Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism.
CYP1B1 and CYP1A1 expression and metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) have been characterized in early-passage human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue of seven individual donors. The level of constitutive microsomal CYP1B1 protein expression was donor dependent (<0.01-1.4 pmol/mg microsomal protein). CYP1B1 expression was substantially induced by exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to levels ranging from 2.3 to 16.6 pmol/mg among the seven donors. Extremely low, reproducible levels of constitutive CYP1A1 expression were detectable in three donors (0.03-0.16 pmol/mg microsomal protein). TCDD inductions were larger for CYP1A1, as compared to CYP1B1, demonstrating substantial variability in the induced levels among the donors (0.8-16.5 pmol/mg). Northern and reverse transcriptase PCR analyses corroborate the donor-dependent differences in protein expression, whereby CYP1B1 mRNA (5.2 kb) was constitutively expressed and was highly induced by TCDD (33-fold). The contributions of CYP1B1 and CYP1A1 to the metabolism of DMBA were analyzed using recombinant human CYP1B1 and CYP1A1, as references, in conjunction with antibody-specific inhibition analyses (anti-CYP1B1 and anti-CYP1A1). Constitutive microsomal activity exhibited a profile of regioselective DMBA metabolism that was characteristic of human CYP1B1 (increased proportions of 5,6- and 10,11-DMBA-dihydrodiols), which was inhibited by anti-CYP1B1 (84%) but not by anti-CYP1A1. TCDD-induced HMEC microsomal DMBA metabolism generated the 8,9-dihydrodiol of DMBA as the predominant metabolite, with a regioselectivity similar to that of recombinant human CYP1A1, which was subsequently inhibited by anti-CYP1A1 (79%). A CYP1B1 contribution was indicated by the regioselectivity of residual metabolism and by anti-CYP1B1 inhibition (25%). DMBA metabolism analyses of one of three donors expressing measurable basal expression of CYP1A1 confirmed DMBA metabolism levels equivalent to that from CYP1B1. The HMECs of all donors expressed similar, very high levels of the aryl hydrocarbon receptor and the aryl hydrocarbon nuclear translocator protein, suggesting that aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator protein expression are not responsible for differences in cytochrome P450 expression. This study indicates that CYP1B1 is an important activator of polycyclic aromatic hydrocarbons in the mammary gland when environmental chemical exposures minimally induce CYP1A1. Additionally, certain individuals express low levels of basal CYP1A1 in HMECs, representing a potential risk factor of mammary carcinogenesis through enhanced polycyclic aromatic hydrocarbon bioactivation.
Larsen
M C
MC
Environmental Toxicology Center, University of Wisconsin, Madison 53706, USA.
Angus
W G
WG
Brake
P B
PB
Eltom
S E
SE
Sukow
K A
KA
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001940:Breast / Q000201:enzymology*; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis*; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D004790:Enzyme Induction; D004847:Epithelial Cells / Q000201:enzymology; D005260:Female; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D011084:Polycyclic Aromatic Hydrocarbons; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D015127:9,10-Dimethyl-1,2-benzanthracene; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1