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Biotransformation of Methoxychlor and Selected Xenobiotics in Channel Catfish (Ictalurus punctatus) and Largemouth Bass ...

Permanent Link: http://ufdc.ufl.edu/UFE0021409/00001

Material Information

Title: Biotransformation of Methoxychlor and Selected Xenobiotics in Channel Catfish (Ictalurus punctatus) and Largemouth Bass (Micropterus salmoides)
Physical Description: 1 online resource (188 p.)
Language: english
Creator: Nyagode, Beatrice
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 3methylcholanthrene, bass, benzoapyrene, bfcod, biotransformation, catfish, cyp3a, cytochrome, fish, hpte, ictalurus, induction, largemouth, methoxychlor, micropterus, ohmxc, p450, punctatus, salmoides, sulfation, sult
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The demethylation of methoxychlor (MXC), an organochlorine pesticide, gives rise to the mono- and bis-demethylated primary metabolites OHMXC and HPTE respectively which are estrogenic and antiandrogenic. In vitro results show that the efficiency of their sulfation is some times as much as ten times lower than their glucuronidation efficiency, both in the liver and intestine. The combined rates of the formation of the glucuronide and sulfate conjugation of OHMXC and HPTE are much lower than those of the cytochrome P450-dependent rates of their formation. This might mean that the expected detoxification pathways are inefficient at aiding the elimination of these potentially harmful metabolites after environmental exposure of the channel catfish to MXC. Sulfotransferases have generally been considered to be non-inducible in fish. We report increased sulfation, especially of 3-hydroxybenzo-a-pyrene, by 3-methylcholanthrene (3MC)-treated channel catfish when compared to control fish. Efficiency of hepatic sulfation increased from 930 ? 366microL/min/mg (mean plus or minus SE) in control to 2976 plus or minus 385 in 3MC-treated fish, while that of intestinal sulfation also went from 4301 plus or minus 1160 to 8602 plus or minus 230. In an in vivo study, the co-exposure of catfish to benzo-a-pyrene and radiolabeled MXC resulted in enhanced elimination of MXC. In control samples, 45.4 plus or minus 8.9% (mean plus or minus SE) of radioactivity was recovered as compared to 30.2 plus or minus 6.8% in benzo-a-pyrene-induced catfish. Of the dose recovered in the tissues analyzed, nearly 90% was found in bile, muscle and fat deposits. Analysis showed that OHMXC, HPTE and their glucuronide conjugates were formed, but no sulfate conjugates were detected. Potentially toxic metabolites of MXC were present in the edible muscle tissue. Probe substrates that yield highly fluorescent metabolites in isozyme-specific P450 reactions are popular because of their convenience compared to other substrates. We report that in the channel catfish and largemouth bass, the 7-benzyloxy-4-(trifluoromethyl)-coumarin O-debenzylase (BFCOD) assay, used successfully in several mammalian systems, does not correlate well with 6'-hydroxylation of testosterone, the standard marker for CYP3A. The BFCOD assay may therefore not be an indicator of CYP3A activity in these two species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Beatrice Nyagode.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: James, Margaret O.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021409:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021409/00001

Material Information

Title: Biotransformation of Methoxychlor and Selected Xenobiotics in Channel Catfish (Ictalurus punctatus) and Largemouth Bass (Micropterus salmoides)
Physical Description: 1 online resource (188 p.)
Language: english
Creator: Nyagode, Beatrice
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 3methylcholanthrene, bass, benzoapyrene, bfcod, biotransformation, catfish, cyp3a, cytochrome, fish, hpte, ictalurus, induction, largemouth, methoxychlor, micropterus, ohmxc, p450, punctatus, salmoides, sulfation, sult
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The demethylation of methoxychlor (MXC), an organochlorine pesticide, gives rise to the mono- and bis-demethylated primary metabolites OHMXC and HPTE respectively which are estrogenic and antiandrogenic. In vitro results show that the efficiency of their sulfation is some times as much as ten times lower than their glucuronidation efficiency, both in the liver and intestine. The combined rates of the formation of the glucuronide and sulfate conjugation of OHMXC and HPTE are much lower than those of the cytochrome P450-dependent rates of their formation. This might mean that the expected detoxification pathways are inefficient at aiding the elimination of these potentially harmful metabolites after environmental exposure of the channel catfish to MXC. Sulfotransferases have generally been considered to be non-inducible in fish. We report increased sulfation, especially of 3-hydroxybenzo-a-pyrene, by 3-methylcholanthrene (3MC)-treated channel catfish when compared to control fish. Efficiency of hepatic sulfation increased from 930 ? 366microL/min/mg (mean plus or minus SE) in control to 2976 plus or minus 385 in 3MC-treated fish, while that of intestinal sulfation also went from 4301 plus or minus 1160 to 8602 plus or minus 230. In an in vivo study, the co-exposure of catfish to benzo-a-pyrene and radiolabeled MXC resulted in enhanced elimination of MXC. In control samples, 45.4 plus or minus 8.9% (mean plus or minus SE) of radioactivity was recovered as compared to 30.2 plus or minus 6.8% in benzo-a-pyrene-induced catfish. Of the dose recovered in the tissues analyzed, nearly 90% was found in bile, muscle and fat deposits. Analysis showed that OHMXC, HPTE and their glucuronide conjugates were formed, but no sulfate conjugates were detected. Potentially toxic metabolites of MXC were present in the edible muscle tissue. Probe substrates that yield highly fluorescent metabolites in isozyme-specific P450 reactions are popular because of their convenience compared to other substrates. We report that in the channel catfish and largemouth bass, the 7-benzyloxy-4-(trifluoromethyl)-coumarin O-debenzylase (BFCOD) assay, used successfully in several mammalian systems, does not correlate well with 6'-hydroxylation of testosterone, the standard marker for CYP3A. The BFCOD assay may therefore not be an indicator of CYP3A activity in these two species.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Beatrice Nyagode.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: James, Margaret O.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021409:00001


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1 BIOTRANSFORMATION OF METHOXYCHL OR AND SELECTED XENOBIOTICS IN CHANNEL CATFISH ( Ictalurus punctatus ) AND LARGEMOUTH BASS ( Micropterus salmoides ) By BEATRICE A. NYAGODE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Beatrice A. Nyagode

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3 To my parents, my brothers and my sisters You are family, my friends and the compass by which I steer my life

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4 ACKNOWLEDGMENTS My utmost gratitude goes to Dr. Margaret O James for introducing me to and then expertly mentoring me through the amazing world of metabolic enzymes. I greatly appreciate the useful suggestions and gui dance of the members of my supervisory committee throughout this work. I would like to thank Laura Rowla nd-Faux for always being willing to help in any and all matters in the laborator y. It would have been a ve ry difficult, boring and unhappy journey without discussions, encouragement, shar ed frustrations and la ughter of many friends, fellow graduate students and members of the Depa rtment of Medicinal Chemistry and Center for Human and Environmental Toxicology here at the University of Flor ida. Finally, I would like to especially acknowledge my family for their supp ort and loving encouragement which motivated me to complete my study. This has not been the work of one person. I have come this far only because I have been surrounded with the help and support of more pe ople than it would be possible for me to individually mention here. I acknowledge each one of you and thank you all very much. It is indeed true: “It takes a whole village to raise a child” – African Proverb

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 LIST OF ABBREVIATIONS........................................................................................................14 ABSTRACT....................................................................................................................... ............16 CHAPTER 1 Biotransformation and the de toxification of xenobiotics.......................................................18 Cytochrome P450 Enzymes....................................................................................................18 Modulation of Cytochrome P450s...................................................................................19 P450s in Fish.................................................................................................................. .20 Fluorometric Assays........................................................................................................23 In Vitro Approaches to Catalytic Specificity...................................................................24 Correlations..............................................................................................................24 Inhibitors..................................................................................................................25 Conjugation Reactions in Metabolism....................................................................................26 Glucuronidation...............................................................................................................2 6 Fish uridine diphosphate-glucu ronosyltransferases (UGTs)....................................27 Regulation of UGTs.................................................................................................28 Sulfation...................................................................................................................... ....28 Fish sulfotransferases...............................................................................................29 Regulation of sulfotransferases................................................................................30 Endocrine Disruption........................................................................................................... ...31 Methoxychlor................................................................................................................... .......31 Metabolism in Humans....................................................................................................34 Metabolism in Other Animals.........................................................................................35 Metabolism in Fish..........................................................................................................37 Regulation of Methoxychlor Metabolism.......................................................................38 Selection of Animal Models...................................................................................................40 Objective of the Study......................................................................................................... ...41 Hypotheses..................................................................................................................... .........41 2 MATERIALS AND METHODS...........................................................................................52 Chemicals...................................................................................................................... .........52 Animal Treatment............................................................................................................... ....53

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6 Catfish........................................................................................................................ ......53 Non-radiolabeled methoxychlor studies...................................................................53 Radiolabeled met hoxychlor studies.........................................................................53 Largemouth Bass.............................................................................................................54 Non-radiolabeled methoxychlor studies...................................................................54 Dieldrin and DDE studies........................................................................................54 Subcellular Fractionation...................................................................................................... ..55 Non-radiolabeled Samples...............................................................................................55 Radiolabeled Samples.....................................................................................................56 Quantitation of the Distribution of Radiolabeled Methoxychlor............................................56 Extraction of Methoxychlor and its Me tabolites from Tissues and Bile................................56 Bile Hydrolysis................................................................................................................ .......57 Lipid Quantitation............................................................................................................. ......58 7-Benzyloxy-4-(trifl uoromethyl)-coumarin (BFC) Synthesis................................................58 Western Blot Analysis.......................................................................................................... ..58 Assays......................................................................................................................... ............59 7-Benzyloxy-4-(trifl uoromethyl)-coumarin O-debenzylation.........................................59 Chemical Modulation of 7-Benzylox y-4-(trifluoromethyl)-coumarin ODebenzylation..............................................................................................................60 Hydroxylation of Testosterone........................................................................................60 Ethoxyresorufin O-Deethylation.....................................................................................61 Sulfonation of Monoand Bisdemethylated Methoxychlor...........................................61 Sulfonation of 3-Hydroxybenzo-[a]-pyrene....................................................................63 Methoxychlor Monooxygenation....................................................................................63 Glucuronidation of Mono-deme thylated Methoxychlor.................................................64 HPLC Analysis.................................................................................................................. .....64 Data Analysis.................................................................................................................. ........65 3 THE 7-BENZYLOXY-4-(TRIFLUOROMETHYL)-COUMARIN O-DEBENZYLASE (BFCOD) ASSAY IN CHANNEL CA TFISH AND LARGEMOUTH BASS......................67 Results........................................................................................................................ .............67 Assay Development.........................................................................................................67 Comparison of Catfish and Largemouth Bass Microsomal Activity..............................68 Validation of the Assay...................................................................................................69 Discussion..................................................................................................................... ..........71 Conclusions.................................................................................................................... .........76 4 SULFATION OF SELECTED XENOBIOTICS.................................................................105 Results........................................................................................................................ ...........105 Sulfation of OHMXC....................................................................................................105 Sulfation of HPTE.........................................................................................................106 Sulfation of 3-Hydroxybenzo-[a]-pyrene......................................................................107 Discussion..................................................................................................................... ........108 Conclusions.................................................................................................................... .......110

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7 5 IN VITRO AND IN VIVO METABOLISM OF METHOXYCHLOR.................................126 In Vivo Radiolabeled Methoxychlor Studies........................................................................126 Distribution of Radioactivity in Fish Tissue and Bile...................................................126 Quantitation of Radiolabeled Metabolites.....................................................................127 Enantioselective Metabolism of Methoxychlor....................................................................128 In Vitro Demethylation of Methoxychlor......................................................................128 In Vitro Glucuronidation of OHMXC...........................................................................129 Bile Hydrolysis..............................................................................................................12 9 Lipid Quantitation............................................................................................................. ....130 Distribution of Non-Radiol abeled Methoxychlor and its Metabolites in Liver...................130 Discussion..................................................................................................................... ........131 Conclusions.................................................................................................................... .......137 LIST OF REFERENCES............................................................................................................. 175 BIOGRAPHICAL SKETCH.......................................................................................................188

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8 LIST OF TABLES Table page 1-1 Effect of different modulators on in vivo CYP3A gene, protein or activity levels of different fish................................................................................................................. ......46 1-2 The 7-benzyloxy-(4-trifluoromethyl)-coumar in-O-debenzylase (BFCOD) assay in different fish................................................................................................................. ......48 1-3 Kinetic constants of zebra fish SULT1 ST1 and ST2 with hydroxychlorobiphenyls and 3,3’,5-triiodo-L-thyronine as substrates......................................................................49 1-4 Estrogen receptor binding activity of demethylated methoxychlor metabolites...............49 1-5 Kinetic parameters for the formation of OHMXC by catfish liver microsomes...............50 1-6 Rates of microsomal formation of OHMXC and HPTE from 14C-MXC in the channel catfish................................................................................................................ ...50 1-7 Rates of glucuronidation of OHMXC by channel catfish microsomes.............................51 1-8 Rates of glucuronidation of HPTE by channel catfish microsomes..................................51 3-1 The BFCOD activity of male and female fish in different treatment groups....................99 3-2 Activity in different assays of channel cat fish microsomes derived from different treatment groups............................................................................................................... ..99 3-3 Activity of products of testosterone meta bolism by hepatic P450s from different largemouth bass treatment groups...................................................................................100 3-4 Activity of products of testosterone meta bolism by hepatic P450s from different catfish treatment groups...................................................................................................100 3-5 Correlation matrix of BFCOD and test osterone metabolism products in the largemouth bass...............................................................................................................1 01 3-6 Correlation matrix of BFCOD and testoste rone metabolism products in the channel catfish. ...................................................................................................................... ......101 3-7 The IC50 values for inhibitors of CYP3A activity in the channel catfish........................102 3-8 The IC50 values for channel catfish BFCOD activity inhibition by ketoconazole...........102 3-9 Correlation coefficients for dealkylation of the fluorometric probe and P450 isoform selective catalytic activities in a pane l of human donors.................................................103

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9 3-10 The 7-benzyloxy-(4-trifluoromethyl)-coumar in-O-debenzylase (BFCOD) assay in different fish................................................................................................................. ....104 4-1 Sulfation rates of OHMXC by hepatic catfish cytosol....................................................123 4-2 Sulfation rates of OHMXC by intestinal catfish cytosol.................................................123 4-3 Sulfation rates of HPTE by hepatic catfish cytosol.........................................................124 4-4 Sulfation rates of HPTE by in testinal catfish cytosol......................................................124 4-5 Rates of Sulfonation of 3 OHBaP by catfish cytosol........................................................125 4-6 Conjugation of hydroxylated PCBs in intestin al fractions of the channel catfish...........125 5-1 Summary of radioactive count distribution in subcellular fr actions of liver of MXCand MXCBaP-treated channel catfish..............................................................................169 5-2 The HPLC method and anal yte retention times of MXC and its metabolites.................169 5-3 Percent of total radioactivity recovered from tissue pellet s after metabolite extraction..170 5-4 Methoxychlor and metabolites detected in liv er of variously treated channel catfish and largemouth bass.........................................................................................................171 5-5 Summary of metabolite concentrations from different tissues and bile of 14Cmethoxychlor-treated channel catfish..............................................................................172 5-6 Comparison of the effect of route of methoxychlor administration on levels of vitellogenin in fish........................................................................................................... 173

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10 LIST OF FIGURES Figure page 1-1 Proposed roles of CYP1A2 and CYP2C9 in methoxychlor O-demethylation by human liver mi crosomes....................................................................................................42 1-2 Relative P450 activities (rate s) in O-demethylation of individual (R)or (S)OHMXC formation of HPTE.............................................................................................43 1-3 Proposed pathway for the metabolism of me thoxychlor by human liver microsomes......44 1-4 Proposed metabolic pathways of methoxychlor by precision-cut rat, mouse Japanese quail and rainbow trout liver slices....................................................................................45 3-1 Proton NMR of 7-benzyloxy-4-(trifluor omethyl)-coumarin.............................................77 3-2 The HPLC chromatogram of 7-benzyl oxy-4-(trifluoromethyl)-coumarin (BFC).............78 3-3 Emission spectra of 7-hydroxy-4-(trifluor omethyl)-coumarin (A) and 7-benzyloxy-4(trifluoromethyl)-coumarin (B) obtained at a 410nm excitation wavelength....................79 3-4 The 7-benzyloxy-(4-trifluoromethyl)-coumar in-O-debenzylase (BFCOD) reaction........80 3-5 Time course for 7-hydroxy-4-(trifluoro methyl)-coumarin production by largemouth bass hepatic microsomes. ..................................................................................................81 3-6 Protein amount optimization for 7-hydr oxy-4-(trifluoromethyl)-coumarin production by hepatic microsomes of the largemouth bass. ...............................................................82 3-7 Stability profiles of BFCOD assay products......................................................................83 3-8 Temperature optimization for HFC production by hepatic microsomes of the channel catfish. ..................................................................................................................... .........84 3-9 Temperature optimization for HFC production by hepatic microsomes of the largemouth bass. ............................................................................................................. .85 3-10 Comparison of kinetic parameters between male and female fish....................................86 3-11 The BFCOD activity of fish in different treatment groups. .............................................87 3-12 Western blot of human CYP3A4 and hepati c microsomes from largemouth bass and catfish. ...................................................................................................................... ........88 3-13 Correlation of protein band intensity and activity of channel catfish samples..................88 3-14 Correlation between BFCOD and 6 -hydroxylation of testoster one activity of hepatic microsomes of channel catfish in different treatment groups............................................89

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11 3-15 Correlation between BFCOD and EROD activit y of hepatic microsomes of channel catfish in different treatment groups..................................................................................90 3-16 Normal phase TLC of testosterone metabol ism by hepatic microsomes of largemouth bass and catfish............................................................................................................... ...91 3-17 Activity of testosterone metabolites in different treatment groups....................................92 3-18 The BFCOD activity of different treatmen t groups for catfish and largemouth bass........94 3-19 Compounds used to modulate hepatic CY P3A activity in catfish microsomes.................95 3-20 The BFCOD Activity of channel catfish microsomes in the presence of chemical modulators..................................................................................................................... .....96 3-21 Chemical Inhibition of hepatic microsom al BFCOD activity of channel catfish by ketoconazole................................................................................................................... ...97 3-22 Testosterone hydroxylation, a useful dia gnostic functional marker for P450s..................98 4-1 Time course for OHMXC sulfate production by channel catfish hepatic and intestinal cytosol........................................................................................................................ ......112 4-2 Sulfation of 100M OHMXC by different amou nts of cytosolic protein from catfish liver and intestine............................................................................................................ .113 4-3 Reverse phase TLC Chromatogram of th e sulfation of 800M OHMXC by catfish liver cytosol. ............................................................................................................... ....114 4-4 Normal phase TLC Chromatogram of th e sulfation of OHMXC by catfish liver cytosol. ...................................................................................................................... ......115 4-5 The OHMXC sulfation activity of hepatic and intestinal catfish cytosol. ......................116 4-6 Time course for HPTE sulfate production by channel catfish hepa tic and intestinal cytosol. ...................................................................................................................... ......117 4-7 Sulfation of 200M HPTE by different amount s of cytosolic protein from catfish liver and intestine............................................................................................................ .118 4-8 Normal phase TLC Chromatogram of the sulfation of OHMXC and HPTE by catfish liver cytosol. Lane 1: 50M OHMXC; Lane 2: 100M HPTE.......................................119 4-9 The HPTE sulfation activity of hepati c and intestinal catfish cytosol.............................120 4-10 Sulfation of 3OHBaP by hepatic catfish cytosol. ...........................................................121 4-11 Plots of 3-hydroxybenzo-[a]-pyrene sulfa tion activity by catfis h liver cytosol..............122

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12 5-1 The EROD activity of MXC and MXCBaP –treated groups...........................................139 5-2 Distribution of radioact ivity in samples of th e channel catfish. .....................................140 5-3 Percent distribution of recove red radioactivity in different tissues and bile of MXCand MXCBaP-treated channel catfish..............................................................................141 5-4 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the liver of 14C-MXC-treated fish. .....................................................142 5-5 Concentration of methoxychlor a nd its metabolites in liver of 14C-methoxychlortreated channel catfish. ...................................................................................................14 3 5-6 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the intestinal mucosa of 14C-MXC-treated fish. ................................144 5-7 Concentration of methoxychlor and its metabolites in intestinal mucosa of 14Cmethoxychlor-treated channel catfish..............................................................................145 5-8 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the blood of 14C-MXC-treated fish. ....................................................146 5-9 Concentration of methoxychlor and its metabolites in blood of 14C-methoxychlortreated channel catfish. ..................................................................................................14 7 5-10 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the muscle of 14C-MXC-treated fish. .................................................148 5-11 Concentration of methoxychlor a nd its metabolites in muscle of 14C-methoxychlortreated channel catfish. ...................................................................................................14 9 5-12 Normal phase TLC Chromatogram of me thoxychlor and its metabolites in fat deposits and gonad of 14C-methoxychlor-treated channel catfish. ................................150 5-13 Concentration of methoxychlor a nd its metabolites in gonads of 14C-methoxychlortreated channel catfish. ...................................................................................................15 1 5-14 Concentration of methoxychlor and its metabolites in fat deposits of 14Cmethoxychlor-treated channel catfish. ...........................................................................152 5-15 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the brain of 14C-MXC-treated fish. ....................................................153 5-16 Concentration of methoxychlor a nd its metabolites in brain of 14C-methoxychlortreated channel catfish. ...................................................................................................15 4 5-17 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the skin of 14C-MXC-treated fish. .....................................................155

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13 5-18 Concentration of methoxychlor and its metabolites in skin of 14C-methoxychlortreated channel catfish. ..................................................................................................15 6 5-19 The HPLC chromatogram of the reverse phase separation of MXC and its metabolites from the bile of 14C-MXC-treated fish. ......................................................157 5-20 Concentration of methoxychlor a nd its metabolites in bile of 14C-methoxychlortreated channel catfish. ....................................................................................................15 8 5-21 The HPLC chromatogram of the revers e phase separation of the products of demethylation of 14C-MXC by largemouth bass hepatic microsomes. ..........................159 5-22 The HPLC chromatogram of the chiral se paration of the products of demethylation of 14C-MXC by channel catfish hepatic microsomes. ...................................................160 5-23 Comparison of enantiomer areas (R /S) after the de methylation of 14C-MXC by microsomes. .................................................................................................................. .161 5-24 Comparison of enantiomer areas (R/S) af ter the glucuronid ation of OHMXC by microsomes. .................................................................................................................. .162 5-25 The HPLC chromatogram of the chiral separation of HPTE and OHMXC from the glucuronidase hydrolyzed bile of 14C-MXC-treated fish. ...............................................163 5-26 The HPLC/UV chromatogram of the separa tion of MXC and its metabolites during method delopment using reverse phase catridges............................................................164 5-27 The HPLC/UV chromatogram of the separati on of MXC and its metabolites from the liver of MXC-treated largemouth bass. .........................................................................165 5-28 Comparison of the amounts of methoxychl or and its metabolites in liver of methoxychlor-treated largemouth bass. ..........................................................................166 5-29 Comparison of the amounts of methoxychl or and its metabolites in liver of methoxychlor-treated channel catfish. ............................................................................167 5-30 Proposed formation of met hoxychlor olefin from methoxychlor....................................168

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14 LIST OF ABBREVIATIONS 3MC 3-methylcholanthrene 3OHBaP 3-hydroxybenzo-[a]-pyrene BaP benzo-[a]-pyrene BSA bovine serum albumin CAR constitutive androstane receptor CB126 3,3’,4,4’,5-pentachlorobiphenyl CYP cytochrome P450 DDE 1,1-dichloro-2,2-bis( p -chlorophenyl)ethene DDT 1,1,1-trichloro-2,2-bis( chlorophenyl)ethane ER estrogen receptor EROD ethoxyresorufin O-deethylase HEPES N-(2-hydroxyethyl)piper azine-N’-2-ethanesulfonic acid HFC 7-hydroxy-(4-trifluoromethyl)coumarin HPLC high performance liquid chromatography HPTE bis-demethylated methoxychlor (2,2-bis( p -hydroxyphenyl)-1,1,1trichloroethane) i.p. intraperitoneal MXC methoxychlor (1,1,1-trichl oro-2,2-bis(4-methoxyphenyl)ethane) MXC olefin 1,1-Dichloro-2, 2-bis(4-methoxyphenyl) ethene NADPH nicotinamide adenine dinucle otide phosphate (reduced form) NMR nuclear magnetic resonance OHMXC mono-demethylated methoxychlor (2-( p -hydroxyphenyl)-2-( p methoxyphenyl)-1,1,1-trichloroethane) PAPS 3’-phosphoadenosine 5’-phosphosulfate

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15 PB phenobarbital PCB polychlorinated biphenyl PICA tetrabutylammonium sulfate PXR pregnane X receptor TMS Tetramethylsilane TLC thin layer chromatography Tris Tris-(hydroxymethyl)-aminomethane UDPGA uridine diphos phoglucuronic acid UGT uridine diphosphate glucuronosyl transferase UV ultraviolet

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOTRANSFORMATION OF METHOXYCHL OR AND SELECTED XENOBIOTICS IN CHANNEL CATFISH ( Ictalurus punctatus ) AND LARGEMOUTH BASS ( Micropterus salmoides ) By Beatrice A. Nyagode August 2007 Chair: Margaret O. James Major: Pharmaceutical Sciences The demethylation of methoxychlor (MXC), an organochlorine pe sticide, gives rise to the monoand bis-demethylated primary metabol ites OHMXC and HPTE respectively which are estrogenic and antiandrogenic. In vitro results show that the efficien cy of their sulfation is some times as much as ten times lower than their glucuronidation efficiency, both in the liver and intestine. The combined rates of the formati on of the glucuronide and sulfate conjugation of OHMXC and HPTE are much lower than those of the cytochrome P450-dependent rates of their formation. This might mean that the expected de toxification pathways ar e inefficient at aiding the elimination of these potenti ally harmful metabolites after environmental exposure of the channel catfish to MXC. Sulfotransferases have generally been considered to be non-inducible in fish. We report increased sulfation, especially of 3-hydroxybenzo-[a]-pyrene, by 3methylcholanthrene (3MC)-treated channel catfish when compared to control fish. Efficiency of hepatic sulfation increa sed from 930 366L/min/mg (mean SE) in control to 2976 385 in 3MC-treated fish, while that of intestin al sulfation also went from 4301 1160 to 8602 230. In an in vivo study, the co-exposure of catfish to be nzo-[a]-pyrene and radiolabeled MXC resulted in enhanced elimination of MXC. In control samples, 45.4 8.9% (mean SE) of

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17 radioactivity was recovered as compared to 30.2 6.8% in benzo-[a]-pyrene-induced catfish. Of the dose recovered in the tissues analyzed, ne arly 90% was found in bile, muscle and fat deposits. Analysis showed that OHMXC, HPTE a nd their glucuronide conjugates were formed, but no sulfate conjugates were detected. Potentia lly toxic metabolites of MXC were present in the edible muscle tissue. Probe substrates that yiel d highly fluorescent metabolit es in isozyme-specific P450 reactions are popular because of th eir convenience compared to other substrates. We report that in the channel catfish and largemouth bass, the 7-benzyloxy-4-(trifluoromethyl)-coumarin Odebenzylase (BFCOD) assay, used successfully in several mammalian systems, does not correlate well with 6 -hydroxylation of testosterone, the st andard marker for CYP3A. The BFCOD assay may therefore not be an indicator of CYP3A ac tivity in these two species.

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18 CHAPTER 1 BIOTRANSFORMATION AND THE DETO XIFICATION OF XENOBIOTICS Xenobiotics that enter the body of most animals are nor mally lipid soluble and cannot therefore be easily eliminated. It is only following their biotransformation, during which a hydrophilic moiety such as a sulfate or glucur onic acid is conjugated to them, that the compounds become less lipophilic an d water soluble enough to enable elimination. Conjugation reactions, normally referred to as phase II reactions, are often but not always, preceded by phase I metabolic reactions such as oxidation or hydrol ysis. The majority of phase I metabolism of xenobiotics occurs through the cytochrome P450 system (P450s). Biotransformation through phase I and II enzymes, though generally a requisite for detoxification and excretion of foreign lipophilic chemicals, is also sometimes res ponsible for the activation of compounds to intermediates that resu lt in adverse effects. Enzymes ma y compete for the conversion of the same and (or) different groups in the same molecu le resulting in an arra y of metabolites that differ in a speciesas well as dose-dependent manner. Cytochrome P450 Enzymes P450s are a superfamily of heme-thiolate pr oteins that catalyze bot h the biosynthesis of molecules of physiological importance as well as catabolic pathways. In mammals, all P450s are membrane bound. Most are found in the endoplasmic reticulum, but five are localized primarily in mitochondria (Guengerich, 2003). P450s pr incipally introduce oxygen into a molecule (Equation 1-1), to increase the hydrophilicity of the product and hence the ease with which the product can be eliminated from the body. NADPH + H+ + RH + O2 NADP+ + H2O + ROH (1-1) (where R = substrate, RO = product). The activity of P450s in the endoplasmic reticulum depend on a flavoprotein, reduced nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase, which transfers

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19 electrons in two separate, one electron reductions, from NA DPH to the P450 (Buhler and Williams, 1989; Guengerich, 2003). These enzymes are expressed in many tissues but in mammals are found at the highest levels in liver. The small intestine is the pr incipal extrahepatic sour ce of P450 isoforms, but smaller quantities are also pres ent in the kidneys, lungs, skin and brain. Cloning and expression studies suggest many similarities among vertebrates P450 genes. The major microsomal gene families, 1, 2, 3 and 4 are found from fish to mammals (Stegeman and Livingstone, 1998). The P450 system has been classified into unique families based on amino acid sequence homology. The gene family name is de noted by an Arabic numeral (e.g. CYP 3 ). P450s within a given family have greater than 40% sequence ho mology. Families are further differentiated into gene subfamilies denoted by an upper case letter (e.g. CYP3 A ). Within a particular subfamily P450s have sequence homology in excess of 55% Gene numbers of individual enzymes are denoted by a second Arabic numeral follo wing the subfamily letter (e.g. CYP3A 4 ). The human genome encodes fifty-seven P450 proteins (Guengerich, 2003). Modulation of Cytochrome P450s Treatment with different compounds or hormone s has been shown to result in different relative concentrations of P450s. Compounds that induce or inhibit activity have been useful in discerning the catalytic specific ity of P450s. Caution is normally exercised during these investigations however since full information about the selectivity of an inhibitor or inducer may not be known (Goksyr and Frlin, 1992; Halpert et al., 1994). While many are expressed constitutively, P450s are inducible. Polycyclic aromatic hydrocarbons (PAHs), such as 2,3,7,8-tetrachlorodibenzop -dioxin (TCDD), 3methylcholanthrene (3MC) and benzo-[a]-pyrene (BaP) induce th e transcription of CYP1A1, a process which is regulated by the aromatic hydr ocarbon (Ah) receptor and the Ah receptor

PAGE 20

20 nuclear translocator (Arnt) prot ein. Pregnane X receptor (PXR) ligands such as the synthetic steroid pregnenolone 16 -carbonitrile (PCN), the antibiotic rifampicin and the anti-inflammatory glucocorticoid dexamethasone, though chemically unrelated, all up-regu late CYP3A activity (Willson and Kliewer, 2002). Phenobarbital (PB) a barbiturate and powerful antiepileptic drug, 1,4-bis(2-(3,5-dichloropyridyl oxy))benzene (TCPOBOP), the organochlorine pesticide 1,1,1trichloro-2,2-bis(chlorophenyl)e thane (DDT) and certain polychl orinated biphenyls (PCBs) are all thought to be ligands of the constitutive a ndrostane receptor (CAR) which regulates CYP2B induction (Whitlock and Denison, 1995; Willson and Kliewer, 2002). A variety of compounds that are structurally dissimilar such as clof ibrate, a hypolipidemic drug, trichloroacetic acid, a solvent and di-(2-ethyl-hexyl)pht halate, a plasticizer are thou ght to act via a peroxisome proliferator activator receptor (PPAR) to i nduce CYP4A activity (Whitlock and Denison, 1995). In the cytochrome P450 catalytic cycle three step s are particularly susc eptible to inhibition: substrate binding, biding of mo lecular oxygen after the first el ectron transfer and the actual oxidation of the substrate. Mechanistically, P450 i nhibitors can be divided into agents that bind reversibly, quasi-irreversibly with the heme ir on atom or irreversibly to protein, heme or accelerate the degradation of the pr osthetic heme group. Species differences have been noted in the selectivity of P450 inhib itors. Inves tigation of 6 -hydroxylation of test osterone by CYP3A showed that ketoconazole was almost 10-fold more potent an inhi bitor in human liver microsomes than rat liver microsomes (Eagli ng et al., 1998). In the CYP2C9-dependent 4hydroxylation of tolbutamide howev er, ketoconazole displayed al most equal potency as an inhibitor in both rat liver microsom es and human liver microsomes. P450s in Fish Different isoforms of P450s have been repor ted in fish. Like in mammals, they are predominantly found in the liver, but are also evid ent at lower concentrat ions in other tissues

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21 (James et al., 1997; Buhler and Wang-Buhler, 199 8; Hegelund and Celander, 2003). It is thought that the rainbow trout P450s are as complex as those seen in ma mmals. Fourteen P450s from seven different P450 families have been docum ented from trout liver, kidney and ovary including CYP1A, CYP2 (K and M), CYP3A CYP4T, CYP11A, CYP17 and CYP19 (Buhler and Wang-Buhler, 1998). In the channel catfish ( Ictalurus punctatus ), there have been reports of CYP1A, at least 4, but possibly 5, CYP2-like isoforms (Schlenk et al., 2002) and also two CYP3A isoforms, 3A86 and 3A87 in liver and in testine respectively (personal communication with D. Barber and M. O. James, 2007). CYP3A 68 and CYP3A69 have also been isolated from the liver and intestine of the la rgemouth bass the intestine. Fish CYP1As are similar to each other a nd to mammalian CYP1A1 proteins (Stegeman, 1989). Like in mammals, PAHs and planar halogenated aromatic hydr ocarbons induce teleost CYP1A genes (Stegeman et al., 1997). Some studies have however documented that ‘phenobarbital-like’ inducer s seem ineffective in fish (Addison et al., 1987; Ankley et al., 1987). In comparison to mammals, there is very little information on the modulation, particularly the inducibility of the CYP3A system in fish. A fe w investigators have repo rted increased protein expression when fish were treated with vari ous compounds including alkylphenols (Hasselberg et al., 2004a; Meucci and Arukwe, 2006; Sturve et al., 2006), ke toconazole (Hegelund et al., 2004), dexamethasone, rifampicin and TCDD (Tseng et al., 2005), DDE and thyroxine (Mortensen and Arukwe, 2006), 3,3',4,4',5-pent achlorobiphenyl (Schlezinger and Stegeman, 2001) as well as pregneolone-16 -carbonitrile (Pathiratne and Geor ge, 1996); (Table 1-1). It has been shown that rifampicin, pregneolone-16 -carbonitrile and dexamethasone, which are known to upregulate mammalian CYP3As, had no effect in the liver and intestin e of the catfish (Lou, 2001). However when catfish fed on a chow diet were compared to those fed on a purified diet,

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22 they had twice the CYP3A c ontent and testosterone 6 -hydroxylation activity of the latter (James et al., 2005). Zebrafish PXR receptor sequences were tested in transient transfection assays against a panel including xenobi otics and endogenous molecules (Moore et al., 2002). Eight compounds, namely nifedipine, phe nobarbital, clotrimazole, 5 -pregnane-3,20-dione, androstanol, DHT, DHEA and n -propylp -hydroxybenzoate resulted in gene activation of between 2.5 and 9.9 times the control sugges ting these compounds may induce CYP3A in zebrafish. Microsomes derived from the sea bass ( Dicentrarchus labrax) treated with some of these compounds however did not show any increas e in the CYP3A-associ ated catalyst activity, testosterone 6 -hydroxylation (Vaccaro et al., 2005). Among fish some differences have been doc umented on the effects of P450 inhibitors. Clotrimazole, an antifungal imid azole, inhibited rainbow trout, Oncorhyncus mykiss hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro but did not affect CYP1A mRNA levels (Levine and Oris 1999). When measured using both the 7benzyloxy-(4-trifluoromethyl)-coumarin-O-d ebenzylase (BFCOD) and EROD assays, ketoconazole was only two times more potent an inhibitor of CYP3A than CYP1A in the Atlantic cod (Hasselberg et al., 2004b). In kill ifish S9 fractions ketoconazole was 60 times less potent an inhibitor of EROD th an BFCOD activity, while in ra inbow trout liver microsomes ketoconazole inhibited EROD and BFCOD activities almost to the same extent (Hegelund et al., 2004). A study evaluating the ability of several inhi bitors of mammalian P450s to affect hepatic P450-mediated monooxygenase activ ities in microsomes from -naphthoflavone (BNF)-treated rainbow trout concluded that i nhibition data from mammalian st udies could not be directly extrapolated to trout and most probably ot her fish studies too (Miranda et al., 1998).

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23 Fluorometric Assays The classical approach for evaluating in vitro enzyme activity is to determine the conversion rate of a probe substr ate into its metabolite over a range of substrate concentrations sometimes with a potential inhibitor. While there are a variety of assays available for this purpose a lot of them are plagued by almost identi cal problems. Most of them are cumbersome and time consuming, involving the use of difficult to obtain radiolabeled substrates and (or) tedious solvent extraction and followed by ch romatographic separati ons that do not lend themselves well to a large volume of assays that n eed to be done in a short time. The need to separate product from substrate prior to analysis also means that, unless multiple time points are taken for each sample, a linear rate of reaction ov er the course of the incubation must be assumed in order to calculate a reaction rate. Finally, many of the curren tly available methods suffer from a lack of sensitivity, often requiring large am ounts of protein for a single determination. Assays with a fluorometric endpoint are partic ularly advantageous because they offer high sensitivity, increased throughput, allow continuous reaction monitoring as well as quantification that does not require chromatographic separati on as well as minimize the amount of enzyme required in the reaction. These assays are ba sed on a P450 catalyzed O-dealkylation reactions which produce an easily detectable fluorescent me tabolite. Several resoru fin derivatives have been used in this manner. 7-Ethoxyresorufin an d 7-methoxyresorufin have been used as probes for CYP1A while 7-benzyloxyresoruf in and 7-pentoxyresorufin were useful for CYP2B isoforms (Burke et al., 1994; Oropeza-Hernandez et al., 2003). Crespi and Stresser (2000) compared the su itability of dibenzylfluorescein (DBF), 7benzyloxy-4-trifluoromethylcoumarin (B FC), 7-benzyloxyquinoline (BQ) and 7benzyloxyresorufin (BzRes) as substrates for CY P3A activity. They reported that relative to BzRes and BQ, DBF and BFC were more soluble in aqueous media. BFC and DBF also gave

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24 improved signal-to-background ratio per unit enzyme It has been reported that the good correlation in IC50 values seen for CYP1 and CYP2 en zymes among fluorometric substrates and traditional substrates does not generally apply to CYP3A4 (Crespi and Stresser, 2000; Miller et al., 2000). These authors therefore recommend the use of multiple probe substrates in evaluating the inhibition potential of CYP3A4 but single out BFC as the most conservative as well as most sensitive choice (Stresser et al., 2000). Stresser et al (2002) investigat ed the catalytic se lectivity of human and rat P450 isoform among several fluorescent substrates. In most cases multiple cDNA-expressed cytochrome P450 isoforms were found to catalyze the formation of the fluorescent product. They reported that both BFC and BQ were less selective than the 6 -hydroxylation of testosterone as CYP3A4 probe substrates. Their results also showed that even though BFC was relatively selective for CYP3A, it was also metabolized by CYP1A2 and (or) other extrahepatic enzymes. In Vitro Approaches to Catalytic Specificity In order to define the cataly tic activity of an individual P450 one or more of different approaches may be used. Those used in th is body of work are detailed below. Correlations Levels of the catalytic activ ity under consideration are measured in a number of different samples and then these correlated with accepted marker catalytic activities of the same samples. A correlation coefficient is a simple statis tical measure of relationship between one dependent and one or more independent variable s. Several correlation coefficients based on different statistical hypothesis are known. The most widely used is the Pearson correlation coefficient. This measures the strength and di rection of the linear relationship between two variables, describing the direction and degree to which one variable is linearly related to another. The problem with the Pearson correlation coeffici ent is that it assumes the variables are well

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25 approximated by a normal distribution (Ott and Longnecker, 2001). If the data under study does not meet this assumption then it is either transfor med in some way or an alternative coefficient is used. The Spearman’s rank correlation coeffici ent is a non-parametric measure of correlation between variables which does not make any assump tions about the frequency distribution or the linearity of variables bei ng compared (Wardlaw, 2000). Frequently the Greek letter (rho) is used to abbreviate the Spearman correlation coeffi cient. The disadvantage of this coefficient is that while it is satisfactory for testing the null hypothesis of no relations hip, it does not aid the interpretation the strength of the relationship between the va riables being measured (Bland, 1995). Both coefficients are interpreted almost the same way. They can take values from -1 to +1. A value of +1 shows that variab les are perfectly correlated by an increasing relationship while a value of -1 means that variable s are perfectly correlated by a de creasing relationship. A value of 0 means that the variables are not correlated to each other. A correlation coefficient that is greater than 0.8 is considered a strong while one less than 0.5 is consider ed a weak correlation. There should be a high correlation if the reacti ons are catalyzed by th e same enzyme. If two P450s are under common regulation however, this approach will not distinguish which one is involved in the reaction. Inhibitors Another approach involves th e inhibition of the catalytic activity under investigation. Experiments are carried out with a range of inhibitors at differe nt concentrations. The maximum effective concentration of inhibitor is viewed, at least in pr inciple, to correspond to the fraction of the reaction resulting from the P450 under consideration. The success of this approach depends largely on the select ivity of the inhibitor.

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26 Conjugation Reactions in Metabolism Conjugation reactions become important duri ng biotransformation when the molecule already has certain functional groups but is still not hydrophilic enough to be eliminated. Common functional groups th at are conjugated include alcohols, phenols, amines, unsaturated carbonyls, carboxylates and thiols. Acetyla tion, sulfation, car bohydrate, glutathione and taurine conjugation are most the common path ways undergone by pesticides and their phase I metabolites in aquatic species (James, 1994) At the phenolic hydroxyl group the major conjugation reactions are glucur onidation and sulfation. Th e two pathways compete for hydroxylated groups on substrates w ith glucuronidation taking ove r at higher concentrations (Pang, 1990). This can be explained on the basis of the differences in Km and Vmax of the two systems. Whereas sulfation is generally a hi gh affinity, low capacity pathway, glucuronidation usually has a low affinity and high capacity for substrates. Glucuronidation Uridine diphosphate-glucuronosyltransferases (UGTs) are a superfamily of membrane bound enzymes that catalyze the conjugation of gluc uronic acid to a nucleophilic substrate. UGTs catalyze an SN2 reaction in which the acceptor gr oup of a substrate attacks the C1 of the pyranose acid ring of uridine diphospha te-glucuronic acid (UDPGA). The -glycosidic bond to which UDP is attached is broken and a glycosidic bond formed to give a -Dglucopyranosiduronic acid conjugate, the glucuronide and UDP. Conjugation occurs at nucleophilic functi onal centers such as oxygen (e.g., hydroxyl groups in phenols and aliphatic alco hols or carboxylic acids), nitr ogen (e.g., amines), sulfur (e.g., thiols) and even carbon. Reactivity of a substr ate depends on its stru cture and includes both electronic and steric factors as well as the lipid solubility of the compound. If more than one

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27 group is available for the reaction, more than one type of glucuronide may be formed (Mulder et al., 1990). All UGT isoforms have broad a nd overlapping substrate specificities. Though the liver appears to be the major or gan involved in gluc uronidation, some UGT isoforms are found in large amounts in both the kidney and intestine, implying that glucuronidation in these organs can be signi ficant too (Tukey and St rassburg, 2000). UGTs are localized within the endoplasmic reticulum; the majority of the protein being lumenal. Two UGT gene families have been identified in mammals. Members of the UGT1 family share more than 50% identity with each other but less than 50% identity with members of the UGT2 family (King et al., 2000). UGT1 isofor ms conjugate bilirubin and phe nolic xenobiotic compounds, the UGT2A subfamily function in olfaction and UGT2B isoforms mainly conjugate steroids, bile acids and thyroid hormones but also some phenols and drugs (George et al., 1998). All UGT1A members share the same last four exons (exons 2–5), and can only be differentiated by their unique first exons. More over, whereas each gene contains a unique 5’ flanking region and N-terminus, all members are identical in their carboxyl terminal 245 amino acids. The UGT2 gene family mRNAs, on the othe r hand, are transcribed from individual genes. All UGTs have highly conserved C-terminals, a region that has been proposed as the UDPGA binding site while the more variable N-terminal half of the protein determines nucleophilic substrate selectivity (K ing et al., 2000; Tukey and Strassburg, 2000). Fish uridine diphosphate-glucu ronosyltransferases (UGTs) Multiple UGT genes have been suggested in zebrafish (George and Taylor, 2002), plaice (Clarke et al., 1992c; George et al., 1998) and channel catfish (Sacco, 2006). A comparison of piscine and mammalian hepatic microsomal gluc uronidation revealed th at both phyla exhibited comparable activity towards planar phenolic compounds but the fish was less effective at conjugating bulky, non-planar as well as endogenous substr ates (Clarke et al., 1992b).

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28 Regulation of UGTs Similar to the P450s, the activ ity of UGTs can be altered by both induction and inhibition (Fisher et al., 2001). In humans oral contraceptives, carbamazep ine and rifampicin have been shown to increase the metabolic clearance of some drugs via glucuronidation (Miners and Mackenzie, 1991). In Caco-2 cells the intestinal cell line, chrysi n has been shown to induce the UGT1A1 (Galijatovic et al., 2001) while t-butylhydroquinone and TCDD have been reported to induce the isoforms 1A6, 1A9 and 2B7 (Munzel et al., 1999). In primary cultures of human hepatocytes, UGT1A1 has been shown to be in duced by phenobarbital, oltipraz, and 3MC (Ritter et al., 1999). The down regulation of UGT2B 17 was documented by Levesque et al (1998). In the rat, UGT1A1 and 2B1 have been shown to be induced by phenobarbital suggesting that they may be controlled by the constitutiv e androstane receptor (CAR) pathway (Bock, 2002). Clofibrate and dexamethasone are also known to induce UGTs but their mechanism of doing so is known. When marine teleost flatfish, the plaice ( Pleuronectes platessa ), were treated with 3MC, Aroclor 1254 and clofibrate the selective induction of UGTs in tissues was reported. 3MC and Aroclor 1254 treatment resulted in a 1.7-fold indu ction of phenol UGT activity in the liver while Aroclor 1254 also showed a slight selective indu ction in the kidney microsomes (Clarke et al., 1992a). The induction of UGT activity however is moderate when compared to the increases observed in P450s (Andersson et al., 1985). Sulfation Sulfotransferases transfer th e sulfonyl moiety from the co factor 3’-phosphoadenosine-5’phosphosulfate (PAPS) to a nucleophilic group of thei r substrates. This electrophilic attack can be on nucleophilic groups such as hydroxyl, amino, sulfhydryl or N -oxide. The reaction is called sulfonation or sulfation, with re ference to the transferred moie ty and the most common product,

PAGE 29

29 which is a sulfate or sulfuric acid ester in the case of O -sulfonation (Glatt, 2002). Double conjugates may be formed subsequent to another conjugation reaction resu lting in disulfates and sulfate-glucuronide conjugates (Mulder and Jakoby, 1990). In mammals two classes of su lfotransferases can be disti nguished. One class metabolizes macromolecular endogenous structures and comp rises mainly membrane-bound forms localized in the Golgi apparatus. No xenobi otic-metabolizing activ ities have been reported for these forms (Glatt, 2000). The other class of enzymes is cytosolic and me tabolizes xenobiotics and small endogenous compounds such as hormones and neurot ransmitters. All cytosolic sulfotransferases studied are members of a single superfamily, now termed SULT, as judged from similarities in the nucleotide sequences of their genes. SULTs in one gene family have no more than 40% similarity with SULTs in other gene families (Nagata and Yamazoe, 2000). SULTs within a subfamily have a 40 – 65% similarity. In humans eleven SULT forms have been detected. Fish sulfotransferases The lamprey, Petromyzon marinus a primitive fish belonging to the class Agnatha, produces large amounts of petromyzonol whose sulf ated derivatives serve as pheromones that cue adults to return to the same breeding ground for spawning. A petromyzonol sulfotransferase has been isolated and characterized from the la mprey larval liver (Ve nkatachalam et al., 2004). This petromyzonol-SULT exhibited a Km of 2.5M for PAPS and 8M for petromyzonol. Tong and James (2000) reported the purification and characterization of a sulfotransferase found both in the liver and intestin e of the catfish. The 41kDa prot ein showed a high affinity for the phenolic substrates 3, 7-, or 9-hydroxybenzo-[ a ]-pyrene, with Km values of 40–100nM and Vmax 125–300 nmol/min/mg of protein. Several cytosolic sulfotransferases have been identified and characterized from Danio rerio the zebrafish (zfSULT) by Yasuda et al (2005b). zfSULT#5, a 34 kDa protein, displayed

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30 substrate specificity for thyr oid hormones and their metabolite s (Yasuda et al., 2005a). zfSULT#4 is a 35kDa protein that significantly sulfated thyroid hormones, estrone and DHEA (Liu et al., 2005); zfSULT#2 displayed much higher activities towards estrone and 17 -estradiol in comparison with thyroid hormones, 3,3’,5triiodothyronine (T3) and thyroxine (T4), dopamine, dihydroxyphenylalanine, and DHEA (Ohki moto et al., 2004). Both zfSULT#1and zfSULT#2 are about 35kDa and have been report ed to sulfate various endogenous as well as xenobiotic compounds including hydr oxychlorobiphenyls (Table 1-3). A 35kDa protein designated zfSULT2 preferen tially sulfated DHEA (Sugahara et al., 2003c). It has also been demonstrated that bisphenol A, 4-n-octyl phenol and 4-n-nonylphenol inhibite the sulfonation of 17 -estradiol by zfSULT#2, zfSULT#3, zfSULT#4 and zfSULT2 in a concentration-dependent manner (Ohkimoto et al., 2003). zfSULT#6 has been documented to display substrate specificity exclusively for endogenous estrogens (Yasuda et al., 2005b). Mishito et al (2004) have also recently reported a zebrafish tyrosylprotein sulfotransferase (TPST ). A 34kDa zfSULT displaying sulfating activity toward dopamine and the thyroid hormones (T3 a nd T4), has been identified and characterized but has not yet been assigned a name since it ap pears to be independent from all known families (Sugahara et al., 2003a). Regulation of sulfotransferases The classical inducers of P450s that seem to co-induce UGTs seem to have little or no effect on SULTs (Mulder and Jakoby, 1990). Though sulfotransferases are generally considered non-inducible, one study recently reported the firs t induction of SULTs in both catfish and in mummichog ( Fundulus heteroclitus ) (Gaworecki et al., 2004 ). Sulfation and glucuronidation of 9-hydroxybenzo-[a]-pyrene was measured in 3MCtreated catfish and in mummichog from the creosote contaminated river. There was a si gnificant induction of both UGT and SULT activity

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31 linked to the expected induction of EROD activ ity in the catfish. In mummichog significant induction was measured for UGT but only a slight increase for SULT activity. Pentachlorophenol, 2,6-dichloro-4 -nitrophenol and dehydroepi androsterone (DHEA) are reported to be selective inhibitors for certain isof orms of sulfotransferases. Since they interact with the substrate binding site and SULTs have a broad substrate selectivit y, there is overlap in the number of isoforms each can affect (Glatt, 2002). Endocrine Disruption There is growing concern regarding envir onmental chemicals that disrupt endocrine function in wildlife and humans (M iyashita et al., 2005). In gene ral endocrine di srupters alter normal hormonal regulation by having agonistic or an tagonistic effects on ster oid receptors. The result of this may be the alte ration of reproductive function and (or) causation of feminization by binding to estrogen or androgen r eceptors. Their binding to the thyroid receptor may dysregulate the neuroendocrine system. Endocrine disrupters ha ve also been shown to alter steroid synthesis and metabolism by inhibiting P450 and sulfotransfe rase isoforms. These effects have been recorded in mammals, fish, birds, reptiles, amphib ia and aquatic invertebra tes but it is not yet clear whether these processes also occur in human beings (Waring and Harris, 2005). Vitellogenin is a protein that is an egg yolk precursor produce d by oviparous female fish in response to circulating plasma es trogens. Induction of and (or) the presence of vitellogenin in the plasma of male fish is used very widely as a biomarker for exposure to compounds with estrogenic properties. Methoxychlor Methoxychlor (MXC) is the common name for 1,1,1-trichloro-2,2-bis(4methoxyphenyl)ethane, an organochlor ine pesticide that is a struct ural analogue of DDT. It was developed to replace DDT due to its low toxicity toward mammals and lower persistence in the

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32 environment. MXC was first registered as a pesticide in the US 1948 and sold under the trade names of Prentox, Methoxcide, Marlate, a nd Metox. This insecticide was used on agricultural crops and livestock, and in animal fee d, barns, and grain storage bins (Krisfalusi et al., 1998; Magliulo et al., 2002). However, MXC appears to bioaccumulate in the environment and there is growing evidence indicating that it induces estrogenic effects in mammals, birds, amphibians, fish and invertebrates. The use of methoxychlor in the United States legally ended in 2004 when the chemical was denied reregistration by the U.S. Environmental Protection Agency; however, this is after more than half a century of widespread use. Concentrations of MXC in surface and drinking water generally are low. Concentrations of 5 – 64ng/L (the mean being 19ng MXC/L) were found in some 11 surface water samples in Belgium (Versonnen et al., 2004). In the Unite d States 0.032 to 15ng/L concentrations were detected in surface waters, up to 50mg/L in some waters near agricultural areas and from 50 to 1,210ng/L in first flush storm water runoff (Versonnen et al., 2004). Worldwide, in both rural and urban areas and irrespective of a sampling re gion’s stage of development, organochlorine pesticides have been documented in human milk (Campoy et al., 2001). This implies that they are passed on through breast-f eeding to the next generation. Demethylation of methoxychlor gi ves its major metabolites: 2-( p -hydroxyphenyl)-2-( p methoxyphenyl)-1,1,1-trichloroet hane (OHMXC) and 2,2-bis( p -hydroxyphenyl)-1,1,1trichloroethane (HPTE). Both metabolites e xhibit significant rat uterus estrogen receptorbinding activity though HPTE is more potent than OHMXC (Ous terhout et al., 1981). Miyashita et al (2005) inves tigated the estrogen receptor (ER) binding affinity of DDTrelated compounds and their metabolites and found that those having methoxy or ethoxy substituents on benzene rings showed enhanced activity after metabolism to phenolic metabolites

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33 by rat liver S9 fractions. Bul ky groups connected to the carbon atom bridging the two benzene rings did not seem to have an effect on activit y. Inhibition of ER binding for racemic, (S)and (R)-OHMXC and HPTE were al so measured (Table 1-4). The (S) enantiomer showed a 3-fold highe r binding affinity than (R)-OHMXC while HPTE had a 1.7-fold higher affinity than (S)-OHMXC Miyashita et al (2004) therefore inferred that the one hydroxyl group and the orientation of the CCl3 group of OHMXC and HPTE are important for the interaction with the estrogen receptor. In vivo metabolic activation of MXC predominantly produces the (S)-OHMXC; this puts into perspective its estrogenic activity since MXC itself has negligible estrogenic activ ity in mammals (Bulger et al., 1978). Versonnen et al (2004) demonstr ated that adult male zebrafi sh are sensitiv e toward the estrogenic effects of MXC. They had elevated le vels of vitellogenin afte r exposure to at least 5ug/L MXC for 14 days. In female catfish liver cytosol the affinities for the hepatic estrogen receptor of MXC and each metabolite were accessed by their ability to displace 17 -[3H]estradiol from specific binding sites. The IC50 values reported increased in the order 17 estradiol (0.0017 M) < HPTE (1.8 M) < OHMXC (3 M) < MXC (78 M) (Schlenk et al., 1998). No stereoselective differences between the (R)or (S)-enantiomers of OHMXC were observed. OHMXC had an approximately 43-fold higher affinity than the parent compound and an IC50 1000-fold greater than that of estradiol. Intraperitoneal administration of MXC to fish does not seem to result in estrogenic effect s (Nimrod and Benson, 1997; Andersen et al., 1999), although exposure via water did (Schlenk et al., 1998; Hemmer et al., 2001; Versonnen et al., 2004). The route of exposure might therefore be a factor in determining effects of toxic substances.

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34 Metabolism in Humans Hu and Kupfer (2002a) reported major differenc es in the formation of enantiomers by one rat and various recombinant human P450 isofor ms as well as human liver microsomes. Following O-demethylation CYP1A1, CYP2B 6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 produced more of the (S)-enantiomer; CYP1A2 and CYP2A6 preferentially formed (R)OHMXC and rCYP2B1 CYP3A4 and 3A5 were not enantioselective. The human liver microsomes samples predominantly formed the (S)-OHMXC. CYP2C19 turned out to be the most active enzyme in the form ation of OHMXC. Subsequent wo rk using inhibitory monoclonal antibodies has however led to the proposal that the pathway of the first O-demethylation of MXC is as depicted in Figure 1-1 (Hu et al., 2004). In supersomes the relative rates of O-demet hylation of (R)or (S)-OHMXC in forming HPTE was used as an indication of enantioselect ivity and gave the order CYP2C19 > 1A2 > 2D6 > 2A6 and 2C9. The relative consumption of the s ubstrate, expressed as a percentage, is shown in Figure 1-2. It has been reported that in human liver microsomes the enzyme isoform that catalyzes this reaction is CYP2 C9 (Hazai and Kupfer, 2005) rather than CYP2C19 (Stresser and Kupfer, 1998; Hu and Kupfer, 2002b) as in supersomes. The proposed pathway for the metabolism of MXC by human liver microsomes is seen on Figure 1-3. CYP1A2 O-demethylat ed (R)-catechol-MXC into 80% (R)-tris-OHMXC in contrast to CYP2C9 and 2C19 which yielded 80 and 77% respectively of the (S)-tris-OHMXC. The ortho -hydroxylation of OHMXC to catechol-MXC and HPTE into tris-OHMXC was primarily catalyzed by CYP3A4 and was not enan tioselective (Hu and Kupfer, 2002a). Since OHMXC was found to be the only primar y metabolite, O-demethylation seems to be the predominant reaction in the metabolic path way of MXC in human liver microsomes. Significantly more HPTE was generated than catechol-MXC during incu bations demonstrating

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35 that a phenolic group is essent ial for the efficient introductio n of the second hydroxyl at the ortho position as was shown previous ly shown with CYP3A4 by Stresser and Kupfer (1997). Hazai et al (2004) reported that on in cubation, both OHMXC and HPTE formed monoglucuronides with human liver microsomes. They pointed out that even though several isoforms catalyze each glucuronidation step, UGT 1A9 is the major contributor to the formation of OHMXCglucuronide, whereas UGT1A3 seemed to be the most active in HPTE-glucuronide formation. There was a relatively small but cons istent enantioselective preference of individual UGT1A1, UGT1A3, UGT1A9 and UGT2B15 enzymes for glucuronidation of the (S)over the (R)-OHMXC. This consistency was not observe d in human liver microsomes. A limitation of this study was that only a single concen tration of OHMXC and HPTE was used. Metabolism in Other Animals Kapoor et al. (1970) compared the metabo lism of radioactive MXC following oral administration in mice and insects. They reporte d very briefly that in houseflies there was both dehydrochlorination to give the corresponding ethyl ene metabolite and O-dealkylation resulting in phenolic compounds that were subsequently ex creted as conjugates. In the Salt Marsh caterpillar, 96% of the parent compound was recove red in excreta but ther e were traces of its ethylene metabolite and phenolic co njugates. A more extensive analysis in mice showed five breakdown products in the urine and feces of mice. The major metabolites were OHMXC, HPTE and its ethylene metabolite. Incubation in m ouse liver microsomes recovered OHMXC and a trace of HPTE. The urine and feces of lactating goats that were fed [14C]-MXC had 17 metabolites (Davison et al., 1982). Most of the fecal and some urinary metabolites were demethylated, dechlorinated, or dehydrochlorinated. Most urinar y metabolites were completely demethylated and glucuronidated. It was suspect ed that a diglucuronic conjugate also existed but could not be

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36 identified for technical reasons. The effect of t opical application of meth oxychlor as well as its metabolites in goat bile were reported by Davison et al. (1983). A total of 7 metabolites were identified. These were similar to those pr eviously reported (Davison et al., 1982) but the glucuronide conjugates were found only in the urine and not the bile. Twenty six metabolites were eith er identified or characterized from the feces, urine or bile of intact, colostomized and bile-fis tulated chickens that were fed [14C]-MXC (Davison et al., 1984). Metabolism included demethylation, ring hydroxylation, dechlorination, conjugation with glucuronic acid, dehydrochlorination, and formation of substituted benzophenones. The metabolism of MXC in rat, mouse, Japa nese quail and rainbow trout was compared using precision-cut liver slic es (Ohyama et al., 2004; Ohyama, 2005) and the metabolites quantified. In the mouse and the quail OHMXC and its glucuronide were the major metabolites. A reductively dehalogenated OHMXC glucuronide wa s observed only in the mouse. In the rat, OHMXC was detected only as a tr ansient intermediate, while HP TE and its O-glucoronide were the major metabolites. A sulfoglucuronide co njugate, 4-O-sulfate 4’-O-glucuronide, was observed only in the rat. In the trout the glucuronides of both OHMXC and HPTE were major metabolites, produced almost in similar quantities. A ratio of OHMXC to all other hydroxylated metabolites and their conjugates for each species wa s computed and showed that the rat was best able to completely demethylate MXC (<5% of intact MXC), followed by mouse and quail (about 50%) and lastly the trout with only 80% (Ohyama, 2005). Species differences in the stereoselectiv e formation of OHMXC have also been documented. The quail and trout ea ch produced more than 85% of the (R)-enantiomer, while the rodents produced predominantly the (S)-enantiomer at yields of about 90 and 75% for the rat and mouse respectively (Ohyama, 2005). A metabolic pa thway by the liver slices of these animals

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37 was then proposed (Figure 1-4). Notably missing from this scheme are the tris-, catecholand ring-OHMXC reported by De hal and Kupfer (1994). Some differences have also been detected betw een the sexes in the rat metabolic profile of MXC (Ohyama et al., 2005b). In both cases metabo lites were extensively glucuronidated. The S-enantiomer of OHMXC was preferably formed but could only be detected in the first half hour of the assay for males. At the 4 hour samp ling point OHMXC was unique to females, its glucuronide and that of HPTE being formed as the main metabolites. Whereas the male metabolic profile did not include the OHMXC glucuronide, the doubly conjugated sulfoglucuronide conjugate was uniquely present. The ratio of bisto mono-demethylated metabolites was about 95:5 for the male and 40:60 for female rats making the authors theorize that the first demethylation is key in the se x-dependent metabolism of MXC in the rats. Metabolism in Fish Microsomes from catfish liver catalyzed the breakdown of 9% of the initial MXC to form two metabolites: OHMXC (87%) and HPTE (13%) (Sch lenk et al., 1998). Stuchal et. al. (2006) report the same metabolites in a study compari ng catfish pretreatment with MXC or 3MC to controls. Hepatic formation of OHMXC by MXC-trea ted fish did not differ significantly from those in the control group, but ha d significantly lower (p<0.05) kinetic parameters than those for catfish treated with 3MC (Table 1-5). Hepatic microsomes formed OHMXC at higher rates than those in the intestine (Table 1-6). The 3MC-trea ted fish produced significantly more HPTE than the control fish. Pretreatment of fish w ith MXC significantly reduced the formation of metabolites in the intestine while 3MC-treatmen t resulted in significantly greater amounts of OHMXC. The Vmax for hepatic microsomal glucuroni dation of both OHMXC and HPTE was significantly (p<0.05) higher in 3MC-treated fish th an in controls (Tables 1-7 and 1-8). Liver

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38 microsomes from both control and 3MC-treate d fish glucuronidated OHMXC and HPTE less rapidly than intestinal microsomes. These Km values are much larger than those found in the P450-dependent formation of these metabolites s uggesting that glucuronidation may not be an efficient pathway of detoxification of MXC by th e catfish at environmen tally relevant exposure levels. Regulation of Meth oxychlor Metabolism MXC has been shown to induce both CYP2B and CYP3A in rats (Li and Kupfer, 1998) mimicking phenobarbital-type inducers. MXC and its metabolites OHMXC, HPTE, ringOHMXC and tris-OHMXC were all found to be poten t activators of the constitutive androstane receptor (CAR) pathway, with tris-OHMXC be ing most potent (Blizard et al., 2001). TrisOHMXC is a much weaker estrogen receptor ag onist than either OHMXC or HPTE. Its estrogenicity was therefore not thought to be a significant fact or in CAR activation. Since the generation of phenolic constituents did not appear to appreciably alter its activation, it was suggested that a common structural motif in the group of compounds is what controls CAR activation. Mikamo et al. (2003) observed that when male rats were given MXC for 7 days, CYP2C11, 2B1/2 and 3A1 were induced in the liver but only CYP2C11 converted MXC to hormonally active metabolites. When rats were injected with 3MC, an inducer of CYP1A, a slight decrease in overall MXC metabolism and shift of the metabolite profile to higher percentages of OHMXC were observed (Dehal and Kupfer, 1994). The effect of CYP1A induction by -naphthoflavone (BNF) treatment did not sign ificantly affect th e metabolite profile or the estrogenic ac tivity of MXC in male catfi sh (Schlenk et al., 1997). When given as a single dose however, BNF significantly reduced ring h ydroxylation but combination doses with MXC or a second BNF treatment failed to affect ring hydroxylation. Schl enk et al. (1997) reported that

PAGE 39

39 these observations indicated that CYP1A is probably not directly involved in MXC biotransformation in catfish and that BNF might have been alte ring the expression of other CYP isoforms responsible for ring hydroxylation. In contrast, Stuchal et al (2006) showed the induction of MXC demethylation in catfish treat ed with 3MC. Inhibition studies however suggested that it was not CYP1A, but rather another CYP1 is ozyme, also induced by 3MC, that was responsible for the observa tion. The results of Smeets et al (1999) s upport the latter findings. Using cultured hepatocytes from a ge netically uniform male carp strain (Cyprinus carpio), the authors studied the effect of CYP1A induction on th e estrogenicity of MXC by coexposing cells to 10pM 2,3,7,8-tetr achlorodibenzo-p-dioxin (TC DD). TCDD caused more than 50-fold increase in EROD activity but vitello genin induction by MXC was not significantly affected. This led the authors to arrive at the conclusion that CYP1A is not involved in the bioactivation of MXC to more pot ent estrogenic metabolites in carp. Pretreatment of catfish with BNF was show n not to affect MXC metabolite profiles, overall rates of MXC biotransformation or mi crosomal proteins recognized by anti-trout CYP2K1 (Schlenk et al., 1997). There was how ever the expected effect of inducing CYP1A and associated increase in EROD activity. In cont rast, pretreatment with MXC, alone or in combination with BNF, significantly reduced ra tes of MXC biotransformation and binding to liver microsomal protein. In catfish it has been shown that ethanol si gnificantly changes kinetic parameters during the demethylation of MXC (Stuchal et al., 2006). The Km for the formation of OHMXC increased from 3.78M to 8.1M in the presence of ethanol. Ethanol concen trations of 1% and 2% in the reaction volume resu lted in a 44% and 71% inhibition of hepatic MXC demethylation respectively.

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40 Chemical inhibition studies with ketoconazole, clotrimazole and -naphthoflavone decreased the production of OHMXC in liver microsomes. Interestingly, -naphthoflavone also stimulated the formation of HPTE (Stuchal, 200 5; Stuchal et al., 2006). The results suggest the involvement of isoforms from CYP1 and CYP3 families in the biotransformation of MXC in catfish. Further investigation is required to help elucidate which specific isoforms are responsible for the differe nt demethylation steps. Selection of Animal Models Due to pesticide run off, chemical waste and sewage effluents the aquatic environment has become a sink for xenoestrogens. Since several fish have been documented to be sensitive to xenoestrogen exposure, they are of interest for th e development of biomarkers of exposure and in environmental monitoring. There is therefore a need to study the func tion and regulation of different enzymes in fish and any species that comes into contact with chemicals in the environment that result from man’s activities. In the mid-1900s marshes surrounding a number of central Florida lakes were drained for farming (muck farms) but were later purchased back for restoration by the State of Florida. Following re-flooding, the Florida Fish and Wildlife Conservation Commission had limited success in establishing reproducing game fish populations, including largemouth bass Micropterus salmoides in these sites in comparison to other sites. Larg emouth bass inhabit numerous fresh water lakes and streams nati onwide. Lipophilic contaminants, even those present initially in low doses, are likely to accumu late in their fatty tissues as they are predatory fish high up in the food chain (Marburger et al., 2002). Due to the demand for Florida largemouth bass in the sports-fis hing industry and its environmen tal relevance as an indicator species it is a fitting model for this study.

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41 Channel catfish ( Ictalurus punctatus) farming is an important industry in the southeastern parts of the USA. To ensure a good harvest, fish farms use various compounds to control infection and infestation by different pests. A lthough some of the pesticides used have already been banned by the US Food and Drug Administrati on, the absence of efficacious alternatives necessitates their continued use (Schlenk et al., 1993). Some of these pesticides may have adverse effects both on the fish and for c onsumers of the fish (James, 1994). Objective of the Study There is a fair amount of documentation of the enzymatic systems involved in the metabolism of xenobiotics in mammalian species but comparatively little work has been focused on piscine species. The overall objective of this proposed study is to inve stigate the extent to which the catfish is able to metaboliz e the model compounds MXC, OHMXC, HPTE, 3hydroxybenzo-[a]-pyrene and BFC with the aid of either hepatic, inte stinal or a combination of both sets of enzymes and whenever possi ble compare this w ith largemouth bass. Hypotheses The hypotheses of this work are: 1) The us e of BFC, a probe substrate used for human liver microsomes and rat liver microsomes can be extrapolated to catfish and largemouth bass; 2) The catfish and largemouth bass are enantiosel ective in their formation and consumption of OHMXC; 3) Pre-treatment of catfish with 3M C or MXC affects sulfation of OHMXC, HPTE and 3OHBaP; 4) Catfish sulfation rates and (o r) kinetic parameters of OHMXC and HPTE are the same as those of glucuronidation previously reported in (Stuchal, 2005). 5) Route of the administration of MXC affects the distribution of MXC or its metabolites in the catfish; 6) Catfish metabolizes and can therefore efficiently eliminate MXC.

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42 H MeO OH CCl3 H O H OMe CCl3 H MeO OH CCl3 MXC (R)-OHMXC (S)-OHMXC CYP1A2 CYP2C9 Figure 1-1. Proposed roles of CYP1A2 and CYP2C9 in methoxychlor O-demethylation by human liver microsomes.

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43 Figure 1-2. Relative P450 activit ies (rates) in O-demethylati on of individual (R)or (S)OHMXC formation of HPTE. [Reprinted with permission from Hu Y and Kupfer D (2002a) Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formati on by various P450 isoforms. Drug Metab Dispos 30:1329-1336 (Page 1333, Figure 4).

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44 H MeO OH CCl3 H MeO OH CCl3 H O H OH CCl3 H OH MeO OH CCl3 H O H OH OH CCl3 MXC OHMXC HPTE Catechol-MXC Tris-OHMXC Figure 1-3. Proposed pathway for the meta bolism of methoxychlor by human liver microsomes. Adapted from Hu and Kupfer (2002b).

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45 Figure 1-4. Proposed metabolic pathways of methoxychlor by precision-cut rat, mouse Japanese quail and rainbow trout liver sl ices. Reprinted with permission from Ohyama K, Maki S, Sato K and Kato Y (2004) In vitro metabolism of [14C]methoxychlor in rat, mouse, Japanese quail and rainbow trout in precision-cut liver slices. Xenobiotica 34:741-754 (Page 751 Figure 5).

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46Table 1-1. Effect of different modulators on in vivo CYP3A gene, protein or activit y levels of different fish Fish Modulator (concentration) Effect Reference Atlantic salmon (Salmon salar) Ethoxyquin increase in gene expression (Bohne et al., 2006) Nonylphenol (30 ppb) decreased CYP3A protein expression North Sea oil (0.5ppm) no effect Atlantic cod (Gadus morhua) Mixture of North Sea oil and alkylated phenols (0.5ppm + 0.1ppm) decreased CYP3A protein expression (Sturve et al., 2006) DDE (10ug/L) 5 days increase in gene expression Atlantic salmon (Salmon salar) Thyroxine (50ug/L) 5 days increase in gene expression (Mortensen and Arukwe, 2006) Atlantic salmon (Salmon salar) 4-nonylphenol (5, 15, 50ug/L) 7 days increase in gene expression (Meucci and Arukwe, 2006) alkylphenol mixture (0.02, 2, 20, 40 and 80ppm) increased CYP3A protein levels; female>males Atlantic cod (Gadus morhua) 17 -estradiol (5ppm) increased CYP3A protein levels in males (Hasselberg et al., 2004a) Rainbow trout (Oncorhynchus mykiss) ketoconazole (25mg/Kg body weight) induction of hepatic protein Killifish (Fundulus heteroclitus) ketoconazole (i.p; 25mg/Kg body wt) 90% reduction in activity (Hegelund et al., 2004) Mummichog (Fundulus heteroclitus) sediment-associated organic contaminants No effect Striped bass (Morone saxatilis). sediment-associated organic contaminants No effect (McArdle et al., 2004)

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47Table 1-1. continued. Fish Modulator (concentration) Effect Reference 10mg BaP /Kg body weight) no effect 12.5mg cortisol/Kg body weight) no effect Arctic charr (Salvelinus alpinus) Stress no effect (Jorgensen et al., 2001) Scup (Stenotomus chrysops) 3,3',4,4',5-pentachlorobiphenyl (1mg /Kg body weight) slight increase in protein (Schlezinger and Stegeman, 2001) Carp (Cyprinus carpio) 500mg/Kg of 17 -ethynylestradiol no effect (Sole et al., 2000) Nile tilapia, (Oreochromis niloticus) pregneneolone 16 -carbonitrile (100mg/Kg in corn oil) 2.2 fold increase in protein (Pathiratne and George, 1996) dexamethasone induced CYP3A65 transcription rifampicin induced CYP3A65 transcription Zebrafish (Danio rerio) 2,3,7,8-tetrachlor o-dibenzo-p-dioxin (TCDD) induced CYP3A65 transcription (Tseng et al., 2005) Turbot (Scophthalmus maximus L.) -naphthoflavone (BNF) 75mg/Kg i.p no effect (Arukwe and Goksyr, 1997) rifampicin no effect on intestinal CYP3A levels Increase in 6 -hydroxylation of testosterone activity pregneneolone 16 -carbonitrile no effect on intestinal CYP3A levels Channel catfish (Ictalurus punctatus) Chow versus purified diet Inte stinal expression higher in chow fed fish (Lou, 2001)

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48Table 1-2. The 7-benzyloxy-(4-trifluoromethyl)-coumarin-O-deb enzylase (BFCOD) assay in different fish Fish Enzyme source Modulator Effect Reference Carp (Cyprinus carpio) Control microsomes ethynyl-estradiol 93% inhibition (T hibaut et al., 2006) clofibrate 46%; IC50 = 1047.0 1.0M gemfibrozil 55%; IC50 = 616.3 1.1M diclofenac 67%; IC50 = 805.3 1.5M (fluoxetine 69%; IC50 = 643.0 1.0M fluvoxamine 78%; IC50 = 274.0 1.3M paroxetine 80%; IC50 = 262.5 1.2M Medaka (Orzias latipes) nonylphenol atypical kinetics; (Kullman et al., 2004) recombinant CYP3A38 baculosomes Vmax = 7.95pmol/min/nmol Km = 0.116M recombinant CYP3A40 baculosomes Vmax = 7.77pmol/min/nmol Km = 0.363M Atlantic cod (Gadus morhua) Microsomes of fish treated with 0.02, 2, 20, 40 and 80ppm alkylphenol mixture. Ketoconazole IC50 =100M (Hasselberg et al., 2004b) Microsomes of fish treated with 5ppm 17 -estradiol. Ketoconazole IC50 =75M Rainbow trout (Oncorhynchus mykiss) Microsomes of fish treated with ketoconazole (i.p; 12 -100mg/Kg body wt) 60 80% reduction in activity (Hegelund et al., 2004) Killifish (Fundulus heteroclitus) Microsomes of fish treated with ketoconazole (i.p; 25mg/Kg body wt) 90% reduction in activity

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49 Table 1-3. Kinetic constants of zebrafish SULT1 ST1 and ST2 with hydroxychlorobiphenyls and 3,3’,5-triiodo-L-thyronine as substrates SULT1 ST1 SULT1 ST2 substrate Km (M) Vmax (nmol.min-1mg-1) Vmax/Km Km (M) Vmax (nmol.min-1mg-1) Vmax/Km 3-chloro-4biphenylol 76 7.7 435 42 5.7 1.3 0.1 66.7 2.9 49.8 3,3’,5,5’-tetrachloro4,4’-biphenyldiol 8.1 1.0 145 13 17.8 1.1 0.1 18.1 0.5 18.8 3,3’5-triiodo-Lthyronine 64.4 4.7 5.4 0.1 0.1 9.4 0.2 9.3 0.2 0.9 data is given as mean SD; n = 3 [Reprinted with permission from Sugahara T, Liu CC, Pai TG, Collodi P, Suiko M, Saka kibara Y, Nishiyama K and Liu MC (2003b) Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and func tional characte rization of zebrafish SULT1 sulfotransferases. Eur J Biochem 270:2404-2411 (Page 2409, Table 3). Table 1-4. Estrogen receptor binding activity of demethyl ated methoxychlor metabolites Compound IC50 ( M) Relative activity Racemic OHMXC 0.20 0.111.0 (S)-OHMXC 0.15 0.051.7 (R)-OHMXC 0.47 0.210.6 HPTE 0.09 0.022.9 IC50 values are mean SD of at least 3 independ ent experiments; Adapted from Miyashita et al. (2004)

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50 Table 1-5. Kinetic parameters for the form ation of OHMXC by catfish liver microsomes. Treatment Km (M) Vmax (pmol/min/mg) Control 3.78 1.33 131 52.8 MXC 3.28 0.78 99 17.4 3MC 5.99 1.07 246 6.13 data is given as mean SD; Adapted from Stuchal et al (2006) Table 1-6. Rates of microsomal formation of OHMXC and HPTE from 14C-MXC in the channel catfish. OHMXC (pmol/min/mg) HPTE (pmol/min/mg) Treatment Liver1 Intestine2 Liver1 Control 118 4532.4 3.855.63 5.17 MXC 87 1815.1 5.54ND 3MC 199 6 72.3 22.315.1 4.02 Activity in pmol/min/mg of protein is expressed as mean SD for n = 4; 1 Incubations were carried out with 25M 14C-MXC; 2 Incubations were carried out with 15M 14C-MXC; ND = not determined. Adapted from Stuchal et al., (2006)

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51 Table 1-7. Rates of gl ucuronidation of OHMXC by cha nnel catfish microsomes. Liver Intestine Km (M) Vmax (pmol/min/mg) Km (M) Vmax (pmol/min/mg) Control 249 9.81 277 42.9175 35.2 536 167 MXC 143 33.4 291 5.95191 17.8 485 182 3MC 252 23.1 426 71.4 309 100 1030 239 data is given as mean SD; Compiled from Stuchal (2005) Table 1-8. Rates of gl ucuronidation of HPTE by channel catfish microsomes. Liver Intestine Km (M) Vmax (pmol/min/mg) Activity (pmol/min/mg) Control 245 23113 19280 134 MXC 180 42112 34 252 74 3MC 403 77193 61435 164 data is given as mean SD; Compiled from Stuchal (2005)

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52 CHAPTER 2 MATERIALS AND METHODS Chemicals N-(2-hydroxyethyl)piperazine-N ’-2-ethanesulfonic acid (HEP ES), bovine serum albumin (BSA), 7-hydroxy-(4-trifluoromethyl)coumarin (HFC), tris-(hydroxymethyl)-aminomethane (Tris), 3-methylcholanthrene (3 MC), nicotinamide adenine di nucleotide phosphate -reduced form (NADPH), phenylmethylsulfonyl fluoride (PMSF), uridine diphosphoglucuronic acid sodium salt (UDPGA) and [14C]-MXC (9.6 mCi/mmole) were al l purchased from Sigma-Aldrich Chemical Company, St.Louis, MO and [14C]-Testosterone from DuPont NENTM (Boston MA). Testosterone metabolite sta ndards were obtained from Steraloids, Inc (Wilton, NH). 7Benzyloxy-4-(trifluoromethyl)-couma rin (BFC) and 7-ethoxyresorufin were synthesized in the laboratory (> 99 % pure). OHMXC and HPTE were prepared ba sed on the method of Hu and Kupfer (2002b) as modified in Stuchal (2005). Resorufin and benzo-[a]-pyrene (BaP) were bought from Aldrich (Milwaukee, WI). Tetrabutylammonium sulfate (PICA) was obtained from Waters Corporation (Milford, MA); 3hydroxybenzo-[a]-pyrene (3OHBaP) and its sulfate conjugate were obtained from the NCI Chem ical Carcinogen Repository through Chemsyn (Lenexa, KS), MXC from ICN (Aurora, OH) and all HPLC grade solvents, sodium chloride, glycerol and hydrochloric acid we re purchased from Fisher Scie ntific. 3’-Phosphoadenosine-5’phosphosulfate (PAPS) was obtaine d from Dr. S.S. Singer, University of Dayton, Ohio. [14C]UDPGA (313mCi/mmole) and [35S]-PAPS (1.82 or 2.67Ci/mmol) were bought from PerkinElmer (Boston, MA). ECL chemilumi nescence detection reag ents, hyperfilm ECL, donkey-anti-rabbit conjugated to horseradish pe roxidase secondary antibody were purchased from Amersham Biosciences, Piscataway, NJ. All gel electrophores is supplies including nitrocellulose membranes, low range molecula r weight markers, G250 coomassie blue, tris-

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53 glycine SDS-PAGE buffers and Tween-20 were purchased from Biorad, Hercules, CA. Polyclonal rabbit-anti-trout CY P3A was a generous gift from Dr. M. Celander of Gteborg University, Sweden. All othe r chemicals used were of pur est grade available and were purchased from commer cial suppliers. Animal Treatment Catfish All channel catfish, Ictalurus punctatus, used were adults. They were maintained on a purified diet as previously described (James et al., 1997) and divided into treatment groups, each with at least four fish. Non-radiolabeled methoxychlor studies Adult catfish weighing between 1100g and 2700g were used. The control group were given an intraperitoneal (i.p.) injection of the ve hicle; the MXC group were given an i.p. injection of 2mg/Kg MXC in corn oil daily for six days before sacrifice on the seventh day and the 3MC group was treated with a single i.p. inje ction of 10mg/Kg 3MC in corn oil 3 to 5 days before sacrifice. Another group of fish weighing between 900 and 1400g were given an intraperitoneal injecti on of 0.1mg/Kg of 3, 3’,4, 4’, 5pentachlorobiphenyl (CB126) 5 days before sacrifice. All fish were sacrificed as approved by the University of Florida Institutional Animal Care and Use Committee. Liver and intestine were removed and their subcellular fractions prepared as detailed below or stored at -80C before use. Radiolabeled methoxychlor studies Male and female adult catfish, weighing between 584g and 701g, were used for studies with radiolabeled MXC. They were fed a purifie d diet as described previously (James et al., 1997). Four catfish were used in each treatmen t group and were dosed by gavage each day for 6 days and harvested on the seventh day. One group received 2mg/Kg per day of 14C-MXC (2Ci)

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54 (MXC group) while the second gr oup was treated with a combin ation of 2mg/Kg per day of 14CMXC (2Ci) and 2mg/Kg BaP (MXCBaP group). Fish were then sacrificed and treated as above. Largemouth Bass Adult largemouth bass, Micropterus salmoides, were purchased from American Sport Fish Hatchery (Montgomery, AL) and were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Non-radiolabeled methoxychlor studies Prior to injections, fish were anaesthetiz ed in water containing MS-222, an aquatic anesthetic agent. All solutions were made up to deliver the treatment in 1L/g of bodyweight. Following injection, the fish were dipped into a saltwater bath to kill any pathogens introduced by handling and to stimulate mucu s layer secretion on the skin. Since they are not sexually dimorphic, adu lt largemouth bass were sexed by massaging the ventral surface of the fish to cau se visualization of semen from the genital opening. Male fish were placed into a separate holding tank for 24 hours before treatment. A total of 81 male largemouth bass were treated with ei ther vehicle (n = 9), 1mg/Kg E2 (n = 9), 2.5mg/Kg MXC (n = 18), 10mg/Kg MXC (n =18), or 25mg/Kg MXC (n =18). Following the injections, the fish were placed in tanks sorted by treatment. On e third of each treatment group was harvested after 24, 48 and 72 hrs, at which time they were anaesth etized and killed by blunt head trauma. The liver that was collected was chopped into small pi eces, frozen in liquid nitrogen and stored at 80C before use. Dieldrin and DDE studies DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethene) and di eldrin were dissolved in menhaden oil and applied to silv ercup floating pellets (Ziegler Bros., Murray, UT). DDE was applied at a rate of 185, 37 and 7ppm to achie ve a whole body concentration of 25ppm (high),

PAGE 55

55 5ppm (medium) and 1ppm (low). Dieldrin was a pplied at the rate of 3, 0.6 and 0.1ppm to achieve whole body concentration of 0.5ppm (h igh), 0.1ppm (medium) and 0.02ppm (low). Menhaden oil alone was used for controls. All fish were given food equiva lent to 1% of their average body weight with adjustments done every 30 days to compensate for growth. Exposure lasted for 120 days. At the end of each experiment 12 fish were euthanized, liver samples snap frozen in liquid ni trogen and stored at 80C until analysis. Subcellular Fractionation Non-radiolabeled Samples The subcellular fractionation of catfish liver and intestine was done as previously described by James and Little (1983). Immediately after removal from the body, liver was rinsed three times with ice-cold liver homogenizing buffer (buffer 1) comprising 0.15M potassium chloride, 0.05M potassium phosphate (pH 7.4), 0.2m M PMSF. The liver was then cut into small pieces and homogenized in 4 times its volume of buffer 1. Following its removal, whole intestine was rinsed with ice-cold intestinal homogeni zing buffer (buffer 2) made up of 0.25M sucrose, 5mM EDTA, 0.05M Tris-Cl (pH 7.4) and 0.2mM PMSF. The whole organ was portioned into distal and proximal portions and sliced open long itudinally to enable the scraping off of the mucosal cells that were then homogenized in 4 volumes of buffer 2. Homogenates were first centrifuged at 10500rpm (20 minutes) to yield a supernatant that was again centrifuged for 45 minutes at 47500rpm. The resulting supernatant c ontained the cytosol whic h was aliquoted into small volumes and stored at -80C until use in enzyme assays. The microsomal pellet was resuspended in buffer 1 and centrifuged at 4750 0rpm for 30 minutes. The supernatant was discarded and the pellet containing washed micr osomes was resuspended in a volume equal to the original wet weight of the liver or intestin al mucosa of a resuspension buffer (buffer 3) containing 0.25M sucrose, 0.01M HEPES (pH 7.4) 5% glycerol, 0.1mM dithiothreitol, 0.1mM

PAGE 56

56 EDTA and 0.1mM PMSF. The washed microsomes were stored under nitrogen at -80C until use in enzyme assays. Microsomal and cytosoli c protein contents were measured by the Lowry assay (Lowry et al., 1951), using BSA as a standard. Radiolabeled Samples Frozen liver samples were thawed on ice and homogenized in four tim es their volume of buffer 1. The homogenate was centrifuged at 2500rpm for 10 minutes to sediment nuclei and cell debris. The supernatant was then centrifuge d at 10,500 rpm for 20 minut es to sediment the mitochondria. The resulting supernatant was ag ain centrifuged at 47,500 rpm for 45 minutes to give the cytosolic fraction (supernatant) and micr osomal fraction (pellet). The microsomal pellet was resuspended in buffer 1 and centrifuged at 47,500 rpm for 30 minutes to yield washed microsomes (pellet) which were resuspended in buffer 3 of a volume equal to the weight of the liver sample. Aliquots were taken at each step to monitor the distribu tion of radioactivity. Quantitation of the Distributio n of Radiolabeled Methoxychlor Each tissue was measured (weight or volume) then treated appropriately before being quantitated. The liquid samples storage buffer (0.5mL), cytosol (0.1mL) and microsomal wash (0.5mL) were added into the sc intillation cocktail and counted di rectly. Bile (0.01mL) was bleached with hydrogen peroxide before counting. Nuclei and cell debris (0.1mL), mitochondria (0.1mL), microsomes (0.05mL), lipid, muscle, br ain and gonad were digested in 0.5mL of 2N sodium hydroxide; neutralized with 0.3mL of 5N hydrochloric acid, then mixed with the scintillation cocktail after being allowed to cool. Liver, skin and blood were digested as above and after cooling were bleached with hydrogen peroxide before counting. Extraction of Methoxychlor and its Metabolites from Tissues and Bile Bile was directly diluted 5 times with mobile phase, filtered and analyzed by HPLC using Method #2 (see section on HPLC analysis). Li ver, intestinal mucosa, muscle, lipid or gonad

PAGE 57

57 were accurately weighed and homogenized in 3 times its volume of 10mM ammonium acetate pH 4.6 buffer and transferred to clean tube. Acet onitrile/ethanol (2/1), 2mL, was then added and then the mixture vortexed and sonicated for 2 mi nutes. The mixture was then centrifuged for 20 minutes at 6,000rpm and the supernatant transferre d to clean vials. The remaining pellet was again extracted with a further 1mL of organic so lvent and the resulting su pernatant added to the first and evaporated to dryness. For liver, intestinal mucosa and muscle the extracted residue was and made up in 200L of mobile phase befo re HPLC analysis (Method #2). For lipid and gonad the residue was reconstituted with 50L of ethanol and spotted on LK5DF TLC plates along with authentic standards. The plates were developed in a mobile phase comprising diethyl ether/n-heptane (1/1) and the correct radiolabeled bands lined up with the standard visualized by UV and quantified as a percentage of the total am ount of radioactivity present. To access the efficiency of extraction the pellet left after cen trifugation was digested in 0.5mL of 2N sodium hydroxide; neutralized with 0.3mL of 5N hydrochlor ic acid, then mixed with the scintillation cocktail after being allowed to cool. The liv er sample was bleached with hydrogen peroxide before counting. Bile Hydrolysis Bile (50L) was added to 450L of 50mM Tr is-Cl buffer (pH 5.0) containing 340 units of -glucuronidase. The tubes were incubated at 37C for 24 hours and th e reaction terminated by the addition of 2mL of ice-cold methanol. The protein was allowed to flocculate for 20 minutes and then precipitated by centrif ugation. The supernatant was then evaporated to dryness, dissolved in 100uL mobile phase and analyzed by HPLC using Method #2 as detailed in the section titled HPLC Analysis.

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58 Lipid Quantitation A modification of the Folch method (Folch et al., 1957) was used. About 1g of tissue was accurately weighed and homogenized in 10mL of chloroform/methanol (2/1). The homogenate was transferred to clean tubes and the original tu bes rinsed out with a further 10mL of solvent. The tubes were then stoppered, vortexed t horoughly and sonicated for 5 minutes then centrifuged. The resulting supernatant was then filtered using filter paper into pre-weighed tubes. To the filtrate, 4mL of 0.01M sodium chloride was added then vortexed for about 30 seconds and centrifuged to separa te the mixture into two phases. The upper phase was then carefully pipetted out and discarded. The e xposed interface was then rinsed twice with methanol/water (1/1) without mi xing the whole preparation. The lower chloroform phase was then evaporated and the remain ing lipid weight recorded. 7-Benzyloxy-4-(trifluoromethyl )-coumarin (BFC) Synthesis BFC was synthesized by reacting HFC with be nzyl bromide in the presence of potassium carbonate in dry acetone. The acetone was evapor ated and the reaction mixture dissolved in chloroform and washed several times with sodi um bicarbonate. Crystals were harvested from the dried chloroform and recrystallized from a 70% aqueous ethanol solution. Spectra (1H NMR) were recorded on a Varian Unity 400 MH z spectrometer (Palo Alto, CA). Chemical shifts are in reference to tetramethylsilane (TMS). Western Blot Analysis An equivalent of 40 g of microsomal protein was fi rst denatured by heating at 95oC for 5 minutes in at least two volumes of SDS-PAGE sample buffer (0.5M tris-HCL, glycerol, 10%w/v SDS, 2mercaptoethanol, 0.05% bromophenol blue) and then loaded on to a discontinuous mini 8.5% resolving and 4% stacking polyacryl amide gel. Molecular weight standards (14,400 – 103,774kDa) and Gentest human CYP3A4 Supersomes were run alongside the samples. Gel

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59 electrophoresis was carried out in a 25mM Tr is/192mM glycine/0.1% SDS running buffer for about 40 minutes at 200V in Mini Protean 3 Biorad apparatus. Using a 25mM Tris/192mM glycine/20% v/v methanol/pH 8.3 transfer buffer, protein in the resulti ng gel was transferred on to a 0.45 M nitrocellulose membrane overn ight (at least 16 hours) at 4oC and 40V in a Biorad Mini-TransBlot setup. The remaining gel wa s stained with Coomassie Blue to monitor the effectiveness of the transfer. Nonspecific sites on the membrane were bloc ked with blocking solu tion (5% (w/v) nonfat dried milk in 500mM NaCl, 0.05% Tween-20, 20mM Tris-HCL pH 7.5 at room temperature for 1 hour. The membrane was then washed with 500mM NaCl, 0.05% Tween-20, 20mM TrisHCL pH 7.5 (T-TBS) for 15 minutes then twice more for 5 minutes each with fresh changes of T-TBS. The membrane was next incubated at room temperature for 2 hours in primary antibody (polyclonal rabbit-antitrout-CYP3A27) diluted 1:1000 in bloc king solution. The membrane was again washed as before then incubated for 1 hour in a 1:25,000 dilution of secondary antibody (donkey-anti-rabbit conjugated to ho rseradish peroxidase) in bloc king solution. The membrane was then washed once for 15 minutes and 3 times for 5 minutes each with T-TBS and then detected using the Amersham ECL Western blo tting detection reagents. The chemiluminescent protein bands were visualized by fluorography on Hyperfilm ECL film, the fluorograms scanned and the protein bands quantified by ScanAnalysis densitometry in comparison with the Gentest human CYP3A4 supersome standards. Assays 7-Benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation 7-Benzyloxy-4-(trifluo romethyl)-coumarin (BFC) was disp ensed from a methanol solution as appropriate in labeled test tubes and allowed to evaporate to dryness then incubated at 35oC for 20 minutes in a reaction mi xture containing 0.1M HEPES pH 7.6, 2% BSA, hepatic

PAGE 60

60 microsomes (0.25mg for catfish and 0.7mg for largemouth bass) and 2mM NADPH (to start the reaction) made up to 1mL with water. Inhibiti on assays were carried out by adding either 10L of dimethylsulfoxide (DMSO) or ketoconazole di ssolved in DMSO to the incubation mixture. The reaction was terminated by adding by addi ng 3mL ice-cold metha nol. The protein was allowed to flocculate and centrifuged to precipitat e protein. The resulting supernatant (2mL) was mixed with 0.75mL of 0.5M Tris base a nd the amount of HFC produced measured with fluorescence at excitation/emission wavelengths of 410/530nm. Quantification was done by comparison with a calibration curve ge nerated with the authentic HFC. Chemical Modulation of 7-Be nzyloxy-4-(trifluoromethyl)-coumarin O-Debenzylation Five chemicals were used to investigate th eir modulation of the BFCOD assay. In each case at least five different con centrations were tested. Ketoconazole and clotrimazole were tested at concentrations ranging from 0.1 – 10M while metyrapone, -naphthoflavone and erythromycin were tested between 1 and 100M. All compounds were dissolved in DMSO and the total organic solvent was kept to 1% of the total volume for all but erythromycin which was 2%. While the assay of other chemicals was exac tly as is described in the section BFC Assay above with the exception of the addition of the modulator, erythromycin was pre-incubated with microsomes for 30 minutes at 35oC in the presence of 1.0mM NADPH. The assay was then started with the addition of th e pre-incubation mixture to tube s with solvent-free BFC. Hydroxylation of Testosterone A radiochemical method previously used by Lou et al. (2002) was used to investigate the 6-hydroxylation of testosterone by largemouth ba ss and catfish liver microsomes. A final concentration of 0.2mM [4-14C]-testosterone (0.13Ci per as say tube) was incubated at 35oC for 10 minutes in a reaction mixture cont aining 0.1M HEPES pH 7.6, 2mM MgCl2, hepatic microsomes (0.25mg for catfish and 1mg for largemouth bass) and 2m M NADPH made up to

PAGE 61

61 0.5mL with water. The reaction was terminat ed by adding 2.5mL ethylacetate and vortexing vigorously. The two resulting phases were allo wed to separate by centrifugation, the organic phase transferred to a clean tube and the extr action repeated. The pooled organic phases were then dried with anhydrous sodium sulfate and bl own to dryness with a stream of nitrogen. The residue was then reconstituted with 50uL of ethanol and 20uL spotted on LK5DF TLC plates along with authentic standards. The plates we re developed three times in a mobile phase comprising diethyl ether:toluene:methanol:aceton e (70:38:0.8:1) and the correct radiolabeled band lined up with the standard visualized by UV and quantified as a pe rcentage of the total amount of radioactivity added. Ethoxyresorufin O-Deethylation To investigate the inducti on of CYP1A activity by treatment with BaP, the ethoxyresorufin-O-deethylase (EROD) assay wa s performed. Ethoxyresorufin (2M) was dispensed into labeled test tube s and allowed to evaporate to dryness. The tubes were then incubated at 35oC for 5 minutes (control microsomes) or 1 minute (induced microsomes) in a reaction mixture containing 0.1M HEPES pH 7. 6, 2% BSA, 0.25mg (control) or 0.025mg (induced) hepatic microsomes and 2mM NADPH (t o start the reaction) made up to 1mL with water. The reaction was terminated by adding 3mL ice-cold methanol, the protein allowed to flocculate and centrifuged. The amount of re sorufin produced was quantified by measuring the fluorescence of 3mL of the resulting supernat ant at excitation/emission wavelengths of 550/585nm. Quantification was done by comparison w ith a calibration curve generated with the authentic resorufin. Sulfonation of Monoand Bi s-demethylated Methoxychlor Sulfotransferase activity was assayed based on a method previously described by James et al. (1994) and was optimized fo r catfish liver and intestinal cytosol. Briefly, various

PAGE 62

62 concentrations of substrate disso lved in methanol were added to assay tubes and the solvent evaporated under nitrogen. An 80L reaction mixt ure consisting of 0.4% BSA, 50mM Tris-HCl (pH 7.0), cytosolic protein and water were added to the tubes which were then placed in a 35oC water bath. The reaction was started by adding 20L 35S-PAPS (1.5Ci) to a final saturating concentration of 20M (Wang et al., 2004). Incubation was allowed to run for a definite amount of time with gentle shaking then the reaction was terminated by adding 400L of an ice-cold 1:1:6 mixture of 2.5% acetic acid : 0.2mM te trabutylammonium dihydrogen phosphate: water. The sulfated product was extracted three times by adding 1.5mL water-saturated ethyl acetate, vortexing then centrifuging for 10 minutes to separate the phases. Total amounts of sulfate conjugate per samp le was determined by reading in duplicate radiolabeled counts in a 500L a liquot of the homogeneous combined organic phase and then extrapolating for the total amount collected. Th e remaining sample (2mL) was then transferred to a clean tube, blown under nitr ogen to dryness and then taken up in 40L of methanol. This was then loaded either on RP-18F254S reversephase TLC plates (Merck, Darmstadt, Germany) and developed using a 4:1 methanol:water (v/v) so lvent system as previously described by Sacco and James (2005); or on LK5D normal-phase TLC plates (Whatman, Florham Park, NJ) and developed using a solvent system of 1:1:4 (v/v ) methanol:ethylacetate:d ichloromethane with 1mL of 5mM tetrabutyl ammonium hydrogen su lfate for 90mL of solvent. Separated radiolabeled spots were visualized and quan tified by autoradiography us ing a Packard Instant Imager (Meriden, CT). The identity of the correct s pot was determined by comparing Rfs with those determined by Sacco and James (2005). The fraction of radio activity representing the sulfate conjugate of the substrate obtained this way was used to accurately quantify it in each sample.

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63 Sulfonation of 3-Hydroxybenzo-[a]-pyrene The sulfonation of 3-hydroxybenzo-[a]-pyrene (3 OHBaP) by catfish liver and intestinal cytosol was studied based on a meth od previously described by James et al. (1997). Various concentrations of the subs trate were incubated at 35oC for 5 minutes in a 0.5mL reaction mixture containing 0.05M Tris–Cl pH 7.0, 0.4% BSA, 20M PAPS to start the reaction and cytosolic protein (2g for intestinal samples; 10g and 2.5g for control and 3MC-induced hepatic samples respectively). The reaction was stoppe d by adding 2mL of ice-cold methanol, the protein allowed to flocculate a nd centrifuged. Some of the resulting supernatant, 2mL, was mixed with 0.5 ml of 1N NaOH and the fluor escence of the BaP-sulfate measured at excitation/emission wavelengths of 294/415nm. The amount of sulfated product formed was calculated by comparison with a standard curve prepared with the authentic sulfate conjugate. Methoxychlor Monooxygenation A modification of the radioche mical method previously reporte d by Stuchal et al (2006) was used. All assays were carried out in a fina l volume of 1mL. Assay tubes contained 5M of 14C-methoxychlor, 0.1M HEPES (pH 7.6), 50mM MgCl2, 1mg of liver microsomal protein and 2mM NADPH. Methoxychlor, made up in ethanol was dispensed into empty tubes and the solvent removed under nitrogen befo re addition of other components. The tubes were then placed in a 35C water bath for 2 minutes, with gentle shaking and then the reaction started with the addition of NADPH. Incubation was stopped after 15 minutes by the addition of 0.5mL of icecold water, 2mL of water-saturated ethyl acet ate and vortexing. Phases were separated by centrifugation and the ethyl acetate layer transferre d to a clean tube. The extraction was repeated twice more with 2mL ethyl acetate each time. Th e pooled ethyl acetate fractions were evaporated to dryness and the extracted compounds dissolved in 200L of mobile phase solvent before being analyzed by HPLC using Method #1 and #3 as detailed in the section titled HPLC analysis.

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64 Glucuronidation of Mono-d emethylated Methoxychlor A glucuronidation assay previously reported by Stuchal (2005) was used to investigate whether catfish and largemouth bass liver micr osomes preferentially used up (R)or (S)OHMXC during glucuronidation of OHMXC. Th e substrate, 500M, was incubated at 35oC for 4 hours in a 1mL reaction mixture containing 1mg of catfish or largemouth bass liver microsomes, 0.1M Tris-Cl pH 7.6, 5mM MgCl2 and 1mM UDP-glucuronic acid to start the reaction. The reaction was terminated by addi ng 0.4mL of 1% acetic acid and vortexing. Unreacted OHMXC was immediately extracted three times; first with 2mL and then twice with 1mL of water-saturated ethyl acet ate. The pooled ethyl acetate fractions were evaporated to dryness and the residue dissolved in 200L of mobile phase solvent before being analyzed by HPLC using Method #4 as detailed in the section titled HPLC Analysis. HPLC Analysis HPLC analysis of MXC and its metabolites wa s done on either of two systems. Methods #1 to #3 were carried out on a Beckman Gold System (Beckman Coulter, Fullerton, CA) equipped with a fluorescence, UV and IN/US radi oactivity detector. Method #4 was carried out with an ISCO model 2350 pump (ISCO, Lincoln, NE), connected to a Dynamax Model UV-1 detector and an IN/US radioactiv ity detector (IN/US Systems In c.,Tampa, FL). Compounds were detected at 245nm or by radioc hemistry and identified by comp arison of retention times with those of authentic standards or by area increase af ter isolation of products by appropriate assays. A BFC sample containing 100nmoles in a 200uL injection loop was analyzed for purity using an isocratic mobile phase of methanol/water (4/1) on a C18 reverse phase column with a flow rate of 1mL/min. UV detection at 254nm was used in conjuncti on with fluorescence at excitation/emission wavelengths of 410nm/530nm.

PAGE 65

65 Two methods were developed using reversed phase (C18) separation, carried out at 40oC, using a 25cm x 4.6 mm Discovery column with a particle size of 5 m fitted with a 2cm x 4mm guard column (Supelco, Bellefonte, PA). Me thod #1 involved an isocratic solvent system comprising 60% acetonitrile in 10mM ammonium acetate/acetic acid buffer system (pH 4.6) flowing at 1mL/min. Method #2, a gradient program operated at 1m L/min flow rate, started with a solution of 30% acetonitrile in 10mM ammonium acetate/acetic acid buffer system (pH 4.6) held for 0.5 minutes then was gradually increa sed to 90% acetonitrile for 20 minutes and to 100% acetonitrile for another 5 minutes. Chiral separation of (R)and (S)-OHMXC was d one at room temperature using a flow rate of 0.9mL/min, using a 10cm x 4.0mm -glycoprotein (AGP) column with a particle size of 5 m fitted with a 2cm x 4mm guard column (ChromTe ch Inc, Apple Valley, MN). Separation was achieved using either a mobile phase of 14% acet onitrile in 10mM ammoni um acetate/acetic acid buffer system (pH 7) (Method #3) or 15% aceton itrile in 10mM ammonium acetate/acetic acid buffer system (pH 7) (Method #4). Data Analysis Activity was calculated using Equation 2-1: Activity (pmol/min/mg) = (concentration (pmo l)) / (protein (mg) time (min)) (2-1) Using Prism 4 software (GraphPad Software, San Diego, CA, USA), kinetic parameters were determined by fitting data (duplicate values) to the appropriate equation; either the Michaelis–Menten hyperbola (Equation 2-2) for one-site binding, the Hill equation (Equation 23) for positive cooperativity or Equation 2-4 for substrate inhibiti on for one site binding (Houston and Kenworthy, 2000). Eadie-Hofstee plots were used to diagnose any atypical kinetics. Values for Km and Vmax derived from Equation 2-2 were used as initial values when

PAGE 66

66 data was fitted to Equation 2-4. The efficien cy of an enzyme was calculated by dividing Vmax by Km. = Vmax [S] / (Km + [S]) (2-2) = Vmax [S]h / (S50 h + [S]h) (2-3) = Vmax [S] / (Km + [S] + ([S]2/Ki)) (2-4) Calculated parameters were compared with in treatments and (or) between organs by ANOVA analysis, followed by the Tukey’s (groups with equal variance) or Dunnett’s T3 (groups with unequal variance) multiple comparis on tests, or by Student’s t-tests as were considered appropriate. Correlati on (Spearman) and regression anal ysis were done to determine relationships between and within variables. All sta tistical analyses were carried out using SPSS (SPSS Inc., Chicago, IL) and SAS 9.1 (SAS Institut e Inc., Cary, NC). All data is presented here as mean SE unless otherwise stated.

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67 CHAPTER 3 THE 7-BENZYLOXY-4-(TRIFLUOROMETHYL)-COUMARIN O-DEBENZYLASE (BFCOD) ASSAY IN CHANNEL CATFISH AND LARGEMOUTH BASS The O-debenzylation of 7-benzyloxy-4-(trif luoromethyl)-coumarin (BFCOD) is an assay whose use in mammalian systems as a CYP3A4 pr obe substrate is accepted even though it is less selective than the 6 -hydroxylation of testosterone (6 OHT), the conventional assay. In this chapter we investigate the suitab ility of BFCOD as a CYP3A marker assay in the channel catfish and largemouth bass. Results Assay Development Following its synthesis, a white crystallin e solid, with a melting point of 101 – 103oC was harvested. The structure was confirmed to be th at of BFC by NMR (Figure 3-1). Proton NMR spectra were recorded at 400 MHz in dimethyl su lfoxide (DMSO) with reference to the residual peak of DMSO (1H NMR 2.50) and added TMS ( 0.00), as internal st andard. The chemical shifts in the -scale with multiplicity (s = singlet, d = doublet, and m = multiplet) and coupling constants (Hz) are presented here. 1 H NMR (DMSO, 400MHz) 7.63 (dd, H, J = 8.8, 1.2), 7.41 (m, 5 H), 7.25 (d, H, J = 2.4), 7.14 (dd, H, J = 8.8, 2.4), 6.87 (s, H), 5.65 (s, 2 H). Its purity was found to be more than 99% by HPLC with detection at 254nm (Figure 3-2). Optimum wavelengths for detection of HFC in the presence of BFC were determined to be an excitation wavelength of 410nm and emission wave length of 530nm using a slit width of 2.5nm (Figure 3-3). Optimum conditions for the BFCOD assay (Fig ure 3-4) were inve stigated by varying different parameters. Starting with 0.5mg of largemouth bass microsomal protein in a 1mL incubation volume, different incubation times (10, 20 and 30 minutes) and substrate amounts

PAGE 68

68 (50, 100, 200M) were investigated (Figure 3-5). There was more product formed when incubations were carried out at 200M than at 50 or 100M. While product formation increased when incubation time was doubled from 10 minutes to 20 minutes, when incubation time was extended to 30 minutes the change was less except in the 50M experiment. Optimization of protein amount wa s therefore done usi ng incubation times of 20 minutes. As was expected, increasing the amount protein result ed in an increase in product formation (Figure 3-6). Due to the limited availability of micros omal protein however, s ubsequent assays were carried out with 0.5mg and 0.7mg of hepatic mi crosomal protein for catfish and largemouth bass respectively. Magnesium ions did not affect HF C production in this assay and were therefore not used for subsequent assays. To increase signal to noise ratio, the pH of the solution with the product was increased using a base. Stability of the product signal in basic solution was compar ed between Tris (tris(hydroxymethyl)-aminomethane) and sodium hydroxi de. Addition of sodium hydroxide resulted in a reduction of signal intensit y over time (Figure 3-7). To optimize temperature conditions, the assay was carried out at 18, 25, 30, 35 and 40oC. With the exception of a decrease in activity from 25 to 30oC for largemouth bass, there was a linea r and proportional relationship between increase in temperature and BFCOD ac tivity (Figure 3-8 and Figure 3-9). Comparison of Catfish and Largemouth Bass Microsomal Activity With just one value each for male largemouth bass samples (Km = 32.63M; Vmax = 8.88pmol/min/mg) and female samples (Km = 41.23M; Vmax = 19.21 pmol/min/mg) it was not possible to include these in multiple comparison statistical analysis. Comparison of catfish samples by sex (N = 4 for each group) using the Students’ t-test did not give a significant difference at the 95% level both for Km values (p = 0.879) or Vmax (p = 0.111) (Figure 3-10).

PAGE 69

69 The BFCOD activity of control fish, largemout h bass treated with dieldrin or DDE and catfish with MXC were compared both by sex and by treatment (Table 3-1). One-way analysis of variance (ANOVA) of this da ta grouped both by treatment and se x reveals that the means are significantly different at the 95% confidence le vel (p = 0.001). Dunnett T3 multiple comparison test, run on all possible pairs, however reveals that most pair s do not have means that are significantly different from each other. Control and MXC-treated catfish were not different from each other but had activity that is significantl y higher than any of the largemouth bass groups. Within each treatment group largemouth bass did not differ by sex. The male fish treated with DDE had the lowest activity and dieldrin-treat ed male largemouth bass the highest activity. When compared by treatment only (Figure 3-11 ), largemouth bass were still less active than catfish. Though neither treatment with DDE nor dieldrin resulted in act ivity different from control fish, dieldrin treated fish had higher activity than DDE treated fish (p = 0.01) To investigate whether the amount of CYP3 A per unit protein amount correlated in any way with the activity or Vmax of the different fish, western blo tting of different samples was done. Figure 3-12 shows a scanned fluorogram of typical results. The human CYP3A4 lane shows a single prominent band about 2.8cm from the origin. Catfish samples have the corresponding CYP3A band at about 2.6cm from the origin wh ile largemouth bass samples have two prominent bands at 2.6 and 3.0cm from the origin. Th e band intensity of different catfish samples (equivalent to 40g of total protein each) correlate well (r2 = 0.97) with the activity of the same samples (Figure 3-13). Validation of the Assay To gain insight into the usef ulness of BFCOD activity as a se lective biomarker in catfish, the results from the BFCOD assay were compared with ethoxyresorufin O-deethylase (EROD) and 6 -hydroxylation of testosterone (6 OHT) activity on the same set of catfish samples. This

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70 data is summarized on Table 3-2 and illustrate d on Figures 3-14 and 3-15. While the table alludes to similar trends between 6 OHT and BFCOD, there was no significant correlation between the two (Spearman’s rho = -0.074, p = 0.703). Surprisingly the correlation between EROD and BFCOD ( = 0.585) was signifi cant (p = 0.002). Further assays were done with hepatic microsomes of catfish and largemouth bass in order to determine whether there is a correlation between the activity of different metabolites of the 6 OHT (or their combinations) and BFCOD. Figure 3-16 shows a representative TLC chromatogram of the 14C-testosterone metabolism profile exhibited by catfish and largemouth bass hepatic microsomes. Major metabolites M1 -M5 were present in largemouth bass while catfish had only M1, M3 and M4. Metabolite s M1, M3, M4 and M5 were identified as androstenedione (Andr), 3 -hydroxytestostereone (3 OH), 6 -hydroxytestosterone (6 OH) and 2 -hydroxytestosterone (2 OH) respectively. Activity of diff erent products of the metabolism of testosterone by P450s are summarized in Table 3-3 for largemouth bass, Table 3-4 for catfish and Figure 3-17, grouped by the di fferent treatment groups. The most abundant metabolite for catfish was 3 -OH while in largemouth bass it depended on treatment. Largemouth bass treated with estradiol and 10mg/Kg of MXC (MXC Me d) were similar to catfish in having 3 -OH as their most abundant metabolite. Largemouth bass treated with 2.5mg/Kg and 25mg/Kg MXC (MXC Low and MXC High groups re spectively) had Andr and 2 -OH respectively as their most abundant metabolites. The corresponding BFCOD activities from these a ssays are illustrated in Figure 3-18. The BFCOD activities of largemouth bass were generally lower than those of catfish liver microsomes. Both fish showed a lot of vari ability. Among the largemouth bass, treatment groups were not different from each other. In catfish, the MXC-treate d group had the lowest

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71 activity while the 3MC exhibited the highest act ivity. The control and the PCB treated groups were not different from each other or the 3M C and MXC-treated groups (p>0.05). Spearman correlation coefficients for all variables compared were very small, implying little or no correlation between the metabolites and each othe r or BFCOD (Table 3-5 and Table 3-6). Several chemical modulators (Fi gure 3-19) were used in an a ttempt to pinpoint the P450s responsible for the O-debenzylati on of BFC. Activity recorded as a percentage of control (no modulator) for each chemical at the highest concen tration used is summarized in Figure 3-20. The highest concentrations used were 20M per reaction volume for ketoconazole (K) and clotrimazole (C) and 150 M for metyrapone (M), -naphthoflavone (N) and erythromycin (E). The IC50 values obtained for each inhibitor tested is listed in Table 3-7. Inhibition studies were con tinued with ketoconazole and the response over several concentrations generally deviated from a sigmoidal dose response equation. The results give a graph that almost seemed as if there were two di fferent curves connected together (Figure 3-21). As a result the EC50s obtained were compared a nu mber of different ways. Since not all sets of data deviated from the equation (p > 0.05), data was analyzed either as ‘all data’ (all results) and ‘edited data’ (only those that s howed significant deviation from th e model). Since there seem to be multiple maxima per curve, parameters were estimated for the upper portion of the curve (lower inhibitor concentrations), lower portions (higher inhibitor concentrations) and for the full set of data points. These results are summarized in Table 3-8. In all categories compared, there was no significant difference between the liver a nd proximal intestinal microsomal activity of BFCOD when inhibited by ketoconazole. Discussion Even though their average Km are the same, visually the mean Vmax values of female and male catfish samples imply that they ought to be different (Figure 3-10). Statistically however

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72 they are not significantly different because of the large variability especially in male samples. Neither body weight, liver weight, nor gonad wei ght were found to be co rrelated significantly with the trend in Vmax of the female or male catfish (d ata not shown). These body parameters would therefore not be considered re sponsible for the variability of Vmax among these fish samples. When largemouth bass treated with various le vels of pesticide are analyzed, pairwise comparison of the different treatments shows no si gnificant differences. Barb er et al (2007) and Garcia-Reyero et al (2006) demonstrated that th ere were significant changes in expression levels of different largemouth bass CYP3As on exposur e to DDE and dieldrin. When fish were exposed to 46ppm p,p'-DDE or 0.8ppm dieldrin for 30 days in their food, there was no significant effect on P450 expressi on in the liver (Barber et al., 2007). However, when fish were exposed to p,p'-DDE for 4 months, there was induction of CYP3A68 and 3A69 expression in the liver of both sexes. In the same experiment, dieldrin produced weak induction of CYP3A68 and suppressed CYP3A69 expression in females, but had little or no effect on males. The lack of differences between treatments reported here su ggests that under the conditions and dosages administered, these chemicals do not have any eff ect on treated largemouth bass as compared to control fish when monitored using the BFC assay. The western blot of CYP3As (Figure 3-12) shows two pr ominent bands for largemouth bass and only one for catfish in the region corr esponding to CYP3A. The largemouth bass bands are also larger than those of catfish which implies that either there is more CYP3A protein per unit weight in largemouth bass or that the cross -reactivity between trout antibody is stronger with the largemouth bass than catfish. If in fact ther e is more of the releva nt protein in largemouth

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73 bass, a higher activity would be expected in largemouth bass than catfish. This however is not the case. A comparison of the percent similarity of available aminoacid sequences of CYP3A from catfish (from personal communication with Dr. David Barber), largemouth bass and rainbow trout (available in GenBank) using BLAST from within the national center for biotechnology information (NCBI) website (http://www.ncbi.nlm .nih.gov) was done. The similarity between liver CYP3A in rainbow trout and largemouth ba ss was 70% (identity) with 82% positive hits. The similarity between the rainbow trout and channel catfish liver CYP3A was 74% (identity) with 87% positive hits. Greater cross-reactiv ity between the rainbow trout and largemouth bass than catfish due to overall similarity of the protei ns is therefore unlikely to be an explanation for the banding pattern seen in the western blotting. This overall result how ever does not directly compare just the similarity of epitopes between largemouth bass, channel catfish and rainbow trout – this might be were the difference lies. There are several fluorometric probe substrates that are cons idered relatively selective for certain isoforms in human and rat P450s but there is little information a bout their suitability for use in assays with fish. One measure of how good a probe substrate is requi res that there is high correlation when results obtained from the new a ssay are compared with those of conventional substrates carried out with the same samples. The metabolites arising from the oxidation of testosterone have been used as diagnostic f unctional markers of P 450s (Figure 3-22). The correlation between BFCOD and any of the metabolit es of testosterone metabolism are very low (Table 3-5 and Table 3-6). Whereas we were not able to identify the 15 -hydroxylated metabolite, both the 6 and 2 were positively identified. Both metabolites however do not correlate well with BFCOD. In largemouth bass where they were both present there is only a

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74 weak correlation ( = 0.55) between them. These results imply, as has been suggested before, that BFCOD activity is not due to one enzyme. Furt hermore, in the case of catfish, CYP3A is not the major enzyme that metabolizes BFC and as such it is not a marker substrate for CYP3A activity in this species. Stresser et al. (2002) concluded that t hough BFC was relatively selective for human CYP3A, CYP1A2 or other extrahepatic enzymes al so contribute to its metabolism (Table 3-9). In earlier work, the same group compared the correlation of IC50 values and concluded that whereas there were good correlations for CYP1 and CYP2 families am ong fluorometric and traditional substrates, the same could not be said for CYP3A4 (Mille r et al., 2000). They suggested the use of multiple probe s for CYP3A4 but still conceded that if a single substrate has to be used then BFC would be a good choice. In both cases however the correlation coefficient values were much higher than those obtained he re and definitely point ed towards BFC being a probe for CYP3A, a conclusion that cannot be made for fish with the results documented in this body of work. This is a disappointing conclusion given that the BFCOD would greatly improve the efficiency of assaying CYP3A activity in fish samples. Survey of literature however reveals that this is might not be altogether too surprising. Most of the work using BFC as a fluorometric substrate that have b een reported have been carried out in mammalian systems and should ther efore not automatically be extrapolated to other P450 systems. The BFCOD assay has however also been used by some investigators as a marker of CYP3A activity in fish (Table 3-10). In all cases no comparison of the data reported was made to those obtained by conventional CYP3A substrates as has been done in this work. Since there is evidence that more than one enzy me is capable of metabolizing BFC, the results

PAGE 75

75 that are obtained by microsomal incubations as compared to results from expressed enzymes, would be particularly difficult to in terpret and draw conclusions from. James et al (2005) reported the IC50s of some modulators of mammalian CYP3As in catfish microsomal incubations. Ketoconazole (0.02M) and metyrapone (2.8M) were more potent than SKF-525A (25M) and eryt hromycin (41M) in inhibiting 6 -hydroxylation of testosterone (James et al., 2005). When the BFC OD was used, the same inhibitors were not as potent but their order of potency rema ined the same (Table 3-7). The IC50s for ketoconazole inhibition of the debenzylation of BFC by channe l catfish microsomes reported here are lower than those reported elsewhere. In the atlantic cod, IC50s ranging from 60 250M were reported (Hasselberg et al., 2004b). Ketoconazole and non ylphenol alone or in combination had no effect on CYP3A expression as analyzed by western blots (Hasselberg et al., 2005). Treatment with ketoconazole and nonylphenol alone resulted in 54% and 35% re duction BFCOD activity respectively. The combined exposure of ketoco nazole and nonylphenol re sulted in 98% decrease in BFCOD activity – a result gr eater than the additive effect of each compound alone. Metabolism of a particular compound is normally described kinetically using the Michaelis-Menten equation, which yields a hyperbolic ra te profile from which estimates of Vmax and Km can be made. Several enzyme-substrate pair s however do not fit th is profile and are better described by atypical or non -Michaelis-Menten models. CYP3A catalysis has been shown in several instances to exhibit unusual kinetic behavior. Kullman et al (2004) demonstrated an indication of negative cooperativity during O-debenzyloxylation of BFC by recombinant CYP3A38 and CYP3A40 from the medaka fish, Orzias latipes. CYP3A metabolizes structurally diverse substrat es with a wide range of molecular sizes, shapes, enzyme affinities and turnover numbers. As a result, the interaction of an inhibitor with

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76 one CYP3A probe may differ from that observed w ith other CYP3A substrat es. Lu et al (2001) attribute these observations to di fferential binding and steric restriction for interacting agents (substrates and inhibitors) which result in both substrateand i nhibitor-dependent inhibition. Stresser et al (2000) demonstrat ed that quantitative and qualitat ive inhibition parameters are substrate-dependent for CYP3A4. The results obtained during this study corroborate this; demonstrating that even in the same species, CYP3A inhibition data obtained with one probe substrate may not be applicable to other probe substrates. Conclusions Fluorescent assays are becoming more popular because they increase throughput, allow continuous reaction monitoring and minimize the amount of the enzyme required in the reaction. To this end significant efforts have been made to develop fluorometric probe substrates that yield highly fluorescent metabolites in isozyme-specific P450 reactions. Most of the reported work to date has been carried out in mammalian system s and should therefore not automatically be extrapolated to other P450 systems. The good correlation in IC50 values seen for other P450 enzymes among fluorometric substrat es and traditional substrates does not generally apply to CYP3A even in mammalian systems (Crespi and St resser, 2000; Miller et al., 2000). Several authors have reported work in which BFC was used as the sole probe substrate for CYP3A activity in different fish species. In this wo rk we report that in the channel catfish and largemouth bass, the BFCOD assay does not correlate well with 6 -hydroxylation of testosterone, the standard mark er for CYP3A activity or any ot her testosterone hydroxylation metabolite. The BFCOD assay may therefore not be an indicator of CYP3A activity in channel catfish and largemouth bass.

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77 Figure 3-1. Proton NMR of 7-benzyloxy-4-(trifluoromethyl)-coum arin. A) Full spectrum B) Expansion of aromatic region. Sample an alyzed in DMSO. Chemical shifts in reference to TMS. a e d f DHO DMSO TMS b c e a B A

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78 Figure 3-2. The HPLC chromatogram of 7benzyloxy-4-(trifluoromethyl)-coumarin (BFC). Reverse phase 25cm x 4.6 mm; 5 m Discovery column, 80% methanol in water was used with UV detection at 254nm.

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79 Figure 3-3. Emission spectra of 7-hydr oxy-4-(trifluoromethyl)-coumarin (A) and 7benzyloxy-4-(trifluoromethyl)-coumarin (B ) obtained at a 410nm excitation wavelength

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80 O O O F F F O O F F F OH CYP3A BFC HFC Figure 3-4. The 7-benzyloxy -(4-trifluoromethyl)-coumarin-O-d ebenzylase (BFCOD) reaction

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81 0 10 20 30 40 0 10 20 30 50 M 100 M 200 M time (mins)pmol/mg Figure 3-5. Time course for 7-hydroxy-4(trifluoromethyl)-cou marin production by largemouth bass hepatic microsomes. Inc ubation done with 0.5mg of protein in 1mL reaction volume.

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82 0.0 0.5 1.0 1.5 2.0 2.5 0 25 50 75 100 125 protein (mg)pmol/min Figure 3-6. Protein amount optimizati on for 7-hydroxy-4-(trifluoromethyl)-coumarin production by hepatic microsomes of the largemouth bass. Incubation conditions: 200M BFC; 20 minutes

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83 Figure 3-7. Stability profiles of BFCOD assay products. Analysis done in the presence of Tris (A), sodium hydroxide (B) and no ba se (C) monitored at excitation/emission wavelength of 410/530nm

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84 Figure 3-8. Temperature optimization for HF C production by hepatic microsomes of the channel catfish. Incubation conditions: 75M BFC; 0.5mg protein; 20 minutes

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85 Figure 3-9. Temperature optimization for HF C production by hepatic microsomes of the largemouth bass. Incubation conditions: 150M BFC; 0.7mg prot ein; 20 minutes

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86 Km (M) Vmax (pmol/min/mg) 0 25 50CF (Female) CF (Male) LMB (Male) 150 400 650 900LMB (Female) Kinetic Parameter Figure 3-10. Comparison of kinetic parameters betw een male and female fish. CF = catfish (N = 4); LMB = largemouth bass (N = 1)

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87 CF (MXC) CF (Con t rol) LMB (Control) LMB ( Dieldrin) LM B (DDE) 0 100 200 300 b ab c c a Treatment GroupActivity (pmol/min/mg) Figure 3-11. The BFCOD activity of fish in diffe rent treatment groups. Means with same letter are not significantly different from each ot her at the 0.05 level; N = 8 for catfish (CF); N = 12 for largemouth bass (LMB)

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88 1 2 3 4 5 6 Figure 3-12. Western blot of human CYP3A4 and hepatic microsomes from largemouth bass and catfish. Lanes 1 and 4 have 40 g catfish hepatic microsomes; 2 and 5 have 2pmole human CYP3A4; 3 and 6: 40 g largemouth bass hepatic microsomes y = 1.4669x 36.88 0 100 200 300 400 500 600 050100150200250300350400450 Activity (pmol/min/mg)Band Area (x1000) Figure 3-13. Correlation of pr otein band intensity and activity of channel catfish samples

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89 2,000 1,500 1,000 500 0 SixbOHT800 600 400 200 MXC PCB 3MC Control Figure 3-14. Correlation between BFCOD and 6 -hydroxylation of testos terone activity of hepatic microsomes of channel catf ish in different treatment groups. BFCOD

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90 2,000 1,500 1,000 500 0 EROD10,000 8,000 6,000 4,000 2,000 0 PCB 3MC Control Figure 3-15. Correlation between BFCOD and EROD activity of hepatic microsomes of channel catfish in different treatment groups. BFCOD

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91 Figure 3-16. Normal phase TLC of testoster one metabolism by hepatic microsomes of largemouth bass (A) and catfish (B); Deve loped three times in a mobile phase comprising diethyl ether:toluene: methanol:acetone (70:38:0.8:1) M2 M3 (3 -OH testosterone) M1 (Androstendione) M4 (6 -OHtestosterone) M5 (2 -OHtestosterone) Testosterone Origin A B

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92 A LMB (Ctrl ) LM B (E 2 ) L MB (Low ) LM B ( M ed ) LMB (High) CF (Ctrl) C F ( 3M C) CF ( M X C) CF ( PC B ) 0 50 100 150 200treatment groupActivity (pmol/min/mg) B LM B (C t r l) LMB (E2) LM B ( Low ) LMB (Me d ) L M B ( Hi gh) 0 10 20 30 40 50treatment groupActivity (pmol/min/mg) Figure 3-17. Activity of testosterone metabo lites in different treatment groups. A) M1 (Andostenedione) B) M2 C) M3 (3 metabolite) D) M4 (6 -hydroxytestosterone) E) M5 (2 -hydroxytestosterone).

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93 C LMB ( C trl ) L M B ( E2) LMB (Low) LMB (Med) L M B (H i gh) CF (Ctrl) CF (3MC) C F (MXC ) C F (P C B) 0 250 500 750 1000treatment groupActivity (pmol/min/mg) L M B (C trl ) L M B ( E 2) LMB (Low) L MB (M ed ) LM B (H i gh) CF (Ctrl) CF (3M C ) C F (M X C ) C F (P C B) 0 250 500 750 1000Dtreatment groupActivity (pmol/min/mg) E LMB (C tr l) LMB (E2 ) LMB (Low ) L M B (Med) L M B (H ig h ) 0 50 100 150treatment groupActivity (pmol/min/mg) Figure 3-17. Continued

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94 Control 3MC PCB MXC 0 1000 2000ab b ab a A treatment groupActivity (pmol/min/mg) E2 MXC Low MXC Med MXC High 0 10 20a a a a B treatment groupActivity (pmol/min/mg) Figure 3-18. The BFCOD activity of different treatment groups for catfish (A) and largemouth bass (B). Groups with same letter in each graph are not significantly different from each other at the 0.05 level

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95 O O O O O H O O O H N OH O O O N O N Cl Cl N N N N O O O O H N N Cl Erythromycin alpha-naphthoflavone Metyrapone Ketoconazole Clotrimazole Figure 3-19. Compounds used to modulate he patic CYP3A activity in catfish microsomes

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96 K C M N E 0 20 40 60 80K C M N E Modulator% of control Figure 3-20. The BFCOD Activity of channel catfi sh microsomes in the presence of chemical modulators. Concentrations per assay used were 20M ketoconazole (K) and clotrimazole (C); and 15 0M for metyrapone (M), -naphthoflavone (N) and erythromycin (E).

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97 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 0 25 50 75 100log10 (inhibitor concentration )% of Control Figure 3-21. Chemical Inhibition of hepatic mi crosomal BFCOD activity of channel catfish by ketoconazole. Conditions: 75M BFC, 0.5mg protein and 20mins incubation;

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98 OH O 6 18 Figure 3-22. Testosterone hydroxylation, a usef ul diagnostic functional marker for P450s.

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99 Table 3-1. The BFCOD activity of male and female fish in different treatment groups Catfish Largemouth bass Treatment Activity Treatment Activity Control MXC 237.1 44.8 (8)a 131.2 23.5 (8)a Control (Male) Control (Female) Dieldrin (Male) Dieldrin (Female) DDE (Male) DDE (Female) 28.0 2.3 (6)ab 24.2 1.3 (6)ab 39.0 6.0 (6)b 30.4 5.1 (6)ab 21.5 1.4 (6)a 24.6 3.3 (6)ab Activity in pmol/min/mg of protein is expressed as mean SE (n); means with same letter are not significantly different from each other at th e 0.05 level; Conditions were catfish: 75M BFC, 0.5mg protein and 20mins incubation; Largem outh bass: 150M BFC, 0.5mg protein and 20mins incubation Table 3-2. Activity in different assays of cha nnel catfish microsomes derived from different treatment groups Treatment BFCOD 6 OHT EROD Control 3MC PCB126 MXC 393 110 (11)b 919 174 (10)ab 1298 336 (4)ab 197 30 (4)a 548.2 54.4 (11)b 403.8 34.4 (10)ab 451.8 68.1 (4)ab 245.9 27.1 (4)a 76 37 (11)a 4082 1903 (10)c 661 116 (4)b Activity is in pmol/min/mg of prot ein is expressed as mean SE (n ); means with same letter are not significantly different from each other at the 0.05 level

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100 Table 3-3. Activity of products of testosterone metabolism by hepatic P450s from different largemouth bass treatment groups Treatment group Metabolite Estradiol MXC (Low)a MXC (Medium)a MXC (High)a M1 (Andr) 60.5 14.1 (3)82.9 10.7 (5)63.8 9.23 (6) 73.7 9.08 (6) M2 18.0 3.51 (3)33.8 1.88 (5)22.6 3.49 (6) 25.1 1.73 (6) M3 (3 OH) 254.7 148 (3) 55.7 4.55 (5)76.4 10.3 (6) 94.2 27.7 (6) M4 (6 OH) 56.3 6.51 (3)80.4 7.83 (5)57.3 6.69 (6) 66.3 4.01 (6) M5 (2 OH) 75.2 3.28 (3)75.4 11.1 (5)63.0 12.5 (6) 100.7 13.7 (6) Activity in pmol/min/mg of protein is expressed as mean SE (n); a MXC (low) fish received 2.5mg/Kg, medium 10mg/Kg and high 25mg/Kg of MXC Table 3-4. Activity of products of testosterone metabolism by hepatic P450s from different catfish treatment groups Treatment group Metabolite Control 3MC PCB MXC M1 (Andr) 127 13.5 (7)115 12.4 (8)95 6.66 (4) 68 7.04 (4) M3 (3 OH) 824 38.3 (7)725 58.2 (8)744 27.8 (4) 425 23.1 (4) M4 (6 OH) 506 68.4 (7)414 39.5 (8)452 68.2 (4) 246 27.1 (4) Activity in pmol/min/mg of protein is expressed as mean SE (n)

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101 Table 3-5. Correlation matrix of BFCOD a nd testosterone metabolism products in the largemouth bass. MI M2 M3 M4 M5 BFCOD M1 (Andr) 1.0 0.22 0.20 0.27 -0.01 0.14 M2 1.0 -0.61** 0.35 -0.21 -0.16 M3 (3 OH) 1.0 -0.44 0.05 0.02 M4 (6 OH) 1.0 0.55* 0.38 M5 (2 OH) 1.0 0.28 BFCOD 1.0 Values are Spearman correlation coe fficients; p < 0.05; ** p < 0.01 Table 3-6. Correlation matrix of BFCOD a nd testosterone metabolism products in the channel catfish. M1 M3 M4 BFCOD M1 (Andr) 1.00.08 0.38* 0.08 M3 (3 OH) 1.0 0.34* 0.05 M4 (6 OH) 1.0 -0.01 BFCOD 1.0 Values are Spearman correlation coefficients; p < 0.05

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102 Table 3-7. The IC50 values for inhibitors of CYP3A activity in the channel catfish Treatment group Inhibitor Control 3MC Ketoconazole 23.84.0 Metyrapone 150.5193.7 Erythromycin 66.677.5 Clotrimazole 11.611.6 Values in M Table 3-8. The IC50 values for channel catfish BFCOD activity inhibition by ketoconazole Upper portion of curve Lower portion of curve Full curve Livera 0.48 0.089 (3)30.9 7.83 (4)23.8 6.84 (4) Liverb 0.18 0.103 (2)38.4 7.42 (2)23.9 8.75 (2) Proximal Intestinea 0.27 0.101 (3)18.3 3.25 (3)6.5 1.11 (3) Proximal Intestineb 0.18 0.060 (2) 18.9 5.55 (2)5.9 0.49 (2) values (M) shown are mean SE (N); a all data collected; b data that did not deviate from the model being tested.

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103 Table 3-9. Correlation coefficients for deal kylation of the fluorometric probe and P450 isoform selective catalytic activit ies in a panel of human donors Correlation coefficient Human P450 Isoform Catalytic activity BFC BFC + 0.5M ketoconazole CYP1A2 Phenacetin O-Deethylase 0.00 0.87** CYP2A6 Coumarin 7-hydroxylase 0.14 -0.15 CYP2B6 (S)-Mephenytoin N-demethylase 0.88** 0.03 CYP2C8 Paclitaxel 6 -hydroxylase 0.58 0.21 CYP2C9 Diclofenac 4’-hydroxylase 0.37 0.50 CYP2C19 (S)-Mephenytoin 4’-hydroxylase 0.29 0.37 CYP2D9 Bufuralol 1’-hydroxylase -0.33 -0.11 CYP2E1 Chlorzoxazone 6-hydroxylase 0.26 0.34 CYP3A4 Testosterone 6 -hydroxylase 0.95** 0.08 CYP4A11 Lauric acid 12-hydroxylase -0.22 0.46 Total P450 (pmol/mg) 0.75** 0.63* p < 0.05 ** p < 0.01 Adapted from (Stresser et al., 2002)

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104 104Table 3-10. The 7-benzyloxy-(4-trifluoromethyl)-coumarin-O-deb enzylase (BFCOD) assay in different fish Fish Enzyme source Modulator Effect Reference Carp (Cyprinus carpio) Control microsomes ethynyl-estradiol 93% inhibition (T hibaut et al., 2006) clofibrate 46%; IC50 = 1047.0 1.0M gemfibrozil 55%; IC50 = 616.3 1.1M diclofenac 67%; IC50 = 805.3 1.5M (fluoxetine 69%; IC50 = 643.0 1.0M fluvoxamine 78%; IC50 = 274.0 1.3M paroxetine 80%; IC50 = 262.5 1.2M Medaka (Orzias latipes) nonylphenol atypical kinetics; (Kullman et al., 2004) recombinant CYP3A38 baculosomes Vmax = 7.95pmol/min/nmol Km = 0.116M recombinant CYP3A40 baculosomes Vmax = 7.77pmol/min/nmol Km = 0.363M Atlantic cod (Gadus morhua) Microsomes of fish treated with 0.02, 2, 20, 40 and 80ppm alkylphenol mixture. Ketoconazole IC50 = 60-250M (Hasselberg et al., 2004b) Microsomes of fish treated with 5ppm 17 -estradiol Ketoconazole IC50 =75M Rainbow trout (Oncorhynchus mykiss) Microsomes of fish treated with ketoconazole (i.p; 12 -100mg/Kg body wt) 60 80% reduction in activity (Hegelund et al., 2004) Killifish (Fundulus heteroclitus) Microsomes of fish treated with ketoconazole (i.p; 25mg/Kg body wt) 90% reduction in activity

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105 CHAPTER 4 SULFATION OF SELECTED XENOBIOTICS While the P450 pathways of MXC metabolis m that form the estrogenic compounds OHMXC and HPTE may be viewed as the bioact ivation routes, the UGT and SULT pathways are viewed as detoxification pathways for the phe nolic metabolites. It has been documented that the glucuronidation of both metabolites is much sl ower than the rate of their formation. This chapter investigates the sulfation of OHMXC HPTE and also 3-hydroxybenzo-[a]-pyrene, a relatively good catfish sulf otransferases substrate. Results Sulfation of OHMXC Optimum conditions for the hepatic and intestin al sulfation of OHMXC were investigated by varying incubation time (0 – 6 hours in one hour increments) and protein amounts (50, 100 and 200g). The production of the sulfate conjugat e remained linear for a bout one hour (Figure 4-1). An incubation time of 60minutes was used fo r subsequent assays. In the liver, the amount of OHMXC sulfate produced by 100 g of protein was almost the same as that from 200g while in the intestine 200g of protein had less product than 100g (Figure 4-2). In both cases 50g of protein gave the most product per unit and was th erefore used for subse quent assays. Typical autoradiograms of TLC chromatograms obtaine d are shown in Figure 4-3 and 4-4. The sulfation activity of cytosolic protein from control, 3MCand MXC-pretreated samples (using 600M for hepatic and 800M OHMXC for intestinal assays) were compared (Figure 4-5). In both organs, activ ity of male fish differed from fe male. In the liver, the female samples had higher activity (p = 0.016), while in the intestine the male activity was higher (p = 0.021). More assays were done to compare th e kinetic parameters of control versus 3MC samples. These results are summarized in Tabl e 4-1 and Table 4-2. For all parameters 3MC-

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106 treated female when compared to male were found not to be significan tly different from each other. When the parameters were compared by treatment group between the two organs, there was no significant difference (p > 0.05) in all three groups. When the same comparison was made within an organ, there was no difference between the Vmax of control and 3MC-treated fish (liver and intestine) and between Km of the two treatments in the intestine. The Km of control samples in the liver were significantly higher (p < 0.05) than that in treated fish. The efficiency of sulfation was also higher in 3MC-treated fish than control fish both in the liver and intestine. Since at no time were there differences between the kinetic parameters of female and male, for further assays the sex of the fish from whic h the cytosol was derived was not taken into consideration. Sulfation of HPTE Optimum conditions for the hepatic and intestin al sulfation of HPTE were investigated by varying incubation time (0 – 4 hours in one hour increments) and protein amounts (50, 100 and 200g). The production of the sulfat e conjugate remained linear for about one hour (Figure 4-6). A one hour incubation time was used for subse quent assays. In both organs, the amount of HPTE sulfate decreased with increase in protein amount (Figure 4-7). For subsequent assays 50g of protein, which gave the highest activity was therefore used. A typical autoradiogram of TLC chromatograms obtained is shown on Figure 4-8. Sulfation activity of cytosolic protein from control, 3MCand MXC-pretreated samples was compared using 400M HPTE for hepatic and 600 M for intestinal assays (Figure 4-9). In both organs activity of MXC and control were similar but lower in activity than 3MC-treated samples. Kinetic parameters of the three tr eatment groups are summarized in Table 4-3 and Table 4-4. Trends in Vmax and Km were similar in both organs. The Vmax of control and MXC samples were similar but lower than that of 3MC samples. The Km of control and 3MC samples

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107 were also similar, but lower than that of MXC-tr eated samples. In the liver the efficiency of MXC samples was less than that of 3MC sample s but both were not different from those of control samples. In the intestine however, th e control and MXC samples had similar efficiency but less than that of 3MC samples. When the parameters were compared by treatment between the two organs, both Vmax and Km in the intestine were generally higher. The efficiency of sulfation of HPTE was not different for any of the groups between the two organs. Sulfation of 3-Hydroxybenzo-[a]-pyrene The sulfation of 3OHBaP by both hepatic and intestinal cy tosol was compared between control and 3MC-treated fish. Optimum conditions for the hepatic sulfation of 3OHBaP were determined to be 10g of protein and 5 minutes in cubation time. Preliminary activity of control, female and male 3MC-treated samples were comp ared using 400M of substrate (Figure 4-10). Male and female fish were not different from each other (p = 0.83) but both had higher activity than control samples. The kinetic parameters for the sulfation of 3O HBaP by both hepatic and intestinal cytosol were then compared between control and 3MC-treated fi sh. The Eadie-Hofstee plot (Figure 4-11a) was characte ristic of positive cooperativity a nd so the Hill plot (Figure 411b) was used to calculate all parameters (Table 4-5). In this assay a very small amount of protein along with a relatively s hort incubation time were necessa ry in order to comply with initial rate conditions that ideally require that substrate turnover be less than 10% (Houston and Kenworthy, 2000). Certain trends were observed in the data. The efficiency of this reaction improved significantly in 3MC as compared to c ontrol samples both in the liver (p = 0.001) and the intestine (p = 0.02). In control samples, intestine had higher Vmax, S50 and efficiency when compared to liver samples. The Vmax and efficiency of 3MC intestin al samples were also higher than liver samples, but their S50 were not different. The Hill co efficient, H, was smallest for intestinal control samples and not signifi cantly different among the other three groups.

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108 Discussion Rates of sulfation of OHMXC and HPTE reporte d here are extremely low compared with those of glucuronidation of the same compounds in the channel catfish (Table 1-7 and Table 18). There have been previous reports of comparatively poor ph ase II metabolism of xenobiotics in the catfish. James and Rowland-Faux (2003) re port results of the conj ugation of hydroxylated polychlorobiphenyls (OHPCBs) which mirrors that documented in this body of work. Both glucuronidation and sulfation of a series of OHPCBs proceeded very slowly with SULT activity being less than UGT when incubated in intestinal preparations in comparison to 3OHBaP (Table 4-6). In van den Hurk and James (2000), the author s report using intestinal mucosa fractions to investigate the conjugation of benzo-[a]-pyr ene-7,8-dihydrodiol. Sulfation had a Vmax of only 2pmol/min/mg while glucuronidation gave a Vmax of 300pmol/min/mg and Km of 23.39M. Sulfation of HPTE, as for OHMXC, showed hi gher activity in 3MC fish than the control group, both in the intestine and liver (p<0. 001). Both OHMXC and HPTE however were not particularly good substrates for su lfation given the relativ ely small activity values reported here. 3OHBaP on the other hand has been documented to be a good substrate for sulfotransferases in the channel catfish (Tong and James, 2000). Cla ssically, metabolism of a particular compound is described kinetically using the Michaelis-Men ten equation (equation 21), which yields a hyperbola. This equation estimates the maximum reaction velocity (Vmax) as well as the apparent Km. An Eadie-Hofstee plot for th is equation should produce a rela tively straight line with a negative slope. The plot depicted in Figure 4-7a is more characteristic of a sigmoidal autoactivation profile resulting from homotropic positive cooperativity – the activator being the substrate itself (Hutzler and Tr acy, 2002). A sigmoidal profile is better described by the Hill equation (2-2) where H is a measure of cooperativity and S50 the substrate concentration resulting in half of Vmax.

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109 The efficiency of the sulfation of OHMXC, HPTE and especially 3OHBaP improved in 3MC as compared to control. For 3OHBaP this improvement was signifi cant both in the liver and intestine. Efficiency is calculated as Vmax divided by S50 and so these results may be better understood in light of the trends in both S50 and Vmax. Within an organ (Table 4-5), Vmax values were higher in 3MC than in control samples and within treatments Vmax was higher for intestine than liver. An increase in Vmax observed without a signi ficant change in S50, is indicative of an increase in the amount of the responsible enzyme responsible. On the othe r hand, an increase in Vmax accompanied with a significant change in S50, is indicative of an increase in the amount of a contributing enzyme, different from the first. Both scenarios arise from increased turnover of substrate which may be a consequence of the indu ction of one or more enzymes responsible for the sulfation of these phenolic metabolites Sulfotransferases have generally been consid ered to be non inducible in fish. There has only been one prior report of induction of fish SULTs resulting in increased sulfation of 9hydroxybenzo-[a]-pyrene by 3MC-treated channel catfish and mummichog from a creosote contaminated river (Gaworecki et al., 2004). The identification and subsequent quantitation of sulfotransferase isoforms in both the liver and inte stine of the channel catfish would be helpful in enabling the elucidation of the sulfotransferases responsible for the conjugation of these metabolites. The Hill coefficient, H, used as an index of cooperativity ranged from 1.6 to 3.3. This is in the normal range for individual enzyme s that exhibit positive cooperativity (CornishBowden, 2004). The Km values reported for both sulfation a nd glucuronidation of HPTE and OHMXC by channel catfish are much larger than those of the P450-dependent formation of these metabolites (Table 1-5). This suggests that even a combin ation of these pathways may therefore not be

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110 efficient in the detoxification of MXC by the catfish at environmentally relevant exposure levels. This is a cause for concern since the effect of MXC on human beings has been directly implied. Monkeys, like humans, undergo a long and comple x period of developmen t during adolescence, making them important models for understandi ng the effects of estrogenic effects from xenobiotics during this period. The consequen ces of treatment with 25 and 50mg/Kg of MXC per day given in the peripubertal period to fema le rhesus monkeys was examined (Golub et al., 2003; Golub et al., 2004). These treatments incr eased estrogen activity of serum as determined with an in vitro estrogen receptor transcription assay. MXC-treatment also led to premature emergence of secondary sex characteristics, redde ning and swelling of skin and retarded growth of the nipple. When evaluated by ultrasound after an 8-month recovery period, uterine size was not affected, but there was some indication of in creased incidence of ovarian cysts or masses. Humans as well as other animals are frequen tly exposed to mixtures of xenobiotics. Concern has been raised that some compounds mi ght be capable of affecting the metabolism of others and thereby altering their t oxicological behavior. In a study to investigate the potential of the phytoestrogen genistein to influence the reproductive developmental toxicity of MXC, Sprague-Dawley rats were exposed to the two compounds, either alone or in combinations, through dietary administration to dams during pr egnancy and lactation and to the offspring directly after weaning (You et al., 2002). The estrogenic responses to their co-administration were shown to be cumulative of the effect s associated with each compound alone. Conclusions Methoxychlor remains an intriguing endocrine disr uptor for several reasons. It is relatively inactive but is biotransformed to more active me tabolites illustrating the importance of metabolic activation for some endocrine-active chemicals. The metabolites, principally HPTE, have estrogen receptor agonist activit y as well as antagonist activity at the androgen receptor.

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111 Exposure to endocrine disrupti ng chemicals in early development can produce changes, some of which might not be evident until much later. Thus, newborns and children may be more vulnerable than adults because of differences in absorption, metabolism, storage and excretion, the sum of which produces higher biologically effective doses in target tissues. Furthermore, children can have greater exposure than adults because of proportionally higher food intake and activities that result in greater cont act with environmental contaminants. Sulfotransferases, generally considered to be no n inducible in fish, have been shown in this work to be induced by the treatment of channel cat fish with 3MC. The rates of sulfation reported for both OHMXC and HPTE are much lower than those for glucuronidation and together are much slower than those found in the P450-dependen t formation of these metabolites. This might mean that glucuronidation and sulfation, the expe cted detoxification pathways, are inefficient at aiding the elimination of these potentially harmful metabolites after environmental exposure of the channel catfish to methoxychlor.

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112 0 1 2 3 4 5 6 7 8 9 0 100 200 300 400 500 Liver Intestine time (hrs)pmol/mg Figure 4-1. Time course for OHMXC sulfat e production by channel catfish hepatic and intestinal cytosol. In cubation conditions: 100M OHMXC; 0.1mg liver protein; 0.05 intestinal protein.

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113 0.00 0.05 0.10 0.15 0.20 0.25 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Liver Intestine protein (mg)pmol/min Figure 4-2. Sulfation of 100M OHMXC by diff erent amounts of cytosolic protein from catfish liver and intestine.

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114 Figure 4-3. Reverse phase TLC Chromatogram of the sulfation of 800M OHMXC by catfish liver cytosol. Developed using a solv ent system of methanol:water (4:1) OHMXC sulfate Endogenous sulfates Origin

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115 Figure 4-4. Normal phase TLC Chromatogram of the sulfation of OHMXC by catfish liver cytosol. Developed using a solvent system of methanol: ethylacetate:dichloromethan e (1:1:4) with tetrabutyl ammonium hydrogen sulfate.; Lane 1: incubation with no substrate; Lane 2: incubation with 800M OHMXC Endogenous sulfates OHMXC sulfate Origin 1 2

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116 A Control MXC 3MC (F) 3MC (M) 0 5 10 15b c d aTreatmentActivity (pmol/min/mg) B Control MXC 3MC (F) 3MC (M) 0 10 20 30a a b cTreatmentActivity (pmol/min/mg) Figure 4-5. The OHMXC sulfation activity of hepatic and intestinal catfish cytosol. A: Liver, 600M OHMXC; B) Intestine, 800M OHM XC; Means with same letter are not significantly different at the 0.05 level.

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117 0 1 2 3 4 5 0 1000 2000 3000 4000 Liver Intestine time (hrs)pmol/mg Figure 4-6. Time course for HPTE sulfate produc tion by channel catfish he patic and intestinal cytosol. Incubation conditions: 200M HPTE; 0.05mg protein.

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118 0.00 0.05 0.10 0.15 0.20 0.25 0 5 10 15 20 25 30 Liver Intestine protein (mg)pmol/min Figure 4-7. Sulfation of 200M HPTE by different amounts of cytosolic protein from catfish liver and intestine.

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119 Figure 4-8. Normal phase TLC Chromatogram of the sulfation of OHMXC and HPTE by catfish liver cytosol. Lane 1: 50M OHMXC; Lane 2: 100M HPTE 1 2 OHMXC sulfate HPTE sulfate Origin Endogenous sulfates

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120 A Control MXC 3MC 0 10 20 30 40a a bTreatmentActivity (pmol/min/mg) B Control MXC 3MC 0 20 40 60 80a a bTreatmentActivity (pmol/min/mg) Figure 4-9. The HPTE sulfation activity of hepati c and intestinal catfish cytosol. A: Liver, 400M OHMXC; B) Intestine, 600M OHM XC; Means with same letter are not significantly different at the 0.05 level.

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121 Control 3MC (Female) 3MC (Male) 0 200 400 600 800a b bTreatmentActivity (pmol/min/mg) Figure 4-10. Sulfation of 3OHBaP by hepatic catfish cytosol. Means with same letter are not significantly different at the 0.05 level.

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122 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 0 50 100 150 200 250 300A v/[S]v 0 100 200 300 400 500 0 100 200 300B [3OHBaP] (nM)v (pmol/min/mg) Figure 4-11. Plots of 3-hydroxybenz o-[a]-pyrene sulfation activity by catfish liver cytosol. A) Eadie-Hoftsee plot; B) Hill plot.

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123 Table 4-1. Sulfation rates of OHMXC by hepatic catfish cytosol Treatment Vmax (pmol/min/mg) Km ( M) Efficiency ( L/min/mg) Control 6.69 1.22 (4)a179.73 8.48 (4)b0.038 0.01 (4)a 3MC (female) 11.01 1.36 (3)a 67.90 7.17 (3)a0.169 0.03 (3)b3MC (male) 12.21 0.53 (3)a 86.46 4.66 (3)a0.141 0.01 (3)b values shown are mean SE (N); for each pa rameter means with the same letter are not significantly different at the 0.05 level Table 4-2. Sulfation rates of OHMXC by intestinal catfish cytosol Treatment Vmax (pmol/min/mg) Km ( M) Efficiency ( L/min/mg) Control 12.64 1.71 (4)a267.30 35.9 (4)a0.048 0.01 (4)a 3MC (female) 22.17 3.61(4)a 171.68 21.8 (4)a0.128 0.01 (4)b3MC (male) 25.73 3.51 (4)a168.58 19.4 (4)a0.152 0.01 (4)b values shown are mean SE (N); for each pa rameter means with the same letter are not significantly different at the 0.05 level

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124 Table 4-3. Sulfation rates of HPTE by hepatic catfish cytosol Treatment Vmax (pmol/min/mg) Km ( M) Efficiency ( L/min/mg) Control 17.5 2.21a32.49 7.47a0.599 0.12ab 3MC 35.4 2.28b74.51 7.30a 0.482 0.03b MXC 22.4 1.28a117.1 5.60b 0.194 0.02a values shown are mean SE (N = 4); for each parameter means with the same letter are not significantly different at the 0.05 level Table 4-4. Sulfation rates of HPTE by intestinal catfish cytosol Treatment Vmax (pmol/min/mg) Km ( M) Efficiency ( L/min/mg) Control 29.6 2.34a119.6 14.9a0.262 0.04a 3MC 63.5 2.31b99.31 5.14a0.644 0.04bMXC 28.5 5.12a148.5 14.7b0.197 0.01a values shown are mean SE (N = 4); for each parameter means with the same letter are not significantly different at the 0.05 level

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125 Table 4-5. Rates of Sulfonati on of 3OHBaP by catfish cytosol Treatment Vmax (pmol/min/mg) S50 (nM) Efficiency ( L/min/mg) H Control (Liver) 103 21a 111 6.0a 930 366a 2.74 0.44b 3MC (Liver) 478 35 b***161 8.1b **2976 385b*** 2.40 0.16b Control (Intestine) 612 102b 141 9.2b 4301 1160b 1.76 0.15a 3MC (Intestine) 1207 43c 140 3.9ab 8602 230c* 2.25 0.25ab values shown are mean SE; N = 4; Significan tly different from control: ***p<0.001; **p<0.01; *p<0.05; for each parameter means with the same le tter are not significantly different at the 0.05 level Table 4-6. Conjugation of hydroxylated PCBs in intestinal fractions of the channel catfish Rate as substrate concentration, pmol/min/mg protein Compound UGT SULT 2-OH-CB77a 13.5 2.5 (3)8.1 2.9 (3) 4’-OH-CB79a 4.6 1.6 (4)2.1 0.3 (3) 5-OH-CB77a 12.6 3.6 (3) 6-OH-CB77a 9.9 0.3 (3) 4’OH-CB72a 12.3 0.3 (3) 3OHBaPb 390 130 (6)1270 490 (6) Adapted from James and Rowland-Faux (2003); a 5M substrate concentration; b 1M substrate concentration

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126 CHAPTER 5 IN VITRO AND IN VIVO METABO LISM OF METHOXYCHLOR Concern about possible adverse effects caused by the inadvertent exposure of humans to endocrine-active chemicals has led to studies of the metabolic pathways of compounds that are persistent in the environment. To determine the in vivo effects of co-exposure, adult channel catfish were treated by gavage for six days with 2mg/Kg 14C-MXC alone or 2mg/Kg 14C-MXC and 2mg/Kg benzo-[a]-pyrene in groups of four. In Vivo Radiolabeled Methoxychlor Studies CYP1A activity, as measured by 7-ethoxyresor ufin O-deethylase (EROD) activity, was significantly greater with microsomes from MXCB aP-treated fish than with those from fish treated with just MXC (Figure 5-1). This conf irms that treatment with BaP induces the CYP1A family of enzymes in the channel catfish. Distribution of Radioactivity in Fish Tissue and Bile On average, bile, muscle, fat deposits, liver and skin each had more than 1% of the radiation present in the initial dose (Figure 5-2A). Due to the overall percentage by mass of the different body parts, the amounts of MXC and metabolites calculated in nmol per gram of tissue follows a different pattern (Figure 5-2B). Bile, fa t deposits, intestine, live r and brain each had at least 25nmol of radioactivity per gram of tissue. Significant differences we re present in liver (p = 0.024) and muscle (p = 0.03). In each case th e MXC-treated fish had hi gher radioactive counts than the MXCBaP-treated fish samples. Overall, more MXC-derived radioactivity remained in MXC (45.4 4.4; mean percent total dose SE) th an the BaP-induced catfish (30.2 3.4). Bile, muscle and fat contained almost 90% of the re maining radioactivity when results from both treatment groups are combined (Fig ure 5-3). Following quantitation of radioactivity in different

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127 liver sub-cellular fractions, it was evident that the overall percent distribution was similar in MXCand MXCBaP-treated fish (Table 5-1). Quantitation of Radiolabeled Metabolites Liquid chromatography was used to quantitat ively analyze radiolabeled MXC and its metabolites in different tissues and bile. Analyt e retention times of MXC and its metabolites for each HPLC method used are summarized in Table 5-2. Results from the liver, (Figure 5-4 and Figure 5-5) and the intestinal mucosa, (Figure 5-6 and Figure 5-7) show the presence of the glucuronides of both HPTE and OHMXC, the phe nolic metabolites OHMXC and HPTE and also some of the parent compound. While in both tis sues the general trend shows a small amount of parent compound and more conjugate than the phenolic compounds, there are more of the phenolic metabolites in the liver than in the intestinal mucosa. Blood samples from the MXC-treated fish we re analyzed individually, but the MXCBaP samples were pooled to get a better sample readi ng. The metabolites seen in blood samples were mainly the phenolic metabolites and the parent compound (Figure 5-8 an d Figure 5-9). While some of the MXC-treated fish chromatograms showed the glucuronides of OHMXC or HPTE, there were none in MXCBaP-tr eated fish blood. There are some unknown compounds in the chromatograms of some of the fish samples. Unknown 1 (U1) was present in one fish from the MXC-treated group and in the MXCBaP pooled blood sample. Unknown 2 (U2) was only evident in the pooled blood of the MXCBaP-treated samples. In muscle, fat deposits, gonads and brain (Figure 5-10 to Figure 5-16), HPTE, OHMXC and MXC were the major metabolites. In muscle and the gonads, HPTE amounts were highest and MXC the least, while it was th e opposite in the fat deposits and br ain. Catfish skin from the different treatments were pooled (by treatment) before analysis. Th ere was evidence of MXC and the glucuronide of HPTE in both treatment groups but the OHMXC glucuronide was present

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128 only in the MXCBaP group (Figure 5-17 and Figure 5-18). In th e bile (Figure 5-19 and Figure 5-20) there were small amounts of the parent co mpound and larger amounts of the conjugates. In both treatment groups the HPTE glucuronide was present in larger quantities than the OHMXC glucuronide. Following the extraction of each tissue sample, the total radioactivity not extracted was monitored by digesting the final pellet in sodium hydroxide to release any resideual radioactivity from cells, then quantifying by sc intillation counting. A summary of these results is shown on Table 5-3. For most of the tissues the extractio n process recovered at least 80% of metabolites. In the case of skin and fat deposits though, the results were very variable, some of the samples not being extracted efficiently. Enantioselective Metabolism of Methoxychlor In Vitro Demethylation of Methoxychlor The demethylation of MXC was compared between the channel catfish and the largemouth bass. Their resulting chromatograms, (Figure 5-21) were almost identical with the exception that catfish gave more product than the bass. Sin ce the incubation protocol was optimized for the first demethylation step, both fish produced a lot of OHMXC and little HPTE. In both profiles there were several peaks that did not co-migrate w ith available authentic standards and therefore were not identified. Both catfish and bass produced a small amount of the methoxychlor olefin, (1,1-dichl oro-2,2-bis(4-methoxyphenyl)ethene). Human CYP1A2 and CYP2C19 have been report ed to preferentially metabolize MXC to its (R)and (S)-enantiomers respectively in human liver microsomes (Stresser and Kupfer, 1998). In the absence of authentic standards for (R)and (S)-OHMXC, the products of these expressed enzymes were used in the development of a chiral separation me thod to investigate the

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129 enantioselective metabolis m of methoxychlor by fish microsomes. A typi cal chromatogram of the chiral separation of microsomal deme thylation products is shown on Figure 5-22. When standards of OHMXC were separated using the chiral-AGP column, the average ratio of the area under the curve for (R)-OHM XC divided by that of (S)-OHMXC for six different injections was 0.768 0.09 (mean SE). This ratio was not significantly different from that resulting from 3MC-treated catfish (1.0 6 0.15, N = 8) but was different (p = 0.016) from that of bass which averaged 1.49 0.17, N = 4 (Figure 5-23). In Vitro Glucuronidation of OHMXC Consumption of either enantiomer was te sted by allowing a mixture of the two enantiomers to be glucuronidated and then comp aring the average ratio of the area under the curve for (R)-OHMXC divided by that of (S)-OHM XC. None of the catfish treatment groups differed from results obtained by injecting standards which averaged 1.001 0.014, N = 9, or from each other. 3MC averaged 0.958 0.02, N = 6 while control samples had a mean of 0.958 0.01 for four samples. The bass samples however had a significantly high er ratio (1.058 0.02, N = 4) than both the catfish samples (Figure 5-24). Bile Hydrolysis Bile was digested with -glucuronidase both to iden tify metabolites as being glucuronides and also to determine the in vivo ratio of (R)and (S)-enantiomers of OHMXC. Despite the presence of bile salts which were shown to inhibit the hydrolysis of 4methylumbelliferyl-D-glucuronide by up to 75% duri ng a 30 minute incubation (data not shown), 24 hours achieved the complete hydrolysis of bile conjugates to give predominantly HPTE and about 30% OHMXC (Figure 5-25). Chiral analysis showed that the R/S ratio for the treatment groups were not different. The MX C-treated group was 1.05 0.25 (N = 4) and MXCBaP-treated fish was 1.02 0.32 (N = 3).

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130 Lipid Quantitation Of the tissues analyzed, as expected, the fat deposits contained the highest percentage of lipids with 81%. The gonads had about 8% and th e liver had the least amount of lipids at nearly 2%. Distribution of Non-Radiolabeled Meth oxychlor and its Metabolites in Liver In the absence of a radioac tive analyte, UV detection at 254nm was used to enable quantitation of MXC and metabol ites in catfish and largemout h bass samples treated with nonradiolabeled MXC. Since there are a lot of other tissue derive d compounds that are detectable under these conditions, a method to clean up sample s was developed that minimized most of the relatively non-polar as well as polar components that might have been extracted. Authentic standards of metabolites that were expected we re then subjected to the same clean up method and monitored by at 245nm (Figure 5-26). This cl ean-up method was used be fore the analysis of catfish and bass samples analyzed by UV alone. A sample chromatogram is shown on Figure 527 and results are summarized on Table 5-4. In the largemouth bass liver the glucur onide of OHMXC was the major metabolite (Figure 5-28) followed by the parent compound. For the three time points, the total amount of conjugates was always more than the phenolic metabolites and there were no significant differences among the three treatments. There did not seem to be a trend over time for any of the metabolites. Since different methods, gavage and intraperit oneal injections, were used to administer MXC to channel catfish, a comparison of amounts of metabolites in liver for the two treatment groups was done. The data obtained by UV detecti on (Figure 5-29) gave an impossibly large reading for the glucuronide of OHM XC in the catfish. This might be due to the co-elution with an unknown compound extracted from the liver of the catfish. This problem was however not

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131 apparent in bass readings. The metabolite amount s in the gavage group we re generally higher than in those fish in which MXC was administer ed using an intraperit oneal injection. The amounts of HPTE and its glucuronide were significantly different be tween the two groups. Discussion Results presented in this work show that, in all samples analyzed, various combinations and ratios of the parent compound MXC, the phenolic metabolites OHMXC and HPTE and their glucuronide conjugates but no sulf ate conjugates were detected. This is consistent with in vitro assays in which it was seen that the rates of gluc uronidation were higher than those of sulfation. Overall, there was less radioactivity remaining in tissues of MXCBaP-treated fish than in fish treated with MXC alone. This implies that co-e xposure of catfish to BaP and MXC resulted in enhanced elimination of MXC. There have not been studies in published lite rature that have quantitatively analyzed the metabolites of methoxychlor after an in vivo treatment. There are a few reports that document the presence of various phenolic and conjugate d metabolites in mice and insects (Kapoor et al., 1970), chicken and goats (Davison et al., 1982; Da vison et al., 1983; Davison et al., 1984). There are however a few reports of MXC metabolit e quantitation in assays using precision cut liver slices, an in vitro technique that is thought to be more similar to in vivo models than in vitro models such as microsomes. The metabolism of 14C-MXC has been studied using prec ision-cut liver slices from the Sprague Dawley male rat, CD-1 male mouse, WE strain male Japanese quail and juvenile rainbow trout (Ohyama et al., 2004). Major metabolites of the mammals were very different. In rat liver slice preparations, MXC was metabolized to HPTE and its glucuronide. In contrast OHMXC and its glucuronide were the main metabolites for the mouse and Japanese quail. A

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132 dechlorinated OHMXC glucuronide, was observed only in mouse pr eparations. In all cases the phenolic forms of these metabolites were detected only as minor products. In rainbow trout, comparative amounts of the glucuronides of both OHMXC and HPTE were formed as the major metabolites. This is slightly different from the results presented here. Whether treated alone or co-exposed with BaP, the channel catfish had comparable amounts of OHMXC and HPTE; the HPTE glucuronide was mo re than HPTE and the OHMXC glucuronide less than OHMXC. In the channel catfish, bile had the highest concentration of conjugated metabolites while fat deposits had the highest concentration of MX C and OHMXC. The highest amounts of HPTE were seen in the liver and fat deposits. The distribution of MXC and the phenolic metabolites mirror each other in the blood and muscle. Th is might be because these metabolites have reached a partitioning equilibrium between th e two tissues. About 70% of the metabolites detected in the gonads are phenolic and another 10% the parent, MXC. The presence of these potentially toxic compounds in the gonads might ha ve dire consequences for future generations of fish as they are likely to be exposed to thes e compounds at what might be very critical stages of development. A lot of compounds, among them organochlorin e pesticides such as MXC, are lipophilic compounds that have been shown to persist in the environment. Due to their lipid solubility and resistance to metabolism, some of these chemicals are believed to accumulate in tissues, especially the fat deposits of different animals that consume them. Several such pesticides, including MXC, have been quantif ied in human serum as well as adipose tissue (Botella et al., 2004; Carreno et al., 2007). In some cases seru m concentrations were significantly correlated with their adipose tissue concentr ations, while in others no sign ificant relationships were found

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133 between the serum and adipose tissue concentrati ons of the compounds determined. Botella et al. (2004) therefore raised doubts about the equi valent use of fat or serum samples, in epidemiological studies, for the exposure assessme nt of some pesticides. In channel catfish fat deposits there was mainly the parent (75%) t ogether with HPTE (2.4%) and OHMXC (12%). Muscle on average contained HPTE (49.8%), OHM XC (28.04%) and 22.16% of MXC. These results are of particular concern because the hydr oxylated metabolites, that are postulated to be responsible for the estrogenic prop erties of MXC, are found in tissues that are most likely to be present in our diet. In the comparative in vitro metabolism of the phenolic me tabolites of methoxychlor in male and female rats using precision-cut liver slices there was a striking difference between the profiles. While in females the OHMXC-glucuroni de was the major metabolite, there were was no detectable amounts of this metabolite in the male profile (Ohyama et al., 2004). HPTE underwent extensive conjugation producing its gl ucuronide and sulfo-gl ucuronide diconjugate (Ohyama et al., 2005a) but, like in the channe l catfish reported here there were no monosulfated conjugates detected. There was also no sulfo-glucur onide conjugate seen in the in vivo experiment with the channel catfish. The in vitro demethylation of methoxyc hlor by catfish hepatic microsomes produced not only OHMXC and HPTE as expected but also revealed several unknown compounds. By using authentic standards it was possible to identi fy the methoxychlor olef in. The presence of methoxychlor olefin has been reported as a metabolite in in vivo studies involving the salt marsh caterpillar (Kapoor et al., 1970), goats (Davison et al., 1982) and chicken (Davison et al., 1984). There was no evidence of the olefin in the channel catfish in vivo studies presented here. Since both dechlorination and dehydrochlor ination products have been doc umented at least in one case

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134 (Davison et al., 1984), it is reasonable to assume th at the olefin may be produced in one step or via a two step process (Figure 530). Incubation of the olefin in hepatic microsomes of catfish and largemouth bass did not however yield a ny products that co-mig rated with the unknown compounds in chromatograms from catfish or bass 14C-MXC in vitro incubations. Human liver microsomes have been shown to generate primarily the (S)-enantiomer of OHMXC (Hu and Kupfer, 2002a) as is the case w ith rats (Ohyama, 2005) while the Japanese quail and rainbow trout produced more of the (R )-enantiomer. Channel catfish microsomal incubations did not show a change in R/S ra tio when compared to OHMXC standards, if anything largemouth bass produced slightly more (R)than (S)-OHMXC (Figure 5-23). It has been documented that in the channel catfish there is evidence of the metabolism of MXC by more than one P450 (Stuchal et al., 2006). In th e presence of several enzymes in a microsomal incubation it is not altogether surprising, as they all compete for the same substrate, that there is no predominant product. The lack of difference in the R/S ratio in the analysis of bile hydrol ysis products from the two radiolabeled MXC-treated fish groups implies that the induction of CY P1A did not affect the proportion of (R)and (S)enantiomers of OHMXC in MXCBaP-treated cha nnel catfish. Both groups also were not significan tly different from the R/S ratio of injected standards which supports the findings of in vitro studies presented here that po inted towards channel catfish not producing either of the enantiomers of OHMXC predominantly. When the glucuronidation of the enantiomers was monitored, none of the groups were different from the control R/S of injected sta ndards. Catfish however had a slightly smaller ratio than bass, which might mean either of tw o things. The catfish mi ght have preferentially been glucuronidating more of the (R)-enantiomer th an the (S)or the bass using up more of the S

PAGE 135

135 than the R-enantiomer. It would however be diff icult to tease out which of the two scenarios is taking place without carrying out inhibition assays in microsomal incubations and (or) using species specific expressed enzy mes to pinpoint the particular enzymes responsible for the production of either (R)or (S )-OHMXC. When OHMXC was gluc uronidated by human hepatic UDP-glucuronosyl transferases, individual UGTs s howed slightly higher activity towards (S)over (R)-OHMXC, whereas in human liver micros omes different results were observed among individual donors (Hazai et al., 2004 ). The results of some donors showed no differences while in others there was a slight preference for (R)-OHM XC. Such variability, as was seen in channel catfish too, may be attributed to the diverse composition, both of UGT isozymes as well as their quantities, in individuals. The amount of lipids recorded here are similar to some found in litera ture. Gaylord et al. (2001) reported channel catfish liver lipid contents of between 3.8 and 4.5% and those of Robinson and Lovell (1978) were even higher at 6.1 to 7.23%. These differences might be attributed to the use of fingerling catfish in both cases in comparison to our use of adult fish as well as a different lipid extraction method in the case of Robinson and Lovell. Katti and Sathyanesan (1984) report gonad lip id contents of 6 – 10% in the testis and 39 – 47% in the ovary. The lack of a trend in the metabolite amounts in the largemouth bass over time is puzzling. It is reasonable to expect that an animal would pr ogressively metabolize a xenobiotic such as methoxychlor resulting in a redu ced body burden over time. The amount of methoxychlor as a percentage of total metabolite s detected in the liver was 22.2%, 9.3% and 28% after 24, 48 and 72 hrs respectively. While these are not the overall amounts in the body, in this study, the sampled amounts are a snap shot of what could be going on in the body. One

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136 explanation for the unusual trend might be a spor adic release of MXC from the injection site, which could be viewed as a sink. A survey of existing literature alludes to th e fact that intraperit oneal administration of MXC to adult male fish did not seem to result in estrogenic effects, us ing vitellogenin induction as a marker, while exposure via wate r did (Table 5-6). When juvenile fish were used the route of administration and the amount administered di d not matter. The result was the same: no detectable vitellogenin levels. Studies involving adults introdu ced several more variables. Either Western blots or ELISA assays were used to quantify the protein under study, for i.p. administration the vehicles in which the test compound was dissolved were different, and the amounts administered were much larger for i.p. inje ctions than for fish trea ted via water. Despite all the different variables, the i. p. treated fish showed little or no vitellogenin i nduction compared to those treated via water. Even discounting the OHMXC glucuronide data in this study using channel catfish, there were some differences noted between intraperitoneal injections and treatment by gavage. The results presented in this work support the hypothesis th at the route of the administration of MXC affects the distribution of MXC or its metabolites in the catfish. While we administered MXC by gavage and not by exposure through water, bot h methods introduce the xenobiotic via the digestive tract and their results mi ght therefore be expected to be similar. The results from the intraperitoneal studies presented here show significantly less phenolic metabolites than the results from the gavage studies Since phenolic compounds, in pa rticular HPTE, are postulated to cause the estrogenic effects that were being measured, this might be the one reason for the two sets of investigators obtaini ng such different outcomes.

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137 There are more cases in literature where the expected outcome was not observed. When trout liver slices were used, Sc hmieder et al (2004) also no ted that while OHMXC bound to an estrogen receptor it did not indu ce vitellogenin mRNA. Such obs ervations led Thorpe et al., (2001) to conclude that only when the mechanism of action of a chemical is well known can a vitellogenic response be accurately predicted. This again serves as a reminder that in studying teleosts, responses once again cannot complete ly be predicted by mammalian literature. Conclusions The different metabolic profiles of MXC in di fferent animals reported here and in other work are most probably due to the diversity of s ubstrate specificities of contributing P450 in the different animals. In view of all the different metabolic profiles possi ble, methoxychlor-induced toxicity of different animals could be very di fficult to understand. Any animal that produces only trace amounts of HPTE, believed to be mo st estrogenic, would be considered less susceptible than one that produces much more of the compound. The amounts of (R)versus (S)OHMXC would also be an important factor since these enantiomer s seem to induce toxicity to different degrees. The rates of c onjugation with glucuronic acid w ould also be important as this is the main detoxification proce ss for MXC’s estrogenic metabolites. Risk assessment data currently available is based only on MXC, the parent compound. This study brings out the need to assess the risk due to metabolites of persistent compounds too. Channel catfish is not considered a fatty fish w ith reported lipid content in muscle ranging from between 4 and 8% (Seo et al ., 1995; Reigh, 1999; Hedrick et al., 2005). Since we do not know the minimum amounts of OHMXC and HPTE it takes to cause an undesirabl e effect, the results of this study bring into light th e importance of looking at the imp lication of our results for highly consumed fish with higher lipid content in musc le than the channel catfish, such as salmon. Varying amounts of pesticide residues, includ ing MXC were detected in soils and ditch

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138 sediments as well as in ditch water leading to salmon streams in several farms in Canada (Wan et al., 2005). Concern about possible adverse effects caused by the inadvertent exposure of humans and wildlife to endocrine-activ e chemicals, alone or in combinati on, has led to the development of several screening methods for endoc rine effects. I believe that inconsistencies in results will continue to be seen throughout literature depending on the rout e of administration of these compounds, the response that is monitored and the organism being stud ied. The study of methoxychlor highlights the need for risk assess ment of the metabolites of any compound that is bioactivated in vivo.

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139 MXCMXC-BaP 0 250 500 750 1000 1250***treatmentpmol/min/mg Figure 5-1. The EROD activity of MXC and MXCBaP –treat ed groups. *** significantly different from MXC group p < 0.001

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140 Live r Intes t ine Bile Fat Deposits Brai n G o n a d M u scl e Sk i n Blood 0 1 2 MXC MXC-BaP 10 20 A% Dose Live r I n t e s t i n e Bi l e Fa t D e p o si ts Brai n Go n ad M u sc le Sk i n B l o o d 0 25 50 75 MXC MXC-BaP 150 300 450 1000 3000 5000 7000 B* *nmol/g tissue Figure 5-2. Distribution of radioactivity in samples of the ch annel catfish. A) percentage of the administered dose B) nmol/g of sa mple; significantly different from each other p < 0.05

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141 Liver 3.73% Intestinal Mucosa 1.06% Bile 35.63% Fat Deposits 19.07% Brain 0.20% Gonad 0.70% Muscle 33.97% Skin 3.82% Blood 1.80% Figure 5-3. Percent distribution of recovere d radioactivity in different tissues and bile of MXCand MXCBaP-treated channel catfish

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142 Figure 5-4. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the liver of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; A = HPTE glucuronide, B = OHMXC glucuronide, C = HPTE, D = OHMXC, E = MXC A B C D E

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143 HPTEgln OHMXCgln HPTE OHMXC MXC 0 10 20 30MXCBaP MXC **metabolitemetabolite concentration (nmol/g of tissue) Figure 5-5. Concentration of methoxychl or and its metabolites in liver of 14C-methoxychlortreated channel catfish. N = 4 for both MXC and MXCBaP treatment groups; ** Significantly different from each other p < 0.01

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144 Figure 5-6. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the intestinal mucosa of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; A = HPTE glucuronide, B = OHMXC glucuronide, C = HPTE, D = OHMXC, E = MXC D E C B A

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145 HPTEgln OHMXCgln HPTE OHMXC MXC 0 10 20 30MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-7. Concentration of methoxychlor and its metabolites in intestinal mucosa of 14Cmethoxychlor-treated channel catfis h; N = 4 for both MXC and MXCBaP treatment groups

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146 Figure 5-8. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the blood of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; C = HPTE, D = OHMXC, E = MXC, U1 and U2 = unknowns E U1 D U2 C

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147 HPTEgln OHMXCgln HPTE OHMXC MXC 0 1 2 3MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-9. Concentration of methoxyc hlor and its metabolites in blood of 14C-methoxychlortreated channel catfish. N = 4 fo r both MXC and MXCBaP treatment groups

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148 Figure 5-10. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the muscle of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; C = HPTE, D = OHMXC, E = MXC C D E

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149 HPTE OHMXC MXC 0 1 2 3 4 5MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-11. Concentration of methoxychl or and its metabolites in muscle of 14Cmethoxychlor-treated channel catfish. N = 4 for both MXC and MXCBaP treatment groups

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150 Figure 5-12. Normal phase TLC Chromatogram of methoxychlor and its metabolites in fat deposits and gonad of 14C-methoxychlor-treated channel catfish. A: fat deposits B: gonad; developed using a solvent system of 1:1 n-heptane/diethyl ether OHMXC MXC HPTE Origin A B

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151 HPTE OHMXC MXC 0 2 4 6 8 10 12MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-13. Concentration of methoxychl or and its metabolites in gonads of 14Cmethoxychlor-treated channel catfish. N = 3 for both MXC and MXCBaP treatment groups

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152 HPTE OHMXC MXC 0 25 50 100 250 400MXC MXCBaP *metabolitemetabolite concentration (nmol/g of tissue) Figure 5-14. Concentration of methoxychlor and its metabolites in fat deposits of 14Cmethoxychlor-treated channel catfish. N = 3 for MXC treatment group; N = 4 MXCBaP treatment groups; Significantly different from each other p < 0.05

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153 Figure 5-15. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the brain of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; C = HPTE, D = OHMXC, E = MXC, U3 = unknown D C E U3

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154 HPTEgln HPTE OHMXC MXC 0 5 10 15 20 25MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-16. Concentration of methoxychl or and its metabolites in brain of 14C-methoxychlortreated channel catfish. N = 4 fo r both MXC and MXCBaP treatment groups

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155 Figure 5-17. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the skin of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; A = HPTE glucuronide, B = OHMXC glucuronide, E = MXC A B E

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156 HPTEgln OHMXCgln MXC 0.0 2.5 5.0 7.5 10.0MXC MXCBaP metabolitemetabolite concentration (nmol/g of tissue) Figure 5-18. Concentration of methoxychl or and its metabolites in skin of 14C-methoxychlortreated channel catfish. N = 4 fo r both MXC and MXCBaP treatment groups

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157 Figure 5-19. The HPLC chromatogram of th e reverse phase separation of MXC and its metabolites from the bile of 14C-MXC-treated fish. For HPLC conditions see Method #2 in Materials and Methods; A = HPTE glucuronide, B = OHMXC glucuronide A B

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158 HPTEgln OHMXCgln MXC 0 25 50 500 1500 2500 3500 4500MXC MXCBaP *metabolitemetabolite concentration (nmol/g of tissue) Figure 5-20. Concentration of methoxychl or and its metabolites in bile of 14C-methoxychlortreated channel catfish. N = 4 for MXC treatment group. N = 3 MXCBaP treatment groups; Significantly di fferent from each other p < 0.05

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159 Figure 5-21. The HPLC chromatogram of the reverse phase separation of the products of demethylation of 14C-MXC by largemouth bass hepatic microsomes. For HPLC conditions see Method #1 in Materials and Methods; C = HPTE, D = OHMXC, E = MXC, F = MXC Olefin C D E F

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160 Figure 5-22. The HPLC chromatogram of the chiral separation of the products of demethylation of 14C-MXC by channel catfish hepatic microsomes. For HPLC conditions see Method #3 in Materials and Methods; A = HPTE B = (R)OHMXC, C = (S)-OHMXC, D =MXC A B C D

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161 Sta n da rd s C o n tr o l C a t f i s h 3MC Catfish C ontr o l B a s s 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 ab a a b treatmentArea of R over S Figure 5-23. Comparison of enantiomer ar eas (R/S) after the demethylation of 14C-MXC by microsomes. Groups with same letter ar e not significantly different from each other at the 0.05 level

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162 Stan d a r ds C on t rol Catf i sh 3 M C Ca t fi s h Cont r ol B a ss 0.00 0.25 0.50 0.75 1.00 1.25 ab a a b treatmentArea of R over S Figure 5-24. Comparison of enantiomer areas (R/S) after the glucur onidation of OHMXC by microsomes. Groups with same letter ar e not significantly different from each other at the 0.05 level

PAGE 163

163 Figure 5-25. The HPLC chromatogram of the chiral separation of HPTE and OHMXC from the -glucuronidase hyd rolyzed bile of 14C-MXC-treated fish. For HPLC conditions see Method #3 in Materials and Methods; A = HPTE B = (R)OHMXC, C = (S)-OHMXC A B C

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164 Figure 5-26. The HPLC/UV chromatogram of the separation of MXC and its metabolites during method delopment using reverse pha se catridges. Chromatograms: A) Wash: 20% acetonitrile in buffer B) El ute: 90% acetonitrile in buffer C) Confirmation wash: 100% acetonitrile in buffer; For HPLC conditions see Method #2 in Materials and Methods; Metabolites: A = HPTE glucuronide, B = OHMXC glucuronide, C = HPTE, D = OHMXC, E = MXC B A C A B C D E

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165 Figure 5-27. The HPLC/UV chromatogram of th e separation of MXC and its metabolites from the liver of MXC-treated largemouth ba ss. For HPLC conditions see Method #2 in Materials and Methods; B = OHMXC glucuronide E = MXC E B

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166 H PTEg ln OH M XC-gl n HPTE O H M X C M XC 0 600Bass (24hrs) Bass (48hrs) Bass (72hrs) 2,500 6,000 10,000 70,000metabolitemetabolite concentration (pmol/g of tissue) Figure 5-28. Comparison of the amounts of methoxychlor and its metabolites in liver of methoxychlor-treated largemouth bass. Tr eatment with a single dose of 25mg/Kg MXC injected intraperitonea lly then sacrificed afte r 24, 48 and 72 hours; N = 3 for each group;

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167 H PT Egln O HM X C -gln HP T E O HM X C M X C 0 30i.p injection gavage 500 15,250 30,000 100,000 300,000*** ***metabolitemetabolite concentration (pmol/g of tissue) Figure 5-29. Comparison of the amounts of methoxychlor and its metabolites in liver of methoxychlor-treated channel catfish. Tr eated with 2mg/Kg of MXC per day for 6 days administered by intraperitoneal in jection or in diet by gavage; N = 4; *** Significantly different from each other p < 0.001

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168 H OMe MeO Cl Cl Cl H OMe MeO Cl Cl H OMe MeO Cl Cl MXC dechlorinated MXC metabolite MXC Olefin Figure 5-30. Proposed formation of methoxychlor olefin from methoxychlor

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169 Table 5-1. Summary of radioac tive count distribution in subce llular fractions of liver of MXCand MXCBaP-treated channel catfish Treatment Groups MXC MXCBaP Nuclei and cell debris 9.9 1.387.6 1.29 Mitochondria 46.0 2.2245.5 0.59 Cytosol 27.8 1.2230.8 1.25 Microsomes 16.3 1.1816.2 0.46 values are mean percentages SE; N = 4 Table 5-2. The HPLC method and analyte re tention times of MXC and its metabolites Reverse phase (C18) chromatographyChiral chromatography Method #1 Method #2 Method #3 Method #4 HPTEgln 6.4 OHMXCgln 9.2 HPTE 5.113.312.6 (R)-OHMXC 20.3 13.9 (S)-OHMXC 8.517.1 30.4 20.1 MXC 18.320.939.2 MXC Olefin 22.221.8 Retention times given in minutes by UV detection

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170 Table 5-3. Percent of total radioactivity recovered from tissue pellets after metabolite extraction Tissue Percent range Mean SE (N) Liver 7 – 1610.4 1.00 (8) Muscle 7 – 1410.5 0.87 (8) Intestinal mucosa 5 – 2011.4 1.79 (8) Fat deposits 8 – 3924.9 4.58 (7) Gonad 2 – 8 5.5 1.32 (4) Brain 2 – 11 5.1 1.16 (8) Blood 2 – 14 6.6 1.41 (8) Skin 8 – 62 27.0 7.72 (8)

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171 Table 5-4. Methoxychlor and metabolites detected in liver of va riously treated channel catf ish and largemouth bass. Bassa Catfishb Metabolite 24hrs 48hrs 72hrs MXCc 14C-MXCd 14C-MXC/BaPd HPTEgln 118 76.2 28.2 28.2 204 204 2389 704 24657 163612343 1532 OHMXCgln 23440 14567 19002 8262 19017 12403 153826 110449 7241 18423724 1067 HPTE 24.2 24.2 NDND 10.4 10.4 13705 14137954 2262 OHMXC 86.9 36.3 44.8 26.2 154 111 18.4 10.7 10819 43996333 2922 MXC 2724 963 3366 2252 4349 1060 ND1695 1264 1104 962 Values are mean SE (N = 4) in pmol/g of tissue; ND = not detected; a 25mg/Kg MXC injected once peritoneally; b 2mg/Kg given on 6 consecutive days; c non radiolabeled MXC injected peritoneally; d radiolabeled MXC ad ministered by gavage

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172Table 5-5. Summary of meta bolite concentrations from different tissues and bile of 14C-methoxychlor-treated channel catfish Source Treatment HPTE glucuronide OHMXC glucuronide HPTE OHMXC MXC MXC (N = 4) 3379 805 1126 82 NDND14.1 7.04 Bile MXCBaP (N = 3) 2526 1185 645 91 NDND6.95 4.02 MXC (N = 4) 0.76 0.33 0.70 0.101.73 0.292.19 0.451.44 0.37 Blood MXCBaP(N = 4) NDND0.76b1.12b1.85bMXC (N = 4) 0.50aND8.94 1.919.61 3.0317.7 2.40 Brain MXCBaP (N = 4) 1.02aND4.38 0.396.09 1.7115.0 4.01 MXC (N = 3) NDND17.7 3.8443.5 7.89 241 81 Fat Deposits MXCBaP (N = 4) NDND7.81 0.2221.0 4.64 157 24 MXC (N = 3) NDND8.65 1.471.85 1.071.46 0.47 Gonad MXCBaP (N = 3) NDND4.72 0.582.85 0.991.45 1.22 MXC (N = 4) 26.8 2.7710.9 2.234.73 2.025.72 2.831.24 0.86 Intestinal mucosa MXCBaP (N = 4) 17.2 4.957.78 3.814.18 1.045.14 3.042.58 1.07 MXC (N = 4) 24.7 1.647.24 1.8413.7 1.4110.8 4.401.70 1.26 Liver MXCBaP (N = 4) 12.3 1.533.72 1.077.95 2.266.33 2.921.10 0.96 MXC (N = 4) NDND3.56 0.912.05 0.671.63 1.07 Muscle MXCBaP (N = 4) NDND2.53 0.551.62 0.470.67 0.32 MXC (N = 4) 2.85bNDNDND9.48b Skin MXCBaP (N = 4) 2.93b1.14bNDND3.09b Values are mean concentration SE in nmol/g of tissue; a detected in only one sample; b from pooled sample

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173Table 5-6. Comparison of the effect of route of methoxychlor administrati on on levels of vitellogenin in fish Fish Route Treatment Vitellogenin response Sheepshead minnows (Cyprinodon variegatus) Adult male (n=32) (Hemmer et al., 2001) Via water Dose: 0, 1.1, 2.5, 5.6, 12.1, and 18.4 g/L MXC Exposure: 2, 5, 13, 21, 35, and 42 days Quantitation: ELISA rapid, dose-dependent increase in hepatic vitellogenin mRNA up to day 5 of exposure then relatively constant dose-dependent expression from 5 to 42 days of exposure dose-dependent increase in plasma vitellogenin over the entire time course of exposure, Channel catfish (Ictalurus punctatus) 2 year old male (n = 5-7) (Nimrod and Benson, 1997) Injected intraperitoneally Dose: 95mg/Kg MXC; Vehicle control 2.5mL/Kg 0.25% agar Exposure: days 1, 4, and 7 then killed on day 10 Quantitation: ELISA Vitellogenin induction not significantly different from agar control zebrafish (Danio rerio) Adults approximately 10 months old (n=5) (Versonnen et al., 2004) Via water Dose:0, 0.5, 5, and 50g MXC/L Solvent control (0.01% ethanol) Exposure: 14 days. Quantitation: Western Blots Vitellogenin induction dete cted at 5 and 50 g MXC/L. zebrafish (Danio rerio) juveniles 4 week old (n=2) (Versonnen et al., 2004) Via water Dose: 0, 0.05, 0.5, and 5g MXC/L Solvent control (0.01% ethanol) Exposure: 14 and 33 days. Quantitation: Western Blots No vitellogenin induction detected

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174Table 5-6. continued Fish Route Treatment Vitellogenin response Rainbow trout Juveniles (n=6) (Andersen et al., 1999) Injected intraperitoneally Dose: 100mg/Kg MXC Control (pre-exposure blood) Exposure: single injection then killed on day 9. Quantitation: ELISA No detectable serum vitellogenin Channel catfish (Ictalurus punctatus) Adults (n=2-3) (Schlenk et al., 1998) Injected intraperitoneally Dose: 100mg/Kg OHMXC 250mg/Kg MXC Exposure: Days 1 and 3 then killed on day 6 Quantitation: ELISA 100-fold vitellogenin induction (OHMXC) Slight vitellogeni n induction (MXC)

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188 BIOGRAPHICAL SKETCH Beatrice Adhiambo Nyagode was born on April 9, 1971 in Kisumu, a beautiful city on the shores of Lake Victoria, in Kenya. She a ttended Egerton University and in 1994 graduated with a Bachelor of Science ( honors) degree in Chemistry and Z oology. In 1998, she received a Master of Science degree in Chemistry from th e same institution. While conducting her thesis research in 1996, she joined the International Center for Insect Physio logy and Ecology (ICIPE) in Nairobi, Kenya, as a volunteer intern. She stayed on and worked at ICIPE for several years conducting research in various capacities. This afforded her opportunitie s to travel and work with researchers from different parts of the worl d. In early 2002, she represented ICIPE at the “Gardening for Food around the World” festival at EPCOT in Walt Disney World, Orlando FL. In August 2002, she joined the Department of Me dicinal Chemistry as a doctoral student. She is passionate about the chemical fate of xenobiotics and plans to pursue a career in metabolism and toxicology upon completion of her program.