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Role of Human Microsomal and Human Complementary DNA-expressed Cytochromes P4501A2 and P4503A4 in the Bioactivation of Aflatoxin B₁¹

Departments of Environmental Health and Institute for Environmental Studies [E. P. G., P. L. S., D. L. E.], and Department of Medicinal Chemistry, [L. C. W., K. L. K], University of Washington, Seattle, Washington 98195

² To whom requests for reprints should be addressed, at Department of Environmental Health, SC-34, University of Washington, Seattle, WA 98195.

The metabolism of the carcinogenic mycotoxin aflatoxin B₁ (AFB₁) was examined in microsomes derived from human lymphoblastoid cell lines expressing transfected CYP1A2 or CYP3A4 complementary DNAs and in microsomes prepared from human liver donors (n = 4). Lymphoblast microsomes expressing only CYP1A2 activated AFB₁ to AFB₁-8,9-epoxide (AFB₁-8,9-epoxide trapped as the glutathione, conjugate) at both 16 µM and 128 µM AFB₁ concentrations, whereas activation of AFB₁ to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 µM AFB₁). AFB₁ epoxidation was strongly inhibited in CYP1A2 but not CYP3A4 lymphoblast microsomes pretreated with furafylline, a specific mechanism-based CYP1A2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited AFB₁ epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M₁ (AFM₁) was observed only in the CYP1A2 microsomes whereas aflatoxin Q₁ (AFQ₁) production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB₁-8,9-epoxide formation at 16 /am AFBi, whereas incubation of HLM with furafylline at 16 µM AFB₁ resulted in an average 72% inhibition of AFB₁-8,9-epoxide formation at 16 µM AFB₁. TAO was slightly more effective than furafylline in inhibiting AFB₁ epoxidation at 128 µM AFB₁ (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB₁-8,9-epoxide formation was inhibited by 89% at low substrate concentration and 85% at high substrate concentrations when HLM were inhibited with a ftirafylline/TAO mixture. AFM₁ formation was strongly inhibited by furafylline, whereas AFQ₁ formation was strongly inhibited by TAO, in all HLM regardless of substrate concentration. Analysis of R-6- and R-1O-hydroxywarfarin activities (respective markers of CYP1A2 and CYP3A4 activities) in the complementary DNA-expressed microsomes demonstrated that TAO was less effective than furafylline as a selective P450 isoenzyme inhibitor (60% inhibition of CYP3A4 by TAO as compared to 99% inhibition of CYP1A2 by furafylline). The rates of AFB₁ epoxidation and AFQ₁ formation in HLM were increased 7- and 18-fold, respectively, at high versus low substrate concentrations. These results are consistent with the hypothesis that CYP1A2 is the high-affinity P450 enzyme principally responsible for the bioactivation of AFB₁ at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB₁ epoxidation and is primarily involved in AFB, detoxification through AFQ₁ formation in HLM. The present study also extends the use of the selective CYP1A2 inhibitor furafylline to studies of AFB₁ oxidation in human liver microsomes.

¹ This research was supported in part by NIH Grants ES-05780, ES-03933, ES-04696, GM 47850, and GM 32165. E. P. G. is supported in part by an NIH Postdoctoral Fellowship in Environmental Pathology and Toxicology (T32ES-07032).

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 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 5/93. Accepted 11/ 5/93.

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