Human studies: In collaboration with Dr. Maria Rugeles (Universidad de Antioquia, Medellin, Colombia) we have obtained a tissue collection that includes: frozen human placental and umbilical cord tissues (n=150);frozen extracted placental DNA (n=145);frozen umbilical cord mononuclear cells (n=77);and formalin-fixed placenta and umbilical cord (n=125). There are 3 groups: HIV-1-uninfected mothers with no ARV drugs;HIV-1-infected mothers receiving no ARV drugs until delivery;and HIV-1-infected mothers receiving ARV drugs during pregnancy. We have extracted DNA from the frozen tissue samples and have analyzed all the samples for mitochondrial DNA (mtDNA) quantity using the HC-CA. Currently the statistical analysis is in progress. Monkey studies: We are investigating the effects of transplacental NRTI exposures in fetal Erythrocebus patas monkeys taken at birth, 1 and 3 yrs of age from dams exposed to human-equivalent antiretroviral drug protocols. The drugs used included 3TC, AZT, AZT/3TC, AZT/ddI, 3TC/d4T, AZT/3TC/ABC and AZT/3TC/NVP. Pregnant patas dams (n=3 to 4/group) were dosed daily during the last half (10 weeks) of gestation, and neonates were given the same drugs for the first 6 weeks after birth. This is an ongoing investigation, and data have been reported for heart, skeletal muscle, brain, liver and umbilical cord in infants taken at birth or 1 yr of age. Notable findings include: incorporation of AZT and 3TC into nuclear and mitochondrial DNA of multiple newborn fetal organs;persistence, up to 3 yr of age, of mesenchymal cell chromosomal abnormalities induced by in utero NRTI exposures;mitochondrial (mt) morphological damage and mtDNA abnormalities in heart, skeletal muscle, and liver of patas offspring at birth and 1yr;progressive depletion of mtDNA in patas fetal brain up to 1 yr;mitochondrial morphological damage in 3 yr patas exposed to NRTIs only in utero and for the first 6 wk of age;demonstration that NRTI exposures can induce mitochondrial toxicity and genotoxicity regardless of maternal HIV-1 status. Currently we are investigating further persistence of NRTI-induced damage in 3 yr old patas exposed only in utero and for 6 wk after birth. We have focused on the 3-drug combinations AZT/3TC/NVP and AZT/3TC/ABC, and the studies have revealed persistent (up to 3 yr) genotoxicity in the bone marrow mesenchymal cells, as well as mitochondrial toxicity in heart and brain, of 1 and 3 yr old offspring. Mesenchymal cells, cultured from bone marrow, were examined for genotoxic end points including: centrosomal amplification (CA);micronuclei (MN);and MN containing whole chromosomes (MN+C). The patas offspring exposed in utero to NRTIs, had significant increases (p0.05) in all three genotoxic end points in cultured bone marrow cells taken at birth, 1 and 3 yr of age. In other experiments, pregnant patas dams were given daily doses of the combination drug Truvada (Tenofovir and Emtricitabine) for the last 10 wk of gestation. This drug is of interest because it is now being given in the clinic for HIV-1 prophylaxis, and is used both before and during pregnancy. In the patas, 2 of 5 Truvada pregnancies ended in fetal loss, accompanied by evidence of disseminated maternal and fetal liver hemmorhage. Currently we are examining the pharmacokinetics of Tenofovir and Emtricitabine in the patas, to elucidate the source of this toxicity, as similar findings have not been reported in the literature. Genotoxicity in Cultured Cells: Using mesenchymal-derived fibroblasts cultured from bone marrow cells of C57BL/6J mice, both null and wild type (WT) for the Xpa gene, and null, haploinsufficient and WT for p53, we explored the processing of AZT-induced genotoxic damage in cells exposed for 24 hr to AZT. Anti-pericentrin antiserum was used to identify CA, nuclei and micronuclei were localized using DAPI, and kinetochore staining (CREST antiserum) revealed the presence of intact chromosomes within MN (MN+C). CA formation increased with dose (0-200 micro molar AZT, for 24 hr) for all genotypes except the Xpa(+/+) p53(+/-) cells, which had very low levels of CA damage, and the Xpa(-/-) p53(-/-) cells, which had very high levels of damage. For CA incidence there was a significant three-way interaction for Xpa, p53 and AZT concentration. However, Xpa(-/-) cells had significantly higher levels of CA damage than their Xpa(+/+) counterparts, only for p53(+/-) cells. The Xpa(-/-) cells, with p53(+/+) or (+/-) genotypes, had levels of MN and MN+C higher than the corresponding Xpa(+/+) mice, but the damage was not correlated with AZT dose. This was further evidenced in the two way interaction observed for Xpa and p53 pooled over AZT concentration. These data show that both aneuploidy (MN+C) and alterations in centrosomal function (CA) occur as a consequence of exposure of mesenchymal cells to AZT. Whereas NER is typically not considered to impact NRTI intracellular processing, these data demonstrate that lack of intact NER enhances genomic instability in AZT-exposed cells. Mitochondrial Toxicity in Cultured Cells: Studies in H9c2 rat cardiomyocytes were designed to evaluate mitochondrial integrity under conditions of chronic NRTI dosing, where different antioxidants were added and evaluated for ability to attenuate mitochondrial toxicity. Mitochondrial integrity was examined by: Seahorse XF24, which measures cellular oxidative and glycolytic capacity;electron microscopy (EM), which reveals mitochondrial morphology;and mitochondrial biogenesis, which reflects proliferation. Cells were dosed for up to 16 passages (P) with AZT/ddI at doses allowing approximately 80% cell survival, in the presence and absence of the stable free radical nitroxides Tempol, and its metabolite Tempol-H. Mitochondrial function was measured by Seahorse, as oxygen consumption rate (OCR) or extracellular acidification rate (ECAR). The Seahorse studies showed that OCR was impaired in H9c2 cells treated with AZT/ddI at most passages. However, addition of Tempol or Tempol-H (200 micro molar), concomitant with AZT/ddI, increased the OCR levels by 161.8+/-24.1% and 197.3+/-34.9% respectively, above values seen with AZT/ddI alone. Electron microscopy (EM) of cells at P16 confirmed the protective effects of Tempol on mitochondrial morphology, and Western blots (at P5) showed that both nitroxides upregulated expression of mitochondrial uncoupling protein-2 (UCP-2). Complex I protein was decreased in AZT/ddI-exposed cells. Superoxide levels were increased in the presence of AZT/ddI, and significantly decreased in cells exposed to AZT/3TC/Tempol at P3, 7 and 10. Overall, the data show that Tempol and Tempol-H protect cardiomyocytes from NRTI-induced mitochondrial compromise, and UCP-2 may play a role through mild uncoupling.
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