Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1
Gallagher, E.P.; Wienkers, L.C.; Stapleton, P.L.; Kunze, K.L.; Eaton, D.L.
Cancer Research 54(1): 101-108
1994
ISSN/ISBN: 0008-5472 PMID: 8261428 Document Number: 432170
The metabolism of the carcinogenic mycotoxin aflatoxin B-1 (AFB-1) 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 AFB-1 to AFB-1-8,9-epoxide (AFB-1-8,9-epoxide trapped as the glutathione, conjugate) at both 16 mu-M and 128 mu-M AFB-1 concentrations, whereas activation of AFB-1 to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 mu-M AFB-1). AFB-1 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 AFB-1 epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M-1 (AFM-1) was observed only in the CYP1A2 microsomes whereas aflatoxin Q-1 (AFQ-1) production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB-1-8,9-epoxide formation at 16 mu-M AFB-1, whereas incubation of HLM with furafylline at 16 mu-M AFB-1 resulted in an average 72% inhibition of AFB-1-8,9-epoxide formation at 16 mu-M AFB-1. TAO was slightly more effective than furafylline in inhibiting AFB, epoxidation at 128 mu-M AFB-1 (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB-1-8,9-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. AFM-1 formation was strongly inhibited by furafylline, whereas AFQ-1 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 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 AFB-1 epoxidation and AFQ-1 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 AFB-1 at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB-1 epoxidation and is primarily involved in AFB-1 detoxification through AFQ-1 formation in HLM. The present study also extends the use of the selective CYP1A2 inhibitor furafylline to studies of AFB-1 oxidation in human liver microsomes.