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. Information on DNA adduct processing in nuclear and mitochondrial DNA are correlated with specific effects of exposure, including tumorigenesis, clinical response, specific toxicities and functional alterations in target organs and organelles. We are particularly interested in searching out themes that are common to 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 both human toxicity and human clinical response. The compounds of intensive investigation include the antiretroviral nucleoside analogs zidovudine (AZT) and lamivudine (3TC), cisplatin, tamoxifen (TAM) and polycyclic aromatic hydrocarbons (PAHs). Most HIV-1-infected pregnant women in the United States receive either AZT alone or the combination of AZT plus 3TC to inhibit maternal-fetal viral transmission. Genotoxicity and mitochondrial toxicity were determined in fetal patas monkeys taken at term after in utero exposure to both drugs. Three pregnant patas monkeys were given 40.0 mg AZT/day for the last 10 wk (50%) of gestation, and 25.0 mg 3TC/day for the last 4 wk of gestation. A new RIA was developed and validated to measure 3TC-DNA incorporation and values in testis, liver, spleen and skeletal muscle ranged from 23-560 molecules of 3TC/106 nucleotides. Measurement of AZT-DNA levels in nuclear and mitochondrial DNA from multiple organs has demonstrated that infant monkeys exposed in utero to both drugs sustain about twice the level of DNA damage as those given only AZT. In addition, the mitochondrial toxicity in heart and skeletal muscle from infant monkeys exposed in utero to AZT plus 3TC was much more severe than in infants exposed only to AZT. Mitochondrial toxicity was evidenced by morphological damage, alterations in oxidative phosphorylation (OXPHOS) enzyme specific activities and depletion in mitochondrial DNA levels. Genotoxicities and mitochondrial toxicities during the first year of life are being followed in patas monkeys exposed in utero to human equivalent combinations of antiretroviral nucleoside analog drugs. Drug-induced toxicities are being assessed by echocardiograms, and muscle biopsies provide tissues for morphology, mtDNA quantitation and measurement of OXPHOS enzymes. Blood will be examined for chemistry, lactic acid levels and concentrations of the various drugs incorporated into DNA. Drug combinations under investigation include AZT plus 3TC, AZT plus ddI, D4T plus 3TC and D4T plus ddI. For the future, these studies will have the flexibility to address other clinically-relevant drug combinations as the need arises. We are performing biomarker studies of samples from Linxian, China where the esophageal/stomach cancer mortality rate is 20% and high levels of PAHs have been measured in ingested food. A highly sensitive immunohistochemical staining method has been developed for PAH-DNA adducts using the benzo[a]pyrene (BP)-DNA antiserum. The intensity of specific nuclear staining has been semi-quantified using the Chroma Vision Automated Cellular Imaging System (ACIS). The dose-response observed in cultured human keratinocytes by ACIS has been reproduced using extracted DNA with adduct quantitation by Chemiluminescence Immunoassay (CIA), and the two methods are highly correlated over a 10-fold range of BP doses. Several esophageal biopsies from Linxian, taken in both 1985 and 1995, showed positive nuclear staining for PAH-DNA adducts and much lower staining with an antigen-absorbed antiserum, indicating that in the esophagus the squamous epithelial DNA has been damaged by PAH exposure. In contrast, 6 esophageal samples taken at autopsy from smokers and non-smokers in the US were negative. Further experiments designed to investigate the role of esophageal PAH-DNA adduct formation in the etiology of esophageal cancer in Linxian are underway. The potential formation of TAM-DNA adducts in human endometrium is a topic of some importance due to the increase (~20%) in endometrial cancer risk in women receiving TAM for breast cancer prevention. To investigate this, 21 human endometrial DNA samples were assayed by TAM-DNA CIA. After the subjects' exposure status was revealed the data demonstrated that two of 12 DNA samples from TAM-exposed women appeared to be positive with values of 45-60 adducts/106 nucleotides, while 9 DNA samples from unexposed women were negative. The assay of these samples will be repeated and the study will be extended to include more TAM-exposed patients. Since Waf1 is induced downstream of p53, mice lacking p21,are hypothesized to likely to retain more DNA damage compared to the repair-competent p21 (+/+) cells. When p21 ( -/-) cultured mouse keratinocytes were compared to normal mouse keratinocytes [p21 (+/+) ] with respect to cisplatin-DNA adduct formation, higher levels of cisplatin DNA adducts were observed in keratinocytes from the null transgenics as compared to those from normal mice. Ongoing experiments in mice of differing genotype will determine the time course for cisplatin-DNA adduct level formation, cell proliferation and apoptosis in the presence and absence of p21.
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