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Vol. 43, No. 4, November 1997

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages839-846

DIFFERENTIAL INHIBITION OF AFLATOXIN B1 OXIDATION BY GESTODENE ACTION ON HUMAN LIVER MICROSOMES Bok Ryang Kim,* Hyun Sook Oh,** and Dong-Hyun Kim~t'l

*Department of Biochemistry, School of Medicine, Won Kwang University, Iksan **Department of Clinical Pathology, Won Kwang Junior College, Iksan, and rDoping Control Center, Korea Institute of Science and Technology, SeouL Korea Received July 8, 1997 Received after revision September 9, 1997

SUMMARY Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin Bl-exo-8,9-epoxide (exo-epoxidation) and aflatoxin Ql (3~hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFBt metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB~ inhibRed both exo-epoxidation and 3~-hydroxylation whereas simultaneous incubation of gestodene with AFBt only inhibited 3r These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB~ binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1. whereas it does not affect the formation of exo-epoxide via the other binding site. Key words: Aflatoxin Bb Gestodene, P450 3A4, Inhibition INTRODUCTION

Cytochrome P450 (P450) enzymes play a major role in the oxidation of a wide variety of chemicals including drugs, environmental contaminants, and endogenous compounds (1,2). P450 consists of multiple forms and each displays unique substrate and catalytic activity profiles.

Among them, P450 3A4 is of special interest because this enzyme metabolizes

structurally diverse substrates ranging from small molecules to cyclosporin (3,4), and its activity can be enhanced in vitro by a number of compounds (5-7).

P450 3A4 enzyme

activities may be directly activated by 7,8-benzoflavone (6,7) whereas some reactions such as

Abbreviations used are: P450, cytochrome P450; AFBI, aflatoxin B6 AFQI, aflatoxin Q1; AFM~, aflatoxin M1. 1To whom correspondence should be addressed 1039-9712/97/040839~08505.00/0 839

Copyright 9 1997 by Academic Press Australi~t. All rights of reproduction in any form reserved.

Vol. 43, No. 4, 1997

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

alfentanil hydroxylation are inhibited (8,9). for P450 3A4 (10).

7,8-Benzoflavone itself may serve as a substrate

These phenomena may be due to the multiple substrate binding sites in

the active site of the enzyme or the existence of different protein conformations. Recently, Koley et al. (11) demonstrated that P450 3A4 is composed of multiple kinetically distinguishable conformers using a CO binding kinetic analysis.

Moreover, studies

by (12) dearly showed direct evidence for the simultaneous binding of two substrates in the P450 3A4 active site by a kinetic analysis of 7,8-benzoflavone and phenanthrene metabolism. In the present investigation we report additional evidence of two substrate binding sites in the P450 3A4 active center by showing the differential effects of gestodene on the inhibition kinetics of AFB1-8,9-epoxidation and 3ct-hydroxylation. MATERIALS AND METHODS

.Chemicals. AFB1,troleandomycin, glucose-6-phosphate, NADP+, glutathione and glucose6-phosphate dehydrogenase were purchased from Sigma Co. (St. Louis, MO). HPLC solvent was obtained from Merck Co. (Darmstadt, Germany). Gestodene was kindly donated by Dr. F.P. Guengenich (Vanderbilt University, USA). All other reagents were of the highest grade commercially available. Preparation of human liver mierosomes. The human liver sample 0IL 110) was donated by Dr. F.P. Guengerich (Vanderbilt) and the microsomal fraction was prepared using the method described of (13). The content of P450 was estimated as 0.87 nmol/mg protein as measured by the method of(14). AFBI metabolism. Measurement of AFB1 metabolism was made using the method described in (15). Briefly, incubation mixtures consisted of 50 mM potassium phosphate buffer (pH 7.5) containing 2 mM glutathione, 0.6 mg mouse liver cytosol, 50 ktM AFBt, 5 mM glucose 6phosphate, 1.0 mM NADP ~, and 1.0 IU glucose 6-phosphate dehydrogenase. The mixtures were incubated for 10 min at 37 ~ and the reaction was terminated by the addition of 36 p / o f formic acid. The reactants were centrifuged at 3000 rpm for 10 min and the supernatant fraction was taken for analysis. Treatment of Gestodene. Treatments with gestodene were carried out in two different ways. In the preincubation experiment, gestodene was incubated with microsomes in the presence of an NADPH generating system for 30 rain prior to the addition of AFBt. In the other type of experiment, gestodene was simultaneously added to the incubation mixture with AFB~. Chromatography. Exo-epoxide and AFQ~ were quantified by HPLC with UV detection according to the modified procedure of (7). The supernatants were loaded onto the octadecylsilyl (C18) column (4.6 x 25cm, 5 #m, Beckman) with a mobile phase of solvent A (20 mM ammonium acetate, pH 5.4) containing 10% solvent B (acetonitrile: methanol: H20 = 4.5: 4.5:1, v/v/v). Elution was achieved using a gradient of 10-70 % solvent B over 16 min at a flow rate of 1.5 me/min. The eluate was monitored at 362 nm. Quantitation of exoepoxide-glutathione conjugate and AFQ1 was made by measuring the peak intensity and making comparisons with the response of an external standard.

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RESULTS AFBt metabolism by human liver microsomes.

The incubation of AFBI with HL 110

microsomes only generated exo-epoxide and AFQx at the ratio of 2.4.

AFM1 that is

predominantly generated by P450 1A2 (16,17) was not detected under the present experimental conditions (Fig. 1).

The product ratio by the microsomes is consistent with the

value range of two to three found for the reconstituted P450 3A4 system (18), and the failure to detect AFM1 suggests that AFB~ metabolism in the incubation system used here was predominantly mediated by P450 3A4. Inhibition of AFB1 metabolism by gestodene.

Gestodene is known to be a selective

inhibitor of P450 3A4 through the inactivation of heme (19).

Preincubation of gestodene

with microsomes for 30 min inhibited both AFB~-8,9-epoxidation and 3 ~z-hydroxylation in a dose dependent manner (Fig. 2A).

The degree of inhibition was linear up to 20 I.tM

gestodene and saturated at concentrations above 50 I-tM. The simultaneous addition of gestodene with AFBI to the microsomes exhibited a different inhibition pattern (Fig 2B). Little inhibition was observed in the case of 3 a -hydroxylation up to t00 [aM gestodene.

On

the other hand, AFBl-8,9-expoxidation was inhibited in a dose dependent manner within the concentration range of 10-100 [aM, suggesting that different inhibition mechanisms exist in AFB1 metabolism. In order to characterize the inhibition kinetics of gestodene, AFB~ metabolism was measured by varing the preincubation time of gestodene with the microsomes (Fig. 3). AFB~ exo-epoxidation was inhibited by 10% in the no preincubation condition whereas the 3 a-hydroxylation reaction was inhibited by 58%.

AFBl-exo-epoxidation was gradually

inhibited with increasing preincubation time, and the magnitude of the inhibition o f exoepoxidation and 3 ct -hydroxylation became the same after 40 min of preincubation. DISCUSSION The P450 3A4 enzyme has complex substrate binding characteristics (2 and references therein).

P450 3A4 plays a major role in both AFBt-8,9-epoxidation and 3~-

hydroxylation (7).

The above two reactions were reported to be regulated differently by an

in vitro activator and/or a selective inhibitor for P450 3A4 and were interpreted in terms of allosteric effects or different protein configurations (7).

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BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

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tR, min Fig. 1. HPLC analysis of the oxidation products of AFBI formed by human liver microsomes. Human liver microsomes were incubated with AFB1 and an NAPPH-generating system in the absence ofgestodene (A) or in the presence of 20 ~tM gestodene 03) for 10 rain at 37~ The indicated peaks were identified as the glutathione conjugate of AF.Bl-exo-8,9-epoxide (1), AFQ1 (2) and AFB1 (3) by comparison with authentic samples of these compounds.

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Vol. 43, No. 4, 1997

BIOCHEMISTRYond MOLECULAR BIOLOGYINTERNATIONAL

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G e s t o d e n e (~tM) Fig. 2. Effect of gestodene on the oxidation of AFB1 by human liver microsomes. (A) gestodene was added to the reaction mixtures and preincubated for 30 min at 37~ prior to the addition of AFB1. (B) gestodene was immediately added to the reaction mixtures containing AFB1. The incubation for AFB~ oxidation was then carried out for 10 min at 37~ The formation of AFBl-exo-8,9-epoxide ( 0 ) and AFQi (Q)) were measured by HPLC. The results are shown as mean + SE for triplicate experiments.

Several lines of evidence suggest that there exist at least two different binding sites in the active center of P450 3A4.

7,8-Benzoflavone stimulated several P450 3A4-mediated

oxidative reactions such as testosterone-613-hydroxylation and AFBl-epoxidation (5-7). Some reactions such as alfentanil hydroxylation were reported to be inhibited by 7,8benzoflavone (8,9), suggesting that different regulatory mechanisms acted on the P450 3A4 reactions.

Recently, more direct evidence for the simultaneous binding of two substrates in

the P450 3A4 active site was reported by (12).

P450 3A4 catalyzes the oxidative

metabolism of 7,8-benzoflavone and phenanthrene, and these two substrates affected the Vmax of each reaction without changing their respective Km values.

This kinetic data may

be interpreted as an instance of independent binding within the active site, because the compounds were shown not to be competitive inhibitions.

Similar results were seen in the

selective inhibition of AFBI metabolism by AFB2 and dehydronifedipine.

AFB2, the

saturated derivative of AFB1, preferentially inhibited the 8,9-epoxidation (9,20) while

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BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

Vol. 43, No. 4, 1997

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