ROLE OF HUMAN MICROSOMAL AND HUMAN COMPLEMENTARY DNA-EXPRESSED CYTOCHROME-P4501A2 AND CYTOCHROME-P4503A4 IN THE BIOACTIVATION OF AFLATOXIN-B(1)

The metabolism of the carcinogenic mycotoxin aflatoxin B1 (AFB1) was e xamined in microsomes derived from human lymphoblastoid cell lines exp ressing transfected CYP1A2 or CYP3A4 complementary DNAs and in microso mes prepared from human liver donors (n = 4). Lymphoblast microsomes e xpressing only CYP1A2 activated AFB1 to AFB1-8,9-epoxide (AFB1-8,9-epo xide trapped as the glutathione, conjugate) at both 16 muM and 128 muM AFB1 concentrations, whereas activation of AFB1 to the epoxide in lym phoblast microsomes expressing only CYP3A4 was detected only at high s ubstrate concentrations (128 muM AFB1). AFB1 epoxidation was strongly inhibited in CYP1A2 but not CYP3A4 lymphoblast microsomes pretreated w ith furafylline, a specific mechanism-based CYP1A2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited A FB1 epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M1 (AFM1) was observed only in the C YP1A2 microsomes whereas aflatoxin Q1 (AFQ1) production was observed e xclusively in the CYP3A4 microsomes. Treatment of individual human liv er microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB1-8,9-epoxide formation at 16 muM AFB1, whereas incubation of HLM w ith furafylline at 16 muM AFB1 resulted in an average 72% inhibition o f AFB1-8,9-epoxide formation at 16 mum AFB1. TAO was slightly more eff ective than furafylline in inhibiting AFB1 epoxidation at 128 muM AFB1 (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB1-8 ,9-epoxide formation was inhibited by 89% at low substrate concentrati on and 85% at high substrate concentrations when HLM were inhibited wi th a furafylline/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 -10-hydroxywarfarin activities (respective markers of CYP1A2 and CYP3A 4 activities) in the complementary DNA-expressed microsomes demonstrat ed that TAO was less effective than furafylline as a selective P450 is oenzyme inhibitor (60% inhibition of CYP3A4 by TAO as compared to 99% inhibition of CYP1A2 by furafylline). The rates of AFB1 epoxidation an d AFQ, formation in HLM were increased 7- and 18-fold, respectively, a t high versus low substrate concentrations. These results are consiste nt with the hypothesis that CYP1A2 is the high-affinity P450 enzyme pr incipally responsible for the bioactivation of AFB1 at low substrate c oncentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB1 epoxidation and is primarily invol ved in AFB1 detoxification through AFQ1 formation in HLM. The present study also extends the use of the selective CYP1A2 inhibitor furafylli ne to studies of AFB1 oxidation in human liver microsomes.