In the area of PAH carcinogenesis we have performed studies using cultured normal human mammary epithelial cells (NHMECs), cancer-susceptible mice null for the DNA repair gene XPA and heterozygous for the tumor promoter p53, and human subjects. In chemoprevention studies using NHMECs exposed to benzo[a]pyrene (BP)we have found interindividual variability in BP-DNA adduct formation, induction of CYP1A1 and 1B1 expression, and induction of CYP1A1/1B1 enzyme (EROD) activity, all of which were reduced in the presence of chlorophyllin (CHLN) and delayed in the presence of tetramethoxystilbene (TMS). Because CHLN reduced BP-DNA damage in all of the 19 cell strains studied it appears this compound may be an appropriate chemoproventive agent. In contrast, TMS delayed BP metabolism but did not reduce the amount of BP-DNA damage generated during 96 hr of exposure. Therefore TMS does not appear to have potential usefulness as a chemopreventive agent. Using XPA(-/-) p53(+/-) mice to study genotoxic and epigenetic factors associated with BP-induced tumorigenesis, we have shown that the transgenic mice have 2-fold higher BP-DNA adduct levels compared to their wild type (WT) counterparts, and that ingestion of diet containing CHLN resulted in reduced BP-DNA adduct levels in liver and lung but not in esophagus. These studies, which are still in progress, have yielded important insights into the activity of CHLN in the whole animal, which are necessarily different than the activity of this compound in cultured cells. In human subjects we have shown, by immunohistochemistry (IHC) with the Automated Cellular Imaging System (ACIS), that PAH-DNA adducts localize preferentially in cytotrophoblast cells and syncytiotrophoblast knots lining the chorionic villi of the human placenta. This study showed substantial DNA damage in placentas taken from a highly-polluted area of the Czech Republic, regardless of the smoking status of the mother. In a study of 184 non-smoking young adults living in Mexico City, we showed that levels of PAH-DNA adducts were significantly higher in the dry season (winter-spring), compared to the rainy season (summer-autumn), and correlated with the seasonal levels of pollution as measured by chemcials extracted from PM10 (10 micron) filters. The levels of PAH-DNA adducts were significantly higher (p less than 0.001) in the dry season (10.663.05 per 10E9 nucleotides) than during the rainy season (9.502.85 per 10E9 nucleotides). Therefore, there was a correlation with level of ambient PAH levels and BP-DNA adducts in peripheral blood. The formation of TAM-DNA adducts in human endometrium is a controversial topic of interest, as TAM-exposed women are at risk for endometrial cancer. We have examined this question in multiple tissues of aging Erythrocebus patas (patas) monkeys given oral TAM dosing, as well as in human endometrial biopsy samples. Adducts were determined by TAM-DNA chemiluminescence immunoassay (CIA) in 3 female patas monkeys given oral TAM dosing for 3 months and 2 controls. The highest TAM-DNA adduct levels were found in liver, uterus, cerebellum and esophagus, but none were detectable in cortex, ovary and kidney. Eight samples of normal endometrium or endometrial tumor from patients receiving TAM therapy had measurable TAM-DNA values, while no TAM-DNA adducts were detected in 8 patients not receiving TAM therapy. Therefore, TAM-DNA adducts are formed in primate reproductive organ tissues as well as human endometrial tissue and tumor. Interestingly, the TAM-DNA adduct levels in patas uterus, after the monkeys were given a daily dose 5-fold higher than the human daily dose, were about 3-fold higher than the levels found in human endometrium. The data suggest that TAM-DNA adducts are likely formed in endometrium of at least some human patients receiving the drug long-term.
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