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Effect of cimetidine on hepatic cytochromes P450 1A and P450 2C in male rats Law, Eva Yuk Wa 1995

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E F F E C T O F CJJVIETJJJINE O N H E P A T I C C Y T O C H R O M E S P450 1A A N D P450 2C IN M A L E R A T S  By  EVA Y U K WA LAW B.Sc. (Pharm.), Pharm.D., The University of Minnesota, 1989  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCES in T H E F A C U L T Y O F G R A D U A T E STUDIES (Faculty of Pharmaceutical Sciences, Division of Clinical Pharmacy)  We accept this thjsis_as_£onforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A October 1995 © Eva Y u k W a Law, 1995  In  presenting  degree freely  at  the  available  copying  of  department publication  this  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  /^UA  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  I  I  further  purposes  gain  the  shall  requirements  agree  that  agree  may  representatives.  financial  permission.  Bepartme^J of  study.  of  It not  be is be  that  the  for  Library  an shall  permission for  granted  by  understood allowed  advanced  the that  without  make  it  extensive  head  of  copying my  my or  written  ii  Abstract Cimetidine, an H2-antagonist, is an inhibitor of hepatic cytochromes P450 in humans and male rats, both in vivo and in vitro. Results of early in vitro studies with rat hepatic microsomes showed that cimetidine, at concentrations in the millimolar range, inhibits many cytochrome P450mediated enzyme activities and that the inhibition appears to be reversible. Thus, it has been thought that cimetidine is a general and reversible inhibitor of the cytochromes P450. However, these early in vitro studies were performed at cimetidine concentrations that were 100-1000-fold higher than the serum cimetidine concentrations present in rats and humans.  As well, there is  indirect evidence from in vivo studies in both species to suggest that cimetidine does not inhibit certain cytochromes P450. In male rats, in vivo administration of cimetidine results in inhibition of hepatic CYP2C11 and some unidentified cytochrome(s) P450, but not CYP2A1, CYP2B1/2 or CYP3A1/2. In addition, preincubation of rat microsomes in vitro with cimetidine and N A D P H prior to the intiation of substrate oxidation increases the potency of its inhibition of CYP2C11 and results in a pattern of inhibition similar to that observed in vivo. These observations indicate that cimetidine exerts its inhibitory effect in vivo by acting as a catalysis-dependent inhibitor. It has been of interest to identify other rat cytochromes P450 that are affected by cimetidine and there is indirect evidence from the literature to suggest that cimetidine is an inhibitor of rat hepatic CYP1A1/2.  The primary objective of the present study was to investigate the effect of cimetidine on other hepatic cytochromes P450 in male rats, in particular CYP1A1/2.  The effects of in vivo  cimetidine on methoxyresorufin and ethoxyresorufin O-dealkylase (MROD and EROD) activities were examined in microsomes from P-napthoflavone (BNF)-induced and uninduced rats that had been administered cimetidine (150 mg/kg) or saline 90 min before decapitation.  Phenacetin  inhibition and immunoinhibition studies were conducted to determine the contribution of CYP1A and CYP2C enzymes to these activities. The effects of in vitro cimetidine on these activities were  iii  investigated in the absence or presence of a preincubation step. In vivo cimetidine had no effect on M R O D or EROD activity in microsomes from BNF-induced rats.  In microsomes from  uninduced rats, in vivo cimetidine inhibited EROD activity, but appeared not to have an effect on M R O D activity. Results of the phenacetin inhibition and antibody inhibition studies suggest that, in microsomes from BNF-induced rats, these activities are mainly due to CYP1A1 (with some contribution from CYP1A2) and that, in microsomes from uninduced rats, they are largely mediated by a CYP2C enzyme(s) other than CYP2C11.  The addition of cimetidine in vitro to  microsomes from BNF-induced or uninduced rats immediately prior to the initation of substrate oxidation resulted in inhibition of these activities with IC50 values ranging from 0.25 - 4 mM. Preincubation of rat liver microsomes with cimetidine in the presence of N A D P H for 15 min prior to the initiation of substrate oxidation had no effect on inhibition of M R O D or E R O D activity in BNF-induced rats. However, under the same conditions, preincubation enhanced the potency of the inhibition of EROD activity in uninduced rats by cimetidine 16-fold, but had no effect on M R O D activity. These results suggest that, in male rats, cimetidine does not inhibit CYP1A1 and has little or no effect on CYP1A2, but does inhibit another CYP2C enzyme(s) in addition to CYP2C11. As in the case of CYP2C11, the inhibition of the CYP2C enzyme(s) responsible for E R O D activity in uninduced rats appears to be mediated by a catalysis-dependent mechanism.  In humans, cimetidine inhibits the hepatic clearance of theophylline. Because CYP1A2 has been implicated as being responsible for at least part of the metabolism of theophylline in human microsomes, it is possible that cimetidine is an inhibitor of human CYP1A2.  Another  objective of the present study was to determine whether cimetidine was a catalysis dependentinhibitor of human hepatic CYP1A2. EROD activity was used an indicator of human CYP1A2. Preincubation of human liver microsomes with cimetidine in the presence of N A D P H prior to the initiation of substrate oxidation had no effect on inhibition of EROD activity. Although results of this preliminary experiment suggest that cimetidine has no catalysis-dependent effect on human CYP1A2, further studies are required to investigate the possible effects of cimetidine on this  iv  enzyme. Based on the observation from the present investigation that cimetidine inhibits another C Y P 2 C enzyme(s) (possibly CYP2C6) in addition to CYP2C11 in male rats and the fact that human and rat cytochromes P450 are immunochemically related, it is hypothesized that cimetidine also inhibits one or more of the CYP2C enzymes in humans.  Future studies with human  cytochromes P450 should investigate the possible effects of cimetidine on CYP2C9 and CYP2C19.  Table of Contents Section  Page no.  Abstract  ii  Table of Contents  v  List of Tables  . . .  List of Figures  ix x  List of Abbreviations  xiii  Acknowledgments  xiv  I. Introduction  1  1.1. Cytochrome P450  1  1.1.1. Background  1  1.1.2. Mechanism of action  1  1.1.3. Nomenclature  2  1.1.4. Major forms of cytochrome P450 in rats and humans  5  1.1.5. Induction  6  1.1.6. Inhibition  8  1.1.6.1. Type of inhibition  8  1.1.6.1.1. Competitive inhibition  9  1.1.6.1.2. Noncompetitive inhibition  10  1.1.6.1.3. Mixed inhibition  10  1.1.6.2. Mechanism of inhibition  11  1.1.6.2.1. Mechanisms of reversible inhibition  11  1.1.6.2.2. Mechanism of quasi-irreversible and irreversible inhibition  13  1.1.6.2.2.1. Metabolite intermediate complexation  13  1.1.6.2.2.2. Autocatalytic inactivation  14  . . .  1.1.7. Catalytic specificity of cytochrome P450  15  1.2. Inhibition of hepatic cytochromes P450 by cimetidine  18  1.2.1. Cimetidine  v  18  1.2.2. Selective inhibition of hepatic cytochromes P450 by cimetidine . . .  19  1.2.3. Mechanism(s) of inhibition of cytochromes P450 by cimetidine . . .  22  1.2.4. Effect of cimetidine on CYP1A2 in male rats and humans and on CYP1A1 in male rats 1.2.5. Isozyme-selective enzyme activities for rat and human CYP1A2  24 . .  28  .  30  1.3. Objectives II. Materials and Methods  32  2.1. Chemicals  32  2.2. Animals  34  2.3. Animal pretreatment  34  2.4. Human liver microsomal samples  35  2.5. Preparation of hepatic microsomes  .  36  2.6. Determination of cytochrome P450 content  38  2.7. Determination of protein content  38  2.8. Microsomal enzyme assays  39  2.8.1. Methoxyresorufin O-demethylase (MROD) and ethoxyresorufin O-  39  deethylase (EROD) assays 2.8.2. Testosterone 2a-and 16a-hydroxylase assays  39  2.8.3. Assay conditions  41  2.9. Enzyme kinetic studies of M R O D and EROD activities  41  2.10. Chemical inhibition studies on M R O D and EROD activities with phenacetin  41  2.11. Immunoinhibition studies  47  2.12. In vitro inhibition by cimetidine of M R O D , EROD, testosterone 2a- and 16a-hydroxylase activities  48  2.12.1. Without preincubation  48  2.12.2. With preincubation  48  2.13. Data analysis  49  III. Results  50  3.1. Effect of B N F on total hepatic microsomal cytochrome P450 content and enzyme activities in male rats  50  3.2. Effect of in vivo cimetidine  50  3.2.1. Total hepatic microsomal cytochrome P450 content  50  3.2.2. Microsomal enzyme activities  51  3.2.2.1. Microsomes from BNF-induced rats  51  3.2.2.2. Microsomes from uninduced rats  52  3.3. Phenacetin inhibition studies 3.3.1. Characterization of M R O D and EROD enzyme kinetics  59 . . . .  3.3.2. Inhibition of microsomal M R O D and EROD activities by phenacetin  3.4.  59 60  3.3.2.1. Microsomes from BNF-induced rats  60  3.3.2.2. Microsomes from uninduced rats  61  Immunoinhibition studies  74  3.4.1. Microsomes from BNF-induced rats  74  3.4.2. Microsomes from uninduced rats  76  3.5. In vitro inhibition by cimetidine of MROD, EROD, testosterone 2a- and 16a-hydroxylase activities  82  3.5.1. Without preincubation  82  3.5.1.1. Microsomes from BNF-induced rats  82  3.5.1.2. Microsomes from uninduced rats  82  3.5.2. With preincubation  83  3.5.2.1. Microsomes from BNF-induced rats  83  3.5.2.2. Microsomes from uninduced rats  84  3.5.2.1. Human liver microsomes 3.6.  85  Summary of results  94  IV. Discussions  96  4.1. Role of CYP1A2 and CYP1A1 in microsomal M R O D and E R O D activities  96  4.1.1. Microsomes from BNF-induced rats  96  4.1.1.1. Induction studies  96  4.1.1.2. Phenacetin inhibition studies  97  4.1.1.3. Immunoinhibition studies  101  4.1.2. Microsomes from uninduced rats  104  4.1.2.1. Phenacetin inhibition studies  104  4.1.2.2. Immunoinhibition studies  106  4.1.3. Summary 4.2. Effect of in vivo cimetidine on microsomal M R O D and E R O D activities  108 .  109  4.2.1. Microsomes from BNF-induced rats  109  4.2.2. Microsomes from uninduced rats  Ill  4.2.3. Summary  114  4.3. Effect of in vitro cimetidine on microsomal M R O D and E R O D activities .  114  4.3.1. In the absence of a preincubation step  114  4.3.2. In the presence of a preincubation step  116  4.3.3. Summary  118  4.4. Effect of preincubation on EROD activity in human liver microsomes 4.5. Future studies  . . 118 121  V . Summary and Conclusions  124  VI. References  126  ix  List of Tables  Table  Page no.  1  Trival names and catalytic specificities of some of the rat hepatic cytochromes I^''! 5 0 • • • • • • • • • •  2  Major classes of rat hepatic cytochrome P450 inducers  3  Estimated Michaelis-Menten kinetic parameters for high affinity and low affinity phenacetin O-deethylase activities in microsomes from uninduced and 3-MC-induced rats . . . . . . . .  29  4  Clinical data for patients from whom the liver samples were obtained  .  37  5  Summary of the assay conditions for MROD, EROD, testosterone 2a- and 16a-hydroxylase activities in rat microsomes . .  46  Effect of B N F on total hepatic microsomal cytochrome P450 content, M R O D , EROD, testosterone 2a-and 16a-hydroxylase activities .  53  Estimated Michaelis-Menten parameters for M R O D and E R O D activities in microsomes from BNF-induced and uninduced rats . . . .  64  Estimated K i values for inhibition of the microsomal M R O D and EROD activities in BNF-induced and uninduced rats by phenacetin .  69  6  7  8  9  .  .  .  ICso values for the inhibition of the various activities by in vitro cimetidine  7  .  87  List of Figures Figure  Page no.  1  The catalytic cycle of cytochrome P450  3  2  Structures of cimetidine and its major metabolites in rats  20  3  Optimization of conditions for M R O D assay in microsomes from BNF-induced rsts • • • • • • • • • •  42  Optimization of conditions for M R O D assay in microsomes from uninduced r&ts • • • • • • • • • •  43  Optimization of conditions for EROD assay in microsomes from BNF-induced r3.ts • • • • • • • • • •  44  Optimization of conditions for testosterone 2a- and 16a-hydroxylase assays in microsomes from BNF-induced rats . . . . . .  45  Effect of in vivo cimetidine on total hepatic microsomal cytochrome P450 content in BNF-induced and induced rats . . . . .  54  Effect of in vivo cimetidine on M R O D and EROD activities in microsomes from BNF-induced rats. Activities were calculated as the rate of product formation (A) per mg of microsomal protein or (B) per nmol of total P450  55  Effect of in vivo cimetidine on testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats Activities were calculated as the rate of product formation (A) per mg of microsomal protein or (B) per nmol of total P450 . . . . . . . . . .  56  Effect of in vivo cimetidine on M R O D and EROD activities in microsomes from uninduced rats. Activities were calculated as the rate of production formation per mg of microsomal protein. (A) 4 rats per group. (B) N = 3 rats in the cimetidine-treated group and 4 rats in the saline-treated group .  57  Effect of in vivo cimetidine on M R O D and EROD activities in microsomes from uninduced rats. Activities were calculated as the rate of production formation per nmol of total P450. (A) 4 rats per group. (B) N = 3 rats in the cimetidine-treated group and 4 rats in the saline-treated group . .  58  4  5  6  7  8  9.  10  11  xi  12  13  14  15  16  17  18  19  20  21  22  Effect of substrate concentration on M R O D and EROD activities in microsomes from BNF-induced and uninduced rats . . . .  63  Lineweaver-Burk plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from BNF-induced rats by phenacetin . . . .  65  Effect of phenacetin on (A) M R O D and (B) EROD activities in microsomes from BNF-induced rats . . . . . . .  66  Comparison of the effect of phenacetin on M R O D and E R O D activities in microsomes from BNF-induced rats. . . . . .. •  67  Dixon plots of inhibition of (A) M R O D and (B) EROD activities in microsomes from BNF-induced rats by phenacetin. . . . . .  68  Lineweaver-Burk plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from uninduced rats by phenacetin . . . .  70  Effect of phenacetin on (A) M R O D and (B) EROD activities in microsomes from uninduced rats . . . . . . . .  71  Comparison of the effect of phenacetin on M R O D and E R O D activities in microsomes From uninduced rats . . . . . .  72  Dixon plots of inhibition of (A) M R O D and (B) EROD activities in microsomes from uninduced rats by phenacetin . . . . . .  73  Effect of preimmune IgG, polyspecific anti-CYP2C11 and anti-CYP 1 A l IgG, and M A b C-8.1 on (A) M R O D and (B) EROD activities in microsomes from BNF-induced rats . . . . . . .  78  Effect of preimmune IgG and monospecific anti-C YP2C11 IgG on testosterone (A) 2a-hydroxylase and (B) 16a-hydroxylase activities in microsomes B N F induced rats . . . . . . . . .  79  23  Effect of preimmune IgG, polyspecific and monospecific anti-C YP2C11 IgG and polyspecific anti-CYP 1A1 IgG on testosterone (A) 2a-hydroxylase and (B) 16ahydroxylase activities in microsomes from uninduced rats . . . 80  24  Effect of preimmune IgG, polyspecific anti-CYP 1 A l , monospecific and polyspecific anti-CYP2Cl 1 IgG on (A) M R O D and (B) E R O D activities in microsomes from uninduced rats . . . . .  81  xii  25  26  27  28  29  30  31.  Effect of in vitro cimetidine (in the absence of a preincubation step) on M R O D , EROD, testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats . . . . . . . .  86  Effect of in vitro cimetidine (in the absence of a precubation step) on M R O D and EROD activities in microsomes from uninduced rats . . .  88  Effect of preincubation with cimetidine in the presence of N A D P H on (A) M R O D and (B) EROD activities in microsomes from BNF-induced rats  89  Effect of preincubation with cimetidine in the presence of N A D P H on testosterone (A) 2a-hydroxylase and (B) 16a-hydroxylase activties in microsomes from BNF-induced rats . . . . . .  90  Effect of preincubation with cimetidine in the presence of N A D P H on (A) M R O D and (B) EROD activties in microsomes from uninduced rats  91  Effect of preincubation with cimetidine in the presence of N A D P H on EROD activities in microsomes from individual uninduced rats . Effect of preincubation with cimetidine in the presence of N A D P H on E R O D activity in human liver microsomes . . . . .  .  92  93  xiii  List of Abbreviations BNF:  P-naphthoflavone  cm:  centimeter  EDTA:  ethylenediaminetetraacetic acid  ELISIA:  enzyme-linked immunosorbent assay  EROD:  ethoxyresorufin O-deethylase  g:  gram  h:  hour  HCB:  3A5,3\4\5'-hexachlorobiphenyl  HEPES:  (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])  i.p.:  intraperitoneal  kg:  kilogram  M:  molar  3-MC:  3-methylcholanthrene  mg:  milligram  min:  minute  mL:  milliliter  mM:  millimolar  MROD:  methoxyresorufin O-demethylase  NADPH:  (3-nicotinamide adenine dinucleotide phosphate  nm:  nanometer  TRIS:  (Tris[hydroxymethyl]amino-methane)  ug:  microgram  u,L:  microliter  uM:  micromolar  TCDD:  2,3,7,8-tetrachlorodibenzo-p-dioxin  xiv  Acknowledgements  I would like to thank my supervisor Dr. Marc Levine for his supervision and for being an excellent teacher. I also wish to thank Dr. Gail Bellward, for her helpful advice and for being very supportive, and to express my appreciation to Dr. Stelvio Bandiera for generously providing the polyclonal antibodies and for his enlightened discussions. I would also like to thank Dr. Thomas Chang for his technical assistance and insightful discussions, and Hong L i for her excellent technical support and caring.  Special thanks are also expressed to Dr. Paul Thomas for  generously providing the monoclonal antibody, Dr. Ronald Reid for chairing my committee and my former supervisor Dr. Alan Mitchell for his helpful guidence and encouragement.  1  Introduction  1.1. Cytochromes P450 1.1.1. Background After their introduction into the body, most drugs are eliminated either by direct renal excretion or undergo biotransformation in the liver to increase their polarity before excretion. The chemical reactions involved in drug biotransformation are classified as Phase I (oxidation, reduction, hydrolysis) and Phase II (conjugation) reactions. The major enzymes involved in drug oxidation and reduction are a family of closely related hemoproteins known as cytochromes P450 (Alvares and Pratt, 1990).  Cytochromes P450 are named this way as they yield absorption difference spectra with peaks at 450 nm following carbon monoxide binding to the reduced heme iron (Omura and Sato, 1964). They are found in almost every kind of cell in the body (Guengerich, 1993) and are present in especially high concentrations in the liver. Cytochromes P450 are embedded in the phospholipids of the endoplasmic reticulum membrane, and are coupled to a flavoprotein, N A D P H cytochrome P450 reductase, which transfers electrons to the cytochrome P450 enzymes during the oxidation reaction. Fragments of endoplasmic reticulum containing the cytochrome P450 enzymes can be isolated from liver homogenates by differential ultracentrifugation and are referred to as microsomes (Alvares and Pratt, 1990).  1.1.2. Mechanism of action The active sites of these enzymes contain a single iron protoporphyrin I X prosthetic group.  The mechanism by which cytochrome P450 oxidizes its substrate is depicted in the  catalytic cycle as shown in Fig. 1.  After the initial binding of the substrate to the ferric (Fe^ ) +  hemoprotein, an electron is transferred from the N A D P H cytochrome P450 reductase to reduce the heme iron to its ferrous state (Fe^ ). This is followed by the coupling of an oxygen molecule +  2  to the substrate-enzyme complex and the transfer of a second electron for the reduction of the complex. When the oxygen-oxygen bond is cleaved, one of the oxygen atoms is incorporated into a molecule of water and the other oxygen atom is transferred for oxidation of the substrate. Following the dissociation of the oxidized product, the ferric (Fe^ ) hemoprotein is regenerated +  (Guengerich, 1993; Alvares and Pratt, 1990).  1.1.3. Nomenclature While there is one only kind of cytochrome P450 reductase, there are multiple forms of cytochrome P450 (Alvares and Pratt, 1990), which can accommodate the catalysis of compounds with diverse structures.  As the various cytochromes P450 are products of discrete genes, the  P450 Nomenclature Committee has standardized the classification and nomenclature of these enzymes on the basis of their amino acid sequences as encoded by the cytochrome P450 genes (Nelson et al, 1993). Each human or rat cytochrome P450 gene is designated with an italicized root symbol "CYP" followed by an Arabic numeral, a letter and then another Arabic numeral e.g. CYP1A2. The corresponding cytochrome P450 enzyme carries the same name as its gene but its designation is not italicized. Cytochromes P450 with more than 40% similarity in their amino acid sequences belong to the same family and are given the same first Arabic numeral (with a few exceptions). Within each family, hemoproteins are divided into subfamilies as indicated by the assigned letters and members within a defined subfamily are more than 55% identical in their sequences.  Within each subfamily, proteins with more than 3% divergence in their amino acid  sequences are considered distinct enzymes and each of them is identified with a different second Arabic numeral. Listed in Table 1 are those rat cytochromes P450 that are discussed in the thesis and their trival names.  3  Fig. 1. The catalytic cycle o f cytochrome P450. (Adapted from Alvares and Pratt, 1990)  NADR^r  "^reduced V  NADPH—  flavoprotein.  Cyt P 4 5 0 ( F e ) 3+  CyrP450/«</wctese"" \^^ >  r  u  9^  R  H  )  c  o  m  P' ^\ e  V*>xidized flavoproteir  RH  / ^CO C y t P 4 S 0 ^=5  I CO  Absorbs light at 450 nm  o,  I  V.  Cyt P450(Fe ) 3+  Cyt P450(Fe /  ,eT  xidized drug  (ROH)  )  IRH IRH  "A  J ) r u g (RH)  Cyt P 4 5 0 ( F e ) 3 +  RH  I  Cyt P450(Fe *) 2  RH  I Cyt P450(Fe *)  ^  2  I , 2H  +  o,-  JZ  ^ " 2  4  Table 1 Trival names and catalytic specificities of some of the rat hepatic cytochromes P450. Catalytic specificity Enzyme  Trival name (1)  Inducer  Enzyme activity  % Contribution (Reference)  CYP1A1  c, pNF-B  None 3-MC HCB  Ethoxyresorufin O-deethylase Ethoxyresorufin O-deethylase Ethoxyresorufin O-demethylase  6(2) 82 (2), > 90 (3) 75 (4)  CYP1A2  P-448, d, H C B  None None 3-MC 3-MC HCB  Ethoxyresorufin O-deethylase High affinity phenacetin O-deethylase Ethoxyresorufin O-deethylase High affinity phenacetin O-deethylase Methoxyresorufin O-demethylase  78 (2)* 80 (3) 27 (2) 76 (3) ~ 100 (4) #  #  CYP2A1  al, a, 3, UT-F, RLM2b, IF-3  None DEX PB  Testosterone 7a-hydroxylase Testosterone 7ot-hydroxylase Testosterone 7a-hydroxylase  > 97 (6), 80 (7) > 96 (5), > 98 (6) > 98 (5)  CYP2B1  b, PB-4, PB-B, PBRLM5 3, PB-5, PB-D, PBRLM6  None None PB PB PB  Pentoxyresorufin O-dalkylase Testosterone 16a-hydroxylase Pentoxyresorufin O-dealkyase Testosterone 16p-hydroxylase Testosterone 16a-hydroxylase  0(8) 14(12), 0(9, 11) > 90 (8, 9, 10) > 90 (9), 89(11) 60-70 (8), 77(11), 66 (12)  CYP2C6  PB1, K , PB-C, pTF2, RLM5a, PB2  PB  Progesterone 21-hydroxylase  80 (13)  CYP2C11  h, M - l , 16 , 2c, UT-A, RLM5, male, UT-2  None None None PB  Ethoxyresorufin O-deethylase Testosterone 2a-hydroxylase Testosterone 16a-hydroxylase Testosterone 16a-hydroxylase  77 (14)* > 85 (9, 15), 100 (16) > 85 (15), 90(16) 30 (9, 15), 60 (16)  CYP3A1  pcnl, PCNa, 6(3, pIGC2 pcn2, PCBb/c, PB-1, 6P-1/3  None DEX DEX PB  Testosterone Testosterone Testosterone Testosterone  > 85 (18) > 85 (18) 55 - 6 0 (17) > 85 (18)  CYP2B2  CYP3A2  2P- & 6p-hydroxylase 2P- & 6p-hydroxylase 2p- & 6p-hydroxylase 2p- & 6p-hydroxylase  Abbreviations: 3-MC, 3-methylcholanthrene; PB, phenobarbital; D E X , dexamethasone; ISF, isosafrole; H C B , 3,4,5,3',4',5'-hexachlorobiphenyl (I) Nelson et. al., 1993; *(2) Kelley et. al., 1987; (3) Sesardic et. ai, 1990; (4) Nerurkar et. al.,. 1993; (5) Arlotto and Parkinson, 1989; (6) Levin et. ai, 1987; (7) Waxman et. ai, 1988; (8) Dutton and Parkinson, 1989; (9) Waxman et. al., 1987; (10) Lubet et. ai, 1985; (II) Reik et. al., 1985; (12) Thomas et. al., 1981; (13) Swinney et. al, 1987; *(14) Nakajima et. al., 1990; (15) Waxman et. al., 1984; (16) Chang et. al., 1992a; (17) Wrighton et. al., 1985; (18) Halvorson et. al., 1990. * There is a discrepancy between these two articles on the major enzyme that contributes to most of the ethoxyresorufin O-deethylase activity in uninduced male rat hepatic microsomes. Phenacetin concentration = 4 u M #  #  5  1.1.4. Major forms of cytochrome P450 in rats and humans About 50 different cytochromes P450 have been identified in rats (Nelson et. al, 1993). The major ones found in the liver of untreated male rats are CYP2C6, CYP2C11, CYP2C13 and CYP3A2. These cytochromes P450 have been reported to account for 13 - 33% , 31 - 100%, 9 19%> and 22 - 33% of the total hepatic cytochrome P450 content in untreated male rats, respectively (Bandiera, 1986; Dannan et. al., 1983; Guengerich et. al, 1982; Imaoka et. al., 1990). CYP2C11, CYP2C13 and CYP3A2 are male-specific cytochromes P450 (Kamataki et. ai, 1983, Waxman et. al, 1984; Ryan et. ai, 1984).  Except for these male-specific forms,  untreated adult female rats have similar forms of hepatic cytochrome P450 as adult male rats and, in addition, have the female-specific CYP2C12 (Kamataki et. al, 1983). In humans, about 30 different cytochromes P450 have been identified (Nelson et. al, 1993) and the major enzymes expressed in liver are CYP3A3/4, with levels approaching as high as 50 - 60% of total microsomal P450 content (Guengerich et. al, 1990, Forrester et. al, 1992). Other major cytochromes P450 found in human liver are CYP1A2, CYP2A6, CYP2C8, 9 & 19, CYP2D6 and CYP2E1 (Wrighton et. al, 1993). Unlike rats, there is no sex-specific hepatic cytochrome P450 present in humans (Guengerich, 1989) and the observed differences in hepatic drug clearance between males and females are small (Giudicelli and Tillement, 1977). There is also considerable interindividual variability in the microsomal levels of the various human cytochromes P450 due to genetic variability and exposure to chemicals in the diet and the environment (Guengerich, 1989).  In addition to their involvement in drug oxidation and reduction, cytochrome P450 enzymes also function to process a wide range of exogenous compounds such as environmental toxins (Wislocki et. al,  1980), as well as endogenous compounds such as fatty acids and  prostaglandins (Kupfer, 1982). Because of the toxicological and physiological significance of these cytochrome P450 enzymes, various factors affecting the catalytic activities and levels of these enzymes, such as exposure to xenobiotics and pathological alternations in endocrine factors, are the subject of detailed investigation (Thummel and Schenkman, 1990). The following section  6  is focused on discussing the mechanisms of induction and inhibition of cytochromes P450 following exposure to various xenobiotics.  1.1.5. Induction When cytochromes P450 are induced by certain chemicals such as drugs and environmental pollutants, there are increases in the absolute microsomal cytochrome P450 levels. As shown in Table 2, the inducers are classified on the basis of the major family of cytochrome P450 induced in rats.  In most cases, the primary induction mechanism for these prototype  inducers involves an increase in de novo protein synthesis resulting from transcriptional activation (Tukey and Johnson, 1990). In the case of induction of CYP2E1 by ethanol and CYP3A1 by triacetylomeandomycin, the increase in microsomal enzyme concentrations is partly due to decreased protein degradation as a result of protein stabilization (Watkins et. al, 1986; Eliasson et. al, 1988). Because the most common mechanism involved in induction of cytochromes P450 is transcriptional activation, it is discussed in greater detail here.  The mechanism of induction of CYP1A1 and CYP1A2 by 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and related compounds is most well understood. After its entry into the body, T C D D binds to the Ah receptor (dioxin receptor) in the hepatic cytosol (Poland et. al, 1976). This is followed by the release of the heat shock protein (hsp 90) from the Ah receptor and the binding of TCDD-Ah receptor complex to the Ah receptor translocation protein (Armt) (Hoffman et. al, 1991; Reyes et. al, 1992). This complex then enters the nucleus and acts on specific regions of CYP1A1 and CYP1A2 genes called Ah-receptor responsive elements (AhREs) (Denison et. al, 1989; Quattrochi and Tukey, 1989) and stimulates the transcription of D N A into messanger R N A for synthesis of CYP1A1 and CYP1A2.  Other unidentified proteins may be  involved in the functional transformation of the TCDD-Ah receptor complex and the binding of the complex to the AhREs (Perdew, 1992; Watson and Hankinson, 1992).  The mechanism of  Table 2 Major classes of rat hepatic cytohchrome P450 inducers. (Adapted fromTukey and Johnson, 1990)  Cytochrome P450  Inducer  Predominant  Secondary  Tetrachlorodibenzo-p-dioxin 3 -Methy lcholanthrene Benzo[a]pyrene  1A  2A  Phenobarbital  2B  2C, 3A, 4B  Imidazole Ethanol  2E  Pregnenolone 16a-carbonitrile Dexamethasone Troleandomycin Erthromycin  3A  Rifampicin Clofibrate Diethyl 2-hexyphthalate  4A  8  transcriptional activation of cytochrome P450 genes following exposure to other inducers is still not well understood.  Some constitutively expressed cytochromes P450 are resistant to induction by known cytochrome P450 inducers.  Examples of these non-inducible cytochromes P450 are the sex-  specific CYP2C11 (Waxman et. al, 1985; Guengerich et. al, 1982), CYP2C12 (Waxman et. al, 1985) and CYP2C13 (Bandiera et. al, 1986). reduction  (suppression)  in the hepatic  administration of 3-methylcholanthrene  Indeed, it is known that there is an absolute  CYP2C11  microsomal level following  in vivo  (3-MC) or some of the polychlorinated biphenyl  compounds (Yeowell et. al, 1987). Decreases in the hepatic CYP2C11 mRNA level were also observed during suppression of CYP2C11 by these inducers (Yeowell et. al, 1987).  The  mechanisms of suppression of the sex-specific cytochromes P450 by known inducing agents have not been fully elucidated.  1.1.6. Inhibition Like most enzymes, cytochromes P450 can be inhibited by various chemical compounds. Depending on the mechanism of inhibition, the catalytic function of the P450 enzymes is temporarily or permanently impaired. The inhibitors can be classified according to the type of inhibition as determined by the kinetics of enzyme inhibition or the mechanism of inhibition.  1.1.6.1. Type of inhibition In the presence of an inhibitor, the type of inhibition observed is determined by how the enzyme kinetic parameters are altered in the presence of the inhibitor, and can be described as competitive, noncompetitive or mixed inhibition.  In the absence of an inhibitor, the enzyme kinetics for a reaction that is catalyzed by a single enzyme is described by the Michaelis-Menten equation.  9  =  V  (Vmax)(S) S + Km  (2)  V is the initial velocity of the reaction at a substrate concentration of S. The reaction rate is not proportional to the substrate concentration, but reaches a limit at infinite substrate concentration. The limiting rate is the maximum velocity, Vmax. K m is the Michaelis-Menten constant and it is usually assumed that Km is inversely proportional to the affinity of the enzyme for the substrate (Gillette, 1971). Inspection of the Michaelis-Menten equation reveals that Km is equivalent to the substrate concentration at which the initial enzyme velocity is one-half of the maximum velocity (i.e. V = V Vmax) (Lehninger, 1982). Vmax and K m are inherent properties 2  of the enzyme.  1.1.6.1.1.  Competitive inhibition  In the case of competitive inhibition, the inhibitor competes with the substrate for binding to the active site of the enzyme, and the enzyme kinetics are characterized by no change in Vmax but an apparent increase in Km as shown in Equation (2).  V =  Vmax (S) S + K m [ l +(I)/(Ki)]  (2)  I is the concentration of the inhibitor and K i is the inhibition constant. K i is an inherent property of the inhibitor and is inversely proportional to the affinity of the inhibitor for the enzyme. If the inhibitor is also a substrate for the enzyme, then K i will be equal to the MichaelisMenten constant of the reaction that involves the breakdown of the inhibitor by the enzyme. At any given I, V can be increased to Vmax by increasing the substrate concentration.  The  Lineweaver-Burk plot can be used for differentiating competitive inhibition from other types of inhibition (Lehninger, 1982). In the case of competitive inhibition, Lineweaver-Burk plots of 1/ V  10  vs 1/S at several inhibitor concentrations yield a family of lines with similar y-intercepts but different x-intercepts.  The Dixon plot of  1/V vs I is another useful plotting technique for  studying enzyme inhibition (Gillette, 1971). It is mainly used for differentiation between complete and partial inhibition but can also be used for K i estimation.  A Dixon plot of completely  competitive inhibition gives a straight line with an intercept on the x-axis at - K i [ (S)/(Km) + 1 )] and the x-intercept value is used for K i estimation. A Dixon plot of partial competitive inhibition gives a curved line and the x-intercept has a different meaning (Lehninger, 1982; Gillette., 1971).  1.1.6.1.2. Noncompetitive inhibition In the case of noncompetitive inhibition, the inhibitor acts by some mechanism(s) other than competition with the substrate for binding to the active site, and the enzyme kinetics are characterized by an apparent decrease in Vmax but no change in K m as shown in equation (2).  y=  Vmax (SVH + (IVflCni S + Km  (2)  In noncompetitive inhibition, K i is inversely proportional to the potency of the inhibitor. At any given inhibitor concentration, V cannot be increased to Vmax by increasing S. At various I, Lineweaver-Burk plots of completely noncompetitive inhibition give a family of lines with common x-intercepts but different y-intercepts, whereas Dixon plots of the same data give a family of lines with x-intercepts of - Ki. In the case of partial noncompetitive inhibition, Dixon plots give curved lines and the x-intercepts have different meanings (Gillette, 1971).  1.1.6.1.3. Mixed inhibition In the case of mixed (competitive and noncompetitive) inhibition, the inhibitor acts by competing with the substrate for binding to the enzyme and by some other mechanism(s) as well. The enzyme kinetics are characterized by apparent changes in Vmax and Km. The rate equation  11  of mixed inhibition is more complicated and will not be discussed here. Lineweaver-Burk plots of mixed inhibition yield a family of lines with different x- and y-intercepts (Gillette, 1971).  Another way to describe the inhibitory effect of an inhibitor is the ICso value (Chang, 1991).  It is defined as the inhibitor concentration at which 50% of the enzyme activity is  inhibited. Depending of the type of inhibition, the ICso value of an inhibitor may vary with the substrate concentration and therefore, does not necessary reflect the K i value of the inhibitor.  1.1.6.2. Mechanisms of Inhibition As indicated above, classification of inhibitors based on the type of inhibition does not necessarily indicate the mechanism of inhibition involved.  More commonly, inhibitors are  classified according to their mechanisms of inhibition. Agents that interfere with the cytochrome P450 catalytic cycle (Fig. 1) prior to the oxidative events (i.e. binding of substrate to ferric heme iron and binding of oxygen to the ferrous heme iron) are usually reversible inhibitors. Agents that act during or subsequent to the oxidation of the substrate are quasi-irreversible or irreversible catalysis-dependent inhibitor (Oritz de Montellano and Reich, 1986).  1.1.6.2.1.  Mechanisms of reversible inhibition  Reversible inhibitors bind to the lipophilic domain of the active site by hydrophobic binding, or to the prosthetic heme iron by ligand binding.  Cytochromes P450 have broad substrate specificities. When two xenobiotics are both substrates for the same enzyme, one can compete with the other for binding to the lipophilic domain of the active site. A good example is the observed inhibition of dextromethorphan Odemethylation in vivo and inhibition of bufuralol 1-hydroxylation in vitro by flecainide in humans (Haefeli et. al,  1990).  It had been shown in a number of studies that the oxidation of  dextromethorphan, bufuralol and flecainide are all mediated by the same enzyme, CYP2D6 (Dayer  12  et. al, 1987; Kupfer et. al, 1986; Mikus et. al., 1989). Other inhibitors can act by competing with the substrates for binding to the active site but are not substrates themselves. For example, quinidine is a potent reversible inhibitor of CYP2D6 (Kobayashi et. al, 1989) in humans but there is in vivo and in vitro evidence to indicate that CYP2D6 is not involved in the metabolism of quinidine (Mikus et. al, 1986; Otton et. al, 1988). Because there are differences in the optical absorbance between the inhibitor-free and inhibitor-bound cytochrome P450, the interaction between a compound and a cytochrome P450 can be observed spectrophotometrically (Schenkman et. al, 1981). When an inhibitor interacts with a cytochrome P450 by lipophilic binding, it results in a Type I difference spectrum with a peak at 385-390 nm and a trough at 420 nm. Because the inhibitor can be displaced from the enzyme by adding more substrate to the reaction medium, the inhibition observed is usually reversible and competitive. As a result, the in vivo inhibitory effect observed with these inhibitors is usually transient (Murray and Reidy, 1990).  The other class of reversible inhibitors binds to the prosthetic heme iron by coordination and therefore, blocks the access of oxygen to the active site and results in inhibition of substrate oxidation. Binding of a ligand to the ferric form of P450 produces a characteristic Type II difference spectrum with an absorption minimum at 390-405 nm and an absorption peak at 425 435 nm (Schenkman et. al, 1981, Oritz de Montellano and Reich, 1986). Cyanide is an ionic ligand that binds to the ferric form of the hemoprotein (Kitada et. al, 1977). Some ligands bind preferentially to the ferrous form of P450 and carbon monoxide is a classic example of such inhibitors (Omura and Sato, 1964).  The inhibition observed with these inhibitors is usually  noncompetitive but reversible because the ligands can be displaced from the enzymes by increasing the oxygen tension or by other stronger ligands.  Reversible inhibitors that interact with the cytochromes P450 by both liphophilic and ligand binding are usually more effective than inhibitors that act by either mechanism alone (Oritz de Montellano and Reich, 1986).  Some imidazole derivatives and nitrogenous containing  13  aliphatic and aromatic compounds such as metapyrone, inhibit cytochromes P450 by both substrate and ligand binding (Oritz de Montellano and Reich., 1986; Jonen et. al, 1974). Since these inhibitors have high affinity for the heme iron, their binding to cytochromes P450 gives rise to the Type II difference spectra, with absorption troughs at 390 nm and peaks at 430 nm (Jefocate et. al, 1969). The inhibition observed with some of these inhibitors has been described as mixed inhibition, with competitive and noncompetitive components attributed to lipophilic and ligand binding respectively (Testa and Jenner, 1981).  1.1.6.2.2. Mechanisms of quasi-irreversible and irreversible inhibition 1.1.6.2.2.1. Metabolite-intermediate complexation Inhibitors that inactivate cytochromes P450 by forming metabolite-intermediate complexes are relatively inactive by themselves as inhibitors. They have to be catalytically activated by cytochromes P450 to some intermediate species, which then coordinate tightly with the heme iron of the activating cytochromes P450 by ligand interaction.  Once the metabolite-intermediate  complexes are formed, oxygen can no longer bind to the heme iron and the cytochromes P450 then lose their catalytic activities. Compounds that are known to form metabolite-intermediate complexes with cytochromes P450 include methylenedioxy compounds (e.g. 1,3-dibenzodioxole) (Hodgson and Philpot, 1974), alkyl and aromatic amines (e.g. SKF 525-A) (Buening and Franklin, 1976), triacetyleandomycin (Pershing and Franklin, 1982), and 1,1-disubstitiuted and acyl hydralazines (e.g. isoniazid) (Mukkassah et. al, 1981) (Oritz de Montellano and Reich, 1986). The formation of the metabolite-intermediate complex results in a characteristic spectral peak or peaks in the absorption difference spectrum. Because this complex prevents the binding of carbon monoxide to the heme iron, the complexed enzyme can no longer be detected by the standard spectral method. The complex formed between the P450s and the activated inhibitors are quite stable but can be dissociated in vitro with the use of specific chemical agents such as ferricyanide and cyclohexane.  Since the dissociated enzymes regain their catalytic activities, this type of  inhibition is referred to as quasi-irreversible (Oritz de Montellano and Reich, 1986). For example,  14  the unstable isosafrole metabolite-intermediate complex (in its ferric form) is dissociable by lipophilic compounds such as cyclohexane (Dickens et. al, 1978). In the case of SKF 525-A, the displacement of the stable metabolite-intermediate complex (in its ferrous form) from the enzyme requires an oxidant (e.g. potassium ferricyanide) (Buening and Franklin, 1976). The observed in vitro inhibition with agents that form metabolite-intermediate complexes with cytochromes P450 have been described as noncompetitive, competitive or mixed, depending on the experimental conditions (Franklin, 1977; Testa and Jenner, 1981). At high concentrations and in the absence of preincubation, these inhibitors may compete with the substrates for binding to the active sites initially and result in competitive inhibition of the enzyme activities. When these inhibitors are incubated at low concentrations with microsomes and N A D P H prior to the addition of substrates, they are oxidized by the enzymes to some intermediate species which form complexes with the prosthetic heme iron.  Upon the addition of other substrates, non-competitive inhibition is  observed. Mixed inhibition may be observed when microsomes are incubated with these inhibitors at high concentrations in the presence of N A D P H prior to the addition of substrate; and this is attributed to competitive binding of the active sites by the parent compounds and non-competitive ligand binding of the metabolite-intermediates to the heme. Administration of these inhibitors to animals result in the formation of metabolite-intermediate complexes in vivo. The stability of these complexes is indicated by the fact that they remain intact during the preparation of microsomes. Because the complexed cytochrome P450s are catalytically nonfunctional, they are likely to be subjected to enzyme degradation in vivo and replaced by newly synthesized P450 enzymes (Murray and Reidy, 1990). Therefore, the in vivo inhibitory effect observed with these inhibitors usually lasts longer than that observed with reversible inhibitors.  1.1.6.2.2.2. Autocatalytic inactivation The irreversible inhibitors inhibit the cytochrome P450 enzymes by autocatalytic inactivation. They are similar to the agents that act by metabolite-intermediate complexation in that both types of inhibitors are inactive by themselves and have to be catalytically converted to  15  some active intermediate species. They are different, however, in that the intermediate species of the irreversible inhibitors bind covalenty to the protein or the heme and result in permanent destruction of the P450 enzyme (Oritz de Montellano and Reich, 1986).  Agents that inhibit  cytochromes P450 by covalent binding to the protein include thiosulfur compounds such as parathion (Halpert et. al, 1980) and halogenated structures like chloramphenicol (Halpert et. ai, 1985).  Terminal olefins (e.g. secobarbital), acetylenes (e.g. danazol), cyclopropylamines and  diazo-containing compounds (e.g.  1-aminobenzotriazole)  are inhibitors that act by heme  alkylation (Oritz de Montellano and Mico, 1980; Oritz de Montellano and Reich, 1984; Hanzlik et. al, 1979; Oritz de Montellano and Mathews, 1981). Evidence for heme alkylation includes equimolar loss of heme and enzymes, and the isolation and characterization of the heme adducts (Oritz de Montellano and Correia, 1983). The types of inhibition observed with these inhibitors are similar to those described for inhibitors that form metabolite-intermediate complexes. The in vivo inhibitory effect observed with these inhibitors is usually prolonged because the body has to synthesize new cytochrome P450 enzymes to replace the inactivated ones (Murray and Reidy, 1990).  1.1.7. Catalytic specificity of cytochromes P450 Cytochromes P450 have broad but overlapping substrate specificities. This means that one cytochrome P450 can catalyze a wide range of substrates and the same substrate may be metabolized by several cytochromes P450. In toxicological and drug metabolism studies, it is useful to identify the cytochromes P450 responsible for the metabolism of the xenobiotic of interest.  If the factors that influence the expression and catalytic activities of the involved  cytochromes P450 are also known, one can anticipate potential drug-drug interactions and the effects of the various factors on the metabolism of the xenobiotic (Wrighton et. al, 1993). A number of in vitro experimental approaches are available for the determination of the enzymes involved in the oxidation of a xenobiotic.  16  One approach is to measure the metabolism of the xenobiotic of interest with a panel of purified cytochromes P450 or with genetically engineered cell lines, each expressing a single cytochrome P450 (Gonzalez et. al, 1994). This will lead to the identification of those P450s that are capable of oxidizing the xenobiotic. In rats, one can pretreat the animals with an inducer for induction of some specific cytochromes P450 and then use the prepared microsomes to measure the various activities in vitro. If the metabolism of a xenobiotic is greatly induced, it suggests that the induced cytochromes P450 may play a role in the metabolism of that xenobiotic.  In most  cases, several P450 isozymes will be identified to be involved in the oxidation of that xenobiotic. Because the above two approaches do not take into account the relative content of the various cytochromes P450  present in microsomes from uninduced subjects,  other experimental  approaches have to be used to determine the contribution of a particular cytochrome P450 in the metabolism of the xenobiotic in microsomes (Birkett et. al, 1993).  One of the less specific approaches is the use of enzyme-selective  substrates as  competitive inhibitors. If both the xenobiotic and the enzyme-selective substrate are metabolized by the same enzyme, the substrate can competitively inhibit the microsomal metabolism of the xenobiotic. This approach can identify the cytochromes P450 that are potentially involved in the metabolism of the xenobiotic in microsomes. However, the above approach has the drawback that the enzyme-selective substrate may have affinity for other cytochromes P450 in addition to the one that is involved in its metabolism (Birkett et. al, 1993).  The more specific approaches are chemical inhibition studies with known enzyme-selective inhibitors and immunoinhibition studies, particularily with inhibitory monospecific antibodies. Selective inhibitors for some of the major cytochromes P450 have been identified. For example, furafylline has been shown to be a selective inhibitor for human CYP1A2 and it acts by catalysisdependent inhibition (Sesardic et. al, 1990; Clarke et. al, 1994). The degree of inhibition of the metabolism of a xenobiotic in microsomes observed with an enzyme-selective inhibitor indicates  17  the extent to which that enzyme contributes to the oxidation of the xenobiotic (Birkett et. ai, 1993). In antibody inhibition studies, the role of a particular cytochrome P450 in the oxidation of a xenobiotic in microsomes can be determined by the proportion of the activity inhibited by a monospecific antibody directed against that cytochrome P450 enzyme.  The reliability of these  methods depends on the specificities of the chemical inhibitor and antibody used. Table 1 lists the catalytic specificities of the male rat cytochromes P450 that are discussed in this thesis.  The  catalytic specificities of these enzymes have mostly been determined from antibody inhibition studies in microsomes prepared from animals that have been pretreated with the various inducers, and should be considered to apply only under those conditions for which they have been established. When different conditions are used, the specificity of an enzyme activity for a given cytochrome P450 needs to be verified again.  In humans, there is large interindividual variability in microsomal enzyme activities. Correlation analysis has been employed to observe whether the rate of oxidation of the xenobiotic is associated with the level of an enzyme-specific activity or the content of an enzyme in a panel of human liver microsomes (Birkett et. al, 1993).  A negative result usually suggests that a  particular enzyme is not involved in metabolizing the xenobiotic, whereas a positive result suggests the possibility of involvement of that enzyme in the metabolism of the xenobiotic.  Considering that there are limitations to each of the approaches, one should use multiple experimental approaches in investigating the contribution of various cytochromes P450 to an enzyme activity and results of the various studies should be consistent with each other.  18  1.2. Inhibition of hepatic cytochromes P450 by cimetidine 1.2.1. Cimetidine Cimetidine was the first histamine H2 -receptor antagonist that was marketed for the treatment of peptic ulcer disease. It is a substituted imidazole (molecular weight = 252.34) and its chemical structure is shown in Fig. 2.  It blocks the histamine H2 -receptors located on the  parietal cells of the gastric mucosa, and thereby decreases the acid secretion stimulated by histamine, gastrin and acetylcholine (Holt and Ibsenberg, 1985). Since its introduction, cimetidine has been widely used for the treatment and prophylaxis of duodenal and gastric ulcers. In addition, it has also been used for the treatment of Zollinger-Ellison syndrome and prophylaxis against stress-induced ulcer (Feldman and Burton, 1990a and b). The maximum recommended daily dose is 1600 mg/day.  The pharmacokinetics of cimetidine in man have been extensively reviewed in the literature (Richards, 1983; Somogyi and Gugler, 1983; Abate et. al, 1982). The bioavailability of cimetidine tablets has been reported to range from 40 to 70% in healthy volunteers. Cimetidine is distributed to most parts of the body except for adipose tissue. It is not extensively bound to plasma proteins (13 - 25% bound) and its volume of distribution at steady state is approximately 1 L/kg. The peak plasma cimetidine concentration attained at 2 hours following the ingestion of a 400 mg tablet is about 10 u M (3 ug/ml). The mean steady state cimetidine concentration was about 0.3 u.M in patients on a regimen of 1000 mg daily (taken by mouth).  In the liver, the  cimetidine concentration is approximately 2 to 5 times that in serum. Depending on the age of the subject, between 50 to 80% of the dose is excreted into urine as unchanged drug following intravenous administration of cimetidine. The elimination half-life of cimetidine is about 2 hours in healthy subjects with no renal or hepatic impairment. Between 25 to 40% of the dose is metabolized before excretion into urine and the major cimetidine metabolites are shown in Fig. 2. About 25%, 10%, 5% and 2% of the dose are recovered in urine as cimetidine-N'-glucuronide,  19  cimetidine sulfoxide, 5-hydroxymethylcimetidine and guanylurea cimetidine, respectively; and these metabolites are pharmacologically inactive.  In rats, cimetidine is well-absorbed (> 90%) after oral administration (Taylor et. al, 1978). The binding of cimetidine to plasma protein is similar to that observed in man (Taylor et. al,  1978).  Following a single intraperitoneal dose of 120 mg/kg, the serum cimetidine  concentration attained at 1 hr post-dose is about 0.8 u M in rats (Speeg et. al, 1982). There is no major accumulation of cimetidine in the liver (Imaura et. al, 1994).  Cimetidine is eliminated  more rapidly in rats and the elimination half-life has been estimated to be about 45 min (Winer and Roth, 1981). The renal and hepatic elimination of cimetidine in rats is similar to that in man. The major metabolites in rats are cimetidine sulphoxide, 5-hydroxymethylcimetidine and guanylurea cimetidine (Taylor et. al, 1978) (Fig. 2).  1.2.2. Selective inhibition of hepatic cytochromes P450 by cimetidine Since its first introduction in 1977, cimetidine has been reported to interact with a number of drugs in man by decreasing their metabolic clearance.  Because cimetidine has been widely  prescribed for the treatment of peptic ulcer disease, drug interactions involving cimetidine have received much attention and has been reviewed in detail in the literature (e.g. Somogyi and Muirhead, 1987; Smith and Kendall, 1988).  Drugs that are reported to interact with cimetidine  are mostly eliminated by cytochrome P450-mediated oxidative metabolism in the liver.  Some  therapeutically important drugs, such as warfarin, phenytoin, theophylline, debrisoquine and some tricyclic antidepressants, have been reported to interact with cimetidine to a clinically significant extent (Serlin et. al, 1979; Desmond et. al, 1984, Levine et. al, 1985; Steiner and Spina, 1987; Spina and Koike, 1986; Grygiel et al, 1984; Cusack et al, 1985). In rats, cimetidine has been observed to decrease the in vivo clearance of pentoxifylline and theophylline; and the mechanism involved in the interaction is likely to be due to inhibition of hepatic metabolism (Luke et. al, 1986; Majaverian et. al, 1985). The effects of in vitro administration of cimetidine on inhibition  20  Fig. 2. Structures of cimetidine and its major metabolites in rats (Taylor et. al., 1978; adapted from Somogyi and Gugler, 1983).  CH 3  /  CH SCH CH NHCNHCH 2  2  2  3  NCN HN  /N  Cimetidine  qH OH 2  ^ CH SCH CH NHCNHCH /  2  2  p  CH 3  3  /  CH SCH CH NHCNHCH 2  HN.  /N  /N  Guanylurea  Hydroxymethyl cimetidine  CH  XJ^SCH^^NHCNHCHg  -3  NCN HN  2  NCONH  NCN HN.  2  /N  Cimetidine  sulphoxide  cimetidine  3  2  21  of microsomal drug metabolism has also been investigated. When added to human microsomes in vitro without a preincubation step, cimetidine has been observed to inhibit the metabolism of lovastatin (Vyas et. al., 1990), nifedipine, tolbutamide, erythromycin, bufuralol, ethoxyresorufin and aniline (Kondell et. al,  1991).  In rat hepatic microsomes, in vitro administration of  cimetidine (without a preincubation step) results in inhibition of the metabolism of many compounds,  including aminopyrine, meperidine, metoprolol, morphine and  pentobarbital  (Pelkonen and Puurunen, 1980; Knodell et. al, 1982; Lennard et. al, 1986; Reilly and Winzor, 1984). Based on these in vivo and in vitro data on inhibition of cytochrome P450-mediated metabolism, it was presumed that cimetidine was a general inhibitor of hepatic cytochrome P450. However, there is direct and indirect evidence to indicate that cimetidine is a more selective inhibitor of cytochrome P450 than was originally considered.  In humans, the hepatic clearances of some drugs that are eliminated by cytochrome P450mediated oxidative metabolism, such as tolbutamide, carbamazepine and cyclosporine, are not affected by cimetidine (Levine et. al, 1985; Stockley et al, 1986; Jarowenko et. al, 1986). In rats, several investigators have examined the effects of  in vivo cimetidine on the catalytic  activities of hepatic cytochromes P450. After in vivo administration of cimetidine, the animals were sacrificed and in vitro enzymes assays were performed on the prepared microsomes. Drew et al. (1981) found that in vivo administration of cimetidine selectively inhibited aryl hydrocarbon hydroxylase activity but not 7-ethoxycoumarin deethylase or aniline hydroxylase activity in microsomes from uninduced and phenobarbital-induced rats. In another study in uninduced male rats, Galbraith and Jellinck (1989) showed that in vivo cimetidine inhibited estradiol 2a- and 16ahydroxylations and some other enzyme activities but that 7-ethoxycoumarin deethylase activity was not affected. Moreover, the same authors found that in vivo cimetidine had no effect on any of these activities in female rats. To demonstrate the fact that cimetidine is a selective hepatic cytochrome P450 inhibitor in male rats, Chang et al. (1992a) showed that in vivo administration of cimetidine resulted in inhibition of CYP2C11, as monitored by testosterone 2a- and 16a-  22  hydroxylase activities, but had no effect on CYP2A1, CYP2B1/2 or CYP3A1/2 mediated enzyme activities. This is consistent with the apparent lack of inhibition of the studied enzyme activities by in vivo cimetidine in female rats (Galbraith and Jellinck, 1989) as CYP2C11 is a male-specific enzyme (Waxman et. ai, 1984). In an accompanying study, Chang et al. (1992b) also showed that the in vivo selectivity of cimetidine could be reproduced by in vitro cimetidine only when a preincubation step was included.  1.2.3.  Mechanism(s) of inhibition of cytochromes P450 by cimetidine  In usual in vitro inhibition experiments (without a preincubation step), the inhibitor and substrate are added to microsomes at the same time and the reaction is initiated by the addition of NADPH.  Under these conditions, cimetidine inhibits the metabolism of a wide range of  substances in rat and human hepatic microsomes at concentrations in the low m M range and the observed inhibition has been described as competitive, noncompetitive or mixed (Pelkonen and Puurunen, 1980; Speeg et al, 1982; Rendic et al, 1979; Knodell et. al., 1991).  Binding of  cimetidine to purified human cytochromes P450 (Knodell et. al,  1991) or microsomal rat  cytochromes P450 (Pelkonen and Puurunen, 1980, Rendic et. al,  1979).//? vitro has been  characterized by Type II difference spectra, which indicates the binding of a ligand nitrogen atom to the prosthetic heme iron of cytochromes P450 Thus, cimetidine appears to inhibit cytochrome P450 enzyme(s) in vitro by reversible binding to the heme group. Nevertheless, there is evidence that cimetidine does not exert its inhibitory effect on cytochromes P450 in vivo by simple reversible inhibition.  To act as an inhibitor of human or rat hepatic cytochromes P450 in standard enzyme assays, cimetidine must be present in vitro at concentrations in the low m M range (Pelkonen and Puurunen, 1980; Hoensch et al, 1985; Knodell et al., 1991). In humans and rats, the serum cimetidine level achieved in vivo is in the low u M range (Somogyi and Gugler, 1983; Speeg et.  23  al, 1982). At such low cimetidine concentrations, very little inhibition of cytochrome P450 can be observed in vitro.  In rats, the half-life of cimetidine is about 45 min (Winer and Roth, 1981) and the serum drug concentration is in the low u.M range when the animals are sacrificed 90 min after the in vivo administration of a single inhibitory dose of cimetidine (Speeg et al., 1982). During microsome preparation, the isolated microsomes are washed twice with buffer and one would expect that most diffusible cimetidine left in the microsomes would be removed. However, the inhibitory effect of cimetidine is still apparent in those microsomes that have been treated with cimetidine in vivo (Drew et al, 1981; Galbraith and Jellinck, 1989; Chang et al,  1992a and b).  This is  consistent with what would be expected for inhibitors that form metabolite-intermediate complexes with cytochrome P450 enzymes (i.e. the stable complexes remain intact during microsomal preparations and the complexed enzymes are catalytically inactive).  In addition, it has been reported that in rat liver microsomes, preincubation of cimetidine with microsomes in the presence of N A D P H prior to the addition of substrate increases the potency of its inhibition of 7-ethoxycoumarin O-deethylase (Jensen and Gugler, 1985) and testosterone 2a- and 16a-hydroxylase activities significantly (Chang et al, 1992b). In human liver microsomes, the inhibitory effect of cimetidine on dapsone N-hydroxylase (Tingle et al, 1991) and oestrogen 2-hydroxylase (Wild and Back, 1989) was augmented after preincubation with N A D P H . Although not all of these studies were conducted with the appropriate controls, the results do suggest that cimetidine has to be catalytically activated to some reactive species in order to inhibit cytochrome P450 enzyme(s) in vivo.  Lastly, Chang et al. (1992b) have examined the effect of in vitro cimetidine on microsomal testosterone hydroxylase activities in uninduced male rats. As expected, in vitro cimetidine at high concentrations inhibited all the  testosterone hydroxylase activities studied (except 7a-  24  hydroxylase) when a preincubation step was not included.  When in vitro cimetidine was  preincubated at low concentrations with microsomes from uninduced male rats in the presence of N A D P H prior to the addition o f testosterone, selective inhibition o f testosterone 2 a - and 16ahydroxylase activities was observed.  This pattern o f inhibition was similar to that observed  following in vivo administration o f cimetidine (Chang et ai, 1992a).  Taken together, these studies suggest that cimetidine is a catalysis-dependent inhibitor in vivo and that it selectively inhibits certain cytochrome P450 enzyme(s). During the preincubation, cimetidine is presumably catalytically activated by certain cytochrome P450 enzyme(s) (including C Y P 2 C 1 1 ) to some transient species and the activating enzyme(s) are inhibited by the activated form o f cimetidine.  Because low concentrations o f cimetidine are used in the preincubation  studies, reversible inhibition is minimized at such concentrations.  Those cytochrome P450  enzyme(s) that activate cimetidine is(are) affected and the selective inhibitory effect o f in vivo cimetidine is thus elicited.  1.2.4. Effect of cimetidine on C Y P 1 A 2 in male rats and humans and on C Y P 1 A 1 in  male rats C P Y 2 C 1 1 is the only enzyme that has been identified to be inhibited by in vivo cimetidine in male rats (Chang et al., 1992a).  There is evidence, however, that other cytochrome P450  enzymes are also affected by cimetidine.  Chang et al. (1992a) demonstrated that C Y P 2 C 1 1 only  accounted for 3 5 % o f aminopyrine N-demethylase activity in uninduced rat microsomes, whereas in vivo cimetidine inhibited this activity by 62%.  The same authors also showed that  erythromycin N-demethylase activity was inhibited by 4 0 % by in vivo cimetidine in uninduced rats, but that C Y P 2 C 1 1 does not contribute to this activity at all. Several lines o f evidence suggest that C Y P 1 A 2 may be another enzyme that is selectively inhibited by cimetidine.  25  Chang (1991) has examined the effect of in vivo and in vitro cimetidine on microsomal 7ethoxyresorufin O-dealkylase (EROD) activity in uninduced and 3-napthoflavone (BNF)-induced male rats. BNF pretreatment resulted in a 22-fold increase in microsomal EROD activity. //; vivo administration of cimetidine decreased the microsomal EROD activity in the uninduced rats by 84%, but had no effect on the same activity in the BNF-induced rats. E R O D activity was also measured after the addition of varying concentrations of cimetidine to the microsomal incubations in vitro. The experiment was performed without a preincubation step. There was a shift of the dose response curve to the right after BNF pretreatment e.g. at a cimetidine concentration of 0.6 m M , microsomal EROD activity was inhibited by 74% in uninduced rats but no effect was observed in the BNF-induced rats. Both the in vivo and in vitro results suggested that different enzymes are responsible for EROD activity in microsomes from uninduced and BNF-induced rats (Chang, 1991), and that in vivo cimetidine inhibits the uninduced but not the induced enzymes that are responsible for EROD activity.  In uninduced male rats, there has been a discrepancy in the literature as to whether CYP1A2 or CYP2C11 accounts for most of the hepatic microsomal E R O D activity. One study using a monoclonal antibody against CYP2C11 showed that this enzyme accounted for 77% of E R O D activity (Nakajima et. al, 1990), whereas another study using a monospecific polyclonal antibody against CYP1A2 revealed that CYP1A2 was responsible for 78% of E R O D activity (Kelley et. al,  1987).  If CYP2C11 is the major enzyme that catalyzes ethoxyresorufin O-  deethylation in microsomes from uninduced rats, the observation of the in vivo effect of cimetidine on E R O D activity can be readily explained. On the other hand, if CYP1A2 contributes to most of the E R O D activity in microsomes from uninduced male rats, the results of Chang et al. (1991) would suggest that CYP1A2 is inhibited by cimetidine in vivo.  There is also evidence in humans that CYP1A2 is selectively inhibited by cimetidine in vivo. Human CYP1A2 is immunologically related to rat CYP1A2 becasue a polyclonal antibody  26  raised against rat CYP1A2 inhibited the.CYPlA2-mediated E R O D activity in human microsomes by 72% (Murray et al., 1993).  Theophylline, an anti-asthmatic drug, is mainly eliminated by  metabolism in the liver. The formation of two of its major metabolites, 1-methyluric acid and 3methylxanthine, has been shown to be correlated with CYP1A2 content in human liver microsomes (Sarkar et al, 1992).  Polycyclic aromatic hydrocarbons in cigarette smoke can  induce CYP1A2 and smoking is known to increase theophylline clearance by selective induction of 3-methylxanthine and 1-methyluric acid formation (Vestal et al, 1987). Cimetidine has been shown to decrease theophylline clearance in smokers and nonsmokers by 28% and 11% respectively in one study (Grygiel et al, 1984), and by 34% and 31% in another study (Cusack et al, 1985). Grygiel et al. (1984) also showed that the reduction in theophylline clearance by cimetidine was mainly due to a decrease in the formation clearances of 1-methyluric acid and 3methylxanthine in both smokers and nonsmokers.  These in vivo studies in smokers and  nonsmokers indicate that cimetidine may inhibit CYP1A2 in humans.  Studies on the effect of in vivo administration of cimetidine on the catalytic activities of CYP1A1 in male rats have yielded conflicting results. Drew et al. (1981) showed that in vivo administration of cimetidine resulted in 70% and 90% inhibition of aryl hydrocarbon hydroxylase activity in microsomes from uninduced and 3-MC-induced male rats, respectively. Results of immunoinhibition studies have shown that CYP1A1 was responsible for 23% and 84% of microsomal aryl hydrocarbon hydroxylase activity in microsomes from uninduced and 3-MCinduced rats, respectively (Ryan et al., 1982). Taken together, these observations would suggest that cimetidine is an inhibitor of hepatic CYP1A1 in male rats. However, there is conflicting evidence from other studies to suggest that cimetidine does not inhibit CYP1A1. In the same study by Drew et al. (1981), it was observed that in vivo cimetidine inhibited microsomal 7ethoxycoumarin deethylase activity by 34% in uninduced rats but had no effect on the same enzyme activity in 3-MC induced rats. Using a monoclonal antibody that was directed against CYP1A1 but that cross-reacted with CYP1A2,  Hietanen et al. (1987) showed that C Y P 1 A  27  enzymes did not contribute significantly to 7-ethoxycoumarin deethylase activity in uninduced rats but were responsible for > 50% of the same activity in 3-MC induced rats.  If CYP1A1  contributes to 7-ethoxycoumarin deethylase activity in 3-MC-induced rats, these results suggest that CYP1A1 is not affected by in vivo cimetidine. In another study by Chang (1991), it was observed that in vivo administration of cimetidine resulted in 85% inhibition of E R O D activity in microsomes from uninduced male rats but had no effect on the same activity in microsomes from BNF-induced rats. Like 3-MC, B N F preferentially induces CYP1A1 (Guengerich et al, 1982; Thomas et al., 1983). It has been shown in microsomes from 3-MC induced rats that CYP1 A l is the major enzyme that catalyzes ethoxyresorufin O-deethylation (Kelly et. al, 1987). If CYP1A1 is also the major enzyme that is responsible for EROD activity in microsomes from BNF-induced rats, then Chang's results would indicate that CYP1A1 is not inhibited by in vivo cimetidine. Because of the apparent discrepancy in the effect of in vivo cimetidine on aryl hydrocarbon hydroxylase, 7-ethoxycoumarin deethylase and EROD activities, the effect of cimetidine on CYP1A1 needs to be explored further.  To date, CYP2C11 is the only enzyme that has been shown to be inhibited by in vivo cimetidine in the rat model. Preincubation studies suggest that cimetidine exerts its inhibitory effect by forming a metabolite-intermediate complex with CYP2C11 (Chang et al., 1992b). However, since there is no human analog to rat CYP2C11, it is important to initially investigate the effect of cimetidine on CYP1A1 and CYP1A2 (which is an important cytochrome P450 in humans) in the rat model as it may lead to the identification of other enzymes in addition to CYP2C11 that are selectively inhibited by cimetidine in vivo. In addition, it will help to clarify the discrepancy as to which enzyme is responsible for EROD activity in microsomes from uninduced rats.  28  1.2.5. Isozyme-selective enzyme activities for rat and human CYP1A2 The findings of some of the studies on the substrate specificity of CYP1A2 provided the tools for investigating the effect of cimetidine on this enzyme. In microsomes from uninduced and 3-MC induced rats, phenacetin O-deethylation has been shown to be mediated by two groups of enzymes, high affinity and low affinity phenacetin O-deethylase (Sesardic et. al, 1990). The estimated Michaelis-Menten kinetic parameters of these enzymes are listed in Table 3. Using a monoclonal antibody directed against CYP1A2, Sesardic et. al (1990) demonstrated that high affinity phenacetin O-deethylase activity is mediated by CYP1A2 in microsomes from uninduced and 3-MC-induced rats.  Recently, Nerurkar et al. (1993) performed some immunoinhibition studies using monospecific antibodies which suggested that methoxyresorufin O-demethylase (MROD) activity was specific for CYP1A2  and EROD activity was specific for CYP1A1 in 3,4,5,3',4',5'-  hexachlorobiphenyl (HCB)-induced rats.  To confirm this finding, the same investigators also  conducted chemical inhibition studies of M R O D and EROD activities in S-9 fractions from H C B induced rats using phenacetin as a chemical inhibitor for CYP1A2.  The rationale behind the  chemical inhibition studies was that high affinity phenacetin O-deethylase has been shown to be mediated by CYP1A2 in microsomes from 3-MC-induced rats (Sesardic et. al, 1990). Because 3-MC and H C B have been shown to be coinducers of CYP1A1/2 (Thomas et. al, 1983, Parkinson et. al, 1983), it is likely that high affinity phenacetin O-deethylase is also mediated by CYP1A2 in microsomes from HCB-induced rats. If so, then at low concentrations, phenacetin is an isozyme-selective substrate and can competitively inhibit other enzyme activities mediated by CYP1A2 in microsomes from HCB-induced rats. Phenacetin inhibited M R O D activity in samples from HCB-induced rats at concentrations as low as 5 u M (the K i value of phenacetin at a methoxyresorufin concentration of 0.5 u M was 20 uM) but did not affect the E R O D activity even at a concentration of 150 u M (Ki value of phenacetin at an ethoxyresorufin concentration of 0.5 u.M was > 333 uM). Results of these phenacetin inhibition studies suggested that CYP1A2  29  Table 3 Estimated Michaelis-Menten kinetic parameters for high affinity and low affinity phenacetin O-deethylase activities in microsomes from uninduced and 3-MC-induced rats. (Adapted from Sesardic et. ai, 1990)  High affinity phenacetin O-deethylase  Low affintiy phenacetin O-deethylase  Pretratment  Vmax (nmol/min/mg)  Km (u.M)  Km (uM)  Vmax (nmol/min/mg)  None  0.076 + 0.023  4.44+ 1.85  569 + 220  1.760 + 0.490  3-methylcholanthrene  0.678 ±0.139  12.6 ± 3 . 2 8  442+ 179  3.800 + 0.100  These values were estimated from the kinetic data obtained from the pooled liver microsomes of 3 animals, assuming a two component system.  30  contributes to M R O D but not EROD activity in HCB-induced rats.  These findings were  consistent with results of their antibody inhibition experiments, which indicated that M R O D and E R O D activities in HCB-induced rats are mediated by CYP1A2 and CYP1A1, respectively.  The above observations provided a method for investigating the effect of cimetidine on CYP1A2. B N F has been shown to be a co-inducer of CYP1A1/2 (Thomas et. al., 1983a) and has been used in our laboratory for pretreating the animals. Because high affinity phenacetin Odeethylase and M R O D activities have been shown to be mediated by CYP1A2 in microsomes from rats pretreated with a CYP1A1/2 inducer, we hypothesized that these activities would also be mediated by CYP1A2 in microsomes from BNF-induced rats. In this project, studies with in vivo cimetidine were first performed to investigate the effect of in vivo administration of cimetidine on microsomal M R O D activity in BNF-induced rats. This was followed by inhibition studies with phenacetin and antibodies to verify the contribution of CYP1A2 to M R O D activity in microsomes from BNF-induced rats  In humans, results of some recent correlation analysis and immunoinhibition studies have shown that hepatic microsomal E R O D activity is mediated by CYP1A2 (Murray et. al, 1993). In this project, E R O D was used a catalytic specific assay for studying human CYP1A2.  1.3. Objectives The primary objective of this investigation was to examine whether cimetidine, when administered in vivo and in vitro, inhibits hepatic CYP1A2 and CYP1A1 in adult male rats. It was proposed in the present investigation that CYP1A1/2 were responsible for M R O D and E R O D activities in microsomes from BNF-induced rats, and that CYP1A2 and CYP2C enzymes are responsible for these activities in microsomes from uninduced rats. In order to explore the effects of cimetidine on these enzymes, it was necessary to determine the role of CYP1A1/2 and CYP2C enzymes in these alkoxyresorufin dealkylase activities. A second objective of this study  31  was to determine the effect of preincubation on the inhibition of CYP1 A2-mediated EROD activity by in vitro cimetidine in human liver microsomes.  32  Materials and Methods 2.1. Chemicals Dimethyl-suphoxide (DMSO), magnesium chloride and sodium dithionite (all of analytical grade), glycerol, sodium chloride and glass distilled dichloromethane were obtained from B D H Chemicals (Toronto, ONT). Ethylenediaminetetraacetic acid (disodium salt dihydrate) (EDTA) of molecular biology grade and sucrose of enzyme grade were supplied by Life Technologies, Inc. (Gaithersburg, MD). Methanol of HPLC grade, acetonitrile, monobasic and dibasic potassium phosphate  were  provided  by  Fisher  Scientific  (Nepean,  ONT).  Tris(hydroxymethyl)aminomethane (TRIS) of reagent grade, dibasic sodium phosphate, N-[2hydroxyethyl]piperazine-N'-[2-ethanesulfonic  acid]  (HEPES),  phenacetin,  P-napthoflavone  (BNF), testosterone and 16-keto-testosterone were purchased from Sigma Chemical Co. (St. louis, M O ) . Ethoxyresorufin and methoxyresorufin were bought from Molecular Probes Inc. (Eugene, OR). The testosterone metabolites, 16a- and 2a-hydroxytestosterone were obtained from Steraloids, Inc. (Wilton, NH). Resorufin was obtained from Aldrich Chemical Company, Inc. (Milwaukee, Wl). Nicotinamide adenine dinucleotide phosphate (NADPH) in the form of tetrasodium salt was purchased from Boehringer Mannheim Canada ltd. (Dorval, QUE). Cimetidine hydrochloride was provided as a gift from Smith Kline & French Canada ltd. (Mississauga, ONT). Lyophilized bovine serum albumin and Bio-Rad protein assay dye reagent concentrate were bought from Bio-Rad lab. (Hercules, CA). ChromoPure rabbit IgG (preimmune IgG) was supplied by Jackson, Immunoresearch lab. Inc. (West Grove, PA) .  Polyclonal antibodies directed against rat hepatic CYP2C11 or CYP1A1 were supplied by Dr. S. Bandiera (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C.).  Information on the preparation and cross-reactivities of these antibodies was obtained  through personal communication with Dr. S. Bandiera. Polyclonal antibody directed against rat CYP2C11 was prepared from a pool of heat-inactivated sera collected from rabbits immunized  33  with the electrophoretically homogenous protein.  IgG was purified from the sera by a  combination of caprylic acid precipitation followed by ammonium sulfate precipitation and a final purification on a DEAE-Sephacol column, as previously described by McKinney and Parkinson (1987) and Thomas et. al. (1976). The purified antibody recognized CYP2C11 and several other cytochromes P450 in the CYP2C subfamily and is referred to as 'polyspecific anti-CYP2Cl 1 IgG' in the present thesis. The cross-reactivity of this antibody preparation against cytochromes P450 in other subfamilies was not tested. The polyspecific anti-CYP2C 11 IgG was made monospecific by passing the preparation successively through a series of columns containing microsomal proteins from adult female untreated rats, partially purified CYP3A1, CYP2C7, CYP2C13 and epoxide hydrolase, and purified CYP2C13. After removal of the cross-reactive IgG fraction, the specificity of the remaining antibody preparation was tested using noncompetitive enzyme-linked immunosorbent assay (ELISA) and immunoblots with purified cytochromes P450 and microsomal samples from control and induced rats. The antibody did not cross-react with purified CYP1A1, CYP2B1, CYP2C7, CYP2C13, CYP3A1, epoxide hydrolase or any proteins in hepatic microsomes from untreated female rats. This antibody preparation is hereafter referred to as 'monospecific anti-CYP2Cll IgG'.  The procedures for preparation and purification of the  polyclonal antibody directed against rat hepatic CYP1A1 were similar to those described for the polyspecific anti-CYP2Cll IgG, except for immunization of the rabbits with purified rat hepatic CYP1A1 instead of CYP2C11. The specificity of the purified antibody was tested using ELISA and immunoblots, and was found to react with CYP1A1, CYP1A2 and CYP2C13. This antibody preparation is referred to as 'polyspecific anti-CYPlAl IgG in the present thesis.  Monoclonal antibody directed against rat CYP1A1 (MAb C-8.1) was a gift from Dr. P. E. Thomas (Department of Chemical Biology, Rutgers - The State University of New Jersey, Rutgers, New Jersey). The preparation and cross-reactivity of M A b C-8.1 were described by Thomas et. al. (1984).  In brief, spleen cells from mice immunized with purified rat liver  microsomal CYP1A1 were fused with myeloma cells.  The hybridoma cells were tested for  34  production of anti-rat CYP1A1 antibodies and the antibody secreting-cells were subcloned by limiting dilution. The subcloned hybridoma cells in each colony were then injected into the mice and monoclonal antibody was purified from the collected ascites fluid by ammonium sulfate precipitation and column chromatography. When tested using ELISA, M A b C-8.1 did not crossreact with purified rat hepatic CYP1A2, CYP2A1, CYP2B1, CYP2B2, CYP2C7, CYP2C11, CYP2C13, epoxide hydrolase, NADPH-cytochrome P450 reductase or two other enzymes of the endoplasmic reticulum. When tested on immunoblots, M A b C-8.1 recognized only a single band, corresponding to CYP1A1, in liver microsomes from untreated or 3-MC-treated rats. M A b C-8.1 was strongly inhibitory for CYP1A1-mediated catalytic activity (aryl hydrocarbon hydroxylase) in a reconstituted system and in microsomes from 3-MC-treated rats, with maximal inhibition attained at an antibody concentration of about 0.1 mg IgG  per nmol of cytochrome P450  (Thomas et. al, 1984).  2.2. Animals Adult male Wistar rats, 10-11 weeks old, weighing between 225 - 300 g, were ordered from Charles River, Canada (Montreal, QUE). Upon arrival, the animals were kept in our animal facility for at least one week prior to initiation of treatment. The animal room was kept at 22°C with constant lighting cycles (8 am on, 10 pm off). The animals were housed in polyester cages (2 to 3 animals per cage) with The Andersons corncob bedding (Maumee, OH); and were allowed free access to Rodent laboratory Chow #5001 (Ralston Purina Canada, Inc., Longueuil, QUE) R  and tap water until death.  2.3.  Animal treatments To induce CYP1A1/2, the rats were first pretreated with B N F 40 mg/kg/d for 3 days.  This standard induction protocol has been employed by other investigators for induction of the C Y P 1 A emzymes in rats (Guengerich et. al., 1982b, Waxman 1984, Halpert et. al., 1985a). B N F was dissolved in corn oil and was administered by intraperitoneal (i.p.) injection. These rats are  35  referred to as 'BNF-induced rats' throughout the thesis. The control rats were pretreated with an equal volume of corn oil and were included in the study for determining the effect of B N F on the microsomal enzyme activities. These control animals were given i.p. injections of corn oil daily for 3 days and were decapitated 24 hr after the last injection of corn oil.  To study the effect of in vivo administration of cimetidine on CYP1A1/2, the B N F induced rats were administered a single dose of 0.9% saline or cimetidine hydrochloride, 150 mg/kg, by i.p. injection in the treatment phase.  Cimetidine hydrochloride was dissolved in  distilled water and was given 24 hr after the last pretreatment dose. The rats were decapitated 90 min after the last injection and livers were excised for microsomal preparation. This inhibition protocol has been shown to yield maximal inhibition of aminopyrine N-demethylase activity in uninduced rats (Chang, 1991) and was employed for studying the inhibitory effects of in vivo cimetidine on rat hepatic cytochromes P450. Cimetidine administered in this manner is referred to as 'in vivo cimetidine'.  To study the effect of  in vivo cimetidine on the microsomal enzyme activities in  'uninduced rats', the animals were not pretreated at all. They were just given a single i.p. injection of saline or cimetidine (150mg/kg) 90 min before decapitation and excision of liver.  2.4. Human liver microsomal samples A bank of human liver microsomal samples were available in the laboratory. D. Leong and Dr. G. Bellward had prepared these human liver microsomes, determined the microsomal protein and cytochrome P450 content, and measured the E R O D activities of these samples. Information regarding the source of the human liver tissue samples, the preparation of the microsomes and the EROD activity of these microsomal samples were provided by Dr. G. Bellward (personal communication).  36  In brief, human liver tissue samples were obtained from oncology patients undergoing partial hepatetomy at Vancouver General Hospital (Vancouver, B. C ) . After blood supply to the liver was cut off for about 20 min, liver tissue was excised and immediately stored in liquid nitrogen. The healthy portion was sent to the laboratory for preparation of microsomes. The tissue samples were thawed in a water bath at 37°C for about 2 min. Human liver microsomes were prepared from the thawed liver samples in a manner similar to that described for rat microsomes (Section 2.4). Among the liver samples from 8 nonsmokers, those from patients 4, 5 and 6 were found to have the highest microsomal EROD activities. In the present investigation, studies were conducted with pooled microsomes from the liver samples of patients 4, 5 and 6. The clinical data available on these patients are shown in Table 4.  2.5. Preparation of hepatic microsomes Hepatic microsomes were prepared by a standard procedure as described previously by L u and Levin (1972).  Immediately after the animals were killed by decapitation, the livers were  excised and chilled in 25 mL ice-cold 50 mM TRIS/ 1.15% potassium chloride buffer (pH 7.4). Either individual or pooled livers were homogenized with a motor-driven Potter-Elvehjem homogenizer.  The homogenate was centrifuged at 10,000 x g for 20 min. The supernatant  obtained after centrifugation was then filtered through 4 layers of cheesecloth.  The post-  mitochondrial (S-9) fraction contained in the filtrate was spun in a centrifuge at 100,000 x g for 60 min. The pellet obtained after ultracentrifugation contained the microsomal subfraction, and was homogenized in 20 mL 10 mM EDTA/ 1.15% potassium chloride buffer (pH 7.4).  The  homogenate was recentrifuged at 100,000 x g for 60 min. The resulting microsomal pellet was washed twice with 1 mL of 0.25 M sucrose and resuspended in 2 mL 0.25 M sucrose. Aliquots (250 u.L) of microsomal suspensions were transferred to cryotubes and stored at -80°C.  Table 4 Clinical data for patients from whom the liver samples were obtained.  Patient Number  Sex  Age  Smoking status  Liver condition  Medications  4  F  37  Nonsmoker  Non-cirhotic  Not on chemotherapy  5  M  25  Nonsmoker  Non-cirrhotic  Not on chemotherapy  6  F  Unknown  Nonsmoker  Unknown  Not on chemotherapy  38  2.6. Determination of cytochrome P450 content The cytochrome P450 content of the prepared microsomes was determined by the standard method of Omura and Sato (1964) using a SIM-Aminco DW-2C spectrophotometer (Urbana, II ). The microsomes were diluted in 100 mM sodium phosphate buffer (pH 7.4)/ 0.1 M E D T A / 20% glycerol at a 1:50 dilution for microsomes from BNF-induced rats and a 1:20 dilution for microsomes from uninduced (not pretreated) or corn oil-pretreated rats. Both the sample and reference cuvettes (1 cm path length) were filled with 2 mL of diluted microsomes. A few milligrams of sodium dithionite were then added to each cuvette to reduce the samples. This was followed by bubbling of carbon monoxide through the microsomes in the sample cuvette at a rate of 1 bubble per second for 1 min. This allowed binding of carbon monoxide to the reduced heme of the cytochrome P450 enzymes. The difference spectrum was measured and recorded when the samples in both cuvettes were scanned from 325 nm to 625 nm. Using a molar extinction coefficient of 91 cm" mM" , the cytochrome P450 concentration was calculated from 1  1  the difference in absorbance taken at 450 and 490 nm. Duplicate determinations were performed for all the microsomal samples.  2.7. Determination of protein content The protein content of the prepared microsomes was determined by the method of Bradford (1976), which employs the principle of protein-dye binding. Microsomes were diluted 100-fold in distilled water. The Bio-Rad protein assay dye reagent concentrate was diluted (1:4) with distilled water and filtered. Five mL of the diluted dye reagent were added to the diluted microsomes. Protein-dye binding results in a shift in the absorption maximum of the dye from 465 to 595 nm. The absorbance of the samples was measured against the blank at 595 nm within 5 to 60 min after the addition of the dye. Bovine serum albumin was used as a standard for preparing the standard curve, from which the protein concentration of the unknown sample was determined. All determinations were performed in duplicate using a Beckman DU -64 spectrophotometer R  equipped with a protein assay Soft-Pac module (Fullerton, CA).  39  2.8. Microsomal enzyme assays 2.8.1. Methoxyresorufin O-demethylase (MROD) and ethoxyresorufin O-demethylase (EROD) assays Microsomal M R O D and EROD activities were determined by a direct fluorimetric method originally described by Burke and Mayer (1974), with some modifications (Chang, 1991). The assays were performed using a Shimadzu RF-540 spectrofluorometer (Columbia, M A ) . The excitation wavelength was 530 nm (slit width = 5 nm) and the emission wavelength was 582 nm (slit width = 5 nm). The assay mixture contained 1.93 mL of 100 m M HEPES/ 5 m M magnesium chloride (pH 7.8), 50 uL of microsomes diluted in 0.25 M sucrose, 10 uL of ethoxyresorufin or methoxyresorufin dissolved in DMSO. The final substrate concentrations were 1 u M for B N F induced samples and 5 u M for uninduced samples. Ten uL of N A D P H (final concentration = 0.25 mM) dissolved in 100 mM HEPES /5 mM magnesium chloride (pH 7.8) was added to start the reaction. The total reaction volume was 2 mL. The reaction mixture was stirred continuously by a magnetic micro stirring bar in the cuvette. The increase in fluorescence associated with the formation of resorufin during the reaction was monitored and recorded. The amount of resorufin formed was determined from a standard curve of fluorescence vs resorufin concentration. A l l determinations were performed in duplicate.  2.8.2. Testosterone 2a- and 16a-hydroxylase assays Testosterone 2a- and 16a-hydroxylase activities were determined by the method of Wood et. al. (1983) with a slight modification of the mobile phase. In brief, the assay mixture contained 0.5 mL of 0.1 M potassium phosphate buffer (pH 7.4), 0.2 mL of 0.25 M sucrose, 0.1 mL of 0.03 M magnesium chloride, 80 uL of microsomes diluted in 0.05 M potassium phosphate buffer (pH 7.4), 20 u L of testosterone dissolved in methanol (final concentration = 0.25 mM). The assay mixture was prewarmed to 37°C and 100 uL of N A D P H (final concentration = 1 mM) dissolved in 0.05 M potassium phosphate buffer (pH 7.4) was added to start the reaction. The  40  final reaction volume was 1 mL. The reaction were allowed to proceed for 5 min in a shaking water bath at 37°C and 6 mL of dichloromethane were added to stop the reaction. This was followed by the addition of 3 nmol of 16-keto-testosterone (internal standard) dissolved in 100 uL methanol. The contents of the incubation tube were then mixed on a vortex at maximum speed for 30 sec and centrifuged at 800 x g for 2 min. Following aspiration of the aqueous layer by vacuum, 4 mL of the organic layer were transferred to a culture tube (15 x 100 mm), and dried under a stream of nitrogen at 37°C.  The residue was reconstituted with 200 uL of filtered  methanol and stored at - 20°C until required for analysis.  The high performance liquid chromatography system consisted of two Waters Model 501 pumps, a Waters Model 712 autosampler, a Waters Model 486 tunable absorbance detector, a Maxima 820 chromatography workstation (Waters chromatography Division, Millipore Ltd., Wilford, M A ) and a Supelco octyldecylsilane reverse phase column (5 u.m, 4.6 x 150 mm inner diameter) preceeded by a Pelliguard^-  LC-18, 2 cm column (Supelco Inc., Bellefonte, PA).  Following the injection of 10 uL of the sample, the column was eluted with a concave gradient (option no. - 7 in Maxima 820 software program) of 40 % methanol, 60 % water and 1.1 % acetonitrile (solvent A) to 76 % methanol, 22 % water and 1.9 % acetonitrile at a flow rate of 1.5 mL/min for 30 min. With an equilibrium delay of 15 min, the total run time for each sample was 45 min.  The absorbance of the column effluents was monitored at 254 nm. All  chromatographic separations were performed at ambient room temperature.  Testosterone hydroxylase products, 2a- and 16a-hydroxytestosterone, were identified by comparing the retention times to those of authentic standards.  Calibration curves were  constructed by plotting peak height ratios of metabolite : internal standard against the known concentration ratios of metabolite : internal standard. In the standard samples, boiled microsomes were used and N A D P H was not added. The amount of each metabolite formed in the unknown  41  samples was determined from the corresponding standard curve.  A l l determinations were  performed in duplicate.  2.8.3. Assay conditions When appropriate, preliminary experiments were performed with microsomes pooled from the livers of 4 animals to verify that reaction rates were linear with respect to protein concentration and reacton time, and that the substrate and N A D P H concentratons used in the assays were optimal (Fig. 3 - 6). The final conditions for the various assays are listed in Table 5. These standard assay conditions were used for most of the experiments. For those experiments that required modification of the standard assay conditions, the modified conditions are stated in the presentation of the results.  2.9.  Enzyme kinetic studies of MROD and EROD activities The enzyme kinetics of hepatic microsomal M R O D and E R O D activities in uninduced and  BNF-induced rats were studied over a substrate concentration range 0 to 5 u M for estimation of the Michaelis-Menten kinetic parameters, K m and Vmax.  The protein concentrations were  reduced at the lower substrate concentrations to ensure that rate of resorufin formation was linear with respect to reaction time (see Section 3.3.1). Enzyme activity was measured in duplicate at each substrate concentration. 2.10.  Chemical inhibition studies on MROD and EROD activities with phenacetin Chemical inhibition studies with phenacetin were performed according to the method  described by Nerukar et. al. (1993), with slight modifications. In both M R O D and E R O D assays, the reaction mixtures contained 1.92 mL instead of 1.93 mL of reaction buffer (100 m M HEPES/ 5 m M magnesium chloride buffer, pH 7.8).  Phenacetin dissolved in D M S O (10 uL) was added  immediately prior to initiation of the reaction with the addition of N A D P H , and the reaction was  42  (B) R e a c t i o n time (A) Protein concentration  2  3  4  Reaction time (min)  Protein concentration (ug/ml)  (C) Substrate concentration  (D) N A D P H concentration  £ r 1  1  2  3  4  5 0.25  Methoxyresorufin concentration (uM)  0.50  0.75  1  NADPH concentration (uM)  Fig. 3. Optimization of conditions for M R O D assay in microsomes from BNF-induced rats. The standard assay conditions were: incubation time = 3 min, protein concentration = 10 u.g/ml, substrate concentration = 1 u M and N A D P H concentration = 0.25 mM. The above variables were altered one at a time as indicated.  43  (B) R e a c t i o n time  (A) Protein concentration 5  r  0 Protein concentration (ug/ml)  5  10  Reaction time (min)  Fig. 4. Optimization of conditions for M R O D assay in microsomes from uninduced rats. The standard assay conditions were: incubation time = 5 min, protein concentration =150 ug/ml, substrate concentration = 5 u M and N A D P H concentration = 0.25 mM. The above variables were altered one at a time as indicated.  44  (B) Reaction time (A) Protein concentration  Protein concentration (ug/ml)  Reaction time (min)  Fig. 5. Optimization of conditions for EROD assay in microsomes from BNF-induced rats. The standard assay conditions were: incubation time = 3 min, protein concentration = 5 ug/ml, substrate concentration = 1 u M and N A D P H concentration = 0.25 mM. The above variables were altered one at a time as indicated.  45  (B) Reaction time  Reaction time (min)  Fig. 6. Optimization of conditions for testosterone 2a- and 16a-hydroxylase assays in microsomes from BNF-induced rats. The standard assay conditions were: incubation time = 5 min, protein concentration = 300 ug/ml, substrate concentration = 250 u M and N A D P H concentration = 1 mM. The above variables were altered one at a time as indicated.  46 Table 5 Summary of the assay conditions for M R O D , E R O D , testosterone 2a- and 16a-hydroxylase activities in rat microsomes  Assay  Microsomal Samples  [Protein]  [Substrate]  [NADPH]  (ug/ml)  Reaction time (min)  (uM)  (uM)  Verification of assay conditions  MROD  BNF-induced Uninduced  10 150  Up to 3 5  1 5  0.25 0.25  Fig. 3 Fig. 4  EROD  BNF-induced Uninduced  5 150  Up to 3 5  1 5  0.25 0.25  Fig. 5 Chang, 1991  BNF-induced Uninduced  300 500  5 5  250 250  1 1  Fig. 6 Chang et. al.,  3  Testosterone 2a- and 16ahydroxylase  a  The assay conditions for EROD activity in human liver microsomes were the same as those in rat microsomes and have been optimized previously by D. Leong and Dr. G. Bellward (personal communication with Dr. G. Bellward).  1992a  47  allowed to proceed as described in Section 2.8.1. The final reaction volume was 2 mL. The final phenacetin concentrations in the reaction mixture were 0, 5.5, 17, 50, 150 and 450 u M . Inhibition studies were performed with phenacetin at the above concentrations on each enzyme activity at several subsrate concentrations that were predetermined multiples of the estimated K m value. All determinations were performed in duplicate.  2.11.  Immunoinhibition studies The effects of various antibody preparations on M R O D , EROD, testosterone 2a- and 16a  -hydroxylase activities were determined by the method described by Thomas et. al. (1981). Modified phosphate buffer saline (137 mM sodium chloride, 2.6 m M potassium chloride, 8.1 m M dibasic sodium phosphate, 1.5 m M monobasic potassium phosphate and 2 m M E D T A , pH 7.4 ) was used for diluting the various antibodies. Microsomes were incubated at room temperature with diluted antibody for 10 min. This was followed by the addition of the assay mixture and initiation of the reaction with N A D P H .  The reaction was allowed to proceed as described in  Sections 2.8.1 and 2.8.2. The concentrations and volumes of the various components in the assay mixture were adjusted, so that the concentration of the components in the final reaction mixture and the total reaction volume were the same as described previously (Sections 2.8.1 and 2.8.2). All determinations were performed in duplicate.  Since preimmune IgG was observed to have little stimulatory or inhibitory effect (< 10%) on  M R O D , EROD, testosterone 2a- or 16a-hydroxylase activities in microsomes from B N F -  induced and uninduced rats, results of the antibody inhibition experiments preparations are expressed as a percent of control activity (i.e. no IgG).  48  2.12.  In vitro inhibition by cimetidine of MROD, EROD, testosterone 2a- and 16a-hydroxylase activities In the in vitro inhibition experiments, cimetidine hydrochloride was dissolved in distilled  water and was added to microsomes in vitro. Cimetidine administered in this manner is referred to as 'in vitro cimetidine'.  2.12.1. Without preincubation The experimental protocol as described by Chang et. al. (1992b) was followed. Cimetidine hydrochloride or distilled water was added to microsomes in vitro immediately prior to initiation of the reaction with N A D P H addition. The reactions were allowed to proceed as described in Sections 2.8.1 and 2.8.2. Volumes of the reaction buffers were reduced so that the final volumes were still 2 mL for M R O D and EROD assays, and 1 mL for testosterone 2a- and 16a-hydroxylase assays. All determinations were performed in duplicate.  2.12.2. With preincubation The experimental protocol as described by Chang et. al. (1992b) was followed. N A D P H was added in vitro to the preincubation medium containing buffer, microsomes and cimetidine. After a preincubation period of 15 min (at 37°C), distilled water was added (to equalize the reaction volume with the control incubation) just prior to initiation of the reaction with the addition of substrate. The reaction was allowed to proceed as described in Sections 2.8.1 and 2.8.2. Volumes of the reaction buffer were reduced so that the final volumes were still 2 mL for M R O D and E R O D assays, and 1 mL for testosterone 2a- and 16a-hydroxylase assays.  In the  control incubations, microsomes were preincubated with N A D P H and distilled water for 15 min. At the end of the preincubation period, cimetidine was added immediately before the addition of substrate. All determinations were performed in duplicate.  49  A preincubation period of 15 min was used in these studies because this preincubation period was found to be optimal in attaining selective inhibition of testosterone 2a-hydroxylase activity (CYP2C11) in uninduced samples in a time course study with a cimetidine concentration of 0.05 m M (Chang et. al, 1992b).  2.13.  Data analysis The data obtained from the kinetic studies were fitted to an one-enzyme model by  iterative, least- squares, non-linear analysis using the Enzfitter computer software program to determine Km and Vmax.  The data obtained from phenacetin inhibition studies are presented as  dose response  curves, in which the enzyme activity at each inhibitor concentration is plotted as a percent of control activity against the logarithm of the inhibitor concentration. The type of inhibition was investigated by analyzing the inhibition data by Lineweaver-Burk plot of reciprocals of reaction velocity vs reciprocals of substrate concentration (see Section 1.1.6.1). Dixon plots of reciprocals of reaction velocity vs inhibitor concentration were generated for differentiation between partial and complete inhibition, and the x-intercepts of these plots were used for calculation of K i (see Section 1.1.6.1.).  All data are expressed as mean + standard deviation in experiments that were conducted with microsomes from individual animals. Mean values were compared using Student's t-test for paired or unpaired data, as appropriate, with the SigmaStat® computer software program. A difference was regarded as being statistically significant when p was < 0.05.  50  Results  3.1.  Effect of B N F on total hepatic microsomal cytochrome P450 content and enzyme activities in male rats  In the present investigation, B N F was used as a coinducer of rat hepatic CYP1A1 and CYP1A2. As shown in Table 6, BNF pretreatment resulted in a 70% increase (p < 0.005) in total hepatic microsomal cytochrome P450.  This was accompained by a 40-fold induction of  microsomal M R O D activity (p < 0.001) and a 50-fold induction of microsomal E R O D activity (p < 0.001) (Table 6). In contrast to the induction of the alkoxyresorufin O-dealkylase activities, B N F pretreatment resulted in suppression of testosterone 2a- and 16a-hydroxylase activities by 61 and 65%, respectively (p < 0.005) (Table 6).  3.2.  Effect of  in vivo cimetidine  To investigate the effect of in vivo administration of cimetidine on hepatic microsomal cytochrome P450 content and enzyme activities, the BNF-induced or uninduced rats were treated with cimetidine (150 mg/kg) or saline in vivo and were decapitated 90 min after after the treatment (Section 2.3). The microsomal cytochrome P450 content and enzyme activities of the cimetidine-treated rats were then compared with those of the saline-treated rats (control).  3.2.1.  T o t a l hepatic microsomal cytochrome P450 content  The effect of in vivo cimetidine on total hepatic microsomal cytochrome P450 content in BNF-induced and uninduced rats is shown in Fig. 7. In BNF-induced rats, in vivo administration of cimetidine had no effect on the microsomal cytochrome P450 content. In contrast, in vivo treatment with cimetidine appeared to decrease the total microsomal cytochrome P450 content by 18% in uninduced rats (p < 0.05). One of rats in the cimetidine group was not injected properly with cimetidine (Section 3.3.2.2) but had a particularly low cytochrome P450 content. When that particular rat was excluded in the calculation of the mean cytochrome P450 content, in vivo  51  cimetidine appeared to decrease the hepatic microsomal P450 content of uninduced rats by 13% (p < 0.05).  3.2.2. Microsomal enzyme activities Selected enzyme assays were performed with microsomes from BNF-induced and uninduced rats that had been administered saline or cimetidine in vivo as described in Section 2.3. The objectives of the studies were to investigate the effect of in vivo cimetidine on M R O D activity in microsomes from BNF-induced and uninduced rats, and to reproduce Chang's results (1991) on the effect of in vivo cimetidine on EROD activity in the same microsomes.  Because in vivo cimetidine was associated with a decrease in the total microsomal cytochrome P450 content in uninduced rats (Fig. 7), the enzyme activity has been expressed as the rate of product formation relative to the amount of microsomal protein as well as the total cytochrome P450 content, to ascertain the inhibitory effect of in vivo cimetidine.  3.2.2.1. Microsomes from BNF-induced rats When the microsomal enzyme activities in BNF-induced rats were expressed as nmol/min/mg, in vivo cimetidine had no effect on M R O D or E R O D activity (Fig. 8A), but inhibited testosterone 2a- and 16a-hydroxylase activities by 58% and 51%, respectively (p < 0.01) (Fig. 9A ). Because in vivo cimetidine did not change the total microsomal cytochrome P450 content in BNF-induced rats (Fig. 7), the inhibition of these activities was similar when results were expressed as nmol/min/nmol P450 (Fig. 8B and 9B).  The observed lack of inhibition of EROD activity in microsomes from BNF-induced rats by in vivo cimetidine is consistent with Chang's original finding (1991). The observed inhibitory effect of in vivo cimetidine on the testosterone hydroxylase activities indicates that the active form of the inhibitor was still present in the BNF-induced microsomes.  However, it is not known  52  whether testosterone 2a- and 16a-hydroxylase  activities are mediated  by CYP2C11 in  microsomes from BNF-induced rats, as they are in microsomes from uninduced rats.  3.2.2.2.  Microsomes from uninduced rats When the microsomal enzyme activities in uninduced  rats were expressed as  nmol/min/mg, in vivo cimetidine was observed to inhibit M R O D and E R O D activities by 27% (p < 0.05) and 59% (p < 0.01), respectively (Fig. 10A). It was noted that one of the cimetidinetreated rats had microsomal EROD activity as high as the saline-treated rats and it was suspected that this particular rat did not receive in vivo cimetidine because of improper injection technique. When this particular rat was excluded from the calculation of mean activities, in vivo cimetidine was observed to inhibit M R O D and EROD activities by 36% (p < 0.01) and 78% (p < 0.001), respectively (Fig. 10B).  When the activities were calculated as the rate of product formation per nmol of total P450 and with all the rats included in the calculation of mean activities, in vivo cimetidine was observed to have no effect on M R O D activity and the decrease in E R O D activity was not statistically significant (Fig. 11 A). However, when the outlier in the cimetidine-treated group was excluded from the calculation of mean activities, in vivo cimetidine was found to inhibit E R O D activity by 75% (p < 0.001) but the 26% decrease in M R O D activity was not significant (p = 0.07) (Fig. 11B). This suggests that the difference in total cytochrome P450 content observed with in vivo cimetidine does account for the inhibitory effect of in vivo cimetidine on E R O D activity. There was insufficient evidence to conclude that in vivo cimetidine inhibited M R O D activity.  Table 6 Effect of BNF on total hepatic microsomal cytochrome P450 content, MROD, EROD, testosterone 2a- and 16a-hydroxylase activities.  Pretreatment Corn oil  BNF  Total cytochrome P450 content (nmol P450/mg protein)  0.94 + 0.10  1.67 ± 0.26*  MROD activity  0.06 ± 0 . 0 1  2.30 ±0.18**  0.54 ±0.05  25.44 ±2.56**  Testosterone 2a-hydroxylase (nmol/min/mg)  2.97 ± 0.92  1.17 ± 0.22*  Testosterone 16a-hydroxylase (nmol/min/mg)  4.60 ± 1.43  1.59 ± 0 . 1 8 *  (nmol/min/mg)  EROD activity (nmol/min/mg)  Results are expressed as mean ± standard deviations (N = 4 rats per group). *P < 0.005 and ** P < 0.001, compared to the corresponding control group (one-tailed, unpaired t-test).  54  Fig. 7. Effect of in vivo cimetidine on total hepatic microsomal cytochrome P450 content in BNF-induced and uninduced rats. Results are expressed as the mean + standard deviation for 4 rats per group ( N = 3 rats, one of the cimetidine-treated rats was an outlier and was excluded from the calcuation of mean activity). *P < 0.05, compared to the corresponding control group (two-tailed, unpaired t-test). a  55  (A)  MROD  EROD  (B)  MROD  EROD  Fig. 8. Effect of in vivo cimetidine on M R O D and EROD activities in microsomes from BNFinduced rats. Activities were calculated as the rate of product formation (A) per mg of microsomal protein or (B) per nmol of total P450. Results are expressed as the mean ± standard deviation for 4 rats per group . The differences between the mean activities were not statistically significant (one-tailed, unpaired t-test).  56  (A)  2A-Hydroxylase  16A-Hydroxylase  (B)  2A-Hydroxylase  16A-Hydroxylase  Fig. 9. Effect of in vivo cimetidine on testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats (protein concentration was 150 u,g/ml). Activities were calculated as the rate of product formation (A) per mg of microsomal protein or (B) per nmol of total P450. Results are expressed as the mean + standard deviation for 4 rats per group. *P < 0.01 and **P < 0.001, compared to the corresponding control group (one-tailed, unpaired t-test).  57  (A) 1.00 r  MROD  EROD  Fig. 10. Effect of in vivo cimetidine on M R O D and EROD activities in microsomes from uninduced rats. Activities were calculated as the rate of production formation per mg of microsomal protein. Results are expressed as the mean + standard deviation for (A) 4 rats per group. (B) One of the cimetidine-treated rats was an outlier and was excluded from the calculation of mean activity (N = 3 rats in the cimetidine-treated group and 4 rats in the salinetreated group). *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the corresponding control group (one-tailed, unpaired t-test).  58  (A) 1.00 r  MROD  EROD  Fig. 11. Effect of in vivo cimetidine on MROD and EROD activities in microsomes from uninduced rats. Activities were calculated as the rate of production formation per nmol of total P450. Results are expressed as the mean + standard deviation for (A) 4 rats per group. (B) One of the cimetidine-treated rats was an outlier and was excluded from the calculation of mean activity (N = 3 rats in the cimetidine-treated group and 4 rats in the saline-treated group). *P < 0.001, compared to the corresponding control group (one-tailed, unpaired t-test).  59  3.3. Phenacetin inhibition studies In vivo cimetidine was observed to have no effect on M R O D and E R O D activities in microsomes from BNF-induced rats (Fig. 8), but did inhibit E R O D activity in microsomes from uninduced rats (Fig. 1 IB, Section 3.2). In order to investigate the involvement of CYP1A2 in the dealkylation of methoxyresorufin and ethoxyresorufin in microsomes from BNF-induced and uninduced rats, chemical inhibition studies were performed with phenacetin.  In the case of competitive inhibition, the extent of inhibition observed depends on the relative affinity of the substrate and inhibitor for the enzyme, as well as the relative concentration of the substrate and inhibitor in the reaction medium. Equations (2) to (4) in Section 1.1.6.1 describes how these parameters affect the extent of inhibition observed. In order to compare the inhibitory effect of phenacetin on M R O D and EROD activities, kinetic studies were performed to estimate the respective Km values of the enzymes for these substrates . Phenacetin inhibition studies were then performed on these activities at substrate concentrations that were equivalent to multiples of their respective Km values.  3.3.1. Characterization of M R O D and E R O D enzyme kinetics The effects of substrate concentrations on M R O D and E R O D actrivities are shown in Fig. 12.  Since the reaction rates became sub-maximal at higher substrate concentrations, the  enzyme activities obtained at these concentrations were not used for kinetic analysis. The kinetic parameters were estimated by fitting the data obtained from one experiment to a one-enzyme model and the results are summarized in Table 7. It was not possible to fit the data into a twoenzyme model due to self-inhibition of enzyme activities.  The K m values for M R O D activity were similar in microsomes from BNF-induced and uninduced rats, whereas there was a 6-fold difference in the Km value for E R O D activity after BNF-induction,  suggesting that different  enzymes are involved  in the  metabolsim of  60  ethoxyresorufin in microsomes from these groups of rats. The K m value for E R O D activity in microsomes from uninduced rats (1.66 uM) was verified with a second determination because it was different from the reported literature value of 0.23 u M (Burke et. ai, 1974).  3.3.2. Inhibition of microsomal M R O D and E R O D activities by phenacetin 3.3.2.2. Microsomes from BNF-induced rats Lineweaver-Burk plots of inhibition of M R O D and EROD activities in microsomes from BNF-induced rats by phenacetin are displayed in Fig. 13. The lines have similar y-intercepts but different x-intercepts, suggesting that there was a change in the Km, but that the Vmax remained the same in the presence of the inhibitor. The observed patterns of inhibition for M R O D and E R O D activities are consistent with competitive inhibition.  The inhibition data were plotted as dose response curves, as shown in Fig. 14. Low concentrations of phenacetin (5 - 50 uM) were observed to inhibit both M R O D and EROD activities. The inhibition curves for the two activities were compared at substrate concentrations that were equivalent to their respective Km estimates. It can be seen that the inhibition curve for M R O D activity was shifted to the left of that for EROD activity (Fig. 15). For example, at a phenacetin concentration of 17 u M , M R O D and EROD activities were inhibited by 32% and 16%o, respectively, suggesting that CYP1A2 plays a greater role in mediating M R O D activity.  Dixon plots of inhibition of M R O D and EROD activities by phenacetin were linear (Fig. 16).  The K i values estimated for inhibition of M R O D activity using the complete range of  phenacetin concentrations were similar to those values estimated using the lowest four concentrations (Table 8), and the same was true for inhibition of EROD activity. The estimated K i values of phenacetin for inhibition of M R O D activity were approximately half of those for inhibition of E R O D activity.  61  Taken together, these results indicate that in microsomes from BNF-induced rats, low concentrations of phenacetin inhibit both M R O D and EROD activities, but that phenacetin is a more potent inhibitor of M R O D activity.  This suggests that CYP1A2 is involved in the  dealkylation of both methoxyresorufin and ethoxyresorufin in microsomes from BNF-induced rats but that this enzyme plays a greater role in M R O D activity.  3.3.2.1. Microsomes from uninduced rats Lineweaver-Burk plots revealed competitive inhibition of M R O D and E R O D activities by phenacetin in microsomes from uninduced rats (Fig. 17).  The inhibitory effects of phenacetin on M R O D and E R O D activities are shown as a series of dose response curves in Fig. 18. Low concentrations of phenacetin (5.5 - 50 uM) were found to inhibit M R O D activity, but had little effect on EROD activity. The inhibition curves for M R O D and EROD activities were compared at substrate concentrations that were equivalent to the respective K m estimates. It can be seen in Fig. 19 that the inhibition curve for M R O D activity was shifted to the left of that for EROD activity. For example, at a phenacetin concentration of 17 u M , M R O D activity was inhibited by 37%, whereas EROD activity was inhibited by less than 5%, suggesting that CYP1A2 is involved in the dealkylation of methoxyresorufin but not ethoxyresourfin.  Dixon plots were generated for estimation of K i values of phenacetin for inhibition of M R O D and E R O D activities in microsomes from uninduced rats. The Dixon plots for M R O D activity appeared to deviate from linearity at higher phenacetin concentrations (Fig. 20A). The K i values estimated using the complete range of phenacetin concentrations were 3-fold greater than those estimated using the lowest 4 concentrations (Table 8). This observation suggests that more than one enzyme is involved in methoxyresourfin O-deethylation and/or that M R O D activity is too low to be measured accurately at high phenacetin concentrations. The Dixon plots for EROD  62  activity were linear (Fig. 20B). The K i values estimated for inhibition of E R O D activity using the complete range of phenacetin concentrations or the lowest four concentrations were similar in magnitude (Table 8). The average estimated K i value for phenacetin inhibition of M R O D activity was 44% (12% with the reduced data) of that for inhibition of EROD activity.  Taken together, these results indicate that in microsomes from uninduced rats, M R O D activity, but not E R O D activity, is subject to competitive inhibition by low concentrations of phenacetin.  This suggests  that CYP1A2 at leastly partly mediates M R O D activity, but not  E R O D activity, in microsomes from uninduced rats.  63  (B) E R O D ( B N F - i n d u c e d )  (A) MROD (BNF-induced) r  30  0  0  1  2  3  4  5  6  .|  ,  0  1  ,  1  Methoxyresorufin concentration (uM)  O)  ,  1  ,  3  ,  ,  4  ,  ,  5  ,  6  Ethoxyresorufin concentration (uM)  (D) E R O D  (C) MROD (Uninduced) 0.10  1  2  0.60  r  (Uninduced)  r  0.0B |-  0  1  2  3  4  5  Methoxyresorufin concenration (uM)  6  0  1  2  3  4  5  6  Ethoxyresorufin concentration (uM)  Fig. 12. Effect of substrate concentration on M R O D and EROD. activities in microsomes from BNF-induced and uninduced rats. The protein concentrations were reduced at lower substrate concentrations to ensure linearity of reaction rate with respect to time (see Table 7 for the range of protein concentrations). Microsomes were prepared from the pooled livers of 4 BNF-induced or uninduced animals.  64  Table 7 Estimated Michaelis-Menten parameters for M R O D and E R O D activities in microsomes from BNF-induced and uninduced rats.  Enzyme activity  MROD  EROD  Microsomal sample 3  Substrate concentration range'' (uM)  Protein concentration range (u /ml)  Km^ (uM)  Vmax^ (nmol/min/mg)  c  g  BNF-induced  0.125-1  5 - 10  0.50  3.96  Uninduced  0.2 - 0.3  6 0 - 150  0.79  0.10  BNF-induced  0.075 - 1  2 -5  0.27  32.38  Uninduced  0.125-4  30 - 6 0  1.66  0.69  e  Microsomes were prepared from the pooled livers of 4 rats. At substrate concentrations above the stated ranges, there was self-inhibition of dealkylation activities. To measure the enzyme activities at lower substrate concentrations, the protein concentrations were reduced to ensure linearity of product formation with respect to reaction time d The kinetic parameters were estimated from the data shown in Fig. 12 as decribed in Section 3.3.1. The enzyme activity was measured in duplicate at each substrate concentration. In most cases, the estimated kinetic parameters were obtained from a single experiment. Mean of 2 determinations. 3  D  c  e  65  (A) M R O D [Phenacetin] (uM) -•-  o  -  5.5  17  50  150  450  35 ____  o E  c c  30  •  25  •  20  -  'E  E_  >  15 • 10 5 "  o-  2  4  6  10  1/[Substrate] (1/uM)  (B) E R O D [Phenacetin] (uM) 17  50  150  -•-  450  0.80 o E c  0.60 0.40  E  -2  0  2  4  6  10  1 /[Substrate] (1/uM)  Fig. 13. Lineweaver-Burk plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from BNF-induced rats by phenacetin. Substrate concentrations were 1.0, 0.5, 0.25 and 0.125 u.M for both M R O D and EROD assays. Microsomes were prepared from the pooled livers of 4 BNF-induced animals.  66  (A) MROD 120 100  I  80  CO  co o  60  40  20  0  V/ 10  100  1000  Phenacetin concentration (uM)  (B) EROD  Fig. 14. Effect of phenacetin on (A) M R O D and (B) EROD activities in microsomes from BNFinduced rats. Phenacetin concentrations were 0, 5.5, 17, 50, 150 and 450 u M . Substrate concentrations were chosen as multiples of the respective K m for M R O D (0.5 uM) and EROD (0.27 uM) activities. Microsomes were prepared from the pooled livers of 4 BNF-induced animals. Results are expressed as a percent of the respective control activity (i.e. no phenacetin). The control dealkylase activities at substrate concentrations of 1.0, 0.5, 0.25 and 0.125 u M were 1.63, 1.38, 0.87 and 0.52 nmol/min/mg, respectively, M R O D ; and 19.44, 18.30, 14.18 and 9.49 nmol/min/mg, respectively, for EROD.  67  H—  MROD  EROD  120 100  > o  80  (0  c o o  60 40 20 0 YA  *  10  •  •  •  100  1000  P h e n a c e t i n c o n c e n t r a t i o n (uM)  Fig. 15. Comparison of the effect of phenacetin on M R O D and E R O D activities in microsomes from BNF-induced rats. M R O D and EROD assays were performed at substrate concentrations that were equivalent to the respective estimated Km values (i.e. methoxyresorufin concentration = 0.50 uJvl and ethoxyresorufin concentration = 0.27 uM). Microsomes were prepared from the pooled livers of 4 BNF-induced animals. Results are expressed as a percent of the respective control activity (i.e. no phenacetin). Control M R O D and EROD activities were 1.38 and 14.18 nmol/min/mg, respectively.  68  (A) M R O D [Substrate] (uM)  -•-  1  -200  2  0  —A-  4  200  -+-  400  8  600  [Phenacetin] (uM)  (B) E R O D [Substrate] (uM)  [Phenacetin] (uM)  Fig. 16. Dixon plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from BNF-induced rats by phenacetin. Phenacetin concentrations were 0, 5.5, 17,50, 150 and 450 u M . Microsomes were prepared from the pooled livers of 4 BNF-induced animals.  Table 8 Estimated Ki values for inhibition of the microsomal MROD and EROD activites in BNF-induced and uninduced rats by phenacetin  Estimated K i value'  1  (uM) Microsomal sample  Activity  [Phenacetin] range 0 - 450 jiM  [Phenactin] range 0 - 50 u M  BNF-induced  MROD EROD  31 (25 - 34)  31 (25 - 34)  67 ( 3 4 - 6 9 )  71 (56 - 9 4 )  83 (72 - 93)  25 (18 - 3 3 )  189(152 - 2 1 5 )  205 (145 - 3 0 0 )  Uninduced  a  MROD EROD  Phenacetin inhibition studies were performed on each activity at four substrate concentrations. Ki values were estimated from the x-intercepts of the Dixon plots (Fig. 16 and 20), as described in Section 1.1.6.1.1. Ki values were obtained using either the complete range of phenacetin concentrations (0 - 450 uM) or the lowest four concentrations (0 - 50 uM). Shown in the table are the average and range (in parentheses) of Ki values for inhibition of each activity at the various substrate concentrations.  70  (A) M R O D [Phenacetin] (uM)  -2  0  2  4  6  1/[Substrate] (1/uM)  (B) E R O D [Phenacetin] (uM)  -  1  0  1  2  3  1/[Substrate] (1/uM)  Fig. 17. Lineweaver-Burk plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from uninduced rats by phenacetin. Substrate concentrations were 1.6, 0.8, 0.4 and 0.2 u M for the M R O D assay; and 3.2, 1.6, 0.8, and 0.4 u M for the E R O D assay. Protein concentration was lowered to 100 u.g/ml for the EROD assay to ensure linearity of reaction rate with respect to time. Microsomes were prepared from the pooled livers of 4 uninduced animals.  71  (A) M R O D 120 100  I  80  CO  iz c o o  6 0  40 20 o  y/-  10  100  1000  P h e n a c e t i n concentration (uM)  (B) E R O D 120 100  >  TJ  80  CO  2 c o o  60 40 20  o  y/-  10  100  1000  P h e n a c e t i n concentration (uM)  Fig. 18. Effect of phenacetin on (A) MROD and (B) EROD activities in microsomes from uninduced rats. Phenacetin concentrations were 0, 5.5, 17, 50, 150 and 450 u.M. Substrate concentrations were chosen as multiples of the respective Km for MROD (0.79 uM) and EROD (1.66 uM) activities. Microsomes were prepared from the pooled livers of 4 uninduced animals. Protein concentration was lowered to 100 ug/ml for the EROD assay to ensure linearity of reaction rate with respect to time. Results are expressed as a percent of the respective control activity (i.e. no phenacetin). The control MROD activities at substrate concentrations of 1.6, 0.8, 0.4 and 0.2 uM were 0.10, 0.08, 0.06 and 0.04 nmol/min/mg, respectively; and the control EROD activities at substrate concentrations of 3.2, 1.6, 0.8 and 0.4 uM were 0.38, 0.31, 0.23 and 0.13 nmol/min/mg, respectively.  72  MROD  EROD  120  100  80  o  (0  c o u  60  40  20  0 V/  ,1, ,1, I . . L . L ,  10  •  100  1000  P h e n a c e t i n c o n c e n t r a t i o n (uM)  Fig. 19. Comparison of the effect of phenacetin on MROD and EROD activities in microsomes from uninduced rats. MROD and EROD assays were performed at substrate concentrations that were equivalent to the respective estimated Km values (i.e. methoxyresorufin concentration = 0.79 u.M and ethoxyresorufin concentration =1.66 uM). Protein concentration was lowered to 100 u.g/ml for the EROD assay to ensure linearity of reaction rate with respect to time. Microsomes were preparedfromthe pooled livers of 4 uninduced animals. Results are expressed as a percent of the respective control activity (i.e. no phenacetin). Control MROD and EROD activities were 0.08 and 0.31 nmol/min/mg, respectively.  73  (A) M R O D [Substrate] (uM) -•-  0.625  »  -200  1.25  - A - 2.5  0  200  -+—  5  400  600  [Phenacetin) (uM)  (B) E R O D [Substrate] (uM)  E  -400  -200  0  200  400  600  [Phenacetin] (uM)  Fig. 20. Dixon plots of inhibition of (A) M R O D and (B) E R O D activities in microsomes from uninduced rats by phenacetin. Phenacetin concentrations were 0, 5.5, 17, 50, 150 and 450 u M . Protein concentration was lowered to 100 ug/ml for the E R O D assay to ensure linearity of reaction rate with respect to time. Microsomes were prepared from the livers of 4 uninduced animals.  74  3.4.  Immunoinhibition studies In order to investigate the role of a particular cytochrome P450 enzyme in M R O D ,  E R O D , testosterone 2a- and 16a-hydroxylase activities, antibody inhibition studies were performed with polyspecific and monospecific anti-CYP2Cl 1 IgG, polyspecific a n t i - C Y P l A l IgG and M A b C-8.1. The preparation and cross-reactivities of these antibodies have been specified in Section 2.1 under Materials and Methods. The objectives of these experiments were to clarify the contribution of the CYP1A1 and CYP1A2 to microsomal M R O D and E R O D activities in BNFinduced rats, and to determine the role of CYP2C enzymes (in particular CYP2C11) in mediating M R O D and E R O D activities in microsomes from uninduced rats.  Since preimmune IgG was observed to have little stimulatory or inhibitory effect (< 10%) on  M R O D , EROD, testosterone 2a- or 16a-hydroxylase activity in microsomes from B N F -  induced and uninduced rats, results of the antibody inhibition experiments preparations were expressed as a percent of control activity (i.e. no IgG).  3.4.1. Microsomes from BNF-induced rats MROD activity The effects of polyspecific anti-CYPlAl IgG, M A b C-8.1 and polyspecific antiCYP2C11 IgG on M R O D activity in microsomes from BNF-nduced rats are presented in Fig. 21A. At saturating concentrations, polyspecific a n t i - C Y P l A l IgG and M A b C-8.1 inhibited M R O D activity by about 90% and 60%, respectively. The above results indicate that CYP1A1 catalyzes 60% of M R O D activity in microsomes from BNF-induced rats and that the remaining 40% of M R O D activity is likely mediated by CYP1A2.  At the highest concentration, polyspecific anti-CYP2Cll IgG did not inhibit M R O D activity in microsomes from BNF-induced rats (Fig. 21 A), indicating that this antibody preparation does not cross-react with CYP1 A l / 2 .  75  EROD activity The effects of polyspecific anti-CYPlAl IgG, M A b C-8.1 and polyspecific antiCYP2C11 IgG on EROD activity in microsomes from BNF-induced rats are presented in Fig. 21B. At the highest antibody concentrations, the polyspecific anti-CYPl A l IgG inhibited 90% of E R O D activity and M A b C-8.1 inhibition of this activity plateaued at 80%. This suggests that CYP1A1 is responsible for 80% of EROD activity in microsomes from BNF-induced rats. It is likely that the remaining 20% of EROD activity is mediated by CYP1A2  As in the case of M R O D , EROD activity in microsomes from BNF-induced rats was not susceptible to inhibition by polyspecific anti-C YP2C11 IgG at saturating antibody concentrations (Fig. 2IB), indicating that the polyspecific anti-CYP2C11 IgG does not cross-react with CYP1A1/2.  Testosterone 2 a- and 16a-hydroxylase activities In microsomes from uninduced rats, testosterone 2a- and 16a-hydroxylase activities are known to be mediated by CYP2C11 (Waxman, 1984; Waxman et. ai,  1987, Chang et. ai,  1992a). In microsomes from BNF-induced rats, Waxman et. al. (1987) suggested that CYP2C11 mediates testosterone 16a-hydroxylase activity though no data were presented in that study to support their suggestion.  An objective of the present antibody inhibition experiments was to  confirm the contribution of CYP2C11 to microsomal testosterone 2a- and 16a-hydroxylase activities in BNF-induced rats.  At an antibody concentration of 5 mg IgG/nmpl P450, the monospecific anti-CYP2C11 IgG inhibited testosterone 2a- and 16a-hydroxylase activities by 100% and 84%, respectively (Fig. 22). This indicates that these activities continue to be mediated by CYP2C11 after BNFinduction.  76  3.4.2. Microsomes from uninduced rats Results of the antibody inhibition experiments with testosterone 2a- and 16ahydroxylase activities are presented first to facilitate the interpretation of the results of the subsequent antibody inhibition data.  Testosterone 2 a- and 16a-hydroxylase activities Testosterone 2a- and 16a-hydroxylase activities are known to be mediated by CYP2C11 in microsomes from uninduced adult male rats (Waxman, 1984; Waxman et. al, 1987, Chang et. al, 1992a). Antibody inhibition studies were performed with these activities to characterize the inhibitory potency of polyspecific and monospecific anti-CYP2C 11 IgG, and to test the crossreactivity of polyspecific anti-CYPlAl IgG with CYP2C11.  Results of these experiments are  presented in Fig. 23. The polyspecific anti-CYP2Cll IgG inhibited more than 90% of these testosterone hydroxylase activities at concentrations of less than 2.5 mg IgG/nmol P450. At a concentration of 5 mg IgG/nmol P450, the monospecific anti-CYP2Cll  IgG inhibited  testosterone 2a- and 16a-hydroxylase activities by 84% and 89% respectively. The polyspecific a n t i - C Y P l A l IgG had no effect on these activities, indicating that this antibody preparation does not cross-react with CYP2C11.  MROD activity The effects of polyspecific and monospecific anti-CYP2Cll IgG, and polyspecific antiCYP1A1 IgG on M R O D activity in microsomes from uninduced rats are presented in Fig. 24A. M R O D activity was inhibited by approximately 85% by the polyspecific anti-CYP2Cl 1 IgG at the highest antibody concentration (10 mg IgG/nmol P450), indicating that M R O D activity is partly mediated by a CYP2C enzyme(s) or (and) an immunochemically related isozyme(s) in microsomes from uninduced rats. The monospecific anti-CYP2Cll IgG had little or no effect on M R O D activity at concentrations less than 5 mg IgG/nmol P450, suggesting that CYP2C11 is not responsible for this activity in microsomes from uninduced rats. Polyspecific a n t i - C Y P l A l IgG  77  inhibited M R O D activity at both low and high antibody concentrations in a biphasic manner. Taken together, the above results suggest that MROD activity in microsomes from uninduced rats is not mediated by CYP2C11, but possibly by another CYP2C enzyme, with some contribution from CYP1A2.  EROD activity The effects of polyspecific and monospecific anti-CYP2C11 IgG, and polyspecific antiCYP 1A1 IgG on EROD activity in microsomes from uninduced rats are shown in Fig. 24B. Complete inhibition of EROD activity was observed with polyspecific anti-CYP2C11 IgG. At a concentration of 5 mg IgG/nmol P450, the monospecific anti-CYP2C11 IgG had no effect on E R O D activity, but inhibited about 90% of CYP2C11-mediated testosterone 2a-hydroxylase activity in microsomes from uninduced rats (Fig. 24B). These results indicate that E R O D activity is not mediated by CYP2C11. The inhibitory effect observed with monospecific anti-CYP2C11 IgG at concentrations higher than 5 mg IgG/nmol P450 was likely due to cross-reaction of the antibody with other cytochrome P450 enzyme(s).  Inhibition of EROD activity was also observed with polyspecific anti-CYPlAl IgG at higher antibody concentrations.  Since there is very little CYP1A1 present in microsomes from  uninduced rats, the observed inhibition of E R O D activity by polyspecific anti-CYP 1A1 IgG may have been due to inhibition of CYPlA2-mediated enzyme activity and/or cross-reaction with some other cytochrome(s) P450.  The above results suggest that EROD activity in microsomes from uninduced rats is not mediated by CYP2C11, but possibly by another CYP2C enzyme(s) or an immunochemically related enzyme(s).  They also suggest that CYP1A2 is not a major contributor to microsomal  E R O D activity in these animals.  78  (B) E R O D  mg IgG / nmol P450  Fig. 21. Effect of preimmune IgG, polyspecific anti-CYP2Clland a n t i - C Y P l A l IgG, and M A b C-8.1 on (A) M R O D and (B) EROD activities in microsomes prepared from the pooled livers of 4 BNF-induced rats. Results are expressed as a percent of the corresponding control activity (i.e. no IgG). The mean (± standard deviation) control M R O D and EROD activities determined from 3 experiments were 1.58 (± 0.17) and 20.33 (± 1.88) nmol/min/mg , respectively.  79  (A) 2A-Hydroxylase J 120  Preimmune IgG  -Q  Monospecific anti-CYP2C11 IgG  100  to 4=  c o u  60  40  20  0  V/0.4  mg IgG / nmol P450  (B) 16A-Hydroxylase Preimmune IgG  120  -Q  Monospecific anti-CYP2C11 IgG  100 ><  I  80  ro £  60  —  40  c o o  20  oV/-  10  0.4  mg IgG / nmol P450  Fig. 22. Effect of preimmune IgG and monospecific anti-CYP2C11 IgG on testosterone (A) 2ahydroxylase and (B) 16a-hydroxylase activities in microsomes prepared from the pooled livers of 4 BNF-induced rats. Results are expressed as a percent of the corresponding control activity (i.e. no IgG). Control testosterone 2a- and 16a-hydroxylase activities were 0.91 and 1.42 nmol/min/mg, respectively.  80 (A) 2A-Hydroxylase  mg IgG / nmol P450  (B) 16A-Hydroxylase  mg IgG / nmol P450  Fig. 23. Effect of preimmune IgG, polyspecific and monospecific anti-CYP2Cll IgG and polyspecific anti-CYPlAl IgG on testosterone (A) 2a-hydroxylase and (B) 16a-hydroxylase activities in microsomes prepared from the pooled livers of 4 uninduced rats. Rsesults are expressed as a percent of the corresponding control activity (i.e. no IgG). The mean (+ standard deviation) control testosterone 2a- and 16a-hydroxylase activities determined from 4 experiments were 1.91 (+ 0.37) and 2.16 (+ 0.82) nmol/min/mg, respectively.  81 (A)  MROD  mg IgG / nmol P450  (B)  EROD  mg IgG / nmol P450  Fig. 24. Effect of preimmune IgG, polyspecific anti-CYP 1 A l , monospecific and polyspecific antiCYP2C11 IgG on (A) M R O D and (B) EROD activities in microsomes prepared from the pooled livers of 4 uninduced rats. Results are expressed as a percent of the corresponding control activity (i.e. no IgG). The mean (+ standard deviation) control M R O D and E R O D activities determined from 4 experiments were 0.07 (± 0.002) and 0.35 (+ 0.01) nmol/min/mg, respectively. (Single measurements were performed on M R O D activity using the monospecific anti-CYP2C11 antibody.)  82 3.5. In vitro inhibition by cimetidine of MROD, EROD, testosterone 2a- and 16ahydroxylase ativities Results of the above sections showed that in vivo cimetidine had no effect on CYPlAl/2-mediated M R O D or EROD activity in microsomes from BNF-induced rats, but inhibited CYP2C-mediated (not CYP2C11) EROD activity.  Studies were conducted to  determine whether in vitro cimetidine, in the absence or presence of a preincubation step, would affect these activities in a manner similar to in vivo cimetidine.  3.5.1. Without preincubation 3.5.1.1. Microsomes from BNF-induced rats In microsomes from BNF-induced rats, in vitro cimetidine inhibited M R O D (substrate concentration = 3 u.M), EROD, testosterone 2a- and 16a-hydroxylase activities, with ICso values ranging from 0.25 - 4 mM. (Fig. 25, Table 9).  This suggests that, in the absence of a  preincubation step, in vitro cimetidine inhibits CYP1A1/2 and CYP2C11. This is in contrast to the observed lack of inhibition of CYPlAl/2-mediated M R O D and E R O D activities by in vivo cimetidine (Fig. 8), but is consistent with the observed inhibition of CYP2C11-mediated testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats by in vivo cimetidine (Fig. 9).  3.5.1.2. Microsomes from uninduced rats In vitro cimetidine inhibited M R O D and EROD activities in microsomes from uninduced rats with ICso values of 1 m M and 0.25 mM, respectively (Fig. 26, Table 9). This indicates that, in the absence of a preincubation step, in vitro cimetidine inhibits the cytochrome P450 enzyme(s) that mediates these activities in microsomes from uninduced rats.  E R O D activity was also  inhibited by in vivo cimetidine but it was not clear whether there was an inhibitory effect on M R O D activity (Fig. 1 IB, Section 3.2.2.2).  83  3.5.2. With preincubation In the absence of a preincubation step, the effects of in vitro cimetidine on the various rat hepatic microsomal activities did not agree with those of in vivo cimetidine (Section 3.5.1). There has been a discrepancy in the literature regarding the selectivity and inhibitory potency of in vivo and in vitro cimetidine (Sections 1.2.2 and 1.2.3). Recently, Chang et. al. (1992b) provided evidence in their studies to suggest that in vivo cimetidine inhibits cytochrome P450-mediated activities by a catalysis-dependent process. By preincubating microsomes in vitro with low doses of cimetidine and N A D P H for 15 min at 37°C, these authors could produce selective inhibition of cytochrome P450 enzyme activities similar to that seen in vivo. Preincubation studies were therefore performed in the present investigation to determine whether this would result in enhancement of the selectivity and potency of the inhibitory effect of cimetidine.  In humans, cimetidine inhibits theophylline metabolism (Grygiel et. al, 1984; Cusack et. al, 1985) and there is indirect evidence to suggest that the metabolism of theophylline is partly mediated by CYP1A2 (Sarkar et. al, 1992). As there is also evidence that E R O D activity is largely mediated by CYP1A2 in human microsomes (Murray et. al, 1993), preincubation studies were performed to determine whether preincubation of low concentrations of cimetidine in vitro with human microsomes and N A D P H would increase the potency of its inhibition of E R O D activity. If so, this would provide evidence that cimetidine acts as a catalysis-dependent inhibitor of human CYP1A2. The experimental protocol for preincubation studies with human microsomes was the same as that described for rat microsomes (Section 2.12.2).  3.5.2.1. Microsomes from BNF-induced rats MROD and EROD activities Preincubation of microsomes in vitro with cimetidine and N A D P H did not increase the potency of its inhibition of M R O D or EROD  activity in microsomes from BNF-induced rats  84  (Fig. 27). This is consistent with the observed lack of inhibition of these activities in microsomes from BNF-induced rats by in vivo cimetidine (Fig. 8).  Testosterone 2 a- and 16a-hydroxylase activities Preincubation of microsomes in vitro with cimetidine and N A D P H increased the potency of its inhibition of testosterone 2a- and 16a-hydroxylase activities by more than 40-fold (Fig. 28). This is consistent with the observed inhibition of these activities in microsomes from BNF-induced rats by in vivo cimetidine (Fig. 9). At a cimetidine concentration of 0.05 mM, these hydroxylase activities were 25 - 30% lower in microsomes preincubated with cimetidine and N A D P H than in those preincubated with N A D P H alone.  Because microsomal testosterone 2a- and 16a-  hydroxylase activities have been shown to be mediated by CYP2C11 in uninduced rats (Chang et. al., 1992a) and are mediated by the same enzyme in BNF-induced rats (Fig. 22), results of the present study are consistent with the reported inhibition of these microsomal testosterone hydroxylase activities in uninduced rats under similar conditions (Chang et. al, 1992b).  3.5.2.2. Microsomes from uninduced rats MROD activity Preincubation of microsomes from uninduced rats in vitro with cimetidine and N A D P H did not affect the potency of its inhibition of M R O D activity (Fig. 29A). This is consistent with the apparent lack of inhibition of M R O D activity observed in microsomes from uninduced rats by in vivo cimetidine (Fig. 11B, Section 3.2.2.2).  EROD activity Preincubation of cimetidine with microsomes and N A D P H increased the potency of its inhibition of E R O D actiivty 16-fold (Fig. 29B). For example, at a cimetidine concentration of 0.05 mM, EROD activity was inhibited by 66% when cimetidine was preincubated with microsomes and N A D P H , but by only 6% when cimetidine was added after preincubation of  85  microsomes with N A D P H . This is consistent with the observed inhibition of E R O D activity by in vivo cimetidine in microsomes from uninduced rats (Fig. 1 IB, Section 3.2.2.2).  The above experiments were performed with pooled liver microsomes.  Additional  experiments were then performed with microsomes from individual uninduced rats to confirm that there was a significant increase in the inhibitory effect of cimetidine after preincubation with N A D P H . A single cimetidine concentration of 0.05 mM was used in the experiment. As shown in Fig. 30, E R O D activity was 53% lower (p < 0.001) in microsomes preincubated with cimetidine in the presence of N A D P H than in those preincubated with N A D P H alone and with cimetidine added after preincubation.  3.5.2.3. Human liver microsomes Results of the preincubation studies are shown in Fig. 31. When cimetidine was added in vitro after preincubation and prior to initiation of the reaction with substrate addition, it inhibited E R O D activity by approximately 13% at a cimetidine concentration of 1 m M and this degree of inhibition is similar to that observed with in vitro cimetidine in the absence of a preincubation step (Knodell et. al,  1991).  Preincubation of human microsomes with low concentrations of  cimetidine (up to 1 mM) and N A D P H did not increase the potency of its inhibition of E R O D activity.  86  —I—  MROD  — e —  EROD  — * —  2A-  — 1 6 A -  Hydroxylase  Hydroxylase  120  100  80  60  40  20  0  -i  v/—  0.02  i  0.1  Cimetidine concentration  10  20  (mM)  Fig. 25. Effect of in vitro cimetidine (in the absence of a preincubation step) on M R O D (substrate concentration = 3 uM), EROD, testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats. Cimetidine was added in vitro immediately prior to initiation of substrate oxidation with N A D P H addition. Microsomes were prepared from the pooled livers of 4 BNF-induced animals. Results are expressed as a percent of the respective control activity (i.e. no cimetidine). Control M R O D and EROD, testosterone 2a- and 16a-hydroxylase activities were 1.94, 19.19, 1.44 and 1.77 nmol/min/mg, respectively.  Table 9 IC values for the inhibition of the various activities by in vitro cimetidine. S0  Microsomal sample  BNF-induced  Uninduced  3  b  c  ICso value  Subsrate concentration (uM)  Km value in the absence of cimetidine (uM)  cimetidine (mM)  MROD EROD Testosterone 2aand 16a-hydroxylase  1 1 250  0.50 0.27 90  1 2 4  MROD EROD  3 5  0.79 1.61  1 0.25  Activity  b  b  c  b  b  of in vitro 3  ICso values were determined from graphically from the inhibition curves shown in Fig. 25 and 26 . Apparent Km values were estimated from the enzyme kinetic studies performed in the present investigation (See Table 7) Chang et. ai, 1992b  88  H —  -B—  MROD  EROD  120  >  100  •*—•  CO  80  O »-  O o  60 40  20  •  0 0.02  •  •  •  1  '  10  0.1  Cimetidine concentration  20  (mM)  Fig. 26. Effect of in vitro cimetidine (in the absence of a precubation step) on M R O D and EROD activities in microsomes from uninduced rats. Cimetidine was added in vitro immediately prior to initiation of substrate oxidation with N A D P H addition. Microsomes were prepared from the pooled livers of 4 uninduced animals. Results are expressed as a percent of the respective control activity (i.e. no cimetidine). Control M R O D and EROD activities were 0.09 and 0.38 nmol/min/mg, respectively.  89  (A) M R O D  o  V/  0.04  0.1 Cimetidine concentration (mM)  (B) E R O D  o  V/ 0.04  1  0.1 Cimetidine concentration (mM)  Fig. 27. Effect of preincubation with cimetidine in the presence of N A D P H on (A) M R O D and (B) E R O D activities in microsomes from BNF-induced rats. Microsomes were preincubated with cimetidine in the presence of N A D P H for 15 min, prior to initation of reaction with substrate addition ( — Q — ) . In the controls, microsomes were preincubated with N A D P H alone for 15 min, followed by the addition of cimetidine and initiation of reaction with N A D P H addition (—•—). Microsomes were prepared from the pooled livers of 4 BNF-induced animals. Results are expressed as a percent of the respective control activity (i.e. no cimetidine). Control M R O D and E R O D activities were 1.25 and 16.87 nmol/min/mg, respectively.  90  (A) 2A-Hydroxylase  i YA 0.02  •—•—• 0.1 Cimetidine concentration (mM)  i TS  0.02  1  1  —  1  0.1 Cimetidine concentration (mM)  Fig. 28. Effect of preincubation with cimetidine in the presence of N A D P H on testosterone (A) 2a-hydroxylase and (B) 16a-hydroxylase activties in microsomes from BNF-induced rats. Microsomes were preincubated with cimetidine in the presence of N A D P H for 15 min, prior to initation of reaction with substrate addition (—Q ). In the controls, microsomes were preincubated with N A D P H alone for 15 min, followed by the addition of cimetidine and initiation of reaction with N A D P H addition ( • ). Microsomes were prepared from the pooled livers of 4 BNF-induced animals. Results are expressed as a percent of the respective control activity (i.e. no cimetidine). Control testosterone 2a-hydroxylase and 16a-hydroxylase activities were 1.31 and 1.67 nmol/min/mg, respectively.  91  (A) M R O D  V/  0.04  •  • • • •  1  0.1 Cimetidine concentration (mM)  (B) E R O D  Cimetidine concentration (mM)  Fig. 29. Effect of preincubation with cimetidine in the presence of N A D P H on (A) M R O D and (B) E R O D activties in microsomes from uninduced rats. Microsomes were preincubated with cimetidine in the presence of N A D P H for 15 min, prior to initation of reaction with substrate addition ( Q — ) . In the controls, microsomes were preincubated with N A D P H alone for 15 min, followed by the addition of cimetidine and initiation of reaction with N A D P H addition (—•—). Microsomes were prepared from the pooled livers of 4 uninduced animals. Results are expressed as a percent of the respective control activity (i.e. no cimetidine). Control M R O D and E R O D activities were 0.05 and 0.22 nmol/min/mg, respectively.  92  0.30 r  cn E  0.24 h  (A) Control  (B) Experimental  Fig. 30. Effect of preincubation with cimetidine in the presence of N A D P H on E R O D activities in microsomes from individual uninduced rats. In the control group (A), microsomes were preincubated with N A D P H for 15 min and cimetidine (0.05 mM) was added afterwards. In the experimental group (B), microsomes were preincubated with cimetidine (0.05 mM) in the presence of N A D P H for 15 min. Results are expressed as the mean + standard deviation (N = 4). *P < 0.001, compared to the control group (one-tailed, paired t-test).  93  o V/-  0.02  •— — 1  0.1 Cimetidine concentration  (mM)  Fig 31. Effect of preincubation with cimetidine in the presence of N A D P H on E R O D activity in human liver microsomes. Microsomes were preincubated with cimetidine in the presence of N A D P H for 15 min, prior to initation of reaction with substrate addition ( — Q — ) . In the controls, microsomes were preincubated with N A D P H alone for 15 min, followed by the addition of cimetidine and initiation of reaction with N A D P H addition ( • — ) . Microsomes were pooled from the liver samples of 3 nonsmokers. Results are expressed as a percent of control activity (i.e. no cimetidine). Control EROD activity was 0.07 nmol/min/mg. E R O D activiy was determinated by a single measurement.  94  3.6. Summary of results Results of studies with in vivo and in vitro cimetidine, phenacetin inhibition and immunoinhibition experiments in male rats are summarized below.  In microsomes from BNF-induced rats, in vivo cimetidine had no effect on M R O D or E R O D activity (Fig. 8), but inhibited testosterone 2a- and 16a-hydroxylase activities (Fig. 9). Results of immunoinhibition studies in microsomes from BNF-induced rats suggest that CYP1A1 is responsible for 60% of M R O D and 80% of EROD activity, and that CYP1A2 accounts for only about 40% of M R O D and 20% of EROD activity (Fig. 21). This was consistent with results of phenacetin inhibition experiments (Fig. 15).  In the same microsomes, results of antibody  inhibition experiments showed that testosterone 2a- and 16a-hydroxylase activities were mediated by CYP2C11 (Fig. 22). In the absence of a preincubation step, in vitro cimetidine inhibited all these activities with ICso values ranging from 1 - 4 m M (Table 9). Preincubation of microsomes from BNF-induced rats in vitro with cimetidine and N A D P H did not increase the potency of its inhibition of M R O D or EROD activity (Fig. 27) whereas the potency of its inhibition of testosterone 2a- and 16a-hydroxylase activities was increased 40-fold (Fig. 28).  In microsomes from uninduced rats, in vivo cimetidine inhibited E R O D activity and appeared not to have an effect on M R O D activity (Fig. 11B, Section 3.2.2.2).  Results of  phenacetin inhibition studies indicated that CYP1A2 contributes to M R O D but not E R O D activity (Fig. 19).  Results of immunoinhibition studies suggest that, in uninduced rats, microsomal  M R O D and E R O D activities are possibly mediated by a CYP2C isozyme(s) other than CYP2C11 (Fig. 24). In the absence of a preincubation step, in vitro cimetidine inhibited these activities with ICso values ranging from 0.25 - 1 mM (Table 9). Preincubation of microsomes from uninduced rats in vitro with cimetidine and N A D P H did not increase the potency of its inhibition of M R O D activity whereas the potency on its inhibition of EROD activity was increased 16-fold (Fig. 29).  95  Preincubation of human liver microsomes in vitro with low concentrations of cimetidine and N A D P H did not enhance the potency of its inhibition of EROD activity (Fig. 31).  96  Discussion  In adult male rats, in vivo administration of cimetidine has been shown to result in inhibition of the catalytic activities of CYP2C11 and other unidentified cytochrome(s) P450 (Chang et. al, 1992a). In humans, it has not yet been determined which cytochromes P450 are inhibited by cimetidine. Since there is indirect evidence to suggest that in vivo cimetidine inhibits CYP1A1/2 in rats (Chang, 1991; Kelley et. al, 1987; Nakajima et. al, 1990) and CYP1A2 in humans (Sarkar et. al, 1992; Grygiel et. al, 1984; Cusack et. al, 1985), the present investigation was undertaken to determine the effect of cimetidine on these enzymes. It was proposed in the current study that CYP1A2 and CYP1A1 play a role in microsomal M R O D and E R O D activities in BNF-induced rats, and that CYP1A2 and CYP2C enzymes are responsible for these activities in uninduced rats. Different approaches were used to investigate the contribution of C Y P 1 A and C Y P 2 C enzymes to M R O D and E R O D activities, including induction, chemical inhibition and immunoinhibition studies. The effects of in vivo and in vitro cimetidine on these activities were examined to determine whether cimetidine inhibits CYP1A2 in addition to CYP2C11.  4.1.  Role of CYP1A2 and CYP1A1 in microsomal MROD and EROD activities  4.1.1. Microsomes from BNF-induced rats There is evidence that CYP1A2 contributes to M R O D activity and that CYP1A1 contributes to E R O D activity in samples from rats pretreated with H C B , a CYP1A1/2 inducer (Nerukar et. al, 1993). In the present investigation, B N F was used to pretreat the animals for induction of CYP1A1/2 enzymes and experiments were performed to investigate the contribution of CYP1 A l and CYP1A2 to M R O D and EROD activities in microsomes from these rats.  4.1.1.1. Induction studies B N F pretreatment resulted in a 70% increase (p < 0.005) of the total hepatic microsomal cytochrome P450 (Table 6).  This is consistent with an earlier report that the total hepatic  97  microsomal cytochrome P450 was increased by 130% after B N F pretreatment, with CYP1 A l and CYP1A2 accounting for 71% and 24% of the total P450, respectively (Thomas et. al, 1983).  After pretreatment of rats with BNF, microsomal M R O D and E R O D activities were induced by 40- and 50-fold, respectively (p < 0.001) (Table 5). The effects of pretreatment with a number of CYP1A1/2 inducers on these alkoxyresorufin O-dealkylase activities have been evaluated in previous induction studies.  In microsomes from BNF-induced rats, M R O D and  E R O D activities were 80- and 100-fold higher, respectively, than the corresponding control activities (Burke et. al, 1985). In microsomes from 3-MC-induced rats, these activities were both induced by approximately 60-fold (Burke et. al, 1985). In S-9 fractions from HCB-induced rats, M R O D and EROD activities were 20- and 47-fold higher, respectively, than the corresponding control activities (Nerukar et. al, 1993).  Results of these previous induction  studies are in general agreement with the induction of M R O D and E R O D activities observed in the present investigation. Previous immunoinhibition studies have also shown that CYP1A2 is responsible for M R O D activity in HCB-induced rats, and that CYP1A1 accounts for 75% of E R O D activity in H C B and 3-MC induced rats (Kelley et. al, 1987; Nerukar et. al, 1993). Because CYP1A2 and CYP1A1 are the major enzymes induced by B N F (Thomas et. al, 1983) and both of these purified enzymes are capable of metabolizing methoxyresorufin and ethoxyresorufin (Burke et. al, 1994), the degree of induction of M R O D and E R O D activities obtained in the present study suggests that these activities are mainly due to CYP1A2 and/or CYP1A1, but does not indicate the degree to which these C Y P 1 A enzymes contribute to each activity.  4.1.1.2. Phenacetin inhibition studies To examine the possible role of CYP1A2 in microsomal M R O D and E R O D activities, inhibition studies were then performed using phenacetin as a chemical inhibitor. In microsomes from BNF-induced rats, phenacetin, at low concentrations, inhibited both M R O D and EROD  98  activities (Fig. 14), but it was a more potent inhibitor of M R O D activity (Fig. 15). The average K i values of phenacetin (estimated using the complete range of phenacetin concentrations) for inhibition of M R O D and EROD activities were 31 and 67 u M , respectively (Table 8). High affinity phenacetin O-deethylase has been shown to be mediated by CYP1A2 in microsomes from 3-MC-induced rats (Sesardic et. al, 1990). Since both 3-MC and B N F are CYP1A1/2 inducers and the relative amount of microsomal CYP1A1 and CYP1A2 induced by these chemicals is similar (Thomas et. al,  1983a), it is likely that high affinity phenacetin O-deethylase in  microsomes from BNF-induced rats is also mediated by CYP1A2.  If so, the observation of  competitive inhibition of M R O D and EROD activities by low concentrations of phenacetin in microsomes from BNF-induced rats in the present investigation (Fig. 13) suggests that CYP1A2 plays a role in both activities. The observation that M R O D activity was more sensitive to inhibition by phenacetin than EROD activity (Fig. 15) suggests that CYP1A2 plays a greater role in M R O D activity in microsomes from BNF-induced rats.  Nerukar et. al. (1993) have previously examined the effect of phenacetin on M R O D and E R O D activities in S-9 samples from HCB-induced rats. In their inhibition studies, the phenacetin concentrations were 0, 5.5, 17, 50, 150 and 450 uM, and the substrate concentrations were 5, 1.7 and 0.5 u M for both M R O D and EROD activities.  The protein concentrations used for  measuring M R O D and E R O D activities were 25-100 ug/ml and the reaction time was 5 min. The authors of that study compared the inhibition of M R O D and EROD activities by phenacetin at the same substrate concentrations, without taking into account the fact that the affinity of the enzyme for the substrate will have an effect on the observed inhibition.  Another problem with their  inhibition protocol was the possible self-inhibition of M R O D and E R O D activities at substrate concentrations above 1 u M , which has been observed at these substrate concentrations in the present investigation (Fig. 12A and B). This self-inhibition phenomenon has been previously observed and was proposed to be due to product and/or substrate inhibition of the cytochrome P450 involved in the microsomal metabolism of ethoxyresorufin in BNF-induced rats (Pohl and  99  Fouts, 1980). Thus, if self-inhibition of M R O D and EROD activities also occurs in S-9 fractions from HCB-induced rats, this may have interfered with the inhibition of the O-deethylation of ethoxyresorufin by phenacetin and may partly explain the lack of effect of phenacetin on EROD activity reported by Nerukar et. al. (1993). Moreover, no indication was given by these authors whether the conditions used would result in a linear reaction rate with respect to protein concentration and reaction time. Underestimation of the control activity due to nonlinearity of reaction rates could actually have led to underestimation of inhibition, which was expressed as percent of the corresponding control activity.  This is particularly true when the substrate  concentrations were non-saturating (e.g. 0.5 uM).  Because of these potential limitations, the  experimental protocol employed for phenacetin inhibition studies in the present investigation was modified. The phenacetin concentrations were the same as those used in the previous studies. To take into consideration the effects of the affinities of the enzymes for the substrates on the observed inhibition, enzyme kinetic studies were performed to estimate the K m of M R O D and E R O D activities (Fig. 12A and B , Table 7). Phenacetin inhibition studies were performed on these activities at substrate concentrations that were equivalent to multiples of the respective Km. The effects of phenacetin on M R O D and EROD activities could then be compared at substrate concentrations that were equivalent to the same multiple of their respective Km. The substrate concentrations used in the present phenacetin inhibition studies were 1, 0.5, 0.25 and 0.125 u M for both M R O D and E R O D activities in microsomes from BNF-induced rats, and no selfinhibition of either activity was observed at these substrate concentrations. Finally, optimization experiments were performed on M R O D and EROD activities in microsomes from BNF-induced rats at substrate concentrations of 0.5 u M . The final protein concentration and reaction time were 10 ug/ml and 2 min, respectively, for M R O D activity; and 5 ug/ml and 1 min, respectively, for E R O D activity. These assay conditions were used at all other substrate concentrations as well.  100  Because B N F and H C B are both CYP1 A l / 2 inducers (Thomas et. al, 1983; Parkinson et. al,  1983), results of the phenacetin inhibition experiments from the present study can be  compared to those of Nerukar et. al. (1993). The present observation that low concentrations of phenacetin inhibited M R O D activity (Fig. 14A) is consistent with the findings of Nerukar et. al. (1993). At a substrate concentration of 0.5 u M , Nerukar et. al. (1993) reported that the K i of phenacetin for M R O D activity was ~ 20 u M , which was similar to that obtained in the current study (Ki ~ 34 u M , Table 8). Nerukar et. al. (1993) also obtained evidence to suggest that M R O D activity is mediated mainly by CYP1A2 in S-9 fractions from HCB-induced rats. If phenacetin, at low concentrations, is an inhibitor of CYP1A2 in samples from BNF-induced rats, then the similarity in the effect of phenacetin on M R O D activity in these samples supports the inference that CYP1A2 is involved in the microsomal metabolism of methoxyresorufin in BNFinduced rats. However, as B N F preferentially induces CYP1A1 and H C B preferentially induces CYP1A2 (Thomas et. al, 1983a, Parkinson et. al, 1983), it is not certain whether CYP1A2 contributes to M R O D activity in BNF-induced rats to the same extent as in HCB-induced rats.  Nerukar et. al. (1993) also reported that low concentrations of phenacetin had no effect on E R O D activity in S-9 fractions from HCB-induced rats (Ki > 333 u M at an ethoxyresorufin concentration of 0.5 uM). This is in contrast to the current observation that low concentrations of phenacetin inhibited microsomal EROD activity in BNF-induced rats (Fig. 14B) with a K i value of ~ 64 u.M at a substrate concentration of 0.5 u M (Table 8).  If phenacetin, at low  concentrations, is a specific alternate substrate inhibitor of CYP1A2 in samples from B N F - or HCB-induced rats, then its differential effect on EROD activity in these samples suggests that CYP1A2 plays a greater role in microsomal EROD activity in BNF-induced rats. This will be considered in further detail when results of the immunoinhibition studies are discussed in the following section.  101  4.1.1.3. Immunoinhibition studies To quantitate the contribution of CYP1A1 to M R O D and E R O D activities in microsomes from BNF-induced rats, immunoinhibition studies were performed using polyspecific antiC Y P 1A1 IgG and M A b C-8.1. Polyspecific anti-CYP 1A1 IgG, directed against CYP1A1 but cross-reactive with CYP1A2 (personal communication with Dr. S. Bandiera), inhibited about 90% of both M R O D and EROD activities (Fig. 21). This indicates that these alkoxyresorufin Odealkylase activities are mediated by CYP1A1/2 and is consistent with results of the induction studies that these activities are highly induced after pretreatment of the animals with the CYP1A1/2 inducer, B N F (Table 6). M A b C-8.1, is a monoclonal antibody that recognizes and inhibits CYP1A1 but not CYP1A2 (Thomas et. al., 1984).  At saturating concentrations, this  antibody inhibited 60% of M R O D activity and 80% of EROD activity in microsomes from BNFinduced rats (Fig. 21). This indicates that CYP1 A l accounts for 60% of M R O D activity and 80% of E R O D activity and suggests that CYP1A2 accounts for as much as 40% and 20% of the remaining M R O D and EROD activities, respectively.  This is consistent with results of the  phenacetin inhibition studies from the current investigation, which indicated that CYP1A2 plays a greater role in M R O D than in EROD activity in microsomes from BNF-induced rats (see Section 4.1.1.2).  Nerukar et. al. (1993) performed immunoinhibition studies with monoclonal antibody 7-1-1 (cross-reactive with both CYP1A1 and CYP1A2) and M A b C-8 (equivalent to M A b C-8.1) to determine the possible roles of CYP1A1 and CYP1A2 in M R O D and E R O D activities in S-9 fractions from HCB-induced rats. Monoclonal antibody 7-1-1 inhibited both activities, whereas, at saturating concentrations, M A b C-8 inhibited M R O D activity by < 2% and E R O D activity by about 75%. Taken together with the results of their induction and phenacetin inhibition studies, these authors concluded that in S-9 fractions from HCB-induced rats, CYP1A2 and CYP1A1 appeared to preferentially metabolize methoxyresorufin and ethoxyresorufin, respectively.  102  The observation from the present investigation that CYP1A2 appears to account for 40% of microsomal M R O D activity in BNF-induced rats differs from results of Nerukar et. al. (1993) in S-9 fractions from HCB-induced rats that M R O D activity is relatively selective for CYP1A2. The difference between these two observations may partly reflect the fact that B N F and H C B are different in their relative induction of CYP1A1 and CYP1A2.  Previous investigators have  examined the capacities of BNF, 3-MC and H C B in inducing CYP1A1 and CYP1A2 in mature male rats. Thomas et. al. (1983) have shown that B N F is similar to 3-MC as an inducer and CYP1A1 is the major cytochrome P450 induced. After induction of rats by B N F or 3-MC, they found that the relative amount of CYP1A1 and CYP1A2 present in hepatic microsomes was in the ratio of 3 to 1.  In contrast, Parkinson et. al (1983) found that CYP1A2 is the major  cytochrome P450 induced by HCB. After induction, the ratio of microsomal content of CYP1 A l to CYP1A2 was 1 to 2.  Purified CYP1A1 and CYP1A2 are both capable of metabolizing  methoxyresorufin while the capacity of CYP1A2 is 2 - 3 times greater than that of CYP1A1 (Burke et. al, 1994). Thus, the relative contribution of these enzymes to M R O D activity after induction by a CYP1A1/2 inducer may partly depend on the relative amount of CYP1A1 and CYP1A2 present. It is possible that when CYP1A1 is present in excess of CYP1A2 as in the case of BNF-induced rats, both CYP1A1 and CYP1A2 can contribute to microsomal M R O D activity. On the other hand, when CYP1A2 is present in excess of CYP1A1 as in the case of HCB-induced rats, CYP1A2 may predominate in catalyzing methoxyresorufin O-demethylation.  Recently, Burke et. al. (1994) concluded that, in 3-MC-induced rats, microsomal M R O D activity is mediated by CYP1A2. They based their conclusion on the finding that 75% of M R O D activity in these animals was inhibited by furafylline.  Their interpretation of the results was  supported by the previous findings of Seasardic et. al. (1990) that furafylline preferentially inhibits CYP1A2 relative to CYP1 A l in 3-MC-induced rats. The finding of Burke et. al. (1994) appears to be in conflict with the present observation that CYP1A2 appears to account for only about 40% of the M R O D activity in BNF-induced rats. In view of the similarity between 3-MC and  103  BNF as CYP1A1/2 inducers, it is questionable whether CYP1A2 can mediate most of the MROD activity in microsomes from 3-MC induced rats. The discrepancy in the role of CYP1A2 in catalyzing MROD activity in 3-MC- and BNF-induced rats can be resolved with future immunoinhibition studies performed on microsomal MROD activity in rats, pretreated with these compounds.  In the present investigation, it was observed that CYP1A1 accounted for 80% of the EROD activity in microsomes from BNF-induced rats (Fig. 2 IB). This is in agreement with the results of previous immunoinhibition studies that EROD activity is mainly due to CYP1A1 in microsomes from 3-MC induced rats (Kelley et. al, 1987) and S-9 fractions from HCB-induced rats (Nerukar et. al, 1993). As mentioned previously, CYP1A1 and CYP1A2 are the major cytochromes P450 induced by BNF and HCB, respectively (Thomas et. al, 1983; Parkinson et. al, 1983). Although CYP1A2 is present in excess of CYP1A1 in microsomes from HCB-induced rats, the capacity of purified CYP1A1 is 9-fold greater than that of CYP1A2 in catalyzing ethoxyresorufin O-deethylation (Burke et. al, 1994). This may explain the fact that CYP1A1 accounts for most of the EROD activity in samples from HCB-induced rats.  As discussed in section 4.1.1.2, it was observed in the present investigation that EROD activity in microsomes from BNF-induced rats was inhibited by low concentrations of phenacetin at a substrate concentration of 0.5 uM (Ki ~ 64 uM) (Fig. 14b, Table 8), whereas in the study of Nerukar et. al. (1993), low concentrations of phenacetin were found to have no effect (Ki > 333 uM) on the same activity in S-9 fractions from HCB-induced rats at the same substrate concentration. The differential effect of phenacetin on EROD activity in BNF- and HCB-treated samples, as indicated by a 5-fold difference in the estimated Ki, suggests that CYP1A2 plays a greater role in EROD activity in BNF-treated samples. However, results of the immunoinhibition studies from the present study and that of Nerukar et. al. (1993) indicate that the same enzyme, CYP1A1, is responsible for most of the EROD activity in microsomes from BNF-induced rats  104  (~ 80%) (Fig. 21B) and in S-9 fractions from HCB-induced rats (~ 75%).  It is not fully  understood why there is a discrepancy in the effect of phenacetin on E R O D activity in B N F treated and HCB-treated samples.  One of the possible explanations is that different assay  conditions were used in the two studies. In the present investigation, the assay conditions were optimized at substrate concentrations of 0.5 u M and the control activities were measured correctly.  In the studies by Nerukar et. al. (1993), it not known whether experiments were  performed to optimize the EROD assay at a substrate concentration of 0.5 u M . If not, it could have led to incorrect measurement of the control activity and underestimation of inhibition, as explained in Section 4.1.1.2.  4.1.2. Microsomes from uninduced rats In vivo cimetidine has been observed to inhibit EROD activity in microsomes from uninduced rats by 85% (Chang, 1991) and this finding was verified again in the present investigation (Fig. 11B). However, there has been a discrepancy in the literature as to whether CYP1A2 (Kelley et. al, 1987) or CYP2C11 (Nakajima et. al, 1990) mediates this activity in uninduced rats. In the present investigation, chemical inhibition and immunoinhibition studies were performed to clarify the contribution of CYP1A2 and CYP2C11 to E R O D activity in microsomes from uninduced rats. With regard to M R O D activity, it has not previously been determined which cytochrome P450 is responsible for this activity in microsomes from uninduced rats. Because of the availability of phenacetin as a CYP1A2 inhibitor and the anti-CYP2Cll antibodies, phenacetin inhibition and immunoinhibition studies were performed to determine whether CYP1A2 or CYP2C11 is responsible for microsomal M R O D activity in uninduced rats.  4.1.2.1. Phenacetin inhibition studies Phenacetin inhibition studies were performed on M R O D and E R O D activities at substrate concentrations that were multiples of their respective estimated K m values.  Preliminary  optimization experiments were performed to verify that the reaction rates were linear with respect  105  to the protein concentration and reaction time at each substrate concentration. The effects of phenacetin on M R O D and EROD activity were then compared at substrate concentrations that were equivalent to their respective estimated Km values.  Low concentrations of phenacetin were found to inhibit M R O D activity in microsomes from uninduced rats (Fig. 18A). Dixon plots of phenacetin inhibition of M R O D  activity  appeared to deviate from linearity at high phenacetin concentrations (Fig. 20A). The K i values estimated using the lowest 4 phenacetin concentrations were about 70% lower than those estimated using the complete range of phenacetin concentrations (Table 8), suggesting that part of the M R O D activity is less sensitive to inhibition by phenacetin.  Fligh affinity phenacetin O-  dealkylase has been shown to be mediated by CYP1A2 in microsomes from uninduced rats (Sesardic et. ai, 1990). Results of the present study suggest that there are at least 2 components of M R O D activity in microsomes from uninduced rats, one of which was sensitive to inhibition by low concentrations of phenacetin, suggesting that CYP1A2 contributes to this component of M R O D activity. The other component of M R O D activity was less susceptible to inhibition by phenacetin, suggesting that some other cytochrome(s) P450 is(are) responsible for this component of activity.  Phenacetin inhibition studies were also performed to determine the possible role of CYP1A2 in EROD activity in microsomes from uninduced rats. If E R O D activity is mediated by CYP1A2 in microsomes from uninduced rats, low concentrations of phenacetin should inhibit this activity at a substrate concentration that was equivalent to the estimated K m value. However, no inhibition of microsomal EROD activity was observed in uninduced microsomes at low concentrations of phenacetin (Fig. 18B), suggesting that CYP1A2 is not responsible for this activity.  106  4.1.2.2.  Immunoinhibition studies To determine the contribution of CYP2C11 and CYP1A2 to M R O D activity in  microsomes from uninduced rats, immunoinhibition studies were performed with polyspecific and monospecific anti-CYP2C11 IgG, and polyspecific anti-CYP 1A1 IgG.  The polyspecific anti-  CYP2C11 IgG cross-reacts with other CYP2C enzymes (personal communication with Dr. S. Bandiera) but not with CYP1 A l / 2 , as shown by its lack of inhibition of microsomal M R O D and E R O D activities in BNF-induced rats (Fig. 21).  At saturating concentrations, this antibody  inhibited M R O D activity in microsomes from uninduced rats by about 85% (Fig. 24A), suggesting that M R O D activity is largely mediated by one or more of the CYP2C enzymes.  The  monospecific anti-CYP2C11 IgG recognizes and inhibits CYP2C11 (Fig. 23) but not other CYP2C enzymes (personal communication with Dr. S. Bandiera).  The lack of inhibition of  microsomal M R O D activity in uninduced rats by the monospecific anti-CYP2C11 IgG (Fig. 24A) indicates that CYP2C11 is not involved in this activity. The polyspecific a n t i - C Y P l A l IgG has been shown to cross-react with CYP1A2 and CYP2C13 (personal communication with Dr. S. Bandiera).  It is, therefore, possible that this antibody preparation also cross-reacts with other  enzymes in the CYP2C subfamily. Biphasic inhibition of M R O D activity in microsomes  from  uninduced rats was observed with the polyspecific anti-CYP 1A1 (Fig. 24A). Since there is very little hepatic microsomal CYP1A1 present in uninduced rats (Waxman et. al., 1985), the inhibitory effect observed with polyspecific anti-CYP 1A1 IgG at low antibody concentrations suggests that M R O D activity is partly mediated by CYP1A2 or an immunochemically related enzyme.  This is consistent with results of the phenacetin inhibition studies from the present  investigation that one component of M R O D activity in microsomes from uninduced rats is sensitive to inhibition by the CYP1A2 inhibitor, phenacetin (Fig. 20A). The inhibition observed with polyspecific anti-CYP 1A1 IgG at high concentrations may have been due to cross-reaction of the antibody with the CYP2C enzyme(s) that is(are) responsible for the majority of M R O D activity. Taken together, results of the phenacetin inhibition and immunoinhibition studies from the present investigation suggest that microsomal M R O D activity in uninduced rats is partly due  107  to CYP1A2, but mainly due to a CYP2C enzyme(s) other than CYP2C11. This differs from the suggestion of Burke et. al. (1994) that MROD activity is mediated by CYP1A2 in uninduced rats. These authors based their conclusion on the observation that furafylline (200 uM) inhibited more than 75% of MROD activity but less than 25% of EROD activity in microsomes from uninduced rats. However, the selectivity of furafylline as an inhibitor of CYP1A2 has not been well established in uninduced rats.  In microsomes from uninduced rats, EROD activity was completely inhibited by saturating concentrations of the polyspecific anti-CYP2Cll IgG (Fig. 24B). Because there is indirect evidence to suggest that this antibody preparation does not cross-react with CYP1A1/2 (Fig. 21), it appears that EROD activity in microsomes is not mediated by CYP1A1/2, but by one or more of the CYP2C enzymes. The monospecific anti-CYP2C 11 IgG did not inhibit EROD activity in microsomes from uninduced rats (Fig. 24B), indicating that this activity is not mediated by CYP2C11. The polyspecific anti-CYPlAl IgG inhibited EROD actiivty at high concentrations (Fig. 24B).  This antibody preparation has been shown to cross-react with CYP1A2 and  CYP2C13 (personal communication with Dr. S. Bandiera), and it is possible that it also recognizes some other enzymes in the CYP2C subfamily. Since there is very little CYP1A1 present in uninduced rats (Waxman et. al., 1985) and CYP1A2 does not appear to be involved in ethoxyresorufin O-deethylation in microsomes from uninduced rats, as discussed above, the inhibition observed with polyspecific anti-CYPlAl IgG at high concentrations may have been due to cross-reaction of the antibody with a CYP2C enzyme(s). Taken together, results of the immunoinhibition studies suggest that EROD activity is not mediated by CYP1A2 or CYP2C11, but by another cytochrome P450 enzyme, likely in the CYP2C family. This is consistent with results of the phenacetin inhibition studies that low concentrations of phenacetin, the CYP1A2 inhibitor, did not inhibit EROD activity in microsomes from uninduced rats (Fig. 18B).  108  In contrast to the above, Kelley et. al. (1987) reported that CYP1A2 accounts for 78% of E R O D activity in untreated rats. That particular study was performed with a polyclonal antiC Y P 1A2 antibody at a single antibody concentration; Results of immunoinhibition studies can be misleading when a complete dose response curve is not obtained and when the cross-reactivity and inhibitory potency of the antibody have not been fully characterized.  The present observation that CYP2C11 is not responsible for microsomal E R O D activity in uninduced rats also differs from the finding of Nakajima et. al. (1990) that  CYP2C11  accounted for 77% of this activity but their experiments were performed with an anti-C YP2C11 antibody that cross-reacted with CYP2C6. More recently, Burke et. al. (1994) concluded that E R O D activity was mediated by CYP2C6 in microsomes from uninduced rats.  These  investigators observed inhibition of EROD activity with both anti-CYP2C6 IgG (cross-reactive with CYP2C11) and anti-CYP2C11 IgG (cross-reactive with CYP2C6) in untreated samples. However, their anti-CYP2C6 IgG preparation inhibited the activity with greater potency than the anti-CYP2C11 IgG. One criticism of their findings is that the two antibody preparations were cross-reactive, and the investigators did not test their inhibitory potencies with isozyme-specific enzyme activities as was done in the present study (Fig. 23).  Despite this drawback, their  suggestion that E R O D activity is mediated by CYP2C6 in uninduced rats is consistent with the current observation.  4.1.3. Summary Results of BNF induction, phenacetin inhibition and immunoinhibition studies from the present investigation suggest that, in microsomes from BNF-induced rats, CYP1A1 accounts for 60% of M R O D and 80% of EROD activities, whereas CYP1A2 appears to account for as much as 40% of M R O D and 20% of EROD activities.  Results of the phenacetin inhibition and  immunoinhibition studies from the present investigation also suggest that in microsomes from uninduced rats, a CYP2C enzyme(s) other than CYP2C11 mediates M R O D and E R O D activities,  109  and that CYP1A2 makes some contribution to M R O D activity but not to E R O D activity. It is not known whether M R O D and EROD activities in microsomes from uninduced rats are mediated by the same CYP2C enzyme(s).  4.2. Effect of in vivo cimetidine on microsomal MROD and EROD activities 4.2.1. Microsomes from BNF-induced rats In microsomes from BNF-induced rats, in vivo administration of cimetidine had no effect on the total cytochrome P450 content (Fig. 7). Cimetidine was observed to inhibit testosterone 2a- and 16a-hydroxylase activities by 61 and 65%, respectively (p < 0.01) (Fig. 9A), but had no effect on M R O D or EROD activity (Fig. 8). The magnitude of inhibition of testosterone 2a- or 16a-hydroxylase activity observed with in vivo cimetidine was similar, when the control activity was expressed as the rate of product formation relative to the amount of microsomal protein or cytochrome P450 (Fig. 9B). The inhibition of these hydroxylase activities by in vivo cimetidine indicated the presence of the active form of the inhibitor in microsomes from BNF-induced rats. In vivo cimetidine, therefore, selectively inhibited the cytochrome(s) P450 responsible for these testosterone hydroxylase activities in microsomes from BNF-induced rats, but had no effect on those responsible for M R O D and EROD activities.  Complete inhibition of testosterone 2a- and 16a-hydroxylase activities in microsomes from BNF-induced rats was observed with the monospecific anti-CYP2Cll IgG (Fig. 22), providing evidence that these activities are mediated by CYP2C11, as in microsomes from uninduced rats (Waxman et. al., 1984 & 1987; Chang et. al, 1992a).  BNF-pretreatment also  resulted in suppression of testosterone 2a- and 16a-hydroxylase activities by over 50% (Table 6). This is consistent with published data that BNF-pretreatment resulted in suppression of the absolute content of CYP2C11 by about 75% (Guengerich et. al, 1982). The present observation that in vivo cimetidine inhibited these activities in microsomes from BNF-induced rats (Fig. 9) is consistent with the original findings of Chang et. al. (1992a) that in vivo cimetidine inhibits  110  CYP2C11 in male rats and indicates that cimetidine continues to be active as an inhibitor of CYP2C11 after BNF-pretreatment. The observed lack of inhibition of M R O D or E R O D activity by in vivo cimetidine in microsomes from BNF-induced rats (Fig. 8) suggests that cimetidine does not inhibit the cytochrome P450 enzymes responsible for these activities.  Results of the phenacetin inhibition and immunoinhibition studies suggested that CYP1A2 and CYP1A1 account for 40% and 60% of M R O D activity, respectively (Section 4.1.1). Because in vivo administration of cimetidine was observed to have no effect on microsomal M R O D activity in BNF-induced rats (Fig. 8), it appears that in vivo cimetidine does not have an inhibitory effect on either CYP1A2 or CYP1A1.  However, since CYP1A2 accounts for only 40% of  M R O D activity and the sample size was small in the present study (4 rats per group), it is possible that cimetidine has an inhibitory effect on CYP1A2 but that the in vivo experiments lacked the power to detect it. A power calculation using the observed mean and pooled variance was done, which indicated that, with a sample size of 4 rats per group, the experiment had the power to detect a 20% difference in mean M R O D activity (one tailed unpaired t-test, a = 0.05, p = 0.2). This would be equivalent to a 50% inhibitory effect on CYP1A2 since 40% of the M R O D activity was due to this enzyme. Thus, the results indicate that if in vivo cimetidine inhibits CYP1A2, the effect is likely to be < 50%.  Results of the phenacetin and antibody inhibition studies showed that CYP1A1 accounts for the majority of EROD activity in microsomes from BNF-induced rats (section 4.1.1).  The  lack of inhibition of EROD activity in microsomes from BNF-induced rats by in vivo cimetidine, reported previously by Chang (1991) and confirmed in the present investigation (Fig. 8), indicates that in vivo cimetidine does not inhibit CYP1A1. This is in accord with the observation that in vivo cimetidine did not inhibit CYPlAl/2-mediated M R O D activity in microsomes from BNFinduced rats (Fig: 8). Drew et. al. (1981) found that in vivo cimetidine selectively inhibited aryl hydrocarbon hydroxylase activity in microsomes from 3-MC-induced rats and this activity was  Ill  subsequently shown to be mediated by CYP1A1 in these animals (Ryan et. al, 1982). Chang et. al. (1991) commented that the discrepancy in the effect of in vivo cimetidine on microsomal E R O D activity in BNF-induced rats and microsomal aryl hydroxylase activity in 3-MC-induced may be substrate- or enzyme-related.  They proposed that there could be two different binding  sites on CYP1A1, and that cimetidine inhibited the site bound by ethoxyresorufin but not the one bound by benzo[a]pyene. It is also possible that multiple forms of CYP1A1 exist and that these enzymes have different catalytic specificities but are immunochemically indistinguishable. Cimetidine may selectively inhibit one of them but not the others. Indeed, Burke et. al. (1994) have recently separated CYP1A1* from CYP1A1 by column chromatography and both enzymes were shown to have similar catalytic activities toward the various alkoxyresorufins. At this point, it is not certain whether CYP1A1* is a distinct form of cytochrome P450.  4.2.2. Microsomes from uninduced rats In microsomes from uninduced rats, in vivo administration of a single dose of cimetidine was associated with a lower total hepatic microsomal cytochrome P450 content by (Fig. 7). As discussed in Section 3.2.2.2, one of the rats in the cimetidine group was an outlier, and with the exclusion of this rat from the calculation of mean cytochrome P450 content, in vivo administration of cimetidine was associated with a 13% reduction in the cytochrome P450 content (n = 3 rats in the cimetidine group and 4 rats in the saline group) (Fig. 7). In contrast, Drew et. al. (1981) (n = 4 rats per group) and Chang (1991) (n = 8 rats per group) found no change in the total hepatic microsomal cytochrome P450 content in male rats after administration of a single dose of cimetidine, and these investigator have similar or larger sample sizes in their studies. The apparent discrepancy in the effect of in vivo cimetidine on the microsomal cytochrome P450 content in uninduced rats may be due to experimental variability or erroneous findings of the present investigation. Future studies should be conducted with larger sample sizes.  112  The effects of in vivo administration of cimetidine on M R O D and E R O D activities were examined in microsomes from uninduced rats. As discussed above, the microsomal cytochrome P450 content was lower in the cimetidine-treated rats than in the saline-treated rats.  It was  therefore possible that, even in the absence of an inhibitory effect of cimetidine, expression of the enzyme activity as the rate of product formation per mg of microsomal protein could lead to an erroneous conclusion that in vivo cimetidine was inhibitory. To account for this possibility, enzyme activities in uninduced rats were expressed in two ways: 1) the rate of product formation per mg of microsomal protein and 2) the rate product formation per nmol of microsomal P450.  When enzyme activity was expressed as the rate of product formation per mg of microsomal protein, in vivo cimetidine was observed to inhibit M R O D and E R O D activities by 27% (p < 0.05) and 59% (p < 0.01), respectively (Fig. 10A). As noted earlier (Section 3.2.2.2), there was an outlier in the cimetidine-treated group.  When the data for this animal were  excluded, the inhibition of M R O D and EROD activities by cimetidine was 36% (p < 0.01) and 78% (p < 0.001), respectively (Fig. 10B). When the activity was expressed as the rate of product formation per nmol of P450, in vivo cimetidine was found to have no effect on M R O D or EROD activity (Fig. 11 A). However, when the data for the outlier were excluded, in vivo cimetidine was observed to inhibit EROD activities by 75% (p < 0.001) but the reduction in M R O D activity (26%) was not statistically significant (p = 0.07) (Fig. 1 IB). Based on these results, it is possible that in vivo cimetidine inhibits M R O D activity by about 20 - 30% but the experiment lacked the power to detect this effect. Due to the problems associated with this particular experiment, it would have to be repeated with an appropriate number of animals if it is necessary to determine the effect of cimetidine on M R O D activity in uninduced rats. Using the observed mean activity and variance, it is estimated that a sample size of 7 animals per group is required to detect a 25% difference in mean M R O D activity (one-tailed, unpaired t-test, a = 0.01, P = 0.1).  113  In vivo cimetidine appeared to inhibit EROD activity (nmol/min/nmol P450) by about 75% when the outlier was excluded (Fig. 1 IB). The relatively low probability of committing a Type I error (p < 0.001) and the fact that Chang (1991) also observed this effect provide stronger evidence that in vivo cimetidine inhibits EROD activity in uninduced rats.  Results of immunoinhibition studies from the present investigation have demonstrated that microsomal E R O D activity in uninduced rats is not mediated by CYP1A2 or CYP2C11, but likely by another CYP2C enzyme(s) present in these animals (Section 4.1.2.2).  Because in vivo  administration of cimetidine has been shown to inhibit microsomal E R O D activity in uninduced rats (Chang, 1991) and this was verified in the present study (as discussed above), it is evident that in vivo cimetidine inhibits the CYP2C enzyme(s) responsible for E R O D activity in these animals.  A likely candidate is CYP2C6, since Burke et. al. (1994) have obtained evidence  suggesting that EROD activity is mediated by this enzyme in microsomes from uninduced rats. Unlike CYP2C11, CYP2C6 is not a sex-specific cytochrome P450 and is present at similar levels in microsomes from untreated male and female rats (Waxman, 1985). CYP2C6 has been shown to account for about 13-24% of the total cytochrome P450 in untreated male rats (Dannan et. al, 1983; Imaoka et. al, 1990) and is inducible by phenobarbital (Guengerich et. al, 1982; Waxman, 1985). The purified enzyme has been shown to catalyze the deethylation of ethoxyresorufin in reconstituted systems (Burke et. al, 1994).  If in vivo cimetidine turns out to have an inhibitory effect on M R O D activity in microsomes from uninduced rats, then results of the phenacetin inhibition and immunoinhibition studies on M R O D activity from the present investigation would also suggest that in vivo cimetidine inhibits another CYP2C enzyme in addition to CYP2C11. Results of the phenacetin inhibition and immunoinhibition experiments from the present investigation suggest that CYP1A2 is involved in the metabolism of methoxyresorufin in microsomes from uninduced rats but do not indicate the quantitative contribution of CYP1A2 to this activity (Fig. 18A & 24A).  The  114  polyspecific anti-CYP2C11 IgG used in this project was observed to inhibit about 85% of the M R O D activity in microsomes from uninduced rats (Fig. 24A). Because there is indirect evidence to suggest that this antibody does not cross-react with CYP1A2 (Fig. 21), it is likely that the M R O D activity in microsomes from uninduced rats is largely mediated by a CYP2C enzyme(s). Taken together, the data suggest that CYP1A2 makes a relatively minor contribution to this activity.  Considering that in vivo cimetidine appears to have little or no effect on CYP1A2  (section 4.1.1), then any inhibition of M R O D activity in microsomes from uninduced rats by in vivo cimetidine would likely be due to inhibition of an enzyme(s) in the CYP2C subfamily (not CYP2C11).  It is not known at present whether this CYP2C enzyme is the same as the one that  mediates microsomal EROD activity in uninduced rats.  4.2.3. Summary In microsomes from BNF-induced rats, administration of cimetidine in vivo resulted in inhibition of the CYP2C11-mediated testosterone 2a- and 16a-hydroxylase activities, but had no effect on CYPlAl/2-mediated M R O D and EROD activities.  In microsomes from uninduced  rats, administration of cimetidine in vivo resulted in inhibition of EROD activity. Since EROD activity in uninduced rats appears to be mediated by a CYP2C enzyme(s) other than CYP2C11 (possibily CYP2C6), the results suggests that in vivo cimetidine also inhibits this enzyme. Future studies would be necessary to determine the effect of in vivo cimetidine on microsomal M R O D activity in uninduced rats.  4.3. Effect of in vitro cimetidine on microsomal MROD and EROD activities 4.3.1. In the absence of a preincubation step The effects of in vitro administration of cimetidine on microsomal M R O D and EROD activities were also examined and compared to those following its administration in vivo. In the absence of a preincubation step, in vitro cimetidine inhibited microsomal M R O D and EROD activities in uninduced and BNF-induced rats, and microsomal testosterone 2a- and 16a-  115  hydroxylase activities in uninduced rats, with ICso values ranging from 0.25 to 4 m M (Fig. 25, Table 9). This suggests that in vitro cimetidine inhibits CYP1A1/2, CYP2C11, and the CYP2C enzyme(s) responsible for M R O D and EROD activities in uninduced rats. In contrast, in vivo cimetidine inhibits CYP2C11 and the CYP2C enzyme responsible for E R O D activtiy in uninduced rats, but appears to have no effect on CYP1A1/2 (Section 4.2.3). The ICso values of in vitro cimetidine for the inhibition of the various cytochromes P450 (Table 9) were at least 50-fold higher than the serum cimetidine level likely present (Speeg et. al, 1982; Winer and Roth, 1981) 90 min after in vivo administration of 150 mg/kg cimetidine, when the rats were killed. Moreover, the freely diffusible cimetidine would have been further diluted during the preparation of microsomes; yet at such low concentrations, in vivo cimetidine inhibited microsomal testosterone 2a- and 16a-hydroxylase activities in BNF-induced rats, and microsomal EROD activity in uninduced rats by more than 50% (Fig. 9 and 11B). Similar discrepancies in the selectivity and potency of in vivo and in vitro cimetidine have been reported previously by Chang et. al (1992a&b). These authors postulated that cimetidine acts in vivo and in vitro by different mechanisms and they performed preincubation studies in vitro to further investigate the inhibitory effect of cimetidine. In the present investigation, preincubation studies were also performed to determine whether the selectivity of in vivo cimetidine could be reproduced in vitro and whether its inhibitory potency could be enhanced.  An important observation arising from in vitro studies is the differential effect of cimetidine (in the absence of a preincubation step) on microsomal testosterone 2a-hydroxylase activity in BNF-induced rats (ICso = 4 mM) and EROD activity in uninduced rats (ICso = 0.25 mM) (Table 9). This difference in ICso values occurred at substrate concentrations that were equivalent to 3-4 times the respective estimated Km values (Table 9), suggesting that different enzymes are involved in catalyzing the two reactions. Since testosterone 2a-hydroxylase activity in microsomes from BNF-induced rats has been shown to be mediated by CYP2C11 (Fig. 22), this provides evidence that, in microsomes from uninduced rats, E R O D activity is mediated by an  116  enzyme(s) other than CYP2C11. This is consistent with results of the immunoinhibition studies from the present investigation that microsomal EROD activity in uninduced rats was not inhibited by monospecific anti-CYP2C 11 IgG (Fig. 24B).  4.3.2. In the presence of preincubation step Preincubation of microsomes from BNF-induced rats in vitro with cimetidine for 15 min in the presence of N A D P H had no effect on the potency of its inhibition of M R O D or EROD activity (Fig. 27), whereas the potency of its inhibition of testosterone 2a- and 16a-hydroxylase activities was increased more than 40-fold (Fig. 28). In microsomes from uninduced rats, preincubation of microsomes in vitro with cimetidine in the presence of N A D P H had no effect on its inhibition of M R O D activity, but increased the potency of its inhibition of E R O D activity 16fold (Fig. 29). These results indicate that preincubation of microsomes with cimetidine in the presence of N A D P H enhances its inhibition of the other CYP2C enzyme(s) involved in the deethylation of ethoxyresorufin in uninduced rats and CYP2C11, but not that of CYP1 A l / 2 . This pattern of inhibition was similar to that observed following the in vivo administration of cimetidine (Sections 4.2.1 and 4.2.2).  The clear effect of preincubation on EROD activity in microsomes from uninduced rats suggests that cimetidine inhibits the CYP2C enzyme(s) responsible for this activity in vivo by a catalysis-dependent process.  This is similar to the catalysis-dependent inhibition of CYP2C11  observed with cimetidine by Chang et. al. (1992b) as well as in the present investigation Taken together, these observations suggest that cimetidine inhibits another C Y P 2 C enzyme(s) in addition to CYP2C11 in male rats in vivo by a catalysis-dependent process.  Other investigators have examined the effect of preincubation on the inhibition of the catalytic activities of hepatic microsomal cytochromes P450 by cimetidine.  Ioannoni et. al.  (1986) preincubated rat microsomes with cimetidine (0.5 mM) or distilled water in the abesence  117  or presence of a NADPH-generating system for up to 15 min and observed no enhanced inhibition of morphine N-demethylase activity by cimetidine. The negative result can be explained if the cytochrome(s) P450 responsible for morphine N-demethylase activity is not inhibited by in vivo cimetidine. Jensen and Gugler (1985) observed that preincubation of rat hepatic microsomes with 0.25 m M cimetidine in the presence of N A D P H for up to 20 min resulted in enhanced inhibition of 7-ethoxycoumarin deethylase activity. However, in that particular study, the microsomes in the control incubation were not preincubated with N A D P H .  The decrease in 7-ethoxycoumarin  deethylase activity observed following preincubation may have resulted from degradation of the heme by the reactive oxygen species formed in the presence of N A D P H (Levin et. al, 1973). When Chang et. al. (1992b) performed preincubation studies with in vitro cimetidine, they preincubated rat hepatic microsomes with N A D P H and 0.05 m M cimetidine for 15 min prior to initiation of reaction with substrate addition.  In the control incubations, microsomes were  preincubated with N A D P H alone for 15 min and cimetidine was added after preincubation and prior to initiation of the reaction with substrate addition.  They found that preincubation of  microsomes with cimetidine and N A D P H resulted in enhancement of its inhibition of CYP2C11mediated catalytic activities and that the pattern of inhibition observed with in vitro cimetidine following preincubation was similar to that observed following in vivo administration of cimetidine.  This finding was verified in the present investigation, as mentioned above.  The  preincubation protocol used in the present investigation was the same as that of Chang et. al. (1992b),. Because N A D P H was also present in the control incubations in the present investigation, the decrease in enzyme activities observed following preincubation of microsomes with cimetidine and N A D P H was not due to degradation of the heme by the free oxygen radicals, but resulted from the inhibition of the cytochromes P450 by the activated form of cimetidine. Recent studies carried out by Vage and Svensson (1994) and Wienkers et. al. (1995) have shown enhanced inhibition of CYP2C11-mediated activities following preincubation of hepatic rat microsomes with cimetidine in the presence of N A D P H .  There is also evidence in human  118  microsomes that preincubation enhances the inhibition of cytochrome P450-mediated activities by cimetidine (Wild and Back, 1989; Tingle et. al., 1991).  4.3. Summary In the absence of a preincubation step, in vitro administration of cimetidine resulted in inhibition of the catalytic activities specific for CYP1A1/2, CYP2C11, and the CYP2C enzyme(s) involved in M R O D and E R O D activities in uninduced rats, with ICso values in the low m M range. This is in contrast to the lack of inhibition of the catalytic activities specific for CYP1A1/2 observed with in vivo cimetidine. Preincubation of microsomes with cimetidine in the presence of N A D P H increased the potency of its inhibition of the CYP2C enzyme(s) involved in EROD activity in uninduced rats and CYP2C11. Under the same conditions, preincubation had no effect on the inhibition of those activities specific for CYP1A1/2 by cimetidine.  This pattern of  inhibition is similar to that observed following in vivo administration of cimetidine, suggesting that this compound inhibits CYP2C11 and the other CYP2C enzyme(s) in vivo by a catalysisdependent process.  4.4. Effect of preincubation on EROD activity in human liver microsomes The results of the present study and those of Chang et. al. (1992a & b) indicate that, in male rats, cimetidine inhibits CYP2C11 by a catalysis dependent process. In the current study, there is evidence to suggest that cimetidine inhibits another CYP2C enzyme in addition to CYP2C11 in male rats by a catalysis-dependent mechanism. In humans, there is clinical evidence indicating that cimetidine is a selective inhibitor of hepatic cytochromes P450 in vivo (Levine et. al, 1985; Grygiel et. al, 1984., Steiner and Spina, 1987; Stockley et. al., 1986) but it is not known which human P450 isozymes are affected. In human liver microsomes, the metabolism of theophylline has been shown to be partly mediated by CYP1A2 (Sarkar et. al, 1992) and the decrease in theophylline clearance in smokers and non-smokers during concomitant cimetidine administration (Grygiel et. al, 1984; Cusack et. al, 1985) has provided indirect evidence that  119  cimetidine is an inhibitor of human CYP1A2.  If cimetidine inhibits human CYP1A2 by a  catalysis-dependent mechanism, then preincubation of human microsomes in vitro with low doses of cimetidine in the presence of N A D P H should increase the potency of its inhibition of this enzyme. There is evidence that EROD activity is mediated by CYP1A2 in human microsomes (Murray et. al., 1983; Sesardic et. al, 1990). Preincubation experiments with human microsomes were therefore performed on EROD activity to investigate the effect of in vitro cimetidine on human CYP1A2. It was observed in the present study, however, that preincubation of human liver microsomes with cimetidine (0.025 - 1 mM) in the presence of N A D P H had no effect on its inhibition of E R O D activity (Fig. 31).  The negative result of this experiment should be interpreted cautiously before drawing the conclusion that in vivo cimetidine does not inhibit human CYP1A2. In clinical studies, cimetidine administration decreased theophylline clearance by only 10 to 30% (Grygiel et. al, 1984; Cusack et. al, 1985), suggesting that cimetidine has a mild inhibitory effect on CYP1A2. It is therefore possible to miss the enhanced inhibition of EROD activity expected with preincubation under the conditions used, particularly if the effect of preincubation is relatively small as well.  It is also possible that theophylline metabolism is not solely mediated by CYP1A2.  A  portion of theophylline metabolism may be due to another cytochrome(s) P450 and inhibition of this enzyme by cimetidine may give rise to the decreased theophylline clearance observed in clinical studies.  Alternatively, the negative result can be explained if EROD activity was not be mediated by CYP1A2 in the human microsomal samples examined. Results of immunocorrelation studies have suggested that EROD activity is mediated by CYP1A2 (Murray et. al, 1993). Furafylline, a selective inhibitor of human CYP1A2 (Sesardic et. al, 1990), has been shown to inhibit EROD activity in human microsomes (Burke et. al, 1994; Clarke et. al, 1994) in a recent study,  120  confirming that this activity is catalyzed by CYP1A2 in these microsomes. However, there is large interindividual difference in human cytochrome P450-mediated enzyme activities due to both genetic variation and exposure to various xenobiotics present in the environment. The highest and lowest human microsomal EROD activities have been observed to span a five-fold range (personal communication from Dr. G. Bellward). In the present investigation, we used pooled microsomes from the liver samples of 3 non-smokers (Table 4) and it is possible that microsomal E R O D activities in these samples were mediated by some cytochrome(s) P450 other than CYP1A2.  Another possiblity is that in vivo cimetidine does not inhibit human CYP1A2 by a catalysis-dependent process, but by reversible binding to the heme moiety as it does in vitro (Knodell et. al, 1991). This explanation is very unlikely because in vitro cimetidine inhibited E R O D activity at concentrations in the m M range. For example, when cimetidine was added in vitro after preincubation of the control microsomes with N A D P H , it inhibited E R O D activity by about 13% at a concentration of 1 mM (Fig. 31). This degree of inhibition was similar to that observed with in vitro cimetidine in the absence of a preincubation step (Knodell et. al, 1991). In humans, the serum cimetidine concentration is in the low u M range through most of the dosage interval (Somogyi and Gugler, 1983). Thus, it is unlikely that cimetidine can inhibit CYP1A2 in vivo by the same mechanism as in vitro cimetidine (i.e. reversible binding to heme moiety), unless the substrates (drugs) are present at concentrations far below the K m values of the enzymes inhibited.  Previously studies carried out by Wild and Back (1989) and Tingle et. al. (1991) have shown enhanced inhibition of cytochrome P450-mediated activities following preincubation of human liver microsomes with cimetidine in the presence of N A D P H , but these studies did not identify which human cytochromes P450 were involved in these activities. Though inappropriate controls may have been used in these studies, their results do provide some support that  121  cimetidine inhibits human cytochrome P450 by a catalysis-dependent mechanism when the correct substrates are used. Future preincubation studies in human microsomes should be conducted with appropriate isozyme-selective subtrates.  4.5. Future Studies Because M R O D activity in microsomes from BNF-induced rats was not found to be selective for CYP1A2, it was not possible to fully investigate the effect of cimetidine on this enzyme. Future studies designed to determine the effect of cimetidine on CYP1A2 should be performed with CYP1 A2-specific activities such as M R O D activity in HCB-induced rats (Nerukar et. al, 1993), or high affinity phenacetin O-dealkylase activity in uninduced or 3-MC-induced rats (Sesardic et. ai, 1990).  A major finding of the present investigation was that E R O D activity is mediated by a CYP2C enzyme other than CYP2C11 (Section 4.1.2).  In a recent publication, Burke et. al.  (1994) reported a similar finding and suggested that this CYP2C enzyme is CYP2C6. Since in vivo cimetidine was observed to inhibit EROD activity in microsomes from uninduced rats by Chang (1991) as well as in the current study, it suggests that cimetidine is an inhibitor of CYP2C6 in vivo. Future experiments should test the hypothesis that cimetidine inhibits CYP2C6 using progesterone 21-hydroxylase activity in phenobarbital-induced rats for monitoring CYP2C6 (Swinney et. al, 1987).  Galbraith and Jellinck (1989) have performed studies with in vivo cimetidine in male and female rats. In vivo cimetidine was observed to inhibit some selected activities in male rats, but had no effect on any of the activities studied in female rats. Interestingly, the authors of that study did not examine the effect of in vivo cimetidine on EROD activity in female rats. The level of microsomal E R O D activity in female rats is comparable to that in male rats (Reily et. al., 1987, Paolini et. al, 1988) and the hepatic microsomal level of CYP2C6 is similar in both male and  122  female rat (Waxman, 1985). If EROD activity is mediated by CYP2C6 in male rats, it is possible that this activity is also mediated by this enzyme in female rats. Studies with in vivo cimetidine should be performed on microsomal EROD activity in uninduced female rats and this may lead to the identification of the first enzyme activity that is inhibited by in vivo cimetidine in female rats. If in vivo cimetidine is observed to inhibit E R O D activity in female rats, it can be followed with immunoinhibition experiments to identify the enzyme responsible for this activity.  It has been shown in the present investigation that in vivo cimetidine does not inhibit CYP1A1-mediated EROD activity in microsomes from BNF-induced rats. This differs from the findings of Drew et. al. (1981) that in vivo cimetidine inhibited aryl hydrocarbon hydroxylase activity in microsomes from 3-MC-induced rats, which was later shown to be mediated by CYP1A1 (Ryan et. al, 1982). Further studies with in vivo cimetidine could be performed to verify the inhibition of aryl hydrocarbon hydroxylase activity in microsomes from 3-MC induced rats by in vivo cimetidine. If positive results are obtained with the above experiments, it will provide evidence that there is either more than one substrate binding site on CYP1A1 or more than one form of CYP1A1 (Chang, 1991).  Levine and Bellward (1994) have performed optical difference spectroscopic studies on microsomes prepared from rats treated with cimetidine or saline in vivo. A difference peak at 428-430 nm was observed, suggesting the presence of a metabolite-intermediate complex in the microsomes. This complex could be generated in vitro by preincubation of microsomes with N A D P H . Once it is determine which rat hepatic cytochromes P450 are inhibited by cimetidine, spectroscopic studies should be performed with purified enzymes to characterize the metaboliteintermediate complex.  Future preincubation studies with human liver microsomes should be carried out with nonselective substrates (e.g. theophylline) whose clearances have been shown to be decreased during  123  cimetdine co-administration.  If enhanced inhibition by cimetidne is observed  following  preincubation, the conditions should be optimized to maximize the preincubation effect.  These  conditions can then be used when preincubation experiments are performed with isozymeselective substrates for the identification of the affected cytochrome(s) P450.  Bufuralol 1-  hydroxylase, an isozyme-selective activity for human CYP2D6 (Dayer et. al,  1987), should  probably be tested because indirect evidence from clinical studies suggests that cimetidine inhibits CYP2D6 (Steiner et. al, 1987; Philip et. al, 1989). Based on the observation from the present investigation that cimetidine inhibits another CYP2C enzyme(s) (possibly CYP2C6) in addition to CYP2C11 in male rats and the fact that human and rat cytochrome P450 are immunochemically related, it is also predicted that cimetidine inhibits one of the CYP2C enzyme(s) in humans. Possible candidates include CYP2C19 and CYP2C9. There is evidence that CYP2C6 is the only enzyme in the CYP2C subfamily in rats that catalyzes progesterone 21-hydroxylation (Swinney et. al, 1987) and that in humans, CYP2C19 is the only member of this subfamily that has been shown to catalyze this activity to date (Richardson et. al, 1995). In humans, CYP2C9 has been implicated in the metabolism of phenytoin (Veronese et. al, 1991; Doecke et. al, 1991) and it has been documented that cimetidine interacts with phenytoin by inhibiting its hepatic clearance (Levin et. al, 1985). Therefore, future studies with human cytochromes P450 should investigate the possible inhibitory effects of cimetidine on CYP2C19 and CYP2C9. Once it is determined which human cytochromes P450 are inhibited by cimetidine, preincubation and spectroscopic studies should be performed with purified enzymes to characterize the active form of the inhibitor.  124  S u m m a r y and Conclusions  1. Specificities of MROD and EROD activities in male rats as determined by phenacetin inhibition and immunoinhibition:  A) In microsomes from BNF-induced rats, CYP1A1 accounts for 60% of MROD and 80% of EROD activity. CYP1A2 appears to account for only about the 40% of MROD and 20% of EROD activity. B) In microsomes from uninduced rats, both MROD and EROD activities appear to be mediated largely by a CYP2C enzyme(s) other than CYP2C11. CYP1A2 appears to contribute to MROD but not EROD activity.  2. Effect of in vivo administration of cimetidine (150mg/kg) on microsomal MROD and EROD activities in male rats:  A) In microsomes from BNF-induced rats, in vivo administration of cimetidine had no effect on MROD or EROD activity. This indicates that in vivo cimetidine does not inhibit CYP1A1, and appears to have little or no effect on CYP1A2. B) In microsomes from uninduced rats, in vivo administration of cimetidine resulted in inhibition of EROD activity, suggesting it inhibits another CYP2C enzyme(s) in addition to CYP2C11. In vivo cimetidine appeared not to have an inhibitory effect on MROD activity in microsomes from uninduced male rats but further studies would be necessary to verify this observation.  3. Effect of in vitro cimetidine on MROD and EROD activities in male rats:  A) When cimetidine was added in vitro to rat microsomes immediately prior to the initiation of the reaction, it inhibited MROD and EROD activities in microsomes from uninduced and BNF-induced rats with ICso values ranging from 0.25 - 4 mM. This suggests that, in the  125  absence of a preincubation step, in vitro cimetidine inhibits CYP1A1, CYP1A2 and another CYP2C enzyme(s) other than CYP2C11. This is in contrast to the observed lack of inhibition of CYPiAl/2-mediated MROD and EROD activities in microsomes from BNF-induced rats by in vivo cimetidine. B) The effect of in vitro cimetidine on these activities was also investigated with the inclusion of a preincubation step. Preincubation of microsomes from BNF-induced rats with cimetidine in the presence of NADPH has no effect on its inhibition of MROD or EROD activity. In microsomes from uninduced rats, preincubation had no effect on the inhibition of MROD activity by cimetidine, but enhanced its inhibition of EROD activity. These results indicate that preincubation enhanced the potency of the inhibtion of the CYP2C enzyme(s) involved in EROD activity by cimetidine, but not that of CYP1A1/2. This pattern of inhibition was similar to that observed following in vivo administration of cimetidine, indicating that cimetidine inhibits the CYP2C enzyme(s) responsible for EROD activity in uninduced male rats in vivo by a catalysis-dependent mechanism.  4. Effect of preincubation on the inhibtion of EROD activity by cimetidine in human liver microsomes:  A) Preincubation of human liver microsomes with cimetidine in the presence of NADPH appeared to have no effect on its inhibition of EROD activity. 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