In the area of PAH carcinogenesis we have performed studies using cancer-susceptible mice null for the nucleotide excision repair (NER) gene XPA, and heterozygous for the tumor promoter p53. A short latency for benzo[a]pyrene (BP)-induced tumors has been shown in XPA(-/-)p53(+/-) C57BL/6 mice lacking the first step in NER and haploinsufficient for p53. In this study we evaluated liver, lung and esophagus, taken from wild type (WT) and XPA(-/-)p53(+/-) mice, for DNA adducts induced by oral dosing with the carcinogen BP for 28 days. Not only did we examine mice fed BP alone, but some mice were given the combination BP plus chlorophyllin (CHLN), hypothesizing that CHLN would lower BP-induced DNA damage levels. Mice were fed diet containing no additions, 100 ppm BP, 100 ppm BP plus 0.3% CHLN, or 0.3% CHLN alone for 28 days. The two methods used to measure BP-induced DNA damage were: high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS), which measures only the major DNA adduct of BP with deoxyguanosine (dG);and, BP-DNA chemiluminescence immunoassay (CIA) using antiserum elicited against BP-modified DNA, which, in addition to BPdG, measures all the other stable BP-induced DNA adducts. Both methods showed that in all three organs of mice fed BP alone, the DNA adduct levels at 28 days were higher in the cancer-susceptible Xpa(-/-)p53(+/-) mice, compared to the WT mice. This observation was consistent with the relative differences in BP-induced tumor susceptibility reported previously. In addition, a comparison of methods showed that lung and liver formed more adducts detected by CIA than HPLC-MS/MS, suggesting that these organs formed many more BP-induced DNA adducts than just BPdG. In contrast, the majority of adducts found in esophagus appeared to be BPdG. Based on prior reports we hypothesized that addition of the chemopreventive agent CHLN to BP in the diet would reduce the level of BP-induced DNA damage in all mouse organs. In the Xpa(-/-)p53(+/-) mice fed BP plus CHLN there was evidence for a reduction in BP-DNA adduct formation in the liver, but not in the other organs. In the WT mice fed BP plus CHLN, BP-DNA adducts were increased several-fold in esophagus and liver, contrary to our expectations. The data suggest that administration of dietary CHLN, along with BP, may not result in reduced BP-induced DNA damage in all organs, and therefore use of CHLN as a chemopreventive agent in the human population should be approached with caution. In the area of Tamoxifen (TAM) carcinogenesis, 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. TAM-DNA adducts were determined by TAM-DNA CIA in 3 female patas monkeys given oral TAM dosing for three months. The highest TAM-DNA adduct levels were found in liver, uterus, and brain, and none were detectable in ovary and kidney. An additional 3 female patas were given TAM oral dosing for three months, followed by no drug for two months, in order to evaluate persistence of the TAM-DNA damage. As expected, liver, uterus and brain from these animals had no persistent TAM-DNA adducts at the end of the 5 month period. In humans, samples of normal endometrium or endometrial tumor from 8 patients receiving TAM therapy all 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. Interestingly, the TAM-DNA adduct levels in uterus of patas monkeys given a daily TAM dose that was 5-fold higher than the human daily TAM dose, were about 3-fold higher than the TAM-DNA adduct levels found in human endometrium. The data suggest that TAM-DNA adducts can be formed in endometrium of human patients and that DNA damage may contribute to the risk of endometrial cancer. Because TAM is an estrogen antagonist in the breast but an estrogen agonist and carcinogen in the uterus, we hypothesized that comparison of gene expression patterns in cultured normal human breast and endometrial cells may elucidate TAM-induced mechanisms. Gene expression studies in TAM-exposed normal human mammary epithelial cells (NHMECs) showed TAM-induced up-regulation of interferon signaling and complement pathways. To contrast breast with endometrium, we examined TAM-exposed human endometrial stromal cells (HESC cells), and found the up-regulated genes to be primarily involved with biosynthesis of steroids and proliferation. In both breast and endometrial cells, microarray expression changes of the most highly altered genes were confirmed by quantitative RT-PCR. Pathway analysis showed that each cell type (breast and endometrium) induced different genes when both were exposed to the same dose of TAM for 48 hr. Immune-response pathways were predominantly induced in human breast, and steroidal and proliferative pathways were predominantly induced in human endometrium.
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