Cytochromes P450 involved in thebioactivation of cocaine in the rat.

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Authors Poet, Torka Sue.

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CYTOCHROMES P450 INVOLVED IN THE

BIOACTIVATION OF COCAINE IN THE RAT

by

Torka Sue Poet

A Dissertation Submitted to the Faculty of the

COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)

In Partial Fulfillment of the Requirements For the Degree

DOCTOR OF PHILOSOPHY

in the Graduate College

THE UNIVERSITY OF ARIZONA

1995

UMI Number: 9603361

OMI Microform 9603361 Copyright 1995, by OMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

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THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have

2

read the dissertation prepared by~T~o~rk~a~S~u~e~P~o~e~t ____________________ ___

entitled Cytochromes P450 Involved in the Bioactivation of Cocaine

in the Rat

and recommend that it be accepted as fulfilling the dissertation

Date I

6-Z~-q~

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Date

3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED' ~ ~t-

4

ACKNOWLEDGMENTS

There are a number of people to whom lowe a dept of gratitude. I will

be forever indebted to Dr. James Halpert for his expert direction, guidance,

support, encouragement and confidence in me. I would also like to thank the

other members of my committee, Dr. Klaus Brendel, Dr. A. Jay Gandolfi, Dr.

Hugh Laird II, and Dr. John Regan for their patience and support, and Dr.

Charlene McQueen for her expert counsel on isolated hepatocytes and in

addition Dr. Roger Johnson for his statistical advice and Dr. Michael Mayersohn

for his expert help with the kinetic analYSis. This work could not have been

completed without the help of all of these people.

I would also like to acknowledge all of the members of Dr. Halpert's

laboratory whose assistance and fellowship was invaluable, especially

Stephanie Born, Dr. Vicki Burnett, and Dr. James Kraner, as well as the

members of Dr. McQueen's laboratory, especially Leslie Lemke and Greg

Stevens. All of these people supported me through their knowledge and

friendship.

5

DEDICATION

I would like to dedicate this dissertation to my father, because I

dedicated my Master's thesis to my mother and it is his tum.

6

TABLE OF CONTENTS Page

LIST OF TABLES ..................................................................................... 9

LIST OF FIGURES... ......... ... ..... ...... .......... ...... .... ... ........ ... .............. ...... ... 10

ABSTRACT.................... ... ....... ............ ..... ... ... ....... .............. ........ .... .... .... 11

1. INTRODUCTION ................................................................. ~ ................ 13 1.1 BIOTRANSFORMATION ............................................................... 13 1.2 CYTOCHROMES P450.... ...... ....... .... ........ ................................. .... 15 1.2.1 Cytochrome P450 Catalysis........... ........ .......... ... .... ..... ............ ... 16

1.2.2 Cytochrome P450 Nomenclature............................................ 17 1.3 REGULATION OF P450 LEVELS .................................................. 21

1.3.1 Induction .......... II ••••••••••••••••••••••••••••• ••• ••••••••••••••••••••••••••••••••• •••• 21 1.3.2 Inhibition.......... ............ ...................... ...................... ............... 22

1.4 STRATEGIES TO IDENTIFY INDIVIDUAL P450s INVOLVED IN DRUG METABOLiSM ..................................................................... 25

1.5 RESEARCH GOALS.................................................... ...................... 26 1.5.1 Manipulation of P450 Enzyme Activity Levels ............................. 26 1.5.2 Cytochromes P450 Responsible for the Bioadivation of Cocaine

......................................................... ~.............................. 27 1.5.3 Marker Activities and Purified Proteins....................................... 28

1.6 COCAINE.................... .......................... ........ ... ............................... ... 29 1.5.1 Cocaine Hepatotoxicity ........................................................... 30 1.5.2 Cocaine Bioactivation.................................................... ......... 30

2. MECHANISM·BASED INACTIVATION OF CYTOCHROMES P450 2B PROTECT AGAINST TOXICITY IN RAT LIVER SLiCES ........................ 34

2.1 BACKGROUND .............................................................................. 34 2.2 MATERIALS AND METHODS ........................................................ 34

2.2.1 Chemicals ............ II ••••• II •••••• II ••• II ••••••••••••• II ••• II II II II. II •••••• II........ 36 2.2.2 Animals ............................... II ••• II ••••••••• 1,.,1 II II •• II •••••••••••••••••••• II' 36 2.2.3 Slices......................... ........................... .... ... ..... ...................... 37 2.2.4 Incubation ........ ........................................... ... ......... ............... 37 2.2.5 Intracellular K+ Content.. ......................................................... 38 2.2.6 Microsomes................................................. ....... .............. ...... 38

7

TABLE OF CONTENTS - continued

Page 2.2.7 Catalytic Assays.................................................................... 39 2.2.8 Statistical Analysis .................................................................. 40

2.3 Results ......................... 1 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 41 2.3.1 Cocaine Toxicity ..................................................................... 41 2.3.2 In vitro Inactivation of 2B1/2 and Protection Against Cocaine

Toxicity .................................................................................... 43 2.3.3 In vivo Inactivation of 2B 1/2 and Protection Against Cocaine

Toxicity ...................................... II •••••••••••••••••••••••••••••••••••••••••••• 45 2.3.4 Cytochrome P450 2B1/2 Inactivation with pN02CIFA ............ 46 2.3.5 Cocaine Toxicity in WM Rat Liver Slices ................................ 47

2.4 DiSCUSSiON ................................................................................. 52

3. PARTICIPATION OF CYTOCHROMES P450 2B AND 3A IN COCAINE TOXICITY IN ISOLATED RAT HEPATOCYTES ..................................... 55

3.1 BACKGROUND .............................................................................. 55 3.2 MATERIALS AND METHODS ........................................................ 57

3.2.1 Chemicals............................................... ................................ 57 3.2.2 Animals ................................................................................. II 57 3.2.3 Isolated Hepatocyte Culture....... ......... .................................... 58 3.2.4 Toxicity Determination ............................................................ 59 3.2.5 Microsomes............... ......... ...... ............. ................. ... ...... .... ... 60 3.2.7 Catalytic Assays...................................................... ............ ... 60 3.2.8 Statistical Analysis .................................................................. 61

3.3 RESUL TS..................................... ............................................ ...... 62 3.3.1 Maintenance of Cytochrome P450 Activity in Isolated

Hepatocytes............................................................................ 62 3.3.2 Microsomal Hydroxylation in Hepatocytes from Phenobarbital-

Induced Rats ........................................................................... 62 3.3.3 Toxicity in Hepatocytes from Phenobarbital-Induced Rats ..... 63 3.3.4 Protection Against Toxicity by Inactivators in Hepatocytes from

Phenobarbital-Induced Rats .................................................... 64 3.3.5 Microsomal Hydroxylations in Hepatocytes from

Dexamethasone-Induced Rats ................................................ 65 3.3.6 Toxicity in Hepatocytes from Dexamethasone-Induced Rats. 66

8

TABLE OF CONTENTS - continued

Page 3.3.7 Toxicity Following Treatment with Inadivators in Hepatocytes

from Phenobarbital-Induced Rats............ ................... ............ 66 3.3.8 Cocaine N-Demethylation Rates............................................ 67 3.3.9 Cocaine N-Demethylation in PB- and Dex-Induced Female Rat

Liver Microsomes .................................................................... 69 3.4 Discussion...................................................................................... 77

4. COCAINE N-DEMETHYLATION BY MICROSOMES FROM PHENOBARBITAL-INDUCED RAT, DOG, AND GUINEA PIG AND BY PURIFIED P450 2B PROTEINS FROM RATS, DOGS AND RABBITS .............................................................................................................. 81 4.1 BACKGROUND .............................................................................. 81 4.2 MATERIALS AND METHODS ........................................................ 83

4.2.1 Chemicals............................................................................... 83 4.2.2 Animals ................................ 1 ..................................... 1............ 83 4.2.3 Purified Proteins......................... ............................................ 84 4.2.4 Lymphoblastoid Cell Culture ................................................... 84 4.2.5 Toxicity Assessment in Lymphoblastoid Cell Culture ............. 84 4.2.6 Microsomes ................................................................... : ........ 85 4.2.7 CatalytiC Assays..................................................................... 86 4.2.8 Statistical Analysis .................................................................. 86

4.3 RESULTS....................................................................................... 87 4.3.1 Cocaine N-Demethylation in PB-Induced Microsomes ........... 87

. 4.3.2 Cocaine N-Demethylation by Purified 2B Enzymes from Rat, Dog, and Rabbit ...................................................................... 87

4.3.3 Cocaine and Norcocaine Toxicity in Lymphoblastoid Cells Expressing Human 2B6.......................................................... 89

4.4 DiSCUSSiON ................................................................................. 91

5. SUMMARY AND CONCLUSiONS...... ........ ............ ............................. 93

APPENDIX 1- Abbreviations .................................................................... 1 00

REFERENCES ......................................................................................... 1 01

9

LIST OF ILLUSTRATIONS

Figure 1.1, Cytochrome P450 Catalytic Cycle......................................... 17 Figure 1.2, Bioactivation Scheme for Cocaine......................................... 33

Figure 2.1, Effect of Preincubation with CAP on Intracellular K+ Loss in Liver Slices from a PB-Lewis Rat Following Exposure to Cocaine........ 48 Figure 2.2.A and B, Comparison of Toxicity in Slices and AD 16B-OH in

Microsomes Made from Slices Incubated in Parallel.. .......................... 49 Figure 2.3.A. and B, Toxicity in Slices Exposed to Cocaine After In Vitro or

In Vivo Inactivator Pretreatment.. ......................................................... 50 Figure 2.4, Inactivation of P450 2B 1 After One or Two Injections with pN02CIFA ................................................................................................ 51

Figure 3.1, Androstenedione Hydroxylation Rates in Microsomes Prepared from Hepatocytes Isolated from a PB-Induced Rat.............................. 71

Figure 3.2.A. and B, Toxicity of Cocaine and Norcocaine in Hepatocytes Isolated from a PB-Induced Rat ............................................................ 72

Figure 3.3, Androstenedione Hydroxylation Rates in Microsomes Prepared from Hepatocytes Isolated from a Dex-Induced Rat............................. 73

Figure 3.4.A and B, Toxicity of Cocaine and Norcocaine in Hepatocytes Isolated from a Dex-Induced Rat .......................................................... 74

Figure 3.5, Cocaine N-Demethylation Rates in Microsomes from Hepatocytes Isolated from Dex- and PB-Induced Rats............................ 75 Figure 3.6, Troleandomycin Inhibition of Cocaine N-Demethylation in Microsomes........................................ ..................... ........ .................. ....... 76

Figure 4.1, Lymphoblastoid Cell Survival Following Exposure to Cocaine and Norcocaine ..................................................................................... 90

10

LIST OF TABLES

Table 1.1. Selected 2B enzymes and their relative abilities to metabolize marker substrates.................................................................................. 19

Table 1.2. Comparison of amino acid sequences of 2B enzymes from selected species ....................................................................................... 20

Table 2.1. K+ Retention in liver slices following exposure to cocaine ..... 42

Table 3.1. EC25 Values for toxicity in hepatocytes isolated from Dex- and PB-induced rats ................................................................................... 68

Table 3.2. Microsomal cocaine N-demethylation Kinetics ...................... 70

Table 4.1. Cocaine N-Demethylation Rates in Microsomes and Purified P450s ....................................................................................................... 88

11

ABSTRACT

Cytochrome P450 enzyme inducters and inhibitors were used to

elucidate the isoforms involved in the bioactivation of cocaine in the rat.

Phenobarbital and dexamethasone induction were used to induce P450s 2B

and 3A, among others, respectively. Isoform-specific inhibitions of P450s 2B

and 3A were then used to determine the relative contributions of these

enzymes toward cocaine bioactivation. The Site-specific hydroxylation of

androstenedione was compared to cocaine toxicity in rat liver slices and

cocaine and norcocaine toxicity in isolated hepatocytes. Cocaine toxicity

determined in rat liver slices, and androstenedione 16B-hydroxylations in

microsomes prepared from coincubated slices, revealed a correlation

between P450 2B marker activities and cocaine-mediated toxicity following

inhibition of P450 2B activities by chloramphenicol. While cocaine N­

demethylation rates were increased by both phenobarbital- and

dexamethasone-indudion, toxicity in isolated hepatocytes from phenobarbital­

induced rats were much greater than in hepatocytes from dexamethasone­

induced rats. Further, P450 2B inactivation by chloramphenicol protected

against cocaine- and norcocaine-mediated toxicity in hepatocytes from

phenobarbital-induced rats, whereas P450 3A inhibition by troleandomycin

12

had no protective effects. While P450s 3A have been observed by other

researchers to be important to the oxidative metabolism of cocaine in mice

and humans, the importance of P450s 3A in rats is shown here to be limited

to the first oxidation step of cocaine which produces norcocaine. Strong

differences in the abilities P450s 28 to N-demethylate cocaine in other

species were observed. Phenobarbital induction of rats and dogs resulted in

increased microsomal cocaine N-demethylation rates commensurate with

results from reconstituted purified 28 enzymes from these species. Cocaine

N-demethylation rates were low in purified rabbit 284 and expressed human

286 enzyme was unable to oxidize cocaine. In the guinea pig, which

constitutively express P450 28 but is only moderately induced by

phenobarbital treatment, the rate of cocaine N-demethylation was high in

microsomes from non-induced animals and was not remarkably increased by

phenobarbital induction. In agreement with this, neither cocaine nor

norcocaine exerted any toxicity in Iymphoblastoid cells expressing human

P450286.

CHAPTER 1

INTRODUCTION

1.1 BIOTRANSFORMATION

13

Biotransformation is the sum of the processes by which drug

metabolizing enzymes alter the chemical nature of substances. Organisms are

exposed to a plethora of drugs and environmental contaminants. The ability

to metabolize foreign compounds, or xenobiotics, to which an organism is

exposed is essential for survival. The lipophilicity of many xenobiotics

facilitates their uptake into the body across the lipid barriers of the

gastrointestinal tract, the skin, or the lungs. Lipophilicity also hinders excretion

which can lead to the build-up of the xenobiotic within the body. Drug

metabolizing enzymes are involved in processes which result in increased

water solubility of the metabolites and thus aid in their elimination from the

body, which is primarily mediated by hydrophilic mechanisms (Sipes and

Gandolfi 1991). Metabolism-mediated changes in chemical structures can

also alter the clinical activity of drugs, either decreaSing or increasing activity.

The enzymes which carry out drug metabolism have been divided into

two classes, designated phase I and phase II. Those enzymes which carry out

oxidations, reductions, or hydrolysis reactions are classified as phase I. Phase

14

I reactions include C- and N- hydroxylations, N-, 0-, and S- oxidations or

dealkylations, and epoxidations. Enzymes which are involved in conjugation

or synthetic reactions belong to the phase II class. Phase II reactions include

glucuronidation, sulfation, acetylation and amino acid conjugation.

Phase I metabolism generally results in one or two outcomes, a

decrease in the lipophilicity of the parent compound, and/or the exposure of

a functional group which can serve as a substrate for phase II conjugations.

Phase II conjugation usually further increases the water solubility of the

metabolite. Toxicity, therefore, is due in a large part, to the extent of exposure

and half-life of the substance in the body. Both of these are intimately related

to individual enzyme activities, levels, and fonns (Gonzalez and Gelboin

1993).

For many xenobiotics, metabolism can lead to the produdion of reactive

intermediates which can produce toxicity, a process referred to as

bioactivation. Generally, the reactive products exert their toxicity by binding

to either cellular macromolecules or DNA (Guengerich and Liebler 1985;

Gonzalez and Gelboin 1993). Alternatively, the electrophilic nature of the ,

products can lead to a build-up of free radicals, which can lead to the cross-

linking of membranes as well as the depletion of protective molecules, such

as glutathione.

15

1.2 CYTOCHROMES P450

Cytochromes P450 are the most abundant phase I metabolizing

enzymes. These enzymes comprise a superfamily of oxidizing enzymes, and

over 200 different cytochrome P450 (P450) 1 genes have been identified

(Nelson, Kamataki et al. 1993). Cytochromes P450 are ubiquitous and have

been found in insects, plants, and animals. Much of the importance of

cytochromes P450 is due to their ability to metabolize a wide array of

substrates (Waxman, Lapenson et al. 1991; Kato and Yamazoe 1992;

Wrighton and Stevens 1992). Although many of these enzymes are involved

in the synthesis of endogenous substrates, such as steroids and bile acids

(Gonzalez, Crespi et al. 1992), P450s are also of major significance to the

metabolism of xenobiotics (Guengerich and Shimada 1991; Coon, Ding et al.

1992; Wrighton and Stevens 1992).

Cytochromes P450 play an influential role in the toxicology and

carcinogenicity of many xenobiotics (Guengerich and Shimada 1991; Iwasaki,

Darden et al. 1994). Toxicants often require activation to more reactive

species to exert their toxic effects, and cytochromes P450 are frequently the

enzymes responsible for the bioactivation of otherwise inert compounds to

toxic metabolites. Thus, the levels and activities of the individual P450 forms

involved can dictate the balance between detoxification and bioactivation of

a given compound (Guengerich and Shimada 1991). Elucidating the

16

specificities and activities of different members of the cytochrome P450

superfamily toward various compounds will help to explain strain and

individual variations in drug metabolism and aid in the design of clinical

interventions for exposures to toxic substances (Halpert 1995).

1.2.1 Cytochrome P450 Catalysis.

The adive site of P450s contains heme, the fifth ligand of which is the

thiol of a conserved cysteine residue (Ryan and Levin 1990). The sixth ligand

of the heme mOiety binds a water molecule, which can be exchanged with

an oxygen molecule upon redudion of the heme iron (Porter and Coon 1991).

Cytochrome P450-mediated catalysis includes sequential steps of substrate

binding, electron transfer, and oxygen binding (Figure 1.1). Cytochrome P45O­

mediated metabolism requires NADPH-cytochrome P450 reductase which

transfers electrons from NADPH to the enzyme. For oxidative metabolism to

occur, O2 is also required (Koymans, Donne-Op Den Kelder et al. 1993).

Generally, P450s are bound to the endoplasmic reticulum of virtually all

cell types throughout the body. In mammals, the primary organ location of

drug metaboliZing P450s is the liver. Thus, many of the adverse affects due

to bioactivation are exerted in the liver, at or near the site of metabolism.

SOH

-A .. Fe+++ S-OH-Fe+++ ,,, yS

P450 t r F +++S

17

F.e+++-S e - NADPH

6 }- e- CADPH-CylOchrOme P450 H20 ~ Redudase

NADP+ Fe++-S

Fr+++-S ~ 0-2

2 Y ~e~++- 02

e- ~

n NADP+ NADPH

NADPH-Cylochrome P450 Redudase

Figure 1.1: Cytochrome P450 Catalytic Cycle. The scheme for P450

electron transport and substrate oxidation. Fe represents the iron on the heme

prosthetic group, S represents the substrate.

1.2.2 Cytochrome P450 Nomenclature.

The amino acid sequences of the cytochromes P450 investigated to

date have been aligned and classified on the basis of proposed evolutionary

divergence (Nelson, Kamataki et al. 1993), On this basis, P450s have been

placed into 12 families which includes more than 22 subfamilies. In this

18

nomenclature, an Arabic number represents the family and is followed by a

letter denoting the subfamily and another Arabic number indicating the

individual gene within the subfamily. For example, 281 would be the first gene

sequenced from the 8 subfamily of the second family of P450 genes.

The P450 enzymes of primary interest to this research are of the 28 and

3A subfamilies. Cytochromes P450 of the 28 and 3A subfamilies are in

included in two of the three families most important to the metabolism of

xenobiotics (families 1 through 3) (Wrighton and Stevens 1992).

Cytochromes P450 from a single subfamily exhibit greater than 55%

amino acid sequence identity. Such that enzymes within the 28 subfamily

exhibit many similar catalytiC abilities (Henderson and Wolf 1992) (Table 1.1).

The 28 enzymes from the species investigated are listed, with their sequence

similarities noted (Table 1.2).

Changes in the relative concentrations and/or activities of P450s can

be brought about through treatment with a diverse array of compounds.

These compounds can affect many, a few, or a single P450 form (Murray and

Reidy 1990). Experimental manipulations of P450 activities can be used as

tools to study the P450-mediated metabolism of substrates. The effects of

P450 induction and inhibition toward toxicity can indicate whether P450s are

involved in bioactivation or detoxification and can lead to descriptions of

catalytic activities of particular P450s or P450 families.

19

Table 1.1

Selected Cytochromes P450 28 and there relative abilities to metabolize marker substrates.

The relative abilities of selected members of the 28 subfamily of P450 enzymes to metabolize some typical marker substrates is compared on an arbitrary scale, as ++ (highly adive), and other activities ranked based on this as either + (active) or - (not active).

Enzyme

Substrate 281 a

·AD

Test

Prog

7-EC

++

++

++

++

a ref. (Waxman 1988).

+

+

+

+

b ref. (Grimm, Dyroff et al. 1994)

+

eref. (Waxman, Lapenson et al. 1991). d ref. (Duignan, Sipes et al. 1988). e ref. (Oguri, Kaneko et al. 1991) .

. '

+

2811 d 28 e

++ ++

++ ++

+

++

20

Table 1.2

Comparison of amino acid sequence of 28 enzymes from selected species.

The amino acid sequences of selected 2B enzymes were compared to the sequence of 2B1 using Intemational Biotechnologies Incorporated Pustell Sequence Analysis computer program version 2.04 (Kodak Co., New Haven, CT). Amino acid identity indicates percent change of any amino acid at a given position relative to 2B 1, similarity takes into account only those amino acid changes which have different properties from the amino acid in 2B1 at the same location. Differences in amino acids from 2B 1 only in the putative substrate recognition sites (amino acids which potentially come into contact with substrates) (Gotoh 1992; He, Luo et al. 1994) are also noted.

Enzyme

2B1 a 2B2b 2B4c 2B6d

identity 100 97 79 78

similarity 97 86 85

SRS regions 92 73 72

• comparison of amino terminal sequence only. a sequence ref. (Guengerich 1978). b sequence ref. (Ryan, Thomas et al. 1982). c sequence ref. (Gasser, Negishi et al. 1988). d sequence ref. (Mimura, Baba et aJ. 1993). e sequence ref. (Graves, Elhag et al. 1990). f sequence ref. (Oguri, Kaneko et aJ. 1991).

2B11 9 2BGP f.

77 83

97 93

68

21

1.3 REGULATION OF P450 ACTIVITIES

The levels and activities of individual P450 fonns are controlled by both

abundant endogenous and exogenous compounds. Endogenously, P450

levels are controlled by regulatory mechanisms which include hormonal up or

down regulation, particularly in the rat (Ron is and Cunny 1994). Diverse

exogenous compounds have been shown to increase or decrease P450

activities through several different mechanisms (Okey 1990; Knickle and Bend

1992; Murray 1992; Larsen, Brake et al. 1994; Halpert 1995).

1.3.1 Induction.

Induction of P450 activities can be brought about by transcriptional

activation, mRNA stabilization, increased translational efficiency or protein

stabilization (Paine 1990; Waxman and Azaroff 1992; Koymans, Donne-Op

Den Kelder et al. 1993; Kraner, Lasker et al. 1993). Numerous exogenous

compounds, which include 3-methyl cholanthrene, phenobarbital (PB), and

dexamethasone (Dex), can also produce increases in P450 levels (Okey

1990; Hodgson 1994).

Phenobarbital is a commonly used P450 inducer. The levels of several

P450s are increased by 2- to 5-fold after PB induction, with 2B enzymes

exhibiting the greatest increase. The level of P450 2B2 is low but detectable

in uninduced rats, P450 2B1 levels are 15-fold lower, but PB induction

22

increases 281 levels by 100-fold or more and 282 twenty times or more

(Waxman and Azaroff 1992).

Dexamethasone treatment of rats leads to an induction of several

P450s, but the most profound effects are on P450s of the 3A subfamily

(Schuetz, Wrighton et al. 1986; Hammond and Fry 1990; Oaujat; Pichard et

al. 1991). Rats induced with dexamethasone exhibit an increase in 3A mRNAs

as well as a 12-fold induction of 3A activity, as measured by testosterone 6B­

hydroxylation (Gonzalez, Song et al. 1986).

1.3.2 Inhibition.

The administration of many compounds can lead to the inhibition of

P450 activities (Murray 1992). Inhibition can be caused by one of three

different general mechanisms; reversible inhibitor binding, metabolic­

intermediate complex formation with the heme iron, or irreversible binding to

the P450 protein or the prosthetiC heme group (Ortiz de Montellano and Reich

1986). Since competitiye inhibitors block the active site to prevent the access

of the substrate they sterically hinder substrate-enzyme interactions in a

competitive fashion. Irreversible binding can be brought about with the use of

mechanism-based inactivators (suicide substrates). Mechanism-based

inactivators are activated by the target enzyme to products which inactivate

the enzyme without leaving the active site (Ortiz de Montellano 1988).

23

Inhibition is usually competitive and less specific than mechanism-based

inactivation. The formation of metabolic-intermediate complexes with the

heme iron produces a pseudo-irreversible inhibition of the enzyme.

The antibiotic, chloramphenicol (CAP) inhibits the activity of several

P450 forms, including P450s in the 2B and 3A subfamilies (Halpert, Balfour

et al. 1985). Chloramphenicol rapidly and irreversibly inactivates P450 2B1

through the enzymatic production of a reactive oxamyl chloride, which can

bind to a lysine residue in the protein, blocking the essential transfer of

electrons to the enzyme (Halpert, Miller et al. 1985). Studies with CAP

analogues have led to the identification of N-(2-p­

nitrophenethyl)chlorofluoroacetamide (pN02CIFA) as a specific inactivator of

P4502B1 and 282 (Halpert, Jawet al. 1990).

The macrolide antibiotic, troleandomycin inhibits 3A enzymes through

the formation of a metabolic intermediate generated by oxidative metabolism

of the P450, leading to an enzyme-metabOlite intermediate complex

(Delaforge, M.J. Jaquin et al. 1983). The oxidized product of TAO metabolism

forms a stable ferrous heme complex, which renders the P450 non-functional.

The TAO metabolite is unable to move and bind to other P450s, making the

inhibition specific to enzymes which are capable of metabolizing TAO

(Franklin 1991).

24

Isoform-selective inhibitors and mechanism-based inactivators of

cytochromes P450 may be used both in vivo and in vitro to attenuate the

adivities of specific P450s (Halpert 1995). The inhibition of P450s responsible

for the bioadivation of certain compounds has the potential to protect against

toxicity and can aid in the determination of enzyme specificity of xenobiotic

metabolism. Of greater potential is the use of specific enzyme inhibition or

inactivation to allow targeting of enzymes responsible for the metabolism of

particular xenobiotics while allowing alternative detoxification pathways to

prevail (Halpert 1995).

25

1.4 STRATEGIES TO IDENTIFY INDIVIDUAL P450S INVOLVED DRUG

METABOLISM.

To identify specific P450 isoforms involved in the metabolism of

compounds, particularly for toxicants and clinacally used drugs, various

strategies have been used. These strategies usually involve the use of a

group of approaches. These include correlation of metabolic activity toward

the experimental substrate and known marker activities for a specific P450,

correlation with immunochemically-detected levels of the specific P450, the

use of purified or heterologously expressed enzyme, and selective inhibition

or induction of specific P450s.

Chemical inducers can be used to assess the effects of increased

adivity of a subset of P450s toward the bioactivation of toxicants, but a lack

of isozyme specificities limits induction as a sole means of identifying P450

isofonns. Immunoinhibition retains specificity, but the antibodies must have

direct access to the P450 of interest, this prevents the ability to test toxicity in

whole cells. Inhibitors exhibit variable specificities, which are improved in

metabolism-requiring inhibition systems, such as mechanism-based ,

inactivation or metabolite-intemediate complexation.

In order to detennine the role(s) of specific individual P450s, a cross-

section of tests must be used. In this dissertation, indudion, inhibition, marker

26

substrate metabolism, toxicity assessment, and purified proteins have all been

used to identify the P450s involved in cocaine bioactivation in the rat.

1.5 RESEARCH GOALS

The objective of the research presented in this dissertation was to

investigate the ability of isoform-specific mechanism-based inactviators to

identify the rat cytochromes P450 that promote hepatotoxicity through

oxidative metabolism of cocaine. The hypothesis was that cytochromes P450

28 are responsible for the bioactivation of cocaine in the rat which leads to the

production of hepatotoxicity. The objectives were met by; 1) using P450

inducers to determine the roles of inducible P450s in cocaine bioactivation,

and 2) using specific P450 inhibitors and inactivators to confirm the

identification of specific inducible enzymes as involved in the bioactivation.

The results indicated that members of the P450 subfamily 28 are the major

enzymes responsible for cocaine bioactivation. To confirm this identification,

two other methods, marker substrate correlation and the use of purified P450s

28, were used to demonstrate the involvement of specific P450s in cocaine­

oxidation.

27

1.5.1 Manipulation of P450 Enzyme Activities

Experimental manipulations of P450 activities, using induction and

inhibition schemes, were used to show the applicability of these procedures

to determinations of P450-mediated bioactivation. Employing induction and

inhibition protocols, the relative contributions of certain P450s 2B and 3A in

the bioadivation of cocaine were determined. Mechanism-based inactivation,

and metabolic-intermediate complex inhibitions were used both in vivo and in

vitro, demonstrating their potential usefulness for both cell culture and whole

body applications.

1.5.2 Cytochromes P450 Responsible for the Bioactivation of Cocaine.

The bioadivation of cocaine is mediated by members of the cytochrome

P450 (P450) superfamily of phase I enzymes. An understanding of the specific

members of the P450 superfamily involved in the oxidation of cocaine and its

metabolites gives insight into the specific relationships between enzymes and

substrates, and into the roles which P450s can have toward promoting the

hepatotoxicity of parent compound which is only hepatotoxic, without

bioactivation, at very high doses (Jover, Ponsoda et al. 1993).

28

1.5.3 Marker Activities and Purified Proteins

It is essential to verify enzyme activities following inhibition or induction

protocols, and marker activity determinations were used to assess the effects

of the resulting alterations in the activities of the enzymes of interest.

Strict verification of isozyme oxidation of cocaine necessitates the use

of purified or expressed proteins. Once enzyme manipulations led to the

identification of P450s 28 as major cocaine bioactivating forms of cocaine in

rats, the abilities of purified and expressed P450s 28 to N-demethylate

cocaine were confirmed.

29

1.6 COCAINE

Cocaine is an alkaloid derived from the leaves of Erythroxylon coca, a

plant which grows naturally in South America. Cocaine was used as much as

1350 years ago by South American Indians, probably to help combat altitude

sickness and fatigue (Mallat and Dhumeaux 1991). In the United States,

cocaine was formerly used as a local and regional anesthetic, but its clinical

use is now limited to rare cases for eye surgery due to its toxic and abuse

potential (Loper 1989). Cocaine has extensive euphoric and stimulant effects

when taken through a variety of routes, including intravenous or intranasally.

During the last twenty years, the incidence of cocaine abuse in the United

States has shown some dramatic increases separated by periods of shallow

decline (Jatlow 1987). The increase in cocaine use in this country is probably

due to lower cost and ease of procurement. Cocaine has been shown to cause

a number of medical complications, including; myocardial infarctions,

hyperpyrexia, arrhythmias, hemorrhages, ischemias, seizures and

rhabdomyolysis (Ennen hom and Barcelous 1988; Roth, Alarcon et al. 1988).

Treatments for cocaine abuse are controversial, and no known treatment is

effective for crack cocaine abuse (Higgins, A.J. et al. 1994).

30

1.6.1 Cocaine Hepatotoxicity.

The liver toxicity of both acute and chronically administered cocaine in

laboratory animals is well documented (Thompson, Shuster et al. 1979; Kloss,

Rosen et al. 1984; Shuster, Garhart et al. 1988). Evidence that chronic

cocaine use may also be associated with abnormal liver function in humans

has also been reported (Marks and Chapple 1967; Perino, Warren et al.

1987; Wanless, Dore et al. 1990; Kanel, Cassidy et al. 1991). In rodents

marked elevations of serum transaminases and liver histology changes have

been observed (Mallat and Dhumeaux 1991). Although all rodent studied have

been highly susceptible to cocaine hepatotoxicity, the extent of susceptibility

and the topography of histological necrosis vary according to species, strain

and sex (Thompson, Shuster et al. 1979; Evans 1983; Thompson, Shuster et

al. 1984). An early discovery was that severity and extent of cocaine-mediated

hepatotoxicity correlated with the extent of oxidation by liver microsomes,

increased by P450 inducing agents, and decreased following the use of P450

inhibitors (Evans and Harbison 1978; Evans 1983; Kloss, Rosen et al. 1984;

Thompson, Shuster et al. 1984; Shuster, Garhart et al. 1988)

1.6.2 Cocaine Bioactivation.

The major route of cocaine metabolism in man is through

cholinesterase and auto-hydrolysis that results in the production of ecgonine,

31

benzoylecgonine, and ecgonine methylester (Inaba, Stewart et al. 1978).

These products of cocaine hydrolysis are believed to be non-psychoactive and

non-toxic (Kambam, Mets et al. 1992; Jeong, Jordan et al. 1995). In man,

these metabolites comprise about 90% of the excreted dose of cocaine

(Evans 1983; Jeong, Jordan et al. 1995). Inhibition of esterase activity leads

to increased levels of hepatotoxicity following cociane exposure in mice,

probably by relocating cocaine metabolism to the cytochrome P450 system

in the liver (Hoffman, Henry et al. 1992; Kambam, Mets et al. 1992).

The hepatotoxicity of cocaine is proposed to be mediated through a

sequential oxidation of cocaine in the liver (Shuster, Garhart et al. 1988). The

increased hepatotoxicity of cocaine following P450 indudion is consistent with

the proposal that an inducible form of P450 may bioactivate cocaine.

Treatment with ethanol significantly increases cocaine hepatotoxicity in mice

(Lieber 1990; Boyer and Petersen 1991), but the major ethanol-inducible form

of P450, P450 2E1, has not been shown to be involved in the metabolism of

cocaine (Boelsterli, Atanasoski et al. 1991). Recent evidence for the novel

production of an ethanol ester of cocaine may be responsible for the

potentiation of hepatotoxicity resulting from administration of these two

compounds (Hearn, Rose et al. 1991; Dean, Harper et al. 1992; Roberts, L.

et al. 1992).

32

Investigations have suggested that cocaine undergoes cytochrome

P45O-mediated bioactivation to produce hepatotoxicity in laboratory animals

(Shuster, Garhart et al. 1988). This bioactivation consists of a series of

sequential oxidations possibly carried out by several P450s and flavin­

containing monooxygenases (Boelsterli and Goldlin 1991). First, cocaine is

N-demethylated to norcocaine by cytochrome P450. Norcocaine is then

oxidized by a flavin-containing monooxygenase to N-hydroxynorcocaine,

which is metabolized by a P450 to norcocaine nitroxide. This pattern of

metabolism produces toxicity through an unknown mechanism that may

involve the depletion of cellular reducing equivalents during redox cycling of

cocaine metabolites or the produdion of another, as yet unidentified, reactive

metabolite (Rauckman, Kloss et al. 1982; Shuster, Garhart et al. 1988;

Boelsteni and Goldlin 1991; Boelsteni, Wolf et al. 1993; Goldlin and Boelsteni

1993; Lloyd, Shuster et al. 1993) (Figure 1.2). The mechanism of toxicity in

human liver is likely to involve a similar metabolic scheme (Roberts, Harbison

et al. 1991; LeDuc, Sinclair et al. 1993).

The muHi-step mechanism of cocaine bioactivation provides a system

in which the nature of P450-mediated oxidation leading to hepatotoxicity

incorporates many questions regarding enzyme structure/activity

relationships. The study of the selectivity of P450 isoforms toward the

bioactivation of cocaine and its metabolites provides examples of P450

33

specificities and the toxic consequences of metabolism by enzymes exhibiting

variations in strain and individual expression.

H3~C'N ___ Non-enzymaticHydrolysis

I I ~H3 _-- Enzymatic Hydrolysis

O~-o~ Nitrosonium Ion?

FMO ,/ ~ ~ I l

_ ~450

H3C -J -.f H 'N Norcocaine I , P450

..... -------.. ~ ~450 FMO~

N-Hydroxynorcocaine

.. Norcocaine Nitroxide /

HO O· i

~N P!50 _ ~ FMOI ~c

P450 Reductase

Figure 1.2: Bloactlvation scheme for cocaine. Cocaine undergoes sequential oxidations in the liver, leading to hepatotoxicity. The steps indicated by dotted arrows are proposed. Steps indicated by solid arrows lead to metabolites which have been isolated.

CHAPTER 2

MECHANISM-BASED INACTIVATION OF CYTOCHROMES P450 2B PROTECT AGAINST COCAINE­

MEDIATED TOXICITY IN RAT LIVER SLICES

2.1 BACKGROUND

34

Because of the enhancement of cocaine toxicity obtained with PB pre-

treatment, a possible role of P450 2B 1 in cocaine metabolism was recently

investigated (Boelsterli, Lanzotti et al. 1992). A polycJonal rabbit anti-rat 28

antibody significantly decreased the rate of cocaine N-demethylation in liver

microsomes from P8-treated rats, and a correlation between phenobarbital

pretreatment and cocaine-mediated cytotoxicity in isolated hepatocytes was

observed. While evidence was presented that compounds which inhibit P450

281 catalytic activity also inhibit cocaine N-demethylation and covalent

binding in liver microsomes, the ability of the inhibitors to protect against

cocaine-mediated toxicity in hepatocytes was not assessed. Therefore, no

direct relationship between P450 281 and hepatotoxicity of cocaine could be

demonstrated. In addition, the experimental design could not differentiate

P450 281 from P450 2821 (8oelsterli, Lanzotti et al. 1992).

Chloramphenicol and pN02CIFA were used to substantiate the

identification of P450 281/2 as P450s involved in cocaine bioactivation.

35

Specific inactivation of cytochromes P450 28 ~lIowed direct assessment of

the role of these enzymes in cocaine-mediated hepatotoxicity. Since in tissue

slices the stratification of cells is maintained, and normal cellular order and

associations are not disrupted, biotransformation and subsequent toxic injury

to the tissue can be measured (Smith, McKee et al. 1989). Either rats or slices

from single rat livers were treated with the inadivators and slices were treated

with cocaine and assessed for toxicity by the loss of intracellular K+. In this

study, inadivation of P450 281/2 and the subsequent cytotoxicity of cocaine

were determined.

1Cytochromes P450 281 and 282 ixhibit greater than 97% amino acid sequence identitiy (Suwa, Mizukami et al. 1985) and generally exhibit similar substrate specificities (Henderson and Wolf 1992). Both enzymes primarily metabolize androstenedione at the 16B position, although P450 281 is approximately 1O-fold more active than 282 (Waxman, Ko et al. 1983; Waxman and Azaroff 1992). In liver microsomes from PB-induced rats of most strains including Lewis, P450 281 is the more prevalent fonn. Neither CAP nor pN02CIFA distinguish completely between P450 281 and 282, although 281 is more rapidly inactivated in vitro (Halpert, Jaw et al. 1990). In this chapter, no distinction is made between the tenn P450 28 and P450 281/2.

36

2.2 METHODS

2.2.1 Chemicals

Sodium phenobarbital was obtained from Mallinckrodt, Inc. (St. Louis,

MO). Waymouth's Medium MB 75211 powder (without sodium bicarbonate)

was purchased from Gibco Laboratories (Grand Island, NY). HEPES (N-2-

Hydroxyethyl-piperazine-N'-2-ethanesulfonic acid) was purchased from

Calbiochem (La Jolla, CA). Gentamicin sulfate, cocaine hydrochloride, and

chloramphenicol were obtained from Sigma Chemical Co. (5t Louis, MO).

N(2-Jrnitrophenethyl)chlorofluoroacetamide was synthesized as previously

described (Halpert, Jawet al. 1990). All other chemicals were obtained from

Sigma Chemical Co. (St Louis, MO.)

2.2.2 Animals

Adult male Lewis (Harlan Sprague-Dawley, Inc., Indianapolis, IN.) and

Munich Wistar (WM) rats (Simonsen Laboratories, Inc., Gilroy, CA) weighing

from 200 - 250 g, were fed Wayne Lab Chow and water ad libitum. Animals

were housed 3 per cage on metal mesh with a 12 hr light/dark cycle. Hepatic ,

mixed-function oxidases were induced by treating the animals with sodium

phenobarbital, 0.1 % (w/v) in drinking water for 4 days, 5 days prior to the

experiments. The phenobarbital solution was replaced with fresh water 18 hr

37

prior to the experiments. For in vivo inactivation, 300 mg/kg CAP or 200

mg/kg pN02CIFA in 0.8 ml propylene glycol was injected intraperitoneally, 2

hr before slice preparation.

2.2.3 Slices

Animals were killed by cervical dislocation. Livers were excised and

rinsed in cold Krebs-HEPES buffer. A sharpened metal tube of 0.8 em

diameter was used to core cylindrical tissue plugs from the liver lobes. Tissue

slices, approximately 250 ~m thick, were prepared using a mechanical tissue

slicer (Brendel, Gandolfi et al. 1987), in cold Krebs-HEPES buffer-(pH 7.4).

Slices were placed in cold Waymouth's media until incubation (less than 15

min.).

2.2.4 Incubation

The liver slices were placed on a polypropylene mesh, two per screen.

The saeens were fitted into the mouth of a 20-ml scintillation vial containing

2.5 ml HEPES-buffered Waymouth's medium supplemented with 84 ~g/ml

gentamicin. The scintillation vials were loaded horizontally on a heated (37°C)

vial roller rack, which rotated at 2 rev/min. Slices were pre-incubated for 2 hr,

to allow them to become equilibrated with the system. Stock solutions of CAP

38

or pN02CIFA dissolved in dimethyl sulfoxide (DMSO) were added to the

medium (1 % v/v). After 1 hr the medium was replaced with fresh medium. To

remove all inhibitor and DMSO, medium was replaced 2 additional times at

15 "min intervals. Fifteen minutes after the final wash, cocaine, dissolved

directly into the medium, was added at final concentrations between 100 and

1000 ~M.

2.2.5 Intracellular K+ Content

Slices were removed from incubation, blotted, and placed individually

in 1 ml distilled water. Slices were sonicated to homogeneity (Model 350,

Branson Sonic Power, Danbury, Conn.). Protein content was determined with

the BCA assay from Pierce (Rockford, IL.), using bovine serum albumin as a

standard. Proteins were precipitated by the addition of 20 ~I of 70% perchloric

acid. The samples were centrifuged for 5 min at 1200 x g (Beckman Microfuge

B, Palo Alto, CA). The supernatant fradion was assayed for K+ using a Perkin

Elmer flame photometer (Model CA-51 , Danbury, CN.). A K+ standard curve

was produced from a 40 mM stock solution of KCI in distilled H20.

2.2.6 Microsomes

Slices were frozen in liquid nitrogen and maintained at -70°C.

Microsomes were prepared from the slices by differential ultracentrifugation

39

much as described previously (Graves, Kaminisky et a!. 1987). Each

microsomal preparation was from two slices from a single vial (approximately

25 mg wet weight), which were placed in buffer (250 mM sucrose, 1 mM

(ethylenedinitrilo)-tetraacetic acid disodium salt (EGTA» and sonicated on ice

to homogeneity.' The homogenate was centrifuged first at 1 0,000 x g for 15

min, then the supernatant was again centrifuged at 100,000 x g for 60 min.

The pelleted microsomes were washed once in buffer (50 mM potassium

phosphate buffer, pH 7.4 with 0.2 mM EGTA), centrifuged at 100,000 X g for

60 min and resuspended in 400 ~I of buffer (10 mM Tris-acetate, 1 mM EDTA,

and 20% glycerol with 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4), and

stored at -70°C.

2.2.7 Catalytic Assays

Androstenedione hydroxylase activity was determined as previously

described (Graves, Kaminisky et a!. 1987). Briefly, 10 ~g of microsomal

protein was incubated with 25 ~M AD in 1 00 ~I at 37°C. The reaction was

started by the addition of NADPH and was allowed to proceede for 5 min,

before the addition of tetrachloracetic acid to stop the reaction.

Androstenedione metabolites were resolved by thin layer chromatography

with two cycles of development in ethylacetate:chloroform (2:1 v/v) , and

visualized by autoradiography. Metabolite bands were identified with

40

standards and quantitated by scintillation counting. The N-demethylation of

cocaine was measured spectrophotometrically through the formation of

formaldehyde (H2CO) by a method modified from Nash (Nash 1953). Briefly,

0.4 to 0.5 mg miaosomal protein was incubated with 0.2 mM cocaine and an

NADPH generating system for 25 min at 37°C in 0.4 ml total volume, and

H2CO production was determined upon addition of the Nash reagent and

compared with a standard curve.

2.2.8 Statistical Analysis

Data were analyzed by a one-way analysis of variance and differences

from controls determined with the Newman-Keuls test. Values are considered

statistically significant at p < 0.01.

41

2.3 RESULTS

2.3.1 Cocaine Toxicity

Cytochromes P450 2B 1 and 282 are constitutively expressed at very

low levels and are induced 50-100-fold, and 20-fold by phenobarbital,

respectively (Guengerich, Dannan et al. 1982; Waxman and Azaroff 1992).

Indirect evidence for 281/2 bioactivation of cocaine comes from the great

increase in toxicity observed following P8 induction (Boelsterli, Lanzotti et al.

1992). Intracellular K+ content is commonly used to assess toxicity in tissue

slices. Because a high t<+ gradient is maintained in healthy cells by the Na+/K+

ATPase system, efflux of K+ provides an early and accurate indication of

cytotoxicity (Azri, Gandolfi et al. 1991). Accordingly, K+ loss was determined

in liver slices from PB-pretreated and non-pretreated rats. In experiments with

liver slices from non-induced Lewis rats, the K+ loss following exposure to 500

fJM cocaine was not statistically significant (173.4 ± 25.2 nmol K+/mg protein

in cocaine-exposed slices, 220.0 ± 33.2 nmol K+/mg protein in controls, n =

4 slices from a single animal). In contrast, in slices from PB-induced animals,

cocaine toxicity was manifested within 4 hr and K+ was essentially at its

minimal level 6 hr after exposure to 500 fJM cocaine. Previously, similar

cocaine-mediated toxicity in rat liver slices was demonstrated following PB

pretreatment of Sprague Dawley rats and cocaine-mediated toxicity was not

evident without PB pretreatment (Connors, Rankin et al. 1991).

42

A concentration-response curve was established for K+ loss in liver

slices from induced rats following exposure to cocaine concentrations of 100,

250, and 500 ~M. After 6 hr the K+ content remaining in the slices from PB-

induced rats exposed to 500 ~M cocaine was 26% of control, and after 9 hr

the K+ level was not significantly lower (22% of control) (Table 1). Therefore,

all subsequent experiments were carried out for 6 hr.

Table 2.1. ~ Retention in liver slices fol/owing exposure to cocaine

Cores were punched in the livers and tissue slices cut using a mechanical slicer. Slices were incubated in Waymouth's media on a rOiling rack at 37°C. Cocaine was added at the concentrations indicated. Potassium contents of individual slices were determined at the times indicated. Data are expressed as nmol K+ Img protein ± SO for n = 6 slices from one PB-induced rat. *Significant difference from noncocaine-exposed control, p < 0.01.

nmol K+/mg protein

Cocaine concentration (~M)

hr control 100 250 500

3 305 ± 8.04 299± 7.9 245 ±4.65 223 ± 16.5*

6 296 ± 33.8 263 ±25.9 195 ± 32.3* 79.0 ± 17.7*

9 314 ± 10.4 233 ± 23.3* 152 ± 13.9* 67.8 ± 13.9*

43

2.3.2 In vitro inactivation of 281/2 and protection against cocaine toxiCity.

Chloramphenicol inactivates a number of PB-inducible cytochromes

P450 (Halpert, 8alfour et al. 1985) .. Therefore, the ability of CAP to protect

against the toxicity of 1 mM cocaine in slices was evaluated at inhibitor

concentrations of 100, 250, or 500 ~M (Figure 2.1). Pretreatment of the slices

with CAP decreased cocaine-mediated K+ loss, even at the lowest

concentration. In DMSO vehicle pretreated slices, the K+ content remaining

6 hr after cocaine exposure was 24% of non-cocaine exposed vehicle­

pretreated slices. One-hundred ~M CAP reduced the extent of cocaine

mediated K+ loss, such that the remaining K+ was 60% of non-cocaine treated

controls, and 500 ~M CAP completely blocked cocaine-mediated K+ loss

after 6 hr. Chloramphenicol itself caused no K+ loss at any concentration

tested.

To assess the relationship between P450 inactivation and cocaine

toxicity, microsomal androstenedione hydroxylation rates were determined

following in vitro treatment with CAP. Androstenedione is specifically

hydroxylated at the 16B position by P450 281/2, while hydroxylation at the 6B

position is indicative of P450 3A1/3A2 activity (Waxman, Ko et al. 1983).

Cytochromes P450 281,282, 3A1 and 3A2 are all phenobarbital inducible

(Waxman and Azaroff 1992). Microsomes were made from liver slices from

PB-induced rats treated with 100, 250 and 500 ~M CAP. A concentration-

44

response for inhibition of androstenedione 16B-hydroxylation (16B-OH) by

CAP was demonstrated (Figure 2.2.A). The extent of inactivation of AD 1611.­

OH correlated with the level of protection against K+ loss (Figure 2.2.B).

The initial P450-mediated oxidation of cocaine produces norcocaine

through N-demethylation. The concomitant production of formaldehyde was

used to measure the first step in the metabolism of cocaine in microsomes

from slices. As with androstenedione 16B-OH, the rate of cocaine N­

demethylation significantly decreased with in vitro CAP pretreatment, from 2.1

± 0.31 nmollminlmg in controls to 0.76 ± 0.16 nmol/minlmg after treatment

with 500 ~M CAP (p < 0.01).

In a separate experiment, treatment of slices from PB-induced rats with

100 ~M CAP or 250 ~M of the P450 2B1/2-specific CAP analogue,

pN02CIFA, protected against toxicity following exposure to 500 ~M cocaine

(Figure 2.3.A). Potassium levels in CAP, pN02CIFA, or vehicle pretreated,

noncocaine-exposed controls remained constant over the incubation period.

At the time of cocaine addition, K+ levels were ~75 ± 33.3, 261 ± 19.5, and

274 ± 20.3 for vehicle, CAP and pN02CIFA, respectively. Chloramphenicol

treatment resulted in a reduction in microsomal androstenedione 16B­

hydroxylation (16B-OH) to 35% of control, while pN02CIFA treatment

decreased the formation of 16B-hydroxyandrostenedione to 51% of control.

45

2.3.3 In vivo inactivation of 281/2 and protection against cocaine toxicity

For clinical utility. inhibitors of cytochromes P450 must be specific and

must function in vivo. Pretreatment of the rats. 2 hr before sacrifice. with

either 300 mg/kg CAP or with 200 mg/kg pN02CIFA totally blocked cocaine­

mediated toxicity measured in slices (Figure 2.3.8). At the time of cocaine

addition K+ levels were 320 ± 30.S • 319 ± 22.4. and 312 ± 1S.2 for propylene

glycol. CAP. and pN02CIFA. respectively. Microsomes prepared from slices

from the same experiments showed a typical androstenedione metabolite

profile. In confirmation of previous work (Stevens and Halpert 1988).

intraperitoneal treatment with CAP. 2 hr before the experiment. resulted in a

substantial decrease in androstenedione 1SB-OHase activity (to 31 % of

control). Treatment with CAP has also been demonstrated to cause a

decrease in androstenedione SB- and 1Sa-OH (Stevens and Halpert 1988).

Androstenedione 16a-hydroxylation is indicative of P450 2C11 as well as 281

adivity (Waxman 1988). and 2C11 is also inhibited by CAP (Halpert. Balfour

et al. 1985). Following treatment with pN02CIFA. a specific decrease in

androstenedione 1SB-hydroxylation has been described. with no effect on SB­

OH (Halpert. Jaw et al. 1990). In microsomes made from liver slices from rats

treated in vivo with pN02CIFA. androstenedione 1SB-OHase activity was

specifically reduced (to 39% of control). confirming the selectivity of the

46

inactivators in this system (data not shown).

In vivo treatment of PB-induced rats with either pN02CIFA or CAP also

resulted in a decrease in the rate of cocaine N-demethylation in the

microsomes made from the liver slices. Treatment with either of these

compounds resulted in an approximately 60% lower rate of cocaine N­

demethylation compared with vehicle-treated controls (three microsomal

preparations from a single rat for each inactivator treatment). Thus, treatment

with either pN02CIFA or CAP obstructed the first step in liver microsomal

metabolism of cocaine following in vivo administration.

2.3.4 Cytochrome P450 2B1/21nactivation with pN02CIFA

Upon direct treatment of microsomes with pN02CIFA, most of the

androstenedione 16B-hydroxylase activity is lost (Halpert, Jaw et al. 1990;

Kedzie, Balfour et al. 1991). Therefore, the consistent lack of inactivation of

a significant portion of androstenedione 16B-OHase activity with pN02CIFA

following treatment of slices or in vivo treatment was intriguing. An attempt

was made to reduce P450 2B1/2 activity with in vivo pN02CIFA below about

40% of control. Rats were injected either once or twice, with 200 mg/kg

pN02CIFA, the first injection 4 hr prior to sacrifice and the second 2 hr later.

In the single injection group, androstenedione 16B-OH was reduced to 45%

47

of control, and after 2 doses androstenedione 16B-OH was reduced to 41 %

of control. Two doses of pN02CIFA was thus not significantly more effective

in inactivating P450 281/2 than one dose. Likewise, the rate of cocaine N­

demethylation was not significantly different in the two dose group (Figure

2.4).

2.3.5 Cocaine Toxicity in WM Rat Liver Slices

To provide further evidence for a major role of P450 281 as opposed

to P450 282 in cocaine bioadivaiion, hepatotoxicity was determined in slices

from P8-treated WM rats, which lack P450 282 (Rampersaud and Walz

1987). In WM slices treated with 0.5 mM cocaine, K+ levels were reduced to

31.8 ± 6.9% of control after 6 hr (p < 0.01) and to 23.5 ± 1.4% of control (p <

0.01) after 9 hr (n = 4 slices from a single PB-induced rat). Likewise, liver

microsomal cocaine N-demethylation was the same in both strains (2.8 ± 0.5

in Lewis and 2.7 ± 0.3 nmol/minlmg in WM rats, n = 3 preparations from

individual animals for each strain). Hence the rate of N-demethylation and

toxicity of cocaine were the same in P8-treated rats of the two strains,

regardless of the lack of P450 282 in the WM rats.

48

300

250 c: .-m 200 ..... e a. 0) 150 E ........

~ 100 -0 E c: 50

o o 1 2 3 4 5 6

Time after Cocaine Exposure (hr)

Figure 2.1: Effect of preincubation with CAP on Intracellular K+ loss In liver slices from a PB·lnduced Lewis rat following in vitro exposure to cocaine. Slices were preincubated with OM SO ( •• 0) (final volume 1% w/v). 100 ~M CAP (~). 250 ~M CAP (0). or 500 ~M CAP ( •• 0). To remove unbound inactivator. after 1 hr the media was replaced with fresh media (at 37°C) three times at 15 min intervals. Slices were exposed to 1 mM cocaine (open symbols) or left unexposed to cocaine (filled symbols). Values are expressed as x :i: SO of n = 4 slices from a ~ingle rat. *Significant difference from noncocaine-exposed controls. p < 0.01.

~ 110 -,--------------, 110 c:: - 100 100 a a

. - J:; 90 90 co c:: 80 80 - a ~ 0 70 70 a a.. 60 60 -c « 50 ~t) 40 :; a 30 30 c:!! c:: 20 20 <0 ~ A ~ ~ 10 10 o -1---~-__,_--_r_-_r--__1 a

a 100 200 300 400 500 CAP Concentration (IJM)

~ 240 C')

E 200 ........ c:: .- 160 E :::::

120 a E

80 c:: "'-"

+~ 40 B 0

234 567 8

49

==-e ..... c:: 8 Q) c:: + .-

~m o 8 a c:: '#-"'-"

Androstenedione 16B-OH (nmol/min/mg) Figure 2.2: Comparison of toxicity In slices and AD 16B-OH In mlcrosomes made from slices Incubated In parallel. (A) Microsomes were made from slices which were preincubated with 0, 100, 250, or 500 IJM CAP. Microsomes were incubated with NADPH and androstenedione for 3 min at 37°C to determine the rate of AD 16B-OH formation (.). Values represent % of non-cap-treated controls. Protection against K+ loss in the slices, 6 hr after exposure to 1 mM cocaine, is also noted (e). Three microsomal preparations were made from each treatment. Two slices from a single scintillation vial were used to prepare each microsomal preparation by ultracentrifugation. (8) The means of the values for AD 16B-OH formation plotted against the means for K+ content of slices, 6 hr after exposure to 1 mM cocaine (r2 = 0.977).

50

..-.... 100 "'0 CD 80 en 0 60 c.. X 40 0).

0) 20 A c .- 0 ctS U 0 100 U

I 80 c 0 60 c ~ 40 0 "'-" 20 + ~ 0

0 1 2 3 4 5 6

Time (hr) Figure 2.3: Toxicity in slices exposed to cocaine after in vitro or in vivo inactivator pretreatment. (A) Slices were pretreated with inactivator prior to exposure to cocaine. Slices were incubated with OMSO vehicle (e), 100 IJM CAP (_), or 250 IJM pN02CIFA (.6.) for 1 hr and the inactivator was removed as described in the text. Slices were exposed to 0.5 mM cocaine. Intracellular K+ content of the liver slices was then determined at the times indicated. Values represent % x ± SO of noncocaine-exposed slices pretreated with the corresponding inactivator or vehicle of n = 4 slices from a single rat.(B) Toxicity in slices exposed to cocaine after in vivo treatment with the inactivators. Rats were pretreated, 2 hr before sacrifice, with propylene glycol (8),300 mg/kg CAP (_), or 200 mg/kg pN02CIFA (.6.). Slices were exposed to 0.5 mM cocaine. Values are expressed as % of x ± SO for four slices from one rat. ""Significant difference form noncocaine-exposed controls, p < 0.01. Similar results were obtained in two separate experiments.

14 C)

E 12 --­c: .-E 10

CD :!:! (5 8 ..c CO

CD 6 E -o 4 E c:

2

o -'----7a 6B 16B 16a

Androstenedione

Metabolite

51

14

12

10

8 -o

6 E c:

4

2

_---L... 0

Figure 2.4: Inactivation of P450 281 in microsomes after one or two Injections with pN02CIFA. Lewis rats were injected with propylene glycol vehicle (_), 200 mg/kg pN02CIFA 2 hr before sacrifice (0) or two doses of 200 mg/kg pN02CIFA 2 and 4 hr before sacrifice (r.:a). Microsomes were prepared from the livers of n = 3 rats for each treatment and androstenedione hydroxylation rates and HCOH production from cocaine N-demethylation detennined. ·Significant difference from vehicle controls, p < 0.01.

52

2.4 DISCUSSION

Several prior investigations have revealed that PB induction of hepatic

P450 enzymes greatly increases the toxicity of cocaine in rat liver (Connors,

Rankin et al. 1991; Boelsterli, Lanzotti et al. 1992). This effect of PB induction

was the impetus for investigating a role for P450 2B1/2 in the bioactivation of

cocaine. Chloramphenicol, a mechanism-based inactivator of several PB­

inducible cytochromes P450, and the 2B1/2-specific analogue, pN02CIFA

(Halpert, Jaw et al. 1990), were used to assess in a rigorous fashion the

relationship between the activity of P450 2B forms and cocaine toxicity.

Androstenedione oxidations performed in microsomes made from slices

treated with the inactivators in parallel to assessing toxicity provided the

opportunity to verify the selectivity of the compounds and to quantitate the

relationship between P450 2B 1/2 activity and cocaine toxicity.

In vitro treatment with CAP protected against cocaine toxicity in a

concentration-dependent manner. A similar inactivator-concentration effect

was seen with androstenedione 16B-hydroxylation, a marker for P450 281/2

activity. Thus, a direct relationship between 2B activity toward

androstenedione and cocaine hepatotoxicity was observed, and this

relationship mimicked the effect of CAP on cocaine N-demethylation, giving

further evidence for a P450-mediated process in cocaine hepatotoxicity. The

53

efficacy of pN02CIFA in blocking cocaine-mediated toxicity provides ever

stronger evidence for 281/2 as major cocaine-bioactivating enzymes.

In order to distinguish P450 281 from 282, experiments were perfonned

in WM rats. No P450 282 protein is found in WM rats (Ram persaud and Walz

1987), making this strain an ideal model for studying P450 281 in the absence

of P450 282. The lack of P450 282 in WM rats coinciding with the identical

toxicity of cocaine in slices from the Lewis and WM rats strongly implicates

P450 281 as the major enzyme responsible for the cocaine-mediated

hepatotoxicity in both strains, but does not rule out 282 as a contributor to

toxicity in Lewis rats.

An interesting observation was that complete protection against toxicity

resulted from treatment with inactivator at doses that decreased the rate of

androstenedione 16B-OH to no lower than 35% of the original enzyme activity.

The ability to protect against cocaine toxicity while only inactivating a portion

of 28112 catalytic activity may simply be due to the presence of protective

mechanisms such as glutathione conjugation. Altematively, the maximal

decrease in K+ was only to approximately 20% of control, suggesting a

possible effect of liver lobule morphology, such that a residual population of

cells is not exposed to cocaine. Previously, it was noted that additional

inactivation of P450 281/2 could be obtained after in vitro incubation with

54

pN02CIFA of microsomes made from rats treated in vivo with the compound

(Halpert, Jaw et al. 1990). The inability of pN02CIFA to inadivate all of the

P450 281/2 when administered in vivo or to the slices, in contrast to the

almost complete inadivation observed upon in vitro treatment of microsomes

may also be due to the presence of a subset of hepatocytes which are not

exposed to pN02CIFA in vivo or in liver slices and thus contain a population

of P450 281/2 that is not subject to inactivation.

In human liver microsomes, members of the P450 3A subfamily were

recently reported to metabolize cocaine to norcocaine (LeDuc, Sinclair et al.

1993). However, 3A enzymes were not found to be involved in the further

steps of hepatic cocaine oxidation. The correlation found in the present study

between cocaine N-demethylation and androstenedione 16B-OH suggests

that P450 28 rather than 3A forms are involved in the metabolism of cocaine

to norcocaine in rats. Whether 28112 are also involved in later oxidation steps

of norcocaine has not yet been determined in rats.

55

CHAPTER 3

PARTICIPATION OF CYTOCHROMES P450 2B AND 3A IN COCAINE TOXICITY IN ISOLATED RAT HEPATOCYTES

3.1 BACKGROUND

It has been proposed that P450 2B enzymes catalyze cocaine N-

demethylation in rats (Boelsterli, Lanzotti et al. 1992), and a correlation

between cocaine-mediated toxicity and P450 2B marker activities in rat

liver slices was observed (in chapter 2; (Poet, Brendel et al. 1994).

However, human P450 3As have recently been suggested to playa role in

cocaine N-demethylation (LeDuc, Sinclair et al. 1993), and inhibition of

P450 3A enzymes has been shown to attenuate cocaine-mediated toxicity

in hepatocytes isolated from mice (Pellinen, Honkakoski et al. 1994). Since

previous studies by this laboratory have indicated that 2B enzymes may not

be the sole enzymes responsible for cocaine bioactivation, we investigated

the contributions of cytochromes P450 3A and 2B in cocaine toxicity in

isolated rat hepatocytes.

The use of hepatocytes for toxicity and metabolic studies has

increased in the last decade (McQueen and Williams 1987). Methods have

been developed to isolate intact hepatocytes, the cells which are the

primary constituent of liver. In monolayer culture, these cells maintain their

56

epithelial morphology. Allowing for a 2 hr attachment period results in the

maintenance of the healthy cells, and the loss of cells damaged during the

isolation. By the end of this 2 hr period, hepatocytes have restored much of

their morphology and functionality (McQueen 1989). Cells in monolayer

cultures can be maintained for days.

Although in is well known that the cytochrome P450 content and

profiles change in hepatocytes culture (Guzelian, Bissell et al. 1977;

Steward, Dannan et al. 1985), the qualitative metabolism of substances in

this system has been shown to closely resemble the metabolism observed

in vivo (McQueen 1989). Levels of rodent cytochromes P450 can drop

substantially within the first 24 hr after hepatocyte isolation (Bassell and

Guzelian 1980). Furthermore, differential decline in cytochromes P450 has

been demonstrated to depend on the species and culture conditions

(Maslansky and Williams 1982; Wortelboer, de Kruif et al. 1991).

However, in vivo induction prior to hepatocyte isolation has been shown to

aid in the maintenance of P450 activities for 24 hr in culture (Hammond and

Fry 1990). Accordingly, isolated rat hepatocytes were used to determine

the effects of PB or Dexamethasone (Dex) induction on cocaine and

norcocaine toxicity as well as the abilities to protect against toxicity using

2B or 3A inhibition (CAP and TAO, respectively).

3.2 MATERIALS AND METHODS

3.2.1 Chemicals.

Sodium phenobarbital was obtained from Mallinckrodt, Inc. (St.

57

Louis, MO). Williams' Medium E, Hank's balanced salt solution, insulin,

fetal bovine serum, and gentamicin were purchased from Gibco-BRL

(Grand Island, NY). Hepes (N-2-hydroxyethyl-piperazine-N'-2-

ethanesulfonic acid) was purchased from Calbiochem (La Jolla, CA).

Cocaine and norcocaine were obtained from the National Institute on Drug

Abuse (Research Triangle Park, NC). [4-14C]Androst-4-ene-3,17-dione

(53.9 mCi/mmol) was purchased from Dupont-New England Nuclear

(Boston, MA). Thin layer chromatography plates (silica gel, 250 ~M, Si 250

PA (19C» were obtained from J.T. Baker, Inc. (Phillipsburg, NJ). All other

chemicals were of reagent grade and obtained from Sigma Chemical Co

(St. Louis, MO).

3.2.2 Animals.

Adult male Sprague-Dawley rats (Harlan Sprague Dawley, Inc.

IndianapoliS, IN) weighing from 250-300 g were given food and water ad

libitum and housed three per cage on metal mesh with a 12-hr light/dark

cycle. Phenobarbital induction of hepatic mixed-function oxidases was

performed by treating the animals with sodium phenobarbital (PB), 0.1 %

58

(w/v) in drinking water for 4 days, 5 days prior to the experiments (Halpert,

Balfour et al. 1985). Dex induction was by intraperitoneal injection with 100

mg/kg Dex in 2% Tween 20 for 4 days, 5 days prior to the experiments

(Daujat, Pichard et al. 1991).

3.2.3 Isolated Hepatocyte Culture.

Hepatocytes were isolated using a two-step in situ perfusion with

collagenase as described previously (McQueen 1989). The liver is first

perfused with Ca ++ - and Mg ++ -free Hanks balanced salt solution containing

0.5 mM ethylene glycol bis {B-amino-ethylether)-N, N-tetraacetic acid

(EGT A) to prevent the accumulation of blood clots. The second perfusion

step consists of William's Media E containing 50 J,Jg/ml gentamicin and

collagenase. The animals were anesthetized with phenobarbitone prior to

cannulation of the portal vein, through which the solutions are perfused.

The subhepatic inferior vena cava is cut to allow drainage until the thoracic

vena cava can be severed. Following in situ perfusion, the liver is removed.

Following dissociation of the capsule the hepatocytes are separated by ,

straining the liver through layers of gauze, washing with William's media E

supplemented with 5% fetal bovine serum (FBS), 50 J,Jg/ml gentamicin, and

2 J,Jg/m I insulin. The cell suspensions are washed twice by centifuging at 35

x g and resuspending in William's medium E to remove all collagenase and

59

cell debris. Cell preparations with viability of greater than 90%, as

determined by trypan blue dye exclusion, were seeded at 7.5 x 105

cells/ml, 2 mllwell in 6-well dishes or 25 ml/150 mm diameter tissue culture

dishes. After 2 hr, cultures were washed and medium replaced. Cultures

were then incubated for 18 hr before medium was replaced with serum-free

medium containing the inhibitors or inactivators. Stock solutions of

chloramphenicol (CAP) or troleandomycin (TAO) were dissolved in

dimethyl sulfoxide (DMSO) and added to the culture in concentrations of 75

or 750 ~M CAP, and 50 ~M TAO. The total volume of DMSO did not

exceed 1 %. Isolated hepatocytes were incubated with the inhibitors for 1

hr. Medium containing inhibitor or DMSO vehicle was then aspirated and

the cultures washed for 30 min. The medium was then replaced with

serum-free medium and cultures were exposed to concentrations of

cocaine or norcocaine for the next 20 hr. Toxicants were added in water

(cocaine) or methanol (norcocaine) in 10 ~lIml.

3.2.4 Toxicity Determination.

Medium was collected from each sample, and cells harvested into an

equal volume of 1 mM Tris (pH 7.6). Cells were sonicated on ice (Model

350, Branson Sonic Power, Danbury, Cn. Cytotoxicity was determined by

the measurement of percent lactic acid dehydrogenase (LDH) activity in the

60

medium compared to the combined activity in the medium and cell fraction

using a kit from Sigma (Sigma, St. Louis, MO).

3.2.5 Microsomes.

Microsomes were prepared from isolated hepatocytes (Halpert,

Naslund et al. 1983). Briefly, hepatocytes were harvested in phosphate­

buffered saline (PBS), and hepatocytes pelleted by centrifugation and

resuspended in 2 ml of sonication solution (250 mM sucrose, 1 mM EDTA,

pH 7.0). Cells were sonicated on ice in two cycles of 10 one-sec pulses as

suggested (Wortelboer, De Kruif et al. 1990). The homogenate was

centrifuged at 10,000 x g for 15 min, and the resulting supernatant

centrifuged at 100,000 x g for 75 min. The microsomal pellet was

resuspended in 500 ~I buffer (10 mM Tris-acetate buffer, 0.2 mM EDTA,

and 20% glycerol with 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4), and

microsomes were stored at -75°C. Microsomes were prepared from PB,

Dex, and vehicle-pretreated animals by differential ultracentrifugation as

. described previously (Graves, Kaminisky et al. 1987).

3.2.6 Catalytic Assays.

Androstenedione (AD) hydroxylase activity was determined as

previously described in chapter 2 (Graves, Kaminisky et al. 1987). Cocaine

N-demethylation was measured spectrophotometrically by a method

modified from Nash (Nash 1953), and described in chapter 2.

3.2.7 Statistics.

61

Microsomal kinetic analysis of cocaine N-demethylation was

performed using the SimuSolv, version 3.0, computer program (Dow

Chemical Co. Midland, MI). To calculate values for EC25 (concentrations

that produce 25% increase in LDH activity in the medium over controls) the

concentration-response curves were plotted on log scales and EC25 values

determined mathematically. Data were analyzed by one-way analysis of

variance and differences determined with the Newman-Keuls test using the

SigmaStat computer program version 1.0 (Jandel Scientific Corp. San

Rafael, CA.). Values are considered statistically significant at p < 0.01.

62

3.3 RESULTS

3.3.1 Maintenance of Cytochrome P450 Activity in Isolated Hepatocytes.

Due to the problems with the maintenance of P450 levels in

hepatocyte culture, marker activities of 2B and 3A enzymes were

monitored in microsomes prepared from isolated hepatocytes. In these

experiments, in vivo induction, limiting the amount of FBS to 5%, and the

addition of insulin to the cell culture medium were found to result in doubled

AD 16B and 6B hydroxylation rates (P450 2B and 3A activities (Waxman

1988» in microsomes from cultured hepatocytes. Androstenedione

hydroxylation rates in microsomes from hepatocytes from PB rats cultured

without optimization were approximately 6.2 and after optimization 10.2

nmol/minlmg for 16B; in microsomes from Dex rat hepatocytes, 6B

hydroxylation rates were 2.9 before and 5.8 nmol/minlmg after

optimization. Further, the metabolite profiles of androstenedione

hydroxylations in cultures containing insulin were comparable to AD

hydroxylation profiles in microsomes prepared from freshly excised rat liver

(data not shown).

3.3.2 Microsomal Hydroxylations in Hepatocytes from Phenobarbital­

Induced Rats.

In order to assess P450 activities at the time of toxicant addition, AD

63

hydroxylation rates and profiles were determined in microsomes prepared

from hepatocytes co-incubated with hepatocytes used for toxicity

measurements. As expected, the rate of AD hydroxylation was greatest at

the 16B-position in rats which were pretreated with PB (Figure 3.1).

Microsomes from isolated hepatocytes exposed to 75 ~M CAP, a

mechanism-based inactivator of certain P450 isoforms (Halpert, Balfour et

al. 1985) showed a specific decrease in AD 16B-OH production by 77%.

Pretreatment with the higher concentration of CAP (750 ~M) resulted in

decreases of AD-hydroxylation at the 6ll, 16a, and 16B positions.

Treatment with 50 ~M TAO, a macrolide antibiotic which inhibits 3A

enzymes (Delaforge, M.J. Jaquin et al. 1983), resulted in a specific, but

slight, decrease in AD 6B-OH production (by 30%).

3.3.3 Toxicity in Hepatocytes from Phenobarbital-induced Rats.

Prior to conducting toxicity experiments, a time course for the

appearance of toxicity following exposure to cocaine or norcocaine were

determined. Isolated hepatoyctes were exposed to 100 or 500 ~M cocaine

or norcocaine, 20 hr after hepatocyte isolation. Hepatocytes were

harvested and LDH activity in the media and cells determined 4, 9, 20 and

26 hr later. No significant toxiCity was manifested until 9 hr after exposure.

Following 20 hr of exposure to toxicant, maximal levels of LDH activity was

64

leaked into the media which were not increased at 26 hr (data not shown).

Isolated hepatocytes from phenobarbital-pretreated rats were

exposed to 10, 50, 100 or 500 fJM cocaine or norcocaine. In non-cocaine

exposed controls, the % LDH activity leaked into the medium was 15.3% :I:

1.2, while exposure to 50 fJM cocaine resulted in leakage of 36.1 % :I: 3.3.

Following exposure to 500 IJM cocaine, the LDH activity in the medium

reached a value of 82.0% :I: 3.0 (Figure 3.2.A).

Similar extents of LDH activity were recovered in the medium of

cultures exposed to norcocaine (Figure 3.2.B). The methanol vehicle­

treated controls exhibited 17.0%:1: 1.7 of the LDH adivity in the medium.

After exposure to 50 and 500 IJM norcocaine the LDH activity recovered in

the medium was 55.5% :I: 2.5 and 81.3% :I: 3.7, respectively. At the lower

concentrations, norcocaine exhibited a slightly greater toxicity than did

cocaine.

3.3.4 Protection Against Toxicity by Inactivators in Hepatocytes from PB

Induced Rats.

Pretreatment with either low or high dose CAP greatly attenuated the

extent of LDH activity leakage into the medium following exposure to either

cocaine or norcocaine. For example, 75 or 750 IJM CAP decreased the

extent of LDH activity leaked into the medium by 60% after exposure to

65

500 ~M cocaine. However, pretreatment with TAO had no protective effects

against toxicity (Figure 3.2.A and 8). The medium LDH activity following

exposure to 500 ~M cocaine in hepatocytes pretreated with TAO was

104% of DMSO-pretreated, cocaine-exposed cultures.

The extent of LDH leakage for norcocaine was similar to those

values obtained for cocaine. Again, TAO pretreatment did not protect

against norcocaine-mediated toxicity (94% of no-inhibitor values after

exposure to 500 ~M norcocaine). Chloramphenicol pretreatment, however,

did attenuate norcocaine-mediated toxicity, decreasing the LDH leakage by

55% following pretreatment with either 75 or 750 ~M CAP.

3.3.5 Microsomal Hydroxylations in Hepatocytes from Dexamethasone­

Induced Rats.

In vivo treatment with dexamethasone elevated AD 6B-hydroxylation

rates above those of 16a and 16B, verifying the induction of P450 3A in

microsomes from cultured hepatocytes. Treatment of the isolated

hepatocytes with TAO resulted in a specific decrease in AD 6B-OH

formation by about 50%. Treatment with low dose CAP resulted in a

specific reduction in the rate of 16B-OH formation by 62%, and treatment

with high dose CAP resulted in a decrease in all isofonn activities

measured (Figure 3.3).

66

3.3.6 Toxicity in Hepatocytes from Dexamethasone-induced Rats.

Following in vivo induction with 100 mg/kg Dex, isolated hepatocytes

were exposed to 0,50,100,2500, or 5000 IJM cocaine or norcocaine.

Toxicant concentrations 1O-fold higher than necessary in PB-induced

samples were required before loss of hepatocyte viability was observed.

Control values for LDH activity in the medium were 14.7% ± 1.8 for cocaine

(water vehicle) and 15.6% ± 1.7 for norcocaine (methanol vehicle) (Figure

3.4.A and B). Exposure to 5000 IJM concentrations of toxicant were

required to elevate the level of LDH activity recovered in the medium to

86.7% ± 3.8 and 89.7% ± 4.3 for cocaine and norcocaine, respectively. The

EC25 values for cocaine and norcocaine were 15-20 times greater than the

EC25 values in PB isolated hepatocytes (Table 3.1).

3.3.7 Toxicity Following Treatment with Inactivators in Hepatocytes

Isolated from Dex-Induced Rats.

Protection following P450 inhibition was much less definitive in

hepatocytes isolated from Dex-induced rats than was observed in

hepatocytes from PB-induced rats. Pretreatment with the high

concentration of CAP (750 IJM) attenuated the LDH leakage resulting from

exposure to the lowest concentration of cocaine which was able to induce

LDH leakage, 2.5 mM, while protection against LDH leakage at any other

67

concentration of toxicant with any inactivator was negligible (Figure 3.4.A).

Treatment with low concentration CAP (75 JJM) had little effect on the

extent of LDH leakage into the medium compared to DMSO-pretreated,

toxicant exposed samples such that after exposure to 5 mM cocaine or

norcocaine LDH activity recovered in the media was 75.6% ± 8.3 and

87.1 % ± 4.3, respectively (Figure 3.4.A and B). Treatment with high

concentration CAP did result in a slight decrease in cocaine-mediated LDH

leakage resulting from exposure to 5 mM cocaine (to 60.6% ± 10.4), but not

for norcocaine (85.3% ± 7.1). Troleandomycin pretreatment had no affect

on LDH leakage after exposure to either 5 mM cocaine or norcocaine

(84.4% ± 4.8 and 86.4% ± 5.1, respectively). Pretreatment with low dose

CAP or TAO did not change EC25 values, and high dose CAP resulted in

only a slight increase in the EC25 value following exposure to cocaine

(Table 3.1).

3.3.8 Cocaine N-demethylation Rates.

In microsomes from isolated hepatocytes from phenobarbital­

induced rats, the control cocaine N-demethylation rate, as measured by

formaldehyde release, was 5.9 ± 0.8 nmol/min/mg. Cocaine N­

demethylation rates were decreased following treatment with either 75 or

750 J.lM CAP (to 64 and 32% of control, respectively). Pretreatment with

68

TAO, however, did not affect the rate of cocaine N-demethylation in the

microsomes from the PB-induced sample (Figure 3.5).

Table 3.1

EC25 values for toxicity in hepatocytes isolated from Dex- and PB-induced rats.

Concentrations of toxicant that produce a 25% increase in the level of LDH leakage out of the isolated hepatocytes into the surrounding medium were calculated from linearized concentration-response curves and determined mathematically. Values are expressed from curves from 3 different experiments with individual rats ± S.D. ·Significant difference from no-inhibitor, toxicant-exposed controls.

EC25 (mM)

DMSO 50~MTAO 75~MCAP 750~MCAP

PB cocaine 0.12 ± 0.02 0.12 ± 0.03 0.71 ± 0.04· 0.90 ± 0.05-

norcocaine 0.09 ± 0.01 0.09 ± 0.01 0.31 ± 0.05- 0.69 ± 0.09-

Dex cocaine 1.75 ± 0.16 1.74 ± 0.20 1.76 ± 0.24 2.5 ± 0.39-

norcocaine 1.64 ± 0.15 1.79 ± 0.33 1.86 ± 0.31 2.17 ± 0.45

The rates of cocaine N-demethylation in microsomes from

hepatocytes isolated from Dex-induced rats were similar to PB-induced

69

rats. Cocaine N-demethylation rates were decreased by all inhibitor

treatments in hepatocytes from the Dex-induced animals. Troleandomycin

pretreatment resulted in a decrease in microsomal cocaine N­

demethylation rate, from 6.3 ± 0.7 to 3.3 ± 0.4 nmol/minlmg microsomal

protein. Both concentrations of CAP were as effective at decreasing

cocaine N-demethylation rates in hepatocytes from Dex-induced animals

as they were in PB-induced cultures. Low and high concentrations of CAP

resulted in decreased rates of N-demethylation by 31 and 64%,

respectively (Figure 3.5).

3.3.9 Cocaine N-Demethylation in PB and Oex Induced Female Rat Liver

Microsomes.

Although cocaine N-demethylation rates were the same'in isolated

hepatocytes from PB- or Dex-induced rats, the toxicity data strongly

implicate PB and not Dex induction in potentiating cocaine hepatotoxicity.

Accordingly, the abilities of microsomes from female rats induced with

either PB or Dex to N-demethylate cocaine were detennined. As expected,

treatment of the rats with either PB or Dex greatly increased the rates of

cocaine N-demethylation over vehicle pretreatment (Table 3.2).

Surprisingly, the Km and V max for cocaine N-demethylation may be slightly

greater in Oex-induced female microsomes than in PB-induced. An

analysis of the kinetic data, however, reveals that there is no difference

between the catalytic efficiency (V ma/Km) of microsomal enzymes from

either Dex- or PB-induced rats toward cocaine N-demethylation.

70

Incubation of the microsomes with the 3A inhibitor, TAO, prior to the

performance of cocaine N-demethylation assays did result in an inhibition

of N-demethylation rates. The inhibition was less than 25% in microsomes

from vehicle and PB-treated animals and was more than 50% in

microsomes from Dex-treated animals (Figure 3.6).

Table 3.2

Microsomal cocaine N-demethylation kinetics

Rates of formaldehyde production from cocaine were determined in microsomes prepared from rats treated with the inducers indicated. Cocaine concentrations from 0.01 to 5 mM were used. Km, Vmax and V ma/Km were determined using the SimuSolv computer program. Cocaine N-demethylations were performed in microsomes from 4 rats/group for each concentration.

Km Vmax Treatment (mM) (nmollmlnlmg) Vma/Km

Vehicle 0.42 1.7 4.2

PB 0.52 10.9 20.9

Dex 0.78 15.6 20.2

"""" 12 C)

-E c: ~ 10 o E c: ....,. c: o .-+J tU E ..... o u. Q) +J .­-o .c J9 Q)

8

6

4

2

~ o -1..--_

6B-OH 16B-OH 16a-OH (3A activity) (2B activity) (2B/2C activity)

Androstenedione Metabolite

71

Figure 3.1. Androstenedione hydroxylation rates In mlcrosomes prepared from hepatocytes Isolated from a PB·lnduced rat. Isolated hepatocytes were exposed to inactivators for 1 hr prior to the preparation of microsomes (83-) vehicle, (0) 50 ~M TAO, (12) 75 ~M CAP, and (1:3) 750 ~M CAP. Experiments were repeated with microsomes from 3 separate rats; data presented are from a single representative experiment. Each data group represents activities from n=3 hepatocyte groupsltreatment:t S.D. ·Significant difference from no inhibitor treatment controls, p < 0.05.

72

....-... 100 -co * ~

0 80 ~

~ 0 ......... 60 ~ .s;

40 n « :r: 20 0 0 --I

0 100 200 300 400 500 Cocaine Concentration (IJM)

C' 100 co ~ 8 * 0 ~ 80 * eft. ......... 60 * ~ .- *§ > 40 n « 20 *§ :r: 0 0 --I

o 100 200 300 400 500 Norcocaine Concentration (IJM)

Figure 3.2.A and B. Toxicity of cocaine and norcocaine In hepatocytes Isolated from a PB·lnduced rat. Isolated hepatocytes were exposed to from 10 to 500 J,lM of either cocaine or norcocaine, and LDH activity was measured in the cells and medium after pre-incubation with inhibitors, (e) DMSO, (_) 50 J,lM TAO, (V') 75 J,lM CAP, and (A) 750 J,lM CAP. Data are expressed as % of LDH activity leaked into the medium compared to the total activity in the medium and cell fractions combined. Experiments were repeated with 3 separate rats; data presented are from a single representative experiment. Each data point represents n=3 wellsltreatment ± S.D. ·Significant difference from non-toxicant exposed controls, §significant difference from non-inhibitor treated samples exposed to the same concentration of toxicant, p < 0.05.

..-.. 7 C)

-E .5 6 § o 5 E c: '-' c: 4 o ~ E 3 .... o u. 2 2 .­-o .c ~ 1 =a: o -'--

SB-OH 1SB-OH 1Sa-OH (3A activity) (28 activity) (28/2C activity)

Androstenedione Metabolite

73

Figure 3.3. Androstenedione hydroxylation rates In mlcrosomes prepared from hepatocytes Isolated from a Dexamethasone-lnduced rat. Isolated hepatocytes were exposed to inhibitors for 1 hr prior to the preparation of microsomes (IS) vehicle, (0) 50 J,lM TAO, (e1) 75 J.lM CAP, and (~) 750 J.lM CAP. Experiments were repeated with microsomes from 3 separate rats; data presented are from a single representative experiment. Each data group represents activities from n=3 hepatocyte groupsJtreatment. ·Significant difference from no inhibitor treatment controls, p < 0.05.

74

Q-co 100 * ~

0 80 ~

'#. 60 '-""

~ .- 40 > .-n « 20 :c C 0 ...J

0 1 2 3 4 5 Q- Cocaine Concentration (mM) co 100 * ~

0 ~

80 '#. '-""

~ 60 .-> 40 .-n « 20 J: C 0 ...J

0 1 2 3 4 5 Norcocaine Concentration (mM)

Figure 3.4.A and B. Toxicity of cocaine and norcocalne In hepatocytes Isolated from a Dex-induced rat. Isolated hepatocytes were exposed to from 0.05 to 5 mM of either cocaine or norcocaine and LOH activity measured in the cells and medium after pre-incubation with inhibitors, (e) OMSO, (.) 50 J,lM TAO, (V) 75 J,lM CAP, and (~) 750 J,lM CAP. Data are expressed as % of LDH activity leaked into the medium compared to the total activity in the medium and cell fractions combined. Experiments were repeated with 3 separate rats; data presented are from a single representative experiment. Each data point represents n=3 wellsltreatment ± S.D. ·Significant difference for OMSO-, TAO-, and 75 J,lM CAP-treated from non-toxicant exposed controls, §significant difference from non­inhibitor treated samples exposed to the same concentration of toxicant, p <0.05.

75

..-... 8 0)

E 7 ........

C .-E 6 ::::::: 0 E 5 C ........... Q) 4 en m

3 Q) -Q)

a:: 2

0 () 1

N :c 0

Vehicle 50 IJM 75 IJM 750 IJM TAO CAP CAP

Treatment

Figure 3.5. Cocaine N-demethylation rates In mlcrosomes from hepatocytes Isolated from Oex- or PB-Induced rats. Isolated hepatocytes were exposed to inhibitors as indicated for 1 hr prior to the preparation of microsomes. Formaldehyde release was measured from cocaine metabolism in microsomes from (0) PB-induced rat, (_) Oex­induced rat. Each data group represents activities from n=3 hepatocyte groupsJtreatment ± S.D. ·Significant difference from no inhibitor treatment controls, p < 0.05.

76

8

7

6 -C')

E 5 --c .-E

:::::- 4 0 E c -- 3 0 ()

C\J 2 :::c

1

0 Vehicle PB Dex

Treatment

Figure 3.S. Troleandomycin inhibition of cocaine N-demethylatlon in microsomes. Female Sprague-Dawley rats were induced with PB or Dex, and cocaine N-demethylation rates determined as described in the text. Microsomal samples were pre-incubated with 50 ~M TAO (0) or propylene glycol vehicle (0), and cocaine N-demethylation rates determined.

77

3.4 DISCUSSION

The role(s) of cytochromes P450 2B and 3A in cocaine bioactivation

and cocaine N-demethylation were investigated in the rat. The proposed

mechanism of metabolism for cocaine, which involves several oxidation

steps (Shuster, Garhart et al. 1988), leads to questions concerning the

cytochromes P450 involved. Cocaine is first N-demethylated by P450(s) to

norcocaine, releasing formaldehyde (which may be responsible for the

nasal irritation caused by cocaine (Dahl and Hadley 1991)}. The formation

of formaldehyde was determined to measure the first metabolism step of

cocaine oxidative metabolism.

In previous experiments using rat liver slices, a strong correlation

between AD 16B-OH formation and cocaine toxicity was observed (Poet,

Brendel et al. 1994). However, apparently a population of cells was not

exposed to either inactivator or cocaine, since incomplete inadivation of

AD-hydroxylations was associated with complete protection against a loss

of intracellular K+ in slices from PB-induced rats. The use of isolated

hepatocytes in the present study, instead of slices, allowed the direct

access of compounds in the medium to each individual cell.

To determine the relative contributions of 2B and 3A enzymes in

cocaine bioadivation, induction and inhibition procedures were used.

Although cocaine N-demethylation rates were equivalent in microsomes

78

from hepatocytes isolated from either Dex- or PB-induced rats, the

concentrations of cocaine and norcocaine needed to produce toxicity were

very different. The EC25 values for cocaine and norcocaine were 15 and 20

times lower in hepatocytes from P8-induced rats than from Dex-induced

rats. Specific inactivation of 28 enzymes with the low concentration of

CAP resulted in a 6-fold increase in LD25 for cocaine and a 3-fold increase

for norcocaine in hepatocytes from P8 rats but no increase of LD25 in

hepatocytes from Dex rats. Moreover, 3A inhibition with TAO resulted in no

increase in LD25 for cocaine or norcocaine in either Dex of P8 isolated

hepatocytes. Thus, it appears that cocaine-mediated toxicity in P8- and

Dex-induced hepatocytes may result from different mechanisms.

Different mechanisms of cocaine hepatotoxicity have been proposed

(Jover, Ponsoda et al. 1993). In P8- induced animals, toxicity is thought to

result from enzyme-mediated bioactivation and result in decreased levels

of reduced glutathione and increased lipid peroxidation. In uninduced

animals, toxicity may be associated with an increase in intracellular Ca++,

which is mediated directly by cocaine. In our studies, the concentrations of

cocaine and norcocaine required to produce toxicity in hepatocytes isolated

from Dex-induced rats were comparable to the concentrations reported to

cause an increase in Ca ++ concentrations in uninduced rats. This suggests

that the toxicity observed in Dex-induced hepatocytes may not have been a

result of bioactivation. This is further supported by the inability of TAO to

substantially protect against LDH activity leakage from the isolated

hepatocytes.

79

Sex and strain variations in cocaine hepatotoxicity are known

(Thompson, Shuster et al. 1984; Pellinen, Honkakoski et al. 1994), and

these may be directly related to differences in P450 isoform expression.

Isoforms of P450 2B are expressed variably in male and female mice

(Henderson and Wolf 1992). In rats, 3A enzyme levels are greater in males

than in females (Kato and Yamazoe 1992). Strong evidence is presented

here that both 3A and 2B enzymes N-demethylate cocaine, but that 3A

enzymes are not sufficient to cause toxicity in isolated rat hepatocytes. The

previous work in mice and humans implicating 3A enzymes in these

species may relate to variations in 2B/3A expression levels and to

differences in isoforms with different substrate recognitions.

The efficiency of either Dex- or PB-induced microsomes toward

cocaine N-demethylation was equivalent. Given that cocaine and

norcocaine are both much more toxic in hepatocytes isolated from PB­

induced rats, it appears that cocaine N-demethylation is not the rate­

limiting step in cocaine bioactivation. Further, this strongly suggests that 3A

enzymes are not greatly involved in the metabolism of norcocaine to

promote toxicity in rats. However, potentiation of toxicity by PB

80

pretreatment, and inhibition with CAP, strongly implicates P450s 28 in the

bioactivation of norcocaine in Sprague-Dawley rats.

CHAPTER 4

COCAINE N-DEMETHYLATION BY MICROSOMES FROM PHENOBARBITAL-INDUCED RAT, DOG, AND GUINEA PIG

MICROSOMES AND BY PURIFIED CYTOCHROMES P450 2B FROM RAT, DOG, AND RABBIT

4.1 BACKGROUND

81

Due to the importance of P450s 2B suggested by the slice and

hepatocyte toxicity data presented in Chapters 2 and 3, and the microsomal

data from the co-incubated tissues, the abilities of P450s 2B from a number

of species to N-demethylate cocaine were investigated. Cocaine N-

demethylation rates were determined for microsomes from PB induced rats,

dogs, and guinea pigs, and for purified P450s 2B from rat, dog, and rabbit.

The differential abilities of very similar 28 enzymes to metabolize

substrates (see Table 1.2) suggested that investigations into their abilities to

N-demethylate cocaine might be interesting. Phenobarbital induction was

used to emphasize P450s 2B in animals which express 28 enzymes variably.

Liver cytochromes P450 2B are expressed at low levels in most species

(Waxman and Azaroff 1992). In dogs and guinea pigs, however, high levels

of 2B enzymes are constitutively expressed (Duignan, Sipes et al. 1987;

Oguri, Kaneko et al. 1991). Accordingly, cocaine N-demethylation rates were

compared in vehicle and P8 pretreated rat, dog, and guinea pig microsomes.

82

To truly investigate the abilities of specific isoforms to metabolize

substances, purified enzymes must be used. The purified proteins

investigated are basally expressed at different levels in vivo. Cytochrome

P450 282 is constitutively expressed at low levels in rat liver, but is much less

active toward most substrates than is the non-constitutively expressed 281

(Waxman and Azaroff 1992). Cytochrome P450 284 is expressed in rabbit

liver in a similar fashion as 281 in rat liver. Generally, 284 has higher activity

toward most substrates than the other major (constitutively expressed) rabbit

liver 28 enzyme, 285 (Grimm, Dyroff et al. 1994). Thus, the relative

contributions of P450s 28 toward bioactivation may be associated with

expression levels as well as enzymatic affinity for the substrate.

To study the human P450 28 enzyme's ability to bioadivate cocaine,

lymphoblastoma cells transfected with human cytochrome P450 286 cDNA

were incubated with cocaine or norcocaine.

4.2 MATERIALS AND METHODS.

4.2.1 Chemicals.

83

Sodium phenobarbital was obtained from Mallinckrodt, Inc. (St. Louis,

MO). Williams' Medium E, Hank's balanced salt solution, insulin, fetal bovine

serum, and gentamicin were purchased from Gibco-BRL (Grand Island, NY).

Hepes (N-2-hydroxyethyl-piperazine-N' -2-ethanesulfonic acid) was purchased

from Calbiochem (La Jolla, CA). Cocaine and norcocaine were obtained from

the National Institute on Drug Abuse (Research Triangle Park, NC). All other

chemicals were of reagent grade and obtained from Sigma Chemical Co (St.

Louis, MO).

4.2.2 Animals.

Adult male Sprague-Dawley rats (Harlan Sprague Dawley, Inc.

Indianapolis, IN) weighing from 250-300 g or male Harley guinea pigs

(Simonsen Laboratories, Inc. Gilroy, CA) weighing from 325-375 g, were given

food an~ water ad libitum and housed three per cage on metal mesh with a

12-hr light/dark cycle. Phenobarbital induction of hepatic mixed-function

oxidases was performed by treating the animals with sodium phenobarbital

(PB), 0.1 % (w/v) in drinking water for 4 days, 5 days prior to the experiments

(Halpert, Balfour et al. 1985).

84

4.2.3 Purified Proteins.

Dog P450 2811 was purified from PB-induced canine liver, rat 281 and

282 were purified from PB-induced rat liver, and rabbit 284 was purified from

rabbit liver as described previously (Duignan, Sipes et al. 1987; Graves,

Kaminisky et al. 1987; Grimm, Dyroff et al. 1994).

4.2.4 Lymphoblastoid Cell Culture.

AHH-1 human Iymphoblastoid cells transiently expressing human P450

286 were obtained from Gentest Corporation (Woburn, MA), and cultured as

suggested by the manufacturer. Cells were suspension cultured in RPMI

medium 1640 supplemented with 9% horse serum. Cells were incubated at

37°C in a 5% CO2 incubator. Cytochrome P450 286 activity was measured

by the deethylation of 7-ethyoxy-4-trifJuoromethylcoumarin (7-EFC) (8uters,

Schiller et al. 1993).

4.2.5 Toxicity Assessment in Lymphoblastoid cell culture.

Cell viability was determined in Iymphoblastoid cells expressing P450

286 following exposure to cyclophosphamide, cocaine, or norcocaine for 4 hr

by spectrophotometrically measuring the metabolism of tetrazolium salt,

sodium 3'[1-[{phenylamino)-carbonyl]3,4-tetrazolium]-bis(4-methoxy-6-

nitro)benzene-sulfonic acid hydrate (XTI) as described by Roehm et al.

85

(Roehm, Rodgers et al. 1991). This tetrazolium salt is reduced by

dehydrogenase enzymes in viable cells, and the resultant product is a highly

colored formazan product. The reduction of XTT requires the addition of an

electron coupling agent, accordingly, phenazine methylsulfate (PMS) is added

to the XTT solution. Briefly, XTT is dissolved in warm media (0.2 mg/ml),

along with 25IJM PMS is added to cells in a 96 well plate (4 x 104 cellslwell)

and incubated for 4 hr in the dark. The optical density was then measured at

450 nm, with a reference at 650 nm and blank control values were subtracted.

4.2.6 Microsomes.

Microsomes were prepared from the Iymphoblastoid cells as by

differential ultracentrifugation. Lymphoblastoid cells were washed once in

PBS, centrifuged, and resuspended in 2 ml of sonication solution (250 mM

sucrose,1 mM EDTA, pH 7.0). Cells were sonicated on ice in two cycles of

10 one-sec pulses as suggested. The homogenate was centrifuged at 10,000

x g for 15 min, and the resulting supematant centrifuged at 100,000 x g for 75

min. The microsomal pellet was resuspended in 500 IJI buffer (10 mM Tris­

acetate buffer, 0.2 mM EDTA, and 20% glycerol with 0.1 mM

phenylmethylsolfonyl fluoride, pH 7.4), and miaosomes were stored at -75°C.

86

4.2.7 Catalytic Assays.

Cocaine N-demethylation was measured spectrophotometrically by a

method modified from Nash (Nash 1953), and desaibed in chapter 2. Purified

proteins were reconstituted as described previously (Kedzie, Balfour et al.

1991). Briefly, reconstitution was by the addition of NADPH-cytochrome P450

reductase and 0.03 ~g/~1 dilauryl-L-3-phosphatidylcholine (DLPC) and pre­

incubation for 10 min at room temperature. Aliquots of reconstituted protein

were incubated for 2 min at 37°C in 15 mM MgCI2, 0.1 mM EDTA, 2.5 mM

cocaine, 0.03 ~g/~I DLPC, and 50 ~M Hepes buffer. The reaction proceeded

for 15 min at 37°C following the addition of 1 mM NADPH.

4.2.8 Statistics.

Data were analyzed by one-way analysis of variance and differences

determined with the Newman-Keuls test. Values are considered statistically

significant at p < 0.01.

87

4.3 RESULTS

4.3.1. Cocaine N-demethylation in P8-induced microsomes.

In the pilot study with guinea pig microsomes cocaine N-demethylation

rates were found to be higher than those for either dog or rat liver

microsomes, and PB-indudion had little effect on the rate of N-demethylation

(Table 4.1). Phenobarbital pretreatment increases guinea pig P450 28 levels

only moderately (Oguri, Kaneko et al. 1991). While dog liver microsomes

readily catalyzed the N-demethylation of cocaine, the rate of cocaine N­

demethylation was more than doubled by P8 pretreatment. In the dog, P8

treatment results in a 5-fold increase in 2811 levels (Ciaccio and Halpert

1989). Cocaine N-demethylation in rat microsomes was less than in either dog

or guinea pig microsomes. Induction with P8 did result in an increased rate

of N-demethylation comparable to that of the vehicle pretreated dog.

4.3.2 Cocaine N-demethylation by Purified 28 Enzymes from Rat, Dog and

Rabbit.

The rates of cocaine N-demethylation catalyzed by purified proteins

from rat, dog, and rabbit varied from 1.7 to 6.4 nmol/min/mg. The two 28

enzymes purified from rat liver exhibited different, but expected, rates of

cocaine N-demethylation. Purified rat liver 281 was 3-times more active

toward cocaine N-demethylation than was 282. Purified dog liver 2811 N-

88

demethylated cocaine faster than rat 281. Although cocaine N-demethylation

was detected with purified 284, the rate was much lower than observed for

purified rat or dog proteins (Table 4.1).

Table 4.1

Cocaine N-demethylation rates by microsomes and purified P450s

Formaldehyde release was measured from cocaine in micro somes from the species indicated following either vehicle or PB-induction. Purified proteins were reconstituted with DLPC and NADPH-cytochrome P450 reductase and reactions initiated by the addition ofNADPH.

Rat

Dog

Guinea Pig

Rabbit·

MicrosomesO

Vehicle

3.8 ±0.42

7.2 ± 1.3

13.3

P8

6.4±0.85

12.5 ± 1.5

15

Purified Proteinb

Protein

281 5.05

282 1.70

2811 6.35

284 0.70

a Mean data for rat and dog from n=3 microsomal preparations from individual animals, for guinea pig, from a single microsomal preparation. Data expressed as nmollminlmg ± S.D. b Data from purified preparations of P450 enzymes indicated, expressed in nmol/minlnmol.

89

4.3.3 Cocaine and Norcocaine Toxicity in Lymphoblastoid Cells Expressing

Human 286.

The ability of cocaine, norcocaine, or cyclophosphamide to cause

toxicity in these cells was determined using the XTT assay.

Cyclophosphamide has been shown to be bioadivated by 286 in this cell line,

resulting in toxicity (Chang, Weber et al. 1993). Although exposure of the cells

to 0.05, 0.1 and 0.5 mM concentrations of cyclophosphamide resulted in

losses in spectrophotometrically-determined formazan product (optical

density at 450 nm (OD4W of 0.51 ± 0.03 for control and 00450 of 0.25 ± 0.04

for 0.1 mM cyclophosphamide), treatment with from 0.05 to 1 mM of either

cocaine or norcocaine did not result in any toxicity (00450 after 1 mM cocaine

0.45 ± 0.07 and 0.43 ± 0.06 with 1 mM norcocaine) (Figure 4.1).

Likewise, cocaine N-demethylation was not observed in microsomes

made from Iymphoblastoid cells expressing 286. The catalytic ability of the

P450 286 expressed in the Iymphoblastoid cells was confirmed by measuring

7-EFC deethylation (12.2 ± 0.3 pmollminl106 cells) to further verify that the

inability to catalyze cocaine N-demethylation was not due to a lack of

enzymatic activity in the cells.

90

0.5

-E c:

0.4 0 LO V -~ :t:: 0.3 t/) c: Q)

C co 0.2 0

:.;::::; Co 0

0.1

0.0 -4-------.------r----_r-------,

0.00 0.25 0.50 0.75 1.00

Toxicant Concentration (mM)

Figure 4.1. Lymphoblastold cell survival following exposure to cocaine and norcocalne. The viability of Iymphoblastoid cells expressing human P450 286 was determined by measuring their ability to reduce XTT following exposure to cyclophosphamide (_), cocaine (_), or norcocaine (A) for 4 hr at the concentrations indicated. ·Significant difference from vehicle-exposed controls, p < 0.01.

91

4.4 DISCUSSION

Cytochromes P450 within the 28 subfamily share amino acid sequence

identities of greater than 55%. Regardless of this sequence similarity, they are

regulated differently in vivo, some are constitutively expressed and many are

highly inducible (Henderson and Wolf 1992). Also, they exhibit variable

catalytiC abilities toward many substrates. Since the importance of P450 28-

mediated metabolism of cocaine could vary between different species

expressing different isoforms, the relative abilities of several constitutive and

non-constitutive P450s 28 to N-demethylate cocaine were compared.

Due to the apparent role of P450s 28 toward cocaine bioadivation in

the rat, the abilities of microsomes from species which constitutively express

P450 28 enzymes to variable degrees to N-demethylate cocaine with and

without P8-indudion were compared. In dogs, which exhibit constitutive

expression of P450 2811, cocaine N-demethylation rates were higher than in

Sprague-Dawley rats, which exhibit much lower expression levels for P450s

28. In both of these species, P8 indudion significantly increased the rates of

cocaine N-demethylation. In the rat, P8-indudion can result in increases in

P450 28 levels greater than 100-fold (Waxman and Azaroff 1992), in the dog

PB-mediated indudion of 28 enzymes is greater than 5-fold. In the guinea pig,

P8 induction of 28 enzymes, which are constitutively expressed at high levels,

is much less pronounced (Oguri, Kaneko et al. 1991). Cocaine N-

92

demethylation rates in guinea pig microsomes were greater than in

microsomes from either the dog or the rat, and were not increased by PB­

induction.

The 28 enzymes from rats, dogs, and rabbits all catalyzed the N­

demethylation of cocaine with very different rates. Expressed human 286 did

not catalyze the N-demethylation of cocaine, nor was there any observable

. cocaine or norcocaine-mediated toxicity in lymphoblastoma cells. Thus, the

relative contributions of species specific 28 enzymes toward cocaine

bioactivation may be highly variable.

CHAPTER 5

SUMMARY AND DISCUSSION

93

The objective of this research was to demonstrate the usefulness of

isoform-selective mechanism-based inactivators of cytochromes P450 to

identify the specific enzymes involved in the bioactivation of cocaine. The

multi-step oxidation scheme which leads to cocaine-mediated hepatotoxicity

provides an interesting system for the study of P450-mediated bioactivation

and P450 isofonn specificity. In these studies, isoform-selective inhibitors and

mechanism-based inactivators were used, along with enzyme induction, to

accumulate evidence for the roles of P450s 28 and 3A in cocaine

bioactivation in the rat. At the same time, marker substrate metabolism was

used to compare to cocaine N-demethylation, as well as cocaine and

norcocaine toxicity.

These studies highlight the value of mechanism-based inactivators of

cytochromes P450 as mechanistic probes. Here, the inactivators provided a

unique opportunity to control enzyme activities and observe toxicity in the

same system. The protection against cocaine toxicity with pN02CIFA, the

P450 2B-selective CAP analogue, illustrates another plausible use for

mechanism-based inadivators. Once P450( s) are identified, the inactivators

94

can be used to reduce toxicity following in vivo exposure to a compound that

would otherwise be bioactivated.

The concurrent use of site-specific metabolism of androstenedione

allowed the assessment of the effects of induction and inhibition on P450

activities. It was shown in chapter 2 that AD 16B-hydroxylase activity (a

marker for P450 2B adivity) correlated strongly to the level of toxicity resulting

from exposure to 500 fJM cocaine. However, in the studies with slices

incomplete inactivation of concurrent androstenedione 16B-hydroxylase and

protedion against cocaine-mediated were observed. Depletion of K+ content

below 20% following exposure to cocaine, and inhibition of androstenedione

hydroxylase adivity below 30% following inactivator pretreatment may reflect

limitations of diffusion parameters due to slice morphology.

Isolated hepatocytes allowed the direct access of both the inactivators

and the toxicants to the individual cells, without the constraints of cut surfaces,

cell-cell interactions, or diffusion hindrance through the 250 fJM thickness of

the slice. Because of the literature reports on the roles of P450s 3A in mice

and man, the metabolite-intermediate complex-mediated inhibition of P450s

3A by TAO and the indudion with the P450 3A inducer, Dex were included in

the hepatocyte toxicity and N-demethylation studies. Also, the addition of

norcocaine to the battery of toxicity assessments allowed the first P450-

95

mediated oxidation of cocaine to be bypassed, so that the role of P450s 28

in further oxidation steps could be determined.

The studies using isolated hepatocytes outlined in Chapter 3 showed

a reliance on P450 28 bioadivation for both cocaine and norcocaine-mediated

toxicity. This was an apparent contrast to the findings that both Dex and P8

induction resulted in the same rate of cocaine N-demethylation in microsomes

prepared from the isolated hepatocytes. The N-demethylation of cocaine is

not the rate limiting step in the bioactivation of cocaine as evidenced by the

equivalent toxicities obtained following exposure of hepatocytes to either

cocaine or norcocaine. Cocaine N-demethylation rates in microsomes from

female rats pre-treated with Dex and P8 were equivalent. It would appear that

although both P450s 28 and 3A catalyze the N-demethylation of cocaine,

P450s 28 playa much more important role in the later oxidation steps which

mediate cocaine bioactivation in the rat.

The comparisons of the abilities of microsomes from species which

constitutively express P450 28 enzymes to variable degrees with and without

PB-induction reported in Chapter 4 revealed that PB-induced increased levels

of P450s 28 do result in increased rates of cocaine N-demethylation. The

variable regulation of P450s 28 in rats, dogs, and guinea pigs was mirrored

by the effects of PB on the rates of formaldehyde release from cocaine.

Phenobarbital-induction of P450s 2B is greatest in the rat, slightly less in the

96

dog, and marginal for guinea pigs. Likewise, the rates of cocaine N-

demethylation inaeased the most following P8-induction the most in rat liver

microsomes and the least in guinea pig microsomes. In dogs the rates of

cocaine N-demethylation were high both in microsomes and in reconstituted

purified 2811 protein, which is constitutively expressed. Thus, it appears that

P450s 28 from rats, dogs, and guinea pigs all oxidize cocaine. The roles P450

isozymes in cocaine bioactivation may be related to the basal expression

levels of the enzymes.

The rate of cocaine N-demethylation catalyzed by purified rabbit P450.

28 was much less than in P450s 28 enzymes from rats and dogs. Expressed

human P450 286 was not able to bioactivate either cocaine or norcociane.

The importance attributed to P450 3A enzymes in mice and humans by other

researchers (Lloyd, Shuster et al. 1993; Pellinen, Honkakoski et al. 1994) may

be related to differences in P450 3A and 28 expression and catalytiC abilities.

Future studies should address additional uses of mechanism-based

inactivators. Isoform-selective inactivators are being used to aid in

structure/activity studies of enzyme active site structure and binding as well

as enzyme mechanisms. Enzyme inadivation kinetics for various isoforms can

be used to determine metabolism/inactivation selectivity between functional

variants within P450 subfamilies. Specific inactivation of cytochromes P450

has the potential to be used in clinical settings to protect against toxic

97

exposures when the isoform responsible for the bioactivation of a compound

is known. This dissertation presents an example of how mechanism-based

inactivators can be used, along with other techniques, to identify the P450

isoforms responsible for the oxidative metabolism of a compound.

lsoform-specific inactivators can be used to further clarify the relative

contributions of P450s 28 and 3A in cocaine bioactivation. The increased

levels of hepatotoxicity in laboratory animals over humans may relate to the

inconsistent constituent expression of human P450 286 (Mimura, 8aba et al.

1993) compared to the P450s 3A. Although 90% of the administered dose of

cocaine is excreted as one of the hydrolyzed metabolites in man, this

percentage can be as low as 50% in mice (Inaba, Stewart et al. 1978;

Thompson, Shuster et al. 1979; Evans 1983; Roberts, Harbison et al. 1991;

Roth, Harbison et al. 1992; Jeong, Jordan et al. 1995). This difference may

relate to disparate enzyme constituents in humans and rodents. In rats, P450

282 is present at low levels and P450 281 is highly inducible. The

consequences of phenobarbital induction in animals will depend on the

resultant expression levels of inducible P450s. In humans P450 286 mRNA

has been found in 12 of 64 human liver samples, but only at very low levels

(Mimura, 8aba et al. 1993). In contrast, several P450s of the 3A family are

found in fairly high levels in human liver samples (Gonzalez, Crespi et at

1992; Kato and Yamazoe 1992; Nelson, Kamataki et al. 1993; Guengerich

98

1994; Shou, Grogan et al. 1994). Inhibition of P450s 3A does not protect

against norcocaine bioactivation in the rat. It may be interesting to probe the

importance of P450s 3A in the bioactivation of norcocaine in man.

Investigations into cocaine-mediated hepatotoxicity in various species,

related to expression levels of P450s 28 and 3A, and relative substrate

specificities of these enzymes toward cocaine and its proximal oxidated

metabolites will reveal many interesting aspects of enzyme-substrate

specificity and enzyme regulation, and how they affect bioactivation. The

studies presented here make it clear that cytochromes P450 from both the 28

and 3A subfamilies catalyze the N-demethylation of cocaine. Further, P450s

28 are involved in the bioactivation of norcocaine. Phenobarbital-induced rat

liver microsomes can be used for in vitro studies of cocaine metabolism.

Analysis of the production of specific metabolites from norcocaine, and the

effects of mechanism-based inactivation of P450s 28 could corroborate the

28-mediated oxidation of norcocaine and N-hydroxynorcocaine.

Interesting subsequent studies could be performed to identify the

metabolite directly responsible for cocaine-mediated hepatOtoxicity.

Experiments to identify the specific mechanism of cocaine-mediated

hepatotoxicity, either redox cycling between N-hydroxynorcocaine and

norcocaine nitroxide or the production of a reactive nitrosonium ion, could be

performed. The characteristics of toxicity following cocaine exposure will give

99

evidence as to the mode of action. The production of lipid peroxidation or the

depletion of glutathione would suggest a role of redox cycling, whereas

binding of radiolabeled cocaine metabolite to cellular macromolecules would

suggest the production of a reactive metabolite. Inhibition of these

manifestations of cocaine-mediated toxicity with P450 inactivators could also

help to identify specific P450-mediated oxidation steps.

APPENDIX 1

ABBREVIATIONS

100

P450, cytochrome P450; AD, [4-14C]androst-4-ene-3,17 dione; PB,

phenobarbital; Dex, dexamethasone; DMSO, dimethyl sulfoxide; CAP,

chloramphenicol; TAO, troleandomycin; LDH, lactic acid dehydrogenase; xrr,

sod ium 3'[ 1-[ (phenyl amino )-carbonyl]3,4-tetrazolium]-bis( 4-methoxy-6-

nitro)benzene-sulfonic acid hydrate; FBS, fetal bovine serum; 7-EFC, 7-

ethyoxy-4-trifluoromethylcoumarin; DLPC; dilauryl-L-3-phosphatidylcholine

101

REFERENCES

Azri, S., A. J. Gandolfi and K. Brendel (1991). "Carbon tetrachloride toxicity in precision-cut rat liver slices." In Vitro Toxicol. 3: 309-320.

Bassell, D. M. and P. S. Guzelian (1980). "Phenotypic stability of adult rat hepatocytes in primary monolayer culture." Ann. N. Y. Acad. Sci. 349: 85-98.

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