A high level of variability was previously observed for Zidovudine (AZT)-DNA incorporation in human leukocytes from exposed mothers and infants. Here we used normal human mammary epithelial cell(NHMEC) strains to investigate variability in human AZT metabolism. Nineteen NHMEC strains were exposed for 24 hours to 0 or 200 uM AZT. AZT-DNA incorporation, determined by RIA, ranged from 6 to >100 AZT molecules/10E6 nucleotides in 12 strains, with no detectable AZT-DNA in 7 strains. Because the active form of the enzyme thymidine kinase (TK1) performs the initial AZT phosphorylation on the pathway to AZT-DNA incorporation, we examined TK1levels by Western blot. The data showed that NHMEC strains with no AZT-DNA incorporation had little or no TK1, while the majority of those with measurable AZT-DNA showed evidence of constitutive or inducible TK1 activity. Therefore, the high variability for AZT-DNA incorporation levels in human samples may be at least partially due to variability in levels of active TK1. In this study we followed genotoxic events in human lymphoblastoid MOLT-3 cells exposed continuously to 800uM AZT for 14 passages, and exposed to no drug for an additional 19 passages. Prior to treatment, cells were passaged once daily, but during the first 5 passages of AZT exposure cells grew more slowly and required passaging only every 3-6 days. AZT-DNA incorporation levels were measured at passages 1, 4 and 14, respectively. During the first 5 passages of AZT exposure, the levels of active TK1 enzyme (quantified by Western blot) became completely depleted and this was not reversed even at passage 19 after removal of AZT from the medium. During all passages in the presence of AZT (1-14) flow-cytometry showed an accumulation of cells in S-phase that was only partially reversed after AZT was removed. To investigate causes for the S-phase arrest, MOLT-3 cells cultured with 800M AZT for 14 passages were examined by NCI oligo-Microarray, Cell Cycle-specific Super Array, and Western Blot. At passage 5, when doubling time was longest, there were increases in Cyclin A, Cyclin D3 and Rb binding protein and micronucleus formation, and PCNA was decreased at both passages 5 and 14. Overall, the AZT-induced S-phase arrest appears to be consistent with changes in expression of various cell cycle-related proteins. Aneuploidy and chromosomal instability are important events in malignant transformation, and here we have shown significant AZT-induced abnormalities in kinetochore and spindle proteins, that may lead to aneuploidy, in CHO cells and in NHMECs. The centrosome (identified by a pericentrin antibody) directs chromosomal migration by a complex process of tubulin-chromatin binding. In these studies centrosomal amplification and fragmentation, evidenced by % of cells containing ≥2 centrosomal positive bodies, occurred in CHO cells exposed to 200, 400 or 800 μM AZT and in NHMECs exposed to 200 μM AZT. Electron microscopy (EM) revealed centrosomal fragmentation with electron-dense pericentriollar satellites and loss of active tubulin in some cells. Because aberrations in centrosome morphology are associated with chromosomal mis-segregation, micronuclei bearing intact chromosomes (identified by CREST antibody) were scored revealing increased positive signal for centromeric kinetochores within micronuclei of cells exposed to AZT. Therefore, AZT-induced genomic instability that is associated with alterations in proteins involved in centrosomal activation may be related to the carcinogenic potency of this compound. The aminothiol drug WR2721 (amifostine), and its free-thiol metabolite WR1065, are cytoprotective agents. Amifostine is licensed by the FDA to attenuate toxicity in cancer patients receiving chemo- and radiation-therapy, and we hypothesized that aminothiols might protect against genotoxicity induced by NRTI exposures. We first needed to ascertain that NRTI antiretroviral activity was not altered in the presence of these aminothiols. WR1065, WR2721 and/or AZT were incubated with fresh human peripheral blood mononuclear cells (PBMCs) previously activated by phytohemagglutinin (PHA) and infected with HIV-1BZ-167. At 72 hr cells were assayed for cytotoxicity and HIV-1 replication. These drugs did not alter the antiretroviral efficacy of AZT, but they did have antiretroviral properties. WR1065 concentrations of 26 and 52 M blocked virus replication by 65.0 % and 88.7 % with cell survival at 78% and 49%, respectively. A dose-response for AZT showed 50% Inhibition of virus replication at 3.5 0.7 M. A dose-response for WR1065 showed 50% Inhibition of virus replication at 3.1 0.6 nM. Therefore, when used at concentrations present in human plasma aminothiols may substantially inhibit HIV-1 replication. Pharmacokinetics of NRTIs have been examined in numerous monkey species and shown to be similar to the human. There are, however no such data for the patas monkey. In this study adult patas monkeys were given a single oral dose of 40 mg AZT plus 24 mg 3TC, essentially equivalent to human daily dosing with Combivir. Blood was drawn at 30 min before exposure, and after drug administration at 1, 2, 4, 6, 8, 12 and 24 h. The unchanged 3TC, the unchanged AZT, and the major AZT metabolites, AZT-glucuronide (AZTG) and 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite, were detected in serum using a triple quadropole mass spectrometer. AZT and 3TC were rapidly absorbed, with peak serum concentrations occurring at 30-60 minutes. AZT had a half life (t1/2) of 1.02 hrs, while 3TC showed a half life of 2.27 hrs. The values (μg/ml hr) for area under the curve (AUC) were as follows: AZT, 2.99 1.37; 3TC, 6.97 3.94; AMT, 0.002 0.001; and AZTG, 20.50 11.65. By 8 hrs, both drugs were 95% eliminated from serum. The pharmacokinetic profile in patas, other monkey species and humans were all very similar, supporting the choice of patas monkeys for these NRTI studies. We are investigating mitochondrial toxicity in fetal Erythrocebus patas monkeys taken at birth and at 1 year of age from dams exposed to human-equivalent protocols containing 3TC, AZT, AZT/3TC, AZT/Didanosine (ddI), 3TC/Stavudine (d4T), AZT/3TC/abacavir (ABC) or AZT/3TC/nevirapine. The antiretroviral drugs were given to pregnant patas dams during the last half of gestation and to their neonates for the first 6 weeks after birth. Mitochondrial morphology, examined by EM, revealed mild changes in heart and skeletal muscle from fetuses exposed to 3TC or AZT alone, while the drug combinations showed substantial mitochondrial EM damage in these organs at birth and 1 year of age. mtDNA quantity was substantially (>50%) depleted at birth in heart and skeletal muscle from infants exposed to the drug combinations. Alterations in OXPHOS enzyme specific activities were not extensive in heart or skeletal muscle from the NRTI-exposed monkeys. T [summary truncated at 7800 characters]
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