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

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 CYPIA2 activated AFB1 to AFBl-8,9-epoxide (AFB1-8,9-epoxide trapped as the glutathione, conjugate) at both 16/tM and 128/tM AFB1 concentrations, whereas activation of AFB~ to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128/~M AFBI). AFB~ epoxidation was strongly inhibited in CYPIA2 but not CYP3A4 lymphoblast microsomes pretreated with furafylline, a specific mechanism-based CYPIA2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited AFB~ epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M~ (AFM1) was observed only in the CYPIA2 microsomes whereas aflatoxin Qt (AFQ0 production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resuited in an average 20% inhibition of AFB~-8,9-epoxide formation at 16 /.tM AFB1, whereas incubation of HLM with furafylline at 16 pM AFBI resulted in an average 72 % inhibition of AFB1-8,9-epoxide formation at 16 /tM AFBI. TAO was slightly more effective than furafylline in inhibiting AFB 1 epoxiclation at 128 pM AFB~ (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFBt-8,9-epoxide formation was inhibited by 89% at low substrate concentration and 85% at high substrate concentrations when HLM were inhibited with a furafylline/TAO mixture. AFM1 formation was strongly inhibited by furafylline, whereas AFQt formation was strongly inhibited by TAO, in all HLM regardless of substrate concentration. Analysis of R-6and R-10-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 AFBI epoxidation and AFQt formation in HLM were increased 7and 18-fold, respectively, at high v e r s u s low substrate concentrations. These results are consistent with the hypothesis that CYP1A2 is the high-affinity P450 enzyme principally responsible for the bioactivation of AFB~ at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFBt epoxidation and is primarily involved in AFBt detoxification through AFQI formation in HLM. The present study also extends the use of the selective CYP1A2 inhibitor furafylline to studies of AFBt oxidation in human liver microsomes.