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Inhibition of CYP1A2 and CYP3A4 by Oltipraz Results in Reduction of Aflatoxin B1 Metabolism in Human Hepatocytes in Primary Culture'

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Inhibition of CYP1A2 and CYP3A4 by Oltipraz Results in Reduction of Aflatoxin B1 Metabolism in Human Hepatocytes in Primary Culture' ICANCER RESEARCH55. 5574-5579. December I. 19951 Inhibition of CYP1A2 and CYP3A4 by Oltipraz Results in Reduction of Aflatoxin B1 Metabolism in Human Hepatocytes in Primary Culture' Sophie Langouët,2Brian Coles,3 Fabrice Morel, Laurent Becquemont, Philippe Beaune, F. Peter Guengerich, Brian Ketterer, and AndréGuillouzo INSERM U49, Centre Hospitalier Regional Universitaire Ponchaillou, 35033 Rennes Cedex. France IS. L. F. M.. A. G.J: Biochemistry and Molecular Biology Department. University College London. London WIP6DB, United Kingdom [B. C., B. K.J: INSERM U 75. Centre Hospitalier Universitaire Necker. 75015 Paris. France IL B., P. B.!: and Deparunent of Biochemistrs and Center in Molecular Toxicology, Vanderbilt University, Nashville. Tennessee 37232 (F. P. G.J ABSTRACT AFB1-associated carcinogenesis (8, 9), apparently by inducing ex pression of GST 10 (10, 11), the rat homologue of mouse GST Yc. Dithiolethiones are thought to act as potent chemoprotective agents It has been assumed that FB1 is also a human hepatocarcinogen against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by because hepatocellular carcinoma is common in many communities inducing glutathione S-transferases (GSTs). To determine whether these antioxidants can be similarly effective in human beings, we have investi that consume AFB 1-contaminated diets (12). Recently, this associa gated metabolism of AFB1 in primary human hepatocytes with or without tion was clearly established by an epidemiological study showing that pretreatment by oltipraz (OPZ), a synthetic derivative of the natural AFB1 is not only a hepatocarcinogen per se in humans, but is also a 1,2-dithiole-3-thione. Aflatoxin M1 (AFM1), glutathione conjugates of powerful cocarcinogen in combination with hepatitis B virus, which is AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cul often endemic in communities exposed to AFB1 (13). tures derived from eight human liver donors. Parenchymal cells obtained The question arises whether inducers, so effective in the rat, might @ from the three GST Mi-positive livers metabolized AFB1to AFM1and to be of value for the chemoprevention of the -associated hepato AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no cellular carcinogenesis in humans. The potential for GST-dependent AFBSGs were formed in the GST Mi-null celis, Pretreatment of the cells detoxication in the human liver is not as great as in the rat liver with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and because human liver has about 1 order of magnitude less GST activity CYP3A4, respectively, caused a significant increase in AFB1 metabolism. Although OPZ induced GST A2, and to a lesser extent GST Al and GST toward exo AFBO (1 1) and no apparent homologue for rat GST Ml, it decreased formation ofAFM1 and AFBSG, which involves CYP1A2 10—10and mouse GST Yc-Yc. The most active GST toward exo and CYP3A4. Inhibition by OPZ of AFB1 metabolism by reducing AFBO is human GST Ml—l (14), the benefit of which is limited by CYP1A2 and CYP3A4 was also demonstrated by decreased activity of a genetic polymorphism involving a null allele, so abundant that about their monooxygenase activities toward ethoxyresorufin and nifedipine, 50% of the human population has a GST M 1-null phenotype (15). respectively. The significant inhibition by OPZ of human recombinant Human hepatocytes in primary culture have proved to be useful in yeast CYP1A2 and CYP3A4 was also shown. These results demonstrate past studies of GST induction (16). In the present work, the use of that AFBSG can be formed by GST Mi-positive human hepatocytes only, hepatocytes in culture has been extended to studies of the effects of and suggest that chemoprotection with OPZ is due to an inhibition of inducers on the metabolism of FB1. Particular importance is at activation of AFB1, in addition to a GST-dependent inactivation of the tached to OPZ, a pharmaceutical derived from D3T, which has been carcinogenic exo-epoxide. reported to be a natural product occurring in cruciferous vegetables (17). In human hepatocytes, OPZ proves to be not only an inducer of INTRODUCTION GSTs but also an inhibitor of exo AFBO biosynthesis by CYPIA2 and CYP3A4. The mechanism of this effect has been investigated, and the @ I a product of the mold Aspergillus flavus, which infests significance of these findings for chemoprevention of FB1-depend grain and other food stuff stored under warm, moist conditions, is ent hepatocellular carcinoma in humans is discussed. metabolized by certain CYPs to a powerful genotoxin, exo AFBO (1—3).AFB1 is therefore a potential carcinogen; however, its carci nogenicity varies from one species to another (4). In the mouse, its MATERIALS AND METHODS hepatocarcinogenicity is negligible (5); this has been attributed to the particularly effective detoxication of exo AFBO by the GST Yc-Yc (6, Chemicals. Culture media and FCS were obtained from GIBCO (Paisley, 7). On the other hand, it is a powerful hepatocarcinogen in the rat Scotland). Collagenase, MC, RIF, bovine albumin, bovine insulin, AFB1,and apparently due to the absence, in any quantity, of a GST that utilizes AFB1 metabolites were products from Sigma Chemical Co. (St. Louis, MO). exo AFBO very effectively (7). However, certain inducers prevent D3T was synthesized by Dr T. W. Kensler, and OPZ was kindly supplied by Dr C. G. Caillard (Rhône-PoulencRorer,Anthony, France). Cell Isolation and Culture. Humanliver sampleswereobtainedin France Received 6/19/95: accepted 10/2/95. from eight patients undergoing liver resection for primary or secondary hep The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with atomas (Table 1). Hepatocytes were isolated by a two-step collagenase perfu 18 U.S.C. Section 1734 solely to indicate this fact. siom procedure, and the experimental procedures used were approved by the I This work was supported by the Institut National de Ia Sante et de Ia Recherche National Ethics Committee (18). Cell viability was 70—85%, as estimated by Medicale,the liguecontreIc cancercomitéd'Illeet vilaine.the AssociationpourIa the trypan blue exclusion test. Cell yields varied with the size of the liver Recherche comtrele cancer. the BIOAVENIR program. the Cancer Research Campaign (United Kingdom). and USPHS Grants CA44353 and ES0O267. S. Langouet was a fragment, and the design of each experiment (time points and treatments) recipient of a fellowship from the Ministère de Ia Recherche et de l'Espace and was depended on the number of viable cells obtained. granted a short-term visiting fellowship from the European Science Foundation to carry Liver parenchymal cells were seeded at a density of 106viable cells/35-cm2 out part of the work in the London group. dish in 2 ml of a nutrient medium consisting of 75% MEM and 25% Medium 2 To whom requests for reprints should be addressed. 3 Present address: National Center for Toxicological Research, Jefferson, AZ 199, supplemented with 10 @xgbovineinsulin/mI, 0.2% BSA, and 10% FCS. 72079-9502. This medium, supplemented with 0. 1 p.M dexamethasone but lacking serum, 4 The abbreviations used are: . aflatoxin : AFBO, aflatoxin 8,9 epoxide; was renewed daily. AFBSG. glutathiome conjugate of FBI oxide: CYP, cytochrome P450; GSH, glutathione; Inducers were added 36—48 h after cell seeding and then 24, 48, and 72 h GST, glutathiome S-transferase: MC, 3-methylcholanthrene; D3T, l,2-dithiole-3-thione; OPZ, oltipraz: RIF, rifampicim; HPLC, high-pressure liquid chromatography; EROD, later, with medium renewal. MC, D3T, OPZ, and RIF were dissolved in 7-ethoxyresorufin 0-deethylase. DMSO before addition to the culture medium to give final concentrations of 5 5574 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. OLTIPRAZ EFFECtS ON A@1 METABOLISM Table I Characteristics of human liver samples and AFB1 biotransfonnation in corresponding prinror@ hepatocyte cultures mycotoxin.HumanI metabolism was quantified by HPLC in untreated cellsafter 2—5daysof culture and8 h of exposure Io the liver of FB1 (%)HL-lsamplesPathologyGST MlDays cultureAFBSGExo/endo ratioAFM1@xM remainingMetabolism (69: M)―Carcinoid tumor of the small bowel+2 76.00HL-2 30.04 0.431.0 1.00.060 0.3601.17 1. 1776.00 (unknown)unknownNull2 3 — — 0.089 0.24 95.20 4 — — 0.094 1.37 72.44 67.95HL-3 5— —— —0.036 0.1941.70 1.6065.90 (69; M)Colon adenocarcimomaNull2 3 — — 0.064 1.55 69.00 4 — — 0.060 0.73 85.40 80.20HL-4 5— —— —0.250 0.1253.17 0.9936.60 (59; M)Colon ademocarcimomaNull2 3 — — 0.115 1.68 66.40 87.80HL-5 4— —— —0.065 0.0474.50 0.6110.00 metastasis+30.0171.00.0751.7864.50HL-6(68; F)Hepatic (I;M)None+30.021.00.1002.9241.60HL-7 areasNull2——0.0123.9920.20HL-8(48; F)Necrotic (49: M)Colon adenocarcimomaNull2 3 — — 0.047 4.07 18.60 4— —— —0.040 0.0334.66 3.227.00 35.60 —:not detected a (Age in years; sex). @.LMforMC and 50 ,xM for D3T, OPZ, and RIF (in 0.2% DMSO, v/v). Control method of Prough er a!. (26). Samples of yeast microsomes containing 50 pmol cultures received the same concentration of solvent. of CYP1A2 were incubated at 28°Cwith variable concentrations of After different times of treatment with the inducers, the cells were exposed ethoxyresorufin (0.2—1.6 @xM)added in 0.1 M potassium phosphate buffer. The to 5 p.M for 8 h. At the end of the incubation with the mycotoxin, both reaction was initiated by the addition of I25 MMNADPH.
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