This research effort has concentrated on mechanisms of interaction between chemical carcinogens, some of which are commonly used drugs, and DNA. Topics under study include both the extent of DNA adduct formation and persistence, and the biological consequences of DNA damage in cultured cells, animal models, and human tissues. DNA adduct processing in specific structural (mitochondrial, telomeric) genomic regions, and in functional (transcribing nuclear matrix) regions, are also subject to investigation. The DNA adduct data are correlated with specific effects of carcinogen/drug exposure, including tumorigenesis, clinical response, specific toxicities, and functional impairment of target organs and organelles. We are particularly interested in searching out themes that appear to be common in both animal models and human subjects, with the intention of applying the knowledge gained to either enhance or reduce a specific effect in humans. The work encompasses issues relevant to human toxicity as well as human clinical response. The compounds of intensive investigation include the antiretroviral nucleoside analog drugs zidovudine (AZT) and lamivudine (3TC), tamoxifen (TAM), and polycyclic aromatic hydrocarbons (PAHs). The antiretroviral nucleoside analog drugs AZT and 3TC become incorporated into nuclear and mitochondrial DNA causing arrest of the replicating DNA strand. Most HIV-1-infected pregnant women in the U.S. receive either AZT alone or the combination of AZT plus 3TC to inhibit maternal-fetal viral transmission. Long term consequences to the fetus include genotoxicity and mitochondrial toxicity, which are expected to be more severe with the drug combination, compared to AZT alone. In organ DNA of fetal Erythrocebus patas monkeys exposed to human equivalent protocols of either AZT or AZT plus 3TC for the last 50% of gestation, both drugs were found incorporated into fetal organ DNA, while shortened telomeres were found in all organs from 3 fetuses exposed to both drugs but in no organs of 3 fetuses exposed to AZT alone. Mitochondrial toxicity also appears to be more severe in heart, skeletal muscle and brain of fetal patas monkeys exposed to both drugs, compared to those exposed to AZT alone. Mitochondrial toxicity has been evidenced by morphological damage, dose-related abnormalities in oxidative phosphorylation enzyme activities, depletion in mitochondrial DNA quantity and mitochondrial DNA degradation. These data, obtained in virus-free monkeys, clearly demonstrate the potential of AZT plus 3TC in combination to cause similar toxicities in HIV-1-uninfected human infants. The estrogen analog tamoxifen (TAM) is administered in the clinic to prevent breast cancer. However, oral TAM administration causes formation of TAM-DNA adducts and liver tumors in rats, and increased incidence of endometrial cancers in women. Controversy has surrounded the issue of whether or not TAM-DNA adducts form in humans. We have elicited a TAM-DNA antiserum and used it to: a) establish a quantitative Chemiluminescence Immunoassay (CIA) that detects 3 TAM-DNA adducts in 109 nucleotides, and b) stain for TAM-DNA in nuclei by immunohistochemistry. Semi-quantitative values for the nuclear staining intensity have been generated by Chromavision Automated Cellular Imaging System (ACIS). Results obtained using CIA and ACIS to measure TAM-DNA adducts in rat liver are highly correlated (R2 = 0.94). When 21 human endometrial DNA samples were assayed as unknowns by TAM-DNA CIA, 9 from unexposed women were negative and 2 of 12 from TAM- exposed women gave values in the range of 5 adducts/109 nucleotides. Sample accrual will continue, but these studies support the notion that TAM-DNA adducts are formed in human endometrium. Molecular dosimetry studies of individuals in China exposed to high levels of PAHs and experiencing high esophageal cancer mortality are in progress. By ACIS, semi-quantitation of PAH-DNA adducts in 5 human esophageal biopsies from China has been observed and validated. The staining appears to be specific for PAH-DNA adducts because it is abrogated in the presence of specific antiserum absorbed with the immunogen. These studies will be expanded in an attempt to correlate PAH-DNA adduct levels and esophageal cancer risk.
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