Role of Human Microsomal and Human Complementary DNA-expressed Cytochromes P4501A2 and P4503A4 in the Bioactivation of Aflatoxin B₁

Abstract

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.