The genetics of aflatoxin B1 metabolism. Association of the induction of aflatoxin B1-4-hydroxylase with the transcriptional activation of cytochrome P3-450 gene.

The association between murine cytochrome P3-450 and hepatic aflatoxin B1-4-hydroxylase, a cytochrome P-450-dependent enzyme which converts aflatoxin B1 (AFB1) to aflatoxin M1 (AFM1), was examined by (a) purification of the cytochrome P-450 which preferentially metabolizes AFB1 to AFM1; (b) isolation of the specific cDNA clone; and (c) correlating induction of transcriptional activation of the specific message with the enzyme activity in the hepatic microsomes. Isolation of cytochromes P-450 from C57BL/6 mice, an Ah-responsive strain, pretreated with a 150 mg/kg dose of beta-naphthoflavone resulted in the partial purification of the cytochrome P-450 with preference for the metabolism of AFB1 to AFM1. Antibodies raised against this cytochrome P-450 were used to enrich hepatic mRNA for cDNA cloning. A cDNA library screened with a rat cytochrome P-450c gene probe yielded only two types of cDNA clones that contained inserts corresponding to cytochrome P1-450 and cytochrome P3-450. Specific restriction fragments of near full-length P1-450 cDNA and full-length P3-450 cDNA, hybridizing only with their respective messages, were isolated and used to assess transcriptional activation of these messages in liver and extrahepatic tissues from C57BL/6 mice treated with 3-methylcholanthrene, beta-naphthoflavone, indolylacetonitrile, and Aroclor-1254. Dose-dependent induction of the two messenger RNAs, when compared with the induction of specific enzyme activities, demonstrated the association of cytochrome P1-450 with aryl hydrocarbon hydroxylase activity and the association of cytochrome P3-450 with AFB1-4-hydroxylase activity. This supports our earlier hypothesis that AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase, although regulated by the Ah locus, are the products of two separate genes (Gurtoo, H.L., Dahms, R.P., Kanter, P., and Vaught, J.B. (1978) J. Biol. Chem. 253, 3952-3961).