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Comparative study of cytochromes P450 in three avian species: induction of cytochrome P450-dependent… Li, Hong 1994

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COMPARATIVE STUDY OF CYTOCHROMES P450 IN ThREE AVIANSPECIES: INDUCTION OF CYTOCHROME P450-DEPENDENTMONOOXYGENASE ACTIVITIES AND IMMUNOCHEMICALRELATEDNESS OF THE AVIAN PROTEINS TO RAT CYTOCHROMES P450byHONG LIB.Sc., Xiamen University, Xiamen, Fujian, P.R. China, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENT FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESFACULTY OF PHARMACEUTICAL SCIENCESDivision of Pharmaceutical ChemistryWe accept this thesis as conformingthe required standardTHE UNIVERSITY OF BRITISH COLUMBIAJune 1994© Hong Li, 1994In presenting this thesis in partial fulfilment of the requn-ements for an advanceddegree at the University of British Columbia, I agree that the library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of________________The University of British ColumbiaVancouver, CanadaDate_______DE-6 (2188)ABSTRACTThe overall goal of the present study was to investigate the effects of threeprototype inducers, phenobarbital (PB), 3-methyicholanthrene (MC) and Aroclor 1254(AR), on hepatic cytochromes P450 in three avian species by means of enzymatic assays,immunoblot analysis and immunoinhibition of enzymatic activities. Accordingly, femalechickens, female quail as well as male and female pigeons were treated with corn oil (2mi/kg body weight i.p once daily for 3 to 4 days), PB (80 mg/kg body weight, i.p., oncedaily for 3 to 4 days), MC (25 mg/kg body weight, i.p., once daily for 3 to 4 days) or AR(300 mg/kg body weight, one i.p. injection). Male Long Evans rats receiving similartreatments were included for the purpose of comparison.Pooled hepatic microsomes were prepared from control and treated animals. Totalhepatic cytochrome P450 content and four cytochrome P450-dependent monooxygenaseactivities, namely 7-ethoxyresorufin 0-deethylase (EROD), benzo[a]pyrene hydroxylase(AHH), 7-pentoxyresorufin 0-depentylase (PROD) and benzphetamine N-demethylase(BPND), were examined. Treatment with PB, MC or AR resulted in 2.2-, 1.5- and 3.6-fold induction of total cytochrome P450 content, respectively, in the rats. It was foundthat total hepatic cytochrome P450 contents in the three avian species were increased 1.3-to 4-fold by PB, approximately 5- to 6-fold by MC, and 2- to 4-fold by AR. In rats,treatment with PB or AR increased EROD, AHH, PROD and BPND activities (PB: 3.6-,2.1-, 101- and 3.1-fold, respectively; AR: 74-, 4.3-, 42- and 3.0-fold, respectively),while treatment with MC induced EROD, AHH and PROD activities (56-, 6.9- and 1.2-fold, respectively) but suppressed BPND activity (33%). In the three avian species,treatment with PB appeared to increase AHH (1.3- to 3.9-fold) and BPND activities (1.3-to 4.3-fold) but suppress EROD (22% in chickens and 57% in quail) and PROD (24% to75%), treatment with MC induced EROD, AHH, PROD and BPND activities (72-, 13-,16- and 3.6-fold, respectively, in chickens; 40-, 14-, 13- and 8.3-fold, respectively, in11quail; 1.2- to 5.7-fold in pigeons), and treatment with AR also increased EROD, AHH,PROD and BPND activities (30-, 4.7-, 7.6- and 1.8-fold, respectively, in chickens; 36-,7.0-, 26- and 7.7-fold, respectively, in quail; 1.3- to 5.7-fold in pigeons except for a 29%decrease in AHH). Flepatic cytochromes P450 are inducible in the avian species by PB,MC and AR. Species differences exist in terms of the inducibility of cytochrome P450-dependent monooxygenase activity. Slight decreases in EROD activity and moderateinduction of AHH activity in the birds treated with PB imply that hepatic EROD and AHHactivities may be mediated by different forms of cytochrome P450. Similarly, PB-treatment suppressed hepatic PROD activity and induced BPND activity, suggesting thathepatic PROD and BPND activities should be mediated by different forms of cytochromeP450 in PB-treated birds.Immunoblot analysis was undertaken to determine the presence of different formsof hepatic cytochrome P450 in the three avian species and to compare theimmunochemical relatedness of avian and rat cytochromes P450. Immunoblots containinghepatic microsomes from control and treated birds were probed with antibodies generatedagainst several purified rat cytochrome P450 enzymes. Analysis of the immunoblotsdemonstrates that (1) the three avian species contain hepatic cytochromes P450 that areimmunorelated to rat CYP1A1 and are inducible by MC and AR, (2) chickens have acytochrome P450 that is immunorelated to rat CYP1A2 and is also inducible by MC andAR, (3) weak cross-reactions between polyclonal anti-rat CYP2B1 IgG and the avianmicrosomes indicate the presence of avian cytochromes P450 that are weaklyimmunologically related to rat CYP2B isozymes, (4) avian cytochromes P450immunorelated to CYP2C 11 are present in both male and female chickens and quail butnot pigeons, (5) these avian species contain hepatic cytochromes P450 that areimmunorelated to other rat CYP2C isozymes (but not to CYP2C7 and CYP2C13), and (5)the three avian species contain hepatic cytochromes P450 immunorelated to rat CYP3A2but not CYP3A1 and they are inducible by PB.111Immunoinhibition of EROD, PROD and BPND activities were conducted in anattempt to find out which forms of avian cytochrome P450 contribute to thesemonooxygenase activities. It was found that avian cytochrome P450 enzymesimmunorelated to rat CYP1A1 mediate not only hepatic EROD activity but also PRODactivity in the MC- or AR-treated birds. Avian cytochrome P450 enzymes that areimmunorelated to rat CYP2B1, CYP2C11 or CYP3A2 contribute to PROD and BPNDactivity to varying extents.The present study provides enzymatic and immunological evidence to demonstratethat the three avian species contain many forms of hepatic cytochrome P450 and treatmentof the birds with PB, MC or AR results in either induction or reduction in expression ofavian cytochromes P450 and their catalytic activities.Stelvio Bandiera, Ph.D.Assistant ProfessorFaculty of Pharmaceutical SciencesThesis SupervisorivTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS VLIST OF TABLES ixLIST OF FIGURES xiLIST OF ABBREVIATIONS xivACKNOWLEDGEMENTS xvii1. INTRODUCTION 11.1. Background and History 11.2. Nomenclature of Cytochrome P450 Genes and Enzymes 51.2.1. Naming Cytochrome P450 Genes and Enzymes 71.2.2. The “40% Rule” 71.3. The Cytochrome P450 Superfamily 81.3.1. The CYP1A Subfamily 81.3.2. The CYP2B Subfamily 121.3.3. The CYP2C Subfamily 151.3.4. The CYP3A Subfamily 201.3.5. Other Cytochrome P450 Subfamily and Inducers 231.3.5.1. CYP2A Subfamily 231.3.5.2. CYP2E Subfamily 241.3.5.3. Mixed-Type Inducers 251.4. Review on Avian Cytochrome P450 Enzymes 251.4.1. Purified Avian Cytochrome P450 281.4.1.1. Purified avian cytochromes P450 induced by MC-type inducers 281.4.1.2. Purified avian cytochromes P450 induced by PB-type inducers 291.4.1.3. Purified avian cytochromes P450 induced byethanol-type inducers 301.4.1.4. Avian CYP2H subfamily 301.4.1.5. Avian CYP19 subfamily 311.4.2. Induction of Cytochromes P450 by MC, PB and AR in AvianSpecies 311.4.2.1. MC-Type induction 321.4.2.2. PB-Type induction 341.4.2.3. PCN-Type induction 361.4.2.4. Ethanol-Type induction 361.4.2.5. Mixed-Type induction 371.5. Purpose of the Present Investigation 392. EXPERIMENTAL 402.1. Chemicals 402.2. Animals 43V2.2.1. Untreated rats and birds 442.2.2. Treatment of rats and birds with corn oil 442.2.3. Treatment of rats and birds with phenobarbital (PB) 452.2.4. Treatment of rats and birds with 3-methyicholanthrene (MC) 462.2.5. Treatment of rats and birds with Aroclor 1254 (MC) 462.3. Microsome Preparation 482.4. Determination of Microsomal Cytochrome P450 Concentration 482.5. Determination of Microsomal Protein Concentration 492.6. Enzymatic Assays 492.6.1. 7-Ethoxyresorufin 0-Deethylase (EROD) Assay 492.6.2. 7-Pentoxyresorufin 0-Depentylase (PROD) Assay 502.6.3. Aryl Hydrocarbon Hydroxylase (AHH) Assay 512.6.4. Benzphetamine N-Demethylase (BPND) Assay 532.6.5. Antibody Inhibition Study of the Four Enzymatic Assays 542.7. SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Assay 542.7.1. Electrophoresis 542.7.2. Immunoblots 553. RESULTS 573.1. Total Hepatic Cytochrome P450 Content 573.2. Cytochrome P450-Dependent Monooxygenase Activities 593.2. 1. The Preliminary Study---Optimizing Assay Conditions 603.2.1.1. 7-Ethoxyresorufin 0-Deethylase (EROD) Assay 603.2.1. 1.1. Resorufin formation versus reaction time.. 603.2.1.1.2. Rate of 7-ethoxyresorufin 0-deethylationversus microsomal protein concentrations 613.2.1.1.3. EROD activity versus 7-ethoxyresorufinconcentration 623.2.1.1.4. EROD activity versus NADPHconcentration 623.2.1.1.5. Summary of EROD assay conditions 673.2.1.2. Aryl Hydrocarbon Hydroxylase (AHH) Assay 673.2.1.2.1. Formation of 3-hydroxy-benzo[a]pyreneversus reaction time 673.2.1.2.2. Rate of 3-OH-BaP formation versusmicrosomal protein concentrations 673.2.1.2.3. AHH activity versus benzo[a]pyreneconcentration 683.2.1.2.4. AHH activity versus NADPHconcentration 683.2.1.2.5. Summary of AHH assay conditions 683.2.1.3. 7-Pentoxyresorufin 0-Depentylase (PROD) Assay . 733.2.1.3.1. Resorufin formation versus reaction time.. 733.2.1.3.2. Rate of pentoxyresorufin 0-depentylationversus microsomal protein concentrations 74vi3.2.1.3.3. PROD activity versus pentoxyresorufinconcentration 743.2.1.3.4. PROD activity versus NADPHconcentration 743.2.1.3.5. Summary of PROD assay conditions 753.2.1.4. Benzphetamine N-Demethylase (BPND) Assay 803.2.1.4.1. Formaldehyde formation versus reactiontime 803.2.1.4.2. Rate of benzphetamine N-demethylationversus microsomal protein concentrations 803.2.1.4.3. BPND activity versus benzphetamineconcentration 813.2.1.2.4. BPND activity versus NADPHconcentration 813.2.1.3.5. Summary of BPND assay conditions 813.2.2. Measurement of Monooxygenase Activities of Rat and AvianHepatic Microsomes 863.2.2.1. 7-Ethoxyresorufin 0-Deethylase Activity (EROD) 863.2.2.2. Aryl Hydrocarbon Hydroxylase Activity (AHH) 883.2.2.3. 7-Pentoxyresorufin O-Depentylase Activity(PROD) 893.2.2.4. Benzphetamine N-Demethylase Activity (BPND) ... 913.3. SDS-Polyacrylamide Gel Electrophoresis and Immunoblots 933.3. 1. SDS-Polyacrylamide Gel and Immunoblots of Rat HepaticMicrosomes 933.3.2. SDS-Polyacrylamide Gel of Avian Hepatic Microsomes 963.3.2.1. Induced Protein Bands in Pigeon Microsomes 963.3.2.2. Induced Protein Bands in Quail Microsomes 973.3.2.3. Induced Protein Bands in Chicken Microsomes 973.3.3. Immunoblot Analysis of Avian Cytochromes P450 1003.3.3.1. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP1A1 IgG 1003.3.3.2. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP2B1 IgG 1033.3.3.3. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP2C7 IgG 1053.3.3.4. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP2C11 IgG 1073.3.3.5. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP2C13 IgG 1103.3.3.6. Immunoblot of Avian Hepatic Microsomes Probedwith Anti-Rat CYP3A1 IgG 1123.4. Antibody Inhibition of Monooxygenase Activities 1153.4.1. Antibody Inhibition of Hepatic EROD Activities 1153.4.2. Antibody Inhibition of Hepatic PROD Activities 117vii3.4.3. Antibody Inhibition of Hepatic BPND Activities 1194. DISCUSSION 1244.1. Comparison of the Effects of PB, MC and AR on Total HepaticCytochrome P450 Content 1244.2. Comparison of the Effects of PB, MC and AR on Rat and AvianHepatic Microsomal Monooxygenase Activities 1264.2.1. EROD Activities 1264.2.2. AHH Activities 1284.2.3. PROD Activities 1304.2.4. BPND Activities 1314.2.5. Summary of the Effects of PB, MC and AR on Avian HepaticMicrosomal Monooxygenase Activities 1334.3. Immunochemical Relatedness between Avian Cytochrome P450 andRat Cytochrome P450 1344.3.1. Avian Cytochrome P450 Immunochemically Related toCYP1A1/2 1344.3.2. Avian Cytochrome P450 Immunochemically Related toCYP2B1 1354.3.3. Avian Cytochrome P450 Immunochemically Related toCYP2C7 1364.3.4. Avian Cytochrome P450 Immunochemically Related toCYP2C11 1364.3.5. Avian Cytochrome P450 Immunochemically Related toCYP2C Members Other Than CYP2C13 1384.3.6. Avian Cytochrome P450 Immunochemically Related toCYP3A 1394.3.7. Summary of the Immunochemical Relatedness of Avian andRat Cytochromes P450 1404.4. Immunoinhibition of Avian Cytochrome P450-DependentMonooxygenase Activities 1414.4.1. EROD Activity and Avian Cytochromes P450Immunorelated to Rat CYP1A1 1414.4.2. PROD Activity and Avian Cytochrome P450 Enzymes 1424.4.3. BPND Activity and Avian Cytochrome P450 Enzymes 1434.4.4. Summary of Immunoinhibition of Avian Cytochrome P450-Dependent Monooxygenase Activities 1445. CONCLUSIONS 1456. REFERENCES 146APPENDIX I 173APPENDIX II 174vi”LIST OF TABLESTable 1.1. Nomenclature of selected cytochrome P450 genes 6Table 1.2. Properties of the CYP1A subfamily 12Table 1.3. Properties of the CYP2B subfamily 15Table 1.4. Properties of the CYP2C subfamily 18Table 1.5. Properties of the CYP3A subfamily 23Table 2.1. Untreated group of rats and birds 44Table 2.2. Treatment of rats and birds with corn oil 45Table 2.3. Treatment of rats and birds with PB 46Table 2.4. Treatment of rats and birds with MC 47Table 2.5. Treatment of rats and birds with AR 47Table 3. 1. Total cytochrome P450 content of hepatic microsomes prepared fromrats and three bird species 59Table 3.2. 7-Ethoxyresorufin 0-deethylase activities (nmole/mg protein minute) ofhepatic microsomes prepared from rats and three avian species 87Table 3.3. Aryl hydrocarbon hydroxylase activities (nmole/mg protein.minute) ofhepatic microsomes prepared from rats and three avian species 89Table 3.4. 7-Pentoxyresorufin 0-depentylase activities (nmole/mg protein minute)of hepatic microsomes prepared from rats and three avian species 90Table 3.5. Benzphetamine N-demethylase activities (nmole/mg protein minute) ofhepatic microsomes prepared from rats and three avian species 92Table 3.6. Apparent molecular weight (MW) and relative mobility (Rf) on a SDSpolyacrylamide gel of microsomal proteins induced by phenobarbital (PB), 993-methylcholanthrene (MC) and Aroclor (AR) in the three avian species.Table 3.7. Inhibition of 7-ethoxyresorufin 0-deethylase activity (EROD) inmicrosomes from MC-treated rats and birds by monoclonal antibody (MAb)and polyclonal antibody (PAb) to rat CYP1A 116ixTable 3.8. Inhibition of 7-pentoxyresorufin 0-depentylase activities (PROD) inhepatic microsomes from PB-treated rats, MC-treated chickens and MC-treated quail by antibodies against various forms of rat cytochrome P450. .. 118Table 3.9. Inhibition of benzphetamine N-demethylase activity (BPND) in hepaticmicrosomes from PB-treated and AR-treated rats by antibodies againstvarious forms of rat cytochrome P450 120Table 3. 10. Inhibition of benzphetamine N-demethylase activity (BPND) in hepaticmicrosomes from PB-treated, MC-treated and AR-treated chickens byantibodies against various forms of rat cytochrome P450. .. 121Table 3.11. Inhibition of benzphetamine N-demethylase activity (BPND) in hepaticmicrosomes from quail treated with PB, MC and AR by antibodies againstvarious forms of rat cytochrome P450 122Table 3.12. Inhibition of benzphetamine N-demethylase activity (BPND) in hepaticmicrosomes from MC-treated and AR-treated pigeons by antibodies againstvarious forms of rat cytochrome P450 123xLIST OF FIGURESFigure 1.1. Scheme for the reation cycle of cytochrome P450 2Figure 1.2. Scheme of the Ah-receptor-mediated regulation of the 5’ flankingregion of the CYPJAJ gene 10Figure 3.1. The effect of reaction time on resorufin formation from 7-ethoxyresorufin 63Figure 3.2. The effect of microsomal protein concentration on the rate ofresorufin formation from 7-ethoxyresorufin 64Figure 3.3. The effect of 7-ethoxyresorufin concentration on 7-ethoxyresorufin 0-deethylase activity 65Figure 3.4. The effect of NADPH concentration on 7-ethoxyresorufin 0-deethylase activity 66Figure 3.5. The effect of reaction time on the formation of 3-hydroxy-benzo[a]pyrene from benzo[ajpyrene 69Figure 3.6. The effect of microsomal protein concentration on the rate of 3-hydroxy-benzo[a]pyrene formation from benzo{a]pyrene 70Figure 3.7. The effect of benzo[ajpyrene concentration on benzo[a]pyrenehydroxylase activity 71Figure 3.8. The effect of NADPH concentration on benzo[a]pyrenehydroxylase activity 72Figure 3.9. The effect of reaction time on resorufin formation from 7-pentoxyresorufin 76Figure 3.10. The effect of microsomal protein concentration on the rate ofresorufin formation from 7-pentoxyresorufin 77Figure 3.11. The effect of 7-pentoxyresorufin concentration on 7-pentoxyresorufin 0-depentylase activity 78Figure 3.12. The effect of NADPH concentration on 7-pentoxyresorufin 0-depentylase activity 79xiFigure 3.13. The effect of reaction time on formaldehyde formation frombenzphetamine 82Figure 3.14. The effect of microsomal protein concentration on the rate offormaldehyde formation from benzphetamine 83Figure 3.15. The effect of benzphetamine concentration on benzphetamine Ndemethylase activity 84Figure 3. 16. The effect of NADPH concentration on benzphetamine Ndemethylase activity 85Figure 3.17. SDS-polyacrylamide gel and immunoblots of hepaticmicrosomes from male untreated, PB-treated and MC-treated ratsprobed with monoclonal antibody against rat CYP1A1 and withpolyclonal antibody against rat CYP2B1 94Figure 3.18. Coomassie blue stained SDS-polyacrylamide electrophoresis gelof hepatic microsomes from the three avian species 98Figure 3.19. Immunoblot of avian hepatic microsomes probed withmonoclonal antibody against rat CYP1A1 102Figure 3.20. Immunoblot of avian hepatic microsomes probed with polyclonalanti-rat CYP1A1 sera 102Figure 3.21. Immunoblot of avian hepatic microsomes probed withpolyspecifc antibody against rat CYP2B1 104Figure 3.22. Immunoblot of avian hepatic microsomes probed withmonoclonal antibody against rat CYP2B1/2 104Figure 3.23. Immunoblot of avian hepatic microsomes probed withpolyspecifc antibody against rat CYP2C7 106Figure 3.24. Immunoblot of avian hepatic microsomes probed withmonospecifc antibody against rat CYP2C7 106Figure 3.25. Immunoblot of avian hepatic microsomes probed withpolyspecifc antibody against rat CYP2C11 108Figure 3.26. Immunoblot of avian hepatic microsomes probed withmonospecifc antibody against rat CYP2C 11 108xiiFigure 3.27. Immunoblot of avian hepatic microsomes probed withpolyspecifc antibody against rat CYP2C13 illFigure 3.28. Immunoblot of avian hepatic microsomes probed withmonospecifc antibody against rat CYP2C13 illFigure 3.29. Immunoblot of avian hepatic microsomes probed withpolyspecifc antibody against rat CYP3A1 114Figure 3.30. Immunoblot of avian hepatic microsomes probed withmonospecifc antibody against rat CYP3A1 114Scheme 1. Illustration of 7-ethoxyresorufin 0-deethylation 49Scheme 2. Illustration of 7-pentoxyresorufin 0-deethylation 50Scheme 3. Enzymatic conversion of BaP to hydroxylated metabolites 51xli’LIST OF ABBREVIATION2-AAF 2-acetylaminofluorene3-OH-BaP 3-hydroxy-benzo[a]pyreneAh aryl hydrocarbonAHH benzo[a]pyrene hydroxylaseAlA 2-allyl-2-isopropylacetamideALAS ö-aminolevulinic acid sythetaseBaP benzo[a]pyrenef3-naphthoflavone I3NFBP-5 PCB mixture containing 50% chlorineBPND benzphetamine N-demethylaseCYP cytochrome P450[CYP2C1 1] avian cytochrome P450 that is immunorelatedto rat CYP2C11[CYP3A] avian cytochrome P450 that is immunorelatedto rat CYP3A subfamilyDDT dichioro-diphenyl-trichioroethaneDEX dexamethasoneDMSO dimethyl sulfoxideDREs dioxin responsive elementsEDTA ethylenediaminetetraacetic acidEROD 7-ethoxyresorufin 0-deethylaseg gramHCHO formaldehydeHC1 hydrochloric acidhr(s) hour(s)xivi. p. intraperitoneal injectionKC1 potassium chloridekD kilodaltonkg kilogramMAb monoclonal antibodyMC 3-methyicholanthrenemg milligrammill minuteml millilitremmole millimoleNADPH nicotinamide adenine dinucleotide phosphatetetra-sodiumNaOH sodium hydroxidenm nanometernmole nanomolePAb polyclonal IgGPAH(s) polycyclic aramatic hydrocarbon(s)PB (Pb) phenobarbitalPBBs polybrominated biphenylsPCB(s) polychiorinated biphenylsPCN pregnenolone- 16c&carbonitrilePenCB 3,31,4t ,41 ,5-pentachlorobiphenylpmole picomolepp’-DDE p,p’ -dichioro-diphenyl-trichioroethanePROD 7-pentoxyresorufin 0-depentylaseRf relative mobilitySDS-PAGE sodium dodecyl sulphatexvTAO triacetyloleandomycinTCB 3,3’ ,4,4’ ‘-tetrachiorobiphenylTCDD 2,3,7, 8-tetrachlorodibenzo-p-dioxinTCDF 2,3,7, 8-tetrachlorodibenzofuranTEMED N, N, N’ ,N’ -tetramethyethylenediaminetg microgrammicrolitreUt untreatedXREs xenobiotic responsive elementsEnzyme induction refers to an increased rate of synthesis of anenzyme, rather than activation of enzymeactivity.xviACKNOWLEDGEMENTSThis thesis is dedicated to my parents, my sister and friends, who have alwaysbeen with me no matter where I am.I would like to thank my supervisor, Dr. S. Bandiera, and my committeemembers, Dr. F. Abbott, Dr. G. Beliward, and Dr. R. Reid. I would also like to thankMs. Caroline Dworschak and Dr. P. Thomas for providing purified rat cytochromes P450and antibodies; and Dr. L. Hart, Dr. K. Cheng, and Ms. C. Nichols for providing thebirds and assisting in treatment of birds. I am thankful to the Faculty for giving me theopportunity to study in Canada.xvii11. INTRODUCTIONThe microsomal cytochrome P450-dependent monooxygenase system has beenthe subject of intense study for more than 30 years. This enzyme system is found inhumans, other mammals, birds, reptiles, amphibians, fish, plants and bacteria.Cytochrome P450 has been extensively characterized in humans as well as in laboratorymammals, especially rats and mice. It functions as a phase I drug metabolizing enzymeand plays an important role in helping the organism cope with the myriad foreignchemicals that are ingested and absorbed daily. The chemicals include pesticides,carcinogens, therapeutic agents and industrial chemicals. Cytochrome P450 alsometabolizes numerous endogenous compounds including certain steroids, fatty acids,vitamins, bile acids, leukotrienes and thromboxanes (Yang and Lu, 1987). The presentinvestigation on cytochrome P450 focuses on three avian species: pigeons, quail, andchickens. By way of background, I will present a brief review of cytochrome P450-mediated monooxygenases in the rat and other mammals and then summarize theinformation pertaining to avian cytochromes P450 reported in the literature.1.1. BACKGROUND AND HISTORYThe presence of a carbon monoxide-binding pigment with an absorption peak atapproximately 450 nm in liver microsomes was first reported in 1958 (Garfinkel, 1958;Klingenberg, 1958). The pigment was subsequently named cytochrome P450 and wasshown to be a hemeprotein by Omura and Sato in 1964. After the first successfulseparation and solubilization of this enzyme in 1968 by Lu et al., cytochrome P450 waspurified in several laboratories and was found to exist as multiple forms. Eachcytochrome P450 form or isozyme exhibits a unique profile of substrate specificity, butmany cytochrome P450 isozymes particitpate in the metabolism of common substrateswith varying degrees of efficiency, showing broad and overlapping substrateiNTRODUCTION /2specificities (Welton and Aust, 1974; Haugen et al., 1975; Thomas ci at., 1976).H20H20Figure 1.1. Scheme for the reaction cycle of cytochrome P450. Fe represents the hemeiron atom at the active site, RH the substrate, RH(H2) a reduction product, ROH amonooxygenation product and XOOH a peroxy compound that can serve as analternative oxygen donor. (From Porter and Coon, 1991)The microsomal cytochrome P450-dependent monooxygenase system is localizedin the membrane of the endoplasmic reticulum and consists of a family of cytochromeP450 enzymes, NADPH-cytochrome P450 reductase and a lipid identified asphosphatidylcholine (Lu et at., 1969; Strobel et al, 1970). This enzyme system worksto transport electrons from nicotinamide adenine dinucleotide phosphate (NADPH) byway of a flavoprotein oxidoreductase, either indirectly through cytochrome b5 ordirectly to cytochrome P450 as the terminal oxidase (Estabrook et at., 1963; Cooper etRHH20e,2H02(RF1240)2I1+jeiNTRODUCTION /3a!., 1965). Cytochrome P450 functions as the binding site for substrate and oxygenmolecules, forming an oxygen-enzyme-substrate trimolecular complex (Remmer et at.,1966). Within the complex, the oxygen molecule is activated by receiving twoelectrons from the heme moiety and the chemical bonds in the bound substrate are thensplit for the insertion of activated oxygen (Ruckpaul et at., 1989) (Figure 1.1). As aconsequence of the oxygenation, lipophilic substrates are converted to hydrophilicmetabolites that are more easily excreted. In most cases these oxygenation reactionslead to detoxication, but for some substrates, catalysis by cytochrome P450 producesactivated carcinogenic metabolites that are more toxic than the parent compounds andbind covalently to macromolecules such as DNA (Anders et at., 1985; Nebert andGonzalez, 1987).A critical property of this hemeprotein family is that some enzymes are able tobe induced by chemicals called cytochrome P450 inducers. The discovery ofcytochrome P450 induction by drugs and other xenobiotics was initiated by the findingthat chronic administration of phenobarbital to rats resulted in a gradual reduction of thesedative effect via enhancement of barbiturate metabolism and clearance (Remmer,1962). Increased phenobarbital metabolism and clearance were later found to be causedby an increase in the amount of hepatic cytochrome P450 (Orrenius et al., 1965;Remmer and Merker, 1963). The induction of cytochrome P450 by xenobiotics is oftenmanifested as an elevation in total cytochrome P450 content and associatedmonooxygenase activities due to increases in the levels of specific cytochrome P450isozymes. However, in some cases, the total cytochrome P450 content may not beincreased because some enzymes are induced while others are suppressed (Ryan andLevin, 1990). The induction of cytochromes P450 requires de novo protein synthesisand is regulated at the level of transcription and posttranscription (Conney et at., 1957;Gelboin and Blackburn, 1964; Alvares et at., 1967; Kuntzman et at., 1969).Cytochrome P450 inducers are generally classified into several categories according toINTRODUCTION /4the pattern of individual forms of cytochrome P450 affected. The categories includephenobarbital-type (PB-type), 3-methylcholanthrene-type (MC-type), ethanol-type,clofibrate-type and pregnenolone 1 6c-carbonitrile-type (PCN-type) inducers (Conney,1967; Guzelian, 1980 and 1988; Koop et a!., 1982; Peng et al., 1982; Remmer andMerker, 1963; Safe et al., 1985; Selye, 1971; Whitlock, 1986).In contrast to induction, some cytochrome P450 enzymes are expressed at arelatively high level in untreated animals. They are called constitutive cytochromesP450. Most of the constitutive enzymes are refratory to all known inducers althoughsome are moderately inducible (Gonzalez, 1989; Ryan and Levin, 1990). Theexpression and hepatic composition of cytochromes P450 are dependent not only uponinduction by enviromental components (e.g. therapeutic agents, pollutants, or diet) butalso upon genetic and physiological factors such as age, gender, diet and disease(Bellward et al., 1988; Gonzalez, 1989). The constitutively expressed cytochromeP450 enzymes are often developmentally and sexually regulated.Cytochromes P450 enzymes are differentially expressed between liver andextrahepatic tissues such as brain, lung, heart, kidney, adrenal, spleen, ovary, testis andsmall intestine (Christous et at., 1987; Keith et al., 1987; Wilson et al., 1987).Isolation and identification of cytochromes P450 are complicated because of theirmultiplicity, particularly with those forms that are present in relatively low amounts inliver and extrahepatic tissues. Using biochemical and immunological approaches suchas protein purification, gel electrophoresis, amino-terminal sequence analysis andimmunoblots, more than twenty forms of rat liver cytochrome P450 have been purifiedand characterized (Ryan and Levin, 1990). During the 1980s, molecular biologyapproaches were introduced into the cytochrome P450 field. With the advent ofmolecular cloning and recombinant DNA technologies, 30 distinct human cytochromeP450 genes and 40 distinct rat cytochrome P450 genes have been identified. Eachcytochrome P450 gene almost always produces a single protein (Nebert et at., 1993).INTRODUCTiON /5To date, there appear to be only a few exceptions to this rule. The exceptions involvedifferential processing of the cytochrome P450 transcript in which entire translatedexons or portions of exons are exchanged to produce a cytochrome P450 enzyme with anew catalytic activity (Lephart et al., 1990; and Miles et a!., 1990) or tissue-specificexpression (Means et al., 1991).1.2. NOMENCLATURE OF CYTOCHROME P450 GENES AND ENZYMESPurification of cytochromes P450 has been the focus in many researchlaboratories, and unfortunately, every laboratory has developed its own system ofcytochrome P450 nomenclature. Different nomenclatures inevitably cause confusionwhen results from different labs are compared. On the basis of the percent similarity inthe primary amino acid sequence of cytochromes P450, a universal system ofnomenclature for cytochromes P450 was first established and later revised by Nebertand his collegues (1987, 1989, 1991a and 1993). The most recent review on thecytochrome P450 superfamily by Nebert et. al. (1993), includes up to 221 cytochromeP450 genes and 12 pseudogenes. These genes have been found in 31 eukaryotes and 11prokaryotes (Nebert et at., 1993). Thirty-six gene families have so far been reportedand twelve of them exist in all mammals (Nebert et al., 1993). The 12 families consistof 22 mammalian subfamilies, of which 17 and 15 have been found in human andmouse genomes, respectively (Nebert et a!., 1993). These numerous cytochrome P450genes and enzymes are classified according to the “40% rule”. Table 1.1 listssubfamilies of cytochrome P450 that are of interest in the present study and disccussion.INTRODUCTION /6Table 1.1. Nomenclature of selected cytochrome P450 genes. (Adapted from Nelson eta!., 1993).Gene Symbol Trivial name SpeciesCYP1A SubfamilyCYPJA] c, f3NF-B RatP1, c, form 6 HumanCYPJA2 P-448, d, HCB RatP3, d, form 4 HumanpP-4501A-6 1 ChickenCYP2B SubfamilyCYP2BJ b, PB-4, PB-B, PBRLM5 RatCYP2B2 3, PB-5, PB-D, PBRLM6 RatCYP2B3 11B3 RatCYP2C SubfamilyCYP2C6 PB1, k, PB-c, pTF2, 2C6 RatCYP2C7 f, RLM5b, pTF1 RatCYP2CJ1 h, M-1, 16cc, 2c, UT-A RatCYP2CJ2 i, 15f3, 2d, UT-i RatCYP2CJ3 +g,-g, UT-5 RatCYP2E SubfamilyCYP2EI j Humanj, RLM6 RatCyp2e-J j MouseCYP2E2 3d, 11E2 RabbitCYP2H SubfamilyCYP2HJ pCHP3, PB15 ChickenCYP2H2 pCHP7 ChickenCYP3A SubfamilyCYP3A1 pcn 1, PCNa RatCYP3A2 pcn2, PCNb/c RatCYP3A3orCYP3A4 HLp HumanCYP11A SubfamilyCYPI1A] scc Human, Rat, Chicken, TroutCYP17 SubfamilyCYPJ7 17c Human, Rat, Chicken, TroutCypl7 17a MouseCYP19 SubfamilyCYPJ9 Arom Human, Rat, Chicken, TroutCypl9 gES-Ml0 MouseINTRODUCTION /71.2.1. Naming Cytochrome P450 Genes and EnzymesFor the purpose of naming a cytochrome P450 gene, the newly revised P450nomenclature (Nebert et al., 1989) recommends that the cytochrome P450 gene bedenoted by a italicized root symbol CYP (Cyp for the mouse), the P450 family bedesignated by an Arabic number, the subfamily be indicated by a letter if more than onesubfamily exists in that family, and the individual gene be represented by an Arabicnumber (with mouse genes the final number is preceeded by a hyphen). Thenonitalicized CYP abbreviation followed by a letter and an arabic number is used for thegene transcript and product. For example, the gene and cDNA that encode ratcytochrome P450c (trivial name) are expressed as CYP1A1 (Cypla-] in mouse), whilethe cytochrome P450c protein and mRNA are denoted by the nonitalicized CYP1A1(Cyp la-i) or simply 1A1. This nomenclature is used throughout the thesis.1.2.2. The “40% Rule”This rule was created by Nebert and Gonzalez (1987) after analyzing over 60cytochrome P450 protein sequences. When a newly found CYP enzyme is identified,the primary amino acid sequence of the protein is aligned with a representative sequencefrom each family and subfamily and by comparisons of overlapping portions of thesequences, excluding the gaps and unmatched ends in the overall length, the percentageof sequence similarity is determined. The rule states that (1) if the sequence of the newprotein is less than 40% identical to all other sequences, the new protein constitutes thefirst member of a new family, (2) if the sequence is at least 40% identical to any othersequence, then the new protein belongs in the same family, (3) if the sequence is from40% to 68% similar to that of any subfamily in that family, the new protein will be thefirst member of a new subfamily, (4) if the sequence has at least 68 % similarity to otherproteins in the same subfamily, the new protein is given the next available number inthe group, and (5) if the sequence is different by only a few (less than 3%) amino acidsINTRODUCTION I 8from a known sequence, it is given the same name and assumed to be the same protein,unless it can be shown to be a product of a distinct GYP gene. So far, there are a fewexceptions to the “40% rule”. These exceptions are (1) inclusion of the CYP2D, CYP2Jand CYP2K gene subfamilies in the CYP2 family despite having less than 40% sequencesimilarity with other CYP2 enzymes, (2) the CYP4C1, CYP4D1 and CYP4E1 genesfrom insects are clearly related to the mammalian CYP4 family, but the amino acidsequences of the proteins they encode are only 26 to 42% similar to the mammalianmembers of this family, (3) the CYP6A1 and CYP6A2 proteins are less than 40%similar to each other but are assigned to the same subfamily since the two genes arerelated, (4) the nuclear genes encoding two mitochondrial proteins, scc and 1 ib, areonly 34 to 39% similar but are included in the same CYP1J family, (5) CYP52A,CYP52B and CYP52C also share only 30 to 44% of their amino acid sequences but arein a single gene family because their genes form a tight cluster, and (6) the bacterialproteins CYP1O5A1 and CYP1O5C1 are 39.6% similar to each other (Nebert andNelson, 1991).1.3. TIlE CvToclmoivw P450 SUPERFAMILYThree of the ten families of mammalian cytochrome P450, that are of interest inthe present study, will be briefly introduced in this section and discussed in terms oftheir amino acid sequence homology, catalytical activities, inducibility and regulation.1.3.1. The CYP1A SubfamilyThe CYP1A subfamily found in rat consist of two isozymes, CYP1A1 andCYP1A2, that are immunochemically related (Reik et al., 1982) and share 68%similarity in amino acid sequence (Kawajiri et al., 1984; Yabusaki et a!., 1984).Purified CYP1A1 exhibits high catalytic activity toward 0-deethylation of 7-ethoxyresorufin and hydroxylation of benzo[alpyrene (Burke and Mayer, 1974;INTRODUCTION /9Guengerich et al., 1982). CYP1A2 displays lower activity toward the metabolism of 7-ethoxyresorufin and benzo[a]pyrene, but is the most efficient catalyst for the metabolismof 2-acetylaminofluorine (Goldstein et al., 1984 and McManus et al., 1984) and for the4-hydroxylation of aflatoxin B1 (Faletto et at., 1988).CYP1A1 and CYP1A2 are highly inducible by “MC-type” inducers that includepolycyclic aromatic hydrocarbons (PAHs) and polychiorinated biphenyls (PCBs). ThePAH group includes the prototype inducer, 3-methyicholanthrene (MC) as well asbenzo[a]pyrene (BaP) and 3-naphthoflavone (Conney, 1967). The most effecaciousinducers of CYP1A enzymes are highly toxic halogenated aromatic compounds such as2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7, 8-tetrachlorodibenzofuran(TCDF) (Poland and Knutson, 1982; Safe et at., 1986). The most effecacious PCBinducers in rats are coplanar biphenyl molecules that are substituted in one para and atleast one meta position of both phenyl rings and do not possess ortho-chlorosubstituents. Examples include 3,3’ ,4,4’-tetra-, 3,3’ ,4,4’ ,5-penta- and 3,3’,4,4’,5,5’-hexachiorobiphenyl (Goldstein et al., 1977).The regulation of CYP1A1 induction is mediated by the TCDD or Ah receptor(Okey, 1990; Poland and Knutson, 1982; Whitlock, 1987). The 5’-flanking region ofCYP1AJ gene contains at least three regulatory elements (Figure 1.2, from Okey 1990):(1) ‘dioxin responsive elements’ (DREs) or ‘xenobiotic responsive elements’ (XREs)that function as transcriptional enhancers when Ah receptor ligands such as TCDD areadded to Ah-responsive cells in culture, (2) a transcriptional promoter that activatesgene expression even in the absence of Ah receptor ligands, and (3) an inhibitorycomponent upstream of the promoter that blocks promoter function when a repressor ispresent. The role of the Ah receptor is to first recognize and bind MC-type inducers.The receptor-ligand complex then translocates into the nucleus and associates withtranscriptional enhancers to stimulate mRNA production. The DREs or XREs bearsome similarity to the glucocorticoid-responsive elements, and the Ah receptorINTRODUCTION / 10resembles the glucocorticoid receptor but it does not have zinc finger DNA bindingdomains and thus does not belong to the steroid receptor superfamily (Okey et at.,1988; Brubach et a!., 1992)). The regulation of the CYPJA2 gene and the mechanismcontrolling the induction of the CYPJA2 gene in liver is not well understood.(-400 to -800) > (-1000)____________,@E•fFiSCrnPOjL5(4jä: jEcP45OIA1 mRNA OTHERSPECIFICmRNAsFigure 1.2. Scheme of the Ah-receptor-mediated regulatin of the 5’-flanking region ofthe CYPJAI gene. At least three ‘dioxin responsive elements’ (DREs) appear to lieapproximately 1000 or more base pairs upstream from the CYPJA1 transcriptional startsite. The DREs have all the properties of transcriptional enhancers. An inhibitoryelement (‘negative control element’) is believed to reside between 400 and 800 basesupstream. The negative control element may function to inhibit the CYPIAJ promoteronly when the negative control element is bound by a postulated (but yet undentified)repressor protein. (Adapted from Okey, 1990)The expression and induction of these two cytochrome P450 isozymes are tissue-dependent in the rat (Goldstein and Linko, 1984; Degawa er a!., 1987). In livers ofINTRODUCTION! 11untreated rats, CYP1A2 is a minor constitutive form while CYP1A1 is not detected.Both isozymes are markedly induced by TCDD and MC in rat liver, however, inextrahepatic tissues, only CYP1A1 is inducible by the two inducers (Kimura et at.,1988). The expression of the CYP1A genes is also tissue-specific. For example, theCYPJa-] gene is not expressed in the liver and other tissues of control mice, but isreadily inducible by TCDD in all tissues tested. In contrast, the CYPJa-2 gene isconstitutively expressed in the liver of control mice and is also inducible by MC-typeinducers (Kimura et at., 1986).CYP1A enzymes are found in humans, rodents, fish, birds, insects, plants andbacteria and their associated monooxygenase activities are well conserved amongdifferent species. For example, CYP1A1-mediated benzo[a]pyrene hydroxylase activityhas been found in many organisms, ranging from fungi to humans. It is suggested thatCYP1A1 is needed for a critical life function in the organism and does not functionsolely as a catalyst for the biotransformation of foreign compounds (Nebert, 1991b).CYP1A enzymes are believed to metabolically activate procarcinogens such as aromatichydrocarbons (e.g. benzo[a]pyrene) and heterocyclic amines (e.g. 2-amino-1-methyl-6-phenylimidazo) and the expression of CYP1A1 has been correlated with development ofpolycyclic aromatic hydrocarbon-associated cancers in rodents (Nebert, 1989). Inhumans, CYP1A1 is highly induced in lungs by cigarette smoking (McLemore et al.,1990) and is also induced by omeprazole, an inhibitor of gastric acid secretion.CYP1A2 is also found in mammals,but the presence of CYP1A2-associated enzymeactivities has not been directly evidenced in nonmammalian species such as birds andfish (Gonzalez, 1988). Some properties of the CYP1A subfamily are summarized onTable 1.2.INTRODUCTiON / 12Table 1.2. Properties of the CYP1A subfamily.RelativeCYP1A amino acid Characteristic Inducers Tissueenzymes sequence catalytic activities distributionsimilarityCYP1A1 100% Benzo[a]pyrene Polycyclic Rat liver andhydroxylation, aromatic lung,7-Ethoxyresorufin hydrocarbons rat olfactory0-deethylation including MC, mucosa,j3NF, TCDD; human colon,AR. etc.CYP 1 A2 68% 2-Acetylaminofluo- Same as above Human liver,rene N-hydroxylation, Rabbit olfactoryEstradiol-1713 mucosa, etc.2-hydroxylation1.3.2. The CYP2B SubfamilySo far, sixteen genes have been identified in the CYP2B subfamily (Nebert etal., 1993). Four CYP2B genes, namely CYP2B1, CYP2B2, CYP2B3 and CYP2B8 havebeen characterized in the rats. CYP2B1 and CYP2B2 have been purified from hepaticmicrosomes of rats treated with phenobarbital. These two proteins exhibit 97% aminoacid sequence similarity but display distinct chromatographic and electrophoreticproperties (Guengerich et al., 1982; Rampersaud and Walz, 1983; and Waxman andWalsh, 1982). The cDNA-deduced amino acid sequence of CYP2B3 is 77% similar tothe sequences of CYP2B1 and 2B2 (Labbe et al., 1988).CYP2B1 and 2B2 have similar substrate specificities but with different turnoveriNTRODUCTION I 13rates. Benzphetamine is a substrate for several different cytochrome P450 enzymes,among which purified CYP2B1 shows the highest catalytic activity for benzphetamineN-demethylation. CYP2B2 also catalyzes this reaction but at a rate that is 15% of therate obtained with CYP2B1 (Guengerich et al., 1982; Ryan and Levin, 1990). PurifiedCYP2B1 and 2B2 are efficient catalysts for 7-pentoxyresorufin 0-depentylation (Duttonand Parkinson, 1989; and Lubet et at., 1985) and CYP2B1 displays 100-fold higheractivity relative to CYP2B2 (Wolf et al., 1988). In addition, CYP2B1 catalyzes themetabolism of testosterone to produce 16a-hydroxytestosterone, 1613-hydroxy-testosterone and androstenedione (Waxman et al., 1983). CYP2B2 also hydroxylatestestosterone with similar regioselectivity but at only 5 to 10% the catalytic rate ofCYP2B1 (Waxman et a!., 1983). It was proposed that the catalytic differences betweenthese two highly homologous isozymes may relate to their heme environment (Wolf etat., 1988). The catalytic properties of CYP2B3 have not been reported.CYP2B1 and CYP2B2 are inducible by “phenobarbital-type” inducers includingphenobarbital (PB) and other barbiturates (Remmer and Merker, 1963; Conney 1967),5,5-diphenyihydantoin and 5-ethyl-5-phenylhydantoin (Conney, 1967; Diwan et at.,1988), some polychlorinated pesticides such as DDT and dieldrin (Conney, 1967), andPCBs containing ortho-chlorine substituents such as 2,2’ ,4,4’-tetrachlorobiphenyl (Safeet at., 1985).CYP2B1 and CYP2B2 are expressed differentially in liver, lung, kidney, adrenalgland and small intestines (Christou et a!., 1987), while CYP2B3 is constitutivelyexpressed as a minor form only in rat liver and is not induced by phenobarbital (Labbeet al., 1988). In untreated rats, the content of CYP2B1 is highest in lung compared toliver and other extrahepatic tissues, while CYP2B2 content is highest in liver (Christouet at., 1987; and Wilson et at., 1987). After phenobarbital treatment, the amounts ofboth CYP2B1 and CYP2B2 are increased to their highest levels in rat liver with thehepatic level of CYP2B1 reported to be approximately two fold higher than that of 2B2INTRODUCTION I 14(Yamazoe et al., 1987). However, in extrahepatic tissues, CYP2B1 content is inducedto a modest degree and the level of CYP2B2 is only induced in the adrenal gland(Christou et at., 1987).The differential distribution of CYP2B isozymes in rat liver and extrahepatictissues is due to the tissue-specific regulation of CYP2B genes (Gonzalez, 1989). Theinduction of CYP2B isozymes requires de nova protein synthesis which is primarilyregulated by CYP2BJ and CYP2B2 genes at the transcriptional level (Adesnik andAtchison, 1985). Two mechanisms have been hypothesized to explain phenobarbitalinduction of CYP2B enzymes. One mechanism suggests that there is a receptor forphenobarbital-type inducers (Okey, 1990). Another proposes that phenobarbital and itsrelated compounds act in a manner analogous to the mechanism of general anesthesia orthat the induced cytochrome P450 enzymes act as ‘receptor’ sites (Polland et at., 1980).So far, neither mechanism has been proven.The CYP2B subfamily is also found in other mammals such as rabbits and mice,as well as in humans. At least three CYP2B genes, designated CYP2B4, CYP2B4P(pseudogene) and CYP2BS, are found in rabbits (Komori et at., 1988; Zaphiropoulos etal., 1986). The deduced amino acid sequences of CYP2B4 and CYP2B5, which havebeen found in phenobarbital-induced rabbit liver, exhibit 95 % similarity (Komori at el.,1988). Three CYP2B genes, CYP2b-9, CYP2b-1O and CYP2b-13, are found in themouse (Noshiro et at., 1988; Lakso et at., 1991). CYP2b-9 and CYP2b-10 enzymesshare 83 % amino acid sequence similarity with each other and display 82% similaritywith rat CYP2B1 (Noshiro et at., 1988). Two CYP2B genes, CYP2B6 and CYP2B7P(pseudogene), are found in human liver (Miles et al., 1988; Yamano et at., 1989). ThecDNA deduced amino acid sequence for CYP2B6 exhibits more than 75% similaritywith the rat CYP2B1 and CYP2B2 enzymes (Miles et at., 1988).INTRODUCTION! 15Table 1.3. Properties of the rat CYP2B subfamilyRelative TissueCYP2B amino acid Characteristic Inducers distributionenzyme sequence catalytic activities in the ratsimilarityCYP2B 1 100% 7-Pentoxyresorufin Phenobarbital-type: Liver,0-depentylation, barbiturates, nasal mucosa,Benzphetamine ortho-chlorinated olfactoryN-demethylation, PCBs, mucosa,Testosterone DDT and dieldrin lung,1 6f3 -hydroxyation brainMixed-type:Aroclor 1254CYP2B2 97% Same as above Same as above. Liverbut with lowerefficiencyCYP2B3 77% Unknown Unknown LivercDNAdeducedprotein1.3.3. The CYP2C SubfamilyTo date, twenty-three members of this subfamily have been found to exist inhumans, rats and other mammals (Table 1.1). Most of the CYP2C isozymes areconstitutively expressed and, at least in the rat, are under developmental and sexualregulation.Seven genes have been identified in the rat CYP2C subfamily. The genes areCYP2C6, 2C7, 2C11, 2C12, 2C13, 2C22 and 2C23 (Nelson et al., 1993). FiveCYP2C enzymes, namely CYP2C6, 2C7, 2C11, 2C12 and 2Cl3, have been purifiedand characterized in rats. CYP2C6 and 2C7 bear 75% amino acid sequence similarityINTRODUCTION / 16to each other and 50% resemblance to CYP2B1 and 2B2 (Gonzalez et at., 1986a). Theamino acid sequence of rat CYP2C1 1, a male-specific cytochrome P450, is 70% and71 % similar to the amino acid sequence of CYP2C6 and 2C7, respectively (Yoshioka etat., 1987). The cDNA-deduced amino acid sequence of the female-specific CYP2C12exhibits 68%, 68% and 71 % similarity to CYP2C6, 2C7 and 2C 11, respectively(Zaphiropoulos et at., 1988). CYP2C13, another male-specific form, has 66% aminoacid similarity to CYP2C1 1 (Zaphiropoulos et at., 1988).The CYP2C enzymes show different regioselectivity and stereoselectivity towardsteroid substrates and distinct turnover numbers. Microsomal CYP2C6 contributessignificantly to the 21-hydroxylation of progesterone on the basis of induction andimmunochemical studies (Swinney et at., 1987; Waxman et al., 1985). PurifiedCYP2C7 catalyzes the 2c-hydroxylation of progesterone and 16c-hydroxylation oftestosterone and progesterone but at a relatively low catalytic rate compared toCYP2C1 1 (Ryan et at., 1984; Swinney et al., 1987). It has also been reported toefficiently metabolize the 4-hydroxylation of retinol (Leo and Lieber, 1985). CYP2C11is an effective catalyst for the 16c& and 2c-hydroxylation of testosterone (Morgan andGustafsson, 1987). The purified enzyme also catalyzes the N-demethylation ofbenzphetamine with an efficiency that is second only to that of purified CYP2B1, and ata rate that is approximately 39% of that obtained with CYP2B 1. In addition, it is activein the metabolism of ethylmorphine, warfarin, hexobarbital and benzo[ajpyrene (Ryanand Levin, 1989). The female-specific CYP2C12 enzyme exclusively catalyzes the1513-hydroxylation of androstanediol disulfate, an important physiological pathway forthe excretion of corticosterone metabolites in female rats (Morgan and Gustafsson,1987). Purified CYP2C12 also mediates the lcL- and l5cL-hydroxylation of testosteronebut at a low rate (Ryan and Levin, 1990). Two major metabolites, 6f3- and l6c-hydroxylated steroids are formed when testosterone or progesterone serve as thesubstrate for purified CYP2C13, however, this enzyme does not appear to catalyze theseiNTRODUCTION /17reactions in intact hepatic microsomes (Swinney et at., 1987). Purified CYP2C13 alsoshows low activity toward N-demethylation of benzphetamine, at about 10% of theturnover number obtained with CYP2C11 (Ryan and Levin, 1990).CYP2C6 is developmentally regulated in male and female rat liver (Gonzalez etal., 1986). The level of hepatic microsomal CYP2C6 mRNA increases with age up to 4weeks of age, and no sex difference in the expression of this isozyme is found in adultrats (Gonzalez et at., 1986; Kimura et at., 1988). CYP2C7, which is alsodevelopmentally regulated, increases from a level of less than 1 % of total cytochromeP450 in immature rats to approximatelly 7% and 14% of total hepatic cytochrome P450in adult males and females, respectively (Bandiera et al., 1986). Levels of CYP2C7mRNA are maximal at 12 weeks of age and are two to three fold higher in female thanin male liver (Gonzalez et at., 1986).CYP2C1 1 and 2C13 are male-specific and developmentally regulated. Hepaticmicrosomal CYP2C 13 content rises from less than 1 % of total cytochrome P450 inyoung male rats to an average value of 17% of total cytochrome P450 in adult males at6 weeks of age (Bandiera et al., 1986). However, variable expression of CYP2CI3 hasbeen reported among individual, outbred, adult male rats (Bandiera et al., 1986;McClellan-Green et al., 1987). CYP2C1 1 is not detectable in the liver of newborn rats,but the hepatic content of CYP2C 11 develops rapidly at 4 to 6 weeks of age and thenremains at a plateau level in adult male rats, but disappears in the liver of senescent rats(>2 years old) (Kamataki et at., 1985a). The age-differentiated expression ofCYP2C1 1 is indirectly dependent on testosterone and may be mediated through themale-specific pattern of growth hormone release (Kamataki et at., 1985b; Morgan etat., 1985).INTRODUCTiON I 18Table 1.4. Properties of the rat CYP2C subfamilyRelativeCYP2C amino acid Characteristic GenderSubfamily sequence catalytic activities specificitysimilarityCYP2C6 70% Progesterone none21 -hydroxylationCYP2C7 69% Retinol More in4-hydroxylation, female thanProgesterone in male2cx-hydroxylation,Testosterone1 6ct-hydroxylationCYP2C 11 66% Testosterone Male1 6x-hydroxylation, specificTestosterone2cx-hydroxylation,BenzphetamineN-demethylation,Benzo[ajpyrenehydroxylationCYP2C 12 79% Androstanediol Femaledisuiphate specific15 3-hydroxylationCYP2C13 100% Testosterone Male1 6c-hydroxylation, specificTestosterone613-hydroxylation,BenzphetamineN-demethylationCYP2C12 is a female-specific isozyme and is also subject to hormonalregulation. This isozyme is absent from the liver of neonatal female rats, developsINTRODUCTION / 19between 4 to 6 weeks of age and thereafter remains relatively constant throughoutadulthood (Kamataki et al., 1985a; Waxman et at., 1985). A low level of this isozymeis detected in immature and very old male rats but not in adult males (Kamataki et at.,1985a). Hypophysectomy results in a loss of CYP2C12 expression in females andtreatment of male rats with exogenous growth hormone to mimic the female pattern ofsecretion causes the appearance of this isozyme (Kamataki et at., 1985b; Waxman etal., 1985). It is suggested that growth hormone regulates the expression of bothCYP2C1 1 and 2C12 at the pretranslational level (MacGeoch et at., 1987;Zaphiropoulos et at., 1988).Extrahepatic expression of members of the CYP2C subfamily has been reported.The adult male-specific rat CYP2C1 1 mRNA has been found exclusively in the liver butnot in the lung, kidney or testis (Yoshioka et at., 1987). However, Ryan et at. (1993)detected CYP2C1 1 protein in microsomal preparations from lung, kidney and testis ofadult male rats by immunoblot analysis. Besides being found in the livers of male andfemale rats, mRNA for CYP2C6 and 2C7 are also expressed in adult female rat brainbut not in kidney or lung of either sex (Kimura et al., 1988).CYP2C isozymes are relatively refractory to induction by MC, PB and otherknown inducers except that Kepone® is a weak inducer of CYP2C7 (Bandiera et at.,1986). In addition, the level of CYP2C6 mRNA is increased two to three fold aftertreatment of rats with PB (Friedberg et al., 1986). However, CYP2C11 content isrepressed in livers of rats treated with MC or PB (Yeowell et al., 1987).A total of nine CYP2C genes have been identified in rabbits and none of theCYP2C cytochromes P450 are under gender-specific regulation (Nebert et at., 1993).Six CYP2C genes have been identified in humans by cDNA cloning, among whichCYP2C8 and CYP2C9 have been purified (Nebert et a!., 1993). CYP2C9 was found tobe the major human cytochrome P450 that metabolizes S-warfarin, an therapeuticanticoagulant (Rettie et at., 1991). Human CYP2C19 is believed to be the principalINTRODUCTION /20determinant of S-mephenytoin polymorphism (Wilkinson et al., 1989; Goldstein et al.,1994).1.3.4. The CYP3A SubfamilyThe rat CYP3A subfamily consists of CYP3A1 and CYP3A2. The nucleotidesequence of the cDNA for CYP3A1 is 90% similar and the cDNA-deduced amino acidsequence is 89% similar to those of CYP3A2 (Gonzalez, 1989).CYP3A1 and 3A2 also have similar substrate specificity. They catalyzeethylmorphine N-demethylation, erythromycin N-demethylation, testosterone 6f3-hydroxylation and benzo[a]pyrene hydroxylation (Waxman et al., 1985; Wrighton eta!., 1985a).The CYP3A1 enzyme was the first member in this subfamily that wascharacterized as being induced by pregnenolone 16cx-carbonitrile (PCN) (Selye, 1971;Lu et al., 1972; Elshourbagy and Guzelian, 1980). CYP3A1 is also inducible by othersteroid compounds such as dexamethasone but not by estrogen, testosterone orpregesterone (Heuman et al., 1982). Besides the glucocorticoids, several other classesof compounds have been found to induce CYP3A isozymes, including “phenobarbitaltype” inducers that are known to be CYP2B inducers (Schuetz et al., 1986), macrolideantibiotics such as triacetyloleandomycin (TAO) and rifampicin (Wrighton et al.,1985b) and antifungal agents such as clotrimazole (Guzelian, 1987).It was recently reported that CYP3A1 is mainly an inducible cytochrome P450enzyme as it is not found in untreated rats, while CYP3A2 is a constitutive form that isdevelopmentally regulated (Cooper et al., 1993). CYP3A1 is not detectable in hepaticmicrosomes from untreated male and female rats at any age but the level of CYP3A 1 inall groups is significantly enhanced following PCN or dexamethasone treatment.CYP3A2 is expressed in untreated adult males and untreated immature males andfemales but not in adult females. It is slightly inducible by dexamethasone in immatureINTRODUCTION /21rats of both sexes and in adult males (Cooper et al., 1993). The differentiatedexpression and induction of the CYP3A1 and 3A2 proteins closely parallels theregulation of the CYP3AJ and 3A2 genes. Gonzalez et al. (1986b) reported thatCYP3A1 mRNA was not detectable in immature or adult male or female rats, whileCYP3A2 mRNA was present at low levels in newborn rats and at elevated levels in bothmales and females at one week of age. The level of CYP3A2 mRNA reaches its highestvalue in male rats at 12 weeks of age and thereafter remains relatively constant, whereasit reaches its highest level in the females at 2 weeks of age and decreases withmaturation. CYP3A1 mRNA is also significantly induced by PCN, dexamethasone andphenobarbital, while CYP3A2 mRNA is not markedly increased by the steroid-inducersbut is readily induced by PB (Gonzalez et al., 1986b). Dexamethasone and PCN appearto be more potent in the induction of CYP3A1 than other inducers (Schutz et al., 1986).The mechanism regulating CYP3A1 induction is complex and diverse.Glucocorticoid-receptors are thought to be involved in induction of CYP3A1 by PCN ina ‘nonclassicaP manner but this mechanism is not yet proven. Increased transcriptionplays an important role in the induction of CYP3A1 by PCN, since PCN anddexarnethasone increase the transcription of the CYP3AJ gene about three- and six-fold,respectively, in rat liver (Simmons et al., 1987). Posttranscriptional regulation is alsoimportant in CYP3A1 induction by steroids. A 12-fold increase in CYP3A1 mRNA asa result of dexamethasone treatment appears to be a consequence of mRNA stabilizationrather than increased transcription (Simmons et al., 1987). Besides the increasedtranscription of CYP3A genes and stabilization of their mRNAs, the decreaseddegradation of CYP3A proteins also plays a crucial role for macrolide antibioticinducers such as TAO and for phenobarbital-like inducers exemplified by someorganochiorine pesticides. Moreover, TAO is a substrate for CYP3A1 and itsmetabolites bind to CYP3A1 to form a complex which can be detected spectrally thatinhibits the degradation of CYP3A1 (Watkins et al., 1986). Although induction ofINTRODUCTiON /22CYP3A1 by PB-type inducers requires de novo synthesis, in hepatocyte culture and inrats treated with chiordane or trans-nonachlor, the increase in accumulation of CYP3A1protein is about 3-fold higher than the augmentation in synthesis of CYP3A 1 (Schuetz etal., 1986).The difference between the structure-activity relationship for induction ofCYP3A isozymes versus induction of CYP2B isozymes by polychlorinated biphenyls(PCB) indicates that a separate recognition mechanism may be involved in the the coinduction of these two subfamilies by PB-type inducers. PCBs with more than twoortho chiorines are found to be the most efficacious inducers of CYP3A1 in hepatocytecultures and in rats (Schuetz et al., 1986), whereas other congeners with two orthochlorine substituents more effectively induce CYP2B 1 and 2B2 in rats (Parkinson et al.,1983).CYP3A gene products are found in the intestine of humans and rats (Watkins etal., 1987). Rat intestinal villus cells have at least two CYP3A isozymes that aredexamethasone-inducible. The iduction of these isozymes in rat intestinal enterocytesparallels increases in the amount of CYP3A 1-related mRNA and erythromycin Ndemethylase activity. In addition, an olfactory specific CYP3A gene, CYP3A9, wasfound in rats (Nef et al., 1990).So far six human CYP3A genes have been found in human liver cDNA libraries(Nebert et al., 1993). CYP3A cytochromes P450 are the most abundant enzymes inhuman livers as they account more than 60% of total cytochrome P450 in some humanliver specimens (Guengerich and Kim, 1990). The expression of CYP3A proteinsvaries greatly among individuals (Nebert et al., 1993). Catalytic studies of CYP3Aenzymes established that CYP3A4, and probably CYP3A3, can metabolize many drugssuch as cyclosporine, erythromycin, 1 7-ethynylestradiol, lidocaine, midazolam,quinidine and warfarin (Guengerich and Shimada, 1991).INTRODUCTION /23Table 1.5. Properties of the rat CYP3A subfamilyCYP3A RelativeSubfamily amino acid Characteristic Inducers Tissuesequence catalytic activity distributionsimilarityCYP3A1 100% Testosterone Pregnenolone Liver,613-hydroxylation, 1 6cL-carbonitrile, intestine,Ethylmorphine Dexamethasone, nasal mucosaN-demethylation, Phenobarbital,Triacetyloleandomycin,ClotrimazoleCYP3A2 89% Testosterone Pregnenolone Same as above613-hydroxylation, 1 6x-carbonitrile,Testosterone Phenobarbital213-hydroxylationBenzphetamineN-demethylation,Benzo[a]pyrenehydroxylation1.3.5. Other Cytochrome P450 Subfamilies and Inducers1.3.5.1 CYP2A SubfamilyThree cytochrome P450 genes in this subfamily, designated as CYP2A1, 2A2 and2A3, have been identified in rats. The CYP2A1 eDNA and CYP2A2 cDNA shares 93%of their nucleotide sequence and 88% of their deduced amino acid sequence (Gonzalez,1988), while CYP2A3 exhibits 71% and 73% similarity in eDNA deduced amino acidsequence with CYP2A1 and 2A2 enzymes, respectively (Kimura et al., 1989).CYP2A1 and 2A2 enzymes exhibit distinct regioselectivity toward the hydroxylation oftestosterone and other steroids (Matsunaga et a!., 1988). CYP2A1 hydroxylatestestosterone mainly at the 7cL position with a small amout of the 6cx metaboliteiNTRODUCTION /24produced, whereas CYP2A2 metabolizes the same substrate preferentially at the l5ctposition, with 7c products comprising only 5% of the total hydroxylated metabolites.CYP2A 1 and 2A2 mRNA are present in rat liver but are not detectable in extrahepatictissues. CYP2A1 is inducible by MC and Aroclor 1254 while CYP2A2 appears to beresistant to induction (Matsunaga et al., 1988). The CYP2A1 gene is expressed inadolescent male and female rats and is specifically repressed in males after puberty(Nagata et al., 1987). In contrast, the expression of the CYP2A2 gene is male-specificand CYP2A2 mRNA levels increase markedly with the onset of puberty (Matsunaga etal., 1988). CYP2A3 is exclusively expressed and inducible by MC in rat lung, and itsmRNA is not detected in liver, kidney or intestines (Kimura et al., 1989).1.3.5.2. CYP2E SubfamilyTo date only one member in this subfamily has been found in rats and designatedas CYP2E1. This cytochrome P450 enzyme is inducible by the anti-tubercular drug,isoniazid, and by ethanol, acetone, pyrazole and other related volatile compounds(Okey, 1990). It is also induced by fasting (Tu and Yong, 1983) and diabetes (Bellwardet al., 1988). Expression of rat CYP2E1 is under developmental regulation (Thomas etal., 1987). CYP2E1 metabolizes many volatile compounds including ethanol, acetone,aniline, diethyl ether, p-nitrophenol, halothane, benzene, pyridine and nitrosamine(Gonzalez, 1989). In addition, it is a very efficient catalyst for the N-demethylation ofthe carcinogen, nitrosodimethylamine (Thomas et al., 1987a).In addition to the cytochrome P450 subfamilies introduced above, several othersubfamilies exist and have been extensively reviewed in the literature (Gonzalez, 1989;Nebert et al., 1991) but are not the focus for the present study and therefore, will not bedisscussed here.INTRODUCTION /251.3.5.3. Mixed-Type inducersA mixed-type inducer is a compound that produces a cytochrome P450 inductionpattern equivalent to that obtained by simultaneous administration of more than oneclass of inducer such as phenobarbital plus 3-methyicholanthrene” (Okey, 1990). Thereare many compounds that simultaneously induce both CYP1A1/1A2 and CYP2B1/2B2in rats. Examples of such compounds are the mono-ortho or di-ortho chlorinatedcongeners of PCBs and PBBs (polychlorinated biphenyls and polybrominated biphenyls,respectively) including 2,3’ ,4,4’ ,5,5’-hexabromobiphenyl (Dannan et al., 1978),2,3’,4,4’,5-pentabromobiphenyl (Robertson et al., 1980), 2,3,3’,4,4’,5,5’-heptachiorobiphenyl (Parkinson et al., 1980).Commercial mixtures of PCB isomers and congeners, such as Aroclor 1254, arealso mixed-type inducers. Some components in the mixture act as MC-type inducers,some act as PB-type inducer, and some act as mixed-type inducers (Safe et al., 1985).Phenothiazines are an additional class of mixed-type inducers which induce bothCYP1A1/1A2 and CYP2B1/2B2 (Thomas et al., 1987b). The carcinogen, 2-acetylaminofluorene (2-AAF) is another agent that produces an induction pattern similarto that obtained with mixed-type inducers. It has been suggested that this inducer is nota mixed-type inducer itself, but that its metabolites contribute to the increased level ofCYP1A1/1A2 and CYP2B1/2B2 enzymes (Astrom et at., 1986). Hexachlorobenzene(HCB) also appears to be a mixed-type inducer (Stewart and Smith, 1986).1.4. REVIEW ON Av1AN CYTOCHROME P450 ENZYMESAs stated above, cytochrome P450-dependent monooxygenases function in thebiotransformation of numerous endogenous and exogenous compounds, and play keyroles in diverse biological pathways. The extent to which these various pathways occurin different species depends upon the complement of cytochromeP450 isozymes present,iNTRODUCTiON /26their catalytic activity and their regulation. Although mammalian cytochromes P450continue to dominate the research field, there is growing recognition of the biologicalsignificance of cytochromes P450 in other vertebrates such as birds and as aconsequence there is increased need for basic information on the diversity and propertiesof cytochrome P450 enzymes in these species.There are about 8,600 avian species including both wild and domestic birds(Farner et al., 1971). Birds form an important part of the terrestrial ecosystem andrepresent an important source of protein for humans. They represent extraordinarydiversity and inhabit virtually every geographical area on earth.Interest in avian cytochromes P450 is growing due to several reasons. First,there is the increasing concern about the fate and the effects of lipophilic environmentalpollutants on avian species and birds are often the victims of exposure to a widespectrum of environmental pollutants. For example, a great blue heron colony atCrofton B. C. showed a decreased reproductive rate in 1989 that was attributed to theuptake of high levels of organochiorine pollutants from the area around Crofton (Elliottet al, 1989). Heron eggs from Crofton colony were found to contain 2,3,7,8-TCDD ata level that was on average 21 times the level present in a control group and a highlysignificant correlation (p < 0.001) was observed between the elevated concentration of2,3,7,8-TCDD and increased ethoxyresorufin 0-dealkylase activity measured in chickshatched from paired eggs (i.e. from the same nest) (Bellward et al., 1990). A declinein bird population has also been reported for some predator species such as theperegrine falcon (Falco peregrinus) and sparrowhawk (Accipiter nisus) in Great Britain,and the bald eagle (Haliaetus leucocephalus) in North America, and this decline inreproduction was attributed to the toxic effects of accumulated organochlorineinsecticide residues in the birds (Ratcliffe, 1980; Newton and Haas, 1984). Theefficient bioaccumulation of compounds such as dieldrin and pp’-DDE in the birds wasfound to be resulted from the slow metabolism of the compounds in these speciesINTRODUCTION /27(Walker, 1981). Very low monooxygenase activity toward organochlorine substrateswas found in the sparrowhawk and certain other predators (Ronis and Walker, 1989). Itappears that the pesticide residues that accumulate in birds also induce certain forms ofavian cytochromes P450 (Walker, 1981).Birds also appear to be more vulnerable to the toxicity of organophosphateinsecticides, such as carbamate insecticides, than mammals, which may be due to thefact that the activity of the monooxygenases responsible for the detoxification ofcarbamate is relatively lower in birds than in mammals (Ronis and Walker, 1989). Theuse of drugs and pesticides on farms may lead to the bioaccumulation of toxic residuesand cause unwanted toxic effects in domestic birds like chickens, geese, ducks andquail. Therefore, an increased knowledge of avian cytochromes P450 and their roles indetoxification may aid not only in the interpretation of existing pollution problems thatimpact on birds but also in the prediction of future environmental problems caused bynew chemical products.Second, there is increasing interest in using inducible forms of cytochromesP450 as bioindicators for monitoring environmental pollution. The study of theinduction of cytochromes P450 in rats and aquatic animals has led to the use of certaininducible forms of cytochrome P450 as bioindicators of environmental contamination.Induction of cytochrome P450 as a result of chemical exposure can be determined bymeasuring the elevation of specific enzymatic activities associated with the inducedcytochromes P450 or by measuring increased levels of the induced protein with specificantibodies (Lubet et al., 1990). This biomonitoring approach can be extended to avianspecies provided that there is sufficient knowledge of avian cytochrome P450 enzymesand their associated monooxygenase activities.Third, cytochrome P450-mediated metabolism of a foreign compound can varyquantitatively and qualitatively among individuals or species as a result of geneticdifferences between individuals or species. With a better understanding of theINTRODUCTION /28biotransformation of various pesticides by different avian species, it is possible tochoose and design pesticides in order to save beneficial bird species while controllingpest damage to valuable agriculture products. Finally, the study of avian cytochromesP450 is essential for the complete knowledge of cytochrome P450 phylogeny.Previous investigations on avian cytochrome P450 can be divided into twogeneral categories. One category emphasizes the purification and characterization ofavian cytochrome P450 enzymes and the other category is concerned with the inductionof avian cytochrome P450 enzymes by various classes of chemicals which have beenwell characterized in mammals such as rats..1.4.1. PurifIed Avian Cytochromes P450Over the last decade a few research laboratories have purified several forms ofavian cytochromes P450 from either chick hepatocyte cultures, chick embryos or adultchickens. These cytochrome P450 enzymes have been characterized with respect totheir catalytic activities, inducibility and immunochemical properties. However, due toa lack of knowledge of their DNA or cDNA sequences or primary amino acidsequences, most of these avian cytochrome P450 enzymes have not been definitivelyassigned to any of the known cytochrome P450 family and subfamily. So far, only fivechicken cytochrome P450 genes can be found in the family tree of cytochrome P450designed by Nebert et al. (1993).1.4.1.1. Purified avian cytochromes P450 induced by MC4ype inducersThree cytochrome P450s were purified from adult White Leghorn hens treatedwith 3-naphthoflavone (f3NF). They were designated as I3NF-A, I3NF-B, and f3NF-C,respectively (Gupta et al., 1990). Cytochrome P450 f3NF-B and 3NF-C wereimmunochemically related but distinct enzymes as their amino-terminal sequences andiNTRODUCTION /29other properties were different. Cytochrome P450 I3NF-B and f3NF-C were present intrace amounts in untreated hen liver and were inducible by f3NF. Cytochrome P450I3NF-C was suggested to be the major enzyme catalyzing 0-dealkylation of 7-ethoxyresorufin and 7-methoxyresorufin, which are predominantly mediated by CYP1Aisozymes in rats (Gupta et al., 1990). Cytochrome P450 I3NF-A was inducible by PBand was different from f3NF-B and f3NF-C.Two cytochromes P450 were isolated from nine-day-old chickens treated with331441,5-pentachlorobiphenyl (PenCB), and designated as P448H and P448L,respectively (Hokama et al., 1988). These two PenCB-inducible P450s were believedto correspond to P450 f3NF-B and I3NF-C, respectively (Gupta et al., 1990).1.4.1.2. Purjfied avian cytochrornes P450 induced by PB-type inducersOron and Bar-Nun (1984a) found that PB treatment induced two proteins incultured chick embryo hepatocytes. They isolated a cytochrome P450 with apparentmolecular weight 56 kD from liver microsomes of PB-treated chickens and named itcytochrome P45OPB (Oron and Bar-Nun, 1984b). PB treatment increased the synthesisof cytochrome P-450PB in both cultured chick embryo hepatocytes and in adult chickenliver (Oron and Bar-Nun, l984b). Chicken cytochrome P-450PB was able to beimmunoprecipitated by using anti-rat CYP2B antibody and anti-chicken P-450PBantibody (Oron and Bar-Nun, 1984b). Gupta et a!. (1990) isolated two forms ofcytochrome P450 from PB-treated chickens which were fasted for 24 hours before beingsacrificed. Sinclair et a!. (1990) found that one of the two chicken cytochrome P450proteins purified by Gupta et a!. (1990) had an N-terminal amino acid sequenceidentical to rat CYP2E and the protein could be induced by acetone and ethanol inchicken liver.INTRODUCTION /301.4.1.3. Purjfied avian cytochromes P450 induced by ethanol-type inducersSinclair et al. (1989) purified a cytochrome P450 with a molecular weight of 50kD from chick embryo hepatocytes using the sedative-hypnotic drug, glutethimide, asan inducer. This chicken cytochrome P450, which was also induced by ethanol, wassuggested to be immunologically homologous to mammalian CYP2E1 since antibodyagainst rabbit CYP2E1 recognized the chicken embryo cytochrome P450. It was alsoreported that both ethanol and glutethimide induced p-nitrophenol hydroxylase activityas well as benzphetamine N-demethylase activity, which are often used as markeractivities for mammalian CYP2E1 and CYP2B1/2B2, respectively. Antibody againstthe glutethimide-induced chick cytochrome P450 inhibited 85-90% of these two enzymeactivities in microsomes from both ethanol- and glutethimide-treated chickenhepatocytes (Sinclair et at., 1989). It has been shown that ethanol produced aphenobarbital-like induction of cytochrome P450 in primary cultures of chicken embryohepatocytes (Sinclair et a!., 1981).1.4.1.4. Avian CYP2H subfamilyThe CYP2H gene subfamily found in chicken embryos and adult hens containsCYP2H1 (or pCHP3) and 2H2 (or pCHP7) genes (May et at., 1987). Each of thesegenes encodes proteins containing 491 amino acid residues that share 92% similarity intheir primary sequences. When compared with rat cytochrome P450 enzymes, thecDNA-deduced amino acid sequences are 30% similar to rat CYP 1 Al, 49% similar torat CYP2B1 and 51% similar to rat CYP2C7 (May et at., 1987). These aviancytochrome P450 genes are inducible by phenobarbital and 2-allyl-2-isopropylacetamide(AlA), an inducer of porphyrin synthesis. In chicken embryos, AlA activates thetranscription of the two genes, but induction of CYP2H1 and 2H2 mRNA by this drugis mainly regulated at the posttranscriptional step. In adult untreated hens, CYP2H1and 2H2 mRNA are detected only in liver and not in heart, kidney or spleen, however,iNTRODUCTION /31they are induced by AlA in all these tissues. Both CYP2H1 and CYP2H2 proteins havebeen isolated from the livers of acetone-treated chickens and in a reconstituted systemthe purified proteins catalyze benzphetamine N-demethylation (Sinclair et al., 1990).1.4.1.5. Avian CYPl9farnilyCYP19, also called cytochrome P450 aromatase, catalyzes the formation ofestrogen from androgen (Fishman and Goto, 1981). This enzyme is found in adultchickens and has been purified from chicken ovary (Leshin et al., 1981; Leshin andNoble, 1986). Chicken CYP19 cDNA was first isolated from a chicken ovary libraryby using a partial cDNA of human placental aromatase (McPhaul et al., 1988). Afterchicken CYPI9 cDNA was transfected into COS cells, which have undetectablearomatase but high levels of NADPH cytochrome P450 reductase, aromatase activitieswere detected in different subcellular fractions from transfected COS cells (McPhaul etal., 1988). The chicken CYP19 cDNA-deduced amino acid sequence shares over 70%similarity with its human counterpart and less than 33 % similarity with the cDNAdeduced amino acid sequence of other cytochrome P450 proteins.1.4.2. Induction of Cytochromes P450 by MC, PB, and AR in Avian SpeciesInterest in the induction of avian cytochromes P450 stems from two differentresearch fields (Okey, 1990). In one line of research, the findings of reproductivefailure caused by chlorinated pesticides in feral birds disclosed the involvement ofcytochrome P450 induction in the reproductive disorder (Peakall, 1967). Exposure ofbirds to the clorinated pesticide DDT or to polychlorinated biphenyls (PCB5) increasedthe activity of hepatic microsomal cytochromes P450 (Abou-Donia and Menzel, 1968)and increased the metabolism of estrogens and androgens (Nowicki and Norman, 1972).In the other field, study of the drug-induced increase of ö-aminolevulinic acid synthetase(ALAS), the rate-limiting enzyme in heme biosynthesis, converged on the induction ofINTRODUCTiON /32cytochromes P450 since it had been found that many of the potent cytochrome P450inducers such as halogenated and non-halogenated polyaromatic hydrocarbons (PAHs)also highly induced ALAS activity (Poland and Glover, 1973; Poland and Knutson,1982). Chick embryo liver proved to be a useful model system to study drug-inducedprophyrin synthesis (Granick, 1966; Racz and Marks, 1969), and this model laterbecame very popular in the study of the induction of cytochromes P450 in chickenssince cytochrome P450-dependent monooxygenase activities are very readily detectedand induced in chick embryo livers (Ronis and Walker, 1989). Inducers can be directlyinjected into the egg yolk sac and diet and living conditions do not affect thedevelopment of chick embryos (Ronis and Walker, 1989). Many of the observations onthe induction of avian cytochromes P450 have been based on chick embryo liver orchick embryo hepatocytes (Okey, 1990; J. Sinclair and P. Sinclair, 1992).1.4.2.1. MC-type inductionPrevious studies on the induction of cytochromes P450 in chick embryos, adultchickens, Japanese quail, pigeons, and some wild bird species revealed that MC-typeinducers produce a pattern of induction of hepatic cytochrome P450 in birds which isquite similar to that in mammals but has some interesting differences (Ronis andWalker, 1989). For example, a single intraperitoneal injection of MC at a dose of 100mg/kg to seven-day-old chicks resulted in a threefold increase in total cytochrome P450content as well as small increase in aminopyrine N-demethylase activity, an activitywhich is not affected by MC-treatment in rats (Powis et al., 1976). Administration ofMC at a dose of 20 mg/kg/day for two days to adult male white leghorn chickensresulted in a fourfold increase in total cytochrome P450 content, a 20-fold increase inAHH activity, a 1.7-fold increase in ethymorphine N-demethylase activity, which isinducible in rats by PB-type inducers but not MC and a 2.5-fold increase in anilinehydroxylase activity, which is inducible in rats mainly by inducers of CYP2E1INTRODUCTION /33(Buynitzky et al., 1978).An excellent study has been done on the development of MC-type inducibility ofcytochrome P450 monooxygenase activity in the chick embryo by Hamilton et al.(1983). The basal level and induced level of benzo[a]pyrene hydroxylase (AHH)activity was examined in the chicken embryo in ovo and in adult chickens (Hamilton etat., 1983). Basal AHH activity was found to be equal in all tissues in chicken embryosand the hepatic activity remained constant during development and maturation(Hamilton et al., 1983). Embryonic AHH activity was inducible by 3,3’,4,4’-tetrachiorobiphenyl (TCB), 2,3,7,8-TCDD, MC and PB. TCDD induced maximalactivity at a dose of approximately 50 nmole/kg of body weight and was about 1000times more potent than MC. MC-treated embryos exhibited no induction until 12 to 15hours after injection, were induced to maximal activity by 18 to 24 hours and rapidlyreturned to control levels 30 to 36 hours after administration (Hamilton et at., 1983).The AHH activity in chick embryo was inducible by TCDD and MC as early as 5 daysafter the start of incubation of the eggs and the degree of induciblity increasedsimutaneously with liver differentiation reaching adult levels by seven days afterincubation. After hatching, chick liver exhibited a sharp increase in AHH activitywhich peaked at one day of age at a level that was 1.5-fold higher than that in adultchickens. This activity declined to adult levels at three days of age.Treatment of adult female Japanese quail with MC at a dose of 150 mg/kg bodyweight/day (i.p. injection) for 4 consecutive days produced a 4.7-fold increase in totalcytochrome P450 content, a 12-fold increase in EROD activity, a 13.4-fold increase inAHH activity and a 6-fold increase in 7-ethoxycourmarin 0-deethylase activity(Carpenter et at., 1985). The activity of 7-ethoxycourmarin 0-deethylase is usuallyinduced by PB-type inducers in rats (Ronis and Walker, 1989). However, Neal et al.(1986) reported that injection of 15 mg of MC/kg body weight/day for 3 consecutivedays to adult male Japanese quail resulted in a 9.1-fold induction of total cytochromeINTRODUCTION /34P450 content accompanied by a 50% decrease in EORD activity.Husain et al. (1981) showed that injection of adult male pigeons Colyrnbidaewith 40 mg of MC/kg body weight/day for two consecutive days resulted in a 2.8-foldinduction of AHH activity in livers.At present, it is not clear whether MC induces avian forms of cytochrome P450that are equivalent to mammalian CYP1A1 and CYP1A2 (Sinclair J. and Sinclair P.,1993).1.4.2.2. PB-type inductionEarlier studies on the induction of cytochromes P450 by PB-type inducers inbirds indicated that cytochrome P450 enzymes are considerably less sensitive to PB-typeinducers in birds than in rats. In birds the evidence for PB-type induction is equivocal.Cytochrome P450 enzymes in some avian species appear to be less sensitive to PB-typeinducers than in other species. In some avian species cytochromes P450 appearcompletely unresponsive to this type of inducer.Schrittmatter and Umberger (1969) first reported the induction of cytochromeP450 by phenobarbital in chickens. They treated chick embryos with PB, 4-day oldchicks and 9-day old chicks at various doses ranging from 5, 10, and 15 mg per day forone or three days. At all stages of development, a four- to fivefold induction was foundin cytochrome P450 specific content. Powis et al. (1976) reported that in 7-day-oldchickens a single dose of PB (100 mg/kg, i.p.) resulted in a three- to fourfold increasein cytochrome P450 specific content and a two- and fourfold induction in anilinehydroxylase and aminopyrine N-demethylase activity, respectively, which reached amaximum level 24 hours after injection.As with MC-type inducers, the chick embryo has proved a valuable model forstudies of cytochrome P450 induction by PB-type inducers in birds (Ronis and Walker,1989). Althaus ci’ a!. (1979) reported that exposure of chick embryo hepatocytes to aINTRODUCTION /35high concentration (0.4 mg/mi) of PB in culture resulted in a threefold increase incytochrome P450 sepcific content. Moreover, they found that with the same treatmentAHH activity was induced by PB to the same level as when the cells were treated withMC (at 0.001 mg/ml). Hamilton et at. (1983) found that treatment with PB at a highdose caused some induction of AHH activity in chick embryo liver exposed in ovo tovarious chemicals.A number of studies have been done on Japanese quail. Two PB-type inducers,DDT and dieldrin, were found to have no inducing effect on cytochrome P450 contentin Japanese quail (Gillette et at., 1966; Gillette, 1969; Gillette and Arscott, 1969). Sifriet al. (1975) reported that DDT inhibited aniline hydroxylase activity by 60% andprolonged pentobarbital-induced sleeping time in adult male and female Japanese quail.Similarly, Buckpitt et al. (1982) found that cytochrome P450 specific content was notaffected by PB in adult male Japanese quail dosed with 100 mg of PB/kg bodyweight/day for 5 days. In addition, Carpenter et al. (1985) compared the effect ofdifferent doses of PB on cytochrome P450 induction in adult female quail. They foundthat a lower dose of PB (i.p. injection at 70 mg/kg body weight/day for 5 days) causedno change in cytochrome P450 specific content but resulted in a 4.6-fold increase inAHH activity and a 3.8-fold increase in 7-ethoxycoumarin 0-deethylase activity, whilea larger dose of PB (i.p. injection at 150 mg/kg body weight/day for 5 days), whichwas sufficient to briefly anesthetize the birds, resulted in only a 1.5-fold increase in thespecific content of cytochrome P450, a 2.3-fold increase in AHH activity and a 1.5-foldincrease in 7-ethoxycoumarin 0-deethylase activity.In addition to studies in the chickens and Japanese quail, induction ofcytochrome P450 by PB-type inducers has been also examined in pigeons. Peakall(1967) reported that DDT and dieldrin (10 ppm and 2 ppm, respectively, in the diet for1 week) increased the rate of testosterone metabolism by approximately 2.6- to 3.9-foldin male one-year-old pigeons and the rate of progesterone metabolism by approximatelyiNTRODUCTION /36threefold in one-year-old female pigeons. Husain et al. (1981) reported that treatmentwith PB at a dose of 40 mg/kg/day for 3 days resulted in a 2.2-fold increase in AHHactivity in adult male pigeons.The studies described above indicate that the avian cytochrome P450 enzymesindued by PB-type inducers had a different metabolic profile from those in rats. It isnot clear whether avian species contain cytochrome P450 enzymes that are analogues ofrat CYP2B1/2B2 which are the main forms induced by PB-type inducers.1.4.2.3. PCN-type inductionThe presence of a PCN-inducible cytochrome P450 in chicken liver was reportedby Lorr et al. (1989). In hepatic microsomes prepared from embroys and neonatalchickens that were untreated or treated with phenobarbital or 3,3’ ,4,4’-tetrachlorobiphenyl, a cytochrome P450 protein could be recognized by a monoclonalantibody (MAb 2-13-2) against PCN-induced rat CYP3A2 (Lorr et al., 1989) and itmigrated identically with rat CYP3A2 on an immunoblot. This chicken cytochromeP450 protein was detected in a higher amount in both untreated and treated chickens atone day posthatching than in the embryos and it was inducible by dexarnethasone andPB (Lorr et al., 1989). Avian microsomal erythromycin N-demethylase, an activitythat is characteristic of PCN-inducible rat cytochrome P450 enzymes, showed a similardevelopmental profile and was inducible by PB and dexamethasone. The level of avianerythromycin N-demethylase activity and the amount of avian immunoreactivecytochrome P450 protein were found to have a good correlation (r2 = 0.86) with eachtreatment and at all ages (Lorr et aL, 1989).1.4.2.4. Ethanol-type inductionEthanol significantly increased microsomal aniline hydroxylase activity andslightly induced p-nitrophenol hydoxylase activity in adult hen livers (Gupta and AbouINTRODUCTiON /37Donia, 1992). Aniline and p-nitrophenol are substrates that are selectively metabolizedby ethanol-induced cytochrome P450 enzymes, CYP2E1, in mammals (Gadeholt,1984). Similarly, in young chicks, p-nitrophenol hydroxylase and Nnitrosodimethylamine demethylase, another CYP2E1 specific activity, were induced byacetone, an ethanol-like cytochrome P450 inducer (Sinclair et at., 1990).1.4.2.5. Mixed-type inductionA number of studies have been done on the induction of cytochrome P450enzymes by various mixed-type inducers such as Aroclor 1254 (AR), BP-5, and 2-acetylaminofluorene in both wild birds and laboratory-reared birds. Bunyan and Page(1978) reported that AR treatment caused a fivefold induction of cytochrome P450content and a 3.8-fold increase in ethylmorphine N-demethylase activity in female four-week-old Japanese quail. Rinsky and Perry (1983) adminstrated AR to adult malechickens at a dose of 10 mg/kg/day in the diet for 2 days and found that the cytochromeP450 content and the activity of aminopyrine N-demethylase were increased fivefoldand 1.3-fold, respectively. Treatment of pigeons with Aroclor 1254 resulted in highlysignificant increases in hepatic cytochrome P450 content (11-fold), in the amount ofcytochrome P450 immunochemically related to rat CYP1A1 (24-fold) and in the activityof 7-ethoxyresorufin 0-deethylase (48-fold) (Borlakoglu et at., 1991). Rinsky andPerry (1981 and 1983) treated nestling barn owls (10-20 days) and adult male andfemale barn owls with a single dose of AR at 30 mg/kg of body weight (injected intoliver parenchymal tissue). They found that cytochrome P450 specific content wasinduced twofold in the nestlings and 4.2-fold in the adults, while aldrin epoxidase andaminopyrine N-demethylase activities were increased two- to threefold and three-tofourfold, respectively, in the nestlings and 3.3-fold and 3.8-fold, respectively, in theadults.Another PCB mixture, BP-5 was given to adult female quail and adult male andINTRODUCTION /38female buzzards at two oral doses five days and three days before sacrifice (Riviere etal., 1985). It was found that there was a 3.6-, 1.3-, and 1.8-fold increase incytochrome P450 specific content, 7-ethoxycoumarin 0-deethylase activity and ERODactivity, respectively, in the quail. However, there was only a 1.3-fold increase incytochrome P450 specific content and 7-ethoxycoumarin 0-deethylase activity and nochange in EROD activity in the buzzard.In chick embryos, 2-acetylaminofluorene (8 mg injected in ovo) was found toincrease cytochrome P450 specific content by 3.6-fold, AHH activity by 2.7-fold, andhexobarbital hydroxylase by 7.6-fold (Darby et al., 1984).The above results indicate that avian species responded to mixed-type inducers,however, the responses varied qualitatively and quantitatively with the species anddevelopmental stage of the treated birds. It seems that mixed-type inducers produced acytochrome P450 induction pattern which was not quite similar to that in rats.As seen above, many excellent studies have been done on the induction of aviancytochromes P450. Tremendous efforts have been put into the characterization of theinduction pattern of avian cytochromes P450 and comparisons of the avian inductionpatterns with the well-characterized patterns in rats. However, it is impossible to drawclear conclusions from the previous findings due to the following reasons: (1) Some ofthe monooxygenase activities used to characterize the induction pattern of aviancytochrome P450 enzymes are not highly specific, (2) there is not sufficient evidence onthe immunochemical relatedness of the induced avian cytochrome P450 enzymes andrats cytochrome P450 counterparts, and (3) there is no evidence on the enzyme-substratespecificities between the monooxygenase activities used and the induced aviancytochrome P450 enzymes.INTRODUCTION /391.5. PURPOSE OF TIlE PRESENT INVESTIGATIONThe overall goal of the present investigation is to examine the effects of threeprototype inducers, 3-methylcholanthrene (MC), phenobarbital (PB) and Aroclor 1254(AR), on cytochromes P450 in three domestic avian species. The specific objectives ofthis thesis are:(1) To examine the induction of hepatic cytochromes P450 by MC, PB and ARin the three bird species by measuring total hepatic cytochrome P450 content and fourcytochrome P450-mediated monooxygenase activities, 7-ethoxyresorufin 0-deethylase(EROD), aryl hydrocarbon hydroxylase also known as benzo[a]pyrene hydroxylase(AHH), 7-pentoxyresorufin 0-depentylase (PROD) and benzphetamine N-demethylase(BPND),(2) To compare the pattern of induction resulting from treatment with MC, PBand AR in the three avian species with that produced in rats,(3) To determine the immunochemical relatedness of individual forms of aviancytochrome P450 to various forms of rat cytochromes P450, and(4) To assess the contribution of individual form(s) of avian cytochrome P450enzymes to EROD, PROD and BPND activities in avian hepatic microsomes.402. EXPERIMENTAL2.1 ChEMicAlsChemicals and reagents which had been used in the present study were obtainedfrom the following sources:Aldrich Chemical Company Inc. (Milwaukee, Wisconsin, U.S.A.).ResorufinBDH Chemicals (Thronto, Ontario, Canada):Acetic acid, acetone, acetylacetone, ammonium acetate, Coomassie Brilliant Blue250, di-potassium hydrogen orthophosphate, di-sodium hydrogen orthophosphate,dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), glycine, hexane,magnesium chloride, methanol, phenobarbitone disodium salt (PB), potassium dihydrogenorthophosphate, potassium chloride, semicarbazide HCI, sodium chloride, sodiumdihydrogen orthophosphate, sodium hydroxide, sucrose, sulphuric acid, trichloroaceticacid.Bio-Rad Laboratories (Mississauga, Ontario, Canada).Acrylamide 99.9%, ammonium persuiphate 98%, BIS (N,N’-methylene-bisacrylamide), 2-mercaptoethanol, low range molecular weight standards (10-100 kD) forgel electrophoresis, sodium dodecyl sulphate (SDS) and TEMED (N,N,N’,N’tetramethylethylenediamine).Boehringer Mannheim Canada Ltd. (Laval, Quebec, Canada):Nicotinamide adenine dinucleotide phosphate tetra-sodium salt (NADPH, reducedform).EXPERIMENTAL /41Chemonics Scientjfic (Anachemia Canada Inc., Richmond, B. C., Canada)Glycerol, hydrochloric acid, hydrogen peroxide 30% solution and isopropanol.Eastman Kodak Company (Rochester, New York, U.S.A.):3-Methyicholanthrene.Fisher ScientJlc Ltd. (Vancouver, B. C., Canada):Bromphenol blue, formaldehyde (37% w/w), sodium bicarbonate and sodiumcarbonate.JCN Biomedicals Canada Ltd. (St. -Laurent, Quebec, Canada):Bovine serum albumin (globulin and fatty acid free, fraction V)J. T. Baker Chemical Co. (Phillipsburg, New Jersey, U.S.A.):Sodium dithionite.Mandel Scienqfic Company Ltd. (Edmonton, Alberta, Canada):Blotting paper and nitrocellulose membrane (Schleicher & Schuell).Medigas Pacific (Vancouver, B. C’., C’anada:Carbon monoxide gas (99.5% purity).Molecular Probes, Inc. (Eugene, Oregon, U.S.A.):7-ethoxyresorufin and 7-pentoxyresorufin.Sigma Chemical Company (‘St. Louis, Mossouri., U.S.A.):Benzo[a]pyrene (98%), 4-chloro-l-naphthol, corn oil, cupric sulfate, Folin andEXPERIMENTAL / 42Ciocalteu’s phenol reagent, HEPES, Pyronin Y (certified), quinine sulphate, sodiumpotassium tartrate, Trizma base, Trizma HC1 and Tween 20.Inter Medico. (Markham, Ontario, Canada):Affinity isolated goat F(ab’)2 anti-rabbit immunoglobulins, gamma and light chainspecific, human Ig absorbed, horseradish peroxidase conjugate and affinity isolated goatanti-mouse immunoglobulin, gamma and light chain specific, horseradish peroxidaseconjugate (Tago Immunologicals).Aroclor 1254 (AR) was kindly provided by Mr. Brock Chittim of WellingtonScientific (Guelph, Ontario) and benzphetamine HC1 was kindly provided by the Bureauof Dangerous Drugs, Health and Welfare Canada (Ottawa, Ontario).Dr. Paul E. Thomas (‘Depaiwzent of Chemical Biology and Pharmacognosy, The StateUniversity of New Jersey, Rutgers, Piscataway, New Jersey, U.S.A.):Rabbit anti-rat CYP1A1 sera (PT-19-154), mouse anti-rat CYP1A1 monoclonalantibody (mixture of C1.0, C4.0, C7.l and C8.0, PT-19-154), mouse anti-rat CYP2B1monoclonal IgG (fraction #9109-21) and mouse anti-rat CYP2B1/2 monoclonal IgG(fraction #28) and rabbit anti-rat CYP2C7 sera.Dr. S. M. Bandiera (Faculty of Pharmaceutical Sciences, UBC):Partially purified rat CYP1A1, purified rat CYP2B1, purified rat CYP2C7,CYP2C11, CYP2C13, CYP3A1 and purified epoxide hydrolase.Rabbit anti-rat CYP2B1 polyspecific IgG, rabbit anti-rat CYP2C7 monospecificIgG, rabbit anti-rat CYP2C7 polyspecific IgG, rabbit anti-CYP2C11 monospecific IgG,rabbit anti-rat CYP2C11 polyspecific IgG, rabbit anti-rat CYP2C13 monospecific IgG,rabbit anti-rat CYP2C13 polyspecific IgG, rabbit anti-rat CYP3A1 polyspecific IgG andEXPERIMENTAL /43extensively backabsorbed rabbit anti-rat CYP3A1 IgG.2.2. ANIMALsSince the overall goal of the present investigation was to examine the effects ofPB, MC and AR on the induction of avian cytochrome P450 enzymes, it was important toobtain birds whose diet and environment could be controlled under laboratory conditionsso as to preclude exposure to pollutants or other factors that might cause induction orinhibition of the avian cytochrome P450 system. Three domestic bird species weretherefore chosen for the study. White Leghorn chickens (Gallus gallus dornesticus),Japanese quail (Coturnix coturnix japonica) and domesticated Silver King pigeons(Columba livia) were bred at the Avian Teaching and Research Center (ATRC) at UBC.Adult female and male White Leghorn Chickens (Gallus gallus domesticus) were obtainedfrom Dr. Leslie Hart, Department of Animal Sciences, UBC. The chickens wereapproximately 30 to 40 weeks old and weighed about 1.5 to 2.0 kg each. Female andmale Japanese quail (Coturnix cotumix japonica) were hatched and raised in the QuailGenetic Stock Center (ATRC) at UBC and obtained from Dr. Kim Cheng, Department ofAnimal Sciences, UBC. Adult quail (approximately 6 weeks old) weighed approximately100 to 160 g each. Adult Silver King pigeons (Columba livia) were also obtained fromDr. Leslie Hart, Department of Animal Sciences, UBC. The pigeons were 6 to 16 monthsold and weighed from 420 to 600 g each. Since the gender of each pigeon could not bedetermined until they were sacrificed, each treatment group contained an unknown numberof both female and male birds. Approximately eight weeks before we were to begintreatment, the pigeons were diagnosed with a disease called chiamydiofif and were treatedwith tetracycline antibiotics for 2 weeks. After the antibiotic treatment was stopped, thesepigeons were fed normally for one month and then were used in our study. All birds werekept at the ATRC during the course of treatment. The birds were housed in wire meshcage units, maintained at constant temperature and were fed commercial diets.EXPERIMENTAL /44Adult Long Evans rats (both male and female), approximately 40 to 50 days oldexcept where indicated, were obtained from Charles River Canada Inc. (Montreal,Quebec). The rats were maintained at constant temperature (23 °C) on 12-hour light and12-hour dark cycles on corncob bedding in clear, polycarbonate cages. Rats were allowedfree access to Ralston-Purina Certified Rodent Chow 5001 and water.2.2.1. Untreated rats and birdsThe following table outlines the untreated groups of rats, chickens, quail andpigeons.Table 2.1. Untreated rats and birds.Species Sex Number Per TreatmentGroupRat male 6 nonefemale >30 noneChicken male 5 nonefemale 5 noneQuail male 5 nonefemale 6 nonePigeon female and 6 nonemale none2.2.2. Treatment of rats and birds with corn oilThe corn oil treatment groups were used as a vehicle because MC and AR weredissolved in it. The following table outlines the treatment of three avian species with cornEXPERIMENTAL /45oil. Corn oil was administered by intraperitoneal injection at the doses indicated below.The time between last dose of corn oil and sacrifice was one day.Table 2.2. Treatment of rats and birds with corn oil.Species Sex Number Per TreatmentGroupRat -------- noneChicken female 6 2 ml/kg body weight/dayfor three consecutive daysQuail female 6 2 mi/kg body weight/dayfor four consecutive daysPigeon female and 6 2 mi/kg body weight/daymale for four consecutive days2.2.3. Treatment of rats and birds with phenobarbital (PB)The following table outlines the treatment of rats and birds with PB. Phenobarbitalsodium salt was dissolved in distilled water and the resulting solution was administered byintraperitoneal injection at the doses indicated below. The time between last dose of PBand sacrifice was one day. Originally, there were six pigeons in the PB-treatment groupbut four pigeons in this treatment group were dead by the third treatment day and in orderto prevent any further deaths the remaining two were not given the fourth dose. Theywere sacrificed on the same day as the pigeons in the other treatment groups.EXPERIMENTAL /46Table 2.3. Treatment of rats and birds with PB.Species Sex Number Per TreatmentGroupRat male 5 75 mg/kg body weight/dayfor four consecutive daysChicken female 6 80 mg/kg body weight/dayfor three consecutive daysQuail female 6 80 mg/kg body weight/dayfor four consecutive daysPigeon female and 2 80 mg/kg body weight/daymale for three consecutive days2.2.4. Treatment of rats and birds with 3-methyicholanthrene (MC)The following table outlines the treatment of rats and birds with MC. MC wassuspended in corn oil and administered by intraperitoneal injection at the dose indicatedbelow. The time between last dose of MC and sacrifice was one day.2.2.5. Treatment of rats and birds with Aroclor 1254 (AR)Table 2.5 outlines the treatment of rats and birds with AR. Aroclor 1254 wasdissolved in corn oil and administered by intraperitoneal injection at the dose shownbelow. The rats in the AR-treatment group were 30 to 35 days old at the time oftreatment. The time between the last dose of AR and sacrifice was four days.EXPERIMENTAL /47Table 2.4. Treatment of rats and birds with MC.Species Sex Number Per TreatmentGroupRat male 5 25 mg/kg body weight/dayfor four consecutive daysChicken female 6 25 mg/kg body weight/dayfor three consecutive daysQuail female 6 25 mg/kg body weight/dayfor four consecutive daysPigeon female and 6 25 mg/kg body weight/daymale for four consecutive daysTable 2.5. Treatment of rats and birds with AR.Species Sex Number Per TreatmentGroupRat male 32 300 mg/kg bodyweight/day for one dayChicken female 6 300 mg/kg bodyweight/day for one dayQuail female 6 300 mg/kg bodyweight/day for one dayPigeon female and 6 300 mg/kg bodymale weight/day for one dayEXPERIMENTAL /482.3. MICROSOME PREPARATIONRats were killed by decapitation and birds were sacrificed by cervical dislocation.The livers were rapidly removed, weighed and minced in ice-cold 0.05 M Tris-HC1,1.15% KC1 buffer, pH7.5. Livers from the same treatment group were pooled together.Livers of each group were homogenized in Tris/KC1 buffer in a Potter-Elvehjem tissuegrinder by 5 slow-speed passes with a loose-fitting pestle and 5 high-speed passes with atight-fitting pestle. The homogenates were centrifuged at 9,000 x g for 20 minutes at 5 0Cin a Beckman centrifuge and the supernatants were collected by filtration through 4 layersof cheesecloth. The 9,000 x g supernatants were centrifuged at 105,000 x g for 60minutes at 5 0C and the pellets were saved. The 105,000 x g pellets were resuspended inice-cold 10 mM EDTA, 1.15% KC1 solution using a homogenizer apparatus (5 passeswith a loose-fitting pestle). The glycogen portion that accumulated on the bottom of thecentrifuge tubes and was discarded. The resuspended 105,000 x g pellets werecentrifuged at 105,000 x g for 1 hour at 5 0C and the pellets were saved and suspended ina small volume of ice-cold 0.25 M sucrose solution by gentle homogenization (theglycogen portion of the pellet was discarded). The resulting 105,000 x g microsomalpreparations were aliquoted into a series of labeled vials and frozen at -80 0C.2.4. DETERMINATION OF MICROSOMAL CYTOCHROME P450 CONCENTRATIONThe microsomes were diluted ten times in 0.1 M phosphate buffer containing 20%glycerol and 0.1 M EDTA (pH 7.4). The total cytochrome P450 concentration wasdetermined from the carbon monoxide difference spectra obtained by bubbling carbonmonoxide into dithionite-reduced microsomal suspensions and measuring the absorbanceof this suspension against the same dithionite-reduced microsomes as described by Omuraand Sato (1964a and 1964b).EXPERIMENTAL /492.5. DETERMINATION OF MICROSOMAL PiomiN CONCENTRATIONMicrosomal protein concentrations were determined by the method of Lowry et at.(1951) using bovine serum albumin (Fraction V, globulin and fatty acid free) as astandard. Absorbance was measured at 720 nm.2.6. ENZYMATIC ASSAYS2.6.1. 7-Ethoxyresorufin 0-Deethytase (EROD) AssaySince an assay for 7-ethoxyresorufin 0-deethylase was first reported by Burke andMayer in 1974, it has been widely used as a relatively specific marker for hepaticCYP1A1 (Burke et a!., 1985; Lubet et a!., 1990a and 1990b). The cytochrome P450catalyzed deethylation of 7-ethoxyresorufin is shown below:RoXOR — -OCH2CH3.Scheme 1. Illustration of 7-ethoxyresorufin 0-deethylation (adapted from Burke and Mayer, 1983).The EROD assay was based on the fluorometric methods previously described byBurke and Mayer (1974), Burke et a!. (1985), and Pohi and Fouts (1980), with minormodifications. The deethylation of 7-ethoxyresorufin was measured at 37 oc in afluorescence cuvette (1 cm path length) containing 1.93 ml of 0.1 M HEPES buffer (pH7.8, containing 5 mM MgC12 and 0.1 mM EDTA), 50 t1 of the diluted microsomalsolution, and 10 t1 of 1 mM 7-ethoxyresorufin. The mixture was preincubated for 2minutes at 37 OC• Then, the reaction was started by the addition of 10 tl of 50 mMNADPH solution, and the fluorescence was measured one, two or three minutes later byusing a Shirnadzu RF-540 spectrofluorometer with both excitation and emission slits set atEXPERIMENTAL / 505 nm. The excitation and emission wavelengths were set at 530 nm and 584 nm,respectively. The increase in fluorescence due to resorufin formation was recorded with aShiniadzu DR-3 data recorder. Microsomes were diluted in 0.25 M sucrose solution. Theamount of resorufin formed in the cuvette was calculated from the standard curve and theenzymatic activity was expressed as nmole of resorufin formed per mg protein per minute.A standard resorufin curve was obtained by plotting fluorescence against resorufinconcentration. For the standard curve, 7-ethoxyresorufin was replaced by variousconcentrations of resorufin in the same incubation mixture as that described above.Preliminary studies were carried out by using rat and chicken microsomes in orderto optimize the EROD assay conditions.2.6.2. Pentoxyresorufin 0-Depentylase (PROD) AssayThe enzymatic depentylation of pentoxyresorufin is illustrated belowR - o - 0 NA0PFç)ADPHO 0 _- 0O2 H20where R is -0C5H11.Scheme 2. Illustration of pentoxyresorufin 0-depentylation (adapted from Burke and Mayer, 1983)The method described by Lubet et al. (1985) was employed with a slightmodification. The fluorometric assay for the measurement of PROD activity is essentiallythe same as the EROD assay, except that 7-pentoxyresorufin replaces 7-ethoxyresorufin asthe substrate. In a typical reaction mixture, the initial concentrations of NADPH and 7-pentoxyresorufin were 250 jiM and 10 jiM, respectively. A preliminary study wasconducted using microsomes from rats and chickens to optimize the PROD assayconditions.EXPERIMENTAL / 512.6.3. Aly! Hydrocarbon Hydroxylase (AHH) AssayBenzo[a]pyrene (BaP) can be metabolized into many hydroxy- and quinonederivatives. A simplified diagram of BaP metabolism by microsomal cytochrome P450 isshown in Scheme 3 (adapted from Nebert and Gelboin, 1968).12 I7 6 5I Senzo[a]pyreneNADPH02 Enzyme SystemOther hydroxy -cnd Iqwnone Set, vat, i-es— —3 Hydroxybenzo!:o]pyreneScheme 3. Enzymatic conversion of BaP to hydroxylated metabolites (adapted from Nebert and Gelboin,1968).It was reported that BaP hydroxylation catalyzed by hepatic microsomes fromcontrol rats yielded the following metabolites at relative percentage: 3-hydroxy-BaP (3-OH-BaP), 36%; 9-hydroxy-BaP (9-OH-BaP), 3-13%; BaP-9, 1 0-diol, 15-25%; BaP-7, 8-diol, 12-14%; BaP-4,5-oxide, 8%; and BaP quinones, 14-17% (Yang and Kicha, 1978).The indirect spectrofluorometric assay of Nebert and Gelboin (1968) was employedwith slight modifications. A preliminary series of experiment was performed to test theAHH reaction conditions used by Nebert and Gelboin (1968) and to establish the standardenzyme reaction parameters for this assay in the present study. In a typical assay, a totalvolume of 1 ml of the reaction mixture contained 0.93 ml of 0.1 M potassium phosphatebuffer (pH 7.4, with 5 mM MgCl2 and 0. 1mM EDTA), 10 tI of 8 mM of BaP in DMSO,10 p1 of 200 mM NADPH, and 50 p1 of the diluted microsomal solution at a proteinconcentration of 2 mg/mi. Microsomes were diluted in 0.25 M sucrose. The reactionEXPERIMENTAL / 52mixture minus NADPH was preincubated at 37 °C in a water bath for 5 minutes. Thenthe reaction was started by adding NADPH and stopped after 4 minutes by adding 1 ml ofice-cold acetone. The hydroxylated metabolites including the major metabolite, 3-hydroxy-benzo[a]pyrene (3-OH-BP) were extracted in two steps. First, 3.25 ml of hexanewas added to the test tube containing the reaction mixture and the remaining substrates andmetabolites were extracted into the hexane phase by mixing the resulting solution on avortex mixer for 1 minute. Then the solution was centrifuged at 5,000 rpm for 10minutes. The top layer (aqueous phase) of the solution was discarded and 1 ml of thehexane phase (bottom layer) was transferred into a clean test tube. In the second step, themetabolites in the hexane phase were extracted with 3 ml of 1M NaOH solution by mixingthe solution for 1 minute. Then the solution was centrifuged at 5,000 rpm for 10 minutesand the top layer (hexane phase) was discarded. The fluorescence of 3-OH-BP in 1MNaOH was measured in a Shimadzu RF-540 spectrofluorometer with the excitationwavelength set at 394 nm, emission wavelength at 512 nm, excitation slit at 2 nm, andemission slit at 5 nm. The fluorometric parameters were tested to ensure that they wereoptimal for the measurement of 3-OH-BaP. Fluorescence was measured within 1 hourafter the reaction was terminated. A standard curve of 3-OH-BP was constructed byplotting fluorescence against a series of dilution of 3-OH-BaP in 3 ml of 1 M NaOH. Thefluorescence was linear with the amount of 3-OH-BP from 0 to 5 nmoles of 3-OH-BaP per3 ml of 1 M NaOH. The assays were run under dim light and with good ventilation.Optimal reaction time, BaP concentration, NADPH concentration and microsomal proteinconcentration were determined for both rat and avian microsomal preparations beforeassaying the samples. Blank determinations were also included, in which the proceduredescribed above was followed except that NADPH was omitted. The average AHHactivity of each sample was reported. The AHH activity was expressed as nmole of 3-OH-BP formed per rng protein per minute.EXPERIMENTAL /532.6.4. Benzphetamine N-Demethylase (BPND) AssayThe demethylation of benzphetamine catalyzed by microsomal cytochrome P450produces formaldehyde which can react with double strength Nash reagent (containing30% of ammonium acetate, 6% of acetic acid, and 4% of acetylacetone) to generateyellow products. The absorbance of the yellow products can be measuredspectrophotometrically at a wavelength of 412 nm.The procedure for the benzphetamine N-demethylase assay was developed frommethods described by Lu et al. (1969), Yoshimura et al. (1979) and Furuya et al. (1989).In a preliminary study, the enzyme reaction conditions described by Lu et al. (1969) weretested and modified to establish a standard procedure for the BPND assay in the presentstudy. In a typical assay, the reaction mixture contained 150 imo1es of potassiumphosphate (pH 7.5), 5 tmoles of semicarbazide HC1, 10 jimoles of MgCl,, 0.3 to 0.5 mgof microsomal protein, 1.5 tmole of benzphetamine MCI and 0.5 jimoles of NADPH in atotal volume of 1.5 ml. The reaction mixture was preincubated at 37 oc for 5 minutesprior to the addition of NADPH. The reaction was started by the addition of NADPH,and stopped 5 minutes later by adding 0.6 ml of 20% trichloroacetic acid with mixing.The reaction mixture was then centrifuged at 5,000 rpm for 10 to 15 minutes and 2 ml ofthe resulting clear supematant was transferred into a clean tube. The amount offormaldehyde produced was determined by the method of Nash (1953). One ml ofdouble-strength Nash reagent was added to 2 ml of supernatant and the mixture wasincubated at 58 °C in a water bath for 10 minutes. After the solution was cooled for 10minutes in the dark, the absorbance of the solution was measured at 412 nm in a ShirnadzuUV- 160 spectrophotometer. Two different blank determinations were performedfollowing the same procedure. One blank had substrate but no NADPH and the other hadNADPH but no substrate. The absorbance value at 412 nm were the same for bothblanks. The absorbance value of the blanks was subtracted from those of the test samples,and the net absorbance values were used to calculate the amount of formaldehyde formedEXPERIMENTAL / 54from a formaldehyde standard curve. The formaldehyde standard curve was constructedusing various concentrations of formaldehyde in the same incubation mixture as thatdescribed above. The absorbance at max 412 nm was found to be linearly proportional toformaldehyde concentrations ranging from 0.02 to 0.25 mM. The concentrations offormaldehyde formed from benzphetamine by hepatic microsomal samples were all below0.25 mM.2.6.5. Antibody Inhibition Study of the Four Enzymatic AssaysAntibodies against rat CYP1A1 (monoclonal and polyclonal), CYP2B1(polyspecific), CYP2C1 1 (monospecific and polyspecific), and CYP3A1 (polyspecific)were used to investigate the contribution of specific cytochrome P450 enzymes to thecytochrome P450-dependent monooxygenase activities of hepatic microsomes from birdsand rats. Assays for 7-ethoxyresorufin 0-deethylase, 7-pentoxyresorufin 0-depentylaseand benzphetamine N-demethylase were performed as described above, except that hepaticmicrosomes were preincubated with antibody for 30 minutes at room temperature beforeaddition of substrates and NADPH. Two different blank determinations were performedfor each enzyme assay. The activity of one blank was measured under the sameconditions but in the absence of any antibody, while the activity of another blank was alsomeasured under the same conditions but in the presence of control rabbit IgG which ispurified immunoglobulin obtained from rabbits that were not immunized with any purifiedrat cytochrome P450 proteins. The amounts of each antibody and control rabbit IgG usedin each assay are specified in the Results section.2.7. SDS-POLYACRYLAMEDE GEL ELECTROPHORESIS AND IMrVIUNOBLOT ASSAY2.7.1. ElectrophoresisSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed according to the method of Laemmli (1970) using a Hoefer SE 600 verticalEXPERIMENTAL /55slab gel unit. The discontinuous SDS-polyacrylamide gel consisted of a 3.0% acrylamidestacking gel (0.75 mm thick, 1 cm long) and a 7.5 % acrylamide separating gel (0.75 mmthick, 12.5 cm long). The stacking gel contained 0.125 M Tris-HC1 (pH6.8), 0.1% (w/v)SDS, 3 % (wlv) acrylamide-BIS, 0.08% (w/v) ammonium persuiphate and 0.05 % (v/v)TEMED and the separating gel consisted of 0.375 M Tris-HCI (pH6.8), 0.1 % (w/v) SDS,7.5% (w/v) acrylamide-BIS, 0.042% (w/v) ammonium persuiphate and 0.03% (v/v)TEMED. Microsomal samples were solubilized by boiling for exactly 2 minutes insample dilution buffer containing 0.062 M Tris-HC1 (pH 6.8) buffer, 1% (w/v) SDS, 10%glycerol, 0.001 % (w/v) bromophenol blue and 5% (v/v) mercaptoethanol. Microsomalproteins (10 to 20 tg/we11) were separated on the basis of molecular weight and net chargeby running the gels at constant current. In a typical electrophoresis experiment, the gelwas first run at 0.11 mA per gel for approximately 1 hour or until the dye front passedthrough the stacking gel and then at 0.24 mA per gel for approximately 2 hours, until thedye front reached the bottom of the separating gel. Microsomal proteins resolved on SDSPAGE were either stained using Coomassie blue R-250 stain or used for immunoblots. Inthe staining procedure, gels were first fixed for one hour in 25 % (v/v) isopropanol and10% (v/v) acetic acid solution, stained for one hour in 25 % (v/v) isopropanol, 10% (v/v)acetic acid and 0.05% (w/v) Coomassie blue and then destained in 10% (v/v) isopropanoland 10% (v/v) acetic acid for as long as required to produce a clear background.2.7.2. ImmunoblotsImmunochemical detection was performed using the method of Towbin et al.(1979). The proteins were first resolved on SDS-PAGE gel and then transferredelectrophoretically onto a 13 x 14 cm sheet of nitrocellulose using a Hoefer Transphorapparatus (Model TE 52) at a setting of 0.4 A for two hours at 4 °C. The nitrocellulosesheet was then blocked overnight at approximately 4 °C with blocking buffer consisting of1% (w/v) fatty acid-free bovine serum albumin (BSA), and 3% (w/v) skim milk powderEXPERIMENTAL / 56(Carnation brand, commercial product) in 0.1 M phosphate-buffered saline (lx PBS, pH7.4). The blocked nitrocellulose paper was rinsed with distilled water and then incubatedwith 50 ml of antibody dilution buffer (pH 7.4, 1% fatty acid-free BSA, 3% powderedskim milk, 1 % Tween 80) containing primary antibody against rat cytochrome P450 at 37°C with shaking for two hours. The primary antibody solution was discarded and thenitrocellulose sheet was washed with wash buffer (0.05% Tween 20 in 1xPBS, pH 7.4)three times (3 x 10 minutes) and then incubated with antibody dilution buffer containinghorseradish peroxidase-conjugated goat anti-rabbit or anti-mouse F(ab’)2 (1:1000 or1:3000 dilution as specified in the Results section) at 37 °C with shaking for at least twohours. The nitrocellulose sheet was washed three time as described above, and thenincubated with a substrate solution containing 0.018% (w/v) 4-chloro-1-naphthol and0.018% (v/v) 11202 in 1xPBS buffer at room temperature with shaking and then immersedin distilled water to stop the reaction. The length of the enzyme-substrate reaction variedwith from 5 to 20 minutes, depending upon the primary antibody.573. RESULTS3.1. TOTAL HEPATIC CYTocimorviE P450 CONTENTInitial studies were undertaken to determine the effect of a PB-type, MC-type andmixed-type inducer on total hepatic cytochrome P450 levels in the three spcies of birdsand to compare the effectiveness of these compounds among the avian species, as well aswith rats, one of the best studied mammalian model.Total cytochrome P450 was measured in hepatic microsomes prepared from rats,chickens, quail and pigeons treated with corn oil, phenobarbital (PB), 3-methyicholanthrene (MC) and Aroclor 1254 (AR), as described in the Experimentalsection. Each microsomal sample represents the suspended 105,000 x g pellet of liverhomogenates pooled from five to six birds (with one exception), or at least five rats, in thesame treatment group. For PB-treated pigeons, liver homogenates were pooled from onlytwo birds because four birds in this treatment group died. Treatment with corn oil, whichwas the vehicle for MC and AR, was arbitrarily induced as a secondary control group forMC and AR treament groups, while treatment with water, which was the vehicle for PB,was not included due to the limited number of birds available at the time. For all speciesexamined, except pigeons which are very difficult to differentiate sexually prior todissection, hepatic microsomes were also prepared from the male and female untreated, inorder to detect gender differences such as those that are known to occur in rats. The term‘untreated animals’ is used to describe animals that did not receive any compounds, whilethe term ‘control animals’ is referred to untreated animals and those treated with corn oil.The total spectrally determined cytochrome P450 content (nmole of cytochromeP450/mg of microsomal protein) of hepatic microsomes prepared from untreated andtreated rats, chickens, quail and pigeons is presented in Table 3.1.Comparison of total hepatic cytochrome P450 contents among untreated ratsreveals that microsomes from untreated male rats had a greater cytochrome P450 contentRESULTS /58than those from untreated female rats. The cytochrome P450 content was also found to behigher in untreated male chickens arid quail than in untreated female chickens and quail.Relative to the avian species, the cytochrome P450 content of hepatic microsomes fromuntreated male rats was 1.3- to 1.8-fold greater than that in untreated male chicken andquail. Similarly, the specific content in hepatic microsomes from untreated female ratswas 2.0- to 4.4-fold higher than in microsomes prepared from the female birds.Untreated female chickens had the lowest hepatic cytochrome P450 content of the threeavian species examined. The obsevation on total hepatic cytochrome P450 content inuntreated male and female rats and birds indicates that (1) rats had a higher basal level oftotal hepatic cytochrome P450 than chickens, quail, and pigeons, and (2) males had ahigher basal level of total hepatic cytochrome P450 than females in each species.Corn oil administered by intraperitoneal injection did not induce the hepaticcytochrome P450 content in quail and pigeons, but a 30% increase in the cytochromeP450 content was observed in corn oil-treated chickens.In rats, the total hepatic microsomal cytochrome P450 content was induced 2.1-,1.5- and 3.6-fold by PB, MC and AR, respectively. In the avian species, PB, MC andAR increased the total cytochrome P450 content by 4.1-, 5.3- and 2.4-fold, respectivelyin chickens, by 1.3-, 6.4- and 4.5-fold, respectively in quail, and by 1.3-, 5.6- and 3.9-fold, respectively in pigeons.RESULTS /59Table 3.1. Total cytochrome P450 content of hepatic microsomes preparedfrom rats and three bird species.Treatment Rat Chicken Quail PigeonUntreated (male) 0.93* 0.70 0.50(1.0) (4.4) (1.6)0.36*Untreated (female) 0.71 0.16* 0.3 1* (1.0)(1.0) (1.0)Corn Oil 0.21 0.27 0.29(1.3) (0.87) (0.81)PB 2.0 0.65 0.40 0.47(2.2) (4.1) (1.3) (1.3)MC 1.4 0.84 2.0 2.0(1.5) (5.3) (6.4) (5.6)AR 3.4 0.38 1.4 1.4(3.6) (2.4) (4.5) (3.9)Total cytochrome P450 content is expressed as nmole of spectrally determined cytochromeP450 per mg microsomal protein.Values in parenthesis indicate the fold increase by corn oil, PB, MC and AR relative to theappropriate untreated indicated by the symbol of .Treated rats were all male. Treated chickens and quail were all female. Hepatic microsomesfrom male and female pigeons were pooled together. Microsomes were prepared from a pooiof livers from either five to six rats or six birds.3.2. CYT0cEIR0ME P450-DEPENDENT MONOOXYGENASE AcTlvn’wsTo determine whether avian hepatic cytochrome P450 enzymes are induced byMC, PB and AR and whether the pattern of induction is similar to that in rats, birds andrats were treated with these three inducers as described in the Experimental section(Section 2.2.). The induction of individual cytochrome P450 enzymes is usually observedas an elevation in their associated monooxygenase activities. Therefore, fourRESULTS/60monooxygenase activities, specifically 7-ethoxyresorufin 0-deethylase (EROD) and arylhydrocarbon hydroxylase, also known as benzo[a]pyrene hydroxylase (AHH), which areinduced mainly by MC-type compounds in mammals, and 7-pentoxyresorufin 0-depentylase (PROD) and benzphetamine N-demethylase (BPND), which are typicallyinduced by PB-type inducers in rats, were investigated in order to assess the effect of eachinducer on avian cytochromes P450.These four cytochrome P450-dependent enzyme activities were measured in hepaticmicrosomes from untreated and treated rats, chickens, quail and pigeons. To ensure thateach enzyme reaction was run under optimal and reproducible conditions, a preliminarystudy was conducted to test the conditions of each enzyme assay so as to establish zeroorder reaction kinetics in which the rate of metabolite formation was linear with respect toboth microsomal protein concentration and reaction time and at saturating concentrationsof the substrate and cofactor, NADPH.3.2.1. The Preliminary Study---Optimizing Assay ConditionsThe preliminary study included the following aspects; (1) the effect of reactiontime on metabolite formation, (2) the effect of microsomal protein concentration on therate of metabolite formation, (3) the effect of the initial substrate concentration on enzymeactivity, and (4) the effect of the initial NADPH concentration on enzyme activity. Theinitial concentration was the concentration of the substrate or NADPH in the reactionmixture before the enzyme reaction was started. The reaction temperature (37 oc) andbuffer pH used were the same as those previously reported in the literature.3.2.1.1. 7-Ethoxyresorufin 0-Deethylase (EROD) Assay3.2.1.1.1. Resorufin formation versus reaction timeIn order to determine the effect of reaction time on resorufin formation, initialconcentrations of 5 iiM for 7-ethoxyresorufin and 250 iiM for NADPH , which are theRESULTS /61same as those reported by Pohi and Fouts (1980) and Burke et al. (1985), were used here.Figures 3.1. A and 3.1. B illustrate the effect of reaction time on resorufin formation byhepatic microsomes from male untreated chickens and female MC-treated chickens,respectively. When microsomes prepared from male untreated chickens were used in thereaction mixture (2 ml volume) at a protein concentration of 0.25 mg/mi, resorufinformation (nmole resorufin formed per mg protein) increased linearly with time for up to6 minutes (Figure 3.1 .A). When microsomes prepared from female MC-treated chickenswere present in the reaction mixture at a protein concentration of 0.125 mg/mi, theformation of resorufin from 7-ethoxyresorufin was found to be linear with time forapproximately 4 minutes and reach a plateau after 6 minutes (Figure 3.1. B).Consequently, a reaction time of two to three minutes was used for microsomes fromcontrol and PB-treated rats and birds, and a reaction time of two minutes or less was usedfor the measurement of 7-ethoxyresorufin 0-deethylase activity in microsomes from MC-and AR-treated birds and rats.3.2.1.1.2. Rate of 7-ethoxyresorufin 0-deethylation versus microsornal proteinconcentrationThe initial concentrations of 7-ethoxyresorufin and NADPH were the same as thosestated above. Figures 3.2.(A) and (B) show that the rate of 7-ethoxyresorufin deethylationincreased linearly with protein concentration from 0.05 to 0.25 mg/mi for microsomesfrom both male untreated and female MC-treated chickens. Therefore, a proteinconcentration of 0. 125 mg/ml was chosen for microsomes prepared from MC- and AR-treated rats and birds. When microsomes prepared from untreated chickens were assayedat a protein concentration of 0.125 mg/mi, the fluorescence reading was very low andunstable. When the protein concentration of these microsomes was increased to 0.25mg/mi, a more readily measured and stable fluorescence reading was obtained. Thus, afinal protein concentration of 0.25 mg/mi was chosen for microsomes from untreated,RESULTS/62corn oil-treated, and PB-treated rats and birds.3.2.1.1.3. EROD activity versus 7-ethoxyresorufin concentrationThe effect of varying the initial 7-ethoxyresorufin concentration on the deethylationof substrate by hepatic microsomes from male untreated and female MC-treated chicken isshown in Figures 3.3.A and 3.3.B. In these experiments, the initial NADPHconcentration was 250 tM and the microsomal protein concentration was 0.25 mg/mi and0.125 mg/ml for microsomes from untreated and MC-treated chickens, respectively.Figure 3.3. shows that the deethylation of 7-ethoxyresorufin reached its maximal levelwhen substrate concentrations were greater than 3 riM. Therefore, an initial substrateconcentration of 5 liM was chosen for all EROD assays because at this concentration theactivity was independent of the initial concentation of 7-ethoxyresorufin.3.2.1.1.4. EROD activity versus NADPH concentrationNADPH is a cofactor for many cytochrome P450-dependent monooxygenases.Microsomes from MC-treated chickens were used to examine the effect of various initialNADPH concentrations on EROD activities (Figure 3.4). When NADPFI was absentfrom the reaction mixture, there was no change in fluorescence, indicating that no reactionoccurred. When this cofactor was included in the reaction mixture, EROD activityincreased linearly with initial NADPH concentrations up to 50 iiM. At NADPHconcentrations greater than 50 iiM, EROD activity continued to increase but much moregradually. Thus, an initial NADPH concentration of 250 iiM was selected for all ERODassays, as the best compromise between near maximal activity and the high cost ofNADPH.RESULTS /63(A)4.0-3.5/Ao 2.0 /AA/1.5- A10--A male untreatedA chicken0.5 A0.0 I I0 2 4 6 8 10 12 14 16Time (minute)(B)25 I I I I I20-. a //oU) t15-0o.2 10 - --4-// • female MC—treated•chicken0i’’ I I I I I I0 1 2 3 4 5 6 7 8 9 1011Time (minute)Figure 3.1. The effect of reaction time on resorufin formation from 7-ethoxyresorufin.The initial concentrations of the substrate and NADPH in 2 ml of reaction mixture were 5p.M and 250 jiM, respectively. Microsomes from male untreated (A) and female MC-treated (B) chickens were used at final protein concentrations of 0.25 and 0.125 mg/mi ofreaction mixture, respectively. The reaction temperature was 37 oC.4.)o0-. .—C(120) Q)—( C(B).4))CC0.1 0.2 0.3 0.4 0.5 0.6Protein Concentration (mg/mi)Figure 3.2. The effect of microsomal protein concentration on the rate of resorufinformation from 7-ethoxyresorufin. The initial concentrations of the substrate and NADPHin 2 ml of reaction mixture were 5 p.M and 250 p.M, respectively. Microsomes from maleuntreated (A) and female MC-treated (B) chickens were incubated in the presence of thesubstrate and NADPH for 2 minutes. The reaction temperature was 37 0C.(A)I I IRESULTS /640.50.40.30.20.10.04.0.0//A male untreatedc hi eke n//0. 0.2 0.3 0.4 0.5Protein Concentration (mg/mi)5 I I4320 -0.0• female MC—treatedchickenL I-eQ ciiq.)0-4- -eC.) ci)0o0ci)ci)0Figure 3.3. The effect of 7-ethoxyresorufin concentration on 7-ethoxyresorufin 0-deethylase activity (EROD). The initial concentration of NADPH in 2 ml of reactionmixture was 250 p.M. The final protein concentrations of microsomes from maleuntreated (A) and female MC-treated (B) chickens were 0.25 and 0.125 mg/mi of reactionmixture, respectively. The reaction time was 2 minutes. The reaction temperature was 37oc.RESULTS/65(A)(B)0.300.250.200.150.100.050,0060 1 2 3 4 5 6 7 8 9 1011Substrate Concentration (tM)5432100 2 4 6 8 10 12 14 16 18 20Substrate Concentration (jiM)RESULTS /66H00.)1 • female MC—treated chicken0 I I I I I I0 50 100 150 200 250 300 350 400 450NADPH Concentration (tiM)Figure 3.4. The effect of NADPH concentration on 7-ethoxyresorufin 0-deethylaseactivity (EROD). The initial concentration of 7-ethoxyresorufin in 2 ml of reactionmixture was 5 riM. The final protein concentration of microsomes from female MCtreated chickens was 0.125 mg/mi of reaction mixture. The reaction time was 1 minute.The reaction temperature was 37 0C.RESULTS / 673.2.1.1.5. Summary of EROD assay conditionsIn summary, the initial concentrations of 7-ethoxyresorufin and NADPH present inthe reaction mixture (2.0 ml volume) were 5 iM and 250 tM, respectively, and theprotein concentration was 0.125 mg/mi for microsomes from MC- and AR-treated ratsand birds and 0.25 mg/ml for other microsomes. A reaction time of two minutes or lesswas used for microsomes from MC- and AR-treated rats and birds and a reaction time oftwo to three minutes was used for other microsomes. EROD activities were expressed asnmole of resorufin formed per mg protein per minute.3.2.1.2. Aryl Hydrocarbon Hydroxylase (AHH) Assay3.2.1.2.1. Formation of 3-hydroxy-benzo[aJpyrene versus reaction timeFigure 3.5 illustrates the effect of reaction time on the formation of 3-hydroxy-benzo[a]pyrene (3-OH-BaP) from benzo[ajpyrene (BaP) by liver microsomes from maleMC-treated rats in the AHH assay. The initial concentrations of BaP and NADPH in thereaction mixture (1 ml volume) were 80 iM and 120 tiM, respectively. The microsomalprotein concentration was 0.1 mg/mi. As shown, under these conditions the formation of3-OH-BaP was linearly proportional to reaction time for up to 4 minutes.3.2.1.2.2. Rate of 3-OH-RaPformation versus microsomal protein concentrationsFigures 3.6.A and 3.6.B show the effect of increasing microsomal proteinconcentration on the rate of 3-OH-BaP formation from BaP by hepatic microsomes fromfemale untreated chickens and male MC-treated rats. The initial concentrations of BaPand NADPH were 80 tM and 120 jiM, respectively. With hepatic microsomes fromfemale chickens, the rate of 3-OH-BaP formation increased linearly with proteinconcentration up to 0.3 mg/mi (Figure 3.6.A). With hepatic microsomes from MC-treated rats, the rate of 3-OH-BaP formation increased linearly with the proteinconcentration only up to 0. 1 mg/mi (Figure 3.6.B). Therefore, a protein concentration ofRESULTS/680.1 mg/mI was chosen for both untreated and treated microsomes.3.2.1.2.3. AHH Activity versus benzolajpyrene concentrationFigure 3.7 shows the effect of varying the initial BaP concentration in the reactionmixture on benzo[a]pyrene 3-hydroxylase activity (AHH) of microsomes from male MC-treated rats. The initial concentration of NADPH was 120 riM, and the microsomalprotein concentration was 0.1 mg/ml. As shown, AHFT activity was maximal when theinitial concentration of BaP was greater than 30 riM. Therefore, an initial BaPconcentration of 80 tM was selected because this substrate concentration was saturating.3.2.1.2.4. AHH Activity versus NADPH concentrationFigure 3.8. shows the effect of various initial NADPH concentration on AHHactivity of microsomes from female MC-treated chickens. The initial BaP concentrationwas 80 p.M and the microsomal protein concentration was 0.1 mg/ml. The product, 3-OH-BaP, was not detected when NADPH was absent from the reaction system. Asshown, AHH activity was maximal with an initial NADPH concentration of between 100and 200 p.M. Thus, an initial NADPH concentraion of 120 p.M was selected.3.2.1.2.5. Summary ofAHH assay conditionsIn summary, a typical AHH assay was performed under the following conditions in37 oC water bath; (1) initial concentrations of benzo[a]pyrene and NADPH were 80p.M and 120 p.M, respectively, (2) microsomal protein concentration was 0.1 mg/mI, and(3) reaction time was 4 minutes.RESULTS/69201612S-4oS0040S0Figure 3.5. The effect of reaction time on the formation of 3-hydroxy-benzo[a}pyrene (3-OH-BaP) from benzo[a]pyrene (BaP). The initial concentrations of BaP and NADPH in 1ml of reaction mixture were 80 iiM and 120 aiM, respectively. Microsomes from maleMC-treated rats were used at a final protein concentration of 0.1 mg/mi of reactionmixture. The reaction temperature was 37 0C.0 5 10 15 20 25Time (minute)RESULTS /70(A)CCICC/VV female untreatedchicken0.4Protein0.0.300.0250.0200.0150.0100.0050.0000.00.60.50.40.30.20.10.00,00.8 1.2 1.6 2.0Concentration (mg/mi)(B)CSo,0.2 0.4 0.6 0.8 1.0Protein Concentration (mg/mi)Figure 3.6. The effect of microsomal protein concentration on the rate of 3-hydroxy-benzo[a]pyrene (3-OH-BaP) formation from benzo[a]pyrene (BaP). The initialconcentrations of BaP and NADPH in 1 ml of reaction mixture were 80 .tM and 120 iiM,respectively. Microsomes from female untreated chickens (A) and male MC-treatecl rats(B) were incubated in the presence of BaP and NADPH for 4 minutes. The reactiontemperature was 37 0CRESULTS /712.52.0•- D...?C.)S1.00.5S0.0BaP Concentration ([M)Figure 3.7. The effect of benzo[a]pyrene (BaP) concentration on benzo[a]pyrenehydroxylase activity (AHH). The initial concentration of NADPH in 1 ml of reactionmixture was 120 .tM. The final protein concentration of microsomes from male MCtreated rats was 0.1 mg/mi of reaction mixture. The reaction time was 4 minutes. Thereaction temperature was 37 0C.0 20 40 60 80 100RESULTS / 722.52.0Sc 1.5C.)1.0CcD) 0.5CS0.0500NADPH Concentration (tM)Figure 3.8. The effect of NADPH concentration on benzo[a]pyrene hydroxylase activity(AHH). The initial concentration of benzo[a]pyrene in 1 ml of reaction mixture was 80iiM. The final protein concentration of microsomes from female MC-treated chickens was0.1 mg/mi of reaction mixture. The reaction time was 4 minutes. The reactiontemperature was 37 OC.0 100 200 300 400RESULTS/733.2.1.3. 7-Pentoxyresorufin 0-Depentylase (PROD) Assay3.2.1.3.1. Resorufinforination versus reaction timeThe assay is based on the method described by Lubet et al. (1985) and Burke et al.(1985), and the same initial concentrations of 7-pentoxyresorufin (10 M) and NADPH(250 pM) were used here. Though these investigators reported that PROD reaction waslinear with respect to time for up to 5 minutes, they did not specify the proteinconcentration that they used. Chang et al. (1992) used protein concentrations of 0.15 and0.05 mg/mi for hepatic microsomes from control and PB-treated rats, respectively.However, in the present study, the fluorescence of resorufin formation was very difficultto measure when using microsomes from untreated and PB-treated birds due to extremelylow and unstable absorbance readings. This problem persisted until the proteinconcentration of the microsomes was increased to 0.25 mg/mi. Another difficulty thatwas encountered with microsomes from control animals and PB-treated birds was that thefluorescence of the reaction mixture initially decreased for about two to three minutes afterNADPH was added to the mixture and then increased. In order to standardize the assay,the point when fluorescence stopped decreasing and started to increase was assumed to bethe zero time for the start of the reaction for those particular microsomes.Figures 3.9.A and 3.9.B show the effect of reaction time on resorufin formation bymicrosomes from male untreated chickens and male PB-treated rats, respectively. Whenmicrosomes from male untreated chickens were used at a protein concentration of 0.25mg/mi, the formation of resorufin was linear with time up to at least ten minutes (Figure3.9.A). Resorufin formation was found to increase linearly with reaction time forapproximately two minutes when the reaction mixture (2 ml volume) containedmicrosomal protein from PB-treated rats at a concentration of 0.125 mg/mi (Figure3.9.B). Therefore, the change in fluorescence after the first minute was used forcaicuiating PROD activities in microsomes from PB- and AR-treated rats as well as MCand AR-treated birds, while fluorescence measured at 10 minutes or less after theRESULTS /74standardized zero time of reaction was used for other microsomes.3.2.1.3.2. Rate of pentoxyresorifin 0-depentylation versus microsornal proteinconcentrationFigure 3. 1O.A and 3. 10.B show the effect of varying the microsomal proteinconcentration on the rate of resorufin formation from pentoxyresorufin in microsomesfrom PB-treated female chickens (A) and PB-treated male rats (B), respectively. Theinitial concentrations of pentoxyresorufin and NADPH used were the same as above.When microsomes from PB-treated chickens were used, the rate of resorufin formationwas found to be extremely low and it was linearly proportional to microsomal proteinconcentrations up to at least 0.375 mg/mi (Figure 3. l0.A). When microsomes from PB-treated rats were used, PROD activity was found to increase linearly with microsomalprotein concentrations up to 0.25 mg/mi (Figure 3. 10.B). Thus, the protein concentrationin the reaction mixture was routinely set at 0.25 mg/mi for microsomes from control ratsand control birds as well as PB-treated birds, and 0.125 mg/mI for liver microsomes fromall other treated rats and birds.3.2.1.3.3. PROD Activity versus pentoxyresorujin concentrationThe effect of pentoxyresorufin concentration on PROD activity was examined byvarying the initial substrate concentration from 0 to 20 jiM (Figure 3.11) in a reactionmixture containing hepatic microsomes from male PB-treated rats. The increase of PRODactivity was very sharp when the initial concentration was below 5 tM, and it was sloweddown when pentoxyresorufin concentration was above 5 riM. Thus, 10 tM was chosen asthe initial pentoxyresorufin concentration in the reaction mixture for all PROD assays.RESULTS /753.2.1.3.4. PROD activity versus NI4DPH concentrationFigure 3.12 shows the effect of varying the initial NADPH concentration on thePROD activity of microsomes from male PB-treated rats. The 0-depentylation ofpentoxyresorufin could not be achieved without NADPH in the reaction mixture. In thereaction mixtures containing 0.125 mg/mi of microsomal protein and 10 tM ofpentoxyresorufin, PROD activity increased linearly with the initiai NADPH concentrationsup to 50 ElM, and it reached a plateau when the initial concentration was greater than 100iiM. Thus, the initial concentration of NADPH in the reaction mixtures was set at 250iiM since at this concentration NADPH was saturating.3.2.1.3.5. Summary of PROD assay conditionsIn summary, the initial concentrations of pentoxyresorufin and NADPH in thereaction mixtures were kept at 10 ElM and 250 tM, respectively, for all microsomalsamples. The protein concentrations were 0.125 mg/ml for microsomes from PB-treatedrats and MC- and AR-treated rats and birds, and 0.25 mg/ml for microsomes fromuntreated rats and birds as well as corn oil- and PB-treated birds. A reaction time of 1minute was chosen for microsomes from PB- and AR-treated rats as well as MC- and ARtreated birds, while a reaction time of 10 minutes or less after the standardized zero timeof reaction was used for other microsomes.RESULTS /76(A)oci, -G).4.)0CI)00S0.30 I I I I0.25--0.20- _A-0.15- /-/A0.10- A-// A male untreatedA chicken0.05- /-AI I5 10 15 20 25 30 35 40Time (minute)108765432(B)0oCI)0oCFigure 3.9. The effect of reaction time on resorufin formation from 7-pentoxyresorufin.The initial concentrations of the substrate and NADPH in 2 ml of reaction mixture were10 p.M and 250 p.M, respectively. Microsomes from male untreated chickens (A) andfemale PB-treated rats (B) were used at final protein concentrations of 0.25 and 0.125mg/mi of reaction mixture, respectively. The reaction temperature was 37 oC.00 1 2 3 4 5 6 7 8 9 1011Time (minute)RESULTS /77(A)• 0.004.—0.003Co 0.002(I]CCl)o- 0.001c C0.0000,0 0.4(B) 1.50‘ 1.00.50 ciI)0.00.0 0.1 0.2 0.3 0.4 0.5 0.6Protein Concentration (mg/mi)Figure 3.10. The effect of microsomal protein concentration on the rate of resorufinformation from 7-pentoxyresorufin. The initial concentrations of the substrate andNADPH in 2 ml of reaction mixture were 10 jiM and 250 jiM, respectively. Microsomesfrom female PB-treated chickens (A) and male PB-treated rats (B) were incubated in thepresence of substrate and NADPH for 10 minutes and 1 minute, respectively. Thereaction temperature was 37 0C.0.1 0.2 0,3Protein Concentration (mg/mi)RESULTS/782.5 I I I I I I2.0/1.00.5 jD male PB—treated rat0.0LJ I I I I I I I0 2 4 6 8 10 12 14 16 18 20 22Pentoxyresorufin Concentration (,uM)Figure 3.11. The effect of 7-pentoxyresorufin concentration on 7-pentoxyresorufin 0-depentylase activity (PROD). The initial concentration of NADPH in 2 ml of reactionmixture was 250 tM. The protein concentration of microsomes from male PB-treated ratswas 0.125 mg/mi. The reaction time was 1 minute. The reaction temperature was 37 0C.RESULTS /792.5 I I I I I I2.0I D0.5-/LI male PB—treated rat0.OLJ I I I I I I0 50 100 150 200 250 300 350 400 450NADPH Concentration uM)Figure 3.12. The effect of NADPH concentration on 7-pentoxyresorufin 0-deethylaseactivity (PROD). The initial concentration of 7-pentoxyresorufin in 2 ml of reactionmixture was 10 .tM. The protein concentration of microsomes from male PB-treated ratswas 0.125 mg/mi. The reaction time was 1 minute. The reaction temperature was 37 0C.RESULTS / 803.2.1.4. Benzphetamine N-Demethylase (BPND) Assay3.2.1.4.1. Formaldehyde formation versus reaction timeFigures 3. 13.A and 3. 13.B illustrate the effect of reaction time on formaldehydeformation from N-demethylation of benzphetamine (BPND) by microsomes prepared frommale untreated and PB-treated rats, respectively. The initial concentrations ofbenzphetamine and NADPH in the reaction mixture (1.5 ml volume) were 1.0 mM and0.33 mM, respectively. The protein concentrations for microsomes from untreated andPB-treated rats were 0.33 mg/mi. Under these conditions, the formation of formaldehydewas found to increase linearly with time for up to 20 minutes with microsomes fromuntreated rats (Figure 3. 13.A) and for approximately five minutes with microsomes frommale PB-treated rats (Figure 3. 13.B). Thus, a 5 minute reaction time was used formeasuring BPND activities of untreated and treated rat and avian microsomes.3.2.1.4.2. Rate of benzphetamine N-demethyiation versus microsomal proteinconcentrationFigures 3. 14.A and 3. 14.B show the effect of varying protein concentrations onthe rate of formaldehyde formation using microsomes from female PB-treated chickens(Figure 3. 14.A) and male PB-treated rats (Figure 3. 14.B). The initial concentrations forbenzphetamine and NADPH were 1.0 mM and 0.33 mM, respectively. Whenmicrosomes from PB-treated chickens were used, the reaction rate was linearlyproportional to microsomal protein concentrations up to 0.67 mg/mi. With microsomesfrom PB-treated rats, the rate of formaldehyde formation was linear with proteinconcentrations up to 0.33 mg/mi.Because the reaction rate should be linear with protein concentration, a microsomalprotein concentration of 0.2 to 0.33 mg/mi in the reaction mixtures was selected for allmicrosomal samples.RESULTS/813.2.1.4.3. BPND Activity versus benzphetarnine concentrationFigure 3. 15.A and 3. 15.B show the effect of varying initial benzphetamineconcentrations on benzphetamine N-demethylase (BPND) activity of hepatic microsomesfrom male untreated and PB-treated rats, respectively. The initial NADPH concentrationwas 0.33 mM, and the protein concentrations for microsomes from male untreated andPB-treated rats were 0.33 mg/mi and 0.2 mg/mi, respectively. BPND activity for bothmicrosomes was found to reach a maximum when the initial concentration ofbenzphetamine was greater than 0.67 mM. Thus, an initial benzphetamine concentrationof 1.0 mM was chosen.3.2.1.4.4. BPND Activity versus NADPH concentrationFigure 16 shows the effect of varying initial NADPH concentrations on BPNDactivity of microsomes from male PB-treated rats. The initial concentration ofbenzphetamine was 1.0 mM and the microsomal protein concentration was 0.2 mg/mi.BPND activity was maximal when initial NADPH concentrations were greater than 0.13mM. Thus, an initial NADPH concentration of 0.33 mM was selected.3.2.1.4.5. Summary ofBPND assay conditionsIn summary, the initial concentrations of benzphetamine and NADPH were 1.0mM and 0.33 mM, respectively, the microsomal protein concentration was 0.2 mg/mi,and the reaction time was 5 minutes.CC- D— L.)oSC.-I-4-> *>‘.4o)-e- >13>1)440CSTime (minute)Figure 3.13. The effect of reaction time on formaldehyde (HCHO) formation frombenzphetamine. The initial concentrations of the substrate and NADPH in 1.5 ml ofreaction mixture were 1 mM and 0.33 mM, respectively. Microsomes from maleuntreated (A) and PB-treated (B) rats were used at a protein concentration of 0.33 mg/mlof reaction mixture. The reaction temperature was 37 0C.RESULTS / 82(A)(B)2001 751501 251 0075502503503002502001 501 005000 5 10 15Time (minute)20 250 5 10 15 20 25o0o 0L) CL)) c:1)—00C dC4- c)—C040353025201510500.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Protein Concentration (mg/mi)RESULTS / 83(A)(B) 3025201510500.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Protein Concentration (mg/mi)Figure 3.14. The effect of microsomal protein concentration on the rate of formaldehyde(HCHO) formation from benzphetamine. The initial concentrations of the substrate andNADPH in 1.5 ml of reaction mixture were 1 mM and 0.33 mM, respectively.Microsomes from female PB-treated chickens (A) and male PB-treated rats (B) wereincubated in the presence of the substrate and NADPH for 5 minutes. The reactiontemperature was 37 OC.. .C).1 szCC-)0Sci)C; -CC-)a)0SFigure 3.15. The effect of benzphetamine concentration on benzphetamine Ndemethylase activity (BPND). The initial concentration of NADPH in 1.5 ml of reactionmixture was 0.33 mM. The final protein concentrations of microsomes from maleuntreated (A) and PB-treated (B) rats were 0.33 and 0.2 mg/mi of reaction mixture,respectively. The reaction time was 5 minutes. The reaction temperature was 37 oC.RESULTS / 84(A)(B)876543200.0 0.5 1.0 1.5 2.0 2.5 3.0 3,5Benzphetamine Concentration (mM)353025201510500.0 0.5 1.0Benzphetamine1.5 2.0 2.5 3.0Concentration (mM)RESULTS/852520a)S.4.)a)*.)Ca)a)S 500.0 0.6Figure 3.16. The effect of NADPH concentration on benzphetamine N-demethylaseactivity (BPND). The initial concentration of benzphetamine in 1.5 ml of reactionmixture was 1 mM. The final protein concentration of microsomes from male PB-treatedrats was 0.2 mg/mi of reaction mixture. The reaction time was 5 minutes. The reactiontemperature was 37 0C.0.1 0.2 03 0.4 0.5NAIJPH Concentration (mM)RESULTS / 863.2.2. Measurement of Monooxygenase Activities of Rat and Avian HepaticMicrosomes7-Ethoxyresorufin 0-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), 7-pentoxyresorufin 0-depentylase (PROD) and benzphetamine N-demethylase (BPND)activities were measured in hepatic microsomes prepared from untreated and treated rats,chickens, quail and pigeons using the optimal assay conditions determined above.Enzyme activities are expressed as nmole metabolites formed per mg of microsomalprotein per minute.3.2.2.1. 7-Ethoxyresorufin 0-Deethylase Activity (EROD)The effects of treatment with PB, MC and AR on hepatic EROD activity in the ratand three avian species are summarized on Table 3.2.Examination of the data for the untreated groups shows that male chickens had 4-fold greater hepatic EROD activity than female chickens. Male quail also showed 2.6-fold greater hepatic EROD activity than female quail. The gender difference in hepaticEROD activity was much smaller in rats than in the birds.Within the treated groups, EROD activity in hepatic microsomes prepared fromPB-, MC- and AR-treated rats was found to be approximately 3.6-, 56- and 74-foldgreater, respectively, than the activity of microsomes from untreated male rats.For female chickens, EROD activity appeared to be slightly decreased in hepaticmicrosomes prepared from corn oil- and PB-treated birds compared to the untreatedgroup, but it was increased approximately 72- and 30-fold in microsomes from MC- andAR-treated birds, respectively.For female quail, the EROD activity of microsomes from the corn oil-treatedgroup was not markedly different from that of untreated group, but a 57% decrease in thisactivity was observed with the PB-treated group. Hepatic EROD activities of quailmicrosomes were increased 40- and 36-fold after treatment with MC and AR,RESULTS/87respectively.With microsomes pooled from both male and female pigeon livers, hepatic ERODactivities of corn oil- and PB-treatment groups were either slightly lower than or similar tothe activity of the untreated group, while a 5.7-fold increase in EROD activity wasobtained after MC- and AR-treatment.Table 3.2. 7-Ethoxyresorufin 0-deethylase activities (nmole/mgproteinminute) of hepatic microsomes prepared from rats and three birdspecies.Treatment Rat Chicken Quail PigeonUntreated (male) 0.105* 0.302 0.298(1.0) (4.0) (2.6)0.192*Untreated (female) 0.191 0.0749* 0.114* (1.0)(1.8) (1.0) (1.0)Corn Oil 0.0631 0.0940 0.116(0.84) (0.82) (0.60)PB 0.381 0.0583 0.0493 0.183(3.6) (0.78) (0.43) (0.95)MC 5.84 5.42 4.61 1.09(56) (72) (40) (5.7)AR 7.80 2.24 4.08 1.09(74) (30) (36) (5.7)Values in parenthesis indicate the fold of increase by corn oil, PB, MC, and AR relative tothe appropriate untreated group indicated by the symbol of ‘.Treated rats were all male. Treated chickens and quail were all female. Hepatic microsomesfrom male and female pigeons were pooled together. Microsomes were prepared from a poolof livers from either five to six rats or six birds.The activity of each microsomal preparation was determined on at least two separateoccassions and in duplicate each time. Values shown are the average of all determinations.RESULTS/88These results indicate that; (1) corn oil did not markedly affect avian hepaticEROD activity, (2) MC and AR increased hepatic EROD activity in the three avianspecies and in rats, and (3) PB appeared to slightly repress this monooxygenase activity infemale quail.3.2.2.2. Aryl Hydrocarbon Hydroxylase Activity (AHH)The data for hepatic AHH activity of both rat and avian microsomes are presentedin Table 3.3. Comparison of the activity among the untreated groups shows that malechickens had 3.5-fold greater hepatic AHH activity than female chickens, while male quailhad 5.1-fold greater activity than the female birds. The level of basal AHH activity ofpigeon hepatic microsomes was the highest of the four species examined.Treatment of male rats with PB, MC and AR resulted in approximately 2.1-, 6.9-and 4.3-fold greater AHH activities, respectively, relative to the untreated group.For female chickens, AHH activities were similar in the untreated and corn oil-treated birds, whereas PB-, MC- and AR-treatment were found to increase this enzymeactivity by 3.9-, 13- and 4.7-fold, respectively.For female quail, the AHH activities in corn oil-, PB-, MC- and AR-treated birdswere found to be 1.6-, 1.9-, 14-, and 7.0-fold greater, respectively, than in untreatedquail.In contrast, the AHH activity of microsomes prepared from pooled male andfemale pigeon livers was slightly decreased in corn oil- and AR-treated birds relative tountreated pigeons, while PB- and MC-treatment resulted in AHH activities that were notmarkedly different from the activity in the untreated pigeons.These results indicate that hepatic AHH activity is induced by PB, MC, and AR inrats, chickens and quail, but not in pigeons.RESULTS / 89Table 3.3. Aryl hydrocarbon hydroxylase activities (nmole/mgprotein• minute) of hepatic microsomes prepared from rats and three birdspecies.Treatment Rat Chicken Quail PigeonUntreated (male) 0.320* 0.393 0.563(1.0) (3.5) (5.1)0.789*Untreated (female) --- 0.113* 0.110* (1.0)(1.0) (1.0)Corn Oil --- 0.120 0.172 0.479(1.1) (1.6) (0.61)PB 0.661 0.444 0.206 1.05(2.1) (3.9) (1.9) (1.3)MC 2.20 1.44 1.49 0.954(6.9) (13) (14) (1.2)AR 1.37 0.531 0.775 0.562(4.3) (4.7) (7.0) (0.71)Values in parenthesis indicate the fold of increase by corn oil, PB, MC and AR relative to theappropriate untreated group indicated by the synbol of .Treated rats were all male. Treated chickens and quail were all female. Hepatic microsomesfrom male and female pigeons were pooled together. Microsomes were prepared from a pooiof livers from either five to six rats or six birds.The activity of each microsomal preparation was determined on at least two separateoccassions and in duplicate each time. Values shown are the average of all determinations.3.2.2.3. 7-Pentoxyresorufin 0-Depentylase Activity (PROD)Hepatic PROD activities of rat microsomes and avian microsomes are listed inTable 3.4.Examination of the data for the untreated groups reveals that the level of basalPROD activity was slightly higher in untreated male rats and chickens than in theuntreated females. Hepatic PROD activity was found to be 4.6-fold greater in maleRESULTS / 90untreated quail than in female untreated quail.Treatment of male rats with PB and AR increased hepatic PROD activity by 101-fold and 42-fold, respectively. MC-treatment resulted in 1.2-fold increase in PRODactivity in the rats.Table 3.4. 7-Pentoxyresorufin 0-depentylase activities (nmole/mgprotein• minute) in hepatic microsomes prepared from rats and three avianspecies.Treatment Rat Chicken Quail PigeonUntreated (male) 0.0174* 0.0131 0.0881(1.0) (1.5) (4.6)0.0148*Untreated (female) 0.0144 0.00868* 0.0190* (1.0)(0.83) (1.0) (1.0)Corn Oil ND 0.0175 0.0103(0.92) (0.70)PB 1.75 0.00216 0.00648 0.0112(101) (0.25) (0.34) (0.76)MC 0.0208 0.140 0.246 0.0196(1.2) (16) (13) (1.3)AR 0.736 0.0664 0.487 0.0347(42) (7.6) (26) (2.3)Values in parenthesis indicate the fold of increase by corn oil, PB, MC, and AR relative tothe appropriate untreated group indicated by the symbol of .Treated rats were all male. Treated chickens and quail were all female. Hepatic microsomesfrom male and female pigeons were pooled together. Microsomes were prepared from a poolof livers from either five to six rats or six birds.The activity of each microsomal preparation was determined on at least two separateoccassions and in duplicate each time. Values shown are the average of all determination.ND means that the activity was not detected in the hepatic microsomes.RESULTS /91In female chickens, hepatic PROD activity could not be measured in the corn oil-treated group. In PB-treated chickens, this activity was only 25 % of that in the untreatedgroup, while the PROD activity of microsomes prepared from MC- and AR-treatedchickens was increased 16- and 7.6-fold, respectively.In female quail, hepatic PROD activity was slightly changed by treatment withcorn oil, was decreased by 66% after PB-treatment and was increased after MC-treatmentand AR-treatment by factors of 13 and 26, respectively.In microsomes pooled from both male and female pigeon livers, the PRODactivities of corn oil- and PB-treated birds were found to be slightly decreased relative tothe activity of the untreated group, while the PROD activities of MC- and AR-treatedpigeons were increased to 1.3- to 2.3-fold.These results indicate that: (1) hepatic PROD activity in rats was markedly inducedby PB and AR, but not by MC, (2) PB did not induce, but suppressed, PROD activity inthe avian species, (3) hepatic PROD activity was inducible by MC and AR in the avianspecies, and (4) PROD activities were more readily induced by MC in female chickensand quail than in rats and pigeons, while the PROD activity in female quail was moresensitive to the inducing effect of AR than the PROD activities in female chickens andpigeons but less sensitive than that in rats.3.2.2.4. Benzphetamine N-Demethylase Activity (BPND)The data for hepatic BPND activity of untreated and treated rats, chickens, quailand pigeons are summarized in Table 3.5.A comparison of the activities among the untreated groups shows that malechickens and quail had 3.6- and 2.1-fold greater BPND activity than female chickens andquail, respectively. BPND activity was also slightly higher in male untreated rats than infemale untreated rats. Corn oil-treatment did not cause marked change in avian hepaticBPND activity.RESULTS/92Among the treated male rats, an approximately 3-fold increase in microsomalBPND activity was found for both PB-treated and AR-treated rats, while a 33 % decreasewas found for MC-treated rats. In female chickens, treatment with PB, MC and ARresulted in 4.3-, 3.6- and 1.8-fold increase in hepatic BPND activities, respectively. Infemale quail, hepatic BPND activities were increased after PB-, MC— and AR-treatment byfactors of 2.2, 7.3, and 6.7, respectively.Table 3.5. Benzphetamine N-demethylase activities (nmole/mgprotein.minute) in hepatic microsomes prepared from rats and three avianspecies.Treatment Rat Chicken Quail PigeonUntreated (male) 9•73* 10.2 6.90(1.0) (3.6) (2.1)6.90*Untreated (female) 6.90 2.81 * 3.23 * (1.0)(0.71) (1.0) (1.0)Corn Oil 2.30 3.60 5.35(0.82) (1.1) (0.78)PB 30.3 12.2 7.05 9.02(3.1) (4.3) (2.2) (1.3)MC 6.48 10.1 26.9 11.7(0.67) (3.6) (8.3) (1.7)AR 29.1 5.04 25.0 9.16(3.0) (1.8) (7.7) (1.3)Values in parenthesis indicate the fold of increase by corn oil, PB, MC, and AR relative tothe appropriate untreated group indicated by the symbol of ‘.Treated rats were all male. Treated chickens and quail were all female. Hepatic microsomesfrom male and female pigeons were pooled together. Microsomes were prepared from a poolof livers from either five to six rats or six birds.The activity of each microsomal preparation was determined on at least two separateoccassions and in duplicate each time. Values shown are the average of all determinations.RESULTS/93In pigeons, a 30% to 70% increase in the activity was found after PB-, MC- andAR-treatment.The data for BPND activity indicate that hepatic BPND activity was induced by PBand AR but suppressed by MC in rats, while in the avian species it was inducible by PB,AR and MC.3.3. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND IMMUNOBLOTSRat hepatic microsomes and avian hepatic microsomes were subjected to SDSpolyacrylamide gel electrophoresis and the resulting gels were stained with Coomassieblue. The stained gels were used to compare the pattern of induction after treatment withPB, MC and AR. Induced proteins are generally visible as more heavily stained bands onthe gel, provided that the amount of protein applied to each lane of the gel is the same forevery microsomal sample.In addition, hepatic microsomal proteins resolved by SDS-polyacrylamide gelelectrophoresis were electroeluted and transferred onto nitrocellulose, and subsequentlyprobed with various primary antibodies raised against different forms of purified ratcytochrome P450.3.3.1. SDS-Polyacrylamide Gel and Iminunoblots of Rat Hepatic MicrosomesIn order to provide a reference for the subsequent analysis of the SDSpolyacrylamide gel and immunoblots of avian hepatic microsomes, a Coomassie blue-stained gel and two immunoblots of rat hepatic microsomes will be presented first.Figure 3.17 illustrates a Coomassie blue stained polyacrylamide gel of rat hepaticmicrosomes together with immunoblots of rat hepatic microsomes probed with monoclonalantibody against rat CYP1A1 and polyclonal antibody against rat CYP2B1. Theelectrophoresis gel shows that several protein bands in microsomes prepared from PB- andRESULTS / 94MC-treated rats were more heavily stained than those from untreated rats.SDS GEL IMMUNOBLOTS PROBED WITH:ANTI-P450c ANTI-P450bUt Pb MC lit PbMC Ut Pb MCFigure 3.17. SDS-polyacrylamide gel and immunoblots of hepatic microsomes from maleuntreated (Ut), PB-treated (Pb) and MC-treated (MC) rats probed with monoclonalantibody against rat CYP1A1 and with polyclonal antibody against rat CYP2B1. Theconcentrations of the monoclonal antibody and the polyclonal antibody are 0.4 ig/ml and80 tg/ml, respectively. Each lane contained 10 ig of rat microsomal proteins.RESULTS /95Examination of the immunoblots (Figure 3. 17) reveals that monoclonal antibodyagainst rat CYP1A1 recognized only a single band in hepatic microsomes from MC-treated rats and did not recognize any protein in microsomes from untreated or PB-treatedrats. This result suggests that CYP1A1 was highly induced by MC in rats since thereaction of anti-CYP1A1 IgG with microsomes from MC-treated rats was very strong asindicated by the presence of a single, darkly stained band.Polyclonal antibody against CYP2B1 reacted with microsomes from untreated andPB-treated rats and produced three bands, indicating that three proteins in microsomesfrom untreated rats were recognized by anti-CYP2B1 polyclonal IgG. Treatment with PBdramatically induced the upper two protein bands. The uppermost protein band isCYP2B2 while the band immediately beneath it is CYP2B1. Rat CYP2B1 and 2B2exhibit 97% amino acid sequence similarity (Gonzalez, 1988). Therefore, both arerecognized equally well by polyclonal antibody prepared against purified CYP2B 1. Thehepatic level of CYP2B1 has been previously shown to be lower than CYP2B2 inuntreated rats, but after PB treatment, the CYP2B1 content increases more than that ofCYP2B2 in rat liver (Christou et a!., 1987).A third CYP2B member, designated as CYP2B3, has been proposed to be presentin untreated rats. CYP2B3 cDNA has been isolated from a liver cDNA library and itscDNA-deduced amino acid sequence was found to be 77% similar to CYP2B1 and 2B2(Labbe et al., 1988). It was reported that the CYP2B3 gene was expressed constitutivelyin rats and was not inducible by PB (Gonzalez, 1988). The bottom protein band shown inmicrosomes from untreated, PB-treated, or MC-treated rats might represent rat CYP2B3.PB-treatment also induced a protein that was located between CYP2B1 and CYP2B3 andwas not detected in control rat microsomes. MC-treatment slightly suppressed thecontents of the three CYP2B isozymes since CYP2B1 was not detected and the stainingintensities of the bands corresponding to CYP2B2 and 2B3 were decreased.RESULTS/963.3.2. SDS-Polyacrylamide Gel of Avian Hepatic MicrosomesA Coomassie-blue stained gel electrophoretogram of avian hepatic microsomes isshown in Figure 3.18. Lanes 1 and 20 contained molecular weight standards rangingfrom 31 to 116 kilodaltons (kD). Plotting relative mobility (Rf) of each molecular weightstandard against the logarithm value of molecular weights produced a linear relationship(Appendix 1) that was used to calculate the apparent molecular weights of protein bandsthat were well resolved on SDS-PAGE. Lane 2 contained a mixture of purified ratCYP1A1, 2C11, 2C13 and epoxide hydrolase which were included in the gel to indicatethe approximate region of cytochromes P450 in avian hepatic microsomes. Using thestandard curve, the apparent molecular weights for CYP 1 Al, 2C 11, 2C 13 and epoxidehydrolase were calculated to be 78.3 kD, 72.5 kD, 69.6 kD and 68.4 kD, respectively.Lanes 3 to 19 contained avian microsomes that were applied to the gel at 10 ig of proteinper lane except for lane 10, which contained 21 jig of microsomal protein for corn oil-treated quail due to a systematic error in the protein determination of this microsomalpreparation. Unfortunately, this error appears here and on several immunoblots.The stained gel shows that multiple protein bands appeared within the range ofapparent molecular weights from 68 to 80 kD, which might encompass the cytochromeP450 region. Apparent molecular weights (MW) were calculated for induced avianprotein bands that migrated in this region. These data are summarized in Table 3.6.3.3.2.1. Induced Protein Bands in Pigeon MicrosomesLanes 3 to 7 were loaded with microsomes from untreated and treated pigeons.Examination of the gel reveals that a protein band with an apparent MW of 75. 1 kD and aRf value of 0.494 (lane 5) was increased after PB treatment and that three protein bandswith MW and Rf values of 76.6 kD and 0.485, 75.1 kD and 0.494, 69.9 kD and 0.529,respectively (lanes 6 and 7), were more intensely stained in microsomes from MC- andAR-treated pigeons. In particular, the band with an apparent MW of 75.1 kD was stainedRESULTS / 97more heavily than the other two in microsomes from MC- and AR-treated pigeons.3.3.2.2. Induced Protein Bands in Quail MicrosomesHepatic microsomes from untreated and treated quail were loaded in lanes 8 to 13.Note that the bands in lane 10, which contained microsomes from corn oil-treated quail,are darker than protein bands in the other lanes due to the fact that this lane wasunintentionally overloaded, as mentioned above. A microsomal protein with an apparentMW of 68.7 kD and a Rf value of 0.535 (lane 11), was slightly induced after treatment ofquail with PB. Treatment of quail with MC or AR induced five proteins with apparentMW and Rf values of 78.6 and 0.473, 77.2 and 0.481, 75.1 and 0.494, 73.5 and 0.504,68.7 and 0.535, respectively (lanes 12 and 13). The proteins with apparent MW of 78.6and 77.2 kD are not visible in microsomes from untreated or corn oil-treated quail (lanes9 and 10), but are heavily stained only in microsomes from MC- and AR-treated femalequail (lanes 12 and 13).3.3.2.3. Induced Protein Bands in Chicken MicrosomesLanes 14 to 19 contained microsomal proteins from untreated and treated chickens.The staining pattern of the bands in lanes 14 and 15, which contained microsomes fromuntreated male and female chickens, appeared to be quite similar except that two bandscorresponding to 83.0 kD (Rf = 0.448) and 81.9 kD (Rf = 0.454) proteins were darker inmicrosomes from male untreated chickens. PB appeared to induce seven microsomalproteins, while MC and AR each induced nine proteins in female chicken livers (Table3.6). The proteins with apparent MW of 73.5 kD, 71.1 kD and 69.6 kD, that werepresent in microsomes from PB-treated chickens, and proteins with apparent MW of 78.6kD, 76.6 kD and 73.5 kD, that were present in microsomes from MC- and AR-treatedchickens, were not visible in microsomes from untreated female chickens.-—,— _i_RESULTS / 98— — —- — — —-—•-.,.--q..4 b-—*1234 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Figure 3.18. Coomassie blue stained SDS-polyacrylamide electrophoresis gel of hepaticmicrosomes from the three avian species. Lanes 1 and 20 contained molecular weightstandards, which are E.coli. 13-galactosidase (116 kD), rabbit muscle phosphorylase (97.4kD), bovine serum albumin (66.2 kD), hen egg white ovalbumin (42.7 kD) and bovinecarbonic anhydrase (31.0 kD). The standards were resolved on a discontinuouspolyacrylamide gel, containing a 7.5% polyacrylamide separating gel that was 0.75 mmthick and 12.5 cm long, as described in the Experimental section.. Lanes 2 contained amixture of purified rat CYP 1 Al, 2C 11, 2C 13 and epoxide hydrolase at 0.4 .tg of each.Lanes 3 to 7 contained hepatic microsomes from male untreated, female untreated, cornoil-, PB-, MC- and AR-treated pigeons, respectively. Lanes 8 to 13 containedmicrosomes from male untreated, female untreated, corn oil-, PB-, MC- and AR-treatedquail, respectively. Lanes 14 to 19 contained microsomes from untreated, corn oil-, PB-,MC- and AR-treated chickens, respectively. Each microsomal sample was applied at 10tg of protein per lane except for the microsomal sample from corn oil-treated quail thatwas applied at 21 ig of protein per lane.kp.——-- 1IIesrI.— m:— — •• — —RESULTS / 99In general, the results of SDS-polyacrylamicle gel electrophoresis of avianmicrosomal proteins indicate that many microsomal cytochrome P450 proteins wereinduced in the three avian species by PB, MC and AR and the induction pattern producedby PB is different from that produced by MC and AR. The induction pattern produced byMC and AR was also different among species.Table 3.6. Apparent molecular weights (MW) and relative mobility (Rf) on a SDSpolyacrylamide gel of microsomal proteins induced by phenobarbital (PB), 3—methyicholanthrene (MC) and Aroclor 1254 (AR) in three avian species.Rf MW Pigeon Quail(lcD) PB MC AR PB MC AR PB MC AR0.458 81.5 +0.463 80.3 ++ ++0.465 80.0 +0.469 79.3 + +0.473 78.6.±.± .±±: ± .±± .±±0.481 77.2 ++ ++0.485 76.6 + + ± ±0.494 75.1 + +++ +++ ++ ++ ++ + +0.504 73.5 ± ± ± ± ±0.515 71.8 + +0.519 71.1 ++0.523 70.5 ± ±0.529 69.6 ++ ++ ± ± ±0.535 68.7 +++ ++Note: The symbol of “+ is used here as an approximate measure of the degree of induction of theproteins; the more ‘ + ‘, the more intensively stained band. The symbol, ±, means that the inducedproteins were not visible in the untreated microsomes,RESULTS / 1003.3.3. Immunoblot Analysis of Avian Cytochromes P450In order to determine the immunochemical relatedness between avian and ratcytochromes P450 and to examine the induction of specific forms of cytochrome P450 inthe three avian species, avian microsomal proteins were separated by SDS-polyacrylamidegel electrophoresis, transferred onto nitrocellulose membranes, and probed with theantibodies generated against several purified rat cytochrome P450 isozymes. Thespecificity of each antibody has previously been assessed in Dr. Bandiera’ s laboratory,using both enzyme-linked immunosorbent and immunoblot assays, with purified ratcytochrome P450 enzymes and with different rat liver microsomal preparations. Thoseresults will not be repeated here.3.3.3.1. Immunoblot of Avian Hepatic Microsomes Probed with Anti-Rat CYP1A1IgGFigure 3.19 illustrates an immunoblot of avian hepatic microsomes probed withanti-rat CYP1A1 monoclonal antibody. As shown, anti-rat CYP1A1 monoclonal IgG didnot recognize any protein in hepatic microsomes from untreated, corn oil-treated, or PB-treated chickens, quail, and pigeons, but reacted with microsomes from MC-treated andAR-treated birds in all three species. The avian antigenic proteins migrated slightly fasteron the polyacrylamide gel than rat CYP1A1.Polyclonal antibody prepared against rat CYP1A1 sera recognizes both CYP1A1and CYP1A2. An immunoblot of avian microsomes probed with polyclonal anti-ratCYP1A1 sera is shown in Figure 3.20. As expected, anti-CYP1A1 sera reacted stronglywith a partially purified preparation of rat CYP1A1 (lane 1). A broad, faintly stainedband is visible in the lanes containing microsomes from MC- and AR-treated pigeons(lanes 5 and 6), but not in lanes containing microsomes from untreated, corn oil-treatedand PB-treated pigeons (lanes 2 to 4). This band was similar to but weaker than the bandon the blot probed with the monoclonal antibody against CYP1A1 (lane 5 and 6 in FigureRESULTS /1013.19). In addition to the major protein band, a second immunoreactive protein wasdetected in microsomes from control and treated pigeons (lanes 2 to 6 in Figure 3.20).This protein is visible as a very faint lower baid and does not appear to be induced by MCor AR as the density of the band in the lane containing microsomes from control pigeonslooks similar to that from MC- and AR-treated birds.Polyclonal anti-rat CYP1A1 sera also reacted with quail hepatic microsomes (lanes7 to 12 in Figure 3.20), producing a dark band with MC- and AR-treated quail but notwith untreated, corn oil- and PB-treated birds. This dark band appears to be similar to theband on the blot probed with monoclonal anti-CYP1A1 IgG (lanes 11 and 12 in Figure3.19). An extremely faint lower band was also detected in the lanes containingmicrosomes from control and treated quail (Figure 3.20). The similarity in stainingintensity of the faint bands in microsomes from control and treated quail suggests that theprotein in this band is not induced.Examination of the lanes containing chicken liver microsomes (lanes 13 to 18 inFigure 3.20), reveals two intensely stained and closely spaced bands in microsomes fromMC- and AR-treated chickens but not in the other microsomes and one of these twoproteins was not detected by monoclonal anti-CYP1A1 IgG (lanes 17 and 18 in Figure3.19).In summary, the cross-reaction between monoclonal and polyclonal antibodiesagainst rat CYP 1 Al and avian hepatic microsomes demonstrates that all three avianspecies have inducible proteins that are immunologically related to rat CYP1A1.RESULTS / 102e1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.19. Immunoblot of avian hepatic microsomes probed with monoclonal antibodyagainst rat CYP1A1. All microsomal samples were applied to the gel at 40 tg of proteinper lane except for microsomes from corn oil-treated quail that were applied at 84 jig ofprotein per lane. The concentration of anti-CYP1A1 monoclonal IgG was 0.4 jig/mi.Lane 1 contained 5 jig of partially purified rat CYP1A1. Lanes 2 to 6 containedmicrosomes from untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively.Lanes 7 to 12 contained microsomes from male untreated, female untreated, corn oil-, PB-MC- and AR-treated quail, respectively. Lanes 13 to 18 contained microsomes frommale untreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens,respectively.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.20. Immunoblot of avian hepatic microsomes probed with polyclonal anti-ratCYP1A1 sera. All microsomal samples were applied to the gel at 40 jig per lane, exceptfor microsomes from corn oil-treated quail that were applied at 84 jig of protein per lane.Anti-CYP1A1 sera was used at a dilution of 1:1000. Lane 1 contained 5 jig of partiallypurified rat CYP1A1. Lanes 2 to 6 contained microsomes from untreated, corn oil-, PB-,MC- and AR-treated pigeons, respectively. Lanes 7 to 12 contained microsomes frommale untreated, female untreated, corn oil-, PB-, MC- and AR-treated quail, respectively.Lanes 13 to 18 contained microsomes from male untreated, female untreated, corn oil-,PB-, MC- and AR-treated chickens, respectively.RESULTS / 1033.3.3.2. Immunoblot of Avian Hepatic Microsomes Probed with Anti-Rat CYP2BIIgGAn immunoblot of avian microsomes probed with polyclonal antibody against ratcytochrome P450 2B1 is shown in Figure 3.21. The polyclonal antibody used recognizesboth rat CYP2B1 and CYP2B2 (Figure 3.17). As seen on the blot, one dark band and oneweak band appeared in the lane loaded with purified rat CYP2B1 (lane 1). Reactions ofthe antibody with avian microsomes (lanes 2 to 18) were uniformly weak.The bands in the lanes containing hepatic microsomes from MC- and AR-treatedpigeons (lanes 5 and 6) appear moderately darker than the bands from untreated and cornoil-treated pigeons (lanes 2 and 3). Polyclonal antibody against rat CYP2B1 also reactedwith quail microsomes, producing a protein band in microsomes from control and treatedbirds (lanes 7 to 12) and two new protein bands in microsomes from MC- or AR-treatedquail (lane 11 and 12). The cross-reactions between polyclonal antibody against ratCYP2B1 and microsomes from control and treated chickens (lane 13-18) generated asingle band in all the chicken microsomes. This protein band seems to be lighter inmicrosomes from female untreated chickens.Because very weak reactions such as those obtained with polyclonal anti-CYP2B1IgG are difficult to interpret, a blot of avian microsomes was probed initially with amonoclonal antibody that is specific for rat CYP2B1 and then re-probed with a secondmonoclonal antibody that recognizes both CYP2B1 and 2B2 (Figure 3.22). As shown, nocross-reaction was detected between these monoclonal antibodies and the avianmicrosomes.RESULTS / 1041 2 3 4 5 6 7 8 9 10 11 121314151617 18Figure 3.21. Immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2B1. All microsomal samples were applied to the gel at 40 jig of proteinper lane except for microsomes from corn oil-treated quail that were applied at 84 jig ofprotein per lane. The concentration of anti-CYP2B1 polyspecific IgG was 100 jig/mi.Lane 1 contained 0.29 jig of purified rat CYP2B1. Lanes 2 to 6 contained microsomesfrom untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively. Lanes 7 to 12contained microsomes from male untreated, female untreated, corn oil-, PB-, MC- andAR-treated quail, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens, respectively.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.22. Immunoblot of avian hepatic microsomes probed with monoclonal antibodyagainst rat CYP2B1/2. All microsomal samples were applied to the gel at 40 jig ofprotein per lane except for microsomes from corn oil-treated quail that were applied at 84jig of protein per lane. The concentration of anti-CYP2B1 monoclonal IgG was 1.0jig/ml. Lane 1 contained 0.29 jig of purified rat CYP2B1. Lanes 2 to 6 containedmicrosomes from untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively.Lanes 7 to 12 contained microsomes from male untreated, female untreated, corn oil-, PBMC- and AR-treated quail, respectively. Lanes 13 to 18 contained microsomes frommale untreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens,respectively.RESULTS/lOS3.3.3.3. Immunoblot of Avian Hepatic Microsomes Probed with Anti-Rat CYP2C7IgGBlots of avian hepatic microsomes were also probed with polyspecific andmonospecific antibody against rat CYP2C7 (Figures 3.23 and 3.24, respectively). Asshown in Figure 3.23, anti-rat CYP2C7 polyspecific TgG reacted strongly with purified ratCYP2C7 (lane 1). Some additional faint bands are visible above and below the CYP2C7band. Monospecific antibody against rat CYP2C7 also reacted with purified rat CYP2C7and produced a single band (lane 1 in Figure 3.24) that appeared to be less intense thanthat produced by the polyspecific antibody (Figure 3.23), indicating that the monospecificantibody is more specific for CYP2C7 but of lower avidity.The staining pattern and staining intensity produced by the reaction of thepolyspecific antibody with avian hepatic microsomes (Figure 3.23) were similar to thestaining pattern and staining intensity generated by the monospecific antibody (Figure3.24). The similarity in staining intensity was unexpected since the antibody preparationsobviously have different titers. The avian immunoreactive proteins were located higher onthe blots than rat CYP2C7. The immunoreactive protein bands in the lanes containingmicrosomes from PB-, MC- and AR-treated pigeons (lanes 4 to 6) appeared to be fainterthan those from control birds (lanes 2 and 3). Examination of lanes 7 to 12 indicates thatthe immunoreactive protein was present in a lesser amount in male quail than in thefemales (lane 7 and 8) and was suppressed in microsomes from MC-treated female quail(lane 11). The immunoreactive protein was not found in microsomes from untreatedfemale and male chickens (lanes 13 and 14), but it was found in microsomes from treatedchickens.RESULTS/1061 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.23. Immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2C7. All microsomal samples were applied to the gel at 40 tg of proteinper lane except for microsomes from corn oil-treated quail that were applied at 84 tg ofprotein per lane. The concentration of anti-CYP2C7 polyspecific IgG was 40 tg/ml.Lane 1 contained 1.43 tmole of purified rat CYP2C7. Lanes 2 to 6 containedmicrosomes from untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively.Lanes 7 to 12 contained microsomes from male untreated, female untreated, corn oil-, PB-MC- and AR-treated quail, respectively. Lanes 13 to 18 contained microsomes frommale untreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens,respectively.— —. —-— —12345 6789101112131415161718Figure 3.24. Immunoblot of avian hepatic microsomes probed with monospecificantibody against rat CYP2C7. All microsomal samples were applied to the gel at 40 ig ofprotein per lane except for microsomes from corn oil-treated quail that were applied at 84tg per lane. The concentration of anti-CYP2C7 monospecific IgG was 50 ig/m1. Lane 1contained 1.43 imole of purified rat CYP2C7. Lanes 2 to 6 contained microsomes fromuntreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively. Lanes 7 to 12contained microsomes from male untreated, female untreated, corn oil-, PB-, MC- andAR-treated quail, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens, respectively.RESULTS / 1073.3.3.4. Immunoblots of Avian Hepatic Microsomes Probed with Anti-Rat CYP2C11IgGThe immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2C1 1 is shown in Figure 3.25. This antibody reacted strongly withpurified rat CYP2C11 (lane 1) and formed a single band. Anti-CYP2C11 polyspecificIgG also reacted relatively strongly with microsomes prepared from untreated and treatedbirds. As shown on the blot, the immunostaining pattern of the microsomal samples fromthe three avian species were quite different from each other.In microsomes from male untreated chickens (lane 2), two dark bands and a thirdweakly stained lower band are visible, while in microsomes from control female chickens(lanes 3 and 4), only a single protein band is apparent. The mobility of the upper band inlane 2 and the single band in lanes 3 and 4 was similar to that of purified rat CYP2C 11.A similar band that was also found in microsomes from PB-, MC- and AR-treatedchickens (lanes 5 to 7) was darker than that found in lane 3 and 4, suggesting that thisprotein may be inducible by PB, MC and AR in female chickens. In addition to the upperprotein band, the reaction of the anti-CYP2C1 1 polyspecific IgG with microsomes fromfemale PB-treated chickens (lane 5) generated two other dark lower bands. These twoprotein bands have the same mobility as the two lower bands visible in microsomes frommale untreated chickens (lane 2). PB appeared to induce an immunochemical stainingpattern in female chickens that is similar to that in untreated male chickens.Polyspecific antibody against rat CYP2C 11 reacted with microsomes fromuntreated and treated pigeons (lanes 8 to 12), producing one protein band. This proteinband appeares to be darker in PB-, MC- and AR-treated pigeons than in untreatedpigeons, suggesting that the immunoreactive microsomal protein is inducible by the threeinducers in pigeons. Treatment of pigeons with MC or AR induced two additionalmicrosomal proteins as two new upper bands are visible in the lanes containingmicrosomes from MC- and AR-treated pigeons.RESULTS/108— —. w— ——1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.25. Immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2C 11. All microsomal samples were applied to the gel at 40 ig of proteinper lane except for microsomes from corn oil-treated quail that were applied at 84 ig ofprotein per lane. The concentration of anti-CYP2C1 1 polyspecific IgG was 25 tg/m1.Lane 1 contained 0.30 ig of purified rat CYP2C1 1. Lanes 2 to 7 contained microsomesfrom male untreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens,respectively. Lanes 8 to 12 contained microsomes from untreated, corn oil-, PB-, MC-and AR-treated pigeons, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated quail, respectively.— — — — ——-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.26. Immunoblot of avian hepatic microsomes probed with monospecificantibody against rat CYP2C 11. All microsomal samples were applied to the gel at 40 igof protein per lane except for microsomes from corn oil-treated quail that were applied at84 ig of protein per lane. The concentration of anti-CYP2C1 1 monospecific IgG was 20jig/ml.. Lane 1 contained 0.30 .ig of purified rat CYP2C11. Lanes 2 to 7 containedmicrosomes from male untreated, female untreated, corn oil-, PB-, MC- and AR-treatedchickens, respectively. Lanes 8 to 12 contained microsomes from untreated, corn oil-,PB-, MC- and AR-treated pigeons, respectively. Lanes 13 to 18 contained microsomesfrom male untreated, female untreated, corn oil-, PB-, MC- and AR-treated quail,respectively.RESULTS /109The reaction of anti-CYP2C 11 polyspecific IgG with microsomes from male andfemale quail (lanes 13 to 18) produced two clearly visible protein bands with eachmicrosomal sample. The proteins represented by the upper and lower bands in quailmicrosomes may be inducible by PB, MC and AR as they appear to be darker inmicrosomes prepared from quail treated with these compounds than in microsomes fromuntreated female quail. A very faint protein band, that is barely visible immediatelybelow the upper band is also present in microsomes from MC- and AR-treated quail.An immunoblot of avian hepatic microsomes probed with monospecific antibodyagainst rat CYP2C11 is shown in Figure 3.26. The monospecific antibody reactedstrongly with the purified rat CYP2CI 1 (lane 1) and with hepatic microsomes preparedfrom chickens and quail, but not pigeons. In contrast to the blot probed with polyspecificanti-rat CYP2C1 1 IgG (Figure 3.25), a single protein was recognized by the monospecificantibody in all microsomes prepared from untreated and treated chickens (lanes 2-7). Thisprotein had the same relative mobility as purified rat CYP2C11 and is equivalent to theupper band in lanes 2 and 5 and the single band in lanes 3, 4, 6 and 7 in Figure 3.25. Itis referred to chicken-[CYP2C11] hereafter for the purpose of comparison. The stainingintensity of the chicken-[CYP2C11] band in the lane containing microsomes fromuntreated male chickens (lane 2) appeared to be darker than that in female untreatedchickens (lane 3), suggesting that chicken-CYP[2C1 1] is constitutively expressed to agreater extent in the male chicken liver than in the female chicken liver. The chicken[CYP2C1 1] may be inducible by PB, MC and AR since the staining intensity of the bandsin lanes 5 to 7 was greater than that in the female control microsomes.In the lanes (lanes 13 to 18) containing quail hepatic microsomes, a protein withrelative mobility similar to that of purified rat CYP2C 11 was detected by monospecificantibody against rat CYP2C 11. This protein is referred to quail-[CYP2C 1111 hereafter.The staining intensity of this band appeared to be darker in the lanes (17 and 18)containing microsomes prepared from MC- and AR-treated quail than in microsomes fromRESULTS /110untreated quail (lane 14), suggesting that quail-[CYP2C 11] may be inducible by MC andAR in quail. A second protein was detected by the monospecific antibody in microsomesfrom PB-, MC- and AR-treated quail. It is referred to quail-CYP[2C1 112 hereafter. Thisimmunoreactive protein moved faster than quail-CYP[2C11]1and was not detected inuntreated male and female quail microsomes, indicating that quail-CYP[2C1 1]2 may beinducible by PB, MC and AR. The two proteins recognized by monosepcific anti-ratCYP2C1 1 IgG appear to be the same proteins recognized by the anti-CYP2C1 1polyspecific IgG (see lanes 13 and 14 in Figure 3.25).3.3.3.5. linmunoblots of Avian Hepatic Microsomes Probed with Anti-Rat CYP2C13IgGAn immunoblot of avian hepatic microsomes probed with polyspecific anti-ratCYP2C13 IgG is shown in Figure 3.27. Polyspecific antibody against CYP2C13 reactedvery strongly with purified rat CYP2C13 (lane 1). It reacted less strongly but relativelyuniformly with the avian hepatic microsomes. The resulting staining pattern (lanes 2 to18) was similar to that generated by polyspecific antibody against CYP2C11 (Figure3.25). Anti-CYP2C13 polyspecific IgG, like anti-CYP2C1 1 polyspecific IgG, recognizesall members of the CYP2C subfamily in rats.Figure 3.28 shows a blot of avian hepatic microsomes probed with anti-ratCYP2C13 monospecific IgG. The monospecific antibody reacted very strongly andcleanly with purified rat CYP2C13 (lane 1). However, no cross-reaction was found withany of the avian microsomes, indicating that the avian immunoreactive proteins recognizedby polyspecific antibody against CYP2C13 are not related to rat CYP2C13 but may beimmunorelated to other members of the CYP2C subfamily.RESULTS / 111—12 34 5 6 78 9101112131415161718Figure 3.27. Immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2C13. All microsomal samples were applied to the gel at 40 jig of proteinper lane except for microsomes from corn oil-treated quail that wer applied at 84 jig ofprotein per lane. The concentration of anti-CYP2C13 polyspecific IgG was 25 jig/mi.Lane 1 contained 0.20 jig of purified rat CYP2C13. Lanes 2 to 7 contained microsomesfrom male untreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens,respectively. Lanes 8 to 12 contained microsomes from untreated, corn oil-, PB-, MC-and AR-treated pigeons, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated quail, respectively.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.28. Immunoblot of avian hepatic microsomes probed with monospecificantibody against rat CYP2C13. All microsomal samples were applied to the gel at 40 jigof protein per lane except for microsomes from corn oil-treated quail that were applied at84 jig of protein per lane. The concentration of anti-CYP2C13 monospecific IgG was 25jig/ml. Lane 1 contained 0.20 jig of purified rat CYP2C13. Lanes 2 to 7 containedmicrosomes from male untreated, female untreated, corn oil-, PB-, MC- and AR-treatedchickens, respectively. Lanes 8 to 12 contained microsomes from untreated, corn oil-,PB-, MC- and AR-treated pigeons, respectively. Lanes 13 to 18 contained microsomesfrom male untreated, female untreated, corn oil-, PB-, MC- and AR-treated quail,respectively.RESULTS / 1123.3.3.6. Immunoblots of Avian Hepatic Microsomes Probed with Anti-Rat CYP3A1IgGIn order to determine if there are avian hepatic cytochromes P450 that are relatedto rat CYP3A1 or CYP3A2, a blot of avian microsomes was probed with polyspecificanti-rat CYP3A1 IgG (Figure 3.29). This antibody recognizes both rat CYP3A1 andCYP3A2 due to the fact that these two CYP3A isozymes share 89% similarity in aminoacid sequence. Examination of the immunoblot reveals that anti-CYP3A1 polyspecificIgG reacted strongly with purified rat CYP3A1 (lane 1), generating one intensely stainedband and two weaker bands.A single band was visible in the lanes containing microsomes from untreated, cornoil-, PB-, MC- and AR-treated pigeons (lanes 2 to 6). The staining intensity of this bandappeared to be darker in lanes 5 and 6, suggesting that this protein may be inducible byMC and AR in pigeons. For the convenience of comparison, this protein is given anarbitary name as pigeon-[CYP3AJ1 Interestingly, in microsomes from PB-treatedpigeons (lane 4), a second lower band is also evident, suggesting that PB-treatmentinduces a new protein in the pigeon that is immunologically related to rat CYP3Aenzymes. The second protein is referred to pigeon-[CYP3A]2hereafter.The interaction between anti-CYP3A1 polyspecific IgG and microsomal proteinsfrom MC- and AR-treated quail resulted in three weak bands (lanes 11 and 12). Theupper two bands are also present in a weakly stained form in microsomes from untreatedmale, untreated female, corn oil- and PB-treated female quail, but in a darker form inmicrosomes from MC-and AR-treated quail, indicating that these two quail proteins areconstitutively expressed in quail and are inducible by MC or AR. The protein representedby the third band was not detected in untreated and corn oil-treated quail, implying thatthis quail immunoreactive protein may be induced by both MC and AR. The threeproteins present in the top, middle and bottom bands are hereafter referred to quail[CYP3A]1,quail-[CYP3A], and quail-[CYP3A]3respectively.RESULTS / 113The reaction of polyspecific antibody against rat CYP3A 1 with microsomes frommale untreated chickens generated a faint upper band and a dark lower band (Jane 13).The upper band was also visible in the lanes containing microsomes from untreated andtreated female chickens (lane 14), but the lower band was found only in microsomes fromPB-, MC- and AR-treated chickens (lane 16 to 18), which suggests that theimmunoreactive protein in the lower band may be induced by all three compounds infemale chickens. The staining intensity of the lower band was greater in microsomes fromPB-treated chickens than in microsomes from MC- or AR-treated chickens, suggesting thatthis cytochrome P450 may be inducible to a greater extent by PB than by the other twoinducers. The proteins present in the upper and lower bands are named as chicken{CYP3A]1 and chicken-[CYP3A]7,respectively.Figure 3.30 shows an immunoblot of avian microsomes probed with anti-ratCYP3A 1 IgG that has been back-absorbed in an attempt to remove the cross-reaction withCYP3A2. This back-absorbed antibody did not react with any of the avian microsomes,but still reacted quite strongly with purified rat CYP3A1.RESULTS / 114—---—1 2 3 4 5 6 7 8 9 10 11 12131415161718Figure 3.29. Immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP3A 1. All microsomal samples were applied to the gel at 40 ig of proteinper lane except for microsomes from corn oil-treated quail that were applied at 84 p.g ofprotein per lane. The concentration of anti-CYP3A1 polyspecific IgG was 40 ig/ml.Lane 1 contained 0.2 ig of purified rat CYP3A1. Lanes 2 to 6 contained microsomesfrom untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively. Lanes 7 to 12contained microsomes from male untreated, female untreated, corn oil-, PB-, MC- andAR-treated quail, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens, respectively.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Figure 3.30. Immunoblot of avian hepatic microsomes probed with anti-rat CYP3A 1 IgGbackabsorbed against hepatic microsomes from untreated adult female rats and isoniazidtreated male rats. All microsomal samples were applied to the gel at 40 tg of protein perlane except for microsomes from corn oil-treated quail that were applied at 84 ig ofprotein per lane. The concentration of anti-CYP3A1 polyspecific IgG was 100 tg/ml.Lane 1 contained 0.2 tg of purified rat CYP3A1. Lanes 2 to 6 contained microsomesfrom untreated, corn oil-, PB-, MC- and AR-treated pigeons, respectively. Lanes 7 to 12contained microsomes from male untreated, female untreated, corn oil-, PB-, MC- andAR-treated quail, respectively. Lanes 13 to 18 contained microsomes from maleuntreated, female untreated, corn oil-, PB-, MC- and AR-treated chickens, respectively.RESULTS / 1153.4. ANTIBODY INBIBrrION OF MONOOXYGENASE AcTlvrrwsThe immunoblot data disclose the presence of avian cytochromes P450immunorelated to rat CYP1A1/2, 2C1 1, 3A and possibly 2B. Antibodies against ratCYP1A, 2B, 2C and 3A were used in an inhibition study of monooxygenase activities toassess the contribution of several avian cytochrome P450 proteins toward the deethylationof 7-ethoxyresorufin (EROD), the depentylation of 7-pentoxyresorufin (PROD) and the Ndemethylation of benzphetamine (BPND). Unfortunately, due to the limited supply of theantibodies, a single concentration of each antibody was used. Anti-CYP1A1 antibodieswere not used for the inhibition of BPND activity for the same reason.3.4.1. Antibody Inhibition of Hepatic EROD ActivititesCYP1A1 is known to be the major catalyst for the O-deethylation of 7-ethoxyresorufin (EROD) in hepatic microsomes from MC-treated rats. In order todetermine the extent of involvement of the avian cytochrome P450 enzymes that areimmunorelated to rat CYP1A1 in the metabolism of 7-ethoxyresorufin in chickens, quailand pigeons, the effect of antibody against rat CYP1A1 on EROD activity in MC-treatedbirds was investigated. The antibody inhibition data for EROD activity listed in Table 3.7were obtained by measuring enzyme activity in the presence of 3 ig of control rabbit IgG,3 tg of monoclonal antibody against CYP1A1 or 15 tl of polyclonal anti-CYP1A1 serafor all microsomal samples.Monoclonal antibody against rat CYP1A1 and polyclonal anti-CYP1A1 serainhibited the EROD activity of hepatic microsomes from MC-treated rats by 37% and68%, respectively. The same amounts of monoclonal and polyclonal antibody produced80% inhibition and 20% inhibition of EROD activity, respectively, in hepatic microsomesprepared from MC-treated chickens, 85% and 37% inhibition of EROD activity,respectivley, in hepatic microsomes from MC-treated quail, and 18% and 19% inhibitionof EROD activity, respectively, in hepatic microsomes from MC-treated pigeons. ERODRESULTS / 116activity was inhibited to a greater extent by the monoclonal antibody in the chicken andquail, while greater inhibition was observed with the polyclonal antibody in the rat,indicating that polyclonal antibody against rat CYP1A1 is more inhibitory to ERODactivity than the monoclonal antibody in rats treated with MC, while monoclonal anti-ratCYP1A1 IgG is more inhibitory to EROD activity than the polyclonal IgG in femalechickens and quail treated with MC. The degree of inhibition produced by the twoantibodies was nearly equal in MC-treated pigeons.Table 3.7. Inhibition of 7-ethoxyresorufin 0-deethylase activity (EROD) in microsomes fromMC-treated rats and birds by monoclonal antibody (MAb) and polyclonal Antibody (PAb) torat CYP1A1.MC-Treated Rat MC-Treated MC-Treated Quail MC-treatedChicken PigeonEROD EROD EROD EROD EROD EROD EROD ERODAntibodies Activity Inhibition Activity Inhibition Activity Inhibition Activity InhibitionWithoutIgG 3.30 0% 6.66 0% 5.20 0% 0.856 0%Control 3.17 3.9% 6.25 6.2% 5.43 no 0.871 noIgGAnti-1AI 1.96 41% 0.891 87% 0.809 85% 0.702 18%MAbAnti-1A1 0.91 72% 4.94 26% 3.40 35% 0.692 19%PAbNote: The amounts of control rabbit IgG and monoclonal anti-CYP1A1 IgG added to 2 ml of the reactionmixture were 0.03 mg, which was equivalent to the ratio of 84 tg, 140 tg, 61 .ig and 60 ji.g of IgG per nmolecytochrome P450 for microsomes from MC-treated rats, MC-treated chickens, MC-treated quail and MC-treatedpigeons, respectively. Fifteen tl of polyclonal anti-CYP IA 1 sera were added to the reaction mixtures, Foranti-CYP IA I sera, the ratios of IgG to nmole cytochrome P450 cannot be determined because the IgGconcentration of the sera is unknown. EROD activities are expressed as nmol resorufin formed per mg proteinper minute. EROD activity was measured according to the standardized conditions in section 3.2.1.1.5.Inhibition % [(Activity without IgG - Activity with IgG)/Activity without IgG] x 100%.RESULTS /1173.4.2. Antibody Inhibition of Hepatic PROD ActivityMonoclonal anti-rat CYP1A1 IgG (0.03 mg), monospecific and polyspecific anti-rat CYP2C11 IgG (0.5 mg each) and polyspecific anti-rat CYP3A1 IgG (0.5 mg) wereused in immunoinhibition experiments of PROD activity of hepatic microsomes from PB-treated rats, MC-treated chickens and MC-treated quail. The reason for choosing toinvestigate the effects of these antibodies on PROD activity was based on the observationthat treatment with MC and AR, but not PB, resulted in increased PROD activity in theavian species. As the avian cytochrome P450 immunorelated to CYP1A1 appeared to beinduced by MC and AR, and because purified rat CYP2C1 1 and CYP3A1 are capable ofcatalyzing PROD and BPND activities in rats (Ryan and Levin, 1990), it is postulated thatthe observed increase in PROD activity in the birds might have resulted from the inductionof avian cytochrome P450 enzymes related to rat CYP1A1, CYP2C11 or CYP3A1.Antibody against rat CYP2B1 was not used due to the limited amount available.The antibody inhibition data for PROD activity are presented in Table 3.8.Monoclonal antibody against CYP1A1 and monospecific antibody against rat CYP2C11did not inhibit PROD activity in PB-treated rats. The PROD activity of hepaticmicrosomes from PB-treated rats was inhibited 18% and 28% by polyspecific antibodyagainst CYP2C 11 and CYP3A 1, respectively. Control rabbit IgG appeared to suppressedPROD activity (31 %) in hepatic microsomes from MC-treated chickens. In hepaticmicrosomes from MC-treated chickens, monoclonal anti-CYP1A1 IgG produced 60%inhibition of microsomal PROD activity, while the other three antibodies did not affectthis activity. For MC-treated quail, no decrease of PROD activity was found with antiCYP1A1 antibody and slight decrease in the activity was observed with the other threeantibodies.RESULTS /118Table 3.8. Inhibition of 7-pentoxyresorufin 0-depentylase activities (PROD) inhepatic microsomes from PB-treated rats, MC-treated chickens and MC-treatedquail by antibodies against various forms of rat cytochrome P450.PB-Treated Rat MC-Treated Chicken MC-Treated QuailAntibodies PROD PROD PROD PROD PROD PRODActivity Inhibition Activity Inhibition Activity InhibitionWithout IgG 1.66 0% 0.078 0% 0.328 0%ControllgG 1.61 3.0% 0.054 31% 0.328 0%MonoclonalAnti-1AI 1.64 no 0.031 60% 0.417 noIgGAnti-2C11 1.64 no 0.076 2.6% 0.315 4.0%MonospecificIgGAnti-2C11 1.36 18% 0.075 3.8% 0.307 6.4%PolyspecificIgGAnti-3A1 1.20 28% 0.069 12% 0.311 5.2%PolyspecificIgGNote: The amount of monoclonal antibody against rat CYP1A1 added to 2 ml of reaction mixturewas 0.03 mg, which was equivalent to 59 p.g, 140 rig, 61 p.g of IgG per nmole of cytochromeP450 for microsomes from PB-treated rats, MC-treated chickens and MC-treated quail,respectively. The amounts of control rabbit IgG and other anti-cytochrome P450 IgG added to thereaction mixture were 0.5 mg, which was equivalent to 0.98 mg, 2.4 mg and 1.0 mg of IgG pernmole cytochrome P450 for microsomes from PB-treated rats, MC-treated chickens and MC-treated quail, respectively. PROD activities were expressed as nmole resorufin per mg protein perminute and were measured according to the standardized conditions described in section 3.2. 1.3.5.Inhibition % [(Activity without IgG - Activity with IgG) / Activity without IgG] x 100%.RESULTS / 1193.4.3. Antibody Inhibition of Hepatic BPND activityThe effects of polyspecific antibodies against rat CYP2B1, CYP2C1 1 and CYP3A1on BPND activity in rats, chickens, quail and pigeons treated with PB, MC or AR wereinvestigated. The rationale for this experiment was based on the earlier observations thatBPND activity was increased by treatment with PB, MC or AR in rats and the three avianspecies and that the three avian species had cytochrome P450 proteins related to CYP2C 11and CYP3A1. As stated above, CYP2C11 and CYP3A1 or the avian cytochrome P450proteins immunorelated to these rat enzymes may contribute to the BPND activities intreated rats and birds.Table 3.9 lists the antibody inhibition data for hepatic BPND activity ofmicrosomes prepared from rats treated with PB or AR. Polyspecific antibody against ratCYP2B 1 produced 44% to 51 % inhibition of the BPND activities in the both ratmicrosomal samples, while polyspecific anti-2C11 IgG resulted in 20% to 22% inhibitionof BPND activity. Polyspecific antibody against rat CYP3A1 yielded only 5.9%reduction in the BPND activity with microsomes from PB-treated rats and no inhibitionwith microsomes from AR-treated rats. These results suggest that rat CYP2B1,CYP2C 11, and possibly CYP3A 1 contribute to the BPND activity in hepatic microsomesfrom PB- and AR-treated rats.Table 3.10 shows the antibody inhibition data for BPND activity of hepaticmicrosomes prepared from chickens treated with PB, MC or AR. It is found that BPNDactivity was inhibited to an appreciable degree by polyspecific antibodies against CYP2B1,CYP2C 11 and CYP3A 1 in all three treatment groups. These results indicate that theBPND activity in chicken liver may be catalyzed by chicken cytochrome P450 enzymesimmunochemically related to rat CYP2B, 2C, and 3A subfamilies.Table 3.11 summarizes the antibody inhibition data for BPND activity of hepaticmicrosomes from quail treated with MC or AR. Polyspecific anti-CYP2B1 IgG,polyspecific anti-CYP2CI 1 IgG and polyspecific anti-CYP3AI IgG were much lessRESULTS / 120inhibitory in quail than in chickens.Table 3.9. Inhibition of benzphetamine N-demethylaseactivity (BPNI)) in hepatic microsomes from PB-treatedand AR-treated rats by antibodies against various forms ofrat cytochrome P450.PB-Treated Rat AR-Treated RatAntibodies BPND BPND BPND BPNDActivity Inhibition Activity InhibitionWithout IgG 22.85 0% 26.66 0%Control IgG 23.27 26.10 2.0%Anti-2B1 11.56 49% 14.88 44%PolyspecificIgGAnti-2C11 17.91 22% 21.37 20%PolyspecificIgGAnti-3A1 21.51 5.9% 27.08 0%PolyspecificIgGNote: The amounts of control rabbit IgG and anti-rat cytochrome P450IgG added to 1.5 ml of reaction mixture were 1.0 mg, which wasequivalent to 1.7 mg and 0.98 mg of IgG per nmole of cytochromeP450 for microsomes from PB-treated and AR-treated rats,respectively. BPND activity is expressed as nmole HCHO formed permg protein per minute and was measured according to the standardizedconditions described in section 3.2.1.4.5. Inhibition % = [(Activitywithout IgG - Activity with IgG) I Activity without IgGj x 100%.Similarly, Table 3.12 shows the antibody inhibtion data for BPND activities ofhepatic microsomes from MC- and AR-treated pigeons. Hepatic BPND activities wereRESULTS / 121inhibited 21% to 35% by polyspecific antibody against rat CYP2B1, 16% to 18% by antiCYP2C1 1 antibody, and 7% to 16% by anti-CYP3A1 IgG. The results suggest thatpigeon BPND activity may be contributed by pigeon cytochrome P450 enzymesimmunorelated to CYP2B1, CYP2C11 and CYP3A.Table 3.10. Inhibition of benzphetamine N-demethylase activities (BPND) inhepatic microsomes from PB-treated, MC-treated and AR-treated chickens byantibodies against various forms of rat cytochrome P450.PB-Treated Chicken MC-Treated Chicken AR-treated ChickenAntibodies BPND BPND BPND BPND BPND BPNDActivity Inhibition Activity Inhibition Activity InhibitionWithoutlgG 11.0 0% 8.55 0% 6.90 0%Control IgG 12.6 no 11.1 no 7.05 noAnti-2B1 9 16 17% 7 05 18% 5.49 20%PolyspecificIgGAnti-2C11 7.61 31% 7.75 9.3% 5.49 20%PolyspecificIgGAnti-3A1 10.6 3.9% 9.72 no 6.20 10%PolyspecificIgGNote: The amounts of control rabbit IgG and anti-rat cytochrome P450 IgG added to 1.5 ml ofreaction mixture were 1.0 mg, which was equivalent to 5.1 mg, 4.0 mg and 8.8 mg of IgG pernmole cytochrome P450 for microsomes from PB-treated, MC-treated and AR-treated chickens.BPND activity is expressed as nmole HCHO formed per mg protein per minute and was measuredaccording to the standardized conditions described in section 3.2.1.4.5. Inhibition % = [(Activitywithout IgG - Activity with IgG) / Activity without IgG] x 100%.RESULTS/122In summary, antibodies against rat CYP2B, CYP2C and CYP3A enzymes inhibitedBPND activity in avian hepatic microsomes.Table 3.11. Inhibition of benzphetamine N-demethylase activity (BPND) inhepatic microsomes from quail treated with PB, MC and AR by antibodies againstvarious forms of rat cytochrome P450.PB-Treated Quail MC-Treated Quail AR-Treated QuailAntibodies BPND BPND BPND BPND BPND BPNDActivity Inhibition Activity Inhibition Activity InhibitionWithoutlgG 18.1 0% 21.0 0% 22.6 0%Control IgG 17.7 5.5% 20.8 1.0% 22.3 1.0%Anti-2B1 16 4 9.4% 17.9 15% 20.3 10%PolyspecificleGAnti-2C11 17 6 2.3% 20.0 4.6% 22.1 1.8%PolyspecificIgGAnti-3A1 17 1 5.5% 19.0 8.8% 21.7 3.7%PolyspecificIgGNote: The amounts of control rabbit IgG and anti-rat cytochromes P450 added to 1.5 ml ofreaction mixture were 1.0 mg, which was equivalent to 8.3 mg, 1.7 mg and 2.4 mg of IgG pernmole cytochrome P450 for microsomes from PB-treated, MC-treated and AR-treated quail,respectively. BPND activity is expressed as nmole HCHO formed per mg protein per minute andwas measured according to the standardized conditions described in section 3.2.1.4.5. Inhibition% = [(Activity without IgG - Activity with IgG) I Activity without IgG] x 100%.RESULTS / 123Table 3.12. Inhibition of benzphetamine N-demethylaseactivities (BPND) in hepatic microsomes from MC-treatedand AR-treated pigeons by antibodies against variousforms of rat cytochrome P450.MC-Treated Pigeons AR-Treated PigeonsAntibodies BPND BPND BPND BPNDActivity Inhibition Activity InhibitionWithout IgG 10.0 0% 9.44 0%Control IgG 10.3 no 9.58 noAnti-2B1 8.17 18% 6.27 35%PolyspecificlgGAnti-2C11 8.46 15% 8.10 14%PolyspecificIgGAnti-3A1 9.58 4.0% 8.10 14%PolyspecificIgGNote: The amounts of control rabbit IgG and anti-rat cytochromesP450 added to 1.5 ml of reaction mixture were 1.0 mg, which wasequivalent to 1.7 mg and 2.4 mg of IgG per nmole cytochrome P450for microsomes from MC-treated and AR-treated pigeons,respectively. BPND activity is expressed as nmole HCHO formed permg protein per minute and was measured according to the standardizedconditions described in section 3.2.1.4.5. Inhibition %= [(Activitywithout IgG - Activity with IgG) I Activity without IgG] x 100%.1244. DISCUSSIONThe present study examined the effects of treatment with three prototype inducers,PB, MC and AR, on hepatic cytochrome P450 enzymes and their monooxygenaseactivities in adult chickens, quail and pigeons. The purpose of this investigation was toprovide a more direct comparison of the effects of these chemicals on the hepaticcytochrome P450 composition and related enyme activities in three avian species.4.1. C0MPA1us0N OF TILE EFFECTS OF PB, MC AR ON TOTAL HEPATICCYTOCIIROME P450 CONThNTCytochrome P450 content has frequently been used as a crude measure of theoxidative capacity of an animal and is a major factor in determining xenobiotic half-life.The results shown in Table 3. 1 demonstrate that control adult rats have greater hepaticcytochrome P450 content than the control adult chickens, quail and pigeons. This findingagrees with previous reports (Bursian et al., 1983; Chhabra et a!., 1974; Davison et a!.,1976; and Litterst et a!., 1975). In a comparison of sixteen species of birds with ninespecies of mammals, Ronis and Walker (1989) concluded that mammals have a fourfoldgreater hepatic cytochrome P450 content than birds and that the lowest level in mammalswas greater than the highest value for birds. The present study also shows that untreatedadult male chickens and quail have higher hepatic cytochrome P450 content than theirfemale counterparts. Ronis and Walker (1989) found that the average values of hepaticcytochrome P450 content in adult male and female quail were 0.32 nmole/mg protein and0.19 nmole/mg protein, respectively. Compared with the average value given by Ronisand Walker (1989), the cytochrome P450 content of male quail shown in Table 3. 1 ishigher. However, Miranda et al. (1992) reported that the cytochrome P450 content inadult male quail was 0.55 nmole/mg protein, which is very close to the one shown inTable 3. 1. Banton et a!. (1992) found that control male White Leghorn chickensDiSCUSSION / 125contained a hepatic cytochrome P450 content at 0.17 nmole/mg protein. Ronis et al.(1987) reported that microsomes pooled from control male and female pigeons had acytochrome P450 content of 0.41 nmole/mg protein. The values reported by the lattertwo laboratories are very close to the cytochrome P450 contents for female chickens aswell as male and female pigeons that are shown in Table 3.1.Although the hepatic cytochrome P450 content was inducible by PB, MC and ARin the three avian species, the response to treatment with the inducers was quantitativelydifferent between rats and the three avian species, as well as between chickens, quail andpigeons. Total cytochrome P450 contents in the treated rats are generally higher thanthose in treated birds, except for quail and pigeons that were treated with MC. This maybe explained by the higher basal level of hepatic cytochrome P450 in male rats.Comparison of effects of treatment, at the doses used, on hepatic total cytochrome P450among the three avian species and rats indicates that (1) the fold induction effected bytreatment with PB in rats is much lower than that in chickens but slightly higher than thatin quail and pigeons, (2) the fold induction resulting from treatment with MC in rats ismuch lower than that in the three avian species, and (3) the fold induction caused bytreatment with AR in rats is greater than that in chickens but lower than that in quail andpigeons. The marked induction of cytochrome P450 in the chickens receiving similartreatment with PB suggests that this species is more sensitive to the inducing effects of PB,at the dose used, than quail, pigeons or rats. In addition, similar treatment with MC (at adose of 25 mg/kg body weight for 3 days) resulted in greater fold induction and higherlevel of total cytochrome P450 in the birds than in the rats, implying that avian species aremore sensitive to the inducing effect of MC than rats.In rats, treatment with AR, a mixed-type inducer, generated a total hepaticcytochrome P450 content (3.4 nmole P450/mg protein) that is equivalent to the sum of thecytochrome P450 contents produced by respective treatment with MC and PB (i.e. 2.0 +1.4 = 3.4 nmole P450/mg protein). The additive effect of AR on cytochromes P450 wasDISCUSSION / 126not observed in any of the bird species; nevertheless, in quail and pigeons, treatment withMC resulted in total hepatic cytochrome P450 contents (2.0 nmole/mg protein) that areslightly greater than the sum of cytochrorne P450 contents caused by respective treatmentwith PB and AR (1 .80-1 .87 nmole/mg protein).4.2. CoMpARisoN OF THE EFFECTS OF PB, MC AND AR ON RAT AND AVIAN HEPATICMICROSOMAL MONOOXYGENASE AcTivrriEsIn the present study, preliminary experiments on the development of the fourenzyme assays, EROD, AHH, PROD and BPND, ensured that the rates of metaboliteformation were linear with respect to both microsomal protein concentration arid reactiontime at saturating concentrations of the substrates and cofactor, NADPH.Examination of the four monooxygenase activities in untreated rats and birdsrevealed that male chickens and quail had higher basal levels of the monooxygenaseactivities than females birds, whereas marked gender differences in the rat were associatedonly with BPND activity but not with EROD and PROD activities (Table 3.2, 3.4 and3.5). The higher basal levels of monooxygenase activities are in parallel with the greatercytochrome P450 content in the untreated male chickens and quail (Table 3.1).The monooxygenase activities of the inducer-treated rats and birds demonstratedthat the effect of each inducer varied with the individual enzyme activity and species andare discussed more fully below.4.2.1. EROD ActivityThe selective induction of hepatic EROD activity by MC-type inducers has beenwell documented in the rat (Okey, 1988). Burke and Mayer (1974) reported that MCpretreatment of male Sprague-Dawley rats (20 mg/kg body weight i.p., once daily for 3days) induced EROD activity 70-fold while PB pretreatment of rats (80 mg/kg bodyweight i.p., once daily for 5 days) did not increase this enzyme activity. Lubet et al.DISCUSSION / 127(1990b) reported that pretreatment of male F344/NCr rats with AR (500 mg/kg bodyweight one i.p. injection) and PB (sodium salt, 0.05% w/v in drinking water for 14 days)resulted in 84- and 2.2-fold induction in hepatic EROD activity, respectively. In thepresent study, the EROD activity for untreated and treated rats is in agreement with thepreviously reported observations of other investigators (Burke and Mayer, 1974; Lubet etal., 1990b) and was preferentially induced by MC and AR in rats.Earlier studies on the induction of avian hepatic EROD activity by MC-typeinducers and mixed-type inducers have led to the perception that avian hepatic ERODactivity is also selectively induced by these two types of inducer (Ronis and Walker,1989). Banton et al. (1992) reported a 27-fold increase in EROD activity in young malechickens treated with 13-naphthoflavone (50 mg/kg body weight i.p. once daily for 3consecutive days). Lubet et al. (1990b) reported that EROD activity was induced 14-foldin hepatic microsomes from female Japanese quail (8-12 weeks of age) that received asingle dose of AR (200 mg/kg body weight i.p., sacrificed 72 hr after the singleinjection). In the present study, induction of hepatic EROD activity by MC and AR in thethree avian species strongly agree with the previous findings. Unlike the situation in ratswhere a slight increase in EROD activity was observed after treatment with PB, ERODactivity was either suppressed or unchanged by PB in all the three avian species.However, in a previous investigation, a 15% increase was reported for female quailtreated with PB (0.1 % in drinking water for 7 days, sacrificed on day 7 of the treatment)by Lubet et al. (1990b).Moreover, the present results demonstrate species differences in the induction ofhepatic EROD activity in the birds. Hepatic EROD activity was highly inducible infemale chickens, slightly less inducible in female quail and much less inducible (by afactor of 12 relative to chickens) in pigeons after similar treatment with MC. Similarly,after the same treatment with AR, hepatic EROD activity in pigeons was not induced tothe same extent as either chickens or quail. It is interesting to note that hepatic ERODDISCUSSiON / 128activities in untreated and corn oil-treated pigeons were higher than the activities inuntreated and corn oil-treated chickens and quail. These data indicate that pigeons have ahigher basal level of hepatic EROD activity but that this activity in pigeons may not be ashighly inducible by MC and AR as it is in chickens and quail. Alternately, pigeons maysimply require a larger dose of these inducers to produce the same level of induction. Forexample, Borlakoglu et al. (1991) reported a 48-fold induction of hepatic EROD activityin adult male and female pigeons treated with AR (500 mg/kg body weight i.p., sacrificed120 hr after single injection).Hepatic EROD activity has been widely used as bio-indicator for exposure to MC-type environmental pollutants in mammals, aquatic species and wild birds (Burke andMayer, 1974; Kleinow et al., 1987; Stegeman and Kloepper-Sams, 1987). The presentstudy provides evidence that hepatic EROD activity could be a useful indicator forexposure to MC-type and mixed-type inducers in avian species. As stated above, hepaticEROD activity is mainly mediated by CYP1A1 enzyme in MC- or AR-treated rats (Burkeand Mayer, 1974), but it is not known which enzymes contribute to the EROD activity inthe three avian species.4.2.2. AHH ActivityIt is known that hepatic AHH activity can also be induced by MC and AR in rats(Okey, 1990). Luster et a!. (1982) reported 2-, 27- and 8-fold of induction of hepaticAHH activity in male Sprague-Dawley rats treated with PB (75 mg/kg body weight i.p.,once daily for 4 consecutive days), MC (50 mg/kg body weight i.p., once daily for 3days) and AR (50 mg/kg body weight i.p., once daily for 3 days), respectively. Thepresent study shows that the induction of AHH activity in male Long Evans rats by thethree compounds agrees with the previous findings.Earlier studies in other laboratories on the induction of AHH activity in avianspecies suggest that avian AHH activity is also inducible by MC-type inducers and mixed-DISCUSSION / 129type inducers. Buynitzky et al. (1978) reported that AHH activity in adult male WhiteLeghorn chickens was increased 20-fold by MC (20 mg/kg body weight i.p., once dailyfor 2 consecutive days). Carpenter et al. (1985) found a 13.4-fold increase in adultfemale Japanese quail treated with 13-naphthoflavone (150 mg/kg body weight i.p., oncedaily for 4 consecutive days). Similarly, the data shown in Table 3.3 demonstrates thathepatic AHH activity is inducible by MC as well as by AR in female chickens and quail.Treatment with MC induced the AHH activity in female chickens and quail to a similardegree whereas treatment with AR produced higher AHH activity in female quail than infemale chickens.Previous investigations found that PB also induced hepatic AHH activity inchickens, quail and pigeons. Darby et al. (1984) injected 8 mg of PB into White Leghornchicken eggs and found a 2.7-fold induction of AHH activity in chicken embryo.Carpenter et al. (1985) reported a 4.6-fold induction of AHH activity in adult femaleJapanese quail treated with PB (70 mg/kg body weight i.p., once daily for 5 days).Husain et al. (1981) found a 2.2-fold induction of AHH activity in adult male pigeonstreated with PB (40 mg/kg body weight i.p., once daily for 3 days). The present studyshows that PB-treatment induced hepatic AHH activity to a greater extent in chickens thanin quail but slightly suppressed AHH activity in pigeons. As stated in the Experimentalsection, four pigeons in PB-treatment group died after receiving the third dose, and theremaining two were not given the fourth dose. The toxicity of PB in the pigeons mayhave negatively affected induction of AHH activity.The AHH data on the pigeons indicate that this avian species has higher basal levelof hepatic AHH activity and their AHH activity is refractory to the three inducers used.It is well known that hepatic AHH activity in rats, especially in MC-treated rats, ismediated to a larger extent by CYP1A1 (Okey, 1990). However, it is not known whichcytochrome P450 enzyme contributes to avian hepatic AHH activity.DISCUSSiON / 130As with EROD activity, hepatic AHH activity might be useful as an indicator ofenvironmental exposure of birds to MC-type inducers. However, at equivalent doses ofMC and AR, the greater increase in hepatic EROD activity makes it a more powerfulmeasure of exposure. In addition, the EROD assay is much simpler than the AHH assay,and often produced more reliable and better duplicate results than the AHH assay.Moreover, resorufin is the only fluorescent metabolite of the dealkylation of 7-ethoxyresorufin at the excitation and emission wavelengths used. The fluorescence whichis proportional to the amount of resorufin formed in the reaction mixtures is measureddirectly while the deethylation is proceeding. This advantage of the EROD assay avoidserrors caused by measurement of fluorescence produced by other metabolites as well as byincomplete extraction of metabolites in the AHH assay. Thus, compared to the AHHassay, the EROD assay would be a better choice for measurement of exposure of avianspecies to MC- or mixed-type chemicals in the environment.4.2.3. PROD ActivityLubet et al. (1985) reported that treatment of male Fisher rats with PB (75 mg/kgbody weight i.p., once daily for 4 days), MC (50 mg/kg body weight i.p., once daily for3 days) and AR (one i.p. injection of 500 mg/kg body weight) increased hepatic PRODactivity by 94-, 1.4- and 52-fold, respectively. The data in the present study on rathepatic PROD activity agree with the previous reports. Thus, it appears that PRODactivity is greatly inducible by both PB and AR in the rat.The current investigation (Table 3.4.) reveals, for the first time, that hepaticPROD activity, unlike in rats, is suppressed by PB but induced by MC in adult femalechickens, quail and pigeons. The present study also shows that avian hepatic PRODactivity is inducible by AR as in the rat. Once again, pigeons were more resistant to theeffects of PB, MC and AR treatment, at the dosewsed, than chickens and quail. Lubet etal. (1990b) reported that quail hepatic PROD activity was unchanged by PB treatmentDISCUSSION / 131(0.1 % PB sodium salt in drinking water for 7 days), whereas Banton et al. (1992) foundthat treatment of male chickens with PB (60 mg/kg body weight i.p., once daily for 3days) produced a modest decrease in PROD activity. In addition, Bunyan and Page(1978) found that treatment of female Japanese quail (4-7 week old) with a MC-typeinducer, 3,4,5,3’ ,4’ ,5’-tetrachlorobiphenyl, (50 ppm in diet for 20 consecutive days),resulted in a 2.8-fold increase in ethylmorphine N-demethylase activity, which is normallyan indicator for PB-type induction in rats (Thompson and Holtzman, 1973).CYP2B1/2 account for more than 90% of hepatic PROD activity in microsomesfrom PB-treated rats (Lubet et at., 1985) and purified CYP2B1 catalyzes the PRODreaction at a rate that is 100-fold faster than with CYP2B2 (Wolf et at., 1988). However,it is not clear whether these findings are applicable to avian hepatic PROD activity.4.2.4. BPND ActivityHepatic BPND activity has also been used as an indicator for PB-type induction inrats (Lu et at., 1973). Guengerich et at. (1982) reported that hepatic microsomal BPNDactivity was induced 3.6- and 2.8-fold by PB and AR, respectively, while 13-naphthoflavone decreased this activity by about 38%. Yoshimura et al. (1979) reportedthat hepatic microsomal BPND activity was induced 3-fold by PB in adult male Wistarrats (80 mg/kg body weight i.p., once daily for 5 consecutive days) and was suppressedby approximately 20% and 80%, respectively, in rats treated with MC (20 mg/kg bodyweight i.p., once daily for 5 consecutive days) and a potent MC-type inducer,3,4,5,3’,4’,5’-hexachlorobiphenyl (10 mg/kg body weight i.p., once daily for 5consecutive days). The rat hepatic microsomal BPND activity shown in Table 3.5confirms that hepatic BPND activity is inducible by PB and AR but is slightly repressedby MC in rats.Moreover, the present study (Table 3.5) indicates that BPND activity is inducibleby PB, MC and AR in the avian species. Gupta et al. (1990) also reported that treatmentDISCUSSION / 132of adult White Leghorn hens with PB (80 mg/kg body weight i.p., once daily for fourdays) produced a 3.7-fold induction of hepatic microsomal BPND activity. A twofoldincrease in BPND activity in PB-treated 36-day old chickens (Cornell K-strain) wasreported by Lorr et al. (1989). However, the present data reveal, for the first time, thathepatic BPND activity, unlike in the rat, is also induced by MC in the avian speciesexamined. Furthermore, species differences in the induction of hepatic BPND activity byeach inducer are obvious. This activity appears to be more readily induced by MC or ARin female quail than in rats, chickens and pigeons, but seems to be more sensitive to theinducing effect of PB in chickens than in the other species.It is well established that PROD and BPND activities are mainly induced by PB-type and mixed-type inducers in mammals such as rats (Lu et al., 1973; Wolf et al.,1988). However, data presented in Table 3.4 and 3.5 show that the induction pattern ofPROD and BPND activities in the avian species is qualitatively different from that in ratsand is also quantitatively variable among avian species, although the prototype mixed-typeinducer, AR, is a relatively effective inducer for these two enzyme activities in all thespecies. Treatment with PB results in suppression of hepatic PROD activity in all threeavian species, while it induces BPND activity in the same species, indicating that thesetwo activities may be catalyzed by different cytochrome P450 enzymes in PB-treatedbirds. On the other hand, both the PROD and BPND activities are markedly induced byMC in the female chickens and quail, while these two activities are either unchanged orsuppressed in MC-treated rats. In summary, the results demonstrate that PROD andBPND activities in the avian species are not selectively affected by any of the threeinducers used and thus cannot be used as indicators of exposure of birds to these classes ofchemicals.DiSCUSSION / 1334.2.5. Summary of the Effects of PB, MC and AR on Avian Hepatic MicrosomalMonooxygenase ActivitiesOverall, the present study of the induction of cytochromes P450 and theirmonooxygenase activities illustrates that the pattern of induction in rats differs from that inthe avian species. In rats, PB mainly induces PROD and BPND activities, MC mainlyinduces EROD and AHH activities, while AR serves as a mixed inducer since it inducesall the four monooxygenase activities. In the three avian species, PB acts as a modestinducer of AHH and BPND activities but appears to be a suppresser of EROD and PRODactivities, while MC and AR induce all four monooxygenases. The fact that PB-treatmentresults in suppression of EROD activity but induction of AHFI activity in the same avianspecies (i.e. female chickens) indicates that these two activities are mediated by differentcytochrome P450 enzymes. Similarly, PROD and BPND activities are likely to becatalyzed by different enzymes in the three avian species.Comparison of the four monooxygenase activities among the three avian speciesreveals that varying sensitivity to the inducers exists among species. Female chickensappear to be more sensitive to the effect of PB and MC, female quail appear to be moresensitive to the effect of AR, while pigeons are relatively insensitive to all threechemicals, at the doses used in this study.As mentioned above, pigeons have a higher basal level of EROD, AHH and BPNDactivities. Before the present study began, the pigeons developed a disease calledchiamydiofif and were treated with tetracycline antibiotics. Although they were drug-freefor one month before the administration of the inducers, the residual effects of the illnessand the antibiotic treatment might have caused some interference with the effects of theinducers.DISCUSSION / 1344.3. IMMUNOCHEMICAL RELATEDNESS BETWEEN Avu Cyiocmorvw P450 AND RATCYTOcIIRoME P450Analysis of the SDS-polyacrylamide gel of avian hepatic microsomes illustratesthat treatment with PB, MC or AR induced a number of hepatic proteins in the chickens,quail and pigeons (Figure 3.18). Immunoblots of avian hepatic microsomes probed withvarious anti-rat cytochrome P450 enzymes demonstrate that these avian species containedforms of cytochrome P450 that were immunochemically related to rat CYP1A, CYP2B,CYP2C and CYP3A. The immunoblot data is discussed more fully below.4.3.1. Avian Cytochrome P450 Inununochemically Related to CYP1A1/2Both monoclonal anti-rat CYP1A1 IgG and polyclonal anti-CYP1A1 serarecognized proteins in hepatic microsomes prepared from MC- and AR-treated birds(Figure 3.19 and 3.20). These results suggest that (1) each avian species contains ahepatic cytochrome P450 enzyme that is immunochemically related to rat CYP1A1 and (2)that the avian protein is either not constitutively expressed or present at very low levels inthe untreated birds but is induced by MC- or AR-treatment. Due to the fact that ratCYP1A1 and CYP1A2 have 68% amino acid sequence similarity, polyclonal anti-ratCYP1A1 sera recognizes both CYP1A1 and CYP1A2. Consequently, the presence of twoclosely resolved bands on the blot containing microsomes from MC- or AR-treatedchickens and probed with anti-rat CYP1A1 sera (lanes 17 and 18 in Figure 3.20) indicatesthat female chickens contain a cytochrome P450 protein that is immunochemically relatedto rat CYP1A2, but the immunological relationship to rat CYP1A2 cannot be definedfurther because an antibody specific for CYP1A2 is not available in our lab. The faintlower bands in microsomes from pigeon and quail (Figure 3.20) may be caused bynonspecific binding due to the presence of various proteins in the antisera, which is arelatively crude antibody preparation. For the same reason the interaction between theantisera and partially purified rat CYP1A1 produced two faint bands below the intenselyDISCUSSiON / 135stained CYP1A1 band (lane 1 in Figure 3.20). CYP1A1 is widely found in mammals aswell as other species such as insects and birds, whereas CYP1A2 has not been directlydetected in non-mammalian species such as birds (Gonzalez, 1988). Borlakoglu et at.(1991) found a microsomal protein in feral pigeons treated with AR by using monoclonalantibody against CYP1A1. Similarly, Ronis et a!. (1989) reported significant cross-reactivity in six species of sea birds with antibody against rat CYP1A1, however, onlyhepatic microsomes from cormorant (Phalacrocorax carbo) reacted with antibody againstrat CYP1A2. Comparable results were obtained with great blue heron hatchlings bySanderson et al. (1994).There is a considerable variability in the intensity of staining of the protein bandsin the three avian species. This variability might reflect either the degree ofimmunochemical relatedness between rat and the avian proteins or the tissue levels of theavian proteins, as staining on an immunoblot is determined by both the amount of antigenpresent and the affinity of the antibody for that antigen. The different electrophoreticbehavior of the avian antigenic proteins and rat CYP1A1 implies that the primarysequence of the avian immunoreactive proteins differs from that of rat CYP1A1.In summary, the cross-reaction between avian hepatic microsomes and antibodiesagainst rat CYP1A1 indicates that all three avian species contain cytochrome P450proteins that are analogous to rat CYP1A1. In addition, chickens have a secondcytochrome P450 protein that is immunorelated to rat CYP1A2, but the presence of asimilar protein in pigeons and quail remains elusive.4.3.2. Avian Cytochrome P450 Inununochemically Related to Rat CYP2B1The very weak cross-reactions between polyclonal antibody against rat CYP2B1and the avian microsomes (Figure 3.21) suggests that these three avian species containforms of cytochrome P450 that are weakly immunorelated to rat CYP2B enzymes.Previous reports in the literature on the presence of CYP2B proteins in avian species areDISCUSSiON / 136somewhat contradictory. For example, little cross-reactivity was observed betweenantibody against rat CYP2B1 and hepatic microsomes prepared from six species of seabirds (Ronis et al., 1989); yet Oron and Bar-Nun (1984b) reported that antibody againstrat CYP2B1 cross-reacted with a PB-induced cytochrome P450 from chick embryohepatocytes.4.3.3. Avian Cytochrome P450 Immunochemically Related to Rat CYP2C7CYP2C7 is a female-specific cytochrome P450 enzyme in rats that is expressed ina higher amount in the adult female rats than in the adult male rats (Bandiera et al., 1986).The cross-reaction between anti-rat CYP2C7 IgG and the avian microsomal proteinsgenerated very faint bands that migrated more slowly than purified rat CYP2C7 on SDSPAGE. May et al. (1987) isolated chicken CYP2HJ and 2H2 cDNAs and found that thededuced amino acid sequences shared 51% similarity with that of CYP2C7. In addition,May et al. (1987) found that CYP2H1 and 2H2 mRNAs were inducible by PB. It is notknown whether the avian immunoreactive proteins found in the present study are related toeither rat CYP2C7 or chicken CYP2H1/2.4.3.4. Avian Cytochrome P450 Inununochemically Related to Rat CYP2C11The immunoblot of avian hepatic microsomes probed with polyspecific antibodyagainst rat CYP2C1 1 (Figure 3.25) indicates that (1) chickens contain three hepaticmicrosomal proteins that are immunorelated to the rat CYP2C subfamily, (2) pigeons havetwo hepatic microsomal proteins that are immunorelated to the rat CYP2C subfamily, and(3) quail have three hepatic microsomal proteins that are immunorelated to the rat CYP2Csubfamily. However, the immunoblot probed with anti-rat CYP2C 11 monospecificantibody (Figure 3.26) demonstrates that only a single protein in chickens (i.e. hereafterreferred to as chicken-[CYP2C11]) and two proteins in quail (i.e. hereafter referred to asquail-[CYP2C1 1] and quail-[CYP2C 1112) were recognized by the monospecific antibody.DiSCUSSION / 137The differences between the two immunoblots can be explained by the differentspecificities of the two antibodies. It is known that the rat CYP2C subfamily membersshare at least 60% similarity in their amino acid sequences (Gonzalez et a!., 1986a).Polyspecific antibody against CYP2C11 recognizes not only CYP2C11 but also CYP2C6,CYP2C7, CYP2C12 and CYP2C13, whereas the monospecific antibody only recognizesCYP2C 11. Thus, the multiple bands generated by the reaction of the polyspecificantibody with avian hepatic microsomes represent multiple forms of avian cytochromeP450 that are immunorelated to the rat CYP2C subfamily. The immunoblot probed withmonospecific antibody against CYP2C11 (Figure 3.26) demonstrates that aviancytochromes P450 immunorelated to rat CYP2C1 1 are present in chickens and quail, butnot in pigeons.CYP2C 11 is known as a developmentally-regulated male-specific cytochrome P450in rats (Kamataki et al., 1985a). Interestingly, the immunoreactive chicken-[CYP2C11]and quail-[CYP2C1 l] proteins were detected in microsomes from both male and femalebirds. Although the chicken-[CYP2C11] protein appeared to be present in a greateramount in untreated male chickens relative to female chickens, because of the differencein cytochrome P450 content between male and female untreated chickens and because themicrosomes were applied to the gel on the basis of protein concentration, we cannotconclusively say that the males have higher level of chicken-[CYP2C11]. The male-predominant phenomenon was not observed with quail-[CYP2C1 1] (Figure 3.26).In the rat, CYP2C 11 is not inducible by treatment with a variety of xenobioticsincluding PB, MC and AR (Bandiera et a!., 1986). However, the immunoblot in Figure3.26 demonstrates that the chicken-[CYP2CI1] protein appears to be induced by PB, MCand AR, while the quail-[CYP2C 11] and quail-[CYP2C 1 l]2 proteins appear to beinduced by MC or AR since the staining intensity of the bands are more intense inmicrosomes from treated birds than that from control birds. This may be the first timethat monospecific anti-rat CYP2C11 IgG was used to detected the presence of avianDiSCUSSiON / 138cytochromes P450 immunorelated to rat CYP2C 11 in both male and female chickens andquail and to find that these avian cytochromes P450 are inducible by PB, MC or AR(Levin et al., 1993). Ronis et al. (1993) also found a microsomal protein in Bob Whitequail that was immunoreactive with anti-rat CYP2C11 polyclonal antibodies and inducibleby two fungicides, propiconazole and vinclozolin.4.3.5. Avian Cytochrome P450 linmunochemically Related to CYP2C MembersOther Than CYP2C13The immunoblot shown in Figure 3.27 demonstrates that these three avian speciescontained microsomal proteins that were recognized by polyspecific anti-rat CYP2C13IgG. The cross-reaction between polyspecific antibody against rat CYP2C13 and theavian microsomes generated a staining pattern similar to the one found on the blot probedwith anti-rat CYP2C1 1 polyspecific antibody, which is shown in Figure 3.25. This resultis not unexpected as polyspecific antibody against CYP2C 11 and polyspecific antibodyagainst CYP2C13 can both crossreact with all members of the CYP2C subfamily tovarious extents. However, no cross-reaction was found between anti-rat CYP2C13monospecific antibody and the avian hepatic microsomal proteins (Figure 3.28). The lackof interactions between the monospecific antibody against rat CYP2C13 and the avianhepatic microsomes excluded the possible presence of avian cytochromes P450immunorelated to rat CYP2C13.The cross-reaction between polyspecific antibody against CYP2C13 and avianmicrosomes are likely caused by the cross-reactivity of the antibody to the aviancytochrome P450 enzymes immunorelated to CYP2C1 1 and other CYP2C members thanCYP2C7 and CYP2C13. The identity of avian cytochrome P450 proteins that areimmunorelated to other rat CYP2C proteins remains to be resolved.DISCUSSION /1394.3.6. Avian Cytochrome P450 Immunochemically Related to rat CYP3AThe rat CYP3A subfamily consists of at least two isozymes, CYP3A1 and 3A2,having 89% amino acid similarity (Gonzalez, 1988). These two CYP3A isozymes can berecognized by polyspecific antibody against rat CYP3A1.In order to probe for the presence of CYP3A proteins in hepatic microsomes fromthe three avian species, two versions of the same antibody were used. The antibody wasinitially generated by immunizing rabbits with purified rat CYP3A 1, but after purificationof IgG from pooied sera some of the IgG was further fractionated by affinity columnchromatography (see Appendix 1 for details) to render back-absorbed antibody. Bothantibody preparations recognize CYP3A1 as well as CYP3A2 and are thus polyspecific,but the crossreactions with CYP3A2 and especially with other cytochrome P450 proteinsare greatly reduced for the back-absorbed antibody.The blot probed with the cruder (i.e. non back-absorbed) antibody preparation(Figure 3.29) indicates that there are more than two forms of avian cytochrome P450 thatare immunorelated to rat CYP3A isozymes, however, no protein band appeared on theblot probed with back-absorbed anti-CYP3A1 IgG (Figure 3.30). The back-absorbed antiCYP3A1 IgG was still highly reactive towards rat CYP3A1 and yielded cleaner resultsthan the polyspecific antibody against CYP3A1 that was not back-absorbed. The absenceof stained protein bands in the lanes containing avian hepatic microsomes (Figure 3.30)implies that the three avian species do not have cytochrome P450 proteins that are highlyimmunologically related to CYP3A 1 as anti-rat CYP3A 1 IgG that was extensively backadsorbed did not react with any of the avian microsomes. However, the variable antigenicreactivity of microsomes from different avian species toward the non back-absorbedantibody indicates that these avian species may contain cytochrome P450 enzymes that areimmunorelated to other CYP3A members. In addition, Lorr et al. (1989) reported that asingle PB- and DEX-induced chick cytochrome P450 was recognized by a monoclonalantibody against rat CYP3A2 and was found to have the same relative mobility as ratDiSCUSSiON / 140CYP3A2 on SDS polyacrylamide electrophoresis gel. In the present study, the middleband on the blot probed with the non back-absorbed IgG (referred to as pigeon-[CYP3A],and chicken-[CYP3A]2)appear to be induced by PB-treatment in pigeons and chickensand they migrated the same distance on the SDS polyacrylamide electrophoresis gel aspurified CYP3A1. Moreover, the lower band (referred to as quail-[CYP3A]9,quail[CYP3A]3and chicken-[CYP3A]2)were induced by MC and AR.In rats, CYP3A2 was found to be male-specific and inducible by dexamethasone(Cooper et al., 1993). In the present study, the immunoblot probed with non back-absorbed antibody (Figure 3.29) shows that the chicken-[CYP3AJ2band was present inmale chickens and inducible in the female chickens by treatment with PB, MC and ARand treatment with PB appears to induced this protein to a greater extent than the othertwo inducers. These findings indicate that chicken-[CYP3A]2may be a male-specificcytochrome P450 and is inducible in the females by the three inducers.The cytochromes P450 in pigeons that are immunorelated to rat CYP3A2 havedifferent staining pattern from those cytochromes P450 in chickens and quail in terms ofstaining density and relative mobility. This indicates that structural differences are presentin the avian cytochrome P450 proteins that are immunorelated to rat CYP3A2.4.3.7. Summary of the linmunochemical Relatedness of Avian and Rat CytochromesP450In general, the present study demonstrates that the three avian species possesscytochrome P450 proteins that are immunochemically related to the rat CYP1A, CYP2B,CYP2C and CYP3A subfamilies. The avian cytochrome P450 immunorelated to ratCYP1A1 is inducible by MC and AR. Adult female chickens also appear to have acytochrome P450 that is immunorelated to rat CYP1A2 and it is also inducible by MC andAR. Avian cytochromes P450 immunorelated to rat CYP2C1 1 are present in quail andchickens but not pigeons and unlike rat CYP2C 11, these avian cytochrome P450 are notDISCUSSION / 141solely expressed in adult males and are inducible by PB, MC and AR in the female birds.There are several forms of avian cytochrome P450 that are immunorelated to ratCYP3A2. These avian cytochromes P450 are structurally varied among the bird species.The three avian species contain cytochromes P450 that are weakly cross-reactive withpolyclonal antibody against CYP2B1 and also contain cytochromes P450 that are weaklyinteractive with antibodies against rat CYP2C7, but they do not have cytochromes P450that are immunorelated to CYP2C 13 and CYP3A 1.4.4. IMMUNOINIUBITION OF AVIAN CYTOCEIROME P450-DEPENDENT MONOOXYGENASEAcTivrrIEsMeasurement of avian hepatic microsomal monooxygenase activities toward fourxenobiotic substrates establishes that these enzyme activities are present and inducible (orsuppressed) in the avian species. Furthermore, the immunoblot results demonstrate thatseveral distinct forms of avian cytochrome P450 are present and inducible in the liver.The next section is a discussion of the possible contribution of individual aviancytochrome P450 enzymes to EROD, PROD and BPND activities, based on analysis ofthe results of the immunoinhibition experiments.4.4.1. EROD Activity and Avian Cytochromes P450 Iminunorelated to Rat CYP1A1Antibodies against rat cytochrome CYP1A1 were very effective at inhibitingEROD activity in hepatic microsomes from MC-treated chickens, quail and pigeons (Table3.7). Surprisingly, the monoclonal antibody mixture was more inhibitory than thepolyspecific sera. The results imply that hepatic EROD activity in MC-induced birds, asin MC-induced rats, is largely mediated by avian cytochrome P450 enzymes that areimmunorelated to rat CYP1A1. Support for this suggestion is provided by theimmunoblots probed with monoclonal antibody (Figure 3.19) and polyclonal antibody(Figure 3.20) against rat CYP1A1, which demonstrate that the three avian species haveDISCUSSION / 142cytochrome P450 proteins that are immunochemically related to rat CYP1A1, and by theavian hepatic EROD activity data (Table 3.2). Together, the results from theimmunoblot, enzymatic activity and immunoinhibition of enzymatic activity clearlydemonstrate that treatment with MC and AR induces avian hepatic cytochrome P450enzymes immunorelated to rat CYP1A1 and these induced avian enzymes contribute to theinduction of hepatic EROD activity in the three avian species.Compared to chickens and quail, the pigeon data are less explicit. Bands of aviancytochrome P450 immunochemically related to CYP1A1 are more intensively stained inmicrosomes from pigeons treated with MC and AR than in microsomes from quail andchickens treated with MC and AR (Figure 3.19). However, hepatic EROD activity inhepatic microsomes from MC- and AR-treated pigeons is much lower than that inmicrosomes from MC- and AR-treated quail and chickens (see Table 3.2), and is inhibitedto a lesser degree by monoclonal anti-rat CYP1A1 IgG, suggesting that in pigeons theMC-induced cytochrome P450 immunorelated to rat CYP1A1 should be a less effectivecatalyst for 0-deethylation of 7-ethoxyresorufin.In summary, it can be generally concluded that avian cytochromes P450immunorelated to CYP1A1 mediate avian hepatic EROD activity in the three avian speciestreated with MC and AR. Furthermore, avian hepatic EROD activity can be a usefulindicator for the induction or suppression of avian cytochrome P450 enzymesimmunorelated to CYP1A1.4.4.2. PROD Activity and Avian Cytochrome P450 EnzymesThe high level of inhibition of PROD activity (60%) by monoclonal anti-CYP1A1IgG in hepatic microsomes from MC-treated chickens was a somewhat intriguing resultand implies that avian cytochrome P450 enzymes immunorelated to rat CYP1A1 are goodcatalyst of this activity in MC- and, by extension in AR-treated chickens. As expected,the immunoinhibition data indicate that rat CYP1A1 does not contribute to PROD activityDISCUSSiON / 143in microsomes from PB-treated rats. However, avian cytochrome P450 enzymesimmunorelated to CYP1A1 do not appear to be involved in the 0-depentylation of 7-pentoxyresorufin in quail. The lack of inhibition of PROD activity by monoclonal antiCYP1A1 IgG in quail microsomes may be due to an insufficient concentration of theantibody since the amount of the monoclonal anti-CYP1A1 IgG per nmole cytochromeP450 in microsomes from MC-treated quail is less than half of that in microsomes fromMC-treated chickens.Inhibition of PROD activity was also observed when hepatic microsomes wereincubated in the presence of anti-CYP2C11 IgG and anti-CYP3A1/2 IgG. The degree ofinhibition was much greater with hepatic microsomes from PB-treated rats than withhepatic microsomes from MC-treated chickens or quail. The results indicate that ratCYP2C 11, CYP3A 1 and CYP3A2 catalyze PROD activity in PB-treated rats and thatavian cytochrome P450 enzymes immunorelated to these isozymes also contribute to thisactivity in MC-treated chickens and quail. Microsomes from PB-treated birds were notused in this experiment because PB-treatment did not induce this activity in any of thethree avian species.4.4.3. BPND Activity and Avian Cytochrome P450 EnzymesThe antibody inhibition data for BPND (Table 3.9) illustrate that CYP2B1 andCYP2C11 are responsible for most of the N-demethylation of benzphetamine inmicrosomes from PB- and AR-treated rats. This result agrees with the findings by Ryanand Levin (1989). Both anti-CYP2B1 IgG and anti-CYP2C11 IgG were effective atinhibiting BPND activity in microsomes from PB-, MC- and AR-treated chickens and inmicrosomes from MC- and AR-treated pigeons. At the single antibody concentrationsused, BPND activity was inhibited by both anti-CYP2C1 1 IgG and anti-CYP3A1 IgG to alesser degree in hepatic microsomes from quail than in microsomes from chickens andpigeons. A possible explanation may be that the amount of antibody added per nmole ofDISCUSSION / 144cytochrome P450 was less for microsomes from treated quail than for those from treatedchickens and pigeons. These results indicate that avian BPND activity in the inducedbirds is also mediated by avian cytochrome P450 enzymes immunorelated to CYP2B1 andcytochrome P450 enzymes immunorelated to CYP2C 11. The 10-14% inhibition of BPNDactivity by polyspecific anti-CYP3A1 IgG in microsomes from AR-treated chickens andpigeons suggests that BPND activity is partly mediated by avian cytochrome P450enzymes immunorelated to CYP3A2. However, it is not possible to estimate the relativecontribution of each isozymes to BPND activity as various concentrations of each antibodywere not tested.4.4.4. Summary of Inununoinhibition of Avian Cytochrome P450-DependentMonooxygenase ActivitiesIn summary, the present immunoinhibition data demonstrate that (1) aviancytochrome P450 enzymes that are immunorelated to rat CYP1A1 catalyze not only the 0-deethylation of 7-ethoxyresorufin in MC-treated birds but also 0-depentylation of 7-pentoxyresorufin in MC-treated chickens and (2) avian cytochromes P450 immunorelatedto rat CYP2B1, CYP2C11 or CYP3A2 catalyze hepatic BPND activity in the avianspecies treated with PB, MC and AR.CONCLUSIONS / 1455. CONCLUSIONSThe present study, employing four different enzymatic assays and western blotanalysis, demonstrates the presence and induction of avian cytochromes P450 as well asthe immunological and enzymatic relatedness of the avian cytochromes P450 to ratCYP1A1, CYP2B1, CYP2C11 and CYP3A2. The following conclusions are drawn fromthe present study:(1) As in rats, total cytochrome P450 specific content in the three avian species areinduced by PB, MC and AR.(2) As in rats, hepatic EROD and AHH activities in the chickens, quail and pigeons areinduced by MC or AR.(3) As in rats, hepatic PROD activity is inducible by AR in the three avian species;however, unlike in rats, this activity is suppressed by PB but induced by MC in the threeavian species.(4) As in rats, hepatic BPND activity in the three avian species is inducible by PB or AR;unlike in rats, this activity is also inducible by MC.(5) The three avian species contain hepatic cytochrome P450 enzymes that areimmunorelated to rat CYP1A1. These avian cytochromes P450 are inducible by MC orAR. In female chickens, this cytochrome P450 contributes to not only hepatic ERODactivity but also PROD activity.(6) The three avian species contain cytochrome P450 enzymes that are weaklyimmunorelated to rat CYP2B1/2 and these avian enzymes mediate hepatic BPND activityin PB-, MC- and AR-treated birds.(7) Chickens and quail, but not pigeons, contain hepatic cytochrome P450 enzymes thatare immunochemically related to rat CYP2C11 and these avian cytochrome P450 enzymescontribute to PROD and BPND activities. They appear to be inducible by MC or AR. Inaddition, the three avian species also contain hepatic cytochrome P450 enzymes that areCONCLUS’JON/ 146immunorelated to other rat CYP2C isozymes (but not to rat CYP2C13). 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Commun. 134, 499-505.APPENDiX /172APPENDIX IRelative Mobility (Rf)log MW 2.34 — 0.94 x Rf(R2 = 0.998)1 .0Figure 3.19. A plot of relative mobility (Rf) of molecular weight standards versus theirmolecular weights (MW). The molecular weight standards (MW) are E. coil. [3-galactosidase (116 kD), rabbit muscle phosphorylase (97.4 kD), bovine serum albumin(66.2 kD), hen egg white ovalbumin (42.7 kD), and bovine carbonic anhydrase (31.0kD). The standards were resolved on a discontinuous polyacrylamide gel, containing a7.5% polyacrylamide separating gel that was 0.75 mm thick and 12.5 cm long, asdescribed in the Experimental section.1 000.0 0.2 0.4 0.6 0.8APPENDIX /173APPENDIX HBack-Absorption of Polyspecific anti-rat 3A1 IgGThe antibody used for the blot shown in Figure 3.30 was obtained by passing thepolyspecific antibody used for the blot in Figure 3.29 through two different columns. Thefirst column that the polyspecific IgG passed was absorbed with adult untreated femalemicrosomes. The antibody eluted out from the first column and then was put through thesecond column which had been absorbed with ethanol-treated adult male rat microsomes.Since the adult untreated female rat microsomes contained a relatively small amount ofCYP3A2 (40 pmole/mg protein) but no 3A1 (Cooper et al., 1993), the polyspecific IgGagainst CYP3A1 would riot lose its cross-reactivity to CYP3A1 and 3A2, but unwantedcross-reactivities to other cytochrome P450 enzymes could be eliminated from the originalantibody. The ethanol-treated microsomes contained a higher amount of CYP3A2(approximately 170 pmole/mg protein, Cooper et al., 1993). Thus, the antibody thateluted from the second column should have diminished cross-reactivity to CYP3A2 butstill possess reactivity to CYP3A1, since the antibodies recognizing CYP3A2 were boundto the CYP3A2 antigens in the microsomes immobilized in the column. As shown in theblot probed with the back-absorbed anti-CYP3A1 IgG, purified rat CYP1A1 enzymesstrongly interacted with this antibody and produced a single dark band (Figure 3.30). Inaddition, the lower two bands below the CYP3A1 band shown in the blot probed with thepre-absorbed polyspecific IgG (Figure 3.29) disappeared from the blot in Figure 3.30.This indicates that back-absorbed anti-CYP3A1 IgG was still highly reactive towards ratCYP3A1 and yielded cleaner results than the polyspecific antibody against CYP3A1 thatwas not back-absorbed.

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