is especially critical to sturgeon, whose economic viability relies heavily on proper endocrine function, notably the production of eggs (caviar). The purpose of this study is to begin to determine the potential effects of elevated environmental nitrate on endocrine function, and investigate whethe r elevated nitrate alters the stress response in captive female Siberian sturgeon. Methods Fish and Sampling Procedures Siberian sturgeon were collected from four 30,000 liter tanks, from separate commercial recirculating aquaculture systems at Mote Marine Laboratorys Aquaculture Park (Commercial Sturgeon Demonstration Project) in Sarasota, FL. Water chemistry in each of these systems was analyzed weekly for ammonia, nitrite, nitr ate, and pH prior to commencement of the experiments. Dissolved oxygen a nd temperature were monitored continuously with stationary 49

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probes, which were spot-checked bi-weekly fo r calibration with portable probes. Hardness, alkalinity and chloride were analyzed the da y prior to commencement of the experiment. The sturgeon were pulled by hand at the side of the tank and immediately held down on a padded V-shaped surgical table. Pulling th e fish from the tank by hand (versus netting) decreased the likelihood of stressing fish remaining in the tank and allowed for more immediate access to the fish for blood sampling. Blood was ex tracted from the caudal vein (5 ml) with a 10 ml syringe (20 gauge needle) within 1 minute of capture; most captures took 30 seconds for the full sample to be drawn. The blood was placed into lithium heparin Vacutainer tubes, and stored on ice for no more than 30 minutes before centrifugation. The plasma was separated via centrifugation (5 10 min at 2000 g), transferred to cryovials, flash frozen in liquid nitrogen and stored at -80 C for 1 3 weeks prior to analysis. Surgical Sexing For surgical sexing, the fish we re anesthetized in a 5 8 C water bath containing carbon dioxide (CO 2 ) gas; CO 2 was used because it is a low regulatory priority anesthetic for fish that are grown for food production and requires no withdr awal period; the sturgeon used in this study were part of a commercial food production pr ogram. Pure oxygen gas administered through a fine air stone was used to maintain a disso lved oxygen concentration of 9.0 13.0 mg/L, and sodium bicarbonate was added to maintain a pH of 6.8 7.6 in the bath throughout the procedure. Fish generally took 3 5 minutes for full anes thetization. A 2.5 3.5 cm incision was made on the ventral side of the fish, approximately 8 cm anterior to the vent, along the median axis to allow inspection of the gonads on either side of the fish for sex determination. The fish was sutured closed with coated vicryl absorbable su ture (Ethicon Inc., Somerville, New Jersey). 50

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Experiment 1 Experiment 1 was conducted in July of 2004 and consisted of two treatments, which sampled fish from each of four commercial cu lture tanks (30,000 l each) located in separate recirculating systems at Mote Marine Laboratory s Aquaculture Park. Two of the culture tanks were held at a nitrate concentr ation of 11.5 mg/L nitrate-N (50 mg /L total nitrate) for one month, and the other two tanks were held at 57 mg/L nitr ate-N (250 mg/L total nitr ate) for the same time period (two replicates each). Nitrate concentrations were achieved by adjusting the freshwater input to each system, typical of commercial culture practices. Prior to the 1-month exposure, nitrate concentrations in the four study tanks oscillated between 20 60 mg/L nitrate-N routinely. A nitrate concentration of 57 mg/L nitrate-N was chosen as the upper li mit in this study, as this is the maximum concentration deemed safe, defined by feeding behavior and mortality, at Motes Commercial Sturgeon Demonstratio n Project. The lower concen tration of 11.5 mg/L nitrate-N was chosen as this was considered extremely safe, yet realistically achievable under normal aquaculture practices. Although th ese concentrations may be typical of commercial recirculating aquaculture facilities, these levels are elevated relative to environmental levels or approved drinking water limits of 10 mg/L nitrate-N (U.S EPA, 1996). Treatment 1 sampled 15 fish from each of the four commercial recirculating culture tanks (two tanks/nitrate concentration; N = 30 per nitrate treatment). Ea ch fish was sampled at time 0 and was surgically sexed immediately after the blood sample was drawn. Only blood samples from female fish were used in the analyses for this study. Each fish was weighed and placed into a holding tank until treatment 2 fish were removed, to avoid stressing fish remaining in the tank. 51

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Treatment 2 sampled 18 fish from each of the four commercial recirculating culture tanks (N = 36 per nitrate treatment). Fi sh were sampled at time 0, and were then placed into square 0.64 m 3 insulated plastic totes (one tote per nitrat e concentration) filled with 530 L of system water for a 6-h period of confinement stress. A numbered tag (Duflex, St. Paul, MN) was placed on the pectoral fin of each fish for identifi cation. Fish were bled at 1 and 6 h during the confinement period (Fig. 4-1). After the 6-h samp ling period, the fish were surgically sexed as previously described. Experiment 2 Experiment 2 was conducted in May of 2005 and was procedurally identical to experiment 1 with the following exceptions. Two of the culture tanks we re held at a nitrate concentration of 1.5 mg/L nitrat e-N (6.5 mg/L total nitrate) fo r one month, and two tanks were held at 57 mg/L (250 mg/L total nitrate) for th e same time period. It should be noted that although the same tanks and population (different individuals) of an imals was used in this second experiment, the tanks that previously held the low nitrate concentrations in experiment 1, now held the elevated nitrate concentration and vice ve rsa, to reduce the possib ility of tank affect among treatment groups. The exposure in the first e xperiment should not affect the fish in either nitrate group in the second experi ment, since nitrate concentrati ons typically oscillate in the range of the upper limit (57 mg/L nitrate-N) and the lower limit (11.5 mg/L nitrate-N) routinely in recirculating aquaculture settings, includi ng our facility. Although 11.5 mg/L nitrate-N is considered low in commercial aquaculture, this concentration exceeds that which would occur in unpolluted natural environments. Th erefore 1.5 mg/L nitrate-N was chosen in this experiment as it would be more reflective of ecologically relevant exposures. Treatment 1 sampled 15 fish from each of the four commercial recirculating cu lture tanks (N = 30 per nitrate treatment) and treatment 2 sampled 25 fish from each of the f our tanks (N = 50 per nitrate treatment). 52

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Hormone Evaluations Plasma samples were thawed on ice, and th e steroid fraction was extracted twice with diethyl ether. Plasma cortisol (F), E 2 (experiment 1), T and 11-KT were analyzed according to instructions provided with the commercial co mpetitive enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI), specific to each horm one. Each hormone was previously validated for Siberian sturgeon by verifying that serial dilutions were para llel to the standard curve. Samples were run in duplicate and each plate contained duplicate wells for interassay variance and a blank. Individual hormones were all run with plates from the same kit lot number and were completed in the same testing session to reduce testing variance. Sample plates were analyzed with a plate reader (BioRad Hercules CA). Glucose was evaluated with an Amplex Red glucose/glucose oxidation kit (Invitrogen Carlsbad, CA). Radioimmunoassays for E 2 (validated for Siberian sturgeon) in experiment 2 were conducted as described previously by this lab (M ilnes et al., 2004). Br iefly, extracted samples were reconstituted in Borate Buffer (50 ul, 0.05 M, pH 8.0). Antibody (Endocrine Sciences, Tarazana, CA, USA) and radiolabeled steroid (2, 4,6,7,16,173 H) were added at 12,000 cpm per 100 l. Interassay variance tubes were similarly pr epared from pooled Siberian sturgeon plasma. Standards were prepared in duplicate at 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 pg per tube. Assay tubes were incubated at 4 C overnight. Bound free separation was performed by adding charcoal a nd centrifuging for 30-min. The s upernatant was then drawn off and diluted with scinti llation cocktail and counted on a Beck man LS 5801 scintillation counter. 53

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Statistical Analyses Statistical analyses were performed using StatView for Windows (SAS Institute, Cary, NC, USA). Initial comparisons we re made to determine significan ce within treatments. F-tests were conducted to test variances among treatm ent groups for homogeneity. If variance was heterogenous, data were log 10 transformed to achieve homogene ity of variance, however, all reported mean ( 1 SE) values are from non-transformed data. Analyses of variance (ANOVA) of weights and hormone concentrations were used to compare differences among treatment groups. If significance was determined ( p 0.05), Fishers protected least-significant difference was used to determine differences among treatment means. Results Experiment 1 In treatment 1, of the 30 fish sampled and sexed in each nitrate concentration, 19 were females in the 11.5 mg/L nitrate-N group, and 18 were females in the 57 mg/L nitrate-N group. Of the 36 fish sampled and sexed in each nitrat e concentration for treatment 2, 16 were females in the low nitrate group, whereas 13 were females in the high nitrate group. The average weight for females in treatment 1 was 4.16 0.53 kg whereas females sampled in treatment 2 was 4.29 0.36 kg. There were no significa nt differences among the tanks within each nitrate group for any tested parameter. Water chemistry parameters were tested the da y of experimentation and were as follows: unionized ammonia (NH 3 ) 4.35 g/L, nitrite 0.15 mg/L; pH 7.4, alkalinity 230 mg/L, chloride 94 mg/L, total hardness 240 mg/L a nd calcium hardness 140 mg/L. Dissolved oxygen concentrations were maintained at 95% saturation throughout th e trial and temperature was 23.3 C. 54

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Time 0 females in treatment 1 were combined with time 0 females from treatment 2 to evaluate the effects of nitrate exposure for each experiment. Fig. 4-2 and 4-3 illustrates time 0 data for each hormone for experiment 1. Initial c oncentrations of plasma F or glucose were not different between females in the 11.5 and the 57 mg/L nitrate-N groups, averaging 5.95 1.08 ng/ml and 255.9 6.8 pg/ml respectively. Plasma T, 11-KT and E 2 concentrations were significantly elevated in the 57 mg/L nitrate-N group when compared to concentrations observed in females exposed to 11.5 mg/L nitrate-N ( p 0.05). Data for plasma F and glucose concentrations in treatment 2 are shown in Fig. 4-4. There was no significant difference in th e stress response, defined by plasma F concentrations, when the females exposed to the two nitrate concentrat ions were compared. The females in both the 11.5 mg/L and 57 mg/L nitrate-N concentration groups demonstrated a dramatic increase in plasma F concentrations at th e 1-h sampling period averaging 42.0 5.7 ng/ml, followed by a significant decrease at the 6-h sampling period. The 6-h plas ma F concentrations were still significantly elevated when compared to time 0 concentrations (11.5 1.7 ng/ml). Plasma glucose concentrations were similar for both nitrate groups at time 0 and 1-h, averaging 227.5 12.2 pg/ml at time 0, and rising sign ificantly to an average of 428 17.5 pg/ml by 1-h. The 11.5 mg/L nitrate-N concentration group females demonstrated a si gnificant increase in plasma glucose from time 1-h to 6-h (517.6 19 pg/ml at 6-h), whereas the 57 mg/L nitrate-N concentration group females exhibited no increase in plasma glucose between the 1-h and 6-h sampling period (427.9 25.1 pg/ml). During the six hour cap tive stress period, we observed no significant changes in plasma T, 11-KT or E 2 concentrations with plasma concentrations within each respective nitrate con centration averaging 10.9 0.8 ng/ml, 4.4 0.4 ng/ml and 784 16.6 pg/ml respectively. 55

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Experiment 2 In treatment 1, of the 30 fish sampled and sexed in each nitrate concentration, 14 were females in the 1.5 mg/L nitrate-N group, and 12 we re females in the 57 mg/L nitrate-N group. Of the 50 fish sampled and sexed in each nitrat e concentration for treatment 2, 22 were females in the 1.5 mg/L nitrate-N group, and 24 were fe males in the 57 mg/L nitrate-N group. The average weight for females in treatment 1 was 5.84 0.89 kg and the average weight for females sampled in treatment two was 6.14 1.10 kg. There were no significant differences among the tanks within each nitrate group for any tested water parameter. Water chemistry parameters were tested the da y of experimentation and were as follows: unionized ammonia (NH 3 ) 5.35 g/L, nitrite 0.20 mg/L; pH 7.6, alkalinity 240 mg/L, chloride 90 mg/L, total hardness 240 mg/L a nd calcium hardness 135 mg/L. Dissolved oxygen concentrations were maintained at 95% saturation throughout th e trial and temperature was 23.5 C. Time 0 females in treatment 1 were combined with time 0 females from treatment 2 to evaluate the effects of nitrate exposure for each experiment. Fig. 4-5 and 4-6 illustrates time 0 data for each hormone for experiment 2. Plas ma F concentrations were not significantly different among females when the 1.5 mg/L or the 57 mg/L nitrate-N groups were compared at time 0. Plasma T concentrations were signif icantly elevated in the 57 mg/L nitrate-N concentration group ( p = 0.010), with an average of 17.28 4.57 ng/ml for the 1.5 mg/L nitrateN group, and 31.17 4.57 for the 57 mg/L nitrate-N group. Plasma 11-KT concentrations were not significantly different for e ither nitrate group at time 0 ( p = 0.091) with an average of 8.5 2.1 ng/ml for the 1.5 mg/L nitrate-N group, and 13.3 2.9 ng/ml for the 57 mg/L nitrate-N group. 56

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Data for treatment 2 is shown in Fig. 4-7. There was no significant difference in plasma F concentrations between nitrat e groups. Initial plasma F concentrations averaged 6.9 1.1 ng/ml, rose to an average of 68.1 6.2 ng/ml at the 1-h sampling peri od and dropped to an average of 26.8 2.6 ng/ml by 6-h. Plasma F concentrations we re significantly different for each sampling period. There was no significant difference in stress response for plasma T or 11-KT for treatment 2 with plasma c oncentrations averaging 26.4 1.9 ng/ml and 11.7 1.4 ng/ml respectively, across all sampling periods. Discussion Absent from most investigations asse ssing the endocrine di srupting effects of environmental pollutants on aquatic inhabitants, ha ve been studies examining the effects of ions, such as nitrate and nitrite, which are ubiquito us components of most aquatic ecosystems. Anthropogenic activities have dramatically im pacted the amount of nitrogenous compounds entering freshwater systems, and recent reports have identified agricultural non-point source pollution, often caused by nitrate laden fertiliz ers, as the leading cause of water quality deterioration to freshwater systems (Sampat, 2000). This paper describes the effects of a chr onic 30 day exposure of Siberian sturgeon to elevated nitrate on circulating concentrations of plasma glucocorticoids (F and glucose) and sex steroids (T, 11-KT, and E 2 ). Results of the first experiment, in which animals were exposed to concentrations of 11.5 and 57 mg/L nitrate-N (50 mg/L and 250 mg/L total nitrate respectively), revealed significantly elevated concentrations of plasma T, 11-KT and E 2 in animals exposed to the higher nitrate concentration. Experiment 2, which evaluated the effects of animals exposed to 1.5 and 57 mg/L nitrate-N (6.6 and 250 mg/L tota l nitrate respectively), also demonstrated an elevated concentration of plasma T and E 2 in animals exposed to the hi gher nitrate concentration. 57

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Although the results of Experiment 2 did not de monstrate a significant el evation in plasma 11KT concentration ( p = 0.09) as shown in Experiment 1 ( p = 0.05), it should be noted that the second experiment was conducted at a slightly different time of the year, and in animals which were almost 1-yr older. S easonal variation and stage of reproductive development can have significant impacts on steroid profiles of mo st fish species (Stacey et al., 1984). This is the first study to demonstrate a nitr ate-induced elevation in concentrations of plasma sex steroids, using a Casp ian Sea sturgeon species habituated to a warm environment, typical of commercial culture. Since small-s cale trials do not always reflect the scale-up challenges of commercial culture environments, or mimic similar effects on physiologic response, this experiment is unique in that it was conducted at a commercial farm under typical culture conditions. This study is al so distinct in that it used naturally occurring nitrate produced by nitrification, to achieve desired nitrate concen trations, versus altering the nitrate environment by chemical addition (e.g. sodium nitrate). It has been proposed that nitrates and nitrites disrupt endocrine function by entering steroidogenic tissues, where they are metabolized to nitric oxid e (NO). NO possesses the ability to bind to the heme moiety of the cytochrome P450 enzymes, which are present at multiple locations along the steroidogenic pathway. Th e mechanism by which nitrate has led to the elevated concentrations of plasma sex steroids se en in this study is unc lear, and more work is necessary to understand the mechanisms involve d. Nitrate induced el evations in plasma concentrations of sex steroids does not necessarily imply that nitrate is not detrimental to the reproductive health of this species. Concentrations of circulating plasma sex steroids are only one endpoint in the reproductive-en docrine axis, and disruptions can occur which will not be manifest at the level of circulat ing steroids. I offer three potentia l explanations fo r the elevations 58

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in plasma concentrations of sex steroids seen in this study. First, nitrate triggered an upregulation of steroidogenic functi on resulting in increased gonadal synthesis of sex steroids. Second, nitrate induced alterations to transport proteins hamper trans port to the liver and concomitantly affect clearance. And lastly, el evated nitrate may impair liver function, thereby reducing its ability to clear these steroids from the blood. The female fish in this study demonstrated increased plasma concentrations of androgens, as well as E 2 Considerable attention in the literature evaluating the effects of endocrine disrupting contaminants on aquatic animals has b een directed at the estrogenic effects of compounds, because many effects reported in wi ldlife populations are a consequence of the feminization of males (Stoker et al., 2003; Sump ter 2005; Milnes et al., 2006). However, a growing literature recognizes that populations of female fish exposed to environmental contaminants exhibit masculinized features (Parrott et al., 2004). Toft et al. (2004) found that female mosquitofish ( Gambusia holbrooki ) exposed to paper mill effluent exhibited masculinized anal fins, and exhibited lower f ecundity (number of embryos per unit of female size) than reference fish. 17 -trenbolone is an anabo lic steroid used to promote growth in beef cattle and has shown strong androgenic activity, a nd is thought to be the cause of reproductive alterations in fish living downstream from anim al feedlot operations (Jegou et al., 2001; Wilson et al., 2002; Orlando et al., 2002). It is unclear what effects el evated androgens, or estrogens for that matter, have on Siberian sturgeon reproduction, and this lab is currently investigating the mechanisms involved. In aquaculture systems, nitrate has been ne glected as a material water quality hazard. Commercial aquaculture ope rations have traditionally used la rge influxes of water to maintain water chemistry, and it is not uncommon to have water exchanges of 100% or more per day. 59

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Consequently, nitrate has not trad itionally been a concern in comm ercial aquaculture since this flush rate is sufficient to maintain relatively low nitrate concentrations. Water is rapidly becoming recognized as a valuable and limited re source, and legislative mandate is becoming more stringent in its limits of the amount of wa ter which may be consumed or discharged. As aquaculture attempts to keep pace with glob al demand, the growing number of aquaculture operations will be forced to utilize recircula ting aquaculture technology, and significantly reduce the heavy water usage in current practice. Nitrification systems are well understood in aquaculture, and are decidedly effective at re ducing ammonia and nitrite to nitrate (Timmons, 2001). In recirculating aquacultu re systems with limited water ex change, nitrate can rise to concentrations far in excess of t hose of natural environments, and it is unclear what impact these concentrations can have on species residing in these environments Understanding the sublethal effects of exposure to nitrate is especially cr itical to sturgeon, whose ec onomic viability relies heavily on proper egg production and reproductive performance. Fish are highly sensitive to the chemical in fluences in their envi ronment, and negative influences are often reflected in an acute stress response, indicated by elevations in concentrations of glucocorticoid s (Guillette et al., 1997). St ress in fish, and the concomitant increase in plasma F concentrations, has been implicated in numerous physiological maladies, including reproductive impairment (Pankhurst and Van Der Kraak, 1997). Stress induced effects on reproduction include decreased plasma concentra tions of sex steroids, depressed vitellogenin production and decreased gamete quality (P ankhurst and Van Der Kraak, 1997). Although plasma concentrations of sex steroids were signifi cantly elevated in the groups of fish exposed to 57 mg/L nitrate-N, time 0 plasma F and glucos e concentrations were not affected by nitrate 60

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concentration in this study, indica ting that the alterations to concen trations of plasma sex steroids were unlikely to be mediated by glucocorticoid action. Induced stress in both experiments in this study, caused by confinement and associated blood sampling stressors, caused a dramatic increase in plasma F concentrations after 1-h, with a significant decrease by the 6-h sampling period; this response was not influenced by nitrate concentration in this study. Previous studies with gilthead sea bream ( Sparus aurata) have shown a decreased acute stress response in chroni cally stressed fish, speculating that the reduced plasma F response likely resulted from negative feedback of mild but chronically elevated F caused by the confinement stressor on the hypothala mic-pituitary-interrenal axis (Barton et al., 2005). Since the initial blood sample s (time 0) were taken generally within 30 s of capture, it is likely initial concentrations of plasma F seen in this study ( 6 ng/ml) are representative of basal plasma F concentrations of captive sturgeon in ou r facility. Previous studies with Siberian sturgeon exposed to severe hypoxic stress, demons trated peak plasma F concentrations of 35 ng/ml (Maxime et al., 1995). Peak concentrations of plasma F in our study rose to over 40 ng/ml in one experiment, and nearly 70 ng/ml in the second experiment, demonstrating the plasticity of physiological response for this spec ies. Nitrate in this study wa s shown to alter at least one component of the stress response, defined by pl asma glucose concentrations, during a 6-h period of confinement stress. In conclusion, elevated nitrate is capable of altering the steroid profiles of cultured female Siberian sturgeon, and is able to alter the secondary stress resp onse, defined by plasma glucose concentrations. We also show that responses to nitrate can change over time, and more work is necessary to uncover the mechanisms involved in steroid alterations seen in this study, as well as understand the impact these effects ma y have on reproductive performance. 61

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T0 T1-hr T6-hr Treatment 1 Treatment 2 Figure 4-1. Blood sampling times for treatments 1 a nd 2 of fish held under confinement stress for 6-h. 62

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A. 0 2 4 6 8 11.5 mg/l57 mg/lCortisol (ng/ml) B. 4.4 4.6 4.8 5.0 5.2 5.4 5.6 57 mg/L 11.5 mg/L Glucose (mmol/l) Figure 4-2. Plasma cortisol (A) and glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N (n = 35 and n = 31 respectively). Means with no superscript are not si gnificantly different ( p 0.05). 63

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A. B. 0 2 4 6 8 10 12 1411.5 mg/l57 mg/lTestosterone (ng/ml) 0 1 2 3 4 5 6 11.5 mg/l57 mg/l11-Ketotestosterone (ng/ml) b b a a C. 0 100 200 300 400 500 600 700 800 900 11.5 mg/l57 mg/lEstradiol (pg/ml) b a Nitrate-N concentration Figure 4-3. Plasma testosterone (A), 11-ketotestosterone (B) a nd estradiol (C) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N (n = 35 and n = 31 respectively). Superscripts design ate significantly different values ( p 0.05). 64

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Figure 4-4. Plasma cortisol (A) a nd glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 11.5 or 57 mg/L nitrate-N (n = 16 and n = 13 respectively). The fish were bled at time 0, 1-h and 6-h during a 6-h period of c onfinement stress. Means with the same superscript are not si gnificantly different ( p 0.05). 60 b Time 0 Time 1-h r Time 6-hrs A. Cortisol ( n g /ml ) b 50 40 30 c a 20 a c 10 0 11.5 57 mg/L B. Nitrate-N concentration 0 2 4 6 8 10 12 11.5 mg/L 57 c Glucose (mmol/l) b b b a a mg/L 65

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0 2 4 6 8 10 1.5 mg/l57 mg/lCortisol (ng/ml) A. 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 1.5 mg/L 57 mg/L B. Glucose (mmol/l) Nitrate-N concentration Figure 4-5. Plasma cortisol (A), glucos e (B) testosterone concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N (n = 36 for both nitrate groups). Means with no superscript are not significantly different ( p 0.05). 66

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a b 0 2 4 6 8 10 12 14 16 18 A. B. C. Testosterone (ng/ml) 57 mg/L 1.5 mg/L b a 0 5 10 15 20 25 30 35 40 11-Ketotestosterone (ng/ml) 57 mg/L 1.5 mg/L Nitrate-N concentration 0 100 200 300 400 500 600 1.5 mg/L 57 mg/L a b Estradiol (pg/ml) Figure 4-6. Plasma cortisol testosterone (A), 11-ketotest osterone (B) and estradiol-17 (C) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N (n = 36 for both nitrate groups). Superscripts desi gnate significantly different values ( p 0.05). 67

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Time 0 Time 1-h r Time 6-hrs 0 10 20 30 40 50 60 70 80 90 1.5 mg/l 57 mg/lCortisol (ng/ml) c b a c b a c a a c b a 12 10 Glucose (mmol/l) 8 6 4 2 0 1.5 mg/L 57 mg/L Figure 4-7. Plasma cortisol (A) a nd glucose (B) concentrations (mean 1 S.E.M.) in cultured female Siberian sturgeon ( Acipenser baeri) exposed for 30 days to concentrations of 1.5 or 57 mg/L nitrate-N (n = 22 and n = 24 re spectively). The fish were bled at time 0, 1-h and 6-h during a 6-h period of conf inement stress. Means with the same superscript are not si gnificantly different ( p 0.05). 68

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CHAPTER 5 EFFECTS OF NITRATE ON STEROIDOGE NIC GENE EXPRESSION IN CAPTIVE FEMALE SIBERIAN STURGEON Introduction Environmental contaminants capable of a ltering steroidogenic regul ation and function are well documented in the literature for both terrest rial and aquatic inhab itants (Guillette and Gunderson, 2001; Mills and Chiche ster, 2005; Sumpter, 2005; Ed wards et al., 2006c). These endocrine disrupting contaminants (EDCs) can exert their eff ects through numerous physiological mechanisms including mimicking naturally occurring steroids, altering hormone synthesis and degradation and interacting directly with steroid receptors (vom Saal et al., 1995; Rooney and Guillette, 2000). In the latter case, EDCs can either stimulate (Parks et al., 2001) or inhibit (Kelce et al., 1995) the expression of the target genes fo r that receptor. The endocrine system is responsible for numerous physiological processes, and as such, perturbations to this system have the potential to dele teriously affect reproductive a nd developmental performance of the affected organism. Stress has also been shown to alter endoc rine function, and is generally negatively correlated with concentrations of sex steroi ds (Pankhurst and Van De r Kraak, 1997; Orlando et al., 2002). Cortisol, a predominant glucocor ticoid, is the most commonly accepted plasma indicator of the degree to which an animal is st ressed and has been associ ated with inhibitory effects on reproduction (Pankhurst and Van Der Kraak, 1997). Commonly st udied stressors in fishes include capture and confinement or hand ling and alterations to various environmental parameters such as temperature, pH or sa linity (Pankhurst and Dedual, 1994). Certain contaminants, however, have also been shown to increase plasma glucocorticoid concentrations, further contributing to the suppres sion of circulating sex steroids (Schreck and Lorz, 1978). 69

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In the United States, the i nput of nitrogen from terrestrial agriculture has increased 20fold in the past 50 years (Pucket, 1995). Aqua tic nitrate concentrations of over 100 mg/L have been reported in some locations (Kross et al., 1993; Rouse et al., 1999), a ten-fold increase over the U.S. drinking water standards of 10 mg/L NO 3 -N (EPA, 1996). A growing body of literature implicates agricultural non-point source pollution as the leading cause of these elevations in freshwater systems, posing a direct health risk to both humans and wild life (Sampat, 2000). A global pollutant of aquatic habita ts, the ubiquitous presence of n itrate has only recently begun to receive attention for its ability to alter endocrine function, and now joins the list of environmental contaminants implicated in reproductive dysgenesis (see review by Guillette and Edwards, 2005). Unlike most environmental en docrine disrupting contaminants, nitrate is unique in that it exists naturally at low concentrations in the aqua tic environment as the degradative end product of nitrific ation. Therefore, the physiologica l disruptive actions of nitrate stem from its relative con centration, as well as its interactions within the environment in which it persists (Edwards et al., 2006a). The seafood trade deficit in the United States is exceeding eight billion dollars annually, a natural resource deficit second onl y to oil and natural gas in magn itude. With the oceans at or exceeding their maximum sustainable yields fo r 75% of commercially relevant species, aquaculture, or the culture of fish and other aquatic organisms, has been proposed as the only viable alternative to keep pace with global de mand (FAO, 2004). Like seafood, water is also becoming a limited and increasingly valuable resour ce, and the necessary increase in aquaculture operations will not be afforded the liberal quantitie s of water permitted to established facilities. Although recirculating aquaculture facilities, which recycle and reuse a significant portion of their water, are becoming increasingly common, the limiting factor for water exchange 70

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for most of these facilities is nitrate. Work is ongoing to devel op technologies to reduce nitrate in commercial aquaculture, but it is still uncl ear what concentrations of nitrate are safe, especially for sensitive physiological systems such as the endocrine system which have been shown to be vulnerable to the effects of nitrate (Suzuki et al., 2003; van Rijn et al., 2006). Sturgeon species are ideally suited to serve as models to study the endocrine disruptive effects of elevated nitrate e xposure. Many species are commer cially viable, highly endangered and have documented sensitivities to environm ental contaminants, including nitrate (Akimova and Ruban, 1995; Dwyer et al., 2005; Hamlin, 2006). The Caspian Sea, which houses some of the most endangered sturgeon species, is becoming increasingly affected by contaminants (Birstein, 1993; Stone, 2002) many of which are implicated in the disruption of reproduction in sturgeon species (Akimova and Ruban, 1995). It has been proposed that aquaculture, incor porating the development of captive broodstock programs, could be the best solution to reduce fi shing pressures, facilita ting recovery of wild populations (Williot et al., 2002). The economic viabi lity of sturgeon culture rests squarely with the successful production of eggs, or caviar, the commercial hallmark of this family of fishes. Therefore, environmental contaminants, that ha ve the potential to a lter reproductive endpoints such as egg production, are critical areas of investigation for threatened species whose promise in aquaculture relies almost enti rely on proper egg development. In many aquatic animals, including most fis h, nitrate enters the bloodstream by crossing the gill epithelia, either by di ffusion or against a concentratio n gradient by substituting for chloride, and accumulating in extracellular fl uid (Lee and Prichard, 1985; Jensen, 1995). Ingested nitrate is readily absorbed by the pr oximal small intestine in mammals (Walker, 1996), or can also be converted to ni trite, although the degree and mech anism of the latter has been a 71

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significant point of debate (Hartman, 1982). C oncentrations of excess nitrite can cause the potentially fatal methemoglobinemia, or brown blood disease in fishes, caused by an inability to reversibly carry oxygen in the blood (Scott and Cr unkilton, 2000). Both nitrate and nitrite are capable of generating nitric oxide (NO) (Mey er, 1995; Cadenas et al., 2000; Lepore, 2000). Nitric oxide has been shown to inhibit steroidogenesis through its interactions with steroidogenic acute regulatory protein (StAR) or the enzyme cytochrome P450 side chain cleavage (P450 SCC ) (White et al., 1987). In the mitochondria of steroidogenic cells, free cholesterol, the precursor for steroidogenesis, is transported across the mitochondrial membrane by StAR. This cholesterol is then converted to pregnenolone by the P450 SCC enzyme (Stocco, 1999). Pregnenolone is subsequently converted to progesterone by mitochondrial 3 hydroxysteroid dehydrogenase (3 HSD) (Stocco, 1999). Progesterone then exits the mitochondria and depending on the tissue, will be converted to either mineralcorticoids, glucocorticoids, progestins, androgens or estrogens in the smooth endoplasmic reticulum (Norris, 199 7). Noticeably absent from nitrate studies describing the mechanisms of altered steroid concentrations, are studies of enzymes and receptors involved in regulating the earliest stages of steroidogenesis. In fact, the majority of steroidogenic research has focused on enzymes and receptors further downstream from the conversions of cholesterol to pr egnenolone (Goto-Kazeto et al., 2004). The goal of this study is to examine nitrateinduced alterations in endocrine function and identify mechanisms through which environmental exposure to nitrate alters steroidogenesis at the molecular level. These mechanisms will be investigated by comparing the mRNA expression of a regulatory enzyme functioning at an early stage of steroidogenesis (P450 SCC ) as well as receptor proteins at the end of the steroidogenic cascade for both sex steroids and 72

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glucocorticoids, estrogen receptor (ER ) and glucocorticoid receptor (GR), the mRNA expression patterns of which have not been previously characterized in sturgeon. Methods Fish and Experimental Systems Siberian sturgeon were collected from four 30,000 liter tanks, from separate commercial recirculating aquaculture systems at Mote Marine Laboratorys Aquaculture Park (Commercial Sturgeon Demonstration Project) in Sarasota, FL. The fish were 4.5 years old and weighed an average of 6.14 1.10 kg. Water chemistry in each of th ese systems was analyzed weekly for ammonia, nitrite, nitrate, and pH prior to co mmencement of the experiments. Dissolved oxygen and temperature were monitored continuously with stationary probes, which were spot-checked bi-weekly for calibration w ith portable probes. Hardness, alkalinity and chloride were analyzed the day prior to commencement of the experiment. Surgical Sexing and Tissue Collection The sturgeon were pulled by hand at the side of the tank and immediately held down on a padded V-shaped surgical table. Pulling th e fish from the tank by hand (versus netting) decreased the likelihood of stressing fish remaining in the tank and allowed for more immediate access to the fish for sampling. For surgical sexing, the fish we re anesthetized in a 5 8 C water bath containing carbon dioxide (CO 2 ) gas; CO 2 was used because it is a low regulatory priority anesthetic for fish that are grown for food production and requires no withdr awal period; the sturgeon used in this study were part of a commercial food production pr ogram. Pure oxygen gas administered through a fine air stone was used to maintain a disso lved oxygen concentration of 9.0 13.0 mg/L, and sodium bicarbonate was added to maintain a pH of 6.8 7.6 in the bath throughout the procedure. 73

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Fish generally took 3 5 minutes for full anes thetization. A 2.5 3.5 cm incision was made on the ventral side of the fish, approximately 8 cm anterior to the vent, along the median axis to allow inspection of the gonads on either side of the fish for sex determination and tissue collection. A piece of gonad approximately 5 mm 3 was removed with a biopsy forcep (Ethicon Inc., Somerville, New Jersey), flash frozen in liquid nitrogen and stored at -80 C. The fish was sutured closed with coated vicryl absorbable suture (Ethicon Inc., Somerville, New Jersey). Treatments and Experimental Conditions Two treatments were established which samp led fish from each of four commercial culture tanks (30,000 L each) located in separate recirculating systems at Mote Marine Laboratorys Aquaculture Park in Sarasota, FL. Tw o of the culture tanks we re held at a nitrate concentration of 1.5 mg/L nitrat e-N (6.5 mg/L total nitrate) fo r one month, and two tanks were held at 57 mg/L (250 mg/L total nitrate). Nitr ate concentrations were achieved by adjusting the freshwater input to each syst em, typical of commercial aqu aculture practices. A nitrate concentration of 57 mg/L nitrate-N was chosen as the upper limit in this study, as this is the maximum concentration deemed safe, defined by feeding behavior and mortality, at Motes Commercial Sturgeon Demonstratio n Project. The lower concen tration of 1.5 mg/L nitrate-N was chosen because it reflects eco logically relevant exposures. Eight fish were sampled from each of the four commercial recirculating cultu re tanks (N = 16 per nitrate treatment). RNA Isolation and Primer Design Frozen gonadal tissues were weighed and im mediately homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) at a ratio of 1 ml TRIzol / 100 mg tissue. Total RNA was isolated by collecting the aqueous phase of a chloroform/phe nol extraction and precip itated in isopropanol. The pellet was washed in 80% ethanol and then di ssolved in DEPC treated water. An SV Total RNA Isolation System kit (Promega, Madison, W I) was used to purify the samples and perform 74

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a DNase treatment The quality and concentration of the total RNA was determined with agarose gel electrophoresis and spectrophotomet er, respectively. First strand cDNA was synthesized with 2 g total RNA with Oligo (dT) 12-18 Primer (Invitrogen) and SuperScript III RNase H Reverse Transcriptase. Degenerate primers for L8 (a ribosomal prot ein used for normalization of mRNA levels), glucocorticoid rece ptor (GR), P450 SCC and ER were designed from conserved regions of the respective genes from other species. The PCR pr imers were used to amplify fragments of the sturgeon cDNA. Amplified cDNA were purifie d by Wizard SV Gel and PCR Clean-up System (Promega) and cloned by pGEM-T Vector System (P romega). Cloned plasmids were isolated by Wizard Plus SV Miniprep DNA Purification System (Promega). We used the BigDye Terminator Cycle Sequencing Kit (Applied Bios ystems, Foster City, CA) to sequence the amplifed fragments which were anal yzed with an ABI PRISM 3100. BLAST ( http://www.ncbi.nlm.nih.gov/BLAST/ ) was used to check for nucleotide and amino acid homology. Primer Express (Applied Biosystems, Fo ster City, CA) was used to design the realtime PCR primers (Table 5-1). Quantitative Real-Time PCR Quantitative real-time PCR (Q-PCR) was conduced using SYBR Green PCR Master Mix using a MyiQ Single Color Real-Time PCR Detecti on System (Bio-Rad) in a reaction volume of 15 l following the manufacturers protocol as prev iously described by this lab (Katsu et al., 2004). Conditions for Q-PCR for all genes were 3 min at 95 C and 40 cycles of 95 C for 10 seconds and 1 min. at the best annealing temp erature for each gene. The best annealing temperature for P450 SCC was 60.6 C, with L8, ER and GR running at an annealing temperature of 65 C. Starting quantities of cDNA (copies/ml) fo r each gene were calculated according to 75

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(Yin, 2001), based on optical density and molecular weight values. The ex pression of mRNA of the samples was calculated from a standard curve created from a serially diluted sample. Samples were run in triplicate and were normalized for ribosomal L8 expression. Sequence Data The sequence data were analyzed using CLC Free Workbench (CLC Bio A/S, Cambridge, MA), and homologous sequences of their deduc ed amino acid sequences were searched by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The amino acid sequences were aligned using ClustalX (Thompson et al., 1997). Genebank accession numbers for the amino acid sequences of RPL8 are Q6P0V6 (zebrafish), P41116 (X. leaves ), XP_416772 (Chicken), P62918 (Mouse) and P62917 (Human); those of GR are BAE92737 (zebrafish), P49844 ( X. laevis ), XP_420437 (chicken), NP_032199 (mouse) and P04150 (human); those of ER-beta are NP_851297 (zebrafish), NP_001035101 ( X. tropicalis ), NP_990125 (chicken), NP_034287 (mouse) and NP_001428 (human); those of P450 SCC are XP_691817 (zebrafish), NP_001001756 (chicken), Q9QZ82 (mouse) and AAH32329 (human). The Conserved Domains in amino acid sequences were searched by CD-search (http://www.ncbi.nlm.nih.gov/Stru cture/cdd/wrpsb.cgi). Statistical Analyses Statistical analyses were performed using StatView for Windows (SAS Institute, Cary, NC, USA). Initial comparisons we re made to determine significan ce within treatments. F-tests were conducted to test variances among treatm ent groups for homogeneity. If variance was heterogenous, data were log 10 transformed to achieve homogene ity of variance, however, all reported mean ( 1 SE) values are from non-transformed da ta. The relative expression of each gene was computed as a ratio with L8 and then multiplied by a consistent multiplier of 10 to ensure all values were greater than one prio r to analyses of variance (ANOVA). Figures, 76

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however, display original values. If significance was determined ( p 0.05). Fishers protected least-significant difference was used to determine differences among treatment means. Results Sequence Data Nucleotide and deduced amino acid sequences of RPL8 (L8), P450 SCC, ER and GR are shown in Figures 5-1 to 54. Cloned cDNAs are 309, 584, 698 and 845 base pairs encoding 102, 194, 232 and 281 amino acids, and are similar to L8, GR, ER and P450 SCC respectively (Figs. 5-1 to 5-8). These are partial cDNA sequences an d it is 40, 26, 42 and 55% of the length of the zebrafish coding region for L8, GR, ERand P450 SCC respectively. Cloned L8 included a partial conserved domain of ribosomal protein L2 C-terminal domain, and revealed higher than 97% of sequence identity among the ve rtebrates (Fig. 5-5). Cloned P450 SCC encoded a part of conserved region for cytochrome P450s, and rev ealed 77, 67, 49 and 51% of sequence identity compared with zebrafish, chicken, mouse and human, respectively (Fig. 5-6). Partially cloned GR included a complete hinge region, and a partial DNAand ligandbinding domain (Fig. 5-7). St urgeon GR showed 74, 67, 59, 67 and 65% of sequence identity with GR cloned from zebrafish, Xenopus leavis chicken, mouse and human, respectively (Fig. 57). Cloned partial cDNA for ER included a partial hinge re gion and ligand binding domain, and revealed 71, 58, 57, 59 and 57% of sequence identity with ER of zebrafish, Xenopus tropicalis chicken, mouse and human, respectively (Fig. 5-8). Water Chemistry Water chemistry parameters were tested the da y of experimentation and were as follows: unionized ammonia (NH 3 ) 5.35 g/L, nitrite 0.20 mg/L; pH 7.6, alkalinity 240 mg/L, chloride 90 mg/L, total hardness 240 mg/L a nd calcium hardness 135 mg/L. Dissolved oxygen 77

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concentrations were maintained at 95% saturation throughout th e trial and temperature was 23.5 C. Steroidogenic Gene Expression and Hormon e Regressions from Previous Studies Expression levels, normalized to L8 expression, of all genes evaluated were statistically similar between the fish residing in the 1.5 or 57 mg/L nitrate-N (Figs. 5-9 to 5-11). As expected, the expression of L8 was not significant for either treatment. Additionally, there was no tank effect among treatments. Mean expression levels for P450 SCC were 0.027 ( 0.007) and 0.026 ( 0.006) for the 1.5 and 57 mg/L nitrate-N treatments respectively (Fig. 5-8). Mean expression levels for GR averaged 0.359 ( 0.056) and 0.341 ( 0.035) for the 1.5 and 57 mg/L nitrate-N treatments respectively (Fig. 5-9). Mean expression level for ER was 0.440 ( 0.109) and 0.583 ( 0.160) for the 1.5 and 57 mg/L nitrate-N concentrations respectiv ely (Fig. 5-10). Simple regression analyses of mRNA expre ssion levels (normalized to L8) of P450 SCC ER and GR, as well as sex steroid and stress ho rmone plasma concentrations from Chapter 4 are summarized in Tables 5-2 and 5-3 as well as Figs. 5-12 to 5-15. Fish exposed to 1.5 mg/L NO 3 -N demonstrated significant regressions (p 0.05) for the following comparisons: GR vs. ER ; GR vs. glucose; and T vs. 11-KT. Fish exposed to 57 mg/L NO 3 -N demonstrated significant regressions for the following comparisons: ER vs. P450 SCC ; ER vs. 11-KT; P450 SCC vs. T; P450 SCC vs. 11-KT. Discussion This is the first study to successfully clone and describe the mRNA expression patterns of sturgeon P450 SCC ER and GR, key constituents in steroidogenic and stress receptor functioning. These genes represent both early (P450 SCC ) and late (ER and GR) steroidogenic endpoints, with their expressions offering in sight into several st eroidogenic pathways. 78

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In mammals, two ERs have been identified, in co ntrast to teleosts in which there are three known ERs, ER and two isoforms of ER (Filby and Tyler, 2005). Although both ER and ER are found in the gonads of fish and mammals, there is currently no agreement regarding the relative importance of one form ove r the other (Hall et al., 2001). ER has been shown to attenuate the ligand stimulated transcriptional activity of ER and has been shown to heterodimerize with ER in vitro, suggesting that relative expression levels of the receptors could dictate cellular se nsitivities to estrogens (Hall et al., 2001). ER is most strongly expressed in the gonad in most fishes. In a study of largemouth bass ( Micropterus salmoides ) the gonadal mRNA expression of ER was many fold greater than ER however its relative expr ession was strongly dependent upon time of the year (SaboAttwood et al., 2004). This study also showed that ER was more strongly expressed in the liver, but only for certain periods of the year. In rivulus ( Rivulus marmoratus ) the greatest expression of ER is found in the gonad and it has been shown that environmental pollutants can dramatically alter ER expre ssion in this species (Seo et al., 2006). Rivulus has both hermaphroditic and primary male forms, and it has been shown that expression levels of ER can vary dramatically depending on th e form (Orlando et. al., 2006). ER has been shown to be preferentially sensitive to synthetic an tiestrogens and phytoe strogens versus ER (Bodo and Rissman, 2006). Taken together, these da ta demonstrate the plasticity of ER mRNA expression and its capacity to be altere d by environmental variables. The fish in this study were part of a larg er body of work examining several endocrine endpoints associated with nitrate exposure. In Chapter 4, we documented a significant rise in plasma concentrations of sex steroids under condi tions of elevated nitrat e. In that study, I offered three possible explanations for the observed rise in plas ma sex steroid concentrations, 79

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which included increased steroidogenesis and a c oncomitant increase in gonadal synthesis of sex steroid hormones, alterations in transport proteins or reductions in liver clearance. The enzyme P450 SCC is regarded as the chronically regulated rate-limiting step in steroidogenesis (Miller, 2002) and functions at the early stag es of steroidogenesis. The P450 SCC enzyme is expressed very early in development; in mice expression be gins at embryonic day 11 (Hsu et al., 2006). During these early embryonic stages, mice with targeted disruption of the P450 SCC gene produce no steroids and have severe adre nal defects, and die shortly afte r birth; zebrafish with blocked P450 SCC function do not survive as well (Hsu et al., 2006). In general, gonadotropins regulate P450 SCC expression, however, sex steroids have been found to alter its expression in several tissues (Von Hofsten et al., 2002). In Arctic char ( Salvelinus alpinus ) 11-KT has been shown to up-regulate P450 SCC expression in the gonads (Von Hofsten et al., 2002). Although nitrate exposure did not appear to alter the mRNA expression of P450 SCC in sturgeon in this study, there was a significantly positive correlation with P450 SCC and both androgens (Chapter 4) in fish exposed to 57 mg/L NO 3 -N, that was not apparent in fish exposed to 1.5 mg/L NO 3 -N. Given this difference, I hypothesize that the sex steroids at the upper nitrate con centration, that were significantly elevated compared to the population of fish exposed to low nitrate, approa ched a threshold for feed back; that is, the binding of a critical number of receptors sufficient to trigger a response, and this elevated gene expression. It is logical to suggest, that although the fish in this study possessed vitellogenic oocytes, they were nonetheless early in their devel opment, and it is possible that the fish in the 1.5 mg/L NO 3 -N concentration would experience an elevation in sex steroid hormones concomitant with progressive egg development, and once these sex steroids reached a critical concentration, they too would demonstrate similar corr elations. It is also possible that nitrate is 80

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affecting an unknown mechanism, th at itself regulates both P450 SCC and sex steroid expression, and that their correlation is not necessarily directly causative. Interestingly, there was a positive correlation between ER and 11-KT in the fish exposed to 57 mg/L NO 3 -N that was not evident in fish exposed to 1.5 mg/L NO 3 -N. It has been shown in female sturgeon that both T a nd 11-KT rise significantly during vitellogenesis, and often peak just prior to final maturation (Barannikova et al., 2004). It is possi ble that under a normal reproductive cycle, that during a key period of de velopment in Siberian sturgeon, androgens of ovarian origin rise, providing a precursor for estr ogen synthesis, and thus, serving as a signal for the production of aromatase to facilitate th e conversion of androgens to estrogens. The estrogen receptor protein expression examined in this study represents an endpoint regulated far downstream, via negative feedback, in the steroidogenic pathway. That we did not observe an increase in mRNA expression for a ch ronically regulated upstream enzyme, nor for downstream estrogenic receptors, suggests that sex steroid elevations were not likely due to increased gonadal output. It is more likely th en, that the discord betw een plasma sex steroid concentrations and mRNA expression patterns could be explai ned by altered hepatic metabolism, either via alterations in transpor t proteins to the liver, or by direct action on the liver itself. Although these results do not provide a mechanis m for hepatic or transp ort protein failure, they do support the need for future studies clarifying liver perfo rmance under high nitrate conditions. Thibaut and Porte ( 2004) found significantly reduced metabolic liver clearance when carp ( C. carpio ) were exposed to estr ogenic nonylphenol and androgenic fenarimol at concentrations as low as 10 M and 50 M, respectively. Several othe r studies have shown that altered plasma sex steroid con centrations, induced by xenobiotics, could be caused by altered hydroxylase enzyme activity in the liver (see review by Guillette and Gunderson, 2001). 81

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NO, derived from nitrate or nitrite, has been shown to have inhibitory effects on steroidogenesis via its ac tions on StAR or P450 SCC by binding to the heme groups of these compounds (White et al., 1987). Heme groups ch aracterize all enzymes of the P450 family, and have been shown to be susceptible to chemi cal perturbation (White et al., 1987; Walsh and Stocco, 2000; Danielson, 2002). These studies pr ovide a possible mechanism for nitrate induced hepatic alterations by inhibiting en zymatic action of the various P450s in the liver responsible for clearance (Guillette and Edwards, 2005). This study is unique in several regards. It is the first st udy to evaluate the steroidogenic effects of nitrate exposure in a commercially viable and ecologically threatened species, habituated to a warm environment under comm ercial culture conditi ons. Of significant importance is the fact that this study used nitrate produced through nitrification as its source. Most studies examining nitrate exposure use a purified aquatic medium dosed with various nitrate salts (e.g. NaNO 3 KNO 3 ). Nitrate produced through nitrif ication brings with it a host of metabolites and oxidative end products not present in a purified medium, and is more relevant to ecological exposure. This is of particular impor tance because it has been shown that the nitrate medium itself can significantly alter its toxic eff ects, even if the same source of nitrate (i.e. NaNO 3 ) is used. Edwards et al. (2006a), found that Southern Toad ( Bufo terrestris) tadpoles exposed to various concentrations of nitrate responded differently de pending on the source of freshwater used, and this differe nce could not be attributed to differential electrolyte balances since both sources we re equivalent. Although we did not observe nitrate induced alterations in mRNA gene expression patterns of P450 SCC ER or GR in this experiment, it is important to note that these animals were exposed to the nitrate concentratio ns for 30 days, and it is probable that the fish were adapted to 82

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the nitrate concentrations in terms of gene expr ession, since most alterati ons in gene expression are observable hours or days after a disrupting ev ent. However, a goal of this study was to understand the implications of long-term exposure to elevated nitrate, and these adaptive and persistent mRNA expression patterns are relevant to aquaculture environments. It is now known that a major function of glucocorticoids (GCs ), including cortisol, is to protect against over stimulation by host defenses in a stress event (Li and Sanchez, 2005). GCs regulate numerous biological pr ocesses and play diverse role s in growth, development and maintenance of stress related homeostasis (Sapols ky et al., 2000). GCs eff ectuate their responses by their association with glucocorticoid receptors (GRs), and alte red GRs have been implicated as a causative factor in several pathologic stat es (Barden, 2004; Marchetti et al., 2005). That GR-deficient mice die within a few hours after bi rth clearly shows that proper GR function is essential for survival (Cole et al., 1995). Although nitrate did not alter the mRNA expr ession of GR in this study, there was a positive correlation between GR and both ER and glucose. There is no evidence in the literature of an overt regulatory me chanism for GR induction of either ER or glucose, or a mechanism by which glucose alters GR or ER expression, and it is possi ble this relationship is the result of an unknown or unapparent factor that is co-regulating these genes. However, it has been shown recently that glucose has the abil ity to regulate hepatic gene expression in a transcriptional manner, through the carbohydrate responsive element binding protein (ChREBP) (Dentin et al., 2006). In addition, glucose has been shown to direct ly up-regulate the mRNA expression of -defensin-1, an immune system peptide, in cultured human renal cells (Malik and Al-Kafaji, 2006). Therefore, although the relationship between glucose and GR mRNA 83

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expression is not yet clear, given that glucose has been shown to regulate gene expression in other systems, it is possible that glucose could regulate the expression patterns of these receptors. Cortisol bio-synthesis commences with the stimulation of interrenal tissues by adrenocorticotropic hormone, resulting in an enzymatic conversion of cholesterol which progresses through the steroidogeni c cascade through a series of en zymatic steps, including the cytochrome P450 family of proteins. It was recently shown in rainbow trout ( O. mykiss ) that xenobiotic stressors that activate aryl hydrocarbon signaling, im pair the corticosteroid response to stress by inhibiting both StAR and P450 SCC (Aluru and Vijayan, 2006). Other studies have also documented the impairment of the adaptive stress response by decreasing the capacity for interrenal cortisol produ ction (Wilson et al., 1998; Hontela, 2005). In Chapter 4 it was shown that basal cortisol production wa s not increased in animals exposed to elevated nitrate for 30 days. Expectedly, we did not observe a change in mRNA expression for GR in animals exposed to elevated nitrate, i ndicating nitrate may not alter the enzy mes involved in the adaptive stress response long term as these animals are likely adapted to the elevated nitrate at the tissue (interrenal) level, although the question of he patic alteration and clea rance still remains a concern. This study contributes a bette r mechanistic understanding of the endocrine disruptive effects of nitrate exposure. Futu re studies of the endocrinological effects of nitrate should focus on mechanisms of hepatic alteration including examining enzymes involved in clearance, expression of gonadal and hepatic StAR protein and vitellogenin pr oduction, as well as transport protein kinetics. 84

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Table 5-1. Forward and reverse primers used for quantitative real-time PCR Gene Forward Primer (5 3) Reverse Primer (3 5) Product Size (bp) L8 CCGGTGACCGTGGTAAACTG TCAGGGTTGTGGGAGATGACA 67 P450 SCC AGCCTCAGCGTCTCCTTTAT CCCTGTTGTGGACCATGTT 159 ER TGGTCAGCTGGGCCAAA CCAATAGGCATACCTGGTCATACA 69 GR CAAGCAACACCGCTACCAGAT CGTTAGCTGTGGCATCGATTT 66 85

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Table 5-2. Regression data mR NA expression patterns for P450 side chain cleavage enzyme (P450 SCC ), estrogen receptor (ER ), glucocorticoid receptor (GR), testosterone (T), 11-ketotestosterone (11KT), 17 -estradiol (E 2 ) cortisol and glucose in sturgeon exposed to 1.5 and 57 mg/L NO 3 -N. Bold numbers represent significant, positive correlations. 1.5 mg/L NO 3 -N P450 SCC ER GR ER p = 0.4821 r 2 = 0.064 GR p = 0.3927 r 2 = 0.093 p = 0.02 r 2 = 0.471 T p = 0.2849 r 2 = 0.161 p = 0.3249 r 2 = 0.121 p = 0.3923 r 2 = 0.093 11-KT p = 0.3640 r 2 = 0.119 p = 0.9435 r 2 = 0.001 p = 0.1477 r 2 = 0.243 E 2 p = 0.0704 r 2 = 0.512 p = 0.7351 r 2 = 0.021 p = 0.6785 r 2 = 0.031 Cortisol p = 0.7455 r 2 = 0.014 p = 0.8008 r 2 = 0.007 p = 0.5109 r 2 = 0.049 Glucose p = 0.2303 r 2 = 0.198 p = 0.2263 r 2 = 0.074 p = 0.035 r 2 = 0.444 57.0 mg/L NO 3 -N P450 SCC ER GR ER p = 0.0278 r 2 = 0.320 GR p = 0.3069 r 2 = 0.080 p = 0.4833 r 2 = 0.039 T p = 0.0002 r 2 = 0.673 p = 0.0827 r 2 = 0.214 p = 0.9835 r 2 = 0.000 11-KT p = 0.0019 r 2 = 0.567 p = 0.0193 r 2 = 0.378 p = 0.4818 r 2 = 0.042 E 2 p = 0.6361 r 2 = 0.026 p = 0.0678 r 2 = 0.324 p = 0.2330 r 2 = 0.060 Cortisol p = 0.9510 r 2 = 0.000 p = 0.8467 r 2 = 0.004 p = 0.7247 r 2 = 0.013 Glucose p = 0.1735 r 2 = 0.149 p = 0.6392 r 2 = 0.019 p = 0.7834 r 2 = 0.007 86

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Table 5-3. Regression data for testoster one (T), 11-ketotestosterone (11KT), 17 -estradiol (E 2 ) cortisol and glucose in sturge on exposed to 1.5 and 57 mg/L NO 3 -N from Chapter 4. Bold numbers represent significant, positive correlations. 1.5 mg/L NO 3 -N T 11-KT E 2 Cortisol 11-KT p = 0.0588 r 2 = 0.377 E 2 p = 0.9919 r 2 = 0.000 p = 0.8984 r 2 = 0.003 Cortisol p = 0.5528 r 2 = 0.046 p = 7108 r 2 = 0.018 p = 0.4648 r 2 = 0.092 Glucose p = 0.6245 r 2 = 0.031 p = 0.4601 r 2 = 0.070 p = 0.5398 r 2 = 0.066 p = 0.4326 r 2 = 0.079 57.0 mg/L NO 3 -N T 11-KT E 2 Cortisol 11-KT p = 0.0001 r 2 = 0.819 E 2 p = 0.9221 r 2 = 0.001 p = 0.4658 r 2 = 0.061 Cortisol p = 0.5190 r 2 = 0.043 p = 0.4652 r 2 = 0.061 p = 0.1247 r 2 = 0.347 Glucose p = 0.0563 r 2 = 0.271 p = 0.1029 r 2 = 0.223 p = 0.3525 r 2 = 0.109 p = 0.3397 r 2 = 0.091 87

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CTCAGCTGAATATTGGCAATGTTCTCCCAGTTGGCACCATGCCTGAAGGTACCATTATTT GCTGCCTGGAAGAGAAGCCCGGTGACCGTGGTAAACTGGCCCGTGCCTCTGGGAACTACG CCACTGTCATCTCCCACAACCCTGAAACTAAGAAATCCCGCGTGAAGCTGCCATCCGGGT CCAAGAAAGTAATCTCCTCTGCCAACAGAGCCGTAGTCGGTGTTGTTGCTGGTGGTGGTC GTATTGACAAACCAATCCTGAAGGCGGGTCGAGCCTATCACAAATACAAGGCCAAGAGAA ACTGCTGGC Q L N I G N V L P V G T M P E G T I I C C L E E K P G D R G K L A R A S G N Y A T V I S H N P E T K K S R V K L P S G S K K V I S S A N R A V V G V V A G G G R I D K P I L K A G R A Y H K Y K A K R N C W 60 120 180 240 300 309 20 40 60 80 100 102 Figure 5-1. Nucleotide and deduced amino acid sequences of Siberian sturgeon ribosomal protein L8 (RPL8). Partial cDNA of RP L8 was 309 base pairs encoding 102 amino acids. 88

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CACAAACAGATAGAGAGGAGTGGGAAAGGAAGCTGGACGGCAGATCTTTCACATGAGCTC TTCAGATTTGCACTTGAGTCGGTGAGCCACGTGCTGTATGGGGAGCGGCTGGGATTGCTG CAGGACCACATCGACCCTGATACCCAGAAGTTCATCGACTGCATCACCCTGATGTTCAAT ACCACGTCACCCATGCTGTACATCCCGCCTGCCCTACTGCGGAGAGTCGGGGCCAAGGTG TGGCGAGACCACGTGGAGGCCTGGGACGCCATCTTCAGTCACGCTGACCGATGCATTCAG AACATCTACAGGAAGTTACGTCAGTCTCCTGAAAGTGAGGGGAAGTACCCTGGAGTCCTG GCTAGCCTTCTCATGCTGGACAAGCTGTCCATTGAAGACATCAAGGCCAACGTGACTGAG CTAATGGCCGGAGGGGTTGACACTACTTCCATTACCCTGTTGTGGACCATGTATGAACTT GCCAGATACCCCGACCTGCAGGAACAGCTGCGGGCTGAGGTTCAGGATGCCTGGGCCTCT TCACAGGGGGACATGATCAAGATGTTAAAGTCAATTCCTTTGGTTAAAGGAGCCATAAAG GAGACGCTGAGGCTGCACCCAGTTGCTGTGAGCTTGCAAAGGTATATAACTGAGGATATT GTGATCCAAAACTACCACATTCCATCAGGGACTCTGGTGCAGCTAGGGCTCTACGCTATG GGGCGGAATCCACAGATTTTCCCAAGACCTCTGCAATATAACCCGGCCCGCTGGCTCAAA GGGGAGAGCCACTATTTCAAAAGCCTTAGCTTTGGATTCGGTCCCCGGCAGTGTCTGGGC CGCAG H K Q I E R S G K G S W T A D L S H E L F R F A L E S V S H V L Y G E R L G L L Q D H I D P D T Q K F I D C I T L M F N T T S P M L Y I P P A L L R R V G A K V W R D H V E A W D A I F S H A D R C I Q N I Y R K L R Q S P E S E G K Y P G V L A S L L M L D K L S I E D I K A N V T E L M A G G V D T T S I T L L W T M Y E L A R Y P D L Q E Q L R A E V Q D A W A S S Q G D M I K M L K S I P L V K G A I K E T L R L H P V A V S L Q R Y I T E D I V I Q N Y H I P S G T L V Q L G L Y A M G R N P Q I F P R P L Q Y N P A R W L K G E S H Y F K S L S F G F G P R Q C L G R 60 120 180 240 300 360 420 480 540 600 660 720 780 840 845 20 40 60 80 100 120 140 160 180 200 220 240 260 280 281 Figure 5-2. Nucleotide and deduced amino acid sequences of Siberian sturgeon P450 SCC. Partial cDNA of P450 SCC was 845 base pairs encoding 281 amino acids. 89

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GAGCGTTCTAATTATCGCATTGTACGACACAGGCGTCTTTCTCAAGGCCAAGTGCAGCCC AGTAGTAAAGCCAGCAAAACCAATGAAAGTGGCTTACTGCAGACAAGGAGGATTCACTTC ACTTCTCTGAGCCCTGAAATGCTCATGTCTTCAGTAATAGAGGCTGAACCGCCTGAGATT TATTTGATGAGCTATCTCATGAAGCCATTCACTGAGGCCACCGTGATGACATCATTAACC ACCCTTGCAGACAAGGAACTCGTTTACATGGTCAGCTGGGCCAAAAAAATTCCAGGGTTT GTGGAGCTCGGTGTGTATGACCAGGTATGCCTATTGGAGTGTTGCTGGTTAGAGGTGCTG ATGGTAGGGCTGATGTGGAGATCTATTAATCATCCAGGGAATCTCGTCTTTGCATCTGAC CTTATTTTAAACAGGGACGACGGGAACTGCGTGGAAGGATTAGTGGAGGTTTTCGACATG CTTTTGGCTATAACTTCAAAGTTTCGAGAGCTGAATCTGCAGCGAGAGGAGTATCTCTGC CTCAAGGTCATGGTCCTCCTCAACTCCACTATGTTCCCCGGTCCCTCAGAGAAGCCAGAA AAAAGTGAAAGTAGAGATAATCTGCTTAAACTTCTGGATGCAATCACCGATGCTTTAGTC TGGGTTATTTCGAAGAAAGGACTCTCTTTACAGCAGCA E R S N Y R I V R H R R L S Q G Q V Q P S S K A S K T N E S G L L Q T R R I H F T S L S P E M L M S S V I E A E P P E I Y L M S Y L M K P F T E A T V M T S L T T L A D K E L V Y M V S W A K K I P G F V E L G V Y D Q V C L L E C C W L E V L M V G L M W R S I N H P G N L V F A S D L I L N R D D G N C V E G L V E V F D M L L A I T S K F R E L N L Q R E E Y L C L K V M V L L N S T M F P G P S E K P E K S E S R D N L L K L L D A I T D A L V W V I S K K G L S L Q Q 60 120 180 240 300 360 420 480 540 600 660 698 20 40 60 80 100 120 140 160 180 200 220 232 Figure 5-3. Nucleotide and deduced amino acid sequences of Siberian sturgeon ER Partial cDNA of ER was 698 base pairs encoding 232 amino acids. 90

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TGAACTTAGAAGCACGGAAAACAAAGAAGCTCAACAAATTGAAGGGAATTCAGGCGCCCG TTGAGCAAGCAACACCGCTACCAGATGAGCGGTCACAGGCGCTGGTCCCCAAATCGATGC CACAGCTAACGCCAACCATGCTGTCGCTCTTGGAGGCCATCGAGCCAGAAATTATCTACT CGGGATACGACAGCACCATACCTGACACGTCCACGCGCCTTATGAGCACACTGAACAGGC TAGGGGGAAGACAAGTGGTAGCTGCAGTAAAGTGGGCAAAGTCATTACCAGGGTTTAGAA GCCTGCACCTTGATGATCAGATGACCCTGCTGCAGTGTTCCTGGCTGTTTCTCATGTCTT TTAGTCTGGGTTGGAGATCCTACAAGCAGTCTAATGGAAGCATGTTGTGCTTTGCACCAG ACCTAGTCATAAACGACGAGAGAATGAAGCTCCCTTACATGTTTGAACAGTGTGAACAAA TGCTGAAGATTTCAAACGAGTTAGTACGACTTCAGCTTTCATATGATGAATACCTCTGCA TGAAGGTTCTGTTGCTGCTCAGTTCAGTTCCTAAAGAGGGTCTG N L E A R K T K K L N K L K G I Q A P V E Q A T P L P D E R S Q A L V P K S M P Q L T P T M L S L L E A I E P E I I Y S G Y D S T I P D T S T R L M S T L N R L G G R Q V V A A V K W A K S L P G F R S L H L D D Q M T L L Q C S W L F L M S F S L G W R S Y K Q S N G S M L C F A P D L V I N D E R M K L P Y M F E Q C E Q M L K I S N E L V R L Q L S Y D E Y L C M K V L L L L S S V P K E G L 60 120 180 240 300 360 420 480 540 584 20 40 60 80 100 120 140 160 180 194 Figure 5-4. Nucleotide and deduced amino acid se quences of Siberian sturgeon GR. Partial cDNA of GR was 584 base pairs encoding 194 amino acids. 91

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Figure 5-5. Sequence comparison of deduced am ino acid sequences for ribosomal protein L8 (RPL8). Asterisk indicates position, which has a single, fully conserved residue. Colon and period indicates position, which ar e fully conserved s trong and weaker groups. 92

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Figure 5-6. Sequence comparison of deduced amino acid sequences for P450 SCC Asterisk indicates position, which has a single, fully conserved residue. Colon and period indicates position, which are fully conserved strong and weaker groups. 93

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Figure 5-7. Sequence comparison of deduced amino acid sequences for GR. The open and filled box indicates the ligand and DNA binding dom ain of nuclear receptor subfamily, respectively. Asterisk indi cates position, which has a single, fully conserved residue. Colon and period indicates position, which are fully conserved s trong and weaker groups. 94

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Figure 5-8. Sequence comparison of deduced amino acid sequences for ER The open and filled box indicates the ligand and DNA binding domain of nuclear receptor subfamily, respectively. Asterisk indicat es position, which has a single, fully conserved residue. Colon and period indi cates position, which are fully conserved strong and weaker groups. 95

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0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 1.5 mg/L 57.0 mg/L Nitrate-NP450SCC Relative Gene Expression Figure 5-9. Mean ( SE) expression of P450 SCC mRNA in 4.5 year-old Siberian sturgeon. 96

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0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 1.5 mg/L 57.0 mg/L Nitrate-NGR Relative Gene Expression Figure 5-10. Mean ( SE) expression of glucocorticoid (GR) receptor mRNA in 4.5 year-old Siberian sturgeon. 97

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.5 mg/L 57.0 mg/L Nitrate-NER Relative Gene Expression Figure 5-11. Mean ( SE) expression of estrogen receptor(ER ) mRNA in 4.5 year-old Siberian sturgeon. 98

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2 2.5 3 3.5 4 4.5 5 5.5 6 6.5Glucose mmol/l .15 .2 .25 .3 .35 .4 .45 .5 .55 GR/l8*100 Y = 2.15 + 6.562 X; R^2 = .444 Figure 5-12. Linear regression of glucose (mmol/L ) vs GR mRNA (normalized to L8 expression) for fish exposed to 1.5 mg/L nitrate-N. 99

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0 .2 .4 .6 .8 1 1.2 1.4 1.6 1.8ERb/L8*100 .15 .2 .25 .3 .35 .4 .45 .5 .55 GR/l8*100 Y = -.49 + 3.142 X; R^2 = .471 Figure 5-13. Linear regression of ER mRNA and GR mRNA (normali zed to L8 expression) for fish exposed to 1.5 mg/L nitrate-N. 100

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0 .01 .02 .03 .04 .05 .06 .07 .08 .09 .1SCC/L8*100 0 10000 20000 30000 40000 50000 60000 70000 T Y = .002 + 1.178E-6 X; R^2 = .673 Figure 5-14. Linear regression of P450 SCC mRNA (normalized to L8 expression) and T for fish exposed to 57 mg/L nitrate-N. The signifi cance of this regression is driven primarily by the two extraneous points. 101

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0 .01 .02 .03 .04 .05 .06 .07 .08 .09 .1SCC/L8*100 0 5000 10000 15000 20000 25000 30000 35000 11-KT Y = -.002 + 2.59E-6 X; R^2 = .567 Figure 5-15. Linear regression of P450 SCC mRNA (normalized to L8 expression) and 11-KT for fish exposed to 57 mg/L nitrate-N. The significance of this regression is driven primarily by the extraneous point. 102

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CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS Summary The role of pesticides in th e reproductive impairment of w ildlife was first made public in Rachel Carsons Silent Spring in 1962 (Carson, 1962), and since then a host of man-made chemicals ranging from surfactants to polychlorinated biphenyls (PCBs) have been implicated in countless developmental and reproductive a bnormalities (Colborn et al., 1993). Noticeably absent from most of these studies are the a ffects of naturally occu rring compounds, which can become elevated well beyond naturally occurri ng background concentrations from anthropogenic impact. The EPA drinking water limit for nitrate is 10 mg/L NO 3 -N, however, many rural drinking water wells exceed this standard. For example, in Iowa 18% of drinking wells above the EPA standard were recorded (Kross et al., 1993). Natural water bodi es can exceed 100 mg/L nitrate (Rouse et al., 1999) and al though elevated nitrat e is becoming a ubiqu itous component of aquatic ecosystems, only recently has it been consid ered for its role in altering reproductive and developmental physiology (Gu illette and Edwards, 2005). Results from Chapter 2 showed sturgeon to be especially sensitive to nitrate toxicosis, and unexpectedly, this sensitivity increases as the fish grows. This could have serious implications for mature and reproductively active animals that could be far more sensitive than the ontogenetic size classes tested in that study. This is espe cially alarming since nitrate concentrations in natural water bodies in Flor ida have been documented approaching the upper nitrate concentration of 57 ppm NO 3 -N, shown in Chapter 4 to significantly alter plasma concentrations of sex steroids in maturing fe male sturgeon (Katz et al., 1999; Katz, 2004). Although sturgeon were more sensitive to nitrate t oxicosis than many species reported to date, it should be noted that the toxic eff ects of nitrate have been examin ed in only a handful of aquatic 103

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species. Although sturgeon may be well suited to serve as sentinel spec ies for nitrate induced reproductive impairment, it is highly likely that other species will be co ncomitantly affected by elevated nitrate exposure. In Chapter 3 we observed a significant eleva tion in plasma T concentration under periods of greatest stress, defined by plasma cortisol. Pl asma T concentrations have been shown to be uniquely sensitive to environmental alterations (M ilnes et al., 2006). Unexpectedly, the positive correlation between T and cortisol was not apparent in studies outlined in Chapter 4. The reproduction system is characterized by cyclic changes, modulated by hypothalamic releasing hormones, pituitary gonadotropes and gonadal ster oids. These cycles can be influenced by environmental cues such as temperature, photope riod and other factors (Norris, 1997; Kim et al., 1998). Experiments in Chapter 4 were conducted at a different time of the year, in animals that were one year older and more reproductively matu re. It is possible c onditions present in the second series of experiments were not conducive to the stress induced alterations observed in Chapter 3. Consistent between these studies (i.e. Chapte rs 3 and 4), however, wa s that induced stress did not result in reductions in plasma sex steroi d concentrations. Chapter 4 examined the effects of nitrate on various steroid endpoints. These data describe nitrate-induced elevations in plasma concentrations of T, 11-KT and E 2 in animals exposed to 57 mg/L NO 3 -N. Although endocrine disrupting contaminants usually reduce plasma sex steroid concentrations, as opposed to the plasma sex steroid elevations obs erved in this study, other studies of aquatic animals have shown elevations in plasma sex ster oid concentrations following chem ical exposure. A study of American alligators showed significant elevatio ns in plasma T concen trations in alligators exposed to a little as 0.01 ppb toxaphene (Milnes, 2005). It is difficult to predict what the 104

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observed elevations in plasma concentrations of sex steroids would impose on reproductive performance. Altered circulati ng concentrations would likely a lter feedback mechanisms on the hypothalamo-pituitary axis as well as negative feedback on the gona d and other tissues responsive to sex steroids, such as the liver and fe male reproductive tract. Persistent elevation in hormones could be countered by adaptation but the long term implications of chronic elevation in hormones is not favorable. It is unclear if ni trate induced elevations in sex steroids observed in Chapter 4 are due to a genera lized stress response, as Chapte r 3 data suggest, but given that plasma cortisol concentrations remained unaffect ed by nitrate, renders this explanation suspect. In aquaculture altered repr oductive performance, of a species such as sturgeon whose economic viability relies almost entirely on th e successful culture of eggs (caviar), could significantly diminish profit margins and reduce th eir potential value. Even in warm captive environments, sturgeon can take 46 years to reach reproductive matu rity, and even slight delays in maturation can have significant financial impacts. From an ecological perspective, the cost of altered reproductive performance, in a wild stur geon that can take 10-20 y ears or more to reach sexual maturity (Detlaff et al., 1993), is that much greater. Fertilizers applied near wate r bodies coupled with spring rainstorms, contributes to an aquatic nitrate pulse that overlaps the breeding season of many sturgeon species (Detlaff et al., 1993; Barbeau, 2004). Although speculative, gi ven the global increases in aquatic concentrations of nitrate, and th e fact that pollution has been cited as a significant cause of reductions in sturgeon populations in the Caspia n, it is reasonable to hypothesize that nitrate could contribute to observed population declines. Although it is possible that the observed elevations in circulating con centrations of sex steroids se en in these studies imparts a 105

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physiological advantage, accelerati ng the reproductive process, data from Chapter 5 renders this conclusion unlikely. In Chapter 5, mRNA expression patterns of various enzyme and receptor proteins involved in the steroidogenic cascade were examined. These data showed no nitrate-induced alterations in mRNA expression patterns as would be expected given the elevations in hormone end products seen in Chapter 4. This was th e first study to clone and describe the mRNA expression patterns of key upstream and downstream constituents in the steroidogenic pathway. We observed significant correlations with ster oidogenic enzymes and hormone end products, which appeared to be nitrate dependent, notable between P450 SCC and both androgens in fish exposed to 57 mg/L NO 3 -N. I hypothesize that this association may be the result of sex steroids that were sufficiently elevated following exposure to the upper nitrate co ncentration, that they reached the threshold needed to induce feed back mechanisms and alter gene expression. Taken together, these data suggest that the observed elevations in plasma sex steroid concentrations are unlikely due to an up-regulation of gonadal synthe sis. Figure 6-1 revisits the summary figure first introduced in Chapter 1 (Figure 1-1), outlining hormone production and cycling. This updated figure reflects possible mechanisms of disruption based on the data collected for this dissertation. Elevated plasma concentrations of sex steroids could result from several physiological mechanisms, including an upre gulation of gonadal synthe sis, alterations in hepatic biotransformation and clearance, or a ltered plasma storage associated with steroid binding proteins. An upregulation of gonadal synthe sis was predicted to be associated with an upregulation in mRNA expression of various st eroidogenic endpoints, su ch as the enzymes essential for this process. An increase in mRNA concentration was not observed for P450scc, suggesting that our prediction was false. Althoug h this is only one enzyme in the steroidogenic 106

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pathway, it plays an initial role and is cons idered a rate-limiting step. Thus, alternative mechanisms to explain the elevated plasma sex steroid concentrations, such as hepatic biotransformation and clearance or altered concentr ations of steroid binding proteins, need to be examined in the future. As the fish used in this study were part of a commercial aquaculture facility, it was not possible in these series of studies to kill the fish to examine hepatic enzyme activity. The hypothesis that nitrate can influence vert ebrate reproduction by altering nitric oxide (NO) regulation has been propos ed by several authors (Vanvoorhi s et al., 1994; DelPunta, 1996; Panesar and Chan, 2000; Guillette and Edwards, 2005) It appears NO reduces the synthesis of steroid hormones by inhibiting steroidogenic enzymes, notably the P450 family of enzymes. Given that the liver relies heavily on P450 enzymes for proper function, it seems a likely possibility that alterations in hepatic function could explain the discord between elevations in concentrations of sex steroids and the unremarkable mRNA e xpression patterns observed in sturgeon cultured in high and low nitrate environments. Future Directions Determining the cause of nitrate induced incr eases in concentrations of circulating sex steroids will be critic al to understanding the effects of nitrate on reproductive physiology. Due to the pervasive role of P450 en zymes in hepatic function, nitrate induced hepatic alteration is proposed as the most likely cause of elevated se x steroid concentrations in sturgeon cultured in elevated nitrate environments. Therefore, future studies should focus on defining the livers role in sex steroid clearance in sturgeon exposed to ni trate. Since the liver is responsible for the production of vitellogenin, a protein necessary for oocyte growth and development, its production and possible alteration should also be examined. N itrate effects should also be 107

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examined in male sturgeon, as well as other comme rcially relevant species to determine if the results observed in this work can be observed in other species. Perhaps most importantly, understanding the biological significance of nitrate induced elevations in concentrations of sex steroids in terms of repr oductive performance, including time to maturation, egg size, fecundity and larval viab ility is critical to determining the ecological significance of nitrates effects. Conclusions In summary, these data suggest that steroids should not serve as th e exclusive endpoint for evidence of nitrate induced endocrine disrup tions. These endpoints should also include steroidogenic enzymes and steroid receptors, a nd possibly hepatic enzymes and receptors as well. Understanding the role of nitrate in sturgeon reproduction will be dependent upon future studies uncovering the biologica l and reproductive implications of hormonal and molecular effects uncovered in these studies. In aquaculture, nitrate has b een overlooked as a material water quality hazard, largely because most aquaculture facilities use larg e quantities of water, which keeps nitrate concentrations well below that which will elicit obs ervable effects, such as mortality. The data obtained in these studies suggest that indeed ni trate is a material wate r quality hazard, and that easily observable effects such as mortality can no longer be considered valid endpoints to define safe concentrations of nitrate in aquaculture. Sublethal effects of nitr ate exposure, such as endocrine disruption, must now be considered in the effective management of sturgeon populations in both natural and captive environments. 108

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Elevated nitrate results in increased concentrations of sex steroid concentrations + Figure 6-1. Possible alterations in nitrate induced elevations of sex steroid concentrations in Siberian sturgeon. (+) represents a possi ble up-regulation and (--) represents a possible mechanism of disruption. Studies of mRNA expression patterns suggest sex steroid elevations are not the result of incr eased gonadal synthesi s, but that another mechanism is involved. Based on nitrat es documented role in altering P450 enzymes and function, it is hypothesized here that the liver, which relies heavily on P450 enzymes for proper function, is altered. It is also possible, that serum binding proteins, are altered by nitrates effects. 109

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BIOGRAPHICAL SKETCH Heather J. Hamlin was born on November 22, 1972 in Bangor, Maine. She spent much of her childhood by the ocean, pawing through clumps of seaweed in search of ocean life that was unfortunate enough to be stranded by the outgo ing tide. Heather graduated from Hampden Academy High School in 1991, and then began an associates degree in legal technology, followed by a bachelors degree in biology from the University of Maine at Orono. In 1996 she was accepted to the graduate program in Marine Bio-Resources at the University of Maine, where she completed her masters degree examin ing the culture and histology of haddocks early development. After graduating in 1998, she worked as a biologist with the National Oceanic and Atmospheric Administration for a year before being hired as a senior biologist with Mote Marine Laboratory in Sarasota, FL, where she has worked since 1999. 130