IBlood cell PAH-DNA adducts and lung cancer risk. Xuan Wei, in southern China, has the highest lung cancer rates in the country, and the etiologic cause is considered to be exposure to smoky coal, used for heating and cooking.
The aim of this study is to elucidate the interaction between environmental exposure and etiology of lung cancer among women in Xuan Wei by measuring PAH-DNA damage as a biomarker associated with increased risk of disease. Currently we have extracted DNA and measured PAH-DNA adducts in 140 samples and the analysis is in progress. The high incidence of gastro-intestinal cancers in beluga whales inhabiting the mouth of the Saguenay River in the St. Lawrence Estuary (SLE) may be due to dietary PAH exposures. For 50 years, starting in 1926, PAH waste from an aluminum smelter was discarded into the river, and since the 1980s the SLE belugas found stranded presented with high occurrences of gastro-intestinal cancer. Because DNA adduct formation provides a critical link between exposure and tumor induction, we performed immuno-histochemical (IHC) staining with an antiserum specific for DNA modified with several carcinogenic PAHs, to investigate PAH-DNA adduct formation in beluga intestine. Cryostat sections of paraffin blocks, obtained from 37 SLE beluga (median age 43 yr) and 19 control beluga (median age 33 yr, from Sea World, the Eastern Beaufort Sea in the Canadian Arctic, and Alaska near Anchorage), were stained with specific PAH-DNA antiserum and fast red to visualize PAH-DNA adducts. For each slide, the whole tissue section was scanned digitally (into the Aperio system) and examined for intestine. Nuclear staining specific for PAH-DNA adducts varied from no pink color to very intense pink color that was found primarily in small intestine crypt epithelial lining cells. Using a simple scoring paradigm, the mean PAH-DNA score for the SLE beluga (n=37) was 2.1, and for the control beluga (n=19) was 0.6. A more-extensive coded analysis of stained slides is in progress, but this preliminary data suggests that the intestinal cancers observed in SLE beluga may, at least partially, be the result of PAH exposures. Tamoxifen (TAM), used for adjuvant therapy of breast cancer, also increases the risk of endometrial cancer. To compare TAM-induced transcriptional changes in breast and endometrium we examined global 5-methyl cytosine (5-meC), TAM-induced microarray changes, 5-meC in promoter region CpG islands of TAM-upregulated genes, and protein levels for histone H3 lysine di-methylases. Evaluation of uterine DNA from women (n=15) and monkeys (Erythrocebus patas n=5, and Macaca fascicularis n=12), either unexposed or exposed long-term to oral TAM, showed no difference in 5-meC levels. Subsequent studies employed normal human mammary epithelial cells (NHMECs) and human endometrial stromal cells (HESCs) exposed to 10 microM TAM for 48 hr. By Affymetrix microarray, with confirmation by RT-PCR, TAM-exposed NHMECs showed significant up-regulation of interferon signaling and immune response pathways, while the TAM-exposed HESCs showed significant up-regulation primarily of steroid and fatty acid biosynthesis pathways. Several genes, highly up-regulated by TAM in each cell type, were examined for 5-meC levels in promoter region CpG islands. In these studies the 5-meC levels were below 7%, too low to measure accurately, so we turned to histone H3 methylases. The di-methyl histone H3 lysine K4, K27, and K36 methylases, examined in TAM-exposed and unexposed NHMECs and HESCs by Western blot, showed a consistent depletion in both cell types with TAM exposure. This study shows that, whereas TAM exposure had no discernible effect on 5-meC levels in primate uterus or in cultured NHMECs and HESCs, TAM exposure induced up-regulation of different transcriptional pathways in NHMECs and HESCs, and depleted some histone H3 methylase protein levels in both cell types.
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