17β-estradiol hydroxylation catalyzed by human cytochrome P450 1A1 : a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA

Abstract Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17β-estradiol (E 2 ) at the C-2, C-4, C-15α, and C-6α positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E 2 . Hydroxylation at each of these four positions of E 2 was inhibited by P450 1A-subfamily inhibitors, α-naphthoflavone, benzo[ a ]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Antirat P450 1A IgG inhibited the hydroxylation of E 2 at C-2, C-15α, and C-6α, but not hydroxylation at C-4. E 2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15α, and C-6α positions of E 2 ; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low- K m hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E 2 at the C-2, C-15α, and C-6α in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E 2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.