The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17 beta-estradiol 4-hydroxylase.
Rates of microsomal 17 beta-estradiol (E2) hydroxylation at the C-2, -4, -6 alpha, and -15 alpha positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 alpha and -15 alpha positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE2 formation predominantly over 4-MeOE2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.
Spink
D C
DC
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Hayes
C L
CL
Young
N R
NR
Christou
M
M
Sutter
T R
TR
Jefcoate
C R
CR
Gierthy
J F
JF
D001189:Aryl Hydrocarbon Hydroxylases; D001943:Breast Neoplasms / Q000378:metabolism*; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D008861:Microsomes / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D013250:Steroid Hydroxylases / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D000072317:Polychlorinated Dibenzodioxins; D004958:Estradiol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1