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 environmental/occupational exposure and human clinical response. The compounds of intensive investigation include the antiretroviral nucleoside analogs zidovudine (AZT) and lamivudine (3TC) as well as the chemotherapeutic agents cisplatin and tamoxifen (TAM), and the polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants. 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 modeled in fetal Erythrocebus patas monkeys taken at term after in utero exposure to AZT plus 3TC, the doses and duration of which were chosen to mimic human exposures. Measurement of AZT-DNA and 3TC-DNA levels in nuclear DNA from multiple organs indicated that fetuses exposed to both drugs sustained twice the burden of DNA damage as fetuses exposed to AZT alone. In all animals exposed to the combination of AZT and 3TC, there was significant telomere shortening in virtually all organs. Mitochondrial toxicities were also found in the patas monkeys exposed in utero to the AZT/3TC combination. Mitochondrial damage was found in heart, skeletal muscle and brain, by transmission electron microscopy (TEM) and oxidative phosphorylation (OXPHOS) enzyme assays. Slot blot assays indicated depletion of mitochondrial (mt) DNA. The toxicity was more severe with AZT/3TC, compared to AZT alone. Genotoxicity and mitochondrial toxicity are also being followed during the first year of life in infant patas monkeys exposed in utero to human-equivalent nucleoside analog drug combinations. Infant monkeys are assessed by echocardiogram, and monthly muscle biopsies that provide tissues for TEM, cytochemistry, mtDNA quantitation and measurement of OXPHOS enzymes. Lactic acid levels, levels and persistence of drug incorporated into blood cell DNA, and chromosomal aberrations in bone marrow are also being examined. Drug combinations under investigation include AZT plus 3TC, AZT plus ddI, D4T plus 3TC and D4T plus ddI. In an attempt to examine genotoxicity and mitochondrial toxicity in exposed human infants, leukocyte samples were obtained from the Women and Infants Transmission Study (WITS). Telomere length and mtDNA quantity have been examined in cord blood leukocytes and peripheral blood leukocytes at 12 and 24 months from HIV-uninfected children of HIV-infected mothers who received either no drug (n=10) or AZT (n=10) during pregnancy, and from normal pregnancies. There was no difference in telomere length in any of the children. However, there was mtDNA depletion in children of HIV-infected mothers at all time points, and administration of AZT depleted mtDNA levels even further. In Linxian, China the esophageal/stomach cancer mortality rate is 20% and high levels of PAHs have been measured in ingested food. We have developed a semi-quantitative immunohistochemical staining method for PAH-DNA adducts, and found PAH-DNA adducts in 4 out of 5 esophageal biopsies taken in 1995. We have also been successful in finding PAH-DNA adducts in freshly-cut paraffin embedded sections taken in Linxian in 1985. The method will be used to explore a correlation between early (1985) PAH-DNA adduct formation and later (1997) esophageal cancer risk. Studies in the literature have found an association between ingestion of well-cooked meat and colon adenoma incidence, and demonstrated the presence of PAH's in meats cooked at high-temperature. A new, highly-sensitive chemiluminescence immunoassay (CIA) has been established to measure PAH-DNA adducts in human samples, and method is being used to assay DNA from lymphocytes of colon adenoma patients and controls, to investigate potential PAH exposures in the etiology of colon cancer. The formation of TAM-DNA adducts in human endometrium is important due to the increase (~20%) in endometrial cancer risk in women receiving this drug. To investigate a possible association, human endometrial DNA samples were assayed by TAM-DNA CIA, and 3 of 15 DNA samples from TAM-exposed women appeared to be positive with values of 45-60 adducts/1010 nucleotides, while 9 DNA samples from unexposed women were negative. To model human TAM exposures, 3 retired breeder cynomolgus monkeys were exposed to 2 mg TAM/kg body weight/day for 28 days. Measurable levels of TAM-DNA adducts were found in livers, brain cortex and uterus and the data were validated by two collaborators using the same DNA samples and two different methods. A study was designed to explore the potential contribution to DNA repair by Waf1/Cip1 using p21Waf1/Cip1 null, heterozygous and wild type cultured mouse keratinocytes. After exposure to 5 mM cisplatin, flow cytometry analyses indicated that cisplatin-exposed cells of all genotypes experienced a drop in the S-phase population and that, at 24 hr, 47% of the cells null for p21Waf1/Cip1 were in S-phase, while only 15% of the wild type cells were in S-phase. The null cells also had a reduced level of apoptosis (1.9%), compared with the wild type cells (7.4%). The results indicate that p21Waf1/Cip1 influences cisplatin-DNA adduct processing in mouse keratinocytes.
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