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Cytochrome P450 enzymes in channel catfish Ictalurus punctatus, and metabolism of testosterone by catfish intestinal microsomes

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Title:
Cytochrome P450 enzymes in channel catfish Ictalurus punctatus, and metabolism of testosterone by catfish intestinal microsomes
Creator:
Lou, Zhen, 1971-
Publication Date:
Language:
English
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xi, 120 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Catfish ( jstor )
Chemicals ( jstor )
Cytochromes ( jstor )
Enzymes ( jstor )
Intestines ( jstor )
Liver ( jstor )
Metabolism ( jstor )
Metabolites ( jstor )
Microsomes ( jstor )
Testosterone ( jstor )
Cytochrome P-450 Enzyme System ( mesh )
Department of Medicinal Chemistry thesis Ph.D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF ( mesh )
Ictaluridae ( mesh )
Intestines -- physiology ( mesh )
Microsomes -- physiology ( mesh )
Testosterone -- metabolism ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D)--University of Florida, 2001.
Bibliography:
Bibliography: leaves 107-119.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Zhen Lou.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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54809361 ( OCLC )

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CYTOCHROME P450 ENZYMES IN CHANNEL CATFISH,
ICTAL UR US PUNCTA TUS, AND METABOLISM OF
TESTOSTERONE BY CATFISH
INTESTINAL MICROSOMES
















By

ZHEN LOU


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


2001















ACKNOWLEDGMENTS

I would first like to express my appreciation to my advisor, Dr. Margaret 0.

James, for her great deal of guidance, advice, and support through my course of study,

which I would not have completed without her help. I believe everything I learned during

my time in her laboratory is indispensable for my future accomplishments as a scientist. I

was deeply impressed with her diligence and talent as well as kindness.

I would also like to express my gratitude to all my committee members, Dr.

Kenneth Sloan, Dr. Stephen Roberts, Dr. Donghai Wu and Dr. William Dolbier, for their

invaluable guidance and the time they committed to my graduate work. My appreciation

is extended to our lab members for their kind help and cheerful encouragement.

Finally, I want to thank all members of my family for their continuous care and

support.















TABLE OF CONTENTS

page

A CKN OW LED GM ENT S .................................................................................................. ii

LIST OF TABLES ....................................................................................................... v

LIST OF FIGURES .......................................................................................................vi

KEY TO ABBREVIA TION S ........................................................................................ ix

AB STRA CT ......................................................................................................................... x

CHAPTERS

1 INTRODU CTION ............................................................................................................ 1

Phase I Enzym es ........................................................................................................ 3
Cytochrom e P450 .................................................................................................... 3
CYP3A Inhibition ................................................................................................. 11
CYP3A Induction ............................................................................................. 15
CYP3A Stim ulation .......................................................................................... 19
Epoxide Hydrolase ............................................................................................... 20
Phase II Enzym es ...................................................................................................... 21
Glutathione S-Transferase ................................................................................... 21
Sulfotransferase .................................................................................................... 25
UD P-G lucuronosyltransferase ............................................................................... 26
3a-Hydroxysteroid D ehydrogenase ........................................................................... 28
Prehepatic M etabolism and Bioavailability ............................................................. 30
Hypothesis ..................................................................................................................... 35

2 M ATERIALS AND M ETH OD S ............................................................................... 36

Chem icals ...................................................................................................................... 36
Instrum ents .................................................................................................................... 36
Anim als and Pretreatm ent ........................................................................................ 37
Surgical Procedures for Oral Gavage ....................................................................... 38
Enzym es Preparation ................................................................................................. 38
Protein A ssay ................................................................................................................ 39
M easurem ent of Cytochrom e P450 .......................................................................... 40
Steroid Hydroxylation Assay ................................................................................... 40









Chemical Modulation of Testosterone Metabolism ................................................. 41
AHH (Aromatic Hydrocarbon Hydroxylation) Assay ............................................. 42
Western Blot Analyses ............................................................................................. 42
Western Blot Analyses of CYP1A ........................................................................ 42
Immunochemical Analyses of CYP3A ................................................................. 44
HPLC Analysis of Testosterone Metabolism .......................................................... 44
Mass Spectrometric Analysis .................................................................................... 45
Sulfotransferase Activity Assay ............................................................................... 45
UDP-Glucuronosyltransferase Activity Assay ........................................................ 46
Statistical A nalysis ................................................................................................... 46


3 R E SU L T S ....................................................................................................................... 47

Response to Aryl Hydrocarbon Receptor Agonists ................................................. 47
CYP 1A Expression in Channel Catfish Intestine ................................................. 47
UDP-Glucuronosyltransferase Expression in Channel Catfish Intestine .............. 51
Function and Expression of CYP3A and Testosterone Metabolism ........................ 53
TLC Analyses of Steroid Metabolism by Catfish Intestinal Microsomes ............ 53
Expression of CYP3A along Catfish Intestine ...................................................... 57
Regional Expression and Dietary Effects on Intestinal CYP3A ........................... 59
Effects of Modulators on CYP3A Activities ........................................................ 65
Modulation of AHH Activities ............................................................................ 77
Identification of Major Testosterone Metabolite ...................................................... 78
HPLC Analysis of Testosterone Metabolism by Catfish Intestinal Microsomes ..... 78
Mass Spectrometric Analysis of Testosterone Metabolite .................................... 82
Regional Expression of 3ax-Hydroxysteroid Dehydrogenase in Catfish Intestine ........ 88
Catfish Intestinal CYP3A Inducibility Studies ........................................................ 90
Catfish Hepatic CYP3A Expression ........................................................................ 93

4 D ISC U SSIO N ................................................................................................................. 97

Induction of CYP1A and UGT in Catfish Intestine ................................................. 97
In vitro Testosterone Metabolism by Catfish Intestinal Microsomes ....................... 98
CYP3A Expression in Catfish Intestine .................................................................... 99
Chemical Inhibition of in vitro Testosterone Metabolism .......................................... 100
Stimulation of AHH Activities by cx-Naphthoflavone ................................................ 102
Inducibility of Catfish Intestinal CYP3A ................................................................... 103
Identification of 3a-Reduced Metabolite of Testosterone .......................................... 103
Catfish Hepatic CYP3A Expression ........................................................................... 105

5 SUMMARY AND CONCLUSIONS ........................................................................... 106

R EFERE N C E S ................................................................................................................ 107

BIOGRAPHICAL SKETCH ........................................................................................... 120















LIST OF TABLES


Table Page

3-1. Intestinal CYPI A level in control and treated fish ................................................... 49

3-2. Kinetic analysis of testosterone metabolism by catfish intestinal microsomes ...... 57

3-3. CYP3A expression and catalytic activities along catfish intestine ............................ 60

3-4. IC50 values of the four inhibitors for testosterone hydroxylation .............................. 76

3-5. Testosterone metabolism by intestinal microsomes from catfish fed with chow or
sem i-synthetic purified diet ............................................................................. 89

3-6. In vitro testosterone metabolism activities in proximal and distal intestine from
control fish and fish treated with rifampicin for two weeks ............................ 92

3-7. Hepatic CYP3A expression in control and treated fish ............................................. 95















LIST OF FIGURES


Figure -Page

1-1. Catalytic cycle of cytochrome P450 ............................................................................ 4

1-2. Mechanism of induction of CYP1A1 gene transcription ............................................ 8

1-3. Bioactivation of benzo(a)pyrene ................................................................................ 10

1-4. Role of PXR in CYP3A gene induction ..................................................................... 18

1-5. CYP3A gene induction: Cross-talk between foreign chemical and endogenous
regulator pathw ays ..................................................................................................... 18

1-6. Mercapturic acid biosynthesis ................................................................................... 22

3-1. Intestinal P450 content in control and treated fish ................................................... 48

3-2. Intestinal and hepatic CYP1A in control and fish treated with 3MC or TCB ........... 49

3-3. Intestinal and hepatic CYP1A in control and fish treated with TCB ........................ 49

3-4. Intestinal CYP1A content and AHH activity ............................................................ 50

3-5. UGT activity in control (n=12) and 3-MC treated (n=l 1) fish ................................. 51

3-6. Intestinal microsomal AHH and UGT activities ........................................................ 52

3-7. TLC of testosterone metabolism by catfish intestinal microsomes ............................ 54

3-8. Progesterone and testosterone metabolism positions by catfish intestinal microsomes. 55

3-9. TLC of progesterone metabolism by catfish intestinal microsomes ......................... 55

3-10. Lineweaver-Burk plot of testosterone metabolism by catfish intestinal microsomes.. 56

3-11. Western blot of hCYP3A4 and catfish intestinal CYP3A ........................................ 58

3-12. Western blot of CYP3A in catfish intestine ............................................................ 58









3-13. Testosterone 6p-hydroxylation activities in proximal and distal intestine of fish
fed chow or purified diet .......................................... 61

3-14. Testosterone metabolism activities in proximal and distal intestine of fish fed
chow or purified diet ................................................................................................. 62

3-15. Correlation between testosterone 6p-hydroxylation and CYP3A enzyme amount ...... 63

3-16. Ratio of testosterone 6p3-hydroxylation/17-oxidation in proximal and distal intestine
of control catfish ..................................................................................................... 64

3-17. Chemical structures of mammalian CYP3A modulators ....................................... 67

3-18. Effect of testosterone 6p-hydroxylation by addition of troleandomycin ................ 68

3-19. TLC of inhibition of testosterone metabolism by metyrapone ................................. 69

3-20. TLC of inhibition of testosterone metabolism by SKF-525A ................................. 70

3-21. Inhibition of testosterone metabolism by erythromycin ........................................... 71

3-22. Inhibition of testosterone metabolism by SKF-525A ............................................... 72

3-23. Inhibition of testosterone metabolism by ketoconazole .......................................... 73

3-24. Inhibition of testosterone metabolism by metyrapone ............................................ 74

3-25. Determination of IC50 of testosterone 63-hydroxylation by ERM, KET, SKF525A
and M E T ....................................................................................................................... 75

3-26. Modulation of testosterone metabolism by a-naphthoflavone ................................. 76

3-27. Stimulation of AHH activity by a-naphthoflavone ................................................. 77

3-28. HPLC (UV detection) profile of 4-androsten-3a,171-diol standard ....................... 79

3-29. HPLC (radiochemical detection) of [14C] testosterone metabolism catalyzed by
catfish intestinal microsom es ................................................................................... 80
3-30. HPLC analysis of mixture of 4-androsten-3cx,171-diol and [14C] testosterone

assay extract .................................................................................................................. 81

3-31. (+)APCI-MS/MS daughter spectra of m/z 289 ions ................................................. 83

3-32. (+)APCI-MS/MS daughter spectra of m/z 273 ions ................................................ 84

3-33. (+)APCI-MS/MS daughter spectra of m/z 255 ions ............................................... 85

3-34. Fragmentation of 4-androsten-3a,17p-diol in APCI-MS ........................................ 86









3-35. In vitro metabolism of testosterone by catfish intestinal microsomes ..................... 87

3-36. Western blot of intestinal CYP3A from control and PCN treated fish ................... 90

3-37. Intestinal CYP3A expression in control or fish treated with RIF or PCN ............... 91

3-38. Western blot of hCYP3A4 and hepatic microsomes from control fish and fish
pretreated with RIF or PCN (10 mg/kg) ................................................................... 94

3-39. Western blot showing cross-reactivity of catfish hepatic microsomes against
a polyclonal antibody to trout CYP3A27 ................................................................. 94

3-40. In vitro testosterone metabolism activities by hepatic microsomes of fish from
control, rifampicin (RIF) and PCN pretreated groups ............................................ 96















KEY TO ABBREVIATIONS


3-MC: 3-methylcholanthrene

AHH: aryl hydrocarbon hydroxylase

AhR: aryl hydrocarbon receptor

ANF: a-naphthoflavone

BNF: P-naphthoflavone

CYP: cytochrome P450

EH: epoxide hydrolase

ERM: erythromycin

GST: glutathione S-transferase

HEPES: N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]

HSD: hydroxysteroid dehydrogenase

KET: ketoconazole

MET: metyrapone

PCN: pregnenolone 16a-carbonitrile

RIF: rifampicin

ST: sulfotransferase

TAO: troleandomycin

TCB: 3, 3', 4, 4'-tetrachloro biphenyl

UGT: UDP-glucuronosyltransferase















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

CYTOCHROME P450 ENZYMES IN CHANNEL CATFISH,
ICTALURUS PUNCTA-TUS, AND METABOLISM
OF TESTOSTERONE BY CATFISH
INTESTINAL MICROSOMES



By

Zhen Lou

May, 2001


Chairman: Margaret 0. James
Major Department: Medicinal Chemistry

Intestinal cytochrome P450 provides the principal, initial source of

biotransformation of ingested xenobiotics. In humans, an important cause of incomplete

bioavailability is prehepatic metabolism in the GI tract, mainly by the CYP3A enzymes.

The expression and properties of CYP proteins were examined along the intestine of

channel catfish, Ictalurus punctatus, fed commercial chow or semi-purified diets.

Benzo(a)pyrene hydroxylase activity was higher in proximal than distal intestine, and

was stimulated by a-naphthoflavone, suggesting involvement of CYP3A. Polyclonal

antibodies (IgG) generated against trout CYP3A27 reacted strongly with catfish intestinal

microsomes, showing a band with MW of 59 kDa. In catfish fed with standard chow, the

expression of this protein was much higher in the proximal segment than in the distal

part. Testosterone 60-hydroxylation activities were monitored as the catalytic indicator of









CYP3A, which was higher in proximal than distal intestine. The 6[3-hydroxylation

activities in the two segments correlated with the amount of CYP3A. Similar results were

obtained with progesterone as substrate. The amount of CYP3A and steroid-6p-

hydroxylation activities were lower in both segments of intestine from fish fed purified

diet compared with commercial chow, but with the same trend along intestine.

Incubation of catfish intestinal or hepatic microsomes with [4- 14C] testosterone

resulted in three major metabolites: 6P-hydroxy testosterone, androstenedione and

another metabolite. The formation of this unknown metabolite requires NADPH as

cofactor. Comparison of the chromatographic behavior and MS of the unknown

metabolite with that of authentic testosterone derivative suggested that this metabolite

corresponds to 4-androsten-3c, 171-diol. The ratio of testosterone 61-hydroxylation/17-

oxidation was significantly higher in proximal than distal intestine.

Testosterone 6[-hydroxylation was inhibited by specific mammalian CYP3A

inhibitors, ketoconazole and erythromycin, and general P450 inhibitors, metyrapone and

SKF-525A, but was not affected by ax-naphthoflavone. Troleandomycin, a mammalian

CYP3A inhibitor, had no effect on the testosterone metabolism by catfish intestinal

microsomes up to 100 paM. Dietary pretreatment of catfish with rifampicin or

pregnenolone-16ct-carbonitrile (PCN) did not alter the CYP3A enzyme level in proximal

and distal intestine. Distal intestine from fish treated with rifampicin for 2 weeks showed

significantly higher testosterone 6p-hydroxylation and 3a-oxido-reduction activities than

that from control fish.














CHAPTER 1
INTRODUCTION

All organisms are exposed constantly and unavoidably to foreign chemicals, or

xenobiotics, which include both man-made and natural chemicals such as drugs,

industrial chemicals, pesticides, pollutants, pyrolysis products and toxins produced by

molds, plants and animals. As a result of a great variety of human activities, the aquatic

environment is becoming increasingly threatened by an alarming number of foreign

chemicals. This pollution is a threat to the health of organisms inhabiting the waters, as

well as to human consumers of such organisms. Fish populations living in highly polluted

areas often have high incidences of gross pathological lesions and neoplasms, associated

with elevated levels of toxic contaminants in the sediment [0]. Of most concern are

xenobiotics that cannot be readily eliminated because of their lipophilicity.

Biotransformation or metabolism of lipophilic chemicals to more water-soluble

compounds is a requisite for detoxification and excretion. An important consequence of

biotransformation is that the physical properties of a xenobiotic are generally changed

from those favoring absorption (lipophilicity) to those favoring excretion in urine or feces

(hydrophilicity). In addition, certain steps in the biotransformation pathway are

responsible for the activation of foreign chemicals to the reactive intermediates that

ultimately result in toxicity, carcinogenicity and other adverse effects. Many of the

enzyme systems involved in biotransformation are also engaged in critical physiological

functions such as steroid hormone biosynthesis and inactivation or fatty acid metabolism,

making interactions between foreign chemicals and physiological processes possible.









The reactions catalyzed by xenobiotic-biotransforming enzymes are generally

divided into two groups, called phase I and phase II. Phase I reactions involve hydrolysis,

reduction, and oxidation. These reactions expose or introduce a functional group (e.g.,

-OH, -NiH2, -SH or -COOH), and usually result in only a small increase in

hydrophilicity. Phase II biotransformation reactions include glucuronidation, sulfation,

acetylation, methylation, conjugation with glutathione (leading to mercapturic acid

synthesis) and conjugation with amino acids. The cofactors for these reactions react with

functional groups that are either present on the xenobiotic or are introduced/exposed

during phase I biotransformation. Most phase II biotransformation reactions result in a

large increase in xenobiotic hydrophilicity; hence they greatly promote the excretion of

foreign compounds. Phase II biotransformation of xenobiotics may or may not be

preceded by phase I biotransformation.

Xenobiotic-biotransforming enzymes are widely distributed throughout the body,

and are present in several subcellular compartments. The liver is the richest source of

enzymes catalyzing biotransformation reactions. These enzymes are also located in the

skin, lung, gastrointestinal tract, and nasal mucosa (which can be rationalized on the basis

that these are major routes of exposure to xenobiotics), as well as numerous other tissues,

including kidney, heart, brain, etc. Intestinal microflora play an important role in the

biotransformatiton of certain xenobiotics. The enzymes catalyzing xenobiotic

biotransformation reactions are located primarily in the endoplasmic reticulum

(microsomes) or the soluble fraction of the cytoplasm (cytosol), with lesser amounts in

mitochondria, nuclei and lysosomes. Their presence in the endoplastic reticulum can be

rationalized on the basis that those xenobiotics requiring biotransformation for urinary or









biliary excretion will likely be lipophilic and, hence, soluble in the lipid bilayer of the

endoplasmic reticulum.


Phase I Enzymes

Cytochrome P450

Among the phase I biotransformation enzymes, the cytochrome P450 system

ranks first in terms of catalytic versatility and the sheer number of xenobiotics it

detoxifies or activates to reactive intermediates. All P450 enzymes are heme-containing

proteins. The term "cytochrome P450" originates from the observation that the reduced

state of the protein forms a complex with carbon monoxide that exhibits maximal

absorbance at 450 nm [2]. The basic reaction catalyzed by cytochrome P450 is

monooxygenation in which one atom of oxygen is incorporated into a substrate,

designated RH, and the other is reduced to water with reducing equivalents derived from

NADPH, as follows:

Substrate (RH) + 02 + NADPH + H+ --> Product (ROH) + H20 + NADP+

The principal catalytic cycle of cytochrome P450 is shown in Figure 1-1. The essential

steps involve the following: (1) binding of the substrate, (2) reduction of the ferric

(resting cytochrome P450) to the ferrous state, (3) binding of molecular oxygen to give a

ferrous cytochrome P450-dioxygen complex, (4) transfer of the second electron to this

complex to give a peroxoiron (III) complex, (5) protonation and (6) cleavage of the 0-0

bond with the concurrent incorporation of the distal oxygen atom into a molecule of

water and the formation of a reactive iron-oxo species, (7) and (8) oxygen atom transfer

from this oxo complex to the bound substrate, and (9) dissociation of the product. What is

not clear is what steps are rate-limiting in various reactions.












S Fe3+ Q


Fe3+ ROH Fe3+ RH NADPH-P450 reductasered



Fe3+ OHR NADPH-P450 reductasex

( Fe2+ RH
Fe3+O RH /020
2eb Fe2+_2 RH


H + Fe 3+.OOH/Z14

Fe2+- leRHbr
FFee 2RH
or
b Fe3+ 02 ~ xNADPH-P450 reductase0'

NADPH-P450 reductasered
Figure 1-1. Catalytic cycle of cytochrome P450. RH: substrate; ROH: the corresponding
hydroxylated metabolite. (Adapted from Guengerich, F.P., Cytochrome P450 3A4:
Regulation and role in drug metabolism. Annu. Rev. Pharmacol. Toxicol. 39, 1-17(1999)



Cytochrome P450 monooxygenases function in the transformation of endogenous

and exogenous compounds, and serve as catalysts that are significant in numerous and

diverse biological pathways. The highest concentration of P450 enzymes involved in

xenobiotic biotransformation is found in liver endoplasmic reticulum (microsomes), but

P450 enzymes are present in virtually all tissues. The roles played by cytochrome P450 in

endogenous pathways encompass the synthesis and degradation of steroids,

prostaglandins, fatty acids and other biological molecules. In the transformation of

foreign compounds, cytochrome P450 plays key roles in the toxicology and









pharmacology of pollutant chemicals, drugs and therapeutic agents, and in the activation

and inactivation of many chemical carcinogens. The extent to which these various

pathways or functions occur in different animal groups will depend to a large degree on

the complement of different P450 proteins present, their catalytic function and their

regulation. The P450 enzymes are encoded by a superfamily of genes. Currently, more

than 800 P450s have been characterized, inclusive of the many different species of

organisms that have been studied. Knowledge of these features of P450 in different

species is necessary to define the general characteristics of P450s and their functions, and

to indicate the evolution of these proteins. Such knowledge is also necessary to define the

susceptibility of different individuals, populations or species to xenobiotic compounds,

particularly those compounds whose toxicity may depend upon biotransformation.

Currently, these processes are understood far better in rodent models than in wild or

cultivated species that provide food and material resources. Research on mammalian

cytochrome P450 continues to dominate the literature, but there is a growing recognition

of its biological significance in other animals, and of our need to know the diversity and

biochemistry of cytochrome P450 enzymes in these groups. The 20,000 species of fish

extant represent about one-half of the known vertebrate species. The fish present

extraordinary diversity, inhabiting all of the world's aquatic environments. They also

present a significant source of protein for humans. The cytochrome P450 forms in fish

thus acquire importance from evolutionary and toxicological standpoints.

Fish possess microsomal P450, similar to those in mammals [3]. Knowledge of

the multiplicity, function and regulation of cytochrome P450 forms in fish continues to

grow in importance. The first fish CYP to be cloned and sequenced was a CYP1A from









3-methylcholanthrene induced trout [4]. Recently, evidence has been presented to

document that trout possess more than a single member of the 1A family [5]. A key

feature of cytochrome P450 systems in both fish and mammals is their inducibility by

chemical substrate for the enzymes/and by structurally related compounds. Fish respond

to the same classes of xenobiotics as mammals with respect to induction of CYP1 A, i.e.,

3-MC, BNF, polycyclic aromatic hydrocarbons (PAHs), polyhalogenated biphenyls

(PCBs and PBBs), and polychlorinated dioxins (PCDDs) and dibenzofurans (PCDFs) [6].

Fish CYP1A are induced by the above hydrocarbons given by injection, feeding or

waterborne exposure. The induction can be detected by ethoxyresorufin O-deethylase

(EROD) and aryl hydrocarbon (BaP) hydroxylase (AHH) activities. The molecular

mechanism and cellular machinery for aromatic hydrocarbon (Ah) receptor-mediated

CYP1A induction in fish appears to be similar to that of mammals, which is known to

involve the following: (i) binding of the ligand to the Ah receptor, (ii) translocation of the

bound receptor into the nucleus, and (iii) binding of the receptor complex to specific

DNA sequences upstream of the CYP1A1 promoter (Figure 1-2). Prior to occupancy by

a ligand, the inactive Ah receptor resides in the cytoplasm of target cells in a soluble

complex with the heat shock protein Hsp90 (Figure 1-2). It appears that Hsp90

chaperones the AH receptor, maintains it in a ligand binding conformation, and represses

its intrinsic DNA-binding activity [7]. Binding of a ligand triggers translocation of the

ligand-receptor complex into the nucleus. The nuclear form of Ah receptor binds with

high affinity to specific DNA enhancer sequences known as AH-responsive elements

(AHRE) located in the 5'-flanking region of responsive genes. The nuclear DNA-binding

complex is not a monomer but a heterodimer [8]. Several recent lines of evidence









confirm that the form of AH receptor that binds to AHREs consists of at least two

proteins, the Ah receptor and ARNT (Ah-receptor-nuclear-translocator). The process by

which ligand binding transforms the cytosolic Ah rceptor to its functional DNA-binding

state is complicated and still poorly understood. Phosphorylation of both Ah receptor and

ARNT by protein kinse C (PKC) appears to be important for generation of the functional

DNA-binding complex [9]. Some inducers, for example, 3,3',4,4'-tetrachlorobiphenyl,

can inhibit the catalytic activity of induced P450 [9]. In such cases analysis of catalytic

activity alone might show no response, but strong induction can still be seen by

immunochemical analysis of the CYP I AI protein or hybridization studies with CYP1A1

mRNA.The fact that many of the inducers of fish P450 activities (PAHs, PCDDs, PCBs)

are known aquatic pollutants has greatly stimulated research in the P450 system of fish.

However, a number of studies have documented the "phenobarbital-type" inducers to be

ineffective as P450 inducers in fish [11,12]. Intestines of fish are also capable of a variety

of biotransformation reactions, some of which respond to dietary cytochrome P4501A

(CYPlA) inducers [13]. Dietary induction studies with 0-naphthoflavone, a model PAH-

type inducer, in catfish indicate that under conditions of low inducer concentrations,

select biotransformation activities in the intestine may equal or even exceed

corresponding hepatic activities [ 14]. Such induction effects may potentially alter the

degree and pathway of metabolism.

P450 induction has been suggested to indicate the exposure of organisms to

contaminants in the environment [ 15]. Earlier studies on environmental induction of

cytochrome P450 emphasized the analysis of catalytic activity. More recently, antibodies

to the PAH-inducible cytochrome P450 from fish have been used to demonstrate









unambiguously that CYP1A forms are elevated in fish from contaminated regions [16].

Several studies with different fish species revealed correlations between the levels of

induced cytochrome P450 and levels of PCBs either in the organisms or in their

immediate environment.Thus, it seems that CYP 1 A activity or protein expression can be

used to monitor the environmental pollution. Many chemical carcinogens are

procarcinogens, requiring activation to a carcinogenic derivative by P450-dependent

metabolic processes. Due to the predominant role that CYP1A plays in the metabolic

bioactivation of environmental procarcinogens, it is not surprising that modulation of

CYP 1A levels and/or catalytic activity can significantly impact tumor development in

fish models.


Figure 1-2. Mechanism of induction of CYP1A1 gene transcription. AhR, aromatic
hydrocarbon receptor; AIRE, AH-responsive-element; ARNT, AH-receptor-nuclear
translocator; Hsp90, heat shock protein 90.









Most studies of CYP in fish have focused on the PAH-inducible CYP 1A

subfamily. While several P450 enzymes other than CYP1A have recently been cloned

and sequenced from fish, CYP2MI( previously known as LMC1) and CYP2K1

(previously named LMC2) were islated from rainbow trout liver [17,18]. CYP2M1 shows

specific fatty acid hydroxylation at co-6 position. CYP2K1 has been shown to activate

aflatoxin in trout liver to its carcinogenic metabolites. The expression of CYP2K1 has

been confirmed to have major sex-related differences.

The roles and regulation of CYP3A forms in fish have begun to attract growing

attention. Members of the CYP3A subfamily are major constitutively expressed CYP

forms in the liver and in the gastro-intestinal tract of mammals [19]. CYP3As appear to

have an extraordinarily broad substrate specificity and in addition to steroids, also

metabolize pro-carcinogens, therapeutic drugs and dietary chemicals [20]. Cytochrome

P450 3A4 is known to catalyze the metabolism of both endogenous substrate (such as the

6p-hydroxylation of testosterone) and many important therapeutic agents, including the

N-demethylation of erythromycin. Studies have indicated a significant role for human

hepatic P450 3A4 in the 9,10-epoxidation of benzo(a)pyrene-7,8-dihydrodiol, forming

the final carcinogen BPDE [21] (Figure 1-3). Most studies of structure, function and

regulation of CYP3As have been in mammalian systems, whereas relatively little is

known about CYP3A in other vertebrate groups. As a matter of fact, fish are

continuously exposed to CYP3A inducers/substrates in their natural habitat as a result of

food preferences and human activities. It has been shown that rainbow trout LMC5,

rainbow trout P450con, scupP450A, codP450b and mammalian CYP3A (human 3A4, rat









3A1) are all immunochemically related [22]. Buhler's group reported the




00

BaP 0


=a00








OH HO OH
(-)-BP-7,8-dihydrodiol
(+)-BP-7,8-dihrodiol


(-)-BP-7,8-diol-9, 10-epoxide- 1
+



.001

HOe'
OH
(+)-BP-7,8-diol-9,10-epoxide-2


HO O
OH
(-)-BP-7,8-diol-9, 1O-epoxide-2
+






H OH

(+)-BP-7,8-diol-9, I 0-epoxide- 1


Figure 1-3. Bioactivation of benzo(a)pyrene.

first CYP3A family member, CYP3A27, in an aquatic species (rainbow trout) which

encodes an LMC5-like protein [23]. The major extrahepatic expression site for CYP3A27









was upper small intestine, which also expressed smaller amounts of CYP2K1. Actually,

upper small intestine has the highest expression of CYP3A27 in female trout, followed by

the ovary and the liver. The high percentage of identities in alignment of CYP3A27 with

other mammalian CYP3A forms suggest that there was significant sequence retention

during evolutionary divergence between terrestrial and aquatic vertebrates. The fact that

CYP3A proteins are present at significant levels in untreated fish implies that they are

constitutively expressed and they may have important endogenous functions in fish. The

substrate selectivity and the role in xenobiotic toxicity of CYP3A27 are not yet known.

CYP3A30, another CYP3A subfamily protein found in aquatic species, was isolated and

sequenced from killifish [24]. The sequence of CYP3A30 is 77% identical to that of

CYP3A27.


CYP 3A Inhibition

The inhibition of enzyme activity is one of the major regulatory devices of living

cells, and one of the most important diagnostic procedures of enzymology. Three types of

enzyme kinetic inhibition patterns are commonly observed: competitive, noncompetitive,

and uncompetitive. The use of chemical inhibitors is one of the common strategies

employed in determining whether cytochrome P450s are involved in the hepatic and

extrahepatic metabolism of drugs, xenobiotics, and endogenous compounds. Selective

chemical inhibitors play an important role especially in elucidating the contribution of a

particular cytochrome P450 enzyme in catalyzing the metabolism of xenobiotics [25].

CYP3A enzymes are inhibited by a variety of compounds, including

troleandomycin (TAO), clarithromycin, erythromycin, gestodene, ketoconazole,

naringenin, and 6,7-dihydroxy-bergamottin [26]. The only common features are their









lipophilicity and relatively large molecular size. Several mechanisms of inhibition are

possible, with some compounds exhibiting more than one-type, e.g., erythromycin [27].

(1) Rapid reversible inhibition: Direct, rapid reversible binding of an inhibitor or its

metabolite to CYP3A. Reversible inhibition has been found to result in either

competitive or noncompetitive inhibition, the extent of which is determined by the

relative binding constants of substrate and inhibitor for the enzyme and by the

inhibitor's concentration.

(2) Formation of MI-complexes (quasi-irreversible inhibition): N-Alkyl-substituted

compounds--a common feature of many CYP3A drugs--often show reversible

inhibition, and an even greater effect is observed after preincubation with a

metabolically competent in vitro preparation. This is due to oxidation of the inhibitor

to form a nitrosoalkane species that forms a slowly reversible complex (MI-complex)

with reduced heme in the CYP3A molecule. Such compounds include macrolides like

TAO, oleandomycin, erythromycin, clarithromycin and roxthromycin [28].

Formation of an MI-complex may, however, be difficult to demonstrate in vitro

because of its dependency on the rapid and relatively efficient generation of the

causative metabolite.

(3) Irreversible, mechanism-based (suicide) inhibition: The ingestion of 6,7-dihydroxy-

bergamottin, a furanocoumarin, can markedly inhibit the first-pass metabolism of

CYP3A substrates. This effect was recently found to be associated with autocatalytic

destruction of intestinal CYP3A both in vitro and in vivo [29]. The mechanism of

suicide inhibition presumably involves CYP3A-mediated formation of a reactive









metabolite(s) that covalently binds to the enzyme in a fashion leading to its

inactivation.

In mechanistic terms, reversible interactions arise as a result of competition at the

CYP active site and probably involve only the first step of the CYP catalytic cycle. On

the other hand, chemicals that act during and subsequent to the oxygen transfer step are

generally irreversible or quasi-irreversible inhibitors. Quasi-irreversible and irreversible

inhibitors require at least one cycle of the CYP catalytic process, and are thus

characterized by both NADPH- and time-dependent inhibition. Experimentally,

mechanisms of inhibition of inhibitors could be assessed initially by comparing their

inhibitory effects obtained in the presence and absence of NADPH during a preincubation

period.

Inhibitors for CYP3A have been found that are drugs, antibiotics, preservatives,

poisons and toxins. Several human hepatic CYP3A substrates, erythromycin,

testosterone, terfenadine, midazolam, and nifedipine mutually inhibited the metabolism

of each other with complex mechanisms [30,31 ]. Troleandomycin (TAO) has been shown

to inhibit CYP3A enzymes through both competitive inhibition and formation of MI-

complex. It was found to be as effective inhibitor of CYP3A enzymes in microsomal

fractions from goat and cattle and in a cell-line expressing bovine CYP3A [32]. Both

human CYP1A2 and CYP3A4 play important roles in bioactivation of aflatoxin BI

(AFB 1); TAO showed potent and specific inhibition of AFB I epoxidation in CYP3A but

not CYP1A2 microsomes [33]. In pharmacokinetic tests of drug bioavailability, TAO and

ketoconazole have widely been used as selective inhibitors of CYP3A [34,35]. Calcium

channel blockers, nicardipine, verapamil, and diltiazem were shown to inhibit human









hepatic CYP3A via, at least in part, quasi-irreversible inhibition and such findings

provide a rational basis for the pharmacokinetically significant interactions reported when

these calcium channel blockers were co-administered with agents that are cleared by

CYP3A-mediated pathways [36].

Chemical inhibitors may also be useful in identifying the individual P450

enzymes responsible for the metabolism of xenobiotics and endogenous lipophilic

compounds in non-mammalian species such as fish. Several inhibitors of mammalian

P450s have been employed to inhibit fish P450s [37]. Ellipcine and a-naphthoflavone

were found to inhibit benzo(a)pyrene hydroxylase activity of liver microsomes from

flounder (Platichthysflesus) [38,39]. Aminoanthracene has been proposed as a

mechanism-based inactivator of CYP1A in channel catfish [40], but its selectivity as a

P450 inhibitor is not known. In a study of Miranda et al to evaluate chemical inhibitors of

trout cytochrome P450s three monooxygenase activities, lauric acid (co-l)-hydroxylase

(LA-OH), 7,12-dimethylbenz(a)anthracene hydroxylase (DMBA-OH), and progesterone

63-hydroxylase (PROG-OH) activities were used as functional markers for trout hepatic

CYP2K1, CYPlA1, and CYP3A27, respectively [41]. At 100 pM concentration, the

reversible inhibitors ketoconazole, miconazole and clotrimazole were most potent in

inhibiting LA-OH activity. The global inhibitors metyrapone, chloramphenicol, and

allylisopropylacetamidem had very little inhibitory effect on trout LA-OH and DMBA-

OH activities. Troleandomycin, a CYP3A inhibitor in mammals, did not affect PROG-

OH activity catalyzed by trout CYP3A27. None of the three enzyme activities was

selectively inhibited by any of the mammalian chemical inhibitors used at a concentration

of 100 LM. These results suggest that inhibition data from mammalian studies could not









be directly extrapolated to fish species and that care must be observed when mammalian

P450 inhibitors are used to determine the participation of P450s in the metabolism and

toxicity of xenobiotics in nonmammalian species.


CYP 3A Induction

CYP3A inducers include a broad range of steroids and antibiotics. Early studies

of rat liver CYP3A enzyme induction made the important, but seemingly paradoxical,

observation that both glucocorticoids (such as dexamethasone, DEX) and

antiglucocorticoids (such as pregnenolone 16a-carbonitrile, PCN) induce these enzymes

at the transcriptional level [42]. Both this finding and the requirement for a relatively

high glucocorticoid concentration for CYP3A induction were recognized as inconsistent

with the classical glucocorticoid receptor playing a major role in the CYP3A induction

response. It was recently found that CYP3A genes are transcriptionally activated by

foreign chemicals through a PXR-dependent mechanism [43]. PXR (pregnane X

receptor), an orphan nuclear receptor, was believed to mediate the CYP3A induction.

PXR, together with other four P450-regulating nuclear receptors, CAR, PPAR, LXR and

FXR, share a common heterodimerization partner, retinoid X-receptor (RXR). When the

ligand binds to PXR, the nuclear receptor heterodimerizes with RXR and efficiently

transactivates the response elements present in CYP3A genes (Figure 1-4). Important

species differences in the induction response have been described [44]. Most notably,

while rat, rabbit, and human CYP3A genes are all inducible by dexamethasone, the anti-

glucocorticoid PCN is an efficacious CYP3A inducer in the rat but not in humans or

rabbits. By contrast, the antibiotic rifampicin is an excellent CYP3A inducer in humans

and rabbits, but not in the rat. Transfection studies carried out in rat and rabbit









hepatocytes and utilizing CYP3A constructs containing DEX-responsive regulatory

elements derived from rat CYP3A23, rabbit CYP3A6 and human CYP3A4 genes

demonstrated that the species-specific induction responses are due to the different

response element on CYP3A genes. In the case of rat CYP3A23, the dexamethasone-

responsive sequence contains a DR3 motif (direct repeat, separated by 3 bp; AGTTCA-

N3 -AGTTCA) that is also present in rat CYP3A2, whereas in human CYP3A4 gene, the

response element contains an unusual ER6 motif (everted repeat, separated by 6 bp;

TGAACT-N6-AGGTCT) that is conserved in human CYP3A5 and rabbit CYP3A6 [45].

Pregnane-X-receptors have recently been cloned from human, mouse, rat, rabbit and

chicken. However, mouse PXR and human PXR share only -75% amino acid sequence

identity in their COOH-terminal ligand-binding domain region (vs 96% identity between

their DNA-binding domains), and this apparently results in sigificant differences in

ligand-binding specificities: human PXR but not mouse PXR is highly activated by

compounds that preferentially induced human CYP3A genes, such as rifampicin, while

mouse PXR but not human PXR exhibits the strong response to PCN that characterizes

mouse CYP3A gene induction. Thus, the species-dependent ligand specificity for CYP3A

induction seen in vivo can be explained by the corresponding ligand specificity of each

species' PXR receptor. Other CYP3A inducers and PXR activators include anti-

hormones belonging to several steroid classes, the organochlorine pesticide, chlordane,

and various nonplanar chlorinated biphenyls [46]. Both the facts that PXR is responsive

to steroids belonging to several distinct classes (prenanes, estrogens, and corticoids) and

that many CYP3A enzymes catalyze steroid 63-hydroxylation reactions suggested the









mechanism of cross-talk between PXR-dependent CYP3A induction pathways and

intracellular signaling pathways involving endogenous hormones (Figure 1-5).

Pre-exposure of fish to mammalian CYP3A inducers, however, has yielded rather

inconsistent results. In juvenile rainbow trout, levels of CYP3A in hepatic microsomes

were slightly elevated by steroids, i.e., cortisol and PCN [47]. Administration of 25 or

100 mg/kg i.p. doses of PCN to sexually immature rainbow trout caused an increase of

hepatic BND (benzphetamine N-demethylase) and ECOD (7-ethoxycoumarin 0-

deethylase) activities but had no effect on the total P450 content or on EROD (7-

ethoxyresorufin O-deethylase) activity [48]. By contrast, treatment of rainbow trout with

a single i.p. dose of PCN at 25 mg/kg did not alter the hepatic microsomal activities of

different fluorescent substrates or hepatic P450 levels [0]. Moreover, DEX or PCN

treatment failed to affect hepatic CYP3A-like protein levels in rainbow trout [50,51].

These discrepencies in responsiveness to various types of mammalian CYP3A inducers

reflect important differences in CYP3A regulation in different taxa.

The content of CYP3A in some teleost fish appears to be influenced by the

composition of the diet, suggesting that CYP3A may be involved in the metabolism of

dietary natural products as well as anthropogenic xenobiotics [52].









PXR RXR


Xenochemical





5,


(AGGTCA-Nx)2


Retinoids



CYP 3A Transcripstion


3'


Figure 1-4. Role of PXR in CYP3A gene induction. Shown is the structure of a
PXR-RXR heterodimer bound to two copies of a hexameric DNA response
element based on the sequence of AGGTCA spaced by X nucleotides. The
hexameric repeat can be arranged as a DR or ER motif. (Adapted from
Waxman, D.J. P450 gene induction by structurally diverse xenobioticals:
Central role of nuclear recptors CAR, PXR, AND PPAR. Arch. Biochem.
Bionhvs. 369(l):11-23. 1999)


Foreign Chemical --------


Hormones, growth factors


Receptors and
transcription factors


Induction


CYP3A gene expression


CYPA nzmes


Foreign chemical
metabolism


Metabolism of
endogenous steroids,
fatty acids,
prostanglandins


Figure 1-5. CYP3A gene induction: Cross-talk between foreign chemical and
endogenous regulator pathways. (Adapted from Waxman, D.J. P450 gene induction
by structurally diverse xenobioticals: Central role of nuclear recptors CAR, PXR,
AND PPAR. Arch. Biochem. Biophys. 369(1):11-23, 1999)









CYP3A Stimulation

A unique characteristic of the CYP3A subfamily is their ability to be activated by

certain compounds. Flavonoids, e.g., 7,8-benzoflavone (ix-naphthoflavone, a-NF), have

been shown to stimulate some reactions but not others. In systems containing purified

recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several

substrates, including testosterone, 17p-estradiol, amitriptyline, and most notably aflatoxin

B-1 [53]. It was reported that CYP3A4-catalyzed phenanthrene metabolism was

activated by 7,8-benzoflavone and that 7,8-benzoflavone served as a substrate for

CYP3A4. Kinetic analyses of these two substrates showed that 7,8-benzoflavone

increased the Vnax of phenanthrene metabolism without changing the KM and that

phenanthrene decreased the Vmx of 7,8-benzoflavone metabolism without increasing the

KM. These results suggest that both substrates (or substrate and activator) are

simultaneously present in the active site. Both compounds must have access to the active

oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the

other substrate. These data provide the first evidence that two different molecules can be

simultaneously bound to the same P450 active site [54]. Quinidine and hydroquinidine

decreased KM and Vmax of meloxicam hydroxylation, which was consistent with a mixed

type activation. Meloxicam, in turn, decreased both KM and Vmax of quinidine metabolism

by CYP3A4, indicating an uncompetitive inhibition mechanism [55]. These results also

support the assumption that CYP3A4 possess at least two different substrate-binding

sites.

The mechanism of cytochrome P450 activation has not been explored to the same

depth as induction or inhibition phenomena. Enhancement of aniline para-hydroxylation









by acetone was the first reported cytochrome P450 activation interaction [56]. Based on

studies with liver microsomal fractions of the dog, rabbit, mouse and rat, it was proposed

that acetone affected either the formation of the peroxy anion complex of cytochrome

P450 or steps beyond this (such as the formation of the oxene complex) because cumene

hydroperoxide-dependent hydroxylation of aniline was stimulated by acetone [57].

Huang et al [58] demonstrated that the stimulatory effect of 7,8-benzoflavone on

benzo(a)pyrene metabolism in rabbit liver microsomes was mediated by a different

mechanism than that observed with acetone. The effect of 7,8-benzoflavone on

benzo(a)pyrene metabolism was thought to be a result of enhanced interactions between

cytochrome P450 and cytochrome P450 reductase. A third mechanism of activation was

proposed by Johnson et al. [59], who reported that the stimulatory effect of a-

naphthoflavone on rabbit CYP3A6 was a consequence of an allosteric effect, as shown by

an increase in the P450 binding affinity for the substrate. Shou et al. [54] have shown that

there was mutual activation between phenanthrene and 7,8-benzoflavone and suggested

that the two molecules simultaneously occupy the active site, thereby altering active site

geometry and oxidation efficiency. In summary, it appears that cytochrome P450

activation may occur by several mechanisms.



Epoxide Hydrolase (EH)

Carcinogenic polycyclic hydrocarbons such as benzo(a)pyrene are oxygenated in

cytochrome P450 catalyzed reactions to form epoxides. Due to their electronic

polarization and ring tension, epoxides are often chemically reactive. Consequently, such

metabolites can bind covalently to nucleophilic groups in many tissue constituents,









including macromolecules such as RNA, DNA and proteins. Epoxide hydrolase

(EC3.3.2.3) catalyzes the trans-addition of water across the oxirane ring of the epoxides

to chemically less reactive transdihydrodiols [60]. The reaction is stereoselective and

regioselective. Several distinct microsomal and cytosolic isoenzymes exist. Usually,

microsomal epoxide hydrolase catalyze the hydration of cis-epoxide while the cytosolic

EH catalyzes the hydration of trans-epoxide. Although the metabolite dihydrodiol is less

toxic, it might be further metabolized by P450 to the ultimate dihydrodiol-epoxide

carcinogens. A classic example is the metabolic activation of benzo(a)pyrene to 7,8-

dihydrodiol-9, 1 0-epoxide (BPDE), which proceeds via 7,8-epoxide followed by epoxide

hydrolase and another oxidation step [61 ]. In reactions with benzo(a)pyrene 4,5-oxide

and styrene 7,8-oxide as substrates, the general trend of microsomal epoxide hydrolase

activity observed was fish
hydrolase activity was found in several marine species, including spiny lobster, shrimp,

fiddler crabs and stingray [63]. It was shown that EH activity with styrene oxide as

substrate was similar in intestinal and hepatic microsomes from catfish [14].





Phase II Enzymes


Glutathione S-Transferase (GST)

The glutathione S-transferases (GST) are a ubiquitous family of isozymes whose

primary functions are involved in the biotransformation and disposition of many toxic

substances. The chemical function of the enzyme is to catalyze the nucleophilic addition

of the thiol of glutathione (y-L-Glu-L-CysGly) to electrophilic acceptors, the first step in











mercapturic acid biosynthesis (Figure 1-6). In addition, it is proposed that the proteins


also serve as depots for the storage of toxic substances, as high capacity steroid-binding


glutathione
conjugate


H
, N" COO-


>I


Methionine or other receptor



+H3 SCH3


gamma-glutamyl transferase
(transpeptidase)


I H
NH3+ gamma-glutamyl peptide


x
I





H3 Coo-


CoASAc

cysteine conjugate
CoA \ X N-acetyltransferase
CoASH


I
recycling of glutamate for GSH
biosynthesis


X





mercapturic acid
HON co (N-acetylcysteine urinary excretion
conjugate)

0 CH3

Figure 1-6. Mercapturic acid biosynthesis.


SCH3


H3" N









proteins, as heme-binding and transport proteins. Both the abundance of the enzymes,

comprising 3 to 10% of the soluble protein in liver, and the high concentrations (5 to 10

mM) of glutathione attest to the importance of glutathione S-transferase in the

maintenance of health [64].

All eukaryotic species possess multiple cytosolic and membrane-bound GST

isozymes. The cytosolic enzymes are much more important and encoded by at least six

distantly related gene families (designated class alpha, mu, pi, sigma, theta and zeta

GST). The quaternary structure of cytosolic GSTs shows that the enzymes occur as

binary combinations of subunits, including both homodimers and heterodimers. The

membrane-bound GST (microsomal GST) is a trimeric protein, structurally unrelated to

the cytosolic enzymes [65].

The glutathione S-transferases (GST) are an important phase II enzyme system in

the detoxification of electrophilic alkylating agents. As a family of isozymes, the enzyme

system is capable of handling a variety of electrophilic compounds, both from exogenous

and from endogenous origins. Conjugation with GSH can generally be regarded as a

detoxification pathway, although several compounds are known to be activated through

this reaction [66]. The glutathione S-transferases catalyze the nucleophilic addition of

GSH to electrophiles including aryl and alkyl halides, sulfate esters, phosphate and

phosphorothioate triesters, nitrate esters, oxiranes, olefins, lactones, organic peroxides,

disulfides and thiocyanates and quinones. The substrate selectivities exhibited by various

isozymes overlap considerably but are nonetheless distinct. Most of the above reactions

can be classed as simple nucleophilic displacements or Michael additions to unsaturated

systems.









GSTs can be induced by a variety of chemical compounds, including conventional

inducers of drug-metabolizing enzymes, such as phenobarbital, 3-methylcholanthrene,

and TCDD. GST-pi has been shown to be a reliable marker for rat hepatocarcinogenesis

[67].

GST was found in both marine and freshwater fish species. It has been indicated

that GST usually shows higher activity than epoxide hydrolase in both hepatic and

extrahepatic tissue in marine fish [63]. Rainbow trout has GST activity with CDNB( 1-

chloro-2,4-dinitrobenzene) in liver and intestine, while only intestine has substantial y-

glutamyl transpeptidase activity. A cluster of three GST genes, GSTA, GSTA1, and

GSTA2, was isolated from marine flatfish, plaice [68]. GST-A expresses in plaice

intestine as well as in liver. It was also indicated that expression of GST-A mRNA was

increased in plaice intestine by pretreatment with P-naphthoflavone (BNF).

A pi-class GST was isolated from catfish intestinal mucosa with N-terminal

sequence homology >63% to mammalian pi-form GST isozymes [69]. GST including

this pi-class GST play an important role in the intestinal biotransformation of the epoxide

and diol-epoxide metabolites of benzo(a)pyrene formed in catfish intestine.

Fish GST was also shown to be inducible by PAHs. Cytosolic GST activity

towards CDNB was elevated approximately three to four-fold in intestine and liver of

mummichog, collected from a creosote-contaminated site. The intestinal GST activity

was even higher than liver GST, supporting the importance of intestinal metabolism of

foreign compounds [70]. GST activity was slightly induced in intestinal, but not hepatic

cytosol of catfish treated with BNF at 10 mg/kg diet level relative to chow controls. Yet









this induction showed no further increase with higher dose of BNF at 100 mg/kg diet

[14].



Sulfotransferase (ST)

The cytosolic sulfotransferases catalyze the transfer of the sulfuryl group from 3'-

phosphoadenosine 5'-phosphosulfate to nucleophiles such as alcohols, phenols, and

amines. The M-form of the enzyme is thermolabile (TL form), catalyzing the sulphate

conjugation of micromolar concentrations of dopamine and other phenolic monoamines.

The other form, P-form, is more thermostable (TS form) and catalyzes the sulphate

conjugation of micromolar concentrations of simple phenols such as p-nitrophenol. Both

forms of the enzymes are particularly active in the intestinal wall but are also widespread

in the body, including the platelet.

Sulfation is one of the major phase II conjugation reactions for drugs and

environmental chemicals as well as for endogenous compounds such as steroids and

monoamine neurotransmitters [71]. The major physiologic consequences of the

conjugation of a drug or xenobiotic with a charged sulfate moiety are increased aqueous

solubility and excretion. Although the major role of sulfation is detoxification, in some

instances sulfate conjugation results in the bioactivation of a compound to a reactive

electrophilic species since the sulfate is such a good leaving group. The electrophile is

capable of covalently binding DNA and causing a mutagenic, teratogenic, or

carcinogenic response. Metabolic activation of 7,12-dimethylbenzanthacene has been

demonstrated to occur by oxidation to the 7-hydroxymethyl- 1 2-methylbenz(a)anthracene

followed by sulfation and alkylation of DNA following loss of sulfate anion [72,73].









Human intestinal mucosa contains forms of phenol sulfotransferase, similar to

those in other human tissues such as brain, liver, and platelet [74]. In rat hepatocyte

culture, sulfotransferase expression was negatively regulated by xenobiotics such as PB-

like CYP2B/3A inducers or AhR agonist CYPlA inducers [75]. In guppy and medaka

after water-borne exposure to the procarcinogen 2-acetylaminofluorene(AAF), the major

pathway for bioactivation was shown to be N-hydroxylation followed by sulfation. AAF-

treated guppies had higher ST activity than controls, but UGT activity was reduced or

unaffected by AAF exposure [76].

Sulfate conjugate was found as a metabolite of benzo(a)pyrene in an isolated

perfused in situ catfish intestinal preparation [77]. The biotransformation is via oxidation

by CYP1A and rearrangement of the epoxide to the phenolic metabolite. In another study

with catfish, it was shown that ST activities with BaP phenols was high in intestine,

suggesting that low concentrations of hydroxylated polycyclic aromatic hydrocarbon

would be readily conjugated in catfish intestine.



UODP-Glucuronosyltransferase (UGT)

The UDP-glucuronosyltransferases are a group of membrane-bound proteins

responsible for the transfer of the glucuronyl group from uridine 5'-

diphosphoglucuronate to a large number of different nucleophilic acceptors. The enzymes

are located primarily in the endoplasmic reticulum of eukaryotic cells, catalyzing the

glucuronidation of a tremendous number of lipophilic molecules having nucleophilic

functional groups of oxygen, nitrogen, sulfur, and carbon. Substrates for glucuronidation

are typically small hydrophobic molecules that are termed aglycones (lacking

carbohydrate). A wide variety of endogenous and exogenous compounds are









glucuronidated, including bilirubin, steroid hormones, bile acids, biogenic amines, fat-

soluble vitamins, environmental toxins and therapeutic drugs. Phenol, dihydrodiol and

quinol metabolites of polycyclic aromatic hydrocarbons are substrate for the microsomal

and purified UGTs [78]. Glucuronidation is generally considered to be a detoxifying

mechanism that alters the physiological and pharmacological activities of chemicals

within the body. In some cases, however, covalent addition of glucuronic acid may

increase the biological activity of an aglycone [79]. The UGT proteins can be

conceptually divided into two domains with the amino-terminal half of the protein

demonstrating greater sequence divergence between isoforms. This region apparently

determines aglycone specificity. The carboxyl-terminal half, which is more conserved in

sequence between different isoforms, is believed to contain a binding site for the

cosubstrate UDP glucuronic acid (UDPGA).

Multiple isoforms of UGT have been found in aquatic species [80]. In a UGT

study in plaice, phenol UGT activity was found to be ubiquitous in hepatic, renal,

intestinal and branchial tissues, and was induced by 3-MC and Aroclor. The

glucuronidation of testosterone was restricted to liver and intestine, while conjugation of

bilirubin was expressed solely in hepatic tissue [81]. In the southern flounder, BaP-7,8-

diol given by gavage was glucuronidated and then transported as such to liver where that

was efficiently excreted into the bile. In vitro studies showed that flounder liver and

intestine had similar UGT activities [82]. In channel catfish, glucuronide of BaP-9-OH

was readily transported intact from the intestinal lumen to the systemic circulation. 3-

OH-BaP was extensively biotransformed to BaP-3-glucuronide in intestinal mucosa [83].

UGT activities with BaP phenols were high in the catfish intestine, suggesting that low









concentrations of hydroxylated polycyclic aromatic hydrocarbon would be readily

conjugated in catfish intestine. UGT activities with 3-,7- and 9-hydroxy-BaP in catfish

intestine were not induced by treatment with BNF and in fish receiving the higher dose

activity with 7- and 9-hydroxy-BaP was lower than in fish fed other diets. In vitro studies

showed that BNF could inhibit UGT activity, suggesting the residues of BNF retained in

intestinal cells after BNF treatment in diet could directly inhibit UGT activity [ 14].

Treatment with 3-methylcholanthrene, 10 mg/kg diet, did induce UGT in catfish

intestinal microsomes [James unpublished data].



3a-Hydroxy-Steroid Dehydrogenase (3a-Oxido-Reductase)

Testosterone homeostasis is crucial for normal growth, reproduction, and

development in vertebrates [84]. In teleost fish, testosterone serves as a precursor to 11-

ketotestosterone and 1713-estradiol. These hormones play an important role in sexual

maturation in male and female fish, respectively. More than one organ contributes to the

metabolic inactivation and elimination of testosterone [85,86]. Enzymes that contribute to

the metabolic elimination of testosterone include cytochromes P450, oxido-reductases,

and transferases [87].

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy of steroid

hormone receptors by converting active steroid hormones into their cognate inactive

metabolites. HSDs belong to either the short-chain dehydrogenase/reductases (SRSs) or

the aldo-keto reductases (AKRs). 3a-hydroxysteroid dehydrogenase (3a-HSD) was

found in both microsomal and cytosolic liver fractions. In rodents, 3c-Hydroxysteroid

dehydrogenase showed higher activities in cytosolic fraction than in microsomes using









dihydrosteroids as substrates [88,89]. By comparison, rat hepatic microsomal 3a-

hydroxysteroid dehydrogenase activity was 12-fold higher than cytosolic 3a-

hydroxysteroid dehydrogenase in human [90]. It was suggested that the major pathway of

DHT (dihydro-testosterone) metabolism in human liver involves 3ot-hydroxysteroid

dehydrogenase reduction in the liver, followed by subsequent glucuronidation and

clearance via the kidney [91]. Human hepatic 3c-HSD also plays a critical step in the

synthesis of bile acids and is responsible for the production of 5p-cholestane-3ca,7a-diol,

which is a committed precursor of bile acids. In steroid target tissues, the production of

5/5-tetrahydrosteroids catalysed by 3ca-HSD is not without consequence. In the human

prostate, 3o-HSD can regulate the occupancy of the androgen receptor. It catalyses the

reduction of 5a-dihydrotestosterone, a potent androgen to 5a-androstane-3a,173-diol, a

weak androgen and is positioned to regulate normal and abnormal androgen-dependent

growth of this gland [92]. By contrast in the central nervous system, 3a-HSD can

regulate the occupancy of the )-aminobutyric acid (GABA)A receptor by converting 5a-

dihydroprogesterone into 3a-hydroxy-5a-pregnan-20-one (allopregnanolone), a potent

allosteric effector of the GABAA receptor [93,94]. In the presence of GABA,

allopregnanolone will potentiate GABAA-mediated chloride conductance. As a result 3a-

HSD is responsible for the production of anxiolytic steroids, and decreased activity in this

pathway has been implicated in the symptoms of pre-menstrual syndrome [95]. Thus 3a-

HSD isoforms regulate the occupancy of both a nuclear receptor (androgen receptor) and

a membrane-bound chloride-ion gated channel (GABAA receptor) and may have

profound effects on receptor function [96].









Testosterone metabolites produced by juvenile and adult fathead minnows

included 4-androstene-3,17-dione (androstenedione), 171-hydroxy-5a-androstan-3-one

(5a-dihydrotestosterone), 5ca-androstane-3x,I713-diol (3ct-androstanediol), 5a-

androstane-33,17p-diol (3 3-androstanediol), 17f3-hydroxy-4-androstene-3,11-dione (11-

ketotestosterone), 16p-hydroxy-4-androsten-3 -one (16p-hydroxytestosterone), and 6p-

hydroxy-4-androsten-3-one (6p-hydroxytestosterone) [97]. Testosterone and its

metabolites were eliminated from minnows in both free and conjugated form. Adult

females eliminated androstanediols at a significantly greater rate than did males,

suggesting higher 3-oxidoreductase activities in female fish than male.



Prehepatic Metabolism and Bioavailability



The primary function of the intestine is to absorb nutrients and water. This is

achieved by mixing food with digestive enzymes to increase the contact of foodstuffs

with the absorptive cells of the mucosa. In addition to this fundamental role, another

function of the intestine arises from the fact that it also provides a major route for

exposure to xenobiotics via food and liquid, and secondarily by swallowing inhaled

xenobiotics after clearance from the tracheobronchial tree. In human and different animal

species, the percentage weight of intestine is usually significantly smaller than the liver.

When a xenobiotic exhibiting systemic effects is administered orally, its fate is

usually as follows: it comes into contact with the contents of the gastrointestinal system,

is dissolved in intestinal juices, and then brought into contact with intestinal epithelium.

It is then absorbed through the gut wall and the enterocytes lining the gut wall, and









transported by the portal veins through the liver, before reaching the systemic circulation

and hence different parts of the body. When the same drug is given intravenously, it

enters the systemic circulation and is distributed through out the body before reaching the

liver for the first time. The extent of systemic availability is described with the

pharmacokinetic term bioavailability (F). F is theoretically determined in the following

way: the drug is administered to the same individual as a single dose intravenously and

orally on separate occasions; drug concentrations in serum (or plasma, blood) are

measured after each dose and used to determine the area under concentration curve

(AUC) from the time 0 to "infinity". Absolute bioavailability of the oral dosage form is

Foaj=AUCora/AUCi.,. If the oral and i.v. doses are unequal, a correction for the dose

difference must be made. The most significant factor influencing the effect or toxicity is

not necessarily the dose but rather the concentration of a xenobiotic at the site of action.

The fraction of a chemical that reaches the systemic circulation is of critical importance

in determining effect or toxicity. The incomplete bioavailability after oral administration

may principally be a result of an incomplete absorption from the intestine or metabolism

of the drug before it reaches the systemic circulation (presystemic metabolism).

Presystemic metabolism can principally take place anywhere before the drug reaches the

systemic circulation, i.e., in intestine and in liver. The metabolism of xenobiotics before

entering the systemic circulation is referred to as first-pass metabolism. This intestinal

and hepatic first-pass biotransformation alters the physico-chemical properties of

xenobiotics and is likely to change the bioavailability. The first-pass metabolism could

substantially prevent many xenobiotics from being distributed throughout the body.

However, the biotransformation could also potentially activate some xenobiotics. It has









been widely believed that the liver is the major site of such first-pass metabolism because

of its size and its high content of drug-metabolizing enzymes. If large amounts of a

chemical are ingested (e.g., therapeutic drugs), it is usually true as the capacity of the

intestinal biotransformation is likely to be overwhelmed. The compounds will be

absorbed and pass to the liver, which has higher capacity for biotransformation than the

intestine. However, recent clinical studies have indicated that the intestine contributes

substantially to the overall first-pass metabolism of cyclosporin, nifedipine, midazolam,

verapamil, and certain other drugs [98]. Some studies suggested that the role of intestinal

metabolism of these drugs is quantitatively greater than that of hepatic metabolism in

overall first-pass effect [99]. The contribution of intestinal enzymes to xenobiotic

biotransformation is particularly important when relatively low concentrations of

chemicals are present, as is normally the case for high potency drugs and environmental

chemical pollutants, since the low concentrations of xenobiotics are readily metabolized

in the intestine and leaving little to pass to the liver for further metabolism.

Almost all of the xenobiotic-metabolizing enzymes present in the liver also are

found in the intestine, although their total amounts are generally much lower in the latter

due to the lower weight of intestine relative to liver. Unlike the liver in which the

distribution of P450 enzymes is relatively homogeneous, the distribution of these

enzymes is not uniform either along the length of the small intestine or along the villi

within a cross-section of mucosa. Longitudinal distribution of total cytochrome P450 and

its activity have been measured in human intestine [100]. Both the content and activity of

cytochrome P450 was higher in the proximal than that in the distal small intestine. The

major enzymes catalyzing drug-metabolizing reactions in the liver and the GI tract belong









to the microsomal CYP3A subfamily. CYP3A4 is predominantly expressed in human

liver and intestine, where it comprises approximately 30 to 50% of the total cytochrome

P450 population in these tissues [101]. Many of the drugs with significant first-pass

metabolism, like cyclosporine, midazolam, nifedipine, and terfenadine, are substrates of

CYP3A. CYP3A activity is prone to induction or inhibition, which may cause clinically

significant drug interactions. Whenever two or more drugs are administered concurrently,

the possibility of drug interactions exists. The ability of a single CYP to metabolize

multiple substrates is responsible for a large number of documented drug interactions

associated with CYP inhibition. In addition, drug interactions can also occur as a result of

the induction of several human CYPs following long-term drug treatment. CYP3A is

highly inducible in humans by synthetic glucocorticoids (dexamethasone), macrolide

antibiotics (rifampicin), and phenobarbital [102]. It has been demonstrated that an

important cause of incomplete bioavailability of many drugs, which were earlier thought

to be primarily poorly absorbed, is prehepatic metabolism in the GI tract, mainly by

CYP3A subfamily of enzymes. Grapefruit juice, a beverage consumed by the general

population, is an inhibitor of the intestinal cytochrome P450 3A4 system. A 47%

reduction in intestinal CYP3A4 concentration occurs within 4 hours of the ingestion of

grapefruit juice, and grapefruit juice maintains a bioavailability-enhancing effect for up to

24 hours. Grapefruit juice acts on the CYP system at the intestinal level, not at the hepatic

level [103]. Drugs are not the only subgroup of xenobiotics that function as substrates for

activation or deactivation by biotransformation processes, including CYP3A-catalyzed

reaction. These enzymes also play a vital role in the biotransformation of such exogenous

compounds as pesticides, carcinogens and other environmental pollutants. Intestinal









CYP3A4 enzymes were shown to activate dietary aflatoxin B 1 to reactive metabolites

that form macromolecular adducts within enterocytes [104]. It is logical for toxicologists

to look for evidence of biotransformation capabilities in the first line of defense against

ingested toxins or carcinogens, the intestinal mucosa.

In summary, in vitro and in vivo data have clearly demonstrated that the small

intestine plays a significant role in first-pass metabolism in certain situations, especially

when a small oral dose is given. The induction or inhibition of intestinal biotransforming-

enzymes might potentially alter the bioavialability and metabolism pathway of the

chemical exposed.

Both phase I and phase II biotransforming-enzymes have been found in fish liver

and intestine. As in mammals, the major organ involved in xenobiotic metabolism in fish

seems to be the liver. Yet, microsomal cytochrome P450 and cytochrome P450-

dependent activities were found in extrahepatic organs in fish, e.g., kidney, upper small

intestine, gonad and brain [105].









Hypothesis


The hypotheses of the present project are: (1) CYP3A is constitutively expressed

in catfish intestine; (2) CYP3A enzyme is expressed regionally along intestine of catfish;

(3) testosterone is hydroxylated at different positions by different P450 isozymes in

catfish intestinal microsomes; (4) the in vitro testosterone 603-hydroxylation activity by

catfish intestinal CYP3A enzymes is inhibited by mammalian CYP3A inhibitors, e.g.,

erythromycin, troleandomycin and ketonconazole and by general P450 inhibitors, e.g.,

metyrapone and SKF 525A, but not inhibited by specific CYP1A inhibitor, e.g., t-

naphthoflavone; (5) CYP3A expression in catfish intestine is under dietary modulation

and is inducible by mammalian CYP3A inducers, e.g., rifampicin and pregnenolone 16a-

carbonitrile; (6) the intestinal CYP3A enzyme plays an important role in the

biotransformation and bioavailability of both endogenous and exogenous compounds,

including environmental pollutants, which the wild catfish are continuously exposed to.














CHAPTER 2
MATERIALS AND METHODS


Chemicals

[4-14C]-testosterone and [4-14C]-progesterone were purchased from DuPont

NENTM (Boston, MA). Authentic steroid and metabolite standards were obtained form

Steraloids, Inc (Wilton, NH). Benzo(a)pyrene and 3-hydroxy benzo(a)pyrene were

purchased from ChemSyn, through the NCI Chemical Carcinogen Repository. Western

blotting kit was from Amersham Life Sciences, Inc. (Arlinton heights, IL). Ketoconazole

and proadifen (SKF-525A) hydrochloride were gifts from Janssen Pharmaceutica, Inc.

(Piscataway, NJ) and Smith Kline & French Labs (Philadelphia, PA), respectively. 2-

methyl-1, 2-di-3-pyridyl-l-propanone (metyrapone) was purchased from Aldrich

Chemical Co. (Milwaukee, WI). Erythromycin, troleandomycin, tricane and NADPH

were obtained from Sigma Chemical Co. (St. Louis, MO). All HPLC and microsomal

preparation supplies were of the highest grade available from standard commercial

sources.



Instruments

The following instruments were used for this study. The gradient HPLC system

was equipped with a Beckman controller 125 solvent module, an analytical Beckman

UV detector model 166 and an INUS f-RAM detector. Shimadzu UV-VIS

spectrophometer model UV-160U, Perkin-Elmer fluorescence spectrometer model LS-3B









and Chromato-vue UV detector were used. Liquid scintillation counters used were

Packard Tri-Carb liquid scintillation system model 460CD and Beckman liquid

scintillation system model LD 500TD. Beckman ultracentrifuge model L8-80M and

DuPont Sorvall centrifuge model RC2-B were used.



Animals and Pretreatment

Groups of 4-8 catfish each (800-1300 g) were fed a commercially available Silver

Cup chow (Silvercup, Nelson & Sons, Inc., Murray, UT) or a semi-synthetic purified diet

(Dyets Inc., Bethlehem, PA) for at least 2 weeks. The semi-synthetic purified feed was

formulated according to guidelines established for warm-water fish by the National

Research Council [106], composed of casein 32%, dextrin 29.8%, cellulose 19%,

soybean oil 3%, Menhaden oil 3%, gelatin 8%, salt and vitamin mix 5%, and choline

chloride 0.17%.

For the respective pretreatments of fish, both control and treatment groups were

acclimated to experimental conditions and maintained on purified diet at least 2 weeks

prior to pretreatment. Control animals were maintained on semi-synthetic diet coated

with corn oil (1 ml corn oil/100 g of diet) while for the treatment group the chemical

(TCB or 3-MC) was delivered in corn oil applied as a coating on the semi-synthetic diet

(1 ml corn oil/100 g of diet). Both dietary groups (control and chemical exposure) were

maintained on designated experimental diets at 0.5% of fish body weight/day for

specified period prior to sacrifice.

For the study of inducibility of mammalian CYP3A inducers, 0.03% (w/w)

rifampicin was formulated in the semisynthetic purified diet, the treatment group were









fed the rifampicin-formulated diet at 3% of fish body weight/day for specified period of

time. For the treatment of pregnenolone-16oa-carbonitrile (PCN), the fish were acclimated

and maintained on purified diet for at least 2 weeks prior to the surgical process for oral

gavage. PCN (10mg) was dissolved in a mixture of 1 ml of IM KCl and 0.5 ml of corn

oil, and applied on 5 g of purified diet powder. Distilled water (12 ml) was added to the

purified diet to form a slurry. The treated fish were fed this slurry at 0.5% of fish body

weight/day by oral gavage for specified duration of exposure.



Surgical Procedures for Oral Gavage

Fish was anaesthetized with tricane (3-aminobenzoic acid ether ether, 6.4 g) in 12

gallon of water. The fish was then taken out of the water and a hole was drilled (5"/32)

in the middle of nostrils. Tubings (I.D. 0.106", O.D. 0.138") were placed through the

hole to the stomach, the other end tied to the dorsal fin. Fish was kept wet by pumping

water on the gills through the surgical process (3.2 g tricane and 3.2 g pottasium

bicarbonate was dissovled in 16 gallon of water).



Enzymes Preparation

Catfish were sacrificed and dissected. Intestines were removed from the stomach

and rinsed thoroughly with ice-cold buffer A containing 0.25 M sucrose, 5 mM EDTA,

0.05 M Tris-Cl (pH 7.4), 0.2 mM PMSF, and 1mM dithiothreitol to remove contents. The

intestine was bisected evenly into proximal and distal sections, opened, and mucosal cells

removed by scraping into 10 ml of buffer A. Mucosal cells were weighed and

homogenized in 4 volumes of buffer A. Washed microsomes were prepared from

homogenates of each section using the procedure described by James and Little [ 107].









The homogenates were poured into suitably sized Sorvall polycarbonate centrifuge tubes

and centrifuged at 13,300 g for 20 min at 4 C to sediment the nuclei, cell debris and

mitochondria. The supernatant containing microsomes and cytosol was transferred into

polycarbonate ultracentrifuge tubes and then centrifuged at 170,000 g for 45 min at 4 'C.

The supernatant was the cytosol. The microsomal pellets were resuspended in buffer A,

resedimented for preparation of washed microsomes and the microsomal pellets were

suspended in 0.25 M sucrose, 0.01 mM HEPES pH 7.4, 0.1 mM EDTA, 0.1 mM

dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 5%(v/v) glycerol in a volume

equal to half of the weight of intestinal mucosal cells. Livers were removed, rinsed twice

with fresh ice-cold buffer B (1.15% KC1, 0.05 M potassium phosphate pH 7.4, 0.2 mM

PMSF), patted dry on a paper towel, weighed, minced with scissors and homogenized in

4 volumes of buffer B. The hepatic microsomes and cytosol were prepared in the same

way as intestinal cytosol and microsmes. Aliquots of the microsomes were flushed with

nitrogen and stored at -80C until used in assays. Human CYP3A4 in the baculovirus

system (with human NADPH-P450 reductase) was obtained from Gentest Co (Woburn,

MA). Rabbit anti-trout polyclonal CYP3A27 antibody was a gift from Malin Celander in

G6teberg University and was prepared as described [ 108,109].



Protein Assay

Lowry protein assay or Bio-Rad protein assay kit both with BSA as standard were

used to determine the protein concentration of microsomes and cytosol.









Measurement of Cytochrome P450

Cytochrome P450 content was measured by the method of Omura and Sato,

modified by Estabrook [ 110,111 ]. A suspension of fish intestinal or hepatic microsomes

was prepared containing about 1 mg/mi protein in 0.1 M HEPES, pH 7.4 with 0.1%

Emulgen 911 (Kao Atlas, Tokyo, Japan). The purpose of adding Emulgen was to

solubilize the membrane-bound enzymes and prevent the suspension from settling. About

5 mg sodium dithionite was added to the microsomal suspension. The suspension was

divided into two cuvettes, and the spectra recorded between 500 and 380 m. CO was

bubbled through the sample suspension, and the spectrum was recorded from 500 and

380 nm. The change in absorbance from 490 to 450 nm was noted. P450 content was

calculated according to the following equation:

(Abs 450-Abs 490)/0.091=nmole P450/ml. (91 mM-1cm-l is the absorptivity of CYP

under these conditions [ 110])



Steroid Hydroxylation Assay

The steroid hydroxylation assay was described and modified by James and

Shiverick [112]. Assay tubes contained 100 mM HEPES pH 7.4, 2 mM MgC12, 1.0 mM

NADPH, 0.1 mM [14C] progesterone or [14C] testosterone (800,000 dpm added in 0.01 ml

ethanol), and 400 jtg catfish intestine microsomal protein, all in a final volume of 1 ml.

For incubations with human CYP3A4, 40 pmole CYP3A4 was added. Tubes were

incubated at 35C for 10 minutes, with the exception of human CYP3A4, which was

incubated at 37C for 10 minutes. The reaction was stopped by the addition of ice-cold

ethyl acetate (5 ml). The extraction was repeated and the two organic phases combined.









Anhydrous sodium sulfate was added to dry, then the extract was evaporated to dryness

under nitrogen. Separation of the metabolites was achieved by TLC on LK5DF silica gel

150A precoated plates (Whatman Inc., New Jersey). The plates were developed once

(progesterone assay) or three times (testosterone assay) in a solvent containing ethyl

ether:toluene:methanol:acetone (70:38:0.8:1) at room temperature. Authentic standards

were chromatographed on the same plate and visualized by viewing the plates under UV

light. In the case of 5ct-dihydro-steroids, 4-androsten-3a, 170-diol and 4-androsten-33,

170-diol, the developed TLC plates were evaporated with iodine followed by spraying of

70 % methanol. Metabolite bands were located and quantified by electronic

autoradiography with InstantlmagerTM (Packard Instrument Co., CT). The 14C in each

metabolite peak was corrected for blank values which usually were negligible. Various

concentrations of testosterone were used (5, 10, 20, 50, 100 and 200 pM) to give KM and

Vmax values. In the case of testosterone metabolite used for MS analysis, 120 mM non-

radiolabeled testosterone and 1.1 mg catfish intestinal microsomal protein (total assay

volume 11 ml) were used in the incubation. The incubation lasted for 60 min at 35C.

The 3cz-reduced metabolite was isolated and recovered by TLC using the above solvent

system.



Chemical Modulation of Testosterone Metabolism

At least five different concentrations of chemical inhibitors were used (1/5 to 4x

IC50) to calculate the IC50 values. Ethanol (for erythromycin and ketoconazole), acetone

(for ct-naphthoflavone) or DMSO (for troleandomycin) were used as the vehicle controls.

The total organic solvent was kept under 2% of the total volume. Proadifen (SKF-525A)









hydrochloride and metyrapone were dissolved in distilled water. For chemicals that have

been reported as CYP3A inhibitors through a MI-complex, i.e., TAO, ERM and SKF-

525A, the inhibitors were preincubated with catfish intestinal microsomes for 30 min at

35C in the presence of 1.0 mM NADPH. The assay was started with the addition of

testosterone and was further incubated for another 30 min under the same conditions.

Testosterone concentration was 30 jiM.



AHH (Aromatic Hydrocarbon Hydroxylation) Assay

The method of Nebert and Gonzalez [113] as previously optimized for catfish

intestinal microsomes [ 14] was used. Tubes contained 0.2 M HEPES-NaOH buffer (pH

7.6), BaP 10 ptM, 0.5 mg intestinal microsomes and 2 mM NADPH (added last) in a

volume of 1 ml. To investigate the effect of ANF, varying concentrations of ANF (2-100

jtM) were added to assay mixture from acetone solution. The volume of acetone was 1%

of the total. After incubation at 35C for 15 min, the assay was stopped by adding I ml

ice-cold acetone. Tubes were extracted with 3 x 3 ml heptane, the pooled heptane extracts

were back-extracted into 3 ml of 1 N NaOH, and the fluorescence of the NaOH extracts

measured at an excitation of 392 nm, emission 513 nm.



Western Blot Analyses

Western Blot Analyses of CYPlA

Microsomal protein fractions (40 jtg for intestine, 20 jig for liver), incubated in

sample buffer as recommended by BioRad, were resolved in a mini gel format (BioRad)

on 4% stacking gel with 8.5% resolving gel. Unstained and prestained molecular weight









standards in the range of 14,400 to 97,000 (BioRad low molecular weight range) were

resolved at the same time as the SDS-treated microsomes. Gentest SupersomesTM

expressing rat CYPIA were used to develop a standard curve for quantification of the

antibody response. Electrophoresis was carried out using a 25 mM Tris/192 mM

glycine/0. 10% SDS buffer at constant voltage of 200 V. Protein was then transferred to

nitrocellulose membrane at 40 V in a mini Transblot system (BioRad) using a 25 mM

Tris/192 mM glycine/20% v/v methanol/pH 8.3 transfer buffer. The remaining gel was

stained with Coomassie blue as an indication of transfer effectiveness.

Immunodetection was carried out using monoclonal antibodies to scup CYP IA

(courtesy of Dr. J.J. Stegeman). Transblotted nitrocellulose was rinsed in a 20 mM Tris,

500 mM NaC1, pH 7.5 buffer and nonspecific binding sites was blocked with 5% (w/v)

dried milk in 20 mM Tris-HC1, pH 7.5, 500 mM NaC1, 0.05% Tween 20 for 1 hour. The

membrane was washed 4 times with 20 mM Tris, 500 mM NaC1, 0.05% Tween 20, pH

7.5 buffer. The primary antibody, diluted 1:10,000 in 5% (w/v) dried milk in 20 mM

Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20, was incubated with the nitrocellulose

for 2 hours. The unbound antibodies were washed away and further incubated with a

1:1000 dilution of secondary antibody (rabbit anti-rat antibody conjugated to horseradish

peroxidase) in blocking agent for 1 hour. After washing 4 times, the immunoreactive

proteins were detected according to the Amersham Western Blotting kit for

chemiluminescent detection and the protein bands were visualized by fluorography on

Kodak X-OMAT AR films. Fluorograms were subsequently scanned and the protein

bands were quantified by scan-analysis densitometry.









Immunochemical Analyses of CYP3A

Western blot analyses of catfish intestinal or hepatic microsomes (40 jIg intestinal

and 20 jtg hepatic microsomal protein per lane) and standard CYP proteins were

performed in discontinuous (4-8.5%) SDS acrylamide gel. The proteins were

electrotransferred to 0.45 gm nitrocellulose sheet and blocked with 5%(w/v) dried milk in

T-TBS buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20). After

blocking, the membrane was incubated with polyclonal rabbit-anti-trout-CYP3A27

(1:1000 dilution with blocking agent) for 2 hours. The unbound antibodies were washed

away in T-TBS buffer and further incubated with 1:1000 dilution of secondary antibody

(donkey-anti-rabbit antibody conjugated to horseradish peroxidase) in blocking agent for

1 hour. After washing with T-TBS, the immunoreactive proteins were detected according

to the Amersham Western Blotting kit for chemiluminescent detection and the protein

bands were visualized by fluorography on Kodak X-OMAT AR films. Fluorograms were

subsequently scanned and the protein bands were quantified by scan-analysis

densitometry. Gentest SupersomesTM human CYP3A4 was the P450 standard (0.5, 1, 2, 5,

8 pmol each lane for standard curve in quantification).



HPLC Analysis of Testosterone Metabolism

The dried steroid assay extracts were redissolved in methanol and filtered through

a Durapore microporous membrane (0.45 tm pore size, Millipore). Steroid metabolites

were separated on a Beckman programmable HPLC using a 5 i Ultrasphere-ODS

reverse-phase (CI8) analytical column (4.6 x 250 mm) fitted with a precolunm guard

column (Beckman Ultrasphere ODS 4.6 x 45 mm). Elution was conducted with a mixture









of H20: methanol: acetonitrile 50:25:25 (v/v/v) at a flow rate of 1 ml/min. Analysis was

achieved using UV (225 nm) and radiochemical detection (INUS detector). Under these

conditions, 6p-hydroxy-testosterone eluted at 6.2 min, 6-dehydro-testosterone at 17.6

min, testosterone at 21.3 min, androstenedione at 23.5 min, and 4-androsten-3ca, 17P-diol

eluted at 33.5 min.


Mass Spectrometric Analysis

Samples were dissolved in isopropanol and analyzed via ESI (electrospray

ionization)-MS and APCI (atmospheric pressure chemical ionization)-MS. Samples were

injected into the HPLC system (Applied Biosystems, model 400) followed by elution

with a mobile phase consisting of 1% acetic acid in 30:35:35 H20:MeOH:isopropanol.

Finnigan MAT (San Jose, CA) LCQ was used in electrospray ionization mode. The

temperatures of vaporizer and capillary for APCI/MS were 300 C and 230 'C,

respectively.



Sulfotransferase Activity Assay

3-OH Benzo(a)pyrene in methanol solution was added to assay tubes so that the

final concentration would be 1 p.tM. The methanol was evaporated with nitrogen and 0.05

M Tris-Cl pH 7.0, 0.4% BSA, 50 ptg cytosolic protein and water were added up to 0.45

ml. After 2 min pre-incubation at 35C, the reaction was started by the addition of 20 pM

3'-phosphoadenosine-5'-phosphosulfate (PAPS) in 50 I water and stopped after 10 min

with 2 ml methanol. Methanol (2 ml) was added to blanks before PAPS. Tubes were

centrifuged and 2 ml supernatant was mixed with 0.5 ml 1 N NaOH. The fluorescence of









sulfate conjugates (BaP-3-sulfate) was measured at ex294/em415 nm. The sulfate product

was calculated against the standard curve of BaP-3-sulfate conjugate [14].



UDP-Glucuronosyltransferase Activity Assay

3-OH Benzo(a)pyrene in methanol solution was added to tubes so that the final

concentration would be 1 jiM, and the methanol was evaporated under nitrogen. To this

was added 0.1 M Tris-Cl pH 7.6, 5 mM MgC12, and 50 jig microsomal protein solubilized

with 0.5 mg Lubrol/mg microsomes in a final volumn of 0.4 ml. After preincubation for 2

min at 35C, the reaction was started by adding 200 jtM UDP-glucuronic acid (UDPGA)

in 0.1 ml water and terminated after 30 min by addition of 2 ml methanol. Tubes were

centrifuged to precipitate protein, and 2 ml of the supernatant was added to 0.5 ml 1 N

NaOH. After mixing, the fluorescence was measured at ex300/em421 nm. The

glucuronide product was calculated against the standard curve of BaP-3-glucuronide

conjugate.



Statistical Analysis

Data are presented as the mean SD unless specified else. Results were analyzed

by a one-way analysis of variance (ANOVA) and differences between pairs of means

were tested by the student t-test. Differences with ap value of <0.05 were considered to

be statistically significant unless specified else. Correlation analysis was performed using

Microsoft Excel software (Microsoft, Redmond, WA).














CHAPTER 3
RESULTS

Response to Aryl Hydrocarbon Receptor Agonists

CYP1A Expression in Channel Catfish Intestine

Total P450 content, CYP1A content and aryl hydrocarbon hydroxylase (AHH)

activity were measured in both the control fish, TCB-treated and 3MC-treated fish, to

assess the effect of preexposure to aryl hydrocarbon receptor agonists upon the metabolic

capacities of the catfish intestine. Mean P450 concentrations (between 0.02 and 0.20

nmol/mg protein) were not significantly altered with TCB or 3MC treatments (Figure 3-

1) (ANOVA: p>0.05). CYP IA cross reactivity was not detected for either the controls or

animals in the 0.5 mg TCB/kg diet treatments. CYP1A levels were variable for the 5.0

mg TCB/kg diet treatment, with values ranging from 0.14 to 24.11 pmol/mg protein

(Table 3-1). Liver microsomes from catfish induced by dietary TCB (5 mg/kg diet) was

used as a positive control. Composite AHH activities were 2.46 + 1.16, 2.43 1.58 and

11.35 10.25 pmol/min/mg protein for the control, 0.5 and 5 mg TCB/kg diets,

respectively. AHH activities of the 5.0 mg/kg treatment were not significantly greater

than controls or the 0.5 mg/kg diet treatment due to the high standard deviation of the

data (ANOVA: p>0.05). Four animals demonstrated large increases (-7 fold) in AHH

activities, while 3 animals exhibited levels similar to the controls. AHH activity exhibited

a strong correlation (r2=0.96) with CYP1A cross reactivity in fish exposed to TCB at 5

mg/kg diets (y=l.143x+1.026). CYP1A was present (20.8 12 pmol/mg microsomal









protein) and AHH activity was induced (26.9 4.1 pmol/min/mg) in all 3MC-exposed

fish (n= 10). In summary, CYP 1A was not constitutively expressed in catfish intestine or

liver. Yet, the immunoblots of intestine microsomes from catfish treated by dietary 3-MC

or TCB showed a clear band crossing-reacting with the anti scup CYP1A antibody

(Figure 3-2 and Figure 3-3). Table 3-1 shows the CYP1A amount in intestinal

microsomes from control fish and fish pretreated by 3-MC (10 mg/kg diet) or TCB (5

mg/kg diet) for 10 days. CYP1A induction did not response at the pretreatment level of

0.5 mg TCB/kg diet (not shown). The results indicated that catfish intestine CYP1A is

inducible by AhR agonists and the AHH activity is highly correlated to CYP1A amount

(Figure 3-4), suggesting intestinal CYP 1A can be used both as a biochemical and an

exposure biomarker.



0.2
E


EA
C A
0.1 A
A



0.0
CONTROL TCB 0.5 mg/kg TCB 5 mg/kg 3MC 10 mg/kg

Figure 3-1. Intestinal P450 content in control and treated fish. The scatter graph
shows the P450 content in intestinal microsomes from control catfish (n=1 1) or
animals exposed for 10 days to 0.5 mg TCB/kg (n=8), 5 mg TCB/kg (n=6) or
10mg 3-MC/kg diet (n=6). The horizontal bars indicate the mean values for each
group.
















(1) (2) (3) (4) (5)

Figure 3-2. Intestinal and hepatic CYPlA in control and fish treated with 3MC or TCB.
40 jtg intestinal (Lane 1-4) and 20 jig hepatic (Lane5)
microsomal protein were in each lane:
(1)-(3) Three individual fish pretreated with 10 mg 3-MC/kg diet;
(4) Control fish;
(5) Fish pretreated with 5 mg TCB/kg diet.





-0 """- ~ *WmW


Figure 3-3. Intestinal and hepatic CYP1A in control and fish treated with TCB.
40 jig intestinal (Lane 1-4) and 20 jig hepatic (Lane 5) microsomal
protein were in each lane:
(1)-(4) Four individual fish pretreated with 5 mg TCB/kg diet;
(5) Fish pretreated with 5 mg TCB/kg diet.


Table 3-1. Intestinal CYP 1 A level in control and treated fish.


Pretreatment Intestinal CYP1A
pmol/mg microsomal protein

Control (n=10) < D.L.

3-MC (10 mg/kg diet) (n=10) 20.86 12.85

TCB (5 mg/kg diet) (n=4) 14.49 7.73


D.L.: detection limit.





















45-

40-

35-

30-

25-

20-

15-

10-

5
DOo
0 I I I I I I I I I
0 5 10 15 20 25 30 35 40 45 50

CYPIA pmolmg microsomal protein

Figure 3-4. Intestinal CYPIA content and AHH activity. This graph shows that CYPIA
content, pmol/mg microsomal protein correlated in a linear fashion (r=0.947, p<0.001)
with AHH activity in intestinal microsomes of catfish from control, exposed to 0.5 and 5
mg TCB/kg and 10 mg 3MC/kg diets for 10 days. The CYP1A content was determined
by cross-reactivity with a scup CYP1A monoclonal antibody and quantified relative to a
rat CYP1A standard curve.











UDP-Glucuronosyltransferase Expression in Catfish Intestine

UGT activity was determined in control and 3MC treated fish. Intestinal

microsomal UGT activity was significantly higher than that in control fish (Figure 3-5).

In addition, the UGT activity correlates with the AHH activity, showing a r2 of 0.75

(Figure 3-6).











UGT activity in control and 3MC-treated fish


250



200



E-150


100.
E


50 f --


0 !I------


Control


3MC (10mgtkg diet)


*: p<0.001.

Figure 3-5. UGT activity in control (n=12) and 3MC treated (n=l 1) fish.


------------- I -----





















300


U M


0 10 20 30 40 50 60
AHH pmol/min/mg

Figure 3-6. Intestinal microsomal AHH and UGT activities. This graph shows that AHH,
pmol/min/mg microsomal protein correlated in a linear fashion (r=0.866, p<0.001) with
UGT activity in intestinal microsomes of catfish from control, exposed to 0.5 and 5 mg
TCB/kg for 10 days.









Function and Expression of CYP3A and Testosterone Metabolism

TLC Analyses of Testosterone Metabolism by Catfish Intestinal Microsomes

Our results indicate that catfish intestinal microsomes hydroxylate testosterone

and progesterone in a regioselective and stereospecific manner. Shown in Figure 3-7 is a

representative chromatogram of the [14C]-testosterone metabolism profile exhibited by

catfish intestinal microsomes. 4-Androsten-3ca, 17p-diol, 613-hydroxytestosterone and

androstenedione were identified as the three major metabolites of testosterone by their

cochromatography with anthentic standards. There were also trace amounts of 1 lIa-

hydroxytestosterone shown (Figure 3-8). Recombinant CYP3A4 in a baculovirus system

coexpressing NADPH-P450 reductase (SupersomesTM) only hydroxylated testosterone at

6P3-position. Similarly, progesterone was hydroxylated or reduced by catfish intestinal

microsomes to give 3a-hydroxypregn-4-en-20-one, 6p-hydroxy-, 17a-hydroxy-, and

16a-hydroxytestosterone as metabolites (Figure 3-8 and 3-9). There were reduced

amounts of 60- and 17c-hydroxylated metabolites and absolutely no 3a-reduced

metabolite formed for both steroids as substrates without NADPH under the same assay

conditions (not shown). Figure 3-10 depicts the Lineweaver-Burk plots for metabolism of

testosterone to the three metabolites (see above) by catfish intestinal microsomes. Table

3-2 shows the apparent KM and Vm,, values of these three metabolism pathways from

testosterone. The lower Km value of testosterone-60-hydroxylation than those of the other

two metabolites indicates that this may be the most important physiological pathway

because of the low physiological concentration of the substrate.


























Androstendione


Testosterone



3a-OH metabolite


60-OH testosterone







11 Ia-OH testosterone



Origin


Figure 3-7. TLC of testosterone metabolism by catfish intestinal microsomes.









11 a


17 a

- 16 a


3 a 6 Progesterone 3 a 613 Testosterone

Figure 3-8. Progesterone and testosterone metabolism positions by catfish intestinal microsomes.












Progesterone


3a-OH metabolite 0

17a-OH progesterone 0


6P-OH progesterone




16a-OH progesterone




Origin


Figure 3-9. TLC of progesterone metabolism by catfish intestinal microsomes.




































4 -4 -2


* 6f3-OH

6P3-OH


2 4 6 a 10 12


1/S (1O01pM)


0 3a-OH
..... 3a-OH


17-oxidation

-- 17-oxidation


Figure 3-10. Lineweaver-Burk plot of testosterone metabolism by catfish intestinal
microsomes









Table 3-2. Kinetic analysis of testosterone metabolism by catfish intestinal microsomes.

6p- 3oc- 17-oxidation
hydroxylation reduction
KM (p.M) 20.4 100.6 44.4
Vmax (pmol/min/mg protein) 175.4 608.9 223.4





Expression of CYP3A along Catfish Intestine

As shown in Figure 3-11, the polyclonal antibodies (IgG) generated against trout

CYP3A27 reacted strongly with catfish intestinal microsomes, showing a band with

molecular weight of 59 kDa. The rabbit-anti-trout CYP3A27 antibody also recognizes

human CYP3A4, which was used in CYP3A quantification. Human CYP3A4 has

molecular weight of 54 kDa. The CYP3A-like protein was expressed constitutively in

catfish intestine. In both groups of fish fed either commercially available chow or

semisynthetic purified diet, the expression of this protein was much higher in the

proximal segment than in the distal part (Figure 3-12). A breakdown product with MW

44 kDa has been found which also cross-reacted with the rabbit-antitrout CYP3A27. In

addition, the intestinal CYP3A amount in fish fed chow was higher than those fed

purified diet (Figure 3-12). This trend is more obvious in the proximal section of intestine

rather than distal part, probably due to the low expression in distal intestine from both

groups of fish.













54 kDa 10


*59 KDa


-f"O -N


(3) (4) (5) (6) (7)


Figure 3-11. Western blot of hCYP3A4 and catfish intestinal CYP3A.
Lane (1)-(5): 0.5, 1, 2, 3.5, 5 pmol hCYP3A4;
(6) 20 lg proximal intestinal microsomes from catfish fed purified diet;
(7) 20 lig distal intestinal microsomes from catfish fed purified diet.








59 kDa -


44 kDa m


Figure 3-12. Western blot of CYP3A in catfish intestine. Each lane has 20 jig
intestinal microsomal protein. (1) proximal intestinal from fish fed chow; (2)
distal intestine from fish fed chow; (3) proximal intestine from fish fed purified
diet; (4) distal intestinal from fish fed purified diet.









Regional Expression and Dietary Effects on Intestinal CYP3A

Testosterone hydroxylation activities by catfish intestinal microsomes and

CYP3A amount are summarized in Table 3-3. Testosterone 61-hydroxylation activities

were significantly higher in the proximal segment in catfish intestine than in the distal

part for both fish groups whether fed on chow or purified diet (Figure3-13). In addition,

in the proximal half of the catfish intestine, the CYP3A catalytic indicator, testosterone

61-hydroxylation activity, was significantly higher in fish fed chow than those fed semi-

purified diet (Table 3-3 and Figure 3-13). This trend was not observed in the distal part of

the intestine. The total metabolism of testosterone in the proximal segment was slightly

higher but not significantly different from the values found in distal intestine (Figure 3-

14). We have also studied the effect of diet on the CYP3A expression in the catfish

intestine. It indicated that the amount of CYP3A protein was lower in the intestine of

catfish that were fed purified diet than those fed with commercial chow, but the two

groups show the same trend of expression along the intestine (Table 3-3). Testosterone

61-hydroxylation activities, the CYP3A catalytic indicator, correlated with the CYP3A

amount shown by immunoblotting (r=0.88) (Fig. 3-15). On the contrary, the testosterone

17-oxidation and the formation of the reduced metabolite, 4-androsten-3a, 1703-diol,

showed poor correlation with the CYP3A protein amount. The ratio of testosterone 63-

hydroxylation over 17-oxidation was much higher in proximal intestine, ranging from 1

to 4, than that in distal section, which is approximately 0.8 (Figure 3-16). This suggests

that the percentage of CYP3A enzyme in total P450 content was significantly higher in

the proximal part than that in the distal intestine. These results demonstrated that a

CYP3A-like protein, related to CYP3A27, was expressed at higher concentrations in the









proximal than in the distal segment of catfish intestine. In addition, the expression of this

CYP3A-like protein was modulated by the diet.









TABLE 3-3. CYP3A expression and catalytic activities along catfish intestine

Diet Testosterone Metabolism CYP3A

613-OH Total Metabolism Enzyme Amount

pmol/min/mg protein pmol/mg protein
Chow (n=4)
Proximal 262.880.3 ab 986.9363.4 101.0-+31. 1ab
Distal 88.615.6 622.3225.5 32.322.8
Purified (n=8)
Proximal 158.432.6 a 687.6107.0 52.56.9a
Distal 104.138.1 466.1180.8 21.615.2
a significantly higher than the corresponding distal values by one tailed student t-test for
paired samples: p<0.01.
significantly different from the corresponding proximal values for purified diet group
by single factor ANOVA: p<0.01.




















C
0 400
--c3 5 a b
x
300

2', 250
c 200 a
E E 150 M Proximal
CD 100- Distal
0
o 50-
00
1 ( 0- WM
Chow Purified diet
Figure 3-13. Testosterone 60-hydroxylation activities in proximal and
distal intestine of fish fed chow or purified diet.

a significantly higher than the corresponding distal values by one
tailed student t-test for paired samples: p<0.01.
b significantly different from the corresponding proximal values
for purified diet group by single factor ANOVA: p<0.01.



























1600-
0
.2 1400
X>% ""1200
2
*O -EE 1000

rE 800

2 E 600
U) 0..
400
0
U) 200
I--
0


M Proximal
E3 Distal


Chow Purified diet
Figure 3-14. Testosterone metabolism activities in proximal and
distal intestine of fish fed chow or purified diet.




















450

(D 400
U)
> 350
x
2> 300
7 IM 250
c= E
200
(D 0 150

100 y = 1846.2x + 62.692
50 R2 = 0.7563


0 0.05 0.1 0.15
CYP3A (nmol/mg protein)


Figure 3-15. Correlation between testosterone 6p-hydroxylation and CYP3A enzyme
amount.









































11~


m Proximal


SON
nun


Proximal


A Distal


A
*AAAA
AAAA


Distal


Figure 3-16. Ratio of testosterone 60-hydroxylation/17-oxidation in proximal and distal
intestine of control catfish.









Effects of Modulators on CYP3A Activities

The chemical structures of the six CYP3A modulators used in our study are

shown in Figure 3-17. The three mammalian quasi-irreversible CYP3A inhibitors,

troleandomycin, erythromycin and SKF 525A, have an N-alkylated amine required to

form the metabolite-intermediate complex. Surprisingly, the selective CYP3A inhibitor

for mammals, troleandomycin, showed no inhibition of formation of any of the three

testosterone metabolites by catfish intestinal microsomes (Figure 3-18). Figure 3-19 and

3-20 are representative TLC analyses, showing chemical inhibition of testosterone

metabolism activities. Erythromycin, ketoconazole, metyrapone and SKF-525A inhibited

testosterone 60-hydroxylation to different extent {Figure 3-21, 3-22, 3-23, 3-24). Figure

3-25 summarizes the effects of four CYP inhibitors, erythromycin, ketoconazole,

metyrapone and SKF-525A, on the metabolism of testosterone. All four chemicals

showed strong inhibition of testosterone 6p3-hydroxylation. The inhibitory effects of

CYP3A-mediated testosterone 63-hydroxylation were: ketoconazole > metyrapone >

SKF-525A > erythromycin as the inhibitory potency decreased (Table 3-4). None of the

four inhibitors showed significant effect on the testosterone 3-oxidoreduction or 17-

oxidation. Only ketoconazole exhibited a concentration dependent inhibition of the

formation of androstenedione (Figure 3-21 and 3-24). Yet the IC50 value of ketoconazole

for testosterone 6P3-hydroxylation was almost 105 -fold smaller than that for the 17-

oxidation to form androstenedione (Table 3-4). The compound oc-naphthoflavone had no

significant effect on testosterone 6 P-hydroxylation activities (Figure 3-26). At 100 PiM a-

naphthoflavone concentration, which is more than three times the substrate concentration






66

(30 jiM), testosterone 6P-hydroxylation still had 91% activity left in comparison to

control (Figure 3-26).





















OH

0 .

Erythromycin 0


Troleandomycin










'7~~~~ N /\0 OCI
Metyrapone H

Ketoconazole






0

0- /





cc-naphthoflavone
SKF 525A (Proadifen)

Figure 3-17. Chemical structures of mammalian CYP3A modulators. Five inhibitors
(erythromycin, troleandomycin, metyrapone, ketoconazole and SKF 525A) and one
enhancer (cc-naphthoflavone) are shown.






68


















120
0
(U
, 00 .......... -. ......
x
2

0
060
E-O
40 -4


0
o 20-
I-
0 I I
0 20 40 60 80 100 120
Troleandomycin Conc, gM

Figure 3-18. Effect of testosterone 6p-hydroxylation by addition of
troleandomycin.












o l 0 0 0





60-OH-- U. Un mm
Testosterone


OuM


0.5uM luM

Metyrapone


2uM


Figure 3-19. TLC of inhibition of testosterone metabolism by metyrapone.












U" 1


603-OH---o.
Testosterone













0 uM


25 uM


mu'


50 uM


100 uM


SKF525A


Figure 3-20. TLC of inhibition of testosterone metabolism by SKF-525A


00- 1
ONIM


























































-- 6 -o

-0- 17-oxidation

-0- 3*-OH


100

Erythromycin Conc, gM


Figure 3-21. Inhibition of testosterone metabolism by erythromycin.


.............. .............................................. A --------





...............................................................................





.. ..............................................................................





.............. ..................................................................





.................................. ............................................





.................................................................................

























140.00

120.00

100.00

80.00

60.00

40.00

20.00

0.00


0 20 40 60 80
SKF-525A conc (gM)


-1 -- -4- 6-OH

.....- -0- 3ca-OH

--- 17-oxidation






100 120


Figure 3-22. Inhibition of testosterone metabolism by SKF-525A.






73
















10000


-4-- 6"3-OH
0.0
o- 3at-OH
& 17-oxidation
r- 6 0 .0 0 -- - - - - - -
0
0


20.00-


0 50 100 150 200
KET conc (nM)

Figure 3-23. Inhibition of testosterone metabolism by ketoconazole.






74










140

120 ---- -







17-oxidation
0
U
-.- 40 -----------
0
20 .. .
0

0 I I I
0 50 100 150 200 250
MET conc (gM)


Figure 3-24. Inhibition of testosterone metabolism by metyrapone













Inhbltlon of testosterone metabolism
by erythromycin


Inhibition of testosterone metabolism
by ketoconazole


150

i
* 100


u 50
0


ERM conc (pM)


1 10 100


KET conc (LM)


-- 6p-hydroxylation

3a-oxido-reducton

-1-- 17-oxidation


Inhiblion of testosterone metabolism
by SKF-525A


Inhibition of testosterone metabolism
by metyrapone


* 100-


0


SKF-525A conc (pM)


of 100-
s0

50 s-


1 10 100
MET conc (ptM)


Figure 3-25. Determination of IC50 of testosterone 613-hydroxylation by ERM,
KET, SKF-525A and MET. Abbreviations: ERM, erythromycin; KET,
ketoconazole; MET, metyrapone.


1Z.004.0x1l0= 0'lI









Table 3-4. IC50 values of the four inhibitors on testosterone hydroxylation.

Chemical IC50 (gM)
6p-hydroxylation 17-oxidation
Ketoconazole 0.0404 >500
Metyrapone 4.48 n.r
SKF-525A 32.1 n.r
Erythromycin 53.8 n.r

n.r. : no inhibition observed up to 200 tM of the inhibitor concentration






160
1 4 0 - - .- .- - - - - - - - - -

1 2 0 -.- -.-- - - - - - - - -


--!1- 3-OH

------ ---- 17-axidation





20 1

0 50 100 150 200 250
ANF conc ([.M)


Figure 3-26. Modulation of testosterone metabolism by a-naphthoflavone.







77


Modulation of AHH Activities

AHH activity was enhanced by the addition of c-naphthoflavone (Figure 3-27).

Maximal enhancement was achieved at 20 pM.


4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00


y = 0.7094Ln(x) + 0.9048
R2 = 0.9153


[ANF], pM


Figure 3-27. Stimulation of AHH activity by a-naphthoflavone.


. ...... ... ......................... .









Identification of Major Testosterone Metabolite

HPLC Analysis of Testosterone Metabolism by Catfish Intestinal Microsomes

A typical HPLC chromatogram of the ethyl acetate extracts obtained after

incubation of [14C] testosterone with NADPH and catfish intestinal microsomes is shown

in Figure 3-28. Testosterone and its hydroxylated metabolites showed peaks around 240

nm on UV spectrum. However, 4-androsten-3ax,170-diol has maximal absorbancy at 212

nm. The UV detector was set at 225 nm to avoid interference by the mobile phase. The

unknown metabolite isolated with TLC gave a peak at 33.5 min on radiochemical

detection, similar to the 4-androsten-3a, 1 73-diol standard by UV detection (Figure 3-29).

As the molar absorptivity of 4-androsten-3ca,173-diol is much lower than that of

testosterone or its hydroxylated metabolites due to the lack of a 3-keto group to conjugate

with the C4=C5, it is not surprising that signal of the unknown metabolite from assay

extract was below detection limit on UV detector. To further prove the identity of the

unknown metabolite, [14C] testosterone assay extract was mixed with 4-androsten-

3a,170-diol standard. The mixture was dried under nitrogen and reconstituted in

methanol. Figure 3-30 indicated the UV and radiochemical identities of the mixture of

[14C] testosterone assay extract with 4-androsten-3a,17p-diol standard. The standard and

the unknown metabolite eluted at the same time of 33.5 min on UV and radioactivity

detection respectively. Thus, testosterone was converted to 6 P-OH testosterone,

androstenedione and 4-androsten-3a, 1 73-diol by catfish intestinal microsomes. The

HPLC profile also provided valuable information on the identification of another

metabolite, 6-dehydrotestosterone (17f3-hydroxy-4,6-androstadiene-3-one) (Figure 3-28,

3-30). The retention time of this 6-dehydrotestosterone metabolite matched that of the








79



authentic standard. HPLC analysis of [14C] testosterone incubation with catfish intestinal


cytosol was also performed by radioactivity detection (not shown). No direct formation


of the 3a-reduced metabolite was observed in incubation with catfish intestinal cytosol.


000e


0 007









0,004









0,001


5 10 15 20
MOnute


000


25 30 35 40


Figure 3-28. HPLC (UV detection) profile of 4-androsten-3a,17p-diol standard.



























-0.30


,26


rO 22


0 18 c

U
n
0.14 t


014, 61


0 10'


0 06

10 15-


-0
Minutea


25 30


002

15 40


Figure 3-29. HPLC (radiochemical detection) of [C14] testosterone metabolism

catalyzed by catfish intestinal microsomes. Catfish intestinal microsomes (0.4

mg) was used; substrate concentration 30 p.M.

Abbreviations: 6p, 6p-hydroxy-testosterone; 6D, 6-dehydrotestosterone or 17p-

hydroxy-4,6-androstadiene-3-one; T, testosterone; A, androstenedione; 3a, 3a-

hydroxytestosterone or 4-androsten-3a, 1 713-diol.





















r80.080
UV





3a, authentic

.............. ........
r 3W8 14c
-T





613 6D /A 3ot


4 10 16 22 28 34 48


Figure 3-30. HPLC analysis of mixture of 4-androsten-3a,1713-diol and [14C]
testosterone assay extract. Abbreviations: 63, 61-hydroxy-testosterone; 6D, 6-
dehydrotestosterone or 171-hydroxy-4,6-androstadiene-3-one; T, testosterone;
A, androstenedione; 3a, 3a-hydroxy-testosterone or 4-androsten-3t, I 713-diol.









Mass Spectrometric Analysis of Testosterone Metabolite

To confirm the structure of metabolite to be 4-androsten-3c,1713-diol, the

metabolite was isolated and subjected to mass spectroscopy. ESI-MS did not provide

efficient ionization of the sample. With APCI-MS, both the 4-androsten-3c, 17p-diol

standard and the testosterone metabolite gave related m/z 255 and 273 ions and m/z 289,

with only very low abundance of the expected m/z 291 [M+H]+ ion. Among the three

daughter ions, m/z 273 is the most intense ion, followed by m/z 255 and m/z 289. The

(+)APCI-MS/MS daughter spectra of all three of these ions from the testosterone

metabolite matched those obtained from the 4-androsten-3a, 170-diol standard (Figure 3-

31, 3-32, 3-33). Attempts to increase the yield of m/z 291 ions were not successful. The

m/z 255 and 273 ions are probably due to [M+H-2H20]+ and [M+H-H20] ions of the 4-

androsten-3a, 1713-diol (MW 290). It is possible that the MW 290 steroid is thermally

labile, eliminating a H20 molecule forming a MW 272 compound which is then ionized.

The m/z 289 is likely the [M+H]+ ion of another steroid, which is probably testosterone

(Figure 3-34).

In summary, identification of the 3a-reduced testosterone metabolite was based

on a complete agreement of its chromatographic pattern with that of the authentic steroid

during chromatography on the reverse phase HPLC column and on TLC in the system

ethyl ether: toluene: methanol: acetone (70:38:0.8:1) and on a perfect agreement between

the mass-spectrum of the three daughter ions (m/z 255, 273, and 289) of the testosterone

metabolite with that of the authentic steroid (Figure 3-31, 3-32, 3-33).

In vitro metabolism of testosterone by catfish intestinal microsomes is

summarized in figure 3-35.




















253.2


97 1


197.1
109 A
~189.2


2112


142 157.2 1,1 1732 111 -.
M12 2 1330 1132 17T.1 N1 1 2252
,G 1Q 11 t .1 12 L I'


0 9 90 10 110 120 130 140 150 199 170 199 199 200 210 220 230 240 250 20 270 200 290 300
1111


1089

197,2
211.1
17 1 187 1211 2

203 13 1929 1911 20 I 3 24 3
139 1 1 0 .1 1753 1 2252 211 3
140 1313 oo1459 ~~. Ii 29o ioo L 241


271.2


5C2


S73


70 80 90 1 0 110 120 IM M 150 T180 9 200 210 220 20 240 2 260 2




Figure 3-31. (+)APCI-MS/MS daughter spectra of m/z 289 ions. The spectra are from


(a) authentic 4-androsten-3o,17p-diol; (b) metabolite isolated from testosterone


incubation.


924d91 99 1


65


.1
.55

Ab

d.



40

35

20

25






10

5


99.1


--"']I


I I IH r I














































81.1







lay ( I


1591 1 1910 2012 250
33 1 V I 0


0 1, 1 i, I I I I I, I8 9. -0 1 i -0 l 1 1 ." I I" 1 1"' t' I"'' P I i;; "' I f I I I I I I I3 -4 I I6 17 I8 1I I0 1 22 3 4 20 20 ,28'i ,' m0 {
70 00 00 00 It0 120 1 100 1170 10 190 200 10 220 20 240 250 260 270 200 290 300


I, 115,o,91
1450 1512 0 2
I j1 1015* 151 0. 21.4 221


70~~~ 60 9 0 10 0 .. 20 ;161' 10 7 0 0 200 210 220' 22 20 25 40 27 80 20









Figure 3-32. (+)APCI-MS/MS daughter spectra of m/z 273 ions. The spectra are from

(a) authentic 4-androsten-3c,1713-diol; (b) metabolite isolated from testosterone

incubation.


I


I I ,


^_


n .. i t ill .





















1501


010







029
5 009 961
9o 22 4~


121 1
1321


13t.0

1091


W41I


1731


21


2193


3 00 00 100 110 13 130* i 11. 4 150 160 170 10 10 20 10 229


r2r7 1



210 2547


-230 ... 25,, 0 ... 200 ... 7 200 3o" 300


1170


1052


81 0


944




801 9 1 0


1210

1301


'1


1470


17U


1 2131


2371
2272


2191





22&1


f39 2916


la 00 90. .. .. 2'o0 1,, i0" Io I .... 0
0 0 0 9 100 110 10 130 '140 ISO 1 t0 170 S0 190 200 210 220 240 no 2w 2M 2M 2M 30

404




Figure 3-33. (+)APCI-MS/MS daughter spectra of m/z 255 ions. The spectra are from


(a) authentic 4-androsten-3a, 1 7P-diol; (b) metabolite isolated from testosterone


incubation.


D .. . .. . . i ; . .% .%'' ; ':' :-. : ." .- .': . . .. . . .


El


t


= II I IIIl I II III


i


, t ,, I1 II II I II


^


t991


1. 1





























+ m/z 291

H-2
4-androsten-3a, I 7f-diol

-H_



H\+H


-2H20
H



m/z 289
testosterone -H20






H


+-C
m/z 255


Figure 3-34. Fragmentation of 4-androsten-3a, 1 70-diol in APCI-MS.


j H

m/z 273





















11 a OH testosterone


OH 6 3 OH testosteron


CYP


3 a oxido-redu;a x


Testosterone


3 a OH testosterone


CYP3A


Androstenedione


Figure 3-35. In vitro metabolism of testosterone by catfish intestinal microsomes.









Regional Expression of 3a-Hydroxysteroid Dehydrogenase in Catfish Intestine

Table 3-5 summarizes the in vitro testosterone metabolism from catfish fed semi-

synthetic diet or commercial chow. Both groups of catfish showed higher testosterone

3c-oxido-reduction in proximal intestine than distal. In proximal intestine, testosterone

6[-hydroxylation, but not 3ct-oxido-reduction, was significantly higher in fish fed

commercial chow than those fed semi-synthetic diet (Table 3-5). Our previous work has

indicated that testosterone 61-hydroxylase in catfish intestinal microsomes was mediated

by CYP3A-like enzymes. From the above results, we found that diet may play a more

significant part in CYP3A expression than expression of 3c-hydroxysteroid

dehydrogenase in catfish intestine, especially in the proximal section. The formation of 4-

androsten-3cx, 1713-diol from testosterone by catfish intestinal microsomes was not

affected by the two general cytochrome P450 inhibitors, metyrapone and SKF-525A.




















Table 3-5. Testosterone metabolism by intestinal microsomes from catfish fed with
chow or semi-synthetic purified diet

Diet Testosterone Metabolism

6P-OH 3or-reduction 17-oxidation total metabolism

pmol/min/mg protein
Chow (n=4)
Proximal 262.880.3 a,b 346.1210.5 285.8119.5 ,b 989.9363.5
Distal 88.615.6 292.7220.7 154.142.7 622.3225.5
Purified (n=5)
Proximal 158.426.3 a 325.483.9 a 117.015.7 691.813.6 a
Distal 108.844.4 162.038.1 136.656.4 481.6158.6
a significantly higher than the corresponding distal values by one tailed student t-test
for paired samples: p<0.01.
aa significantly higher than the corresponding distal values by one tailed student t-
test for paired samples: p<0.05.
b significantly different from the corresponding proximal values for purified diet
group by single factor ANOVA: p<0.01.




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CYTOCHROME P450 ENZYMES IN CHANNEL CATFISH ICTALURUS PUNCTATUS AND METABOLISM OF TESTOSTERONE BY CATFISH INTESTINAL MICROSOMES By ZHEN LOU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E GRE E OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2001

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ACKNOWLEDGMENTS I would first like to express my appreciation to my advisor Dr. Margaret 0 James, for her great deal of guidance, advice, and support through my course of study, which I would not have completed without her help. I believe everything I learned during my time in her laboratory is indispensable for my future accomplishments as a scie n tist. I was deeply impressed with her diligence and talent as well as kindness I would also like to express my gratitude to all my committee members Dr. Kenneth Sloan, Dr. Stephen Roberts, Dr. Donghai Wu and Dr William Dolbier fo r their invaluable guidance and the time they committed to my graduate work. My apprec i ation is extended to our lab members for their kind help and cheerful encouragement. Finally I want to thank all members of my family for their continuous care and support. 11

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TABLE OF CONTENTS ACKNOWLEDGMENTS . . . ............. . . . . .... . . ... .. ... . . .... ... ...... .. ........... ... . ..... . . . .... .. ii LIST O F TABLES ... ........... . ... . . ............. .......... ... ...... . ......... .. .. .. .... ........................ . V LIST O F FIGURES ........ ......... . ... . . ... . ... . ...... . . ... ........... ... .. .. . .. . .............................. vi KEY TO ABBREVIATIONS .. . ... ... ... . ... . . .... . ... . ........ . .... . .. ..... . .. .. ...... ..... ........ .. ix ABSTRACT .... . ..... ... ..... .... ..... . ... ...... ... ... ..... .. . . . .. . .. . . .. ....... ..... ... .... . ... ... .... .. .. x CHAPTE RS 1 INTRODUCTION ... . ... ... .. .. ....... ... . ... ... . .. ... . ............... ..... . .. .... .... .. ..... .. . .... ... .. ...... 1 Phase I Enzymes ... . .... .. . .. ..... ... ........ . . . ....... .. . . . .... .... . .. ..... .. . . .. ................ .... .... 3 Cytochrome P450 ........ ..................... .. ...... ... .. . . ............ ... .... . .. .. .. . ... ......... .. . ....... . 3 CYP3A Inhibiti on .......................... ............... .... .................................. ..... ...... ..... .. 11 CYP3A Induction ... ... ... . ... ... . ... ... . . .. . . . ........ . .. . .... . ..... .. .. . .... . .. ... . ...... ... ... 15 CYP3A S timulati on ... ... . ..... . ........ . ..... .... . ...... .. .................... ...... ..... ... ... .... .. 19 Epoxide Hydrolase . ......... . ... . . ... . . ... .... . .... . ... .... . .. . .. .... .......... . .... . ... .. .. .... .. 20 Phase II Enzymes .................... ...................................................................................... 21 Glutathione S-Transferase .. ............................................... ........................ .............. 21 Sulfotransferase ... ........................ . ...... . . . . . .. . . .. ...... .. .. ....... . .... .... . . ........... 25 UDP-Glucuronosyltransferase . ... ....... ........ . .... . . .. .... ... .. .. .. . ...... .. . . .. . ........... . 26 3a-Hydroxysteroid Dehydrogenase . . . .......... . . .... ........ ....... .. ......... ....................... 28 Prehepatic Metabolism and Bioa vai labili ty ................................................................ .. 30 Hypothesis . ............... ... ... . .............. . ... ... .. . ........ . . .... .... .. .... .... .... .. ..................... 35 2 MATERIALS AND METHODS ........ . ..... ... .... ............ .................... .... ................ .. .. 36 Chemica ls . . ..... ............. ..................... . . . ...................... . ....... .. . .. .. . .... . ....... . . ... .. .. 36 Instruments .............. . . .... ....................... . ... .... . . . .... ............ .. .... .... ...... . . ... . . . ...... 36 Animals and Pretreatment .. ...... .. ... ... . . . ........ ...... .... ................. .... .... .......... . .... ... .. 37 Surgical Procedur es for Oral Gavage ............ .................... .. .... .... .... ............ ................ 38 Enzymes Prep aration . . . . ... . . ... . . ..... . .... . ............ .. .. . .. . .. . .. .......... ... . ..... . ......... 38 Protein Assay ............. ........... ............ ... . . ... ... . .. ... ......... .. .. . ....... ......... .................... 39 Measurement of Cytochrome P450 ................................. ........... .................................. 40 Steroid Hydroxylation Assay ... .............. .. ...... .. ............ ......................... .. ................ ...... 40 lll

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Chemical Modulation of Testosterone Metabolism ....... . .. ... ......................... .............. 41 AHH (Aromatic Hydrocarbon Hydroxylation) Assay .... .... .... ..................... . ... ... ........ 42 Western Blot Analyses .................................................................................................. 42 Western Blot Analyses of CYPlA ............................................................................ 42 Immunochemical Analyses of CYP3A .... . ... . ...... . ......................... ...... ... . ...... ... ... 44 HPLC Analysis of Testosterone Metabolism ..... ................ .... ....... .. ... .. ......... .. .. .... 44 Mass Spectrometric Analysis . ... . . . . . . . ..................... .. . .... .. .. . .. .... ... ............... ... ... 45 Sulfotransferase Activity Assay .................................................................................... 45 UDP-Glucuronosyltransferase Activity Assay ............................................................. 46 Statistical Analysis . ... . ....... ........... . . . . ..... ...... ..................... ....... .... ........ ............... 46 3 RESULTS ....... . ... ..... . ....... ... . . .............. ... ...... . . .... ... .. . ....... ..... ................. ......... 47 Response to Aryl Hydrocarbon Receptor Agonists .................. .... ....... .. . ...... . ........... 47 CYPlA Expression in Channel Catfish Intestine ..................................................... 47 UDP-Glucuronosyltransferase Expression in Channel Catfish Intestine .. .. . .... . .... .. 51 Function and Expression of CYP3A and Testosterone Metabolism .. .. . .......... ... ....... 53 TLC Analyses of Steroid Metabolism by Catfish Intestinal Microsomes ................ 53 Expression of CYP3A along Catfish Intestine . ........ ............ ....................... . ....... 57 Regional Expression and Dietary Effects on Intestinal CYP3A ......... ........ ...... ... ... 59 Effects of Modulators on CYP3A Activities ............................................................ 65 Modulation of AHH Activities .. ...... ... ...... . . . .................. .................... ....... ...... .. 77 Identification of Major Testosterone Metabolite ..................... .. .. . .. . .. ... .... ... ............ .. 78 HPLC Analysis of Testosterone Metabolism by Catfish Intestinal Microsomes ..... 78 Mass Spectrometric Analysis of Testosterone Metabolite ..................... ....... . . ....... 82 Regional Expression of 3a-Hydroxysteroid Dehydrogenase in Catfish Intestine ........ 88 Catfish Intestinal CYP3A Inducibility Studies ............................................................. 90 Catfish Hepatic CYP3A Expression .......... .. ... . . ...... ................. .......... . . ..... ........ .. 93 4 DISCUSSION ............... ................. ... ... . . ...... ................. .. . .......... .. . .................. . .. ... 97 Induction of CYPlA and UGT in Catfish Intestine ... .. . .... .... ..... . .............. ... ........... 97 In vitro Testosterone Metabolism by Catfish Intestinal Microsomes ........................... 98 CYP3A Expression in Catfish Intestine . .... . ... ...... .. ............ .. .... .. .. .... ...... ..... ... .. .... 99 Chemical Inhibition of in vitro Testosterone Metabolism . . ................ . .............. .... 100 Stimulation of AHH Activities by a-Naphthoflavone ........... .. .. . .. . ... ... ... ............. .... 102 Inducibility of Catfish Intestinal CYP3A .. ... . ... .. ............. . .. .. . ... ............... ..... . . .... 103 Identification of 3a-Reduced Metabolite of Testosterone ... .. . .. .. .............. ......... ... .... 103 Catfish Hepatic CYP3A Expression ........................................................................... 105 5 SUMMARY AND CONCLUSIONS ....... ................. . .. . ............ .... .... ........... ........... .. 106 REFERENCES ..... .. .......... . . . ..... . . . . . . ... ........ . . .. . ........... .. . .. .. ...... .. ..... ..... ...... .. . 107 BIOGRAPHICAL SKETCH ..... . .... ............... .......... . ....... ......... ...... ....... ... .. ...... ........ ... 120 IV

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LIST OF TABLES 3-1. Intestinal CYPlA level in control and treated fish .............................................. ..... ...... 49 3-2. Kinetic analysis of testosterone metabolism by catfish intestinal microsomes ... .......... 57 3-3. CYP3A expression and catalytic activities along catfish intestine ............. .. .................. 60 3-4. IC 50 values of the four inhibitors for testosterone hydroxylation ................................... 76 3-5 Testosterone metabolism by intestinal microsomes from catfish fed with chow or semi-synthetic purified diet. ...................... ...... . .. ....... .. . .. .... .............. ... .......... 89 3-6. In vitro testosterone metabolism activities in proximal and distal intestine from control fish and fish treated with rifampicin for two weeks ... .. .............. .. .... ....... 92 3-7. Hepatic CYP3A expression in control and treated fish ....................................... ..... ...... 95 V

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LIST OF FIGURES 1-1. Catalytic cycle of cytochrome P450 ................. . ... . .. .............. .. .... .. ........ ...................... 4 1-2. Mechanism of induction of CYPlAl gene transcription ....... .. .. .... ............. .... . . .. .. .. .. 8 1-3. Bioactivation ofbenzo(a)pyrene ..... . . ................... ... .... ...... .. . .. .. ............ ............... ..... 10 1-4. Role of PXR in CYP3A gene induction .. . . ... .. ..... . ...................... ........ ................... ...... 18 1-5. CYP3A gene induction: Cross-talk between foreign chemical and endogenous regulator pathways ............... ....... ... . . ... ................ .. . .. . ....................... . ..... . .............. 18 1-6. Mercapturic acid biosynthesis ........ ... . .............. ...................... ....... . ...... ............ . ..... . 22 3-1. Intestinal P450 content in control and treated fish ................... . .. ... ....... ...... ................. 48 3-2. Intestinal and hepatic CYPlA in control and fish treated with 3MC or TCB ... . ... ........ 49 3-3. Intestinal and hepatic CYPlA in control and fish treated with TCB ........... .................. 49 3-4. Intestinal CYPlA content and AHH activity ................... .. .... .......... .............. . . .... .. .... 50 3-5. UGT activity in control (n=l2) and 3-MC treated (n=l l) fish .. ....... ...... ... . .............. 51 3-6. Intestinal microsomal AHH and UGT activities .................................... .. ... ... .... ....... .. 52 3-7. TLC of testosterone metabolism by catfish intestinal microsomes ... . .. . . ....... ... .. .. ..... 54 3-8. Progesterone and testosterone metabolism positions by catfish intestinal microsomes. 55 3-9. TLC of progesterone metabolism by catfish intestinal microsomes .... ....... ... .. .... . .. ..... 55 3-10. Lineweaver-Burk plot of testosterone metabolism by catfish intestinal microsomes . 56 3-11. Western blot ofhCYP3A4 and catfish intestinal CYP3A. ......... .. ... .... ......... .... .. .... .. 58 3-12 Western blot of CYP3A in catfish intestine .... . ............ ...... .......................... .. . ....... 58 Vl

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3-13. Testosterone 6P-hydroxylation activities in proximal and distal intestine of fish fed chow or purified diet. .............................................................................................. 61 3-14. Testosterone metabolism activities in proximal and distal intestine of fish fed chow or purified diet. ..................... .............. .................. .............. .................... ........... .. 62 3-15. Correlation between testosterone 6P-hydroxylation and CYP3A enzyme amount.. .... 63 3-16. Ratio of testosterone 6P-hydroxylation/17-oxidation in proximal and distal intestine of control catfish . ... ............................. . . ........ ..... ............................ ............... ....... .. 64 3-17. Chemical structures of mammalian CYP3A modulators ..................... ........................ 67 3-18. Effect of testosterone 6P-hydroxylation by addition of troleandomycin ..................... 68 3-19. TLC of inhibition of testosterone metabolism by metyrapone ..................................... 69 3-20. TLC of inhibition of testosterone metabolism by SKF-525A ...................................... 70 3-21. Inhibition of testosterone metabolism by erythromycin ..................................... .. .. .. .. 71 3-22. Inhibition of testosterone metabolism by SKF-525A. .................................................. 72 3-23. Inhibition of testosterone metabolism by ketoconazole ........... .. ...... .... ................ ....... 73 3-24. Inhibition of testosterone metabolism by metyrapone ...................................... .... .... ... 74 3-25. Determination oflC 5 o of testosterone 6P-hydroxylation by ERM, KET, SKF525A and MET ....................................................................................................................... 75 3-26. Modulation of testosterone metabolism by a-naphthoflavone ..................................... 76 3-27. Stimulation of AHH activity by a-naphthoflavone ...................................................... 77 3-28. HPLC (UV detection) profile of 4-androsten-3a,17P-diol standard .......... .................. 79 3-29 HPLC (radiochemical detection) of [ 14 C] testosterone metabolism catalyzed by catfish intestinal microsomes . ... ..... ... . ... ...... .......................... .. ....... . ........... .... .. .... 80 3-30. HPLC analysis of mixture of 4-androsten-3a,17P-diol and [ 14 C] testosterone assay extract. ................................................................................................................. 81 3-31. (+)APCI-MS/MS daughter spectra of m/z 289 ions ............................... ............ .......... 83 3 32. (+)APCI-MS/MS daughter spectra of m/z 273 ions .................................................... 84 3-33. (+)APCI-MS/MS daughter spectra of m/z 255 ions ............... ...................... .... ........... 85 3-34. Fragmentation of 4-androsten-3a, 17P-diol in APCI-MS ..... ............ ........... ...... ........... 86 Vll

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3-35. In vitro metabolism of testosterone by catfish intestinal microsomes .. ........ ...... .... ..... 87 3-36. Western blot of intestinal CYP3A from control and PCN treated fish ........................ 90 3-37. Intestinal CYP3A expression in control or fish treated with RIF or PCN ... ................ 91 3-38. Western blot ofhCYP3A4 and hepatic microsomes from control fish and fish pretreated with RIF or PCN (10 mg/kg) ..................... ... .......... ... ................................. 94 3-39. Western blot showing cross-reactivity of catfish hepatic microsomes against a polyclonal antibody to trout CYP3A27 ........................ ........ ............................... .... .. 94 3-40. In vitro testosterone metabolism activities by hepatic microsomes of fish from control, rifampicin (RIF) and PCN pretreated groups .................................................. 96 vm

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KEY TO ABBREVIATIONS 3-MC: 3-methylcholanthrene AHR: aryl hydrocarbon hydroxylase AhR: aryl hydrocarbon receptor ANF: a-naphthoflavone BNF: P-naphthoflavone CYP: cytochrome P450 EH: epoxide hydrolase ERM: erythromycin GST : glutathione S-transferase HEPES: N-[2-hydroxyethyl]piperazine-N' -[2-ethanesulfonic acid] HSD: hydroxysteroid dehydrogenase KET: ketoconazole MET: metyrapone PCN : pregnenolone 16a-carbonitrile RIF: rifampicin ST: sulfotransferase TAO: troleandomycin TCB: 3, 3', 4, 4'-tetrachloro biphenyl UGT: UDP-glucuronosyltransferase IX

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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 CYTOCHROME P450 ENZYMES IN CHANNEL CATFISH, ICTALURUS PUNCTATUS AND METABOLISM OF TESTOSTERONE BY CATFISH INTESTINAL MICROSOMES Chairman: Margaret 0. James By Zhen Lou May 2001 Major Department: Medicinal Chemistry Intestinal cytochrome P450 provides the principal initial source of biotransformation of ingested xenobiotics. In humans an important cause of incomplete bioavailability is prehepatic metabolism in the GI tract, mainly by the CYP3A enzymes. The expression and properties of CYP proteins were examined along the intestine o f channel catfish Ictalurus punctatus fed commercial chow or semi-purified diets. Benzo(a)pyrene hydroxylase activity was higher in proximal than distal intestine, and was stimulated by a-naphthoflavone suggesting involvement of CYP3A. Polyclona l antibodies (IgG) generated against trout CYP3A27 reacted strongly with catfish intestinal microsomes, showing a band with MW of 59 kDa. In catfish fed with standard chow the expression of this protein was much higher in the proximal segment than in the dista l part. Testosterone 6P-hydroxylation activities were monitored as the catalytic indicator of X

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CYP3A, which was higher in proximal than distal intestine. The 6P-hydroxylation activities in the two segments correlated with the amount of CYP3A. Similar results were obtained with progesterone as substrate. The amount of CYP3A and steroid-6P hydroxylation activities were lower in both segments of intestine from fish fed purified diet compared with commercial chow, but with the same trend along intestine. Incubation of catfish intestinal or hepatic microsomes with [ 414 C] testosterone resulted in three major metabolites: 6p-hydroxy testosterone, androstenedione and another metabolite. The formation of this unknown metabolite requires NADPH as cofactor. Comparison of the chromatographic behavior and MS of the unknown metabolite with that of authentic testosterone derivative suggested that this metabolite corresponds to 4-androsten-3a, 17P-diol. The ratio of testosterone 6P-hydroxylation/17oxidation was significantly higher in proximal than distal intestine. Testosterone 6P-hydroxylation was inhibited by specific mammalian CYP3A inhibitors, ketoconazole and erythromycin, and general P450 inhibitors, metyrapone and SKF-525A, but was not affected by a-naphthoflavone. Troleandomycin, a mammalian CYP3A inhibitor, had no effect on the testosterone metabolism by catfish intestinal microsomes up to 100 M. Dietary pretreatment of catfish with rifampicin or pregnenolone-16a-carbonitrile (PCN) did not alter the CYP3A enzyme level in prox~mal and distal intestine. Distal intestine from fish treated with rifarnpicin for 2 weeks showed significantly higher testosterone 6p-hydroxylation and 3a-oxido-reduction activities than that from control fish. Xl

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CHAPTER 1 INTRODUCTION All organisms are exposed constantly and unavoidably to foreign chemicals or xenobiotics, which include both man-made and natural chemicals such as drugs industrial chemicals, pesticides, pollutants, pyrolysis products and toxins produced by molds, plants and animals. As a result of a great variety of human activities, the aquatic environment is becoming increasingly threatened by an alarming number of foreign chemicals This pollution is a threat to the health of organisms inhabiting the waters as well as to human consumers of such organisms. Fish populations living in highly polluted areas often have high incidences of gross pathological lesions and neoplasms associated with elevated levels of toxic contaminants in the sediment [O]. Of most concern are xenobiotics that cannot be readily eliminated because of their lipophilicity Biotransformation or metabolism of lipophilic chemicals to more water-soluble compounds is a requisite for detoxification and excretion An important consequence of biotransformation is that the physical properties of a xenobiotic are generally changed from those favoring absorption (lipophilicity) to those favoring excretion in urine or feces (hydrophilicity). In addition, certain steps in the biotransformation pathway are responsible for the activation of foreign chemicals to the reactive intermediates that ultimately result in toxicity, carcinogenicity and other adverse effects. Many of the enzyme systems involved in biotransformation are also engaged in critical physiological functions such as steroid hormone biosynthesis and inactivation or fatty acid metabolism making interactions between foreign chemicals and physiological processes possible 1

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2 The reactions catalyzed by xenobiotic-biotransforming enzymes are generally divided into two groups, called phase I and phase II Phase I reactions involve hydrolysis reduction and oxidation. These reactions expose or introduce a functional group (e.g. -OH -NH 2 -SH or COOR), and usually result in only a small increase in hydrophilicity. Phase II biotransformation reactions include glucuronidation sulfation, acetylation, methylation conjugation with glutathione (leading to mercapturic acid synthesis) and conjugation with amino acids The cofactors for these reactions react with functional groups that are either present on the xenobiotic or are introduced/exposed during phase I biotransformation. Most phase II biotransformation reactions result in a large increase in xenobiotic hydrophilicity; hence they greatly promote the excretion of foreign compounds Phase II biotransformation ofxenobiotics may or may not be preceded by phase I biotransformation. Xenobiotic-biotransforming enzymes are widely distributed throughout the body and are present in several subcellular compartments The liver is the richest source of enzymes catalyzing biotransformation reactions These enzymes are also located in the skin lung gastrointestinal tract, and nasal mucosa (which can be rationalized on the basis that these are major routes of exposure to xenobiotics), as well as numerous other tissues including kidney heart brain, etc. Intestinal microflora play an important role in the biotransformatiton of certain xenobiotics. The enzymes catalyzing xenobiotic biotransformation reactions are located primarily in the endoplasmic reticulum ( microsomes) or the soluble fraction of the cytoplasm (cytosol) with lesser amounts in mitochondria nuclei and lysosomes. Their presence in the endoplastic reticulum can be rationalized on the basis that those x enobiotics requiring biotransformation for urina ry or

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3 biliary excretion will likely be lipophilic and hence soluble in the lipid bilayer of the endoplasmic reticulum. Phase I Enzymes Cytochrome P450 Among the phase I biotransformation enzymes, the cytochrome P450 system ranks first in terms of catalytic versatility and the sheer number of xenobiotics it detoxifies or activates to reactive intermediates. All P450 enzymes are heme-containing proteins The term cytochrome P450" originates from the observation that the reduced state of the protein forms a complex with carbon monoxide that exhibits maximal absorbance at 450 nm [2]. The basic reaction catalyzed by cytochrome P450 is monooxygenation in which one atom of oxygen is incorporated into a substrate designated RH and the other is reduced to water with reducing equivalents derived from NADPH as follows : Substrate (RH) + 0 2 + NADPH + Ir Product (ROH) + H 2 0 + NADP + The principal catalytic cycle of cytochrome P450 is shown in Figure 1-1. The essential steps involve the following: (1) binding of the substrate, (2) reduction of the ferric (resting cytochrome P450) to the ferrous state, (3) binding of molecular oxygen to g i ve a ferrous cytochrome P450-dioxygen complex (4) transfer of the second electron to t hi s complex to give a peroxoiron (III) complex (5) protonation and (6) cleavage of the 0-0 bond with the concurrent incorporation of the distal oxygen atom into a molecule o f water and the formation of a reactive iron-oxo species (7) and (8) oxygen atom transfer from this oxo comple x to the bound substrate and (9) dissociation of the product. What is not clear is what steps are rate-limiting in various reactions

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4 CD "' ROH Fe 3 + ~RH K.tl \V Fe 3 + ROH FeJ+ l.N0H-P450 reductase" 3/ Fe 3 +0HR \ NADPH-P45O reductase" Fe 2 +RH Fe 3 +0RH /o ,0 -H20A 2+ @)\ Fe -0 2 RH H+Fe 3 +-00H RH '~ 2+ leb red J.J~ Fe -0 2 RH ~ 4 5 or 1tb Fe'+-o,-'RH bs r NADPH-P450 reductase ox ox NADPH-P450 reductasered Figure 1-1 Catalytic cycle of cytochrome P450. RH: substrate; ROH: the corresponding hydroxylated metabolite. (Adapted from Guengerich, F.P., Cytochrome P450 3A4 : Regulation and role in drug metabolism Annu Rev. Pharmacol. Toxicol 39 1-17 ( 1999) Cytochrome P450 monooxygenases function in the transformation of endogenous and exogenous compounds and serve as catalysts that are significant in numerous and diverse biological pathways. The highest concentration of P450 enzymes involved in xenobiotic biotransformation is found in liver endoplasmic reticulum (microsomes), but P450 enzymes are present in virtually all tissues. The roles played by cytochrome P450 in endogenous pathways encompass the synthesis and degradation of steroids prostaglandins, fatty acids and other biological molecules. In the transformation of foreign compounds cytochrome P450 plays key roles in the toxicology and

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5 pharmacology of pollutant chemicals, drugs and therapeutic agents and in the activation and inactivation of many chemical carcinogens. The extent to which these various pathways or functions occur in different animal groups will depend to a large degree on the complement of different P450 proteins present, their catalytic function and their regulation The P450 enzymes are encoded by a superfamily of genes Currently more than 800 P450s have been characterized, inclusive of the many different species of organisms that ha v e been studied. Knowledge of these features of P450 in different species is necessary to define the general characteristics of P450s and their functions, and to indicate the evolution of these proteins. Such knowledge is also necessary to define the susceptibility of different individuals, populations or species to xenobiotic compounds particularly those compounds whose toxicity may depend upon biotransformation. Currently, these processes are understood far better in rodent models than in wild or cultivated species that provide food and material resources. Research on mammalian cytochrome P450 continues to dominate the literature, but there is a growing recognition of its biological significance in other animals and of our need to know the diversity and biochemistry of cytochrome P450 enzymes in these groups. The 20 000 species of fish extant represent about one-half of the known vertebrate species. The fish present extraordinary diversity, inhabiting all of the world's aquatic environments. They also present a significant source of protein for humans. The cytochrome P450 forms in fish thus acquire importance from evolutionary and toxicological standpoints Fish possess microsomal P450 similar to those in mammals (3]. Knowledge of the multiplicity, function and regulation of cytochrome P450 forms in fish continues to grow in importance. The first fish CYP to be cloned and sequenced was a CYPlA from

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6 3-methylcholanthrene induced trout [4]. Recently, evidence has been presented to document that trout possess more than a single member of the IA family [5]. A key feature of cytochrome P450 systems in both fish and mammals is their inducibility by chemical substrate for the enzymes/and by structurally related compounds. Fish respond to the same classes ofxenobiotics as mammals with respect to induction of CYPlA, i.e., 3-MC, BNP, polycyclic aromatic hydrocarbons (PAHs), polyhalogenated biphenyls (PCBs and PBBs), and polychlorinated dioxins (PCDDs) and dibenzofurans (PCDFs) [6]. Fish CYPlA are induced by the above hydrocarbons given by injection, feeding or waterborne exposure. The induction can be detected by ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon (BaP) hydroxylase (AHH) activities. The molecular mechanism and cellular machinery for aromatic hydrocarbon (Ah) receptor-mediated CYPlA induction in fish appears to be similar to that of mammals, which is known to involve the following: (i) binding of the ligand to the Ah receptor, (ii) translocation of the bound receptor into the nucleus, and (iii) binding of the receptor complex to specific DNA sequences upstream of the CYPlAl promoter (Figure 1-2). Prior to occupancy by a ligand, the inactive Ah receptor resides in the cytoplasm of target cells in a soluble complex with the heat shock protein Hsp90 (Figure 1-2). It appears that Hsp90 chaperones the AH receptor, maintains it in a ligand binding conformation, and represses its intrinsic DNA-binding activity [7]. Binding of a ligand triggers translocation of the ligand-receptor complex into the nucleus. The nuclear form of Ah receptor binds with high affinity to specific DNA enhancer sequences known as AH-responsive elements (ARRE) located in the 5'-flanking region ofresponsive genes. The nuclear DNA-binding complex is not a monomer but a heterodimer [8]. Several recent lines of evidence

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7 confirm that the form of AH receptor that binds to AHREs consists of at least two proteins the Ah receptor and ARNT (Ah-receptor-nuclear-translocator). The process by which ligand binding transforms the cytosolic Ah rceptor to its functional DNA-binding state is complicated and still poorly understood. Phosphorylation of both Ah recepto r and ARNT by protein kinse C (PKC) appears to be important for generation of the functional DNA-binding complex [9]. Some inducers for example 3 3 ', 4 4'-tetrachlorobiphen y l can inhibit the catalytic activity of induced P450 [9] In such cases analysis of catalytic activity alone might show no response, but strong induction can still be seen by immunochemical analysis of the CYPlAl protein or hybridization studies with CYPlAl m.RNA.The fact that many of the inducers offish P450 activities (PAHs, PCDDs, PCBs) are known aquatic pollutants has greatly stimulated research in the P450 system of fish However a number of studies have documented the "phenobarbital-type" inducers to be ineffective as P450 inducers in fish [ 11 12] Intestines of fish are also capable of a variety ofbiotransformation reactions some of which respond to dietary cytochrome P4501A ( CYPlA) inducers [13]. Dietary induction studies with P-naphthoflavone a model PAR t ype inducer in catfish indicate that under conditions of low inducer concentrations select biotransformation activities in the intestine may equal or even exceed corresponding hepatic activities [14]. Such induction effects may potentially alter the degree and pathway of metabolism. P450 induction has been suggested to indicate the exposure of organisms to contaminants in the en v ironment [15]. Earlier studies on environmental induction of cytochrome P450 emphasized the analysis of catalytic activity. More recently, antibodies to the P AH-inducible cytochrome P450 from fish have been used to demonstrate

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8 unambiguously that CYPlA forms are elevated in fish from contaminated regions [16]. Several studies with different fish species revealed correlations between the levels of induced cytochrome P450 and levels of PCBs either in the organisms or in their immediate environment.Thus it seems that CYPlA activity or protein expression can be used to monitor the environmental pollution. Many chemical carcinogens are procarcinogens requiring activation to a carcinogenic derivative by P450-dependen t metabolic processes Due to the predominant role that CYPlA plays in the metabolic bioactivation of environmental procarcinogens it is not surprising that modulation of CYPlA levels and/or catalytic activity can significantly impact tumor development in fish models l Cyiochrome Endoplasmic P450 1A1 Aalle\llum m A Olhor AhR.'Amt / .......... H' t a/Amt ResponsNeGenes ++++Tai? ++ ++ ResponsiveGeoes l l Tr Chr ropl>Oll Facior ReGruitmonl? Aemod 009 Facl0r(s)7 HoSlOnO Mod~oca on? Co-AcllYatol'(S)? Figure 1-2. Mechanism of induction ofCYPlAl gene transcription. AhR aromatic hydrocarbon receptor ; ARRE AH-responsive-element; ARNT AH-receptor-nuclear translocator; Hsp90 heat shock protein 90.

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9 Most studies of CYP in fish have focused on the PAR-inducible CYPlA subfamily. While several P450 enzymes other than CYPlA have recently been cloned and sequenced from fish CYP2Ml( previously known as LMCl) and CYP2Kl (previously named LMC2) were islated from rainbow trout liver [17 18]. CYP2Ml shows specific fatty a cid hydroxylation at co-6 position. CYP2Kl has been shown to activate aflatoxin in trout liver to its carcinogenic metabolites. The expression of CYP2Kl has been confirmed to have major sex-related differences The roles and regulation of CYP3A forms in fish have begun to attract growing attention. Members of the CYP3A subfamily are major constitutively expressed CYP forms in the liver and in the gastro-intestinal tract of mammals [19]. CYP3As appear to have an extraordinarily broad substrate specificity and in addition to steroids also metabolize pro-carcinogens therapeutic drugs and dietary chemicals [20] Cytochrome P450 3A4 is known to catalyze the metabolism of both endogenous substrate (such as the 6P-hydroxylation of testosterone) and many important therapeutic agents including the N-demethylation of erythromycin. Studies have indicated a significant role for human hepatic P450 3A4 in the 9 10-epoxidation ofbenzo(a)pyrene-7 8-dihydrodiol forming t he final carcinogen BPDE [21] (Figure 1-3) Most studies of structure function and r egulation of CYP3As have been in mammalian systems, whereas relatively little is known about CYP3A in other vertebrate groups. As a matter of fact fish are continuously exposed to CYP3A inducers/substrates in their natural habitat as a result of food preferenc e s and human activities It has been shown that rainbow trout LMC5 rainbow trout P450con scupP450A codP450b and mammalian CYP3A (human 3A4 rat

PAGE 21

10 3Al) are all immunochemically related [22]. Buhler's group reported the OH HO (-)-BP7 8-dihydrodiol (-)-BP-7,8-diol-9, I 0-epoxidel + OH (+)-BP-7,8-diol-9 10-epoxide-2 OH (+)-BP-7,8-dihrodiol OH (-)-BP7 8-diol-9 I 0-epoxide-2 + OH (+)-BP7,8-diol-9 10-epoxide-l Figure 1-3. Bioactivation ofbenzo(a)pyrene. first CYP3A family member, CYP3A27, in an aquatic species (rainbow trout) which encodes an LMC5-like protein [23]. The major extrahepatic expression site for CYP3A27

PAGE 22

11 was upper small intestine which also expressed smaller amounts of CYP2Kl. Actually, upper small intestine has the highest expression of CYP3A27 in female trout follo w ed by the ovary and the liver. The high percentage of identities in alignment of CYP3A27 with other mammalian CYP3A forms suggest that there was significant sequence retention during e v olutionary divergence between terrestrial and aquatic vertebrates. The fac t that CYP3A proteins are present at significant levels in untreated fish implies that they are constitutively expressed and they may have important endogenous functions in fish. The substrate selectivity and the role in xenobiotic toxicity of CYP3A27 are not yet known. CYP3A30 another CYP3A subfamily protein found in aquatic species was isolated and sequenced from killifish [24]. The sequence of CYP3A30 is 77% identical to that o f CYP3A27. CYP 3A Inhibition The inhibition of enzyme activity is one of the major regulatory devices of l iv ing cells, and one of the most important diagnostic procedures of enzymology. Three types of enzyme kinetic inhibition patterns are commonly observed: competitive noncompet i tive and uncompetitive. The use of chemical inhibitors is one of the common strategies employed in determining whether cytochrome P450s are involved in the hepatic and extrahepatic metabolism of drugs, xenobiotics and endogenous compounds Selecti v e chemical inhibitors play an important role especially in elucidating the contribution of a particular cytochrome P450 enzyme in catalyzing the metabolism of xenobiotics [25 ]. CYP3A enzymes are inhibited by a variety of compounds, including troleandomycin (TAO) clarithromycin, erythromycin gestodene ketocona z ole naringenin and 6 7-dihydroxy-bergamottin [26]. The only common features are their

PAGE 23

12 lipophilicity and relatively large molecular size. Several mechanisms of inhibition are possible, with some compounds exhibiting more than one-type, e.g., erythromycin [27]. (1) Rapid reversible inhibition: Direct, rapid reversible binding of an inhibitor or its metabolite to CYP3A. Reversible inhibition has been found to result in either competitive or noncompetitive inhibition, the extent of which is determined by the relative binding constants of substrate and inhibitor for the enzyme and by the inhibitor's concentration. (2) Formation of MI-complexes (quasi-irreversible inhibition): N-Alkyl-substituted compounds--a common feature of many CYP3A drugs--often show reversible inhibition, and an even greater effect is observed after preincubation with a metabolically competent in vitro preparation. This is due to oxidation of the inhibitor to form a nitrosoalkane species that forms a slowly reversible complex (Ml-complex) with reduced heme in the CYP3A molecule Such compounds include macrolides like TAO, oleandomycin, erythromycin, clarithromycin and roxthromycin [28]. Formation of an MI-complex may, however, be difficult to demonstrate in vitro because of its dependency on the rapid and relatively efficient generation of the causative metabolite. (3) Irreversible, mechanism-based (suicide) inhibition: The ingestion of 6,7-dihydroxy bergamottin, a furanocoumarin, can markedly inhibit the first-pass metabolism of CYP3A substrates. This effect was recently found to be associated with autocatalytic destruction of intestinal CYP3A both in vitro and in vivo [29]. The mechanism of suicide inhibition presumably involves CYP3A-mediated formation of a reactive

PAGE 24

13 metabolite(s) that covalently binds to the enzyme in a fashion leading to its inactivation In mechanistic terms reversible interactions arise as a result of competition at the CYP active site and probably involve only the first step of the CYP catalytic cycle On the other hand chemicals that act during and subsequent to the oxygen transfer step are generally irreversible or quasi-irreversible inhibitors Quasi-irreversible and irreversible inhibitors require at least one cycle of the CYP catalytic process, and are thus characterized by both ADPHand time-dependent inhibition. Experimentally mechanisms of inhibition of inhibitors could be assessed initially by comparing their inhibitory effects obtained in the presence and absence ofNADPH during a preincubation period Inhibitors for CYP3A have been found that are drugs antibiotics preservati v es poisons and toxins. Several human hepatic CYP3A substrates, erythromycin testosterone terfenadine midazolam and nifedipine mutually inhibited the metabolism of each other with complex mechanisms (30 31]. Troleandomycin (TAO) has been shown to inhibit CYP3A enzymes through both competitive inhibition and formation ofMI complex It was found to be as effective inhibitor of CYP3A enzymes in microsoma l fractions from goat and cattle and in a cell-line expressing bovine CYP3A (32]. Both human CYP1A2 and CYP3A4 play important roles in bioactivation of aflatoxin Bl (AFB 1 ); TAO showed potent and specific inhibition of AFB 1 epoxidation in CYP3A but not CYP1A2 microsomes (33]. In pharmacokinetic tests of drug bioavailability TAO and ketoconazole have widely been used as selective inhibitors of CYP3A (34,35]. Calcium channel blockers nicardipine verapamil and diltiazem were shown to inhibit human

PAGE 25

14 hepatic CYP3A via at least in part, quasi-irreversible inhibition and such findings provide a rational basis for the pharmacokinetically significant interactions reported when these calcium channel blockers were co-administered with agents that are cleared by CYP3A-mediated pathways [36). Chemical inhibitors may also be useful in identifying the individual P450 enzymes responsible for the metabolism of xenobiotics and endogenous lipophilic compounds in non-mammalian species such as fish. Several inhibitors of mammalian P450s have been employed to inhibit fish P450s [37). Ellipcine and a-naphthoflavone were found to inhibit benzo(a)pyrene hydroxylase activity ofliver microsomes from flounder (Plat i chth y sflesus) [38,39). Aminoanthracene has been proposed as a mechanism-based inactivator of CYPlA in channel catfish [40), but its selectivity as a P450 inhibitor is not known. In a study of Miranda et al to evaluate chemical inhibitors of trout cytochrome P450s three monooxygenase activities, lauric acid ( co-1 )-hydroxy lase (LA-OH) 7 12-dimethylbenz(a)anthracene hydroxylase (DMBA-OH), and progesterone 6P-hydroxylase (PROG-OH) activities were used as functional markers for trout hepatic CYP2Kl, CYPlAl and CYP3A27 respectively [41). At 100 M concentration the reversible inhibitors ketoconazole, miconazole and clotrimazole were most potent in inhibiting LA-OH activity. The global inhibitors metyrapone chloramphenicol and allylisopropylacetarnidem had very little inhibitory effect on trout LA-OH and DMBA OH activities. Troleandomycin, a CYP3A inhibitor in mammals did not affect PROG OH acti v ity catal yz ed by trout CYP3A27. None of the three enzyme activities was selectively inhibited by any of the mammalian chemical inhibitors used at a concentration of 100 M. These results suggest that inhibition data from mammalian studies could not

PAGE 26

15 be directly extrapolated to fish species and that care must be observed when mammalian P450 inhibitors are used to determine the participation of P450s in the metabolism and toxicity of xenobiotics in nonrnamrnalian species. CYP 3A Induction CYP3A inducers include a broad range of steroids and antibiotics Early stud i es of rat liver CYP3A enzyme induction made the important but seemingly paradoxical observation that both glucocorticoids (such as dexamethasone, DEX) and antiglucocorticoids (such as pregnenolone 16a-carbonitrile PCN) induce these enzymes at the transcriptional level [ 42]. Both this finding and the requirement for a relatively high glucocorticoid concentration for CYP3A induction were recognized as inconsistent with the classical glucocorticoid receptor playing a major role in the CYP3A induction response. It was recently found that CYP3A genes are transcriptionally activated by foreign chemicals through a PXR-dependent mechanism [43]. PXR (pregnane X receptor) an orphan nuclear receptor was believed to mediate the CYP3A induction PXR, together with other four P450-regulating nuclear receptors CAR, PP AR LXR and FXR, share a common heterodimeri z ation partner, retinoid X-receptor (RXR) When t he l i gand binds to PXR the nuclear receptor heterodimerizes with RXR and efficiently transactivates the response elements present in CYP3A genes (Figure 1-4) Important species differences in the induction response have been described [ 44]. Most notably while rat rabbit and human CYP3A genes are all inducible by dexamethasone the anti glucocorticoid PC is an efficacious CYP3A inducer in the rat but not in humans or rabbits. By contrast the antibiotic rifampicin is an excellent CYP3A inducer in humans and rabbits but not in the rat. Transfection studies carried out in rat and rabbit

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16 hepatocytes and u t ili z ing CYP3A constructs containing DEX-responsive regulatory elements derived fr om rat CYP3A23 rabbit CYP3A6 and human CYP3A4 genes demonstrated that the species-specific induction responses are due to the different response element on CYP3A genes. In the case of rat CYP3A23 the dexamethasone responsive sequence contains a DR3 motif ( direct repeat separated by 3 bp; AGTTCA N 3 AGTTCA) that is also present in rat CYP3A2 whereas in human CYP3A4 gene the response element contains an unusual ER6 motif ( everted repeat separated by 6 bp ; TGAACT6 -AGGTCT) that is conserved in human CYP3A5 and rabbit CYP3A6 [ 45]. Pregnane-X-recep t ors have recently been cloned from human, mouse rat rabbit and chicken However mouse PXR and human PXR share only ~75% amino acid sequence identity in their COOR-terminal ligand-binding domain region (vs 96% identity between their DNA-bindin g domains) and this apparently results in sigificant differences in ligand-binding specificities : human PXR but not mouse PXR is highly activated by compounds that preferentially induced human CYP3A genes such as rifampicin while mouse PXR but not human PXR exhibits the strong response to PCN that characteri z es mouse CYP3A gene induction Thus the species-dependent ligand specificity for CYP3A induction seen in v ivo can be explained by the corresponding ligand specificity of each species PXR receptor. Other CYP3A inducers and PXR activators include anti hormones belonging to several steroid classes the organochlorine pesticide chlordane and various nonplanar chlorinated biphenyls [ 46]. Both the facts that PXR is responsive to steroids belonging to several distinct classes (prenanes, estrogens and corticoids ) and that many CYP3A enzymes catalyze steroid 6P-hydroxylation reactions suggested the

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17 mechanism of cross-talk between PXR-dependent CYP3A induction pathways and intracellular signaling pathways involving endogenous hormones (Figure 1-5) Pre-exposure o f fish to mammalian CYP3A inducers however, has yielded rather inconsistent results In juvenile rainbow trout, levels of CYP3A in hepatic rnicrosomes were slightly elevated by steroids i.e. cortisol and PCN [ 4 7] Administration of 25 or 100 mg/kg i.p doses of PCN to sexually immature rainbow trout caused an increase of hepatic BND (benzphetamine N-demethylase) and ECOD (7-ethoxycoumarin deethylase) activities but had no effect on the total P450 content or on EROD ( 7ethoxyresorufin O-deethylase) activity [ 48]. By contrast, treatment ofrainbow trout with a single i.p. dose of PCN at 25 mg/kg did not alter the hepatic microsomal activities of different fluorescent substrates or hepatic P450 levels [O]. Moreover DEX or PCN treatment failed to affect hepatic CYP3A-like protein levels in rainbow trout [50 51]. These discrepencies in responsiveness to various types of mammalian CYP3A inducers reflect important differences in CYP3A regulation in different taxa The content of CYP3A in some teleost fish appears to be influenced b y the composition of the diet suggesting that CYP3A may be involved in the metabolism of dietary natural products as well as anthropogenic xenobiotics [52].

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18 @ PXR Xenochernical ~ RXR Retinoids / CYP 3A Transcrips t io n 5 3 (AGGTCA-N x ) 2 Figure 1-4 Role of PXR in CYP3A gene induction. Shown is the structure of a PXR-RXR heterodimer bound to two copies of a hexameric DNA response element based on the sequence of AGGTCA spaced by X nucleotides The hexameric repeat can be arranged as a DR or ER motif. (Adapted from Waxman DJ. P450 gene induction by structurally diverse xenobioticals: Central role of nuclear recptors CAR PXR AND PP AR. Arch B i och e m B i onh v s. 369(1 ) : 11-23 1999) Foreign Chemical --------Hormones, growth factors Receptors and transcription factors 1 Induction CYP3A gene expression For e ign chemical metabolism [ CYP3A enzymes ] Metabolism of endogenous steroids fatty acids prostanglandins Figure 1-5. CYP3A gene induction: Cross-talk between foreign chemical and endogenous regulator pathways. (Adapted from Waxman DJ P450 gene inductio n by structurally di v erse xenobioticals: Central role of nuclear recptors CAR PXR AND PPAR Ar c h Bioch e m. Bioph y s 369(1):11-23 1999)

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19 CYP3A Stimulation A unique characteristic of the CYP3A subfamily is their ability to be activated by certain compounds Flavonoids, e.g., 7,8-benzoflavone (a-naphthoflavone, a-NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17P-estradiol, amitriptyline, and most notably aflatoxin B-1 [53]. It was reported that CYP3A4-catalyzed phenanthrene metabolism was activated by 7,8-benzoflavone and that 7,8-benzoflavone served as a substrate for CYP3A4 Kinetic analyses of these two substrates showed that 7,8-benzoflavone increased the V max of phenanthrene metabolism without changing the KM and that phenanthrene decreased the V max of 7,8-benzoflavone metabolism without increasing the KM. These results suggest that both substrates ( or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two different molecules can be simultaneously bound to the same P450 active site [54]. Quinidine and hydroquinidine decreased KM and V max of meloxicam hydroxylation, which was consistent with a mixed type activation. Meloxicam in turn, decreased both KM and V ma x of quinidine metabolism by CYP3A4, indicating an uncompetitive inhibition mechanism [55]. These results also support the assumption that CYP3A4 possess at least two different substrate-binding sites. The mechanism of cytochrome P450 activation has not been explored to the same depth as induction or inhibition phenomena. Enhancement of aniline para-hydroxylation

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20 by acetone was the first reported cytochrome P450 activation interaction [56]. Based on studies with liver microsomal fractions of the dog rabbit, mouse and rat, it was proposed that acetone affected either the formation of the peroxy anion complex of cytochrome P450 or steps beyond this (such as the formation of the oxene complex) because cumene hydroperoxide-dependent hydroxylation of aniline was stimulated by acetone [57]. Huang e t al [58] demonstrated that the stimulatory effect of 7 8-benzoflavone on benzo(a)pyrene metabolism in rabbit liver microsomes was mediated by a different mechanism than that observed with acetone. The effect of 7 ,8-benzoflavone on benzo(a)pyrene metabolism was thought to be a result of enhanced interactions between cytochrome P450 and cytochrome P450 reductase A third mechanism of acti v ation was proposed by Johnson et al. [59], who reported that the stimulatory effect of naphthoflavone on rabbit CYP3A6 was a consequence of an allosteric effect as shown by an increase in the P450 binding affinity for the substrate Shou et al. [54] have shown that t here was mutual activation between phenanthrene and 7,8-benzoflavone and suggested t hat the two molecules simultaneously occupy the active site, thereby altering active site geometry and oxidation efficiency. In summary, it appears that cytochrome P450 activation may occur by several mechanisms. Epoxide Hydrolase (EH) Carcinogenic polycyclic hydrocarbons such as benzo(a)pyrene are oxygenated in cytochrome P450 catalyzed reactions to form epoxides. Due to their electronic polarization and ring tension epoxides are often chemically reactive Consequently, such metabolites can bind covalently to nucleophilic groups in many tissue constituents

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21 including macromolecules such as RNA, DNA and proteins. Epoxide hydrolase (EC3.3 2.3) catal yz es the trans-addition of water across the oxirane ring of the epoxides to chemically less reactive transdihydrodiols [60]. The reaction is stereoselective and regioselective. Several distinct microsomal and cytosolic isoenzymes exist. Usually microsomal epoxide hydro lase catalyze the hydration of cis-epoxide while the cytosolic EH catalyzes the hydration of trans-epoxide. Although the metabolite dihydrodiol is less toxic, it might be further metabolized by P450 to the ultimate dihydrodiol-epoxide carcinogens. A classic example is the metabolic activation ofbenzo(a)pyrene to 7 8dihydrodiol-9 10-epoxide (BPDE), which proceeds via 7,8-epoxide followed by epoxide hydrolase and another oxidation step [61] In reactions with benzo(a)pyrene 4 5-oxide and styrene 7 8-o x ide as substrates, the general trend of microsomal epoxide hydrolase activity observed was fish < amphibia < birds < rodent < larger mammals [62] Epoxide hydrolase activity was found in several marine species, including spiny lobster shrimp, fiddler crabs and stingray [63]. It was shown that EH activity with styrene oxide as substrate was similar in intestinal and hepatic microsomes from catfish [14] Phase II Enzymes Glutathione S-Transferase (GST) The glutathione S-transferases (GST) are a ubiquitous family of isozymes w h ose primary functions are involved in the biotransformation and disposition of many tox i c substances The chemical function of the enzyme is to catalyze the nucleophilic add i tion of the thiol of glutathione (y-L-Glu-L-CysGly) to electrophilic acceptors the first st e p in

PAGE 33

22 mercapturic acid biosynthesis (Figure 1-6). In addition, it is proposed that the proteins also serve as depots for the storage of toxic substances, as high capacity steroid-binding i glutathione conjugate 0 s ~~'--./COO" Methionine or other receptor oo~ + H3/ ~SCH3 0 X H2y gamma-glutamyl transferase (transpeptidase) gamma-glutamyl peptide xteinylglycine dipeptidase j recycling of glutamate for GSH biosynthesis + H 3 ~COff X I s H,~coo CoASAc X CoASH cysteine conjugate N-acetyltransferase X I s~ coo O CH 3 mercapturic acid (N-acetylcysteine conjugate) Figure 1-6. Mercapturic acid biosynthesis. urinary excretion

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23 proteins, as heme-binding and transport proteins. Both the abundance of the enzymes, comprising 3 to 10% of the soluble protein in liver, and the high concentrations (5 to 10 mM) of glutathione attest to the importance of glutathione S-transferase in the maintenance of health [64]. All eukaryotic species possess multiple cytosolic and membrane-bound GST isozymes. The cytosolic enzymes are much more important and encoded by at least six distantly related gene families ( designated class alpha, mu, pi, sigma, theta and zeta GST). The quaternary structure of cytosolic GSTs shows that the enzymes occur as binary combinations of subunits, including both homodimers and heterodimers. The membrane-bound GST (microsomal GST) is a trimeric protein, structurally unrelated to the cytosolic enzymes [65]. The glutathione S-transferases (GST) are an important phase II enzyme system in the detoxification of electrophilic alkylating agents. As a family of isozymes, the enzyme system is capable of handling a variety of electrophilic compounds, both from exogenous and from endogenous origins. Conjugation with GSH can generally be regarded as a detoxification pathway, although several compounds are known to be activated through this reaction [66]. The glutathione S-transferases catalyze the nucleophilic addition of GSH to electrophiles including aryl and alkyl halides, sulfate esters, phosphate and phosphorothioate triesters, nitrate esters, oxiranes, olefins, lactones, organic peroxides, disulfides and thiocyanates and quinones. The substrate selectivities exhibited by various isozymes overlap considerably but are nonetheless distinct. Most of the above reactions can be classed as simple nucleophilic displacements or Michael additions to unsaturated systems.

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24 GS Ts can be induced by a variety of chemical compounds, including conventional inducers of drug-metabolizing enzymes, such as phenobarbital, 3-methylcholanthrene, and TCDD. GST-pi has been shown to be a reliable marker for rat hepatocarcinogenesis [67]. GST was found in both marine and freshwater fish species. It has been indicated that GST usually shows higher activity than epoxide hydrolase in both hepatic and extrahepatic tissue in marine fish [63]. Rainbow trout has GST activity with CDNB( chloro-2,4-dinitrobenzene) in liver and intestine, while only intestine has substantial glutamyl transpeptidase activity A cluster of three GST genes, GSTA, GSTAl, and GSTA2, was isolated from marine flatfish, plaice [68]. GST-A expresses in plaice intestine as well as in liver. It was also indicated that expression of GST-A mRNA was increased in plaice intestine by pretreatment with ~-naphthoflavone (BNF). A pi-class GST was isolated from catfish intestinal mucosa with N-terminal sequence homology >63% to mammalian pi-form GST isozymes [69]. GST including this pi-class GST play an important role in the intestinal biotransformation of the epoxide and diol-epoxide metabolites ofbenzo(a)pyrene formed in catfish intestine. Fish GST was also shown to be inducible by P AHs. Cytosolic GST activity towards CDNB was elevated approximately three to four-fold in intestine and liver of mummichog, collected from a creosote-contaminated site. The intestinal GST activity was even higher than liver GST, supporting the importance of intestinal metabolism of foreign compounds [70]. GST activity was slightly induced in intestinal, but not hepatic cytosol of catfish treated with BNF at 10 mg/kg diet level relative to chow controls. Yet

PAGE 36

25 this induction showed no further increase with higher dose ofBNF at 100 mg/kg die t [14]. Sulfotransferase {ST) The cytosolic sulfotransferases catalyze the transfer of the sulfuryl group from 3 phosphoadenosine 5 '-phosphosulfate to nucleophiles such as alcohols, phenols and amines. The M-form of the enzyme is thermolabile (TL form) catalyzing the sulphate conjugation of micromolar concentrations of dopamine and other phenolic monoamines The other form P-form is more thermostable {TS form) and catalyzes the sulphate conjugation of micromolar concentrations of simple phenols such as p-nitrophenol. Both forms of the enzymes are particularly active in the intestinal wall but are also widespread in the body including the platelet. Sulfation is one of the major phase II conjugation reactions for drugs and environmental chemicals as well as for endogenous compounds such as steroids and monoamine neurotransmitters [71]. The major physiologic consequences of the conjugation of a drug or xenobiotic with a charged sulfate moiety are increased aqueous solubility and excretion. Although the major role of sulfation is detoxification in some instances sulfate conjugation results in the bioactivation of a compound to a reactive electrophilic species since the sulfate is such a good leaving group. The electrophile is capable of covalently binding DNA and causing a mutagenic, teratogenic, or carcinogenic response. Metabolic activation of 7 12-dimethylbenzanthacene has been demonstrated to occur by oxidation to the 7-hydroxymethyl-12-methylbenz(a)anthracene followed by sulfation and alkylation of DNA following loss of sulfate anion [72 73].

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26 Human intestinal mucosa contains forms of phenol sulfotransferase similar to those in other human tissues such as brain, liver and platelet [74]. In rat hepatocyte culture sulfotransferase expression was negatively regulated by xenobiotics such as PB like CYP2B / 3A inducers or AhR agonist CYPlA inducers [75]. In guppy and medaka after water-borne exposure to the procarcinogen 2-acetylaminofluorene(AAF), the major pathway for bioactivation was shown to be N-hydroxylation followed by sulfation. AAF treated guppies had higher ST activity than controls, but UGT activity was reduced or unaffected by AAF exposure [76]. Sulfate conjugate was found as a metabolite of benzo(a)pyrene in an isolated perfused in situ catfish intestinal preparation [77]. The biotransformation is via oxidation by CYPlA and rearrangement of the epoxide to the phenolic metabolite In another study with catfish it was shown that ST activities with BaP phenols was high in intestine suggesting that low concentrations of hydroxy lated polycyclic aromatic hydrocarbon would be readily conjugated in catfish intestine. UDP-Glucuronosyltransferase (UGT) The UDP-glucuronosyltransferases are a group of membrane-bound proteins responsible for the transfer of the glucuronyl group from uridine 5 diphosphoglucuronate to a large number of different nucleophilic acceptors. The enzymes are located primarily in the endoplasmic reticulum of eukaryotic cells, catalyzing the glucuronidation o f a tremendous number of lipophilic molecules having nucleophilic functional groups of oxygen nitrogen sulfur, and carbon. Substrates for glucuronidation are typically small hydrophobic molecules that are termed aglycones (lacking carbohydrate) A wide variety of endogenous and exogenous compounds are

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27 glucuronidated, including bilirubin, steroid hormones bile acids, biogenic amines, fat soluble vitamins, environmental toxins and therapeutic drugs Phenol, dihydrodiol and quinol metabolites of polycyclic aromatic hydrocarbons are substrate for the microsomal and purified UGTs [78]. Glucuronidation is generally considered to be a detoxifying mechanism that alters the physiological and pharmacological activities of chemicals within the body. In some cases however covalent addition of glucuronic acid may increase the biological activity of an aglycone [79]. The UGT proteins can be conceptually divided into two doma i ns with the amino-terminal half of the protein demonstrating greater sequence divergence between isoforms This region apparentl y determines aglycone specificity The carboxyl-terrninal half which is more conserved in sequence between different isoforms is believed to contain a binding site for the cosubstrate UDP glucuronic acid (UDPGA). Multiple isoforms of UGT have been found in aquatic species [80]. In a UGT study in plaice, phenol UGT activity was found to be ubiquitous in hepatic renal i ntestinal and branchial tissues and was induced by 3-MC and Aroclor. The glucuronidation of testosterone was restricted to liver and intestine while conjugaf on of bilirubin was expressed solely in hepatic tissue [81]. In the southern flounder BaP7 8diol given by gavage was glucuronidated and then transported as such to liver where that was efficiently excreted into the bile In vitro studies showed that flounder liver and intestine had similar UGT activities [82]. In channel catfish glucuronide of BaP-9-OH was readily transported intact from the intestinal lumen to the systemic circulation. 3OH-BaP was extensively biotransformed to BaP-3-glucuronide in intestinal mucosa [83]. UGT activities with BaP phenols were high in the catfish intestine, suggesting that lo w

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28 concentrations of hydroxy lated polycyclic aromatic hydrocarbon would be readily conjugated in catfish intestine. UGT activities with 3-,7and 9-hydroxy-BaP in catfish intestine were not induced by treatment with BNF and in fish receiving the higher dose activity with 7and 9-hydroxy-BaP was lower than in fish fed other diets. In vitro studies showed that BNF could inhibit UGT activity, suggesting the residues ofBNF retained in intestinal cells after BNF treatment in diet could directly inhibit UGT activity [14]. Treatment with 3-methylcholanthrene, 10 mg/kg diet did induce UGT in catfish intestinal rnicrosomes [James unpublished data]. 3a-Hydroxy-Steroid Dehydrogenase (3a-Oxido-Reductase) Testosterone homeostasis is crucial for normal growth, reproduction, and development in vertebrates [84]. In teleost fish, testosterone serves as a precursor to 11ketotestosterone and 17~-estradiol. These hormones play an important role in sexual maturation in male and female fish, respectively. More than one organ contributes to the metabolic inactivation and elimination of testosterone [85,86]. Enzymes that contribute to the metabolic elimination of testosterone include cytochromes P450, oxido-reductases, and transferases [87]. Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the short-chain dehydrogenase/reductases (SRSs) or the aldo-keto reductases (AKRs). 3a-hydroxysteroid dehydrogenase (3a-HSD) was found in both microsomal and cytosolic liver fractions In rodents, 3a-Hydroxysteroid dehydrogenase showed higher activities in cytosolic fraction than in microsomes using

PAGE 40

29 dihydrosteroids as substrates [88,89]. By comparison rat hepatic microsomal 3a hydroxysteroid dehydrogenase activity was 12-fold higher than cytosolic 3a hydroxysteroid dehydrogenase in human [90]. It was suggested that the major pathway of DHT (dihydro-testosterone) metabolism in human liver involves 3a-hydroxysteroid dehydrogenase reduction in the liver followed by subsequent glucuronidation and clearance via the kidney [91] Human hepatic 3a-HSD also plays a critical step in th e synthesis of bile acids and is responsible for the production of 5P-cholestane-3a,7a-diol which is a committed precursor of bile acids. In steroid target tissues the production of 5a / 5P-tetrahydrosteroids catalysed by 3a-HSD is not without consequence. In the human prostate, 3a-HSD can regulate the occupancy of the androgen receptor. It catalyses t he reduction of 5a-dihydrotestosterone, a potent androgen to 5a-androstane-3a, 17P-diol a weak androgen and is positioned to regulate normal and abnormal androgen-dependent growth of this gland [92]. By contrast in the central nervous system 3a-HSD can regulate the occupancy of the y-aminobutyric acid (GABA) A receptor by converting 5a dihydroprogesterone into 3a-hydroxy-5a-pregnan-20-one (allopregnanolone) a potent allosteric effector of the GABA A receptor [93 94]. In the presence of GABA allopregnanolone w ill potentiate GABA A -mediated chloride conductance. As a resul t 3a HSD is responsible for the production of anxiolytic steroids, and decreased activity in this pathway has been implicated in the symptoms of pre-menstrual syndrome [95]. Thus 3a HSD isoforms regulate the occupancy of both a nuclear receptor (androgen receptor ) and a membrane-bound chloride-ion gated channel (GABA A receptor) and may ha v e profound effects on receptor function [96].

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30 Testosterone metabolites produced by juvenile and adult fathead minnows included 4-androstene-3, 1 7-dione ( androstenedione ), 17P-hydroxy-5a-androstan-3-one (5a-dihydrotestosterone), 5a-androstane-3a, 17P-diol (3a-androstanediol), 5a androstane-3p, 17P-diol (3 P-androstanediol), l 7p-hydroxy-4-androstene-3, 11-dione (11ketotestosterone ), 16P-hydroxy-4-androsten-3-one (16P-hydroxytestosterone) and 6P hydroxy-4-androsten-3-one (6P-hydroxytestosterone) [97]. Testosterone and its metabolites were eliminated from minnows in both free and conjugated form Adult females eliminated androstanediols at a significantly greater rate than did males, suggesting higher 3-oxidoreductase activities in female fish than male. Prehepatic Metabolism and Bioavailability The primary function of the intestine is to absorb nutrients and water This is achieved by mixing food with digestive enzymes to increase the contact of foodstuffs with the absorptive cells of the mucosa. In addition to this fundamental role, another function of the intestine arises from the fact that it also provides a major route for exposure to xenobiotics via food and liquid, and secondarily by swallowing inhaled xenobiotics after clearance from the tracheobronchial tree. In human and different animal species, the percentage weight of intestine is usually significantly smaller than the liver. When a xenobiotic exhibiting systemic effects is administered orally, its fate is usually as follows : it comes into contact with the contents of the gastrointestinal system, is dissolved in intestinal juices, and then brought into contact with intestinal epithelium. It is then absorbed through the gut wall and the enterocytes lining the gut wall, and

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31 transported by the portal veins through the liver before reaching the systemic circulation and hence different parts of the body. When the same drug is given intravenously it enters the systemic circulation and is distributed through out the body before reaching the liver for the first time. The extent of systemic availability is described with the pharmacokinetic term bioavailability (F) F is theoretically determined in the following way: the drug is administered to the same individual as a single dose intravenously and orally on separate occasions; drug concentrations in serum ( or plasma, blood) are measured after each dose and used to determine the area under concentration curve (AUC) from the time Oto "infinity Absolute bioavailability of the oral dosage form is F orai= AUC 0 rai/ AUC i.v If the oral and i v doses are unequal a correction for the dose difference must be made. The most significant factor influencing the effect or toxicity is not necessarily the dose but rather the concentration of a xenobiotic at the site of action. The fraction of a chemical that reaches the systemic circulation is of critical importance in determining effect or toxicity The incomplete bioavailability after oral administration may principally be a result of an incomplete absorption from the intestine or metabolism of the drug before it reaches the systemic circulation (presystemic metabolism). Presystemic metabol i sm can principally take place anywhere before the drug reaches the systemic circulation i.e. in intestine and in liver The metabolism of xenobiotics before entering the systemic circulation is referred to as first-pass metabolism. This intestinal and hepatic first-pass biotransformation alters the physico-chemical properties of x enobiotics and is likely to change the bioavailability The first-pass metabolism could substantially prevent many xenobiotics from being distributed throughout the body. However the biotransformation could also potentially activate some xenobiotics. It has

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32 been widely believed that the liver is the major site of such first-pass metabolism because of its size and its high content of drug-metabolizing enzymes. If large amounts of a chemical are ingested (e.g., therapeutic drugs), it is usually true as the capacity of the intestinal biotransformation is likely to be overwhelmed. The compounds will be absorbed and pass to the liver, which has higher capacity for biotransformation than the intestine However, recent clinical studies have indicated that the intestine contributes substantially to the overall first-pass metabolism of cyclosporin, nifedipine, rnidazolarn, verapamil, and certain other drugs [98]. Some studies suggested that the role of intestinal metabolism of these drugs is quantitatively greater than that of hepatic metabolism in overall first-pass effect [99]. The contribution of intestinal enzymes to xenobiotic biotransformation is particularly important when relatively low concentrations of chemicals are present, as is normally the case for high potency drugs and environmental chemical pollutants, since the low concentrations of xenobiotics are readily metabolized in the intestine and leaving little to pass to the liver for further metabolism Almost all of the xenobiotic-metabolizing enzymes present in the liver also are found in the intestine, although their total amounts are generally much lower in the latter due to the lower weight of intestine relative to liver. Unlike the liver in which the distribution of P450 enzymes is relatively homogeneous, the distribution of these enzymes is not uniform either along the length of the small intestine or along the villi within a cross-section of mucosa Longitudinal distribution of total cytochrome P450 and i t s activity have been measured in human intestine [100]. Both the content and activity of cytochrome P450 was higher in the proximal than that in the distal small intestine. The major enzymes catalyzing drug-metabolizing reactions in the liver and the GI tract belong

PAGE 44

33 to the microsomal CYP3A subfamily. CYP3A4 is predominantly expressed in human liver and intestine where it comprises approximately 30 to 50% of the total cytochrome P450 population in these tissues [101]. Many of the drugs with significant first-pass metabolism, like cyclosporine midazolam, nifedipine, and terfenadine, are substrates of CYP3A. CYP3A activity is prone to induction or inhibition which may cause clinically significant drug interactions. Whenever two or more drugs are administered concurrently the possibility of drug interactions exists. The ability of a single CYP to metabolize multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result o f the induction of se v eral human CYPs following long-term drug treatment. CYP3A i s highly inducible in humans by synthetic glucocorticoids (dexamethasone) macrolide antibiotics (rifampicin) and phenobarbital [102]. It has been demonstrated that an important cause of incomplete bioavailability of many drugs which were earlier thought to be primarily poorly absorbed is prehepatic metabolism in the GI tract mainly by CYP3A subfamily of enzymes. Grapefruit juice a beverage consumed by the general population is an inhibitor of the intestinal cytochrome P450 3A4 system. A 47 % reduction in intestinal CYP3A4 concentration occurs within 4 hours of the ingestion of grapefruit juice and grapefruit juice maintains a bioavailability-enhancing effect for up to 24 hours. Grapefruit juice acts on the CYP system at the intestinal level not at the hepatic level [103]. Drugs are not the only subgroup ofxenobiotics that function as substrates for activation or deactivation by biotransformation processes including CYP3A-catalyzed reaction These enzymes also play a vital role in the biotransformation of such exogenous compounds as pesticides carcinogens and other environmental pollutants Intestinal

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34 CYP3A4 enzymes were shown to activate dietary aflatoxin B 1 to reactive metabolites that form macromolecular adducts within enterocytes [104]. It is logical for toxicologists to look for evidence ofbiotransformation capabilities in the first line of defense against ingested toxins or carcinogens, the intestinal mucosa In summary, in vitro and in vivo data have clearly demonstrated that the small intestine plays a significant role in first-pass metabolism in certain situations, especially when a small oral dose is given. The induction or inhibition of intestinal biotransforming enzymes might potentially alter the bioavialability and metabolism pathway of the chemical exposed. Both phase I and phase II biotransforming-enzymes have been found in fish liver and intestine. As in mammals, the major organ involved in xenobiotic metabolism in fish seems to be the liver. Yet, microsomal cytochrome P450 and cytochrome P450dependent activities were found in extrahepatic organs in fish, e.g., kidney, upper small intestine, gonad and brain [105].

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35 Hy po thesis The hypotheses of the present project are: (1) CYP3A is constitutively expressed in catfish intestine ; (2) CYP3A enzyme is expressed regionally along intestine of catfish; (3) testosterone is hydroxylated at different positions by different P450 isozymes in catfish intestinal microsomes; (4) the i n vitro testosterone 6~-hydroxylation activity by catfish intestinal CYP3A enzymes is inhibited by mammalian CYP3A inhibitors e.g. erythromycin troleandomycin and ketonconazole and by general P450 inhibitors, e g. metyrapone and SKF 525A but not inhibited by specific CYPlA inhibitor e.g ., a. naphthoflavone; (5) CYP3A expression in catfish intestine is under dietary modulation and is inducible by mammalian CYP3A inducers e.g., rifampicin and pregnenolone 16a. carbonitrile; (6) the intestinal CYP3A enzyme plays an important role in the biotransformation and bioavailability of both endogenous and exogenous compounds, including environmental pollutants which the wild catfish are continuously exposed to.

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CHAPTER2 MATERIALS AND METHODS Chemicals [ 414 C]-testosterone and [ 414 C]-progesterone were purchased from DuPont NEN (Boston MA) Authentic steroid and metabolite standards were obtained form Steraloids, Inc (Wilton, NH) Benzo(a)pyrene and 3-hydroxy benzo(a)pyrene were purchased from ChemSyn, through the NCI Chemical Carcinogen Repository. Western blotting kit was from Amersham Life Sciences, Inc (Arlinton heights, IL) Ketoconazole and proadifen (SKF-525A) hydrochloride were gifts from Janssen Pharmaceutica, Inc. (Piscataway, NJ) and Smith Kline & French Labs (Philadelphia, PA), respectively. 2methyl-1, 2-di-3-pyridyl-l-propanone (metyrapone) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Erythromycin, troleandomycin, tricane and NADPH were obtained from Sigma Chemical Co. (St. Louis MO) All HPLC and microsomal preparation supplies were of the highest grade available from standard commercial sources. Instruments The following instruments were used for this study. The gradient HPLC system was equipped with a Beckman controller 125 solvent module, an analytical Beckman UV detector model 166 and an INUS ~-RAM detector. Shimadzu UV-VIS spectrophometer model UV-160U, Perkin-Elmer fluorescence spectrometer model LS-3B 36

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37 and Chromato-vue UV detector were used. Liquid scintillation counters used were Packard Tri-Carb liquid scintillation system model 460CD and Beckman liquid scintillation system model LD 500TD. Beckman ultracentrifuge model L8-80M and DuPont Sorvall centrifuge model RC2-B were used. Animals and Pretreatment Groups of 4-8 catfish each (800-1300 g) were fed a commercially available Silver Cup chow (Silvercup Nelson & Sons Inc., Murray UT) or a semi synthetic purified diet (Dyets Inc ., Bethlehem PA) for at least 2 weeks The semi-synthetic purified feed was formulated according to guidelines established for warm-water fish by the National Research Council [106] composed of casein 32% dextrin 29.8% cellulose 19 %, soybean oil 3 %, Menhaden oil 3% gelatin 8% salt and vitamin mix 5%, and choline chloride 0 17 %. For the respective pretreatments of fish both control and treatment groups were acclimated to experimental conditions and maintained on purified diet at least 2 weeks prior to pretreatment. Control animals were maintained on semi-synthetic diet coated with com oil (1 ml com oil/100 g of diet) while for the treatment group the chemica l (TCB or 3-MC) was delivered in com oil applied as a coating on the semi-synthetic diet (1 ml com oi l/ 100 g of diet) Both dietary groups (control and chemical exposure) were maintained on designated experimental diets at 0.5 % of fish body weight/day for specified period prior to sacrifice For the study of inducibility of mammalian CYP3A inducers, 0.03 % (w / w) rifampicin was formulated in the semisynthetic purified diet the treatment group we r e

PAGE 49

38 fed the rifampicin-formulated diet at 3% of fish body weight/day for specified period of time. For the treatment of pregnenolonel 6a-carbonitrile (PCN), the fish were accl i mated and maintained on purified diet for at least 2 weeks prior to the surgical process for oral gavage. PCN (10mg) was dissolved in a mixture of 1 ml of lM KCl and 0.5 ml of com oil, and applied on 5 g of purified diet powder. Distilled water (12 ml) was added to the purified diet to form a slurry. The treated fish were fed this slurry at 0.5% of fish body weight/day by oral gavage for specified duration of exposure. Surgical Procedures for Oral Gavage Fish was anaesthetized with tricane (3-aminobenzoic acid ether ether 6.4 g) in 12 gallon of water. The fish was then taken out of the water and a hole was drilled (5 "/ 32) in the middle of nostrils. Tubings (1.D. 0.106" O.D. 0.138 ) were placed through the hole to the stomach the other end tied to the dorsal fin Fish was kept wet by pumping water on the gills through the surgical process (3 2 g tricane and 3 2 g pottasium bicarbonate was dissovled in 16 gallon of water) Enzymes Preparation Catfish were sacrificed and dissected. Intestines were removed from the stomach and rinsed thoroughly with ice-cold buffer A containing 0.25 M sucrose 5 mM EDTA 0 05 M Tris-Cl (pH 7.4) 0.2 mM PMSF, and lmM dithiothreitol to remove contents. The intestine was bisected evenly into proximal and distal sections, opened, and mucosa! cells removed by scraping into 10 ml of buffer A. Mucosa! cells were weighed and homogenized in 4 v olumes of buffer A. Washed microsomes were prepared from homogenates of each section using the procedure described by James and Little [107].

PAGE 50

39 The homogenates were poured into suita b ly sized Sorvall polycarbonate centrifuge tubes and centrifuged at 13,300 g for 20 min at 4 C to sediment the nuclei, cell debris and mitochondria. The supernatant containing microsomes and cytosol was transferred into polycarbonate ultracentrifuge tubes and then centrifuged at 170,000 g for 45 min at 4 C. The supernatant was the cytosol. The microsomal pellets were resuspended in buffer A, resedimented for preparation of washed microsomes and the microsomal pellets were suspended in 0.25 M sucrose, 0.01 rnM HEPES pH 7.4, 0.1 rnM EDTA, 0.1 rnM dithiothreitol, 0 1 mM phenylmethylsulfonyl fluoride and 5%(v / v) glycerol in a volume equal to half of the weight of intestinal mucosal cells. Livers were removed, rinsed twice with fresh ice-cold buffer B (1.15% KCl, 0.05 M potassium phosphate pH 7.4, 0.2 mM PMSF), patted dry on a paper towel, weighed, minced with scissors and homogenized in 4 volumes of buffer B. The hepatic microsomes and cytosol were prepared in the same way as intestinal cytosol and microsmes Aliquots of the microsomes were flushed with nitrogen and stored at -80C until used in assays. Human CYP3A4 in the baculovirus system (with human NADPH-P450 reductase) was obtained from Gentest Co (Woburn, MA) Rabbit anti-trout polyclonal CYP3A27 antibody was a gift from Malin Celander in Goteberg University and was prepared as described [108,109]. Protein As sa y Lowry protein assay or Bio-Rad protein assay kit both with BSA as standard were used to determine the protein concentration of microsomes and cytosol.

PAGE 51

40 Measurement of Cytochrome P450 Cytochrome P450 content was measured by the method of Omura and Sato, modified by Estabrook [110,111]. A suspension of fish intestinal or hepatic microsomes was prepared containing about 1 mg/ml protein in 0.1 M HEPES, pH 7.4 with 0.1 % Emulgen 911 (Kao Atlas, Tokyo, Japan). The purpose of adding Emulgen was to solubilize the membrane-bound enzymes and prevent the suspension from settling. About 5 mg sodium dithionite was added to the microsomal suspension The suspension was divided into two cuvettes, and the spectra recorded between 500 and 380 nm. CO was bubbled through the sample suspension, and the spectrum was recorded from 500 and 380 nm. The change in absorbance from 490 to 450 run was noted. P450 content was calculated according to the following equation: (Abs 450-Abs 490)/0.09l=nmole P450/ml. (91 mM 1 cm 1 is the absorptivity of CYP under these conditions [110]) Steroid Hydroxylation Assay The steroid hydroxylation assay was described and modified by James and Shiverick [112]. Assay tubes contained 100 mM HEPES pH 7.4, 2 mM MgCli, 1.0 mM NADPH, 0.1 mM [ 14 C] progesterone or [ 14 C] testosterone (800,000 dpm added in 0.01 ml ethanol), and 400 g catfish intestine microsomal protein, all in a final volume of 1 ml. For incubations with human CYP3A4, 40 pmole CYP3A4 was added. Tubes were incubated at 35C for 10 minutes, with the exception of human CYP3A4, which was incubated at 3 7C for 10 minutes. The reaction was stopped by the addition of ice-cold e t hyl acetate (5 ml). The extraction was repeated and the two organic phases combined.

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41 Anhydrous sodium sulfate was added to dry then the extract was e v aporated to dryness under nitrogen. S e paration of the metabolites was achieved by TLC on LK5DF silica gel 150A precoated plates (Whatman Inc. New Jersey) The plates were developed once (progesterone assay) or three times (testosterone assay) in a solvent containing ethy l ether:toluene:methanol:acetone (70 : 38:0 8 : 1) at room temperature Authentic standards were chromatographed on the same plate and visualized by viewing the plates under UV light. In the case of 5a-dihydro-steroids 4-androsten-3a 17P-diol and 4-androsten-3p 17P-diol the developed TLC plates were evaporated with iodine followed by spraying of 70 % methanol. Metabolite bands were located and quantified by electronic autoradiography with Instantimager (Packard Instrument Co ., CT). The 14 C in each metabolite peak was corrected for blank values which usually were negligible. Various concentrations of testosterone were used (5 10, 20 50, 100 and 200 M) to give K M and V max values In the case of testosterone metabolite used for MS analysis 120 mM non radiolabeled testosterone and 1.1 mg catfish intestinal microsomal protein (total assa y volume 11 ml) were used in the incubation. The incubation lasted for 60 min at 35C. The 3a-reduced metabolite was isolated and recovered by TLC using the above solv e nt system Chemical Modulation of Testosterone Metabolism At least five different concentrations of chemical inhibitors were used (1 / 5 to 4x IC so ) to calculate the IC 5 o values. Ethanol (for erythromycin and ketoconazole) aceto n e (for a-naphthoflavone) or DMSO (for troleandomycin) were used as the vehicle controls The total organic solvent was kept under 2 % of the total volume. Proadifen (SKF-525A)

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42 hydrochloride and metyrapone were dissolved in distilled water. For chemicals that have been reported as CYP3A inhibitors through a MI-complex, i e TAO ERM and SKF525A, the inhibitors were preincubated with catfish intestinal microsomes for 30 min at 35C in the presence of 1.0 mM NADPH. The assay was started with the addition of testosterone and was further incubated for another 30 min under the same conditions Testosterone concentration was 30 AHH (Aromatic Hydrocarbon Hydroxylation) Assay The method ofNebert and Gonzalez [113] as previously optimized for catfish intestinal microsomes [14] was used. Tubes contained 0.2 M HEPES-NaOH buffer (pH 7.6), BaP 10 0.5 mg intestinal microsomes and 2 mM NADPH (added last) in a volume of 1 ml. To investigate the effect of ANF varying concentrations of ANF ( 2 -100 M) were added to assay mixture from acetone solution The volume of acetone was 1 % of the total. After incubation at 35C for 15 min the assay was stopped by adding 1 ml ice-cold acetone. Tubes were extracted with 3 x 3 ml heptane the pooled heptane extracts were back-extracted into 3 ml of 1 N NaOH, and the fluorescence of the NaOH extracts measured at an excitation of 392 nm emission 513 nm. Western Blot Analyses Western Blot Analyses of CYPlA Microsomal protein fractions (40 g for intestine 20 g for liver) incubated in sample buffer as recommended by BioRad were resolved in a mini gel format (BioRad) on 4 % stacking gel with 8.5 % resolving gel. Unstained and prestained molecular we i ght

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43 standards in the range of 14 400 to 97,000 (BioRad low molecular weight range) were resolved at the same time as the SDS-treated microsomes Gentest Supersomes TM expressing rat CYPlA were used to develop a standard curve for quantification of the antibody response. Electrophoresis was carried out using a 25 mM Tris / 192 mM glycine / 0.10% SDS buffer at constant voltage of 200 V. Protein was then transferred to nitrocellulose membrane at 40 V in a mini Transblot system (BioRad) using a 25 mM Tris / 192 mM glycine / 20 % v / v methanol/pH 8.3 transfer buffer. The remaining gel was stained with Coomassie blue as an indication of transfer effectiveness lrnmunodetection was carried out using monoclonal antibodies to scup CYP l A (courtesy of Dr. J.J Stegeman) Transblotted nitrocellulose was rinsed in a 20 mM Tris, 500 mM NaCl pH 7.5 buffer and nonspecific binding sites was blocked with 5% (w /v ) dried milk in 20 mM Tris-HCl, pH 7 5, 500 mM NaCl, 0.05% Tween 20 for 1 hour The membrane was washed 4 times with 20 mM Tris 500 mM NaCl 0 05% Tween 20 pH 7.5 buffer. The primary antibody, diluted 1 : 10 000 in 5% (w / v) dried milk in 20 mM T ris-HCl pH 7.5 500 mM NaCl, 0 05% Tween 20 was incubated with the nitrocellulose for 2 hours. The unbound antibodies were washed away and further incubated with a 1: 1000 dilution of secondary antibody (rabbit anti-rat antibody conjugated to horseradish peroxidase) in blocking agent for 1 hour. After washing 4 times, the immunoreactive proteins were detected according to the Amersham Western Blotting kit for chemiluminescent detection and the protein bands were visualized by fluorography on Kodak X-OMAT AR films Fluorograms were subsequently scanned and the protein bands were quantifi e d by scan-analysis densitometry

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44 lmmunochemical Analyses of CYP3A Western blot analyses of catfish intestinal or hepatic rnicrosomes ( 40 g intestinal and 20 g hepatic microsomal protein per lane) and standard CYP proteins were performed in discontinuous (4-8.5 % ) SDS acrylarnide gel. The proteins were electrotransferred to 0.45 m nitrocellulose sheet and blocked with 5%(w / v) dried milk in T-TBS buffer (20 mM Tris-HCl, pH 7 5 500 mM NaCl, 0.05 % Tween 20). After blocking, the membrane was incubated with polyclonal rabbit-anti-trout-CYP3A27 (1: 1000 dilution with blocking agent) for 2 hours. The unbound antibodies were washed away in T-TBS buffer and further incubated with 1: 1000 dilution of secondary antibody (donkey-anti-rabbit antibody conjugated to horseradish peroxidase) in blocking agent for 1 hour. After washing with T-TBS the immunoreacti ve proteins were detected according to the Amersham Western Blotting kit for chemiluminescent detection and the protein bands were visualized by fluorography on Kodak X-OMAT AR films. Fluorograms were subsequently scanned and the protein bands were quantified by scan-analysis densitometry. Gentest Supersomes human CYP3A4 was the P450 standard (0.5 1 2 5 8 pmol each lane for standard curve in quantification). HPLC Analysis of Testosterone Metabolism The dried steroid assay extracts were redissolved in methanol and filtered through a Durapore microporous membrane (0.45 m pore size, Millipore). Steroid metabolites were separated on a Beckman programmable HPLC using a 5 Ultrasphere-ODS reverse-phase (C1 s ) analytical column (4 6 x 250 mm) fitted with a precolumn guard column (Beckman Ultrasphere ODS 4.6 x 45 mm). Elution was conducted with a mi x ture

PAGE 56

45 ofH 2 O : methanol: acetonitrile 50:25:25 (v/v/v) at a flow rate of 1 ml/min. Analysis was achieved using UV (225 nm) and radiochemical detection (!NUS detector). Under these conditions, 6P-hydroxy-testosterone eluted at 6.2 min 6-dehydro-testosterone at 17.6 min, testosterone at 21.3 min, androstenedione at 23.5 min, and 4-androsten-3a, 17P-diol eluted at 33 5 min Mass Spectrometric Analysis Samples were dissolved in isopropanol and analyzed via ESI ( electrospray ionization)-MS and APCI (atmospheric pressure chemical ionization)-MS. Samples were injected into the HPLC system (Applied Biosystems, model 400) followed by elution with a mobile phase consisting of 1 % acetic acid in 30:35:35 H 2 O:MeOH:isopropanol. Finnigan MAT (San Jose, CA) LCQ was used in electrospray ionization mode. The temperatures of vaporizer and capillary for APCI/MS were 300 C and 230 respectively. Sulfotransferase Activity Assay 3-OH Benzo(a)pyrene in methanol solution was added to assay tubes so that the fmal concentration would be 1 M. The methanol was evaporated with nitrogen and 0.05 M Tris-Cl pH 7 0 0.4% BSA, 50 g cytosolic protein and water were added up to 0.45 ml. After 2 min pre-incubation at 35C, the reaction was started by the addition of 20 M 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS) in 50 l water and stopped after 1 O min with 2 ml methanol. Methanol (2 ml) was added to blanks before PAPS. Tubes were centrifuged and 2 ml supernatant was mixed with 0 5 ml 1 N NaOH. The fluorescence of

PAGE 57

46 sulfate conjugates (BaP-3-sulfate) was measured at ex294 / em415 run. The sulfate product was calculated against the standard curve ofBaP-3-sulfate conjugate [14]. UDP-Glucuronosyltransferase Activity Assay 3-OH Benzo(a)pyrene in methanol solution was added to tubes so that the final concentration would be 1 and the methanol was evaporated under nitrogen To this was added 0.1 M Tris-Cl pH 7.6 5 mM MgCh, and 50 g microsomal protein solubilized with 0.5 mg Lubro l/ mg microsomes in a final volurnn of 0.4 ml. After preincubation for 2 min at 35C the reaction was started by adding 200 M UDP-glucuronic acid (UDPGA) in 0 1 ml water and terminated after 30 min by addition of2 ml methanol. Tubes were centrifuged to precipitate protein and 2 ml of the supernatant was added to 0.5 ml 1 N NaOH After mixing the fluorescence was measured at ex300 / em421 nm. The glucuronide product was calculated against the standard curve ofBaP-3-glucuronide conjugate. Statistical Analysis Data are presented as the mean SD unless specified else. Results were analyzed by a one-way analysis of variance (ANOVA) and differences between pairs of means were tested by the student t-test. Differences with a p value of < 0.05 were cons i dered to be statistically significant unless specified else. Correlation analysis was performed using Microsoft Excel software (Microsoft Redmond WA )

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CHAPTER3 RESULTS Response to Aryl Hydrocarbon Receptor Agonists CYPlA Expression in Channel Catfish Intestine Total P450 content, CYPlA content and aryl hydrocarbon hydroxylase (AHH) activity were measured in both the control fish, TCB-treated and 3MC-treated fish, to assess the effect of preexposure to aryl hydrocarbon receptor agonists upon the metabolic capacities of the catfish intestine. Mean P450 concentrations (between 0.02 and 0.20 nmoVmg protein) were not significantly altered with TCB or 3MC treatments (Figure 31) (ANOVA: p>0.05). CYPlA cross reactivity was not detected for either the controls or animals in the 0.5 mg TCB/kg diet treatments. CYPlA levels were variable for the 5.0 mg TCB/kg diet treatment, with values ranging from 0.14 to 24.11 pmoVmg protein ( Table 3-1 ). Liver microsomes from catfish induced by dietary TCB (5 mg/kg diet) was used as a positive control. Composite AHH activities were 2.46 1.16, 2.43 1.58 and 11.35 10 25 pmoVmin/mg protein for the control, 0 5 and 5 mg TCB/kg diets r espectively. AHH activities of the 5.0 mg/kg treatment were not significantly greater t han controls or the 0.5 mg/kg diet treatment due to the high standard deviation of the data (ANOV A: p>0.05) Four animals demonstrated large increases ( ~ 7 fold) in AHH activities, while 3 animals exhibited levels similar to the controls. AHH activity exhibited a strong correlation (r2=0.96) with CYPlA cross reactivity in fish exposed to TCB at 5 mg/kg diets (y=l.143x+l.026) CYPlA was present (20.8 12 pmoVmg microsomal 47

PAGE 59

48 protein) and AHH activity was induced (26.9 4.1 pmol/min/mg) in all 3MC-exposed fish (n=lO). In summary, CYPlA was not constitutively expressed in catfish intestine or liver. Yet, the immunoblots of intestine microsomes from catfish treated by dietary 3-MC or TCB showed a clear band crossing-reacting with the anti scup CYPlA antibody (Figure 3-2 and Figure 3-3). Table 3-1 shows the CYPlA amount in intestinal microsomes from control fish and fish pretreated by 3-MC (10 mg/kg diet) or TCB (5 mg/kg diet) for 10 days. CYPlA induction did not response at the pretreatment level of 0.5 mg TCB/kg diet (not shown). The results indicated that catfish intestine CYPlA is inducible by AhR agonists and the AHH activity is highly correlated to CYPlA amount (Figure 3-4), suggesting intestinal CYPlA can be used both as a biochemical and an exposure biomarker. 0.2 C) ., E ::::::: 0 E s:: 0.1 0 It) "'l;f' a.. ., ., ., ., 0 0 CONTROL TCB 0.5 mg/kg TCB 5 mg/kg 3MC 10 mg/kg Figure 3-1. Intestinal P450 content in control and treated fish. The scatter graph shows the P450 content in intestinal microsomes from control catfish (n=l 1) or animals exposed for 10 days to 0.5 mg TCB/kg (n=8), 5 mg TCB/kg (n=6) or 10mg 3-MC/kg diet (n=6). The horizontal bars indicate the mean values for each group.

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49 (1) (2) (3) (4) (5) Figure 3-2 Intestinal and hepatic CYPlA in control and fish treated with 3MC or TCB. 40 g intestinal (Lanel-4) and 20 g hepatic (Lane5) microsomal protein were in each lane: (1)-(3) Three individual fish pretreated with 10 mg 3-MC/kg diet; (4) Control fish; (5) Fish pretreated with 5 mg TCB/kg diet. (1) (2) (3) (4) (5) Figure 3-3. Intestinal and hepatic CYPlA in control and fish treated with TCB. 40 g intestinal (Lane 1-4) and 20 g hepatic (Lane 5) microsomal protein were in each lane: (1)-(4) Four individual fish pretreated with 5 mg TCB/kg diet; (5) Fish pretreated with 5 mg TCB/kg diet. Table 3-1. Intestinal CYPlA level in control and treated fish. Pretreatment Control (n = l0) 3-MC (10 mg/kg diet) (n=l0) TCB (5 mg/kg diet) (n = 4) D.L.: detection limit. Intestinal CYPlA pmol/mg microsomal protein < D.L. 20.86 12.85 14.49 7 73

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50 50 45 40 35 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 CYP1 A pmol/mg microsomal protein F igure 3-4. Intestinal CYPlA content and AHH activity. This graph shows that CYPlA content, pmol/mg microsomal protein correlated in a linear fashion (r=0 947 p < 0.001) with AHH activity in intestinal microsomes of catfish from control, exposed to 0.5 and 5 mg TCB/kg and 10 mg 3MC/kg diets for 10 days The CYPlA content was determined by cross-reactivity with a scup CYPlA monoclonal antibody and quantified relative to a rat CYPlA standard curve

PAGE 62

51 U DP-Glucurono sy ltransferase Ex pression in Catfish Intestin e UGT activity was determined in contr ol and 3MC treated fish Intestinal microsomal UGT activity was significantly higher than that in control fish (Figure 3-5) In a d dition the UGT activity correlates with the AHH activity, sh o wing a r2 of 0 75 (Fig ur e 3-6). UGT activity in control and 3MC-treated fish 250 ,-------------------------, 200 c ; i 150 .:!! Cl ti E nl I. E: g : 100 0 E .9: 50 -----0 -t----~ -.....__ __ --, ___ ......._ ___ ___, c__ __ -l Control 3MC (10mg/kg diet) : p < 0 001. Figure 3 5. UGT activity in control (n=l2) and 3MC treated (n=l l) fish.

PAGE 63

300 250 >i C) .E 200 C: .., .E 150 I(!) 0 ::> 100 50 11':. 52 0-----.------,.----~--"""T""-----T----, 0 10 20 30 40 50 60 AHH pmol/min/mg Figure 3-6 Intestinal microsomal AHH and UGT activities. This graph shows that AHH pmol/min/mg microsomal protein correlated in a linear fashion (r=0 866 p < 0 001) with UGT activity in intestinal microsomes of catfish from control, exposed to 0.5 and 5 mg TCB/kg for 10 da y s.

PAGE 64

53 Function and Expression of CYP3A and Testosterone Metabolism TLC Analyses of Testosterone Metabolism by Catfish Intestinal Microsomes Our results indicate that catfish intestinal microsomes hydroxylate testosterone and progesterone in a regioselective and stereospecific manner. Shown in Figure 37 is a representative chromatogram of the [ 14 C]-testosterone metabolism profile exhibited by catfish intestinal microsomes. 4-Androsten-3a, 17P-diol, 6p-hydroxytestosterone and androstenedione were identified as the three major metabolites of testosterone by their cochromatography with anthentic standards. There were also trace amounts of 1 la hydroxytestosterone shown (Figure 3-8). Recombinant CYP3A4 in a baculovirus system coexpressing NADPH-P450 reductase (SupersomesTM) only hydroxylated testosterone at 6P-position. Similarly, progesterone was hydroxy lated or reduced by catfish intestinal microsomes to give 3a-hydroxypregn-4-en-20-one, 6P-hydroxy-, 17a-hydroxy-, and 16a-hydroxytestosterone as metabolites (Figure 3-8 and 3-9) There were reduced amounts of 6Pand 17a-hydroxylated metabolites and absolutely no 3a-reduced metabolite formed for both steroids as substrates without NADPH under the same assay conditions (not shown). Figure 3-10 depicts the Lineweaver-Burk plots for metabolism of testosterone to the three metabolites (see above) by catfish intestinal microsomes. Table 3-2 shows the apparent KM and V ma x values of these three metabolism pathways from testosterone. The lower KM value of testosterone-6p-hydroxylation than those of the other two metabolites indicates that this may be the most important physiological pathway because of the low physiological concentration of the substrate.

PAGE 65

54 Androstendione -. Testosterone _. II 3a-OH metabolite 6P-OH testosterone _.. 11 a-OH testosterone Origin Figure 3-7. TLC of testosterone metabolism by catfish intestinal microsomes

PAGE 66

55 11 a. 1 7 a 0 3a 0 Progesterone 3a + 6~ Testosterone Figure 3-8. Progesterone and testosterone metabolism positions by catfish intestinal microsomes Progesterone 3a.-OH metabolite l 7a.-OH progesterone 6~-0H progesterone 16a.-OH progesterone Origin Figure 3-9. TLC of progesterone metabolism by catfish intestinal microsomes.

PAGE 67

0 E C c i e Cl. C) E )( C e c::: ... -6 -4 6P OH --6P-OH 25 20 15 10 -2 5 56 // / / 2 / / / / / 4 1/5 (100/M) 3a. OH 3a. OH / / / / / / 6 / / / / / 8 / / 10 17 -oxidation 17 -oxidation 12 Figure 3-10. Lineweaver Burk plot of testosterone metabolism by catfish intestinal m1crosomes

PAGE 68

57 Table 3-2 Kinetic analysis of testosterone metabolism by catfish intestinal microsomes. 6~3a1 7-oxidation hydroxylation reduction K M (M) 20.4 100 6 44.4 V max {emol/min/mg erotein] 175.4 608 9 223.4 Expression of CYP3A along Catfish Intestine As shown in Figure 3-11 the polyclonal antibodies (IgG) generated against trout CYP3A27 reacted strongly with catfish intestinal microsomes showing a band with molecular weight of 59 kDa. The rabbit-anti-trout CYP3A27 antibody also recognizes human CYP3A4 which was used in CYP3A quantification. Human CYP3A4 has molecular weight of 54 kDa. The CYP3A-like protein was expressed constitutively in catfish intestine In both groups of fish fed either commercially available chow or semisynthetic purified diet the expression of this protein was much higher in the proximal segment than in the distal part (Figure 3-12) A breakdown product with MW 44 kDa has been found which also cross-reacted with the rabbit-antitrout CYP3A27 In addition, the intestinal CYP3A amount in fish fed chow was higher than those fed purified diet (Figure 3-12) This trend is more obvious in the proximal section of intestine rather than distal part probably due to the low expression in distal intestine from bo t h groups of fish.

PAGE 69

54 kDa -+58 (1) (2) (3) (4) (5) (6) (7) Figure 3-11 Western blot ofhCYP3A4 and catfish intestinal CYP3A Lane (1)-(5): 0.5 1, 2 3.5, 5 pmol hCYP3A4; ( 6) 20 g proximal intestinal microsomes from catfish fed purified diet ; (7) 20 g distal intestinal microsomes from catfish fed purified diet. 59kDa 44kDa (1) (2) (3) (4) -+59KDa Figure 3-12. Western blot of CYP3A in catfish intesti n e Each lane has 20 g intestinal microsomal protein. (1) proximal intestinal from fish fed chow ; (2) distal intestine from fish fed chow ; (3) proximal intestine from fish fed purified diet; ( 4) distal intestinal from fish fed purified diet.

PAGE 70

59 Regional Expression and Dietary Effects on Intestinal CYP3A Testosterone hydroxylation activities by catfish intestinal microsomes and CYP3A amount are summarized in Table 3-3. Testosterone 6P-hydroxylation activities were significantly higher in the proximal segment in catfish intestine than in the distal part for both fish groups whether fed on chow or purified diet (Figure3-13). In addition, in the proximal half of the catfish intestine, the CYP3A catalytic indicator, testosterone 6P-hydroxylation activity was significantly higher in fish fed chow than those fed semi purified diet (Table 3-3 and Figure 3-13). This trend was not observed in the distal part of the intestine. The total metabolism of testosterone in the proximal segment was slightly higher but not significantly different from the values found in distal intestine (Figure 314). We have also studied the effect of diet on the CYP3A expression in the catfish intestine It indicated that the amount of CYP3A protein was lower in the intestine of catfish that were fed purified diet than those fed with commercial chow, but the two groups show the same trend of expression along the intestine (Table 3-3) Testosterone 6P-hydroxylation activities, the CYP3A catalytic indicator, correlated with the CYP3A amount shown by immunoblotting (r=0.88) (Fig 3-15) On the contrary, the testosterone 17-oxidation and the formation of the reduced metabolite, 4-androsten-3a 17P-diol showed poor correlation with the CYP3A protein amount. The ratio of testosterone 6P h ydroxylation over 17-oxidation was much higher in proximal intestine, ranging from 1 t o 4, than that in distal section, which is approximately 0.8 (Figure 3-16) This suggests t hat the percentage of CYP3A enzyme in total P450 content was significantly higher in t he proximal part than that in the distal intestine. These results demonstrated that a CYP3A-like protein, related to CYP3A27, was expressed at higher concentrations in the

PAGE 71

60 proximal than in the distal segment of catfish intestine. In addition, the expression of this CYP3A-like protein was modulated by the diet. TABLE 3-3. CYP3A expression and catalytic activities along catfish intestine Diet Testosterone Metabolism CYP3A 6P-OH Total Metabolism Enzyme Amount pmol/min/mg protein pmol/mg protein Chow (n=4) Proximal 262.8 3 3,b 986.9.4 101.0.13,b Distal 88.6.6 622.3.5 32.3 8 Purified (n=8) Proximal 158.4.6 a 687.6.0 52 5.9a Distal 104 1.1 466.1.8 21.6.2 a significantly higher than the corresponding distal values by one tailed student t-test for paired samples : p<0.01. b significantly different from the corresponding proximal values for purified diet group by single factor ANOVA : p<0.01.

PAGE 72

C 400 0 :;::::; co 350 >. X o300 I.... 0) -o E >__ 250 ..C C c:6.E 200 c.o -Q) 0 150 C E e n. Q) ......... 100 ...... Cl) 0 50 t5 Q) 0 61 a,b Chow a Purified diet ~Distal Figure 3-13. Testosterone 6~-hydroxylation activities in proximal and distal intestine of fish fed chow or purified diet. a significantly higher than the corresponding distal values by one tailed student t-test for paired samples: p<0 01. b significantly different from the corresponding proximal values for purified diet group by single factor ANOVA: p < 0 01

PAGE 73

C 0 :;:; ro 1400 ...--... 1200 0 O') -c 1000 >, C Ia> 800 C 0 e E 600 Q) c.. t, ........, 400 0 Cl) Q) I200 62 Chow Purified diet Distal Figure 3-14 Testosterone metabolism activities in proximal and distal intestine of fish fed chow or purified diet.

PAGE 74

450 a., 400 (J) rn >-c 350 X 0 a., .... 300 "O 0 >, .... .c c.. I 0) 250 ~E
PAGE 75

64 4 3 Proxima l 2 ... Distal 1 ... ......... 0 Prox i ma l Distal Figure 3-16 Ratio of testosterone 6P-hydroxylation/l 7-oxidation in proximal and distal intestine of control catfish.

PAGE 76

65 Effects of Modulators on CYP3A Activities The chemical structures of the six CYP3A modulators used in our study are shown in Figure 3-17. The three mammalian quasi-irreversible CYP3A inhibitors, troleandomycin, erythromycin and SKF 525A, have an N-alkylated amine required to form the metabolite-intermediate complex. Surprisingly, the selective CYP3A inhibitor for mammals, troleandomycin, showed no inhibition of formation of any of the three testosterone metabolites by catfish intestinal microsomes (Figure 3-18). Figure 3-19 and 3-20 are representative TLC analyses, showing chemical inhibition of testosterone metabolism activities. Erythromycin, ketoconazole, metyrapone and SKF-525A inhibited t estosterone 6p-hydroxylation to different extent {Figure 3-21, 3-22, 3-23, 3-24). Figure 3-25 summarizes the effects of four CYP inhibitors, erythromycin, ketoconazole, metyrapone and SKF-525A, on the metabolism of testosterone. All four chemicals showed strong inhibition of testosterone 6P-hydroxylation. The inhibitory effects of CYP3A-mediated testosterone 6P-hydroxylation were: ketoconazole > metyrapone > SKF-525A > erythromycin as the inhibitory potency decreased (Table 3-4). None of the four inhibitors showed significant effect on the testosterone 3-oxidoreduction or 17oxidation Only ketoconazole exhibited a concentration dependent inhibition of the formation of androstenedione (Figure 3-21 and 3-24). Yet the IC 50 value of ketoconazole for testosterone 6p-hydroxylation was almost 10 5 -fold smaller than that for the 17oxidation to form androstenedione (Table 3-4). The compound a-naphthoflavone had no significant effect on testosterone 6P-hydroxylation activities (Figure 3-26) At 100 M naphthoflavone concentration, which is more than three times the substrate concentration

PAGE 77

66 (30 M), testosterone 6~-hydroxylation still had 91 % activity left in comparison to control (Figure 3-26).

PAGE 78

67 0 .. .. , .. ,,,, n .,,_____N/ ~ ~"" H01, ,,, ,, ... .. 1111111110 0 1 , ,, ,, .. . """"'""' ""' ""'O 0 ... ,,,,,o ~ Erythrom y cin Troleandom y cin Metyrapon e Ketoconazole a naphthofla v one SKF 525A (Proadifen ) Figure 3-17. Chemical structures of mammalian CYP3A modulators. Five inhibitors ( erythromycin, troleandomycin metyrapone ketoconazole and SKF 525A) and one enhancer ( a-naphthoflavone) are shown.

PAGE 79

68 120 Cl) Cl) l'O 100 >, >< y--------=-=c.:...:...:~.:..::~.:.::...:.:. ~.:.:~:..:.:..:...:..::..=.;..:~-""T 0 ... 80 "C >, 0 .c .!:; C'.l. C: I 0 60 (0 (.) Cl)C: 0 e~ 40 Cl) Cl) 0 20 Cl) Cl) I0 0 20 40 60 80 100 120 Troleandomycin Cone, M Figure 3-18 Effect of testosterone 6P-hydroxylation by addition of troleandomycin.

PAGE 80

69 6~-0H ----. Testosterone OuM 0.5u l \'1 luM 2uM Metyrapone Figure 3-19 TLC of inhibition of testosterone metabolism by metyrapone.

PAGE 81

70 Testosterone 0uM 25 uM 50 uM l00uM SKF525A Figure 3-20 TLC of inhibition of testosterone metabolism by SKF-525A

PAGE 82

E !!! 0 .cs~ a, g a, 0 C: 0 .! ti) .s ti) 71 140 -,---------------------------, 80 . . . . . . . . . . . . -+6j3-0H --17-oxidation -0-3a-OH 20 0+----------------------...,...-0 50 100 150 200 Erythromycin Cone, M Figure 3-21. Inhibition of testosterone metabolism by erythromycin

PAGE 83

72 140 00 -.------------------, 120 00 0 [ 100 00 ... r'T""-.r-........ >, ;;; 80 00 .:: CJ ns 0 ... r::: 0 CJ .... 0 -,fl. 60 00 40 00 20. 00 0 00 +---------------0 20 40 60 80 100 120 SKF-525A cone (M) Figure 3-22. Inhibit i on of testosterone metabolism by SKF-525A. -+-6 POH -ll-3aOH -ts17-oxidation

PAGE 84

120.00 0 100 00 >. s; 73 80.00 ----------------------------------------------0 l'O 0 '5 60.00 0 .._ 0 ";fl. 40 00 20 00 -+-------r------.---------,----0 50 100 150 200 KET cone (nM) Figure 3-23. Inhibition of testosterone metabolism by ketoconazole. -+6~-0H -e-30.-0H -i:r--17o xi da tion

PAGE 85

74 140 120 0 100 >, s: 80 .:; -+6~0H u ns ---3a-OH 0 60 a.. C: -Ir17-oxidation 0 u 40 0 0 20 0 0 50 100 150 200 250 MET cone (M) Figure 3-24. Inhibition of testosterone metabolism by metyrapone

PAGE 86

>, = i?: ti 150 ta~ 100 0 e~ i: > o0 50 Inhibition of testosterone metabolism by erythromycin 75 >, = i?: ti 150 Inhibition of testosterone metabolism by ketoconazole ta_ 100 0 e~ c> o0 50 0+----,---,------.-----,--,-0--'----~--------.--1 0...,1 0 ... 0 1 10 100 >, i?: ti 150 ta~ 100 ei c> o0 0 50 10 100 KET cone { M) ERM cone {M) 6~-hydroxylation 3a-oxido reduction -.17-oxidation Inhibition of testosterone metabolism by SKF-525A >, = .i?: ti 150 ta_ 100 0 e~ c> o0 50 Inhibition of testosterone metabolism by metyrapone 0+-----~----~~10 100 1 10 100 SKF-525A cone {M) MET cone { M) Figure 3-25. Determination oflC 50 of testosterone 6~-hydroxylation by ERM, KET, SKF-5 25A and MET. Abbreviations: ERM, erythromycin; KET, ketoconazole; MET, metyrapone.

PAGE 87

76 Table 3-4. IC 50 values of the four inhibitors on testosterone hydroxylation. Chemical IC so (M) 6@-hydroxylation 1 7-oxidation Ketoconazole 0 0404 > 500 Metyrapone 4.48 n.r SKF-525A 32.1 n r Erythromycin 53.8 n.r n r. : no inhibition observed up to 200 M of the inhibitor concentration 160 140 120 s:100 .:; () ca -+6~-0H e ao C: ---3u-OH 0 () 0 60 -617-o xi da t ion ';;'?40 20 0 0 50 100 150 200 250 ANF cone (M) Figure 3 26 Modulation of testosterone metabolism by a-naphthoflavone.

PAGE 88

77 Modulation of AHH Activities AHH activity was enhanced by the addition of a-naphthoflavone (Figure 3-27). Maximal enhancement was achieved at 20 M. 4 50 4.00 0 ... 3 50 ... C: 0 (.) 3 00 0 ... 2 50 :;:; ca 2 00 1 50 s;: 1 00 < 0 50 0 00 -i--------.------.----,-.--,-,-"'T""'T--r-----,----,---,---,---.,......,.--,--r-1 y = 0 7094Ln(x) + 0 9048 R 2 = 0 9153 10 [ANF], M Figure 3-27 Stimulation of AHH activity by a-naphthoflavone. 100

PAGE 89

78 Identification of Major Testosterone Metabolite HPLC Analysis of Testosterone Metabolism by Catfish Intestinal Microsomes A typical HPLC chromatogram of the ethyl acetate extracts obtained after incubation of [ 14 C] testosterone with NADPH and catfish intestinal microsomes is shown i n Figure 3-28 Testosterone and its hydroxylated metabolites showed peaks around 240 nm on UV spectrum. However, 4-androsten-3a,17P-diol has maximal absorbancy at 212 nm. The UV detector was set at 225 nm to avoid interference by the mobile phase. The unknown metabolite isolated with TLC gave a peak at 33.5 min on radiochemical detection, similar to the 4-androsten-3a,17P-diol standard by UV detection (Figure 3-29). As the molar absorptivity of 4-androsten-3a,17P-diol is much lower than that of testosterone or its hydroxy lated metabolites due to the lack of a 3-keto group to conjugate with the C 4 =C 5 it is not surprising that signal of the unknown metabolite from assay extract was below detection limit on UV detector. To further prove the identity of the unknown metabolite, [ 14 C] testosterone assay extract was mixed with 4-androsten3a, 17P-diol standard The mixture was dried under nitrogen and reconstituted in methanol. Figure 3-30 indicated the UV and radiochemical identities of the mixture of [ 14 C] testosterone assay extract with 4-androsten-3a, 17P-diol standard. The standard and the unknown metabolite eluted at the same time of 33.5 min on UV and radioactivity detecti on respectively. Thus, testosterone was converted to 6P-OH testosterone, androstenedione and 4-androsten-3a,17P-diol by catfish intestinal microsomes. The HPLC profile also provided valuable information on the identification of another metabolite, 6-dehydrotestosterone (17P-hydroxy-4,6-androstadiene-3-one) (Figure 3-28, 3-30) The retention time of this 6-dehydrotestosterone metabolite matched that of the

PAGE 90

79 authentic standard. HPLC analysis of [ 14 C] testosterone incubation with catfish intestinal cytosol was also performed by radioactivity detection (not shown). No direct format i on of the 3a-reduced metabolite was observed in incubation with catfish intestinal cytosol. oooa l I 0 006 0 007 1 0 007 3a 0 ooti 006 0 000 0 005 A A u u 0 004 0 004 0 003 003 j I r 000 2 I 0 002 j 0 00 1 I 00 1 t . 5 10 1 5 20 25 30 35 40 M inu t es Figure 3-28. HPLC (UV detection) profile of 4-androsten-3a, 17P-diol standard

PAGE 91

80 030 -0 30 I I I 0 2G T K)26 07 022 0 0 1 0 -0 10 C 0 0 u u n I 0 14 n I 0 14 6P t 1 / & 010 3a. 0 1 0 6D I I \ -0 06 0 00 0 02 J/ I 00 2 ~ ,~r., , l' ,~ ~1 5 ?O 25 30 40 ,nvt Figure 3-29 HPLC (radiochemical detection) of [C 14 ] testosterone metabolism catalyzed by catfish intestinal microsomes. Catfish intestinal microsomes (0.4 mg) was used; substrate concentration 30 M. Abbreviations: 6P, 6p-hydroxy-testosterone; 6D, 6-dehydrotestosterone or 17P hydroxy-4 6-androstadiene-3-one; T, testosterone ; A, androstenedione; 3a., 3a. hydroxytestosterone or 4-androsten-3a.,17P-diol.

PAGE 92

81 r 00.000 UV 3a., authentic ..... T.:-:-.-. ~ ~-. 3500 -T 6D 3a. .... .. ..... . ... T .... 1 10 16 22 28 31 10 Figure 3-30 HPLC analysis of mixture of 4-androsten-3a.,17P-diol and [ 14 C] testosterone assay extract. Abbreviations: 6P 6p-hydroxy-testosterone; 6D 6dehydrotestosterone or 17P-hydroxy-4 6-androstadiene-3-one; T testosterone ; A androstenedione; 3a., 3a.-hydroxy-testosterone or 4-androsten-3a. 17P-diol.

PAGE 93

82 Mass Spectrometric Analysis of Testosterone Metabolite To confirm the structure of metabolite to be 4-androsten-3a l 7P-diol, the metabolite was isolated and subjected to mass spectroscopy. ESI-MS did not provide efficient ionization of the sample. With APCI-MS, both the 4-androsten-3a l 7P-diol standard and the testosterone metabolite gave related m/z 255 and 273 ions and m/z 289, with only very low abundance of the expected m/z 29l[M+Ht ion Among the three daughter ions, m/z 273 is the most intense ion, followed by m/z 255 and m/z 289 The ( + )APCI-MS/MS daughter spectra of all three of these ions from the testosterone metabolite matched those obtained from the 4-androsten-3a, l 7P-diol standard (Figure 331, 3-32, 3-33) Attempts to increase the yield of m/z 291 ions were not successful. The m/z 255 and 273 ions are probably due to [M+H-2H 2 Ot and [M+H-H 2 Ot ions of the 4androsten-3a, l 7P-diol (MW 290) It is possible that the MW 290 steroid is thermally labile eliminating a H 2 O molecule forming a MW 272 compound which is then ionized. The m/z 289 is likely the [M+Ht ion of another steroid, which is probably testosterone (Figure 3-34) In summary identification of the 3a-reduced testosterone metabolite was based on a complete agreement of its chromatographic pattern with that of the authentic steroid during chromatography on the reverse phase HPLC column and on TLC in the system e t hyl ether : toluene: methanol : acetone (70:38:0.8:1) and on a perfect agreement between the mass-spectrum of the three daughter ions (m/z 255 273, and 289) of the testosterone metabolite with that of the authentic steroid (Figure 3-31 3-32 3-33). In vitro metabolism of testosterone by catfish intestinal microsomes is summarized in figure 3-35.

PAGE 94

100 95 90 .. .. 75 70 .. 60 Roi ""' ... Ab ....., .. '%5 "' 35 30 25 2ll 15 10 100 95 90 05 00 75 70 65 60 Rel ... 55 Ab ....., ,. '%5 ,o 35 30 25 20 15 .. 90 00 90 97 1 1091 100 110 12ll 130 140 150 100 971 100I 100 110 12!) 130 1<0 150 100 83 253 2 (a) 271 1 1971 189 2 2112 .,2.tJ.1 170 2532 (b) 271 2 197 2 211.1 1711 Ul7 1 221 170 100 190 200 210 220 230 2<0 250 260 270 200 290 300 m/Z Figure 3-31. ( + )APCI-MS/MS daughter spectra of m/z 289 ions. The spectra are from (a) authentic 4-androsten-3a 17P-d i ol ; (b) metabolite isolated from testosterone incubation.

PAGE 95

84 255 2 100 95 90 .. (a) .. 75 70 65 60 Ro '"155 ,. Ab ..,so .. nc45 o 35 30 25 &II 20 15 10 .. 90 100 110 120 130 1,0 150 160 170 l&ll 190 200 210 ml, 2 1 100 95 90 .. (b) .,, 70 65 60 Ro latt55 ,. :50 .. nc45 40 35 30 25 20 &II 15 10 173 1 .. 90 100 110 120 130 l
PAGE 96

85 1 1 1 1 100 .. 2 IO 85 (a) .. 1 I 75 10 21 1 65 .. 22 I Ro ~5 ,,. :50 .. 14] 1 n<45 ,o 1~ 1 35 I 0 I I 1J7 0 30 I I 25 I 9 2 171 20 1&9 1 WI 151'..;! 15 a 12 I 21 3 91.Q 132.J ,;u. 10 0 10 .. .. 100 110 120 130 140 150 .. 170 .. .. 200 210 220 ""' I 0 100 I I 95 .. 21 I (b) 85 .. 15 1 2 10 .. .. I I 1 2 R 111165 "' .. -50 221.2 23 I .. nc45 I 7 0 I .I I J 2 ,o 11 0 13 o 35 17 30 I 2 25 81 0 20 15 10 .. to 100 110 120 130 140 .. 180 190 200 210 220 290 ""' Figure 3-33. ( + )APCI-MS/MS daughter spectra of m/z 255 ions. The spectra are from (a) authentic 4-androsten-3a 17~-diol ; (b) metabolite isolated from testosterone incubation.

PAGE 97

86 m/z 291 4-androsten 3a I 7P-diol + m/z 273 testosterone + m/z 255 Figure 3-34. Fragmentation of 4-androsten-3a,17P-diol in APCI-MS.

PAGE 98

0 3a 87 OH 0 11 a OH testosterone OH 6 OH testosteron OH /YP3A 0 oxido-reductas1/ Testosterone / OH 0 3 a OH testosterone Androstenedione Figure 3-35. In vitro metabolism of testosterone by catfish intestinal microsomes.

PAGE 99

88 Regional Expression of 3a-Hydroxysteroid Dehydrogenase in Catfish Intestine Table 3-5 summarizes the in vitro testosterone metabolism from catfish fed semisynthetic diet or commercial chow. Both groups of catfish showed higher testosterone 3a-oxido-reduction in proximal intestine than distal. In proximal intestine, testosterone 6P-hydroxylation but not 3a-oxido-reduction, was significantly higher in fish fed commercial chow than those fed semi-synthetic diet (Table 3-5) Our previous work has indicated that testosterone 6P-hydroxylase in catfish intestinal microsomes was mediated by CYP3A-like enzymes. From the above results, we found that diet may play a more significant part in CYP3A expression than expression of 3a-hydroxysteroid dehydrogenase in catfish intestine, especially in the proximal section. The formation of 4androsten-3a, 17P-diol from testosterone by catfish intestinal microsomes was not affected by the two general cytochrome P450 inhibitors, metyrapone and SKF-525A.

PAGE 100

89 Table 3-5. Testos t erone metabolism by intestinal microsomes from catfish fed with chow or semi-synthetic purified diet Diet Testosterone Metabolism 6P-OH 3a-reduction 17-oxidation total metabolism pmol/min/mg protein Chow (n=4) Proximal 262.8.3 \ b 346 1.5 285 8.5 aa/ 989.9.5 Distal 88 6.6 292.7.7 154.1.7 622.3.5 Purified (n = 5) Proximal 158.4.3 a 325.4.9 a 117.0.7 691.8.6 aa Distal 108.8.4 162.0.1 136.6.4 481.6.6 a significantly higher than the corresponding distal values by one tailed student t-test for paired samples : p < 0.01. aa significantly higher than the corresponding distal values by one tailed student test for paired samples: p < 0 05 b significantly different from the corresponding proximal values for purified diet group by single factor ANOVA: p < 0 01.

PAGE 101

90 Catfish Intestinal CYP3A lnducibility Studies CYP3A enzyme amount in proximal and distal intestine was measured in control fish and fish treated with rifampicin (RIF) for 2 or 4 weeks or with PCN for 1 or 3 weeks The CYP3A quantification was achieved by immunoblotting using Supersomes human CYP3A4 as standard Figure 3-36 shows a typical Western blot of intestinal microsomes from control and treated fish. There is no significant difference in intestinal CYP3A level between control and any treated groups, either in the proximal or distal segment (Figure 3-37). On the contrary it seemed that testosterone 6p-hydroxylation and 3a-oxido reduction activities but not 17-oxidation were statistically significantly higher in distal intestine from fish treated with rifampicin for 2 weeks than control fish (Table 3-6). (1) (2) (3) (4) (5) (6) (7) (8) Figure 3-36 Western blot of intestinal CYP3A from control and PCN treated fish. Intestinal microsomal protein ( 40 g) was in each lane (1) and (4): fish treated with PCN for 3 weeks; (2) and (3) : fish treated with PCN for 1 week ; (5)-(8): control fish.

PAGE 102

91 140 0 '120 C: 0 (.) .... 0 100 !.C: ::::s 0 E ca C: a, 0 'a. < M ll. >0 80 121 Proximal 60 40 20 Control RIF 2weeks RIF 4weeks PCN 1week PCN 3weeks Figure 3-37. Intestinal CYP3A expression in control or fish treated with RIF or PCN. Both proximal and distal intestinal CYP3A protein levels are shown in fish treated with rifampicin (n=4) or PCN (n=3) compared with control fish (n=4).

PAGE 103

92 Table 3-6. In vitro testosterone metabolism activities in proximal and distal intestine from control fish and fish treated with rifampicin for two weeks. 6P3a-oxido17Total Ratio hydroxylation reduction oxidation Metabolism (6P-OH/17oxidation (pmol/min/mg protein) Proximal Control 107.5 66 156.8 7 73 1 75 395.7 1.47 15 RIF 108.1.4 142.5 3 74 3 75 370 7 1.46 01 treatment Distal Control 40 6.54 66 2 9 50 0 04 178 1 6 0 82.11 RIF 61.3.6* 83 7.24** 65 6 1 231.8 01 0 95.12 treatment : p<0 01 : p<0 05

PAGE 104

93 Catfish Hepatic CYP3A Expression Figure 3-38 indicates Western blotting results of CYP3A expression in catfish liver. More than one band in catfish hepatic microsomes cross-reacted with polyclonal antiCYP3A27 antibody. There were individual differences in the CYP3A bands but no sex differences can be concluded from Western blotting results (Figure 3-39) PCN (10 mg/kg body weight), but not rifampicin (10 mg/kg) pretreatment of catfish resulted in a slight increase in hepatic CYP3A amount (Table 37). Although PCN and rifampicin pretreatment caused a slight increase in testosterone 6p-hydroxylation activities, it is not statistically different (p>0.05) probably due to high intra-group variation caused by small group size However, both PCN and rifampicin pretreatment resulted in significant increases in 3a-oxido-reduction and testosterone total metabolism activities (Figure 3-40).

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94 (1) (2) (3) (4) (5) (6) (7) Figure 3-38. Western blot ofhCYP3A4 and hepatic microsomes from control fish and fish pretreated with RIF or PCN (10 mg/kg). Lane (1)-(3) has 3, 5, 7 pmol human CYP3A4; lane (4)-(7) are catfish hepatic microsomes, 40 g per lane. Lane (4) and (5) are from control fish; lane (6) from fish pretreated with rifampicin for 2 weeks; lane (7) from fish treated with PCN for 1 week. Lane (5) and (6) were from female fish; lane (4) and (7) were from male fish. (1) (2) (3) (4) (5) (6) (7) (8) Figure 3-39. Western blot showing cross-reactivity of catfish hepatic microsomes against a polyclonal antibody to trout CYP3A27. Lane 1-8 has 40 g hepatic microsomes protein per lane. Lane (1) (2): from control fish; lane (3), (4), (5): from fish pretreated with rifampicin (10 mg/kg) for 2 weeks; lane (6): from fish pretreated with rifampicin (10 mg/kg) for 3 days; lane (7) and (8): from fish pretreated with PCN (10 mg/kg) for 1 and 3 weeks, respectively. (1), (3), (4) and (7) were from female fish; (2), (5), (6) and (8) were from male fish.

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95 Table 37 Hepatic CYP3A expression in control and treated fish Treatment Control (n=4) RIF (n=4) PCN (n=3) *: p < 0 05 Body Weight Liver Weight Protein Yield CYP3A (g) (g) (mg/g) (pmol/mg) 1032.8 167.5 20 96 0.26 10.01 2.30 178.96 15.98 1303.5 116.8 23.16 3.44 8 73 2.70 169 02 33.01 1767.3 712 5 28.61 14 31 8 13 1.92 205 76 15.40*

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96 250 ~----------------------200 150 c E -0 E 100 C. 50 0 6beta-OH 6-dehydro 3alpha-reduction 17-oxidalion IZ!Control PCN p<0.05. * ... ... .. ... ... ... m ... m. ... ... !!! ... ::: .. m m ... ... m ... ... ... total Figure 3-40. In vitro testosterone metabolism activities by hepatic microsomes of fish from control, rifiampicin (RIF) and PCN pretreated groups.

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CHAPTER4 DISCUSSION Induction of CYPlA and UGT in Catfish Intestine Intestinal AHH activity and CYPlA content were not affected by the 0.5 mg tetrachlorobiphenyl (TCB)/kg-diet pretreatment and showed induction, but great variability, with the 5 0 mg TCB/kg diet. The consistency of AHH activities at near control values for the 0 5 mg TCB/kg-diet dose, and the lack of detectable CYPlA, suggest that this dosage may be insufficient to induce intestinal AHH activity when fed at approximately 0.5% of body weight. Conversely, detectable CYPlA content and AHH activities ranging from control levels to more than 12-fold higher with the 5 mg/kg d iet dosage indicate that TCB is capable of intestinal induction. Previous studies have demonstrated dose-dependent induction and inhibition of fish hepatic CYPlA catalytic activities by TCB with induction at low and high doses while inhibition occurs only at high doses (13,114]. The differential response seen in the catfish intestine is most likely differential induction resulting from factors associated compound ingestion and bioavailability. Direct correlation of AHH activity with CYPlA content for individual animals suggests that the variability was not related to TCB inhibitory effects Administration by gavage of 10 mg/kg 3MC yielded induction of CYPlA in all fish, though again a varia ble response was observed. This variability may be due to d i fferent uptake by intestine orally, or to individual genetic difference between fish. 97

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98 UGT activity was also induced by 3-MC treatment. The induction of UGT correlates with the induced AHH activity (r2=0.75). The UGT expression in catfish intestine showed similar inducibility as CYPlA by AhR agonists. At least two isoforms (UGT1A6 and UGT1A9) of human UGTs have been shown to be induced by AhR inducers [115]. The AhR agonist 3-MC is a bifunctional inducer, inducing both phase I (CYPlA) and phase II (UGT) enzymes in catfish intestine In vitro Testosterone Metabolism by Catfish Intestinal Microsomes Steroid hydroxylation reactions catalyzed by cytochrome P450s serve numerous physiological functions including catabolism of cholesterol to bile acids, activation of vitamin D 3 and the biosynthesis of all major classes of steroid hormones [116]. Steroid hormones are subject to site-specific hydroxylation reactions catalyzed by many, but not all of the more than 20 distinct P450 enzymes [117]. Studies carried out using hepatic P450s purified from rodent and human resources have established that individual P450 enzymes exhibit unique patterns of steroid hormone hydroxylation. These patterns can be both characteristic of individual P450s and diagnostic of the identity and purity of isolated P450 preparations. Hydroxysteroid metabolite patterns can also be useful in monitoring the relative concentrations of individual P450 forms present in microsomal fractions that simultaneously express multiple cytochrome P450. Our present results show that testosterone was hydroxylated at 6Pand 17a-, and to a lesser extent, I la-positions by catfish intestinal microsomes. The metabolism of testosterone is concentration-dependent on both testosterone and the cofactor NADPH The KM of testosterone 6p-hydroxylation was lower than those of the other two major metabolites, indicating that this may be the most important physiological pathway

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99 because of the low physiological concentration of the substrate. Another major metabolism pathway of testosterone by catfish intestinal microsomes is 3a-reduction probably via 3a-hydroxysteroid dehydrogenase. 3a-Hydroxysteroid dehydrogenase catalyzes the reversible interconversion of hydroxy and carbonyl groups at position 3 of the steroid nucleus. In mammalian tissues 3a-hydroxysteroid dehydrogenase works in concert with Saand 5P-reductase to convert 5a / 5P-dihydrosteroids into 5a / 5P tetrahydrosteroids [118 119]. Human 3a-hydroxysteroid dehydrogenase plays an important role in steroid hormone metabolism and action [96]. In rodents 3a hydroxysteroid dehydrogenase showed higher activities in cytosolic fraction than in microsomes using dihydrosteroids as substrates [88 89]. Our results showed that the 3a oxido-reduction of testosterone by catfish intestinal microsomes is NADPH-dependent and there is no direct formation of 4-androsten-3a l 7P-diol observed using intestinal cytosol (not shown). CY P3 A Ex pr e ssion in C atfish Intestine Small intestinal cytochromes P450 provide the principal, initial source of b i otransformation o f ingested xenobiotics The P4503A subfamily has been demonstrated to be particularly prominent in the human small intestine [19 100]. An important cause of incomplete bioavailability of many drugs is prehepatic metabolism in the GI tract mainly by the CYP3A subfamily of enzymes. Although most previous studies of CYP in fish have focused on th e PAR-inducible CYPlA subfamily CYP3A in fish is clearly an important constituti v e enzyme. The CYP3A-like enzyme has good cross-reactivity with the polyclonal antibody rabbit-anti-trout CYP3A27 showing the same molecular weight

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100 of 59 kDa as trout hepatic CYP3A27 [120 47 23]. This CYP3A-like protein was expressed in untreated catfish intestine at significant levels. Both this fact and results showing that two steroids and benzo(a)pyrene could be metabolized by intestinal microsomes from control fish imply that the CYP3A enzyme is constitutively e x pressed and it may have important endogenous functions in catfish intestine This CYP3A protein was expressed gradually along catfish intestine showing the highest amount at the proximal end. This longitudinal distribution is similar to the expression of most CYP enzymes in mammalian intestine [100]. Correlation between testosterone 6P hydroxylation acti v ities and immunochemically measured CYP3A content ( r2= 0. 79) suggests that testosterone 6P-hydroxylation is a good catalytic marker of CYP3A enzymes in fish species as well as in mammals. Chemical Inhibition of in vitro Testosterone Metabolism Chemical inhibition of a CYP enzyme can go through one or more than one of the following three mechanisms: (1) reversible inhibition ; (2) formation of MI-complex ; ( 3) irreversible mechanism-based (suicide) inhibition [27]. In the present study we used two global CYP enzyme inhibitors (metyrapone SKF-525A) and three specific mammal i an CYP3A inhibitors (ketoconazole erythromycin and troleandomycin) [26 121]. Among these five inhibitors metyrapone and ketoconazole are categorized as reversible inhibitors A reversible inhibitor (or its metabolite) binds to a CYP enzyme in a direc t, rapidly reversible w ay. Reversible inhibition is either competitive or noncompet i ti v e the extent of which is determined by the relative binding constants of substrate and inhib i tor for the enzyme and by the inhibitor's concentration [27]. SKF-525A and troleandom y cin

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101 are classified as quasi-irreversible inhibitors of mammalian P450. The oxidation of these two N-alkyl-substituted compounds forms a nitroso derivative which binds tightly to the ferrous iron of P450 heme to form the MI complex. Erythromycin inhibits human CYP3A in both competitive reversible and quasi-irreversible way. The formation of an inhibitory cytochrome P450 Fe 2 + -metabolite complex can easily be detected in spectral analysis of a peak at 456 nm [28 32 122] Preincubation was performed in our studies for quasi-irreversible inhibitors to examine the possibility that these inhibitors may be metabolized by catfish intestinal microsomal enzymes to products that form complex with the CYP3A enzyme. Surprisingly we found that TAO the selective CYP3A inhibitor for mammals showed no inhibition of formation of any of the three metabolites produced by catfish intestinal microsomes This is in accordance with the literature reports of the lack of inhibitory effect of TAO on trout monooxygenase [ 41]. A possible explanation for the lack of inhibitory effect of TAO is the inability of catfish CYP3A to form a MI comple x with TAO. CYP3A in catfish intestine may be inactive in catalyzing TAO N-demethylation an obligatory step in the formation of the nitroso intermediate SKF-525A also an amine and a quasi-irreversible inhibitor of mammalian P450 was found to inhibit CYP3A-mediated testosterone 6P-hydroxylation even without preincubation with the inhibitor. We concluded that the inhibition of testosterone 6P hydroxylation caused by SKF-525A may be the result of competitive reversible inhibition. Further kinetic study is needed to verify this assumption. Testosterone 6P hydroxylation activities were inhibited by erythromycin, ketoconazole metyrapone and SKF-525A but not affected by a-naphthoflavone (Figure 3-21, 32 2 3-23, 3-24 3-25 326). The inhibition b y erythromycin ketocona z ole metyrapone and SKF-525A was

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102 specific toward 6P-hydroxylation, only ketoconazole showed slight inhibition of testosterone 17-oxidation at high concentration of the inhibitor. In summary the results of the chemical inhibition studies showed which of the reversible and quasi-irreversible inhibitors of mammalian P450s are effective inhibitors of catfish P450. Naphthoflavone, the specific CYPlA inhibitor did not affect CYP3A-mediated testosterone 6p-hydroxylation activities. The inhibitory potency of testosterone 6P hydroxylation is: ketoconazole > metyrapone > SKF-525A > erythromycin. Troleandomycin is inactive toward catfish intestinal CYP3A enzymes. Thus caution must be observed in the use of mammalian P450 inhibitors as probes for the involvement of P450 in the metabolism and toxicity of chemical in fish. Stimulation of AHH Activities by a-N aphthoflavone Our results suggest that CYP3A is important in biotransformation of benzo(a)pyrene in fish that was not exposed to an inducer of CYPlA. No CYPlA was detected in control fish [123], but benzo(a)pyrene was metabolized to fluorescent metabolites and this metabolism was stimulated by a-naphthoflavone. Previous stud i es showed that a-naphthoflavone inhibited CYPlA-dependent metabolism of benzo(a)pyrene [38]. Others have shown that a-naphthoflavone can stimulate CYP3A dependent monooxygenase activity with some but not all substrates [53 55 124 125 126 127] In this regard the CYP3A like protein in catfish intestine appears to behave similarly to other CYP3A proteins in that a-naphthoflavone stimulates activity with some but not all, substrates [127].

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103 lnducibility of Catfish Intestinal CYP3A In contrast to mammals very little is known about the inducibility of the CYP3A enzymes in fish Interestingly it has been found that the pretreatment ofrainbow trout with 3,4,5 3 ,4 5'-hexachlorobiphenyl (1 mg/kg) significantly increases the progesterone 6P-hydroxylase activity of trout liver with no effect on CYP3A levels [128]. Our results indicate that the CYP3A has a lower expression in the intestine of catfish fed purified diet than those fed chow This finding correlates with the fact that the hepatic CYP3A content in teleost fish was under dietary modulation [52]. Mammalian CYP3A enzymes are highly inducible by the synthetic glucocort i coid (dexamathasone) macrolide antibiotic (rifampicin), synthetic steroid (pregnenolone 16a carbonitrile) and phenobarbital. Yet the two mammalian CYP3A inducers we used in catfish study i .e rifampicin and PCN failed to induce intestinal CYP3A protein level at the dose of 10 mg/kg body weight/day While fish treated with rifampicin for two weeks showed significantly higher testosterone 6phydroxylation and 3a-oxido-reduction activities in their distal intestine compared to that in control fish, the 6P-OH/17-oxidation ratio was not changed again indicating no significant induction of CYP3A. Identification of 3a-Reduced Metabolite of Testosterone The present study demonstrated that in addition to hydroxylation testosterone was also reduced at the 3-keto position to form 4-androsten-3a l 7P-diol by catfish intestinal microsomes. The latter pathway is not likely to be a P450-mediated process as it is not affected by any of the cytochrome P450 inhibitors used in this study i .e metyrapone SKF-525A ketoconazole erythromycin, a-naphthoflavone. These 3a

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104 reduction activities were mainly in the microsomes rather than in cytosolic fraction, which is similar to that of human liver [90]. Although testosterone 6p-hydroxylation has the lowest KM value, 3a-oxido reduction has the highest V max (Table 3-2), suggesting that the 3a-oxido reduction may play an important role in endogenous function. Another metabolic pathway for testosterone is 6-dehydrogenation. It was shown that 6-dehydro testosterone was formed directly from testosterone by CYP3A isozyrnes that catalyze 6P hydroxylation of testosterone in rat liver microsomes; the formation of 17P-hydroxy-4,6androstadiene-3-one was enhanced by pretreatment with phenobarbital, pregnenolone 16a-carbonitrile, and dexamethasone [129]. Both 6P-hydroxylation and 6dehydrogenation are likely mediated by CYP3A isozymes in catfish intestine [130]. The (+)APCI-MS/MS daughter spectra of three major daughter ions from the testosterone metabolite matched those obtained from a 4-androsten-3a, 17P-diol standard. The m/z 255 and 273 may be the [M+H-2H 2 Ot and [M+H-H 2 Ot ions, respectively, of the MW 290 steroid or they may be the [M+H-H 2 Ot and [M+Ht ions, respectively, of a MW 272 decomposition product or thermally degraded MW 290 steroid. Similarly, the m/z 289 may be the [M+Ht ion of a MW 288 compound (e.g., testosterone), the [M+H-H 2 Ot ion of an MW 306 compound or be due to thermal dehydrogenation of the MW 290 steroid during the vaporization (220 C and 300 C) process. The daughter spectra from the testosterone metabolite are almost identical to those obtained from the authentic steroid indicating they are most likely the same structures. Methods commonly used for identification of steroid hormone metabolites include high-performance liquid chromatography (HPLC), thin layer chromatography

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105 (TLC) and gasor liquid-chromatography-mass spectrometry (GC-MS and LC-MS) Our identification process of this 3a.-reduced testosterone metabolite suggested that a combination of these methods is perhaps the ideal way to identify a steroid hormone metabolite definitively in laboratories that have access to the specialized equipment required. In mammalian tissues, 3a.-hydroxysteroid dehydrogenase works in concert with Sa.and 5 P-reductases to generate the 3a.,5a.and 3a.,5 P-tetrahydroxysteroids, respectively, thereby acting as a molecular switch in steroid hormone activation [131]. Our finding of high activities of 3a.-hydroxysteroid dehydrogenase in catfish intestinal microsomes and activities of Sa-steroid reductase in intestinal cytosol suggested that these two reductases could work in concert in vivo. The biological significance of this steroid metabolism pathway in fish is not very well known. Catfish Hepatic CYP3A Expression The reason for the inducibility of PCN in hepatic CYP3A enzyme amount quantified by densitometric scans ofimmuno blots, but lack of effect on the CYP3A mediated testosterone 6P-hydroxylation activities is still unknown. Whether a larger dose of PCN or rifampicin would have resulted in both CYP3A enzyme amount and its catalytic activities remains to be determined.

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CHAPTERS SUMMARY AND CONCLUSIONS In conclusion the present study showed that a CYP3A-like protein was expressed in catfish intestine It is expressed gradually along intestine being the highest at the proximal end. Testosterone 6p-hydroxylation is a good biomarker for this CYP3A isozyme and this catalytic activity could be inhibited by P450 inhibitors, metyrapone ketoconazole SKF-525A and erythromycin Intestinal CYP3A expression and its catalytic activities were under dietary modulation, being higher in fish fed commercial chow than those fed semisynthetic purified diet. The mammalian CYP3A inducers rifampicin and PCN were not able to induce CYP3A in catfish intestine at the dose of 10 mg/kg fish body weight/day. In addition, testosterone was metabolized to 4-androsten3a 17P-diol and androstenedione by catfish intestinal microsomes. The former 3a-oxido reduction process is likely mediated by 3a-hydroxysteroid dehydrogenase The activity of 3a-hydroxysteroid dehydrogenase was mainly in the microsomal fraction in catfish intestine which was similar to that of human rather than rodents 106

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118 1 20 Lee, S-J ., Yang, Y-H., Wang, J-L., Miranda, C.L., Cok, I., Lech J.J., Buhler, D.R. Cloning, sequencing and tissue expression of the first CYP3A form from fish (CYP3A27) in rainbow trout. Fed. Proc. 11, A824(1997). 1 21. Ortiz de Montellano P.R., Correia, M.A ., (1995). Inhibition of cytochrome P450 enzymes In Cytochrome P450: Structure, Mechanism and Biochemistry (P.R. Ortiz de Montellano, Ed.), 2 nd ed., pp305-364. Plenum Press, New York. 1 22 Mansuy, D., Beaune, P., Cresteil, T., Bacot, C., Chottard, J.-C., Gans, P., Formation of complexes between microsomal cytochrome P-450-Fe(II) and nitrosoarenes obtained by oxidation of arylhydroxylamines or reduction of nitroarenes in situ. Eur. J Biochem. 86, 573-579(1978). 1 23. Doi, A.M., Lou, Z., Holmes, E., Li, C -L.J ., Venugopal, C.S., James, M.O ., and Kleinow, K.M. Effect of micelle fatty acid composition and 3,4,3 ',4' tetrachlorobiphenyl (TCB) exposure on intestinal [ 14 C]-TCB bioavailability and biotransformation in channel catfish in situ preparations. Toxico/ Sci 55, 8596(2000) 1 24. Cinti, D.L. Agents activating liver microsomal mixed-function oxidase system. Pharmaco/. Ther. 2, 727-749(1978). 1 25 Lee, C.A., Manyike, P.T., Thummel, K.E., Nelson, S.D., Slattery, J.T. Mechanism of cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes. Drug Metab. Dispos. 25, 1150-1156(1997). 1 26. Maenpaa, J., Hall, S.D Ring, B.J., Strom, S C ., Wrighton, S.A. Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone. Pharmacogenetics 8(2), 137-55(1998). 127. Hosea, N.A., Miller, G.P., Guengerich, F.P. Elucidation of distinct ligand binding sites for cytochrome P450 3A4. Biochemistry 39(20), 5929-5939, 2000. 128. Miranda, C.L., Wang, J-L., Chang, H-S ., Buhler, D.R. Multiple effects of 3,4,5,3',4',5'-hexachlorobiphenyl administration on hepatic cytochrome P450 isozymes and associated mixed-function oxidase activities in rainbow-trout. Biochem. Pharmoco/. 40, 387-390(1990) 129. Nagata, K., Liberato, D.J., Gillette, J.R. and Sasame, H.A An unusual metabolite of testosterone: l 7P-hydroxy-4,6-androstadiene-3-one. Drug Metab & Dispos. 14(5), 559-565(1986)

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119 130. Lou Z ., Celander, M ., Rowland-Faux, L. and James, M.O. Substrate inhibitor and activator properties and regional expression of a CYP3A-like protein in channel catfish intestine in preparation. 1 31. Khanna M. Qin, K.N. Wang, R W. Cheng K.C Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3a-hydroxysteroid dehydrogenases J Biol Chem. 270(34), 20162-8(1995) 132. Gibb C ., Glover, V ., Sandler, M. In vitro inhibition of phenolsulphotransferase by food and drink constituents. Biochem. Pharmacol. 36(14) 2325-30(1987). 1 33 Bamforth K.J., Jones, A.L ., Roberts, R.C., Coughtrie M W Common food additives are potent inhibitors of human liver 17 a-ethinyloestradiol and dopamine sulphotransferases. Biochem. Pharmacol. 46(10) 1713-20(1993). 1 34. Eaton E.A., Walle, U.K., Lewis, A.J., Hudson T., Wilson, A.A. Walle T. Flavonoids potent inhibitors of the human P-form phenolsulfotransferase. Potential role in drug metabolism and chemoprevention Drug Metab Dispos 24(2), 232-7(1996). 135 Ghazali, R.A., Waring, R H. The effects of flavonoids on human phenolsulphotransferases : potential in drug metabolism and chemoprevention. Life Sci. 65(16) 1625-32(1999). 136. Galijatovic A., Walle, U.K., Walle, T. Induction of UDP-glucuronosyltransferase by the flavonoids chrysin and quercetin in Caco-2 cells Pharm. Res. 17(1), 216(2000). 137. Siess M .H Guillermic, M., Le Bon, A.M., Suschetet, M. Induction of monooxygenase and transferase activities in rat by dietary administration of flavonoids. Xenobiotica 19, 1379-1386(1989) 138. Canivenc-Lavier M.C. Vemevaut, M.F., Totis M., Siess, M.H., Magdalou, J., Suschetet, M. Comparative effects of flavonoids and model inducers on drug metabolizing enzymes in rat liver. Toxicology 114(1 ), 19-27(1996).

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BIOGRAPHICAL SKETCH Zhen Lou was born on July 28, 1971, in Shanghai and spent her whole life in this city before she came to the United States of America in 1996. She earned her Bachelor of Science degree in medicinal chemistry in July 1993 from Shanghai Medical University. In July 1996 she acquired her Master of Science degree in organic synthesis from Shanghai Institute of Materia Medica, the Chinese Academy of Sciences She spent her first year in America in Massachusetts, working on Na +, K + -ATPase in the Department of Chemistry and Biochemistry Worcester Polytechnic Institute. She joined the research group of Dr. Margaret 0. James in fall 1997 when she transferred to the Department of Medicinal Chemistry College of Pharmacy University of Florida. 120

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy ff),,,.____,_ Margaret 0. ames, Chair Professor of Medicinal Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy ------------Sk -~ S~ Roberts Professor of Veterinary Medicine I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philos~ Kenneth B Sloan Professor of Medicinal Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. UI~ ~ William S. Dolbier, Jr. Professor of Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. ?n~~ Assistant Professor of Medicinal Chemistry

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This dissertation was submitted to the Graduate Faculty of the College of Pharmacy and to the Graduate School and was accept~d as parti lfillment of th requirements for the degree of Doctor of Philosophy. '~4May, 2001 Dean, Graduate School

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