Hepatic microsomal warfarin metabolism in warfarin-resistant and susceptible mouse strains: Influence of pretreatment with cytochrome P-450 inducers

Chem.-BioL Interactions, 75 (1990) 171--184 Elsevier Scientific Publishers Ireland Ltd.

171

H E P A T I C M I C R O S O M A L W A R F A R I N M E T A B O L I S M IN W A R F A R I N - R E S I S T A N T A N D S U S C E P T I B L E M O U S E STRAINS: I N F L U E N C E OF P R E T R E A T M E N T W I T H C Y T O C H R O M E P-450 INDUCERS

FRANCES A. SUTCLIFFE"*, ALAN D. MacNICOLL b and G. GORDON GIBSONL**

°Department of Biochemistry, Molecular Toxicology Group, University of Surrey, Guildford, Surrey, GU2 5XH and bMinistry of Agriculture, Fisheries and Food, Hook Rise South, Tolworth, Surbiton, Surrey, KT6 7NF (U.K.) (Received October 25th, 1989) (Revision received February 20th, 1990) (Accepted February 27th, 1990)

SUMMARY

In the present paper, the heterogeneity of hepatic cytochrome P-450 isoenzymes in the mouse has been probed, using warfarin as the substrate. Both sex and strain differences in the in vitro microsomal metabolism of warfarin have been investigated in male and female warfarin-resistant HC and warfarin-susceptible LAC-grey mouse strains. Animals were either untreated or treated with the cytochrome P-450 inducers phenobarbitone, f3napthoflavone or clofibrate. In both sexes and strains of mice, metabolism of warfarin was stereoselective in favour of the R ( + ) enantiomer. However, regioselectively was different in both strains and sexes of untreated animals. After pretreatment with phenobarbitone, increases in the rate of formation of 4' and 7-hydroxy R ( + ) and S ( - ) warfarin metabolites in HC mice were observed, compared with untreated animals. In LAC-grey mice increases in 4'-, 6-, 7- and 8-hydroxy R ( + ) and S ( - ) warfarin metabolites were noted, compared with untreated animals. This data indicated that different amounts or forms of cytochrome P-450s were responsible for warfarin metabolism after phenobarbitone treatment in the two strains. Pretreatment of animals with f~-napthoflavone resulted in significant decreases in the rate of R ( + ) warfarin metabolism in both strains and sexes of mice indicating that the f3naphthoflavone-inducible cytochrome P-450 isoenzymes were less active in the metabolism of warfarin, as compared to the uninduced isoenzymes. In addition, the cytochrome /)-450 isoenzyme composition in the two mouse strains was different after clofibrate pretreatment, as reflected in reduced *Present address: ICI Pharmaceuticals, Mereside, Alderley Park, Macelesfield, Cheshire, SKIO 4TG, U.K. **To whom correspondence should be sent. 0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

172 levels of some warfarin metabolites and a reduced total metabolism of warfarin, consistent with the narrow substrate specificity of clofibrate-induced cytochrome P450IVA1 for fatty acid hydroxylation. Accordingly, it is clear that both the basal and xenobiotic inducible hepatic cytochrome P-450 isoenzymes in warfarin-resistant and susceptible mice are different and therefore have implications for the in vivo disposition of warfarin.

Key words: Warfarin metabolism - - R e s i s t a n c e -- Cytochrome P-450 Mice INTRODUCTION Numerous reports have indicated that identification of specific warfarin metabolites formed in microsomal incubations may be useful diagnostic aids for assessement of functional cytochrome P-450 isoenzymes, since the relative formation of R ( + ) and S ( - ) warfarin metabolites is altered differentially in hepatic microsomes by inducing agents [1--3]. Thus, warfarin has previously been used as a substrate to probe the heterogeneity of cytochrome P-450 in various species [4-6]. For example, in microsomes prepared from human liver, warfarin is metabolised to dehydro warfarin and 4'-, 6-, 7-, 8- and 10-hydroxywarfarin by cytochrome P-450. The additional observation that warfarin metabolism exhibits substrate stereoselectivity has been used as the basis for evaluating similarities or differences between cytochrome P450 isoenzyme compositions, and the above warfarin metabolites have been detected in incubations of microsomes prepared from livers of rats, rabbits and mice [4-6]. Liver cytochromes P-450 from human, rat and mouse have been partially or completely purified in several laboratories [7]. Warfarin appears to be a substrate for almost all of the cytochrome P-450 isozymes isolated and purified up to the present time, and therefore using the racemates of one substrate, the species, strain and organ differences in cytochromes P-450 may be investigated. In this paper warfarin metabolism, as well as acting as a probe for multiple forms of cytochrome P-450, also serves as a measure of the inherent detoxification capacities of both warfarin-susceptible LAC-grey and warfarin-resistant HC mice, resistance being determined by survival to exposure to 0.025% (w/w) warfarin in a standard 21-day feeding trial. These two strains of mice have previously not been characterised with respect to their hepatic cytochrome P-450 isoenzyme profiles and therefore ability to metabolise warfarin. MATERIALS AND METHODS

Chemicals Racemic warfarin (3-a-acetonyl benzyl-4-hydroxycoumarin) was purchased from Ward Blenkinsop and Co. Ltd (London, U.K.) and R ( + ) and S ( - ) war-

173 farin enantiomers were kindly provided by Dr. B.K. Park (Department of Pharmacology, University of Liverpool). Phenobarbitone sodium,/3-naphthoflavone and clofibrate were obtained from British Drug Houses (BDH, Poole, Dorset, U.K.), Sigma Chemical Company (Poole, Dorset, U.K.) and ICI (Macclesfield, Cheshire, U.K.), respectively. All solvents were purchased from Rathburn Chemicals Ltd (Walkerburn, Scotland), and were of HPLC grade. All other chemicals and reagents were of analytical grade and purchased from BDH. Animals and pretreatment schedules Two strains of mice were used throughout these studies, One warfarinsusceptible (LAC-grey) and the other warfarin resistant (Homozygous Cambridge (HC)), resistance being determined by survival to exposure to 0.025% (w/w) warfarin in a standard 21-day feeding trial. The warfarin-resistant HC strain was bred at the Ministry of Agriculture, Fisheries and Food, following the identification of a major gene on chromosome 7, coding for warfarin resistance in a wild colony of mice at the Plant Breeding Institute in Cambridge and the incorporation of this gene into a warfarin-resistant, wildderived strain of mouse, named PBI. The HC-homozygous strain has the warfarin resistance gene from the PBI, introduced into a laboratory-bred, warfarin-susceptible strain (LAC-grey). All PBI/LAC-grey progeny were known to be heterozygous for warfarin resistance and from this F 1 generation, six further generations of heterozygous animals were mated. All animals were 70--90 days old, housed in plastic cages with sawdust bedding and maintained on Rodent Diet 41B (Oxoid Ltd., Cambridge, U.K.) and tap water ad libitum. A 12-h light/dark cycle was in operation in an ambient temperature of 21°C and 50% humidity. Male and female warfarin-resistant and warfarin-susceptible mice were divided into groups of ten and dosed with either phenobarbitone sodium, (0.1% (w/v) in drinking water for 6 days),/3-naphthoflavone (80 mg/kg i.p. in corn oil for 3 days) or clofibrate (250 mg/kg in 0.9% (w/v) saline i.p. for 3 days). Control animals received the appropriate vehicle i.p. for 3 days. All mice were killed by cervical dislocation 24 h after the last dose, their livers were excised immediately for the preparation of microsomal fractions, and perfused with 0.9% (w/v) saline {10 ml). Preparation of mouse hepatic microsomes Microsomal fractions were prepared by the calcium precipitation method as previously described [8] from pooled livers of ten mice. All procedures were carried out at 4 °C and centrifugation was undertaken using an MSE 18 centrifuge with a model No. 69181 fixed angle, 8 x 50 ml rotor head. After preparation, microsomes were resuspended in 50 mM potassium phosphate buffer (pH 7.25) containing 20% (v/v) glycerol and were stored in 2-ml aliquots at - 8 0 ° C until analysis. Hepatic microsomes could be stored in this manner for several months without loss of cytochrome P-450-dependent activity.

174

Cytochrome P-$50 and protein determinations Cytochrome P-450 concentrations were determined from the reduced carbon monoxide (CO) versus the reduced difference spectra (AE450_490 = 91 mM -1 cm -1) according to the method of 0 m u r a and Sato [9], using a Varian DMS 100 spectrophotometer in the split beam mode at 20 o _+ 1°C. Protein concentrations were determined by the method of Lowry et al. [10], using bovine serum albumin to construct a standard curve.

Microsomal metabolism of warfarin enantiomers The in vitro metabolism of R( + ) and S( - ) warfarin by mouse liver microsomes was performed as previously documented [6], with the following modification. Reaction mixtures contained, in a total volume of 1.65 ml of 50 mM T r i s - H C I buffer (pH 7.4) and 5 mM MgC12, 8 mg of microsomal protein and 0.33/~mol of R ( + ) or S ( - ) sodium warfarin. The mixture was preincubated with shaking at 37 °C for 1 rain and NADPH added to initiate the reaction, such that the final concentration in the reaction mixture was 1 mM. The reaction w a s allowed to proceed for 10 rain (metabolite production under these conditions was linear up to and including 10 min) and was terminated by rapid cooling in an ice-bath and filtering through a pad of millipore filters (1 x 1.2/~m and 2 x 0.22/~m) in a 13-ram Swinnex filter unit. Control and blank experiments were performed where warfarin or NADPH were omitted from the incubation mixture, respectively, all other procedures being identical to those described above. The filtrates were stored at - 2 0 °C for subsequent HPLC analysis. All incubations were performed in triplicate.

Analysis of warfarin metabolites Quantitative HPLC of warfarin, its diastereoisomeric alcohols and 4'-, 6-, 7-, 8- and benzylic hydroxylated metabolites was accomplished using a method based on that described by Fasco et al. [11]. A Waters Associates {Hartford, Cheshire, U.K.) liquid chromatography system was used, which comprised of three model M6000A pumps, a model 710B autoinjector, a model 720 systems controller, a model 740 data module and a model 481 variable wavelength ultraviolet detector, set at 313 nm. The column (300 x 4.5 ram) was packed with 10 ~m reverse phase silica (/~Bondapack C18, Waters Associates). All solvents and buffers were filtered through 0.22-~m membranes (Millipore, MA, U.S.A.) and degassed before use. Chromatographic separation of warfarin and its metabolites was carried out using two solvents: solvent A (water plus 1.5% (v/v) acetic acid, buffered to pH 6.0 with NaOH) and solvent B (acetonitrile plus 1.5% (v/v) acetic acid) at a flow rate of 2 ml/min. A multistep gradient was operated via the system controller, starting with 100% solvent A to concentrate the 200 ~1 sample onto the top of the column. The metabolites of warfarin were eluted with optimum separation with a ratio of 69°/0 solvent A: 31°/0 solvent B. Aliquots (10--200 ~l) of the filtrates from microsomal metabolism studies were injected directly onto the HPLC column and eluted as described above. Metabolite concentrations were estimated from the peak areas of each hydroxylated metabolite of warfarin (0.02 - 4 pg) derived from standard curves.

175 RESULTS

The cytochrome P-450 specific contents in hepatic microsomes from control and pre-treated male and female, warfarin-susceptible LAC-grey and warfarin-resistant HC mice are presented in Table I. Both phenobarbitone and ~-naphthoflavone increased the cytochrome P-450 specific contents to similar extents in the two strains and sexes of mouse, whereas clofibrate administration did not influence this parameter in either strain. Cytochrome P-450 specific contents and R ( + ) and S ( - ) warfarin metabolism in untreated, saline or corn oil-treated animals were identical (data not shown) and therefore data for only untreated mice is presented here. Hepatic microsomal preparations from both mouse strains and sexes exhibited the same overall stereoselectivities for R( + ) warfarin metabolism, with this enantiomer being favoured approximately 9-fold in male mice as compared to metabolism of the S ( - ) enantioner (Fig. 1). All four groups of mice showed different metabolic regioselectivites for R ( ÷ ) warfarin, such that 6- and 7-hydroxywarfarin in the HC female, 6-hydroxywarfarin in the HC male and LAC-grey female and 7-hydroxywarfarin in the LAC-grey male were the major metabolites. Metabolites of S( - ) warfarin were not detected in preparations from female mice of either strain, whereas in the microsomes of males of the two strains, small amounts of 4 J-, 7- and 8-hydroxywarfarin metabolites were formed. The overall rate of metabolism of R ( + ) and S ( - ) warfarin (based on both cytochrome P-450 and protein concentration), was 2.5--2.6-fold higher for microsomes from males and females of the warfarinresistant HC than the warfarin susceptible LAC grey mice. These figures are exemplified by significant increases in 4 J-, 6- and 8-hydroxywarfarin in the male and significant increases in 6-, 7- and 8-hydroxywarfarin in female HC mice, compared with their warfarin susceptible LAC-grey counterparts (Fig. 1). The male and female HC warfarin resistant mice therefore showed the capacity for a significantly greater (P ~ 0.001 and P ~ 0.01, respectively) rate of microsomal R ( ÷ ) warfarin metabolism than did LAC-grey warfarinTABLE I CYTOCHROME P-450 SPECIFIC CONTENTS IN MICROSOMAL PREPARATIONS DERIVED FROM WARFARIN SUSCEPTIBLE AND WARFARIN RESISTANT MICE Results presented as representative values from groups of 10 mice, which did not vary by more than 10%. Treatment

Untreated Phenobarbitone /~-naphthoflavone Clofibrate

Specific content (nmol. cyt. P-450/mg protein) LAC (male)

LAC (female)

HC (malel

HC (female)

Absorption maximum (nm)

0.36 0.92 0.92 0.38

0.28 1.44 1.09 0.37

0.33 0.90 1.09 0.38

0.32 1.23 1.01 0.34

450.0 450.1 448.0 451.9

176

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susceptible mice. In addition to this strain difference, male warfarin-resistant and warfarin-susceptible mice exhibited overall rates of metabolism which were approximately 2-fold higher than those of their female counterparts, showing that a sex d i f f e r e n c e in hepatic microsomal metabolism also occurred in untreated mice.

177

Phenobarbitone-treated mice In all sexes and strains of mice examined, the overall stereoselectivity for R(+) warfarin hepatic microsomal metabolism was approximately 7-11-fold greater than that for S ( - ) warfarin (Fig. 2). The metabolic regioselectivities were very similar for males and females of the HC strains, which showed significant (P < 0.001) increases in 4'-hydroxy R ( + ) warfarin as the major metabolite and smaller increases in 7- and 8-hydroxy R ( + ) warfarin in female and 7-hydroxy R ( + ) warfarin in male HC mice. In addition, the 4'and 7-hydroxylation of S ( - ) warfarin was induced by phenobarbitone pretreatment in HC females, whereas biotransformation remained unchanged in males. For LAC-grey mice the metabolic regioselectivity was slightly different between the sexes after phenobarbitone treatment. Significant increases (P < 0.001) in 4'-, 6-, 7- and 8-hydroxy R ( + ) warfarin were seen in both males and females, the rates of metabolism being the same in females, whilst the rate of 4'-hydroxy metabolite production was approximately twice that of the other metabolites in the male. The production of all of the above 4 metabolites of S ( - ) warfarin was increased by phenobarbitone pretreatment in female LAC-grey mice from non-detectable levels in untreated animals. An increase (P < 0.005) in 4'-hydroxy S ( - ) warfarin was shown for LAC-grey males (Fig. 2). ~Naphthoflavone-treated mice Hepatic microsomal preparations from both strains and sexes of mice exhibited the same overall steroselectivity for R( + ) warfarin with this enantiomer being favoured by approximately 8-fold in comparison to the S ( - ) substrate (Fig. 3). Metabolites derived from S ( - ) warfarin were only produced in small amounts (6- and 7-hydroxywarfarin) in HC male microsomal preparations, metabolite levels being non-detectable in the other 3 groups of mice examined. The regioselectivity in the males and females of both strains of mice were very similar, with 6- and 7-hydroxy R ( + ) warfarin being produced in approximately equal amounts. /3-Naphthoflavone treatment led to significant decreases in the production of 4'-, 6- and 8-hydroxy R ( + ) warfarin in both male and female HC hepatic microsomes (Fig. 3 ) a s well as significant decreases in 8-hydroxywarfarin formation in LAC-grey females and in 4'-hydroxywarfarin formation in LAC-grey males. Clofibrate-treated mice Again, hepatic microsomal preparations from both strains and sexes were stereoselective for the metabolism of the R ( + ) enantiomer of warfarin (approx. 3-fold), and the regioselectivities with male and female microsomes within the same strain were similar {Fig. 4). 6-Hydroxy R ( + ) warfarin was the major metabolite in HC mice, and 7- and 8-hydroxy R ( + ) warfarin were produced as major metabolites by LAC-grey microsomes. Clofibrate pretreatment resulted in significant increases in the production of 7-hydroxy R( + ) warfarin and 7- and 8-hydroxy R( + ) warfarin in female and male LACgrey microsomal preparations, respectively. Similarly, in HC mice, a signifi-

178 Warfarin-Resistant HC Mice Male .E E

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179

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Fig. 3. Influence of ~-naphthoflavone p r e t r e a t m e n t on t h e hepatic m i c r o s o m a l m e t a b o l i s m of warfarin in mice. Metabolite f o r m a t i o n significantly d i f f e r e n t from c o r r e s p o n d i n g r a t e in liver micros o m e s derived from u n t r e a t e d mice at * P < 0.01, * * P < 0.005 or * * * P < 0.001. n.d., not detectable.

180

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181 cant increase in the microsomal production of 4’-hydroxy R( + )warfarin from females and decreases in 4’-, 6- and 8-hydroxy R( + 1 warfarin from males were seen (Fig. 41. DISCUSSION

The metabolism of warfarin has been used by several groups of workers in order to probe the complexities of cytochrome P-450 isoenzyme multiplicity [3,5]. Substrate stereo- and regioselectivities of microsomal bound cytochrome P-450s in the metabolism of warfarin have been used to partially predict the complement of microsomal cytochrome P-450 isoenzymes by comparison with the regio- and stereoselectivities of highly purified cytochrome P-450 preparations. However, it must be emphasised that more than one form of cytochrome P-450 contributes to the hydroxylation of warfarin at a specific position [1,6]. The excellent correlations between the rates oft formation of warfarin metabolites in microsomal fractions and the presence of the corresponding isoenzymes in the same preparations indicated that warfarin metabolism has potential for assessing the functional cytochrome P450 isoenzyme composition of tissues. After pretreatment of mice with phenobarbitone, the most striking difference in metabolite production in male warfarin-susceptible mice was a large increase in 4’-hydroxy R(+ 1 warfarin which was not seen specifically in females, where all hydroxy metabolites were produced in equal quantities. The regioselectivity of S( - 1 warfarin metabolism was however similar in both sexes. A sex-dependence in microsomal cytochrome P-450 induction by phenobarbitone therefore appeared to be in operation in LAC-grey mice. The same was not true for warfarin-resistant HC mice, where the regio- and stereospecificites were the same in males and females, but differed from both sexes of the LAC-grey mice. Studies of microsomal warfarin metabolism in C57BL16 (B61 and DBA12 (D21 mice [S] using antibodies raised to the major inducible forms of rat cytochrome P-450 isozymes, showed that the relatively higher formation rates of R( + 1 8-, 7-, 4’- and 6-hydroxywarfarins catalysed by microsomes from phenobarbitone-treated mice were associated with the presence of cytochromes P-45OIIC1, P-450IIB1, P-450IIB2, and P450IIIAUA2. Although the regioselectivities were not the same in the strains of mouse used in the present study, it is possible that the large increases in 4’- and 7-hydroxy R( + 1and S( - ) warfarin metabolite production may be due to induction of cytochrome P-450 isozymes P-450IIBl and P450IIB2, respectively. In LAC-grey mice, different amounts or forms of cytochrome P-450s were responsible for warfarin metabolism after phenobarbitone treatment. In the female, 4’-, 6-, 7- and 8-hydroxy R( + 1and S( - 1 warfarin metabolites were increased to the same extent as each other. Again, P-450IIBl and P-450IIB2 may have been responsible for the formation of these metabolites. Phenobarbitone was probably able to induce a cytochrome P-450 similar to rat P-450IAl in LAC-grey females and to a smaller extent in males (based on

182 metabolite profiles in the rat, 11), although in this sex, P-450IIB1 seemed to be the most active isoenzyme in the microsomal preparation. Hepatic microsomal preparations from phenobarbital-treated LAC-grey and HC mice exhibited similar regio- and stereoselectivity for warfarin metabolism with the exception of 6- and 8-hydroxy R ( + ) warfarin in LAC-grey mice. Thus, after phenobarbitone treatment the cytochrome P-450 isoenzyme compositions of the 2 mouse strains were similar, and an additional isoenzyme may have been present in LAC:grey hepatic microsomal preparations. /3-Naphthofiavone treatment increased the cytochrome P-450 specific content in microsomal preparations derived from LAC-grey and HC mice (Table I). Phenobarbitone treatment also showed the same effect, but the absorption maximum of the reduced carbon monoxide spectra after the two treatments were different, exhibiting absorption maximum values of 450.1 and 448.0 nm for phenobarbitone and /3-naphthoflavone treatment, respectively, indicating that the major isoenzymes of cytochrome P-450 induced by the two agents were different. Hepatic microsomal preparations from/3-napthoflavone-treated LAC-grey and HC mice (both males and females) exhibited similar regioselectivity and stereoselectivity for warfarin metabolism, indicating that after /3-naphthoflavone treatment, the cytochrome P-450 isoenzyme composition of the two mouse strains was similar. Compared with untreated mice in all four groups of mice studied, significant decreases in the rate of R ( + ) warfarin metabolism were observed, indicating that the /~naphthoflavone inducible cytochrome P-450 isoenzymes did not readily metabolise warfarin. Kaminsky et al. [6] showed that in B6 and D2 mice, treatment with f]-naphthoflavone induced cytochrome P-450 isoenzymes which did not metabolise warfarin, in agreement with the results presented herein. Clofibrate pretreatment also resulted in regioselectivity and stereoselectivity in warfarin metabolism that was different in the two strains of mouse studied, but the cytochrome P-450 specific contents in microsomal preparations were not increased over the control values. Despite this observation, the cytochrome P-450 absorption maxima of 451.9 nm, showed that the major isoenzyme of cytochrome P-450 induced by this agent was different from both untreated microsomes and preparations from phenobarbitone or /3-napththoflavone treated mice. Clofibrate-treated HC mice exhibited metabolite regio- and stereoselectivities which were very similar to those seen in untreated HC female mice (Figs. 4 and 1). Thus, the observed warfarin metabolism by microsomes from clofibrate-treated HC female mice must have been catalysed by other non-clofibrate inducible cytochrome P-450 isoenzymes since the metabolite profiles of clofibrate-treated and untreated HC female mice were similar. In the same way, untreated HC male mice must have non-clofibrate or /3-napthoflavone induced cytochrome po450 isoenzymes, although the relative amounts/composition was different from that of HC females as judged by their different regioselective metabolite patterns (Fig. 1). Hepatic warfarin metabolism, after treatment of rats or mice with clofibrate, has not been reported previously, and although a highly

183

purified cytochrome P-450 isoenzyme induced by this agent has been isolated and characterised in the rat [12] the stereo- and regioselectivities of this isoenzyme for warfarin metabolism has not been reported. Clofibrate produced different metabolite pattcrns in LAC-grey and HC mice indicating that the isoenzyme composition in the two mouse strains was different after clofibrate treatment. In addition, clofibrate pretreatment decreased the overall levels of warfarin metabolites consistent with the observation that, in the rat, clofibrate-induced cytochrome P-450IVA1 exhibits a narrow substrate specificity for the metabolism of fatty acids [12]. In untreated mice, different regioselectivities in warfarin metabolism were observed for microsomal preparations of the 4 groups of mice examined (Fig. 1). Males and females of LAC-grey and HC strains would appear to all have different cytochrome P-450 isoenzyme complements from one another, or different amounts of these isoenzymes. Whether this observation is a result of specific strain or sex difference or whether it is related to warfarinresistance per se is not known at present. In conclusion, this study has demonstrated significant differences in regioselective and stereoselective metabolism of warfarin in resistant and susceptible mice, in both untreated and xenobiotic-induced animals, which may be rationalised by differences in the cytochrome P-450 isoenzyme complements of hepatic microsomal preparations, a conclusion that needs to be further substantiated by the use of specific antibody and gene probes for the cytochrome P-450 isoenzymes present in microsomal fractions derived from control and xenobiotic-pretreated mouse strains. Whether these differences in warfarin metabolism are biochemically or genetically related to warfarin-resistance per se remains to be determined. REFERENCES 1 L.S. Kaminsky, M.J. Fasco and F.P. Guengerich, Comparison of different forms of liver, kidney and lung microsomal cytochrome P-450 by immunological inhibition of regio- and stereoselective metabolism of warfarin J. Biol. Chem., 254 (1979) 9657-9662. 2 M. Ikeda, A.H. Conney and J.J. Burns, Stimulatory effects of phenobarbital and insecticides on warfarin metabolism in rats, J. Pharmacoh Exp. Ther., 162 (1968) 338--343. 3 F.P. Guengerich, G.A. Dannan, S.T. Wright, M.V. Martin and L.S. Kaminsky, Purification and characterisation of liver microsomal cytochrome P-450: Electrophoretic, spectral, catalytic and immunological properties and inducibility of eight isoenzymes isolated from rats treated with phenobarbital or/3-naphthoflavone, Biochemistry, 21 (1982) 6019--6030. 4 M.G.Townsend, E.M. Odam and J.M.J. Page, Studies of the microsomal drug metabolism system in warfarin resistant and warfarin susceptible rats, Biochem. Pharmacol., 24 (1975) 729-735. 5 L.D. Heimark and W.F. Trager, Stereoselective metabolism of conformational analogues of warfarin by /3-naphthoflavone-inducible cytochrome P-450, J. Med. Chem., 28 (1985) 503505. 6 L.S. Kaminsky, G.A. Dannon and F.P. Guengerich, Composition of cytochrome P-450 isozymes from hepatic microsomes of C57BL/6 and DBA/2 mice, assessed by warfarin metabolism, immunoinhibition and immunoelectrophoresis with anti-(rat cytochrome P-450), Eur. J. Biochem., 141 (1984) 141--148. 7 P.P. Tamburini, H. Masson, S.K. Bains, R. Makowksi, B. Morris and G.G. Gibson, Multiple

184 forms of hepatic cytochrome P-450. Purification, characterisation and comparison of a novel clofibrate-induced isoenzyme with other major forms of cytochrome P-450, Eur. J. Biochem., 139 (1984) 235--246. 8 D.L. Cinti, P. Moldeus and J.B. Schenkman, Kinetic parameters of drug metabolizing enzymes in Ca2÷-sedimented microsomes from rat liver, Biochem. Pharmacol., 21 (1972) 3249 --3256. 9 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem., 239 (1964) 2370-2378. 10 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265--275. 11 M.J. Fasco, L.J. Piper and L.S. Kaminsky, Biochemical applications of a quantitative highperformance liquid chromatographic assay of warfarin and its metabolites, J. Chromatogr., 131 (1977) 365--373. 12 P.P. Tamburini, H.A. Masson, S.K. Bains, R.J. Makowski, B. Morris and G.G. Gibson, Multiple forms of hepatic cytochrome P-450: purification, characterisation and comparison of a novel clofibrate-induced isoenzyme with other major forms of cytochrome P-450, Eur. J. Biochem., 139 (1984) 235--246.