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Site-specific impacts on gene expression and behavior in fathead minnows (Pimephales promelas) exposed in situ to stream...
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Title: Site-specific impacts on gene expression and behavior in fathead minnows (Pimephales promelas) exposed in situ to streams adjacent to sewage treatment plants
Series Title: BMC Bioinformatics
Physical Description: Archival
Creator: Garcia-Reyero,Nat
Adelman,Ira
Martinovic,Dalma
Liu,Li
Denslow,Nancy
Publication Date: 2009
 Notes
Abstract: BACKGROUND:Environmental monitoring for pharmaceuticals and endocrine disruptors in the aquatic environment traditionally employs a variety of methods including analytical chemistry, as well as a variety of histological and biochemical endpoints that correlate with the fish fitness. It is now clear that analytical chemistry alone is insufficient to identify aquatic environments that are compromised because these measurements do not identify the biologically available dose. The biological endpoints that are measured are important because they relate to known impairments; however, they are not specific to the contaminants and often focus on only a few known endpoints. These studies can be enhanced by looking more broadly at changes in gene expression, especially if the analysis focuses on biochemical pathways. The present study was designed to obtain additional information for well-characterized sites adjacent to sewage treatment plants in MN that are thought to be impacted by endocrine disruptors.RESULTS:Here we examine five sites that have been previously characterized and examine changes in gene expression in fathead minnows (Pimephales promelas) that have been caged for 48 h in each of the aquatic environments. We find that the gene expression changes are characteristic and unique at each of the five sites. Also, fish exposed to two of the sites, 7 and 12, present a more aggressive behavior compared to control fish.CONCLUSION:Our results show that a short-term exposure to sewage treatment plant effluents was able to induce a site-specific gene expression pattern in the fathead minnow gonad and liver. The short-term exposure was also enough to affect fish sexual behavior. Our results also show that microarray analysis can be very useful at determining potential exposure to chemicals, and could be used routinely as a tool for environmental monitoring.
General Note: Periodical Abbreviation:BMC Bioinformatics
General Note: Start pageS11
General Note: M3: 10.1186/1471-2105-10-S11-S11
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BMC Bioinformatics n.oMed Central



Proceedings
Site-specific impacts on gene expression and behavior
in fathead minnows (Pimephales promelas) exposed in situ
to streams adjacent to sewage treatment plants
Natalia Garcia-Reyero1'5, Ira R Adelman2, Dalma Martinovic3, Li Liu4
and Nancy D Denslow*1

Address: 'Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville,
FL 32611, USA, 2Dept. of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, St. Paul, MN, USA, 3Department of Biology,
University of St Thomas, St Paul, MN, USA, 4ICBR, University of Florida, Gainesville, FL 32611, USA and 5Department of Chemistry,
Jackson State University, Jackson, MS 39217, USA
E-mail: Natalia Garcia-Reyero natalia@icnanotox.org; Ira R Adelman ira@umn.edu; Dalma Martinovi6 dalma@stthomas.edu;
Li Liu liliu@biotech.ufl.edu; Nancy D Denslow* ndenslow@ufl.edu
* Corresponding author


from Sixth Annual MCBIOS Conference. Transformational Bioinformatics: Delivering Value from Genomes
Starkville, MS, USA 20-21 February 2009

Published: 08 October 2009
BMC Bioinformatics 2009, 10(Suppl I 1):SI I doi: 10.1 186/1471-2105-10-SI I-SI I


This article is available from: http://www.biomedcentral.com/1471-2105/10/SI I/S I I
2009 Garcia-Reyero et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Environmental monitoring for pharmaceuticals and endocrine disruptors in the
aquatic environment traditionally employs a variety of methods including analytical chemistry, as
well as a variety of histological and biochemical endpoints that correlate with the fish fitness. It is
now clear that analytical chemistry alone is insufficient to identify aquatic environments that are
compromised because these measurements do not identify the biologically available dose. The
biological endpoints that are measured are important because they relate to known impairments;
however, they are not specific to the contaminants and often focus on only a few known endpoints.
These studies can be enhanced by looking more broadly at changes in gene expression, especially if
the analysis focuses on biochemical pathways. The present study was designed to obtain additional
information for well-characterized sites adjacent to sewage treatment plants in MN that are
thought to be impacted by endocrine disruptors.
Results: Here we examine five sites that have been previously characterized and examine changes
in gene expression in fathead minnows (Pimephales promelas) that have been caged for 48 h in each
of the aquatic environments. We find that the gene expression changes are characteristic and
unique at each of the five sites. Also, fish exposed to two of the sites, 7 and 12, present a more
aggressive behavior compared to control fish.






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Conclusion: Our results show that a short-term exposure to sewage treatment plant effluents
was able to induce a site-specific gene expression pattern in the fathead minnow gonad and liver.
The short-term exposure was also enough to affect fish sexual behavior. Our results also show that
microarray analysis can be very useful at determining potential exposure to chemicals, and could be
used routinely as a tool for environmental monitoring.


Background
Effluents from sewage treatment plants are a big source
of endocrine-disrupting compounds (EDCs) into the
aquatic environment. Exposure of aquatic organisms to
EDCs has been linked to adverse physiological effects
such as reduced fertility [1], intersex fish [2], sex reversal
[3], and immunotoxicity or altered metabolism [4].
Effluents are very complex and variable mixtures of
many different compounds [5,6], as a result their adverse
effects on wildlife are extremely difficult to predict.

Lee et al. [5,7] analyzed the effects and the presence of
EDCs in several streams in Minnesota. Potential sources
of EDCs in Minnesota are treated sewage (domestic and
industrial) and runoff from agricultural or forested land.

In the Lee study [7], female and male carp were collected
at each site and were analyzed for fish health and a series
of biomarkers of endocrine disruption. These included
measurements of gonadosomatic index (GSI), plasma
hormone and vitellogenin (Vtg) levels, and gonad
histopathology. GSI is an indicator of reproductive
status and chemical exposure and it is related to
reproductive success. Gonad histopathology consisted
of microscopic examination for the presence of abnorm-
alities, such as ceroid/lipofuscin deposits in the males.
Cellular level abnormalities are often seen prior to
macroscopic abnormalities and are used as a warning of
sublethal health effects and are correlated with increased
susceptibility to disease [7]. In the present study only
male fish were included.

These studies indicated the presence of EDCs by
analyzing biological characteristics, such as hormone
and vitellogenin levels, in the common carp. The studies
identified sewage treatment plant effluents as a potential
source of EDCs. Additionally, fish located at sites
upstream of sewage treatment plant effluent draining
agricultural and forested land showed indications of
EDCs. The present study was designed to obtain
additional information using genomics for some of
these well-characterized sites in MN that are thought to
be impacted by EDCs.

Microarray analysis has developed in recent years to the
point where it can be used to gain mechanistic


understanding about how fish health is impacted by
contaminants. This method provides an unbiased open
assessment of the health of fish exposed to polluted
aquatic environments. A few studies have used micro-
arrays to analyze the gene expression signature in fish
exposed to polluted field sites [8,9]. If these efforts
continue to efficiently move ahead, the use of micro-
arrays to evaluate contaminants could become an
extremely useful tool in ecological risk assessment.

A variety of anthropogenic chemicals such as industrial
chemicals, surfactants and pesticides are known to have
some estrogenic potency [2,10,11]. But more recently,
among the primary estrogenic components of sewage
discharge the human estrogens 17p-estradiol (E2) and
estrone (Ej), and the pharmaceutical estrogen 17a-
ethinylestradiol (EE2), found in birth control pills,
have been identified [12]. It is also evident that effluent
run off from confined animal feeding organizations
(CAFO) introduces high levels of anabolic androgens
(17a-trenbolone, and 17p-trenbolone) [13], estrogens
(E2 and zearalenone) and progestins (melengestrol
acetate) [3]. Minnesota is impacted by effluents from
sewage treatment plants that deal with domestic and
industrial wastes and by run off from agricultural or
forested land, where there is an abundance of CAFOs.

Here we examine five sites that have been previously
characterized by Lee et al. [5,7] by examining changes in
gene expression in male fathead minnows (Pimephales
promelas FHM) that were caged for 48 h in the different
aquatic environments. We also examined whether 48 h
exposure to water from the five sites of interest affected
the ability of males to compete for nests and females. For
many fish, including the FHM, acquisition of spawning
territory is a competitive process in which more
aggressive males acquire and maintain spawning terri-
tories (i.e., nest sites), whereas subordinate males often
fail to reproduce. Past studies indicated that exposure to
environmental estrogens can lead to reduced ability of
males to acquire nests, whereas exposure to androgens
can increase the performance of aggressive behaviors and
enhance nest acquisition [14].

We hypothesized that fish exposed to water downstream
from sewage treatment plants will have a different gene




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expression signature than those exposed to upstream
waters, probably showing a more estrogenic signature, as
waters downstream of sewage treatment plants have
been shown to be estrogenic in some cases [15,16]. For a
48 h exposure, the gene expression signature will be a
snapshot that depends on the contaminants present in
the effluent during the time of exposure, rather than an
average over time. We also hypothesized that the short
exposure would be long enough to change fish sexual
behavior.


Results
Selection of field sites
Five sites were selected for exposing FHM to possible
EDCs in the natural environment (Figure 1 and Table 1).
These sites were identified in Lee et al. [7] as showing
evidence of EDCs using biological characteristics of
common carp (Cyprinus carpio). The five sites were the
following: Site 7 (4336'02"; 9317'30") is located on
Shell Rock River near Albert, Lea, MN. Site 11 (43 51'54";
9518'47") is on the Des Moines River, upstream of
Windom. Site 12 (4351'27"; 9506'28") is on the Des
Moines River, downstream of Windom and down stream


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.......



number)
IOWA


SSite location downstream of wastewater
treatment plant (number is site location
number)
5,te location dnostream ofwate a r
treatment plant (number is site location
number)

Figure I
Location of the sites. Adapted from Lee et a/. [5,7].


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from a sewage treatment facility. Site 13 (4343'04";
96009'49") is located on Rock River upstream of Luveme,
MN. Although the high percentage of agricultural land
and the number of animal feedlots in the drainage basin
provides the potential for runoff of animal wastes to the
streams, those characteristics do not guarantee that runoff
into the stream has occurred or was present when the
fish were exposed. A fifth site, Site 21 at Jewitt's Creek
(4508'42"; 94031'00") downstream of Litchfield, MN,
was identified in Lee et al. [5] by the presence of a number
of sterols in the water quality analysis, although the effects
in fish were not analyzed.


Microarray results
A 22,000 gene microarray specific for FHM [17] was used
to analyze changes in gene expression patterns in liver
and gonad of caged fish exposed to each of the effluents
for 48 h. Although fish from site 11, a site located up-
stream from a sewage treatment facility, were closest to
laboratory controls (data not shown), gene expression
patterns differed from the laboratory controls, probably
due to differences in water quality and environment.
This site is the closest to a reference site among the sites
tested, yet it was not perfect. Fish at this site presented
low GSI, but no Vtg induction in the males. Although we
are aware that the site might have low concentrations of
EDCs, it did have the lowest impact on fish, compared to
the laboratory controls. Consequently, gene expression
patterns at all of the field sites were compared to that of
fish at site 11.

To examine expression patterns across sites, we plotted
the union of genes identified as significantly regulated
across all sites (2624 total, p-value < 0.05) in relation to
their fold-expression compared to Site 11 (Fig 2 and Fig
[3]). Each field site showed a unique gene expression
pattern that was produced after only 48 h exposure of
caged FHM. In the liver and the gonad, the number and
identity of up- and down-regulated genes varied tre-
mendously from site to site indicating the complexity of
each of the effluents (Table 2).

Hierarchical clustering of all samples using the union of
all differentially expressed genes was conducted to
directly compare all the field samples (Fig 4A and 4B).
It is clear that expression patterns grouped the fish
together by location of exposure. Interestingly, the
clustering patterns are very similar for both liver and
gonad tissues. Sites 11 and 12, up- and down-stream
respectively on the same river, had patterns of gene
expression that were more clearly aligned with each
other. Sites 7 and 12, both downstream locations with
aggressive behavior, clustered together. Sites 21 and 13
clustered together, both sites with little or no impact on


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Table I: Field sites characteristics


Land use (% of drainage basin)

Site # Site Name Drainage Urban Agicultural Wetland Forest Water Other Feed Lots
area (km2) within I or 5 mi

7 Shell Rock River near Albert Lea, MN 388 7.6 81.7 2.6 3.5 4.5 0.1 NA
II Des Moines River upstream of Windom 1432 1.1 89.5 3.9 2.3 3.2 0 0, 3
12 Des Moines River down stream of 2944 1.7 90.4 3.3 1.8 2.8 0 NA
Windom
13 Rock River upstream of Luvern, MN 792 1.3 94.9 2.2 1.5 0.1 0 2, 12
21 Jewitts Creek downstream of Litchfield, 27 7.3 63.9 14.9 4.4 ? ? 0, I
MN*

Study sites and land basin characteristics from Lee et al. [7], Sites 3, 7, I I, 12, 13, and Lee et al. [5], Site 21. Feedlots are the number of "non-
permitted" animal feedlots within approximately 1/4 mile from the stream or its tributaries within and I and 5 miles upstream of the exposure site.
Table was abstracted from Lee et al. [7].
*Data is from Lee et al. [7].
NA, data not available.


Site 7
8.00
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o400
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00.



-8.00

1 171 341 511 81 851 102111911361 15311701 187120412211 2381 2551




Site 13
8.W


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1 171 341 511 181 861 10211191136115311701157120412211231 2551


Site 12
8.00
6.00

2. 00
0.00
.-2.00
-400
.-6.09
-8.00
1 171 341 511 681 851 10211191136115311701 18712041221123812551




Site 21
00U





0.1 -1



1 171 341 611 681 851 10211191136115311701 1871 2041 2211231 2551


Figure 2
Expression fingerprints for the liver at each of the sites. Genes are expressed as fold change over expression at site
#11 (similar to control). All genes were arranged from most highly expressed to most highly repressed for site#12 and the
same order is kept for the other sites. Genes whose fold expression data was not significant at p-value < 0.05 were set to 0.


behavior after four days of testing despite being located
on different rivers.

Functional analysis
While it is interesting to identify individual genes
regulated by each of the effluents, most biological


processes occur through functional pathways. To assess
this, we identified human homologs where possible and
then used this information to find enriched Gene
Ontology (GO) groups at each field site. We were able
to identify human homologs for about 50% of the
differentially expressed genes.



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Site7
200
040

e ~c o---------------- o il-





1 195 389 583 777 971 11651359 153 1747 19412135 2329 25232717 2911





Site 13
6.W
40 4-0

O




-6.0
1 195 389 5B3 777 971 115 1359 1553 1747 1941 2135 2329 223 2717 2911


Site 12
6.00
0 4.0
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1 195 39 583 777 971 11651359165317471941 21352329252327172911





Site 21
0 0




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1 195 389 583 777 971 11651359155317471941 21352329252327172911


Figure 3
Expression fingerprints for the gonad at each of the sites. Genes are expressed as fold change over expression at site
#11 (similar to control). All genes were arranged from most highly expressed to most highly repressed for site#12 and the
same order is kept for the other sites. Genes whose fold expression data was not significant at p-value < 0.05 were set to 0.


We used the functional clustering implemented in the
web-based application DAVID (http://david.abcc.
ncifcrf.gov, [18,19]) to determine significant changes
in processes enriched in the differentially regulated
gene sets in order to look at a higher order of
complexity and determine which biological processes
were altered because of the exposures (see additional
files 1 to 4). The results show that exposure of fish to
Site 7 waters alters processes involved in RNA
splicing, metabolism, protein transport or protein
catabolic process in both tissues, and also apoptosis
in the liver. For site 12 some of the most enriched GO


groups are sterol, cholesterol, and steroid biosynthetic
process in the liver; and catabolism and signal
transduction in the gonad. Another enriched GO
group for the liver of site 12 was immune response,
as previously described for the gonad of these fish [9].
Some of the main groups enriched for site 13 were
metabolism, protein transport, or modification in the
gonad; metabolism, mRNA processing or protein
transport in the liver. For site 21, the main groups
affected were protein transport, or metabolism in the
gonad; and phosphorylation or steroid receptor
signaling in the liver.


Table 2: Gene expression changes

LIVER GONAD

up regulated Down regulated Total up regulated down regulated Total

Site 12 80 133 213 48 64 1 12
Site 7 394 253 647 438 374 812
Site 21 434 431 865 738 388 1 126
Site 13 650 463 I 113 91 1 679 1590

Number of genes significantly altered compared to site I I.


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Figure 4
Hierarchical clustering. Two-way hierarchical clustering for genes differentially expressed following 48 h exposure at each
of the sites. Expression data was analyzed by ANOVA and then z-transformed. Genes used in the cluster were significant
at P < 0.05. Represented are genes that are up-regulated (red) or down regulated (green) by the treatments compared to
controls. (A) Cluster for genes changed in the liver and (B) cluster for genes changed in the gonad.


Behavioral effects
The behavioral test found no effects on behavior in fish
exposed at site 11, which lends support to the gene
expression analyses that fish from the site were the least
affected. Fish from site 13 also presented unchanged
behavior. The fish from site 21 showed initial increases
in aggressive behavior, but were not able to maintain
possession of the nests; by the third day of behavioral
experiments there were no significant differences in nest-
holding ability between exposed and control males.
Fish exposed to Sites 7 and 12 consistently dominated
control fish and actively defended the majority of the
nests over the course of the whole experiment. Figure 5
shows the results of behavioral observations.


Discussion
These studies were designed to re-examine specific
locations in rivers in Minnesota that had previously
been studied by Lee et al. [5,7]. The Lee study design
chose paired sites, targeting locations up stream and
down stream of waste water treatment plants (WWTP) in
Minnesota. The expectation was that up stream sites
would be influenced more by agricultural run off and the
down stream sites by effluent discharge and urban


nsok 11
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SObute 13
0 asa 21

10
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Figure 5
Results of behavioral observations. Asterices (*)
indicate significant differences (t-test with Welch correction,
P < 0.05) in the ability of males to occupy and defend nest
over the period of the whole 4 (**) or just 2 (*) days of the
test following a 48 h exposure period to site water.


runoff. Locations on rivers were chosen that had a
barrier (e. g. dam) that would prevent fish from
swimming up stream. Originally the Lee et al. [7] study
used 22 sites. Of these we picked 4 sites, those that
seemed to have the most impact on fish reproduction.



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BMC Bioinformatics 2009, 10(Suppl 11 ):S11


We picked an additional fifth site at Jewitt's Creek [5],
labeled as site 21 located downstream of a wastewater
treatment plant outfall. Of the original paired sites, we
only have one on the Des Moines River near Windom;
with site 11 (upstream) and site 12 (downstream) from
the WWTP. The other sites selected were not paired.
Effluents and consequently the waterways into which
they disgorge are known to be very variable [5,6],
therefore we expected that effects on fish might be
different over time, and will depend on many factors,
such as duration of exposure, season, or weather, among
others.


Gene expression and functional analysis
The expression fingerprints for fish exposed to five
different field locations showed unique patterns for fish
within each location that differed among the five regions
picked (sites 7, 11, 12, 13 and 21) for both the liver and
the gonad. Fish exposed at the same site showed similar
gene expression profiles. Sites 11 and 12 were closely
related consistent with their locations on the same river,
up-stream and down-stream respectively, from a sewage
treatment facility.

Site 11 male carp had the fewest biological anomalies;
presenting only low GSI [7]. FHM exposed to this
effluent for 48 h clustered most closely with non-
exposed laboratory controls, suggesting that the water
at this upstream location was not heavily contaminated
[9]. At site 12, downstream from the WWTP site, male
carp in the Lee study [7] also presented a low GSI in
addition to lower plasma levels of 11-keto testosterone
(11-KT) with an average of 400 pg/mL in fish from site
12 compared to an average of 600 pg/mL in fish from
site 11. Carp also showed evidence of high ceroid/
lipofuscin staining in their gonads, suggestive of
susceptibility to disease. Some of the most enriched
GO groups in FHM at site 12 were sterol and cholesterol
biosynthetic process and steroid metabolism, all of
which might help explain the decrease in 11-KT levels
in relation to site 11, in endogenous carp. FHM placed in
cages for 48 h at this location showed changes in gene
expression that indicated that apoptosis or immune
response were among the most affected GO groups in
the liver (Additional file 2).

Site 7 is located on the Shell Rock River down stream
from the WWTP near Albert Lea, MN. Male carp at this
location [7] were shown to have plasma Vtg present. The
levels of 11-KT in the carp were also lower than site 11
(400 pg/mL), and E2 levels were slightly higher (250 pg/
mL compared to 150 pg/mL). In the present study,
responses to site 7 appeared very complex, since we had
from 650-800 genes altered by the treatment, but


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distinguishable from other sites by its molecular
fingerprints. We did not find Vtg mRNA up-regulated
after 48 h. We expect this site would be highly variable as
well because it not only receives effluent from the sewage
treatment facility but also because it gets a lot of
agricultural runoff.

Males normally have a balance between estrogen and
androgen hormones. Both types of hormones play a very
important role in homeostasis. In site 7, we found
several affected genes prohibition prohibition 2, and
estrogen receptor-related gamma) that could be decreas-
ing the effects of estrogenic compounds, thereby tipping
the hormonal balance towards more androgenic path-
ways and resulting in more aggressive behavior. Prohi-
bitin and prohibition 2 were up-regulated both in the
gonad and liver of fish exposed to site 7 waters.
Prohibitin, a potential tumor suppressor, has been
shown to function as a potent transcriptional corepressor
for estrogen receptor alpha (ERa) [20]. The repression of
ERa could help explain the aggressive behavior found in
these fish. Estrogen receptor-related gamma (ERRy) is
down-regulated in the gonad of site 7. ERRy is a member
of the orphan nuclear receptor family. It does not bind to
endogenous estrogens but it has been shown to bind to
endocrine disruptors like bisphenol A and is deactivated
by the ER antagonist [21] suggesting the presence anti-
estrogenic compounds that might affect behavior. All
these changes could help explain why Vtg mRNA was
unchanged after a 48 h exposure.

Site 13, located upstream from Luverne, MN, has a
considerable number of animal feeding operations
within 5 miles of the site. Male carp at this location
presented with high plasma E2 and Vtg levels and low
plasma 11-KT levels, suggesting impairment in steroido-
genesis and potential exposure to estrogenic contami-
nants [7]. They also showed evidence of high ceroid/
lipofuscin staining in their gonads. Caged FHM in our
study failed to show Vtg increases but did have the
highest number of differentially expressed genes (1100-
1600 genes). One of the genes up-regulated in the gonad
and the liver of fish exposed to site 13 is progesterone
receptor, which requires estrogen to be induced [22],
suggesting that estrogenic compounds could be coming
from the nearby CAFO facilities. The abnormal hormone
levels found in fish exposed to this site could be related
to the up-regulation of StAR (steroidogenic acute
regulatory protein), a protein in charge of cholesterol
transport into the mitochondria, a key step in steroido-
genesis. Another up-regulated gene in the gonads of site
13 was the retinoic acid-related orphan receptor alpha
(RORa), an orphan member of the subfamily 1 of
nuclear hormone receptors. Cholesterol or cholesterol-
derivatives are suspected to be its natural ligands of


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BMC Bioinformatics 2009, 10(Suppl 11 ):S11


RORa, suggesting that RORa plays a very important role
in the regulation of cholesterol homeostasis in humans
[23]. There is also evidence that RORa participates in the
xenobiotic regulatory network [24]. RORa up-regulation
would also be consistent with the up-regulation of StAR,
as it has been shown to be an important regulator of that
protein in the largemouth bass [25].

Finally site 21, located on Jewitt's Creek near Litchfield,
was chosen based on a separate study by Lee et al. [5]. It
was chosen because it was within one mile of the
discharge source and was the site that presented more
EDCs and organic wastewater compounds from all the
sites examined [5]. Exposure of FHM to this site altered
900-1100 genes. The most highly up-regulated protein
in the liver was retinol binding protein (RBP). RBP up-
regulation has been related to exposure to estrogenic
compounds in Xenopus laevis [26,27]. Signal transducer
and activator of transcription 1 (STAT1) is up-regulated
in the gonad. STAT1 is a critical transcription factor
involved in the JAK-STAT signalling pathway which is
central for innate immunity [28] and apoptosis [29]
among other functions. STAT1 is activated via the
retinoic receptor signalling pathways [30] and it has
been shown to be up-regulated by exposure to EE2 in the
gonad of FHM [31]. This could help support the idea
that exposure to estrogenic compounds decreased the
initial aggressive behavior in fish from this site. The very
dynamic nature of the effluent could have initially
exposed fish to androgenic compounds immediately
followed the day after by estrogenic compounds. The
steroid hormone receptor signaling pathway was one of
the most affected GO groups in the liver of these fish,
which would also support that statement.


Behavioral effects
The fish from Sites 7 and 12 were more successful at
acquiring nests than control males, while fish from Site
13 showed no significant changes in behavior. The
observed increases in the ability of males from sites 7
and 12 to occupy and defend nests are consistent with
findings of others who found that waterborne androgens
increased aggressive behavior and nest acquisition [14].
The fish from site 21 showed initial increases in
aggressive behavior, but were not able to maintain
possession of the nests; by the third day of behavioral
experiments there were no significant differences in nest-
holding ability between exposed and control males.


Unchanged or intermediate behavior
Sites 13 and 21 presented up-regulation of several
thyroid hormone associated proteins (TRAP), both in
the gonad (TRAP5 in both sites and TRAP230 in site 13)
and the liver (TRAP3 in both sites and TRAP4 in site 21).


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The TRAP complex is a coactivator complex that interacts
and modulates the activity of thyroid hormone receptors
[32,33], suggesting the presence at both these sites of
compounds that interact with the thyroid receptor.
Disturbances in the thyroid system are associated with
impaired reproduction in fish [34]. Flame retardants
such as PBDE 47 (2,3,4,4-tetrabromodiphenyl ether) are
known to affect thyroid hormone levels [35]. PBDE 47
has also been shown to decrease the number of mature
spermatozoa in the FHM as well as the overall male
reproductive fitness [35,36]. Although we do not have
the flame retardant levels for site 13, nor the specific
concentration of PBDE 47 in site 21, we do know that
flame retardants were quite abundant in site 21 [5]. That
might be related to the time-dependent decrease of
aggressive behavior in fish from site 21 and the lack of
aggressive behavior in fish from site 13.

CYP20A1 is up-regulated in these sites although that
is not the case of some of the other CYPs involved in
the metabolism of xenobiotics compounds, like CYPA1.
The lack of CYPIA induction could also be related to the
presence of flame retardants, as some have been shown
to be antagonists that can reduce the induction of CYP1A
by a more potent agonist [37,38].

Ornithine decarboxylase antizyme 2, an ornithine dec-
arboxylase (ODC) inhibitor [39], was up-regulated in all
sites. ODC has been identified as a sensitive marker of the
action of androgens and antiandrogens in the testis [40].
Androgens are essential for the maintenance of ODC
activity and administration of androgens increases
synthesis of ODC mRNA [41]. ODC is involved in the
biosynthesis of polyamines many of which (such as
putrescine, spermidine, spermine) play an important role
in reproduction [42]. Our finding that ODC antizyme
2 was up-regulated suggests that androgens were present
in all sites and raise concern that the reproduction of
endogenous fish at these locations may be impacted.


Aggressive behavior
In order to elucidate the causes of the increased nest
acquisition rates (these are mediated by aggressive
behavior), we compared the differentially expressed
genes from sites 7 and 12. We expected these changes
to be related to androgen exposure, as circulating
androgens have been related to aggressive behavior in
fish [43,44]. Thirty-one genes were common between
site 7 and 12. Some of these genes were calbindin 2, Rho
Family GTPase 3, NADH dehydrogenase subunit 2,
caveolin 1 (CAV1), all up-regulated; and calmodulin,
synaptogyrin 2, silencing mediator of retinoic acid and
thyroid hormone receptor (SMRT), and hepatocyte
nuclear factor 4 alpha (HNF4A), all down-regulated.


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BMC Bioinformatics 2009, 10(Suppl 11 ):S11


CAV1, a membrane protein up-regulated at sites 7 and
12, is one of the few proteins known to bind cholesterol,
the precursor of steroid hormones, tightly and specifi-
cally. It has a very important role in cholesterol home-
ostasis. Caveolins have also been linked to reverse
cholesterol transport, where excess free cholesterol is
released into the plasma [45]. CAV1 has been previously
found to respond to androgenic compounds [17],
confirming the importance of this gene in relation to
androgen exposure. RORa is down-regulated in sites 7
and 12. As mentioned earlier, this receptor is an
important regulator for StAR and therefore, very impor-
tant for steroidogenesis.

Conclusion
Our results show that even a short exposure (48 h) to
streams adjacent to sewage treatment plants was able to
induce a site-specific gene expression pattern in the
fathead minnow gonads and livers. The short-term
exposure was also enough to affect the fish sexual
behavior at two of the sites. These findings suggest that
gene array analysis can complement chemical analysis as
a monitoring tool. These findings also suggest that
microarray analysis is relatively robust especially when
used in conjunction with other more established
methods to define contaminated aquatic environments
for risk assessment and environmental monitoring.

Methods
Fish exposures
To conduct the field exposures at each field site, FHM
were transported from the laboratory to the field site in
aerated, insulated tanks. At each site, 25 males and 25
females were placed in separate wire mesh minnow traps
with the entrance funnel plugged. The traps were
anchored to the bottom in the stream current with the
top of the traps submerged. Fish were removed from the
traps 48 h later and transported back to the laboratory in
aerated, insulated tanks containing the stream water.
Immediately upon arrival at the laboratory, four males
and four females were sacrificed as described for the
laboratory exposures. Liver and gonads were removed
and stored in liquid nitrogen until processed for arrays.

All procedures involving live fish were reviewed and
approved by the University of Minnesota Institutional
Animal Care and Use Committee (IACUC).

Preparation of total RNA
Total RNA was isolated from gonadal tissue with the
RNA Stat-60 reagent (Tel-test, Friendswood, TX) as
described previously [46]. RNA pellets were resuspended
in 50 to 150 pl RNA Secure (Ambion, Austin, TX) to
inactivate RNases following the manufacturer's protocol.


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A total of 10 pg of RNA was treated with DNase to avoid
contaminating DNA using DNA-free (Ambion, Austin,
TX) following the manufacturer's protocol. The quality
of total RNA was assessed with the Agilent 2100
BioAnalyzer (Agilent, Palo Alto, CA) and the quantity
was determined on a NanoDrop spectrophotometer
(NanoDrop Technologies, Wilmington, DE). RNA was
stored at -80 C until further use.


Microarrays
Fathead minnow 22,000 gene arrays were designed by
EcoArray (Alachua, FL) and were purchased from
Agilent. Array hybridizations were performed using a
reference design, where each sample was compared to a
reference sample. The reference sample consisted of
equal amounts of RNA from control female and male
tissues (liver, brain and gonad). Four replicates, each
consisting of a different individual, were analyzed for
each of the treatment sites (sites 7, 11, 12, 13 and 21).
cDNA synthesis, cRNA labeling, amplification and
hybridization were performed following the manufac-
turer's kits and protocols (Agilent Low RNA Input
Fluorescent Linear Amplification Kit and Agilent 60-
mer oligo microarray processing protocol; Agilent, Palo
Alto, CA). Gonad and liver samples from the fish at the
sites were labeled with Cy5 while the reference sample
was labeled with Cy3. Consistent with the Minimum
Information about a Microarray Experiment (MIAME)
standards [47], text versions of the Agilent raw data from
this study have been deposited at the Gene Expression
Omnibus website (GEO: http://www.ncbi.nlm.nih.gov/
geo/; Accession series record number GSE16645).


Bioinformatics
Microarray image processing and data pre-processing
were performed using Agilent's Feature Extraction soft-
ware v 9.5 (Agilent, 2007). The intensity of each spot
was summarized by the median pixel intensity. A log2
transformed signal ratio between the experimental
channel and the reference channel was calculated for
each spot, followed by within-array lowess transforma-
tion and between array scale normalization on median
intensities [48].

One-way ANOVA was performed on normalized log2
transformed signal ratios of each probe individually,
followed by Tukey-HSD pair-wise comparisons to
determine genes whose expression was significantly
regulated between sites. Statistical significance was
determined at a p-value of < 0.05 with an FDR threshold
of 16%. FDR was calculated using Benjamini-Hochberg
approach [49]. After testing for significance we also
eliminated from consideration genes whose fold-expres-
sion changes were less than 1.5 fold.


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Behavioral assay
Individual fish were transferred into 10-L glass test
aquaria for behavioral testing, each of which was
supplied with clean well water (50 ml/min). Prior to
placement in tanks, both site water-exposed and control
males were acclimated to the temperature of the
observation tank (25 C) over a 4-h period. For each
one of the sites, we tested 7-10 behavioral replicates.
Each one of these replicates consisted of a control male
added along with the site water-exposed male to an
aquaria containing one unexposed female and a nest.
Male fish were selected so that their total lengths were
within 3 mm of each other and one fish had a upper
caudal fin clip, and the other a lower caudal fin clip
(marking pattern was not treatment-specific).

The behavioral assay followed protocols described by
Martinovic et al. [14]. Briefly, identification of nest-
holders commenced after 24 h had elapsed from the
time the fish were introduced into test aquaria. Each
individual was observed each day between 10.00 h and
14.00 h for 5 min and those fish which spent the
majority of their time in a nest while also exhibiting the
nest-tending (nibble, rub nest etc.) or nest-defense
behaviors (chase, bite, butt etc.) were categorized as
'nest-holders'. After 4 days the experiments were termi-
nated. We calculated the percentage of times an
individual was identified as a nest-holder (number of
times each male was identified as a nest-holder was
divided by total number of observations). These data
were tested using Kolmogorov and Smirnov test and
because none of the datasets violated the normality
assumption, the significance of differences in nest
holding between controls versus exposed males was
evaluated with unpaired t-test with Welch correction. In
addition to examining overall nest-holding ability (over
4 days) we also compared the number of control and
exposed nest holders for each day using Fisher's exact
tests. The behavioral data are reported as significantly
different if p < 0.05.


Competing interests
The authors declare that they have no competing
interests.


Authors' contributions
NGR: RNA extraction, microarrays, drafted the manu-
script; IRA: conceived of the study, and participated in its
design and coordination and helped draft the manuscript;
DM: behavior assays, helped draft the manuscript; LL:
bioinformatics analysis, helped drafted the manuscript;
NDD: conceived of the study, and participated in its
design and coordination and helped draft the manuscript.
All authors read and approved the final manuscript.


Additional material


Additional file 1
functional clustering of enriched GO biological processes altered in site #
7.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-10-S 11-S11-S1.xls]

Additional file 2
functional clustering of enriched GO biological processes altered in site #
12.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-10-S 11-S11-S2.xls]

Additional file 3
functional clustering of enriched GO biological processes altered in site #
13.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-10-S 11-S11-S3.xls]

Additional file 4
functional clustering of enriched GO biological processes altered in site #
21.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-10-S 11-S11-S4.xls]



Acknowledgements
This work is the result of research sponsored by the Minnesota Sea Grant
College Program supported by the NOAA Office of Sea Grant, United
States Department of Commerce, under the grant No NA030AR4170048
to IA and ND and by a fellowship from the Spanish Ministry of Sciences
and Technology (EX-2004-0986), co-funded by the European Union to
NGR. The U.S. Government is authorized to reproduce and distribute
reprints for government purposes, not withstanding any copyright
notation that may appear hereon. This paper is journal reprint No JR
563 of the Minnesota Sea Grant College Program. ND holds equity in
EcoArray, Inc., a company commercializing the microarray technology
used in this study.

This article has been published as part of BMC Bioinformatics Volume 10
Supplement II, 2009: Proceedings of the Sixth Annual MCBIOS
Conference. Transformational Bioinformatics: Delivering Value from
Genomes. The full contents of the supplement are available online at
http://www.biomedcentral.com/1471-2105/10?issue=S I I.

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