Correlation between CYP1A1 RNA transcript, protein level, enzyme activity, and DNA adducts in primary normal human mammary epithelial cells exposed to benzo[a]pyrene.
Benzo(a)pyrene (BP) undergoes metabolic activation and forms DNA adducts. The goal of this study is to identify the key players that contribute to BP-DNA adduct formation in mammary epithelial cells. We quantified RNA copies/ng cDNA (RNA cpn) of Cytochrome P450 1A1 (CYP1A1) and CYP1B1, genes which code for metabolic enzymes that form r7, t8, t9-trihydroxy-c-10-(N2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[ a]-pyrene (BPdG), the major BP-DNA adduct, and NAD(P)H:Quinone Oxidoreductase 1 (NQO1), which codes for NQO1 that converts BP quinones to less toxic hydroquinones. Primary normal human mammary epithelial cell (NHMEC) strains from 16 healthy women and MCF-7 breast cancer cells were used for comparison. We found 56- 836 and 251-13234 CYP1A1, 336-5587 and 4133-57077 CYP1B1, and 5943-40112 and 4456-55887 NQO1 RNA cpn in unexposed and BP exposed (4 uM, 12h) NHMECs, respectively. NHMECs had 7.47 (median; range: 0.85-15.8) BPdG adducts/108 nucleotides while MCF-7 cells had 790 adducts. In the NHMECs, a linear association (p=0.0015) was observed between BPdG adducts and BP-induced CYP1A1, and no correlation with other genes examined. Western blots of 4 NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation (p=0.013) between BPdG and CYP1A1, but none between BPdG and CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1/1B1 together, correlated (p=0.038) with BPdG in NHMECs, and was highly induced by BP in MCF-7 cells. Overall, the data suggest that CYP1A1 is critical for BPdG adduct formation in NHMECs. The 10-fold higher adduct level found in BPexposed MCF-7 cells, compared to NHMECs, appears likely due to high EROD activity.