Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1.
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 microM and 128 microM AFB1 concentrations, whereas activation of AFB1 to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 microM 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 microM AFB1, whereas incubation of HLM with furafylline at 16 microM AFB1 resulted in an average 72% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1. TAO was slightly more effective than furafylline in inhibiting AFB1 epoxidation at 128 microM 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 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 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 AFB1 detoxification through AFQ1 formation in HLM.(ABSTRACT TRUNCATED AT 400 WORDS)
Gallagher
E P
EP
Department of Environmental Health, University of Washington, Seattle 98195.
Wienkers
L C
LC
Stapleton
P L
PL
Kunze
K L
KL
Eaton
D L
DL
D000293:Adolescent; D000328:Adult; D016604:Aflatoxin B1 / Q000378:metabolism*; D001711:Biotransformation; D019388:Cytochrome P-450 CYP1A2; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D014217:Troleandomycin / Q000494:pharmacology
D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D013806:Theophylline; C050131:furafylline; D014217:Troleandomycin; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D042926:Cytochrome P-450 CYP4A