The role of cytochrome P450 1B1 in benzo(a)pyrene (BP) metabolism, DNA damage and protection by chlorophyllin (CHL) in human breast cells.

B57 Our previous studies have shown that CHL (5 μM) exposure of normal human mammary epithelial cells (NHMECs) in the presence of BP (4 μM) reduced BP-DNA adducts, along with CYP1A1 and 1B1 expression. Here, we compared BP-DNA adducts, changes in CYP1A1 and 1B1 expression, and abundance of gene copies in NHMECs and MCF-7 breast cancer cells exposed to 4µM BP in the presence of different concentrations of CHL. BP-DNA adducts were determined by (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[ a ]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA), gene expression by real time (RT)-PCR, and gene copies/ng RNA by quantitative RT-PCR. For each dose of CHL (0, 2, 3, 4, 8, and 16 µM), NHMEC (strain M00012) and MCF-7 cells were incubated with CHL for 24 hr before BP (4µM) was added, and for 24 hr in the presence of 4µM BP. In cells exposed to BP alone, the NHMECs had 42.3 ± 6.32 (mean ± SD, n=6) BP-DNA adducts/10 8 nucleotides, while the MCF-7 cells had 603.5 ± 23.42 (mean ± SD, n=6) BP-DNA adducts/10 8 nucleotides. Incubation of NHMECs with BP in the presence of 2-16 µM CHL caused a dose-response in reduction of BP-DNA adduct levels ( r=0.971 ), with a reduction of 66% at 16 µM CHL. However, in the malignant MCF-7 cells, CHL did not reduce BP-DNA adduct levels at any dose. Gene expression studies showed that, in the absence of CHL, BP-induced fold-increases in CYP1A1 and 1B1 expression were similar in both cell types, with 23- and 38-fold for CYP1A1 , and 8- and 3-fold for CYP1B1, in the NHMECs and MCF-7 cells, respectively. These inductions did not correlate with BP-DNA adduct level. However, the total numbers of gene copies/ng RNA, did correlate with DNA adduct level. In unexposed cells, CYP1A1 abundance (copies/ng RNA) was 13.4 in the NHMECs compared to 26.0 in the MCF-7 cells, while CYP1B1 abundance was 1262 in the NHMECs compared to 11650 in the MCF-7 cells. In the presence of BP and CHL the MCF-7 cells had 16844-33891 CYP1B1 copies/ng RNA. Therefore, although the BP-induced fold-induction for CYP1A1 was greater than for CYP1B1 , the basal and induced expression levels of CYP1B1 were much higher than for CYP1A1 in both cell types. The data suggest that CYP1B1 may be driving the formation ofBP-DNA adducts in human breast cells. In the MCF-7 cells the lack of CHL response, either for attenuation of adducts or CYP expression, and the very high copy numbers for CYP1B1 induced by BP, suggest that if gene abundance is translated into active enzyme, CYP1B1 may be considered a major determinant in the formation of BP-DNA adducts.