Cytochromes CYP1A1 and CYP1B1 in the rat mammary gland: cell-specific expression and regulation by polycyclic aromatic hydrocarbons and hormones.
Cultured rat mammary cells express both CYP1A1 and CYP1B1 in response to polycyclic aromatic hydrocarbons (PAH) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell type-specific manner. The expression of each P450 was determined functionally (regioselective PAH metabolism), as apoprotein (immunoblots) and as mRNA (Northern hybridization). The epithelial rat mammary cells (RMEC) expressed CYP1A1, however only after PAH or TCDD treatment. CYP1B1 protein was scarcely detected in these induced RMEC but was surprisingly active as a participant in 7,12-dimethylbenz[a]anthracene (DMBA) metabolism shown through selective antibody inhibition (40% of total activity). CYP1B1 was selectively expressed in the stromal fibroblast population of rat mammary cells to the exclusion of CYP1A1. In the rat mammary fibroblasts (RMF), CYP1B1 protein and associated activity were each present at low levels constitutively and were highly induced by benz[a]anthracene (BA) to a greater extent than by TCDD (12- versus 6-fold). However, BA (10 microM) and TCDD (10 nM) stimulated the 5.2-kb CYP1B1-specific mRNA equally. These increases are consistent with the involvement of the aryl hydrocarbon (Ah) receptor in the transcription of the CYP1B1 gene and with the additional stabilization of CYP1B1 protein by BA, previously observed in embryo fibroblasts. Exactly this regulation of CYP1B1-dependent activity was seen in RMEC suggesting that this arises from exceptionally active CYP1B1 in a small proportion (5%) of residual RMF. The constitutive expression and PAH inducibility of CYP1B1 and CYP1A1 proteins in RMF and RMEC, respectively, were each substantially decreased (approximately 75%) by a hormonal mixture (17 beta-estradiol (0.2 microM) progesterone (1.5 microM) cortisol (1.5 microM) and prolactin (5 micrograms/ml)). Progesterone and cortisol, added singly to RMF suppressed CYP1B1 protein expression (approximately 80%) in both untreated and BA-induced cells, while cortisol also suppressed the 5.2-kb CYP1B1 mRNA. In contrast, 17 beta-estradiol stimulated constitutive expression of CYP1B1 protein (50-75%) and mRNA level (2- to 3-fold), but did not affect CYP1B1 expression in BA-treated RMF. The expression of CYP1A1 and CYP1B1 is therefore highly cell specific even though each is regulated through the Ah receptor. Each P450 exhibits a surprisingly similar pattern of hormonal regulation even though expressed in different cell types.
Christou
M
M
Department of Pharmacology, University of Wisconsin, Madison 53705, USA.
Savas
U
U
Schroeder
S
S
Shen
X
X
Thompson
T
T
Gould
M N
MN
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004848:Epithelium / Q000201:enzymology; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006728:Hormones / Q000494:pharmacology*; D006854:Hydrocortisone / Q000494:pharmacology; D066298:In Vitro Techniques; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011084:Polycyclic Aromatic Hydrocarbons / Q000494:pharmacology*; D011374:Progesterone / Q000494:pharmacology; D011388:Prolactin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D001551:Benz(a)Anthracenes; D006728:Hormones; D000072317:Polychlorinated Dibenzodioxins; D011084:Polycyclic Aromatic Hydrocarbons; D012333:RNA, Messenger; D011374:Progesterone; D004958:Estradiol; D015127:9,10-Dimethyl-1,2-benzanthracene; D011388:Prolactin; D003577:Cytochrome P-450 Enzyme System; C030935:benz(a)anthracene; D001189:Aryl Hydrocarbon Hydroxylases; C585499:Cyp1b1 protein, rat; D065633:Cytochrome P-450 CYP1B1; D006854:Hydrocortisone