We are performing biomarker studies in esophageal samples from Linxian, China where the esophageal/stomach cancer mortality rate is 20%, high levels of polycyclic aromatic hydrocarbons (PAHs) have been measured in ingested food, and residents excrete high levels of 1-hydroxypyrene. Using a semi-quantitative immunohistochemical staining method (ACIS) for PAH-DNA adducts, studies designed to elucidate a potential risk for esophageal cancer conferred by the presence of esophageal PAH-DNA adducts, within a nested case-control study design, are now in progress. An epidemiological association between ingestion of well-cooked meat and colon adenoma incidence, and the observation that PAH=s are produced during high-temperature cooking of meats, have lead to the hypothesis that PAH-DNA adduct formation may be associated with colon adenoma incidence. Samples of human leukocyte DNA were obtained from 82 individuals with rectal adenoma and 111 controls, all of whom were non-smokers. PAH-DNA adducts, measured by BPDE-DNA Chemiluminescence Immunoassay (CIA) were found to be higher among colorectal adenoma cases (1.4 adducts/108 nucleotides) than polyp-free controls (1.2 adducts/108 nucleotides) (p=0.05). Compared to individuals in the lowest quartile of PAH-DNA adduct level, those in the highest quartile had an odds ratio (OR) of 2.8 for risk of colorectal adenoma formation (p=0.048). The data support a link between PAH-exposure and colorectal neoplasia. Epidemiological studies have shown that, among women with cancer associated HPV infection, smoking may increase the cervical cancer risk an additional 2 4 fold. In an effort to elucidate the contribution of smoking we examined human cervical paraffin-embedded sections for the presence of PAH-DNA adducts using immunohistochemical staining and the ACIS. The study, involving 142 coded samples of human cervix, has shown a broad range - between 25 and 191 adducts/108 nucleotides - for PAH-DNA adduct formation. There was, however, no association between smoking and PAH-DNA adduct levels or between degree of cervical neoplasia and PAH-DNA adduct levels. The data suggest that tobacco-associated cervical cancer in HPV-infected women has multifactorial origins. Normal human mammary epithelial cell (NHMEC) strains, cultured from human breast tissue obtained at reduction mammoplasty, provide a relevant model for investigation of human interindividual differences in carcinogen metabolism and DNA-damage response. PAHs, including benzo[a]pyrene (BP), are activated to DNA binding species by Phase I enzymes (CYP1A1 and 1B1) and are detoxified by Phase II enzymes (NQO1). To elucidate the formation of BP-DNA adducts and understand the underlying metabolism, 15 NHMEC strains and MCF-7 cells were exposed to 4 M BP and assayed for BP-DNA adducts and CYP1B1 and NQO1 gene abundance (transcripts/ng RNA [tpn]) by quantitative real-time PCR (qRT-PCR). BP-DNA adduct levels (adducts/108 nucleotides) were 0.2-10.6 in NHMECs and 790 in MCF-7 cells exposed to BP for 12h. In unexposed cells, CYP1B1 abundance was between 569 and 3,452 tpn in NHMECs, and 12,897 tpn in MCF-7 cells. After 12h of BP exposure, CYP1B1 abundance increased 7- to 8-fold in NHMECs and 2.8-fold in MCF-7 cells. In unexposed cells, NQO1 gene abundance was between 9,004 and 22,530 tpn in NHMECs, and was 4,962 tpn in MCF-7 cells. After 12h of BP exposure, NQO1 abundance increased 1.1- to 1.4-fold in NHMECs, and 2.7-fold in MCF-7 cells. Therefore, before and after BP exposure the ratio of NQO1 to CYP1B1 was higher in NHMECs (9.1 before BP and 1.9 after BP exposure) compared to the low value (0.4) in MCF-7 cells. To compare gene expression with enzyme activity, CYP1A1 and 1B1 enzyme activities (by EROD assay), and dicumorol sensitive NQO1 enzyme activity, were measured in NHMEC strain M98016 and in MCF-7 cells. In unexposed MCF-7 cells, EROD activity was 4 fold higher than that found in the NHMECs. In BP-exposed NHMECs and MCF-7 cells EROD activity was increased 1.6- to 5-fold. NQO1 enzyme activity decreased by 45% and 13% in BP-exposed MCF-7 cells and NHMECs, respectively. Therefore, the high BP-DNA adduct level observed in MCF-7 cells appears to be driven by high levels of CYP1B1 expression and enzyme activity, and low levels of NQO1 enzyme activity. Overall, these results suggest that normal human breast cells are protected from BP-DNA damage, and possibly mutagenesis, by their high capacity for detoxification and comparatively low levels of activation. Previous studies showed that NHMEC strains form BPdG adducts when exposed to benzo[a]pyrene (4 μM BP) and that incubation with 5 μM chlorophyllin (CHLN) causes a 50-80% in reduction of BPdG adducts. In this study NHMECs were exposed to BP and CHLN, and expression of the BP-metabolizing enzymes CYP1A1 and CYP1B1 was determined by microarry and real time (RT)-PCR, while BPdG adducts were measured by BPDE-DNA CIA. CYP1A1 and CYP1B1 expression levels were increased in cells exposed to BP and decreased 70-85% in cells incubated with CHLN either with BP, before BP, or before and with BP exposure. Formation of BPdG adducts was reduced up to 85% in cells treated with CHLN, and the extent of adduct reduction correlated directly with the inhibition of CYP1A1 and CYP1B1 expression (p<0.01). The data suggest that CHLN may act not only by altering CYP1A1 and CYP1B1 expression levels but also by lowering the effective dose of carcinogen. The potential formation of tamoxifen (TAM)-DNA adducts in human endometrium is a controversial topic of interest, as TAM-exposed women are at risk for endometrial cancer. Modeling of chronic human TAM exposures in female monkeys revealed TAM-DNA adduct formation in ovary and uterus, but our current efforts to measure TAM-DNA adducts in human endometrium have been inconclusive. Hypothesizing that TAM-induced gene expression changes may provide insight into breast tissue response, TAM-exposed cultured normal human mammary epithelial cell (NHMEC) strains, derived from reduction mammoplasty tissue taken from 3 individuals, were subjected to microarray. We found up-regulation of the same immune response genes participating in the STAT1/JAK-interferon pathway in all 3 NHMEC strains, suggesting a novel mechanism of TAM activity in the breast. In a second project we are using immunohistochemistry (IHC), with antiserum elicited against TAM-modified DNA, to investigate TAM-DNA adduct formation in human reproductive organ tissues taken from women at hysterectomy
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