Cell Culture Studies Studies in rat and mouse cardiomyocytes were designed to evaluate mitochondrial integrity under conditions of chronic dosing with relatively non-toxic levels of NRTIs. Mitochondrial integrity was examined by: Seahorse XF24, which measures cellular oxidative and glycolytic capacity;electron microscopy (EM), which reveals mitochondrial morphology;and mitochondrial biogenesis kit, which measures proliferation. We chose doses of AZT and AZT/ddI that allowed good (more than 80%) cell survival in the H9c2 rat and HL-1 mouse cardiomyocytes, and followed cells for 36 passages in the presence and absence of drug. The Seahorse measures oxygen consumption rate (OCR), as an indicator of oxidative phos-phorylation, and extracellular acidification rate (ECAR), as an indicator of glycolysis. At most passages OCR was impaired in both rat and mouse cardiomyocytes treated with AZT or AZT/ddI, compared to unexposed controls. Also, compared to unexposed controls, ECAR, a measure of lactic acid production, was elevated at passage 20 and above in NRTI-exposed cells. In an attempt to protect mitochondria from this damage we used the stable free radical Tempol and its metabolite Tempol-H, cyclic nitroxides with antioxidant properties. H9c2 cells were exposed to AZT/ddI for 15 passages in the presence and absence of Tempol or its metabolite Tempol H. The AZT/ddI combination caused moderate growth inhibition (<30 %), and co-exposure with Tempol or Tempol H did not restore cell survival. The Seahorse analysis showed a reduction in OCR with AZT/ddI, and co-incubation with Tempol or Tempol H resulted in substantial restoration in OCR (22 to 91%). Western blotting showed that Tempol and Tempol H increased the expression of mitochondrial uncoupling protein-2 (UCP-2), suggesting that mild uncoupling may contribute to the protective mechanism. We are continuing to investigate Tempol mechanisms in cardiomyocytes, and have begun to test this compound in monkeys. Previous studies in cultured cells have demonstrated that NRTIs terminate DNA replication resulting in genomic instability, manifested as centrosomal amplification (CA, greater than 2 centrosomes/nucleus), micronuclei (MN), and MN containing whole chromosomes (MN+C). Here we examined these end points in cultured bone marrow cells grown from WT C57BL/6J mice and their counterparts having genotypes of Xpa(-/-)p53(+/+), Xpa(-/-)p53 (+/-), and Xpa(-/-)p53 (-/-), where Xpa is a DNA repair gene and p53 is a tumor suppressor gene. Cells were grown to confluence and exposed for 24 hr to non-toxic doses of AZT. Immunohistochemical staining with pericentrin revealed CA in 12, 15 and 20% of WT cells exposed to 0, 10 or 100 microM AZT, respectively. CA values were further elevated in AZT-exposed cells from the transgenic mice, compared to WT mice (ANOVA, p= 0.0025 for WT vs. each transgenic group). MN, revealed by DAPI staining, and MN+C, revealed using anti-CREST staining, were both significantly lower in WT cells compared to Xpa(-/-)p53(-/-)cells (p<0.05). Therefore, spindle abnormalities and aneuploidy (whole chromosomal loss) both occur in mouse fibroblasts cultured in the presence of AZT. The data show that both nucleotide excision repair and tumor suppressor capacity help to protect cells against genoxicity induced by AZT exposure. Monkey studiesWe are investigating the effects of transplacental NRTI exposures in fetal Erythrocebus patas monkeys taken at birth, 1 and 3 years of age from dams exposed to human-equivalent protocols. The NRTIs used included Lamivudine (3TC), Zidovudine (AZT), AZT/3TC, AZT/Didanosine (ddI), 3TC/Stavudine (d4T), and AZT/3TC/abacavir (ABC), as well as the NRTIs AZT/3TC combined with the non-NRTI Nevirapine (NVP). To mimic the use of these drugs in the clinic, the ARV drugs were given daily to pregnant patas dams (n=3/group) during the last half (10 weeks) of gestation, and daily to their neonates for the first6 weeks after birth. In this ongoing investigation we have published data for heart, skeletal muscle, brain and liver in patas at birth and 1 year of age. The 3 year study for the AZT/3TC/NVP and AZT/3TC/ABC infants is complete and samples are being analyzed. Mitochondrial morphology, examined in heart and brain by electron microscopy (EM), has revealed that AZT/3TC/NVP induces substantial damage in the heart that is persistent for up to 3 years of age. AZT/3TC/ABC also causes damage to cardiac mitochondria, but to a lesser extent than AZT/3TC/NVP. Breeding continues to be successful and we are currently dosing with Truvada (Tenofovir plus Emtricitabine). Using the human-equivalent dose we have encountered some mild and unexpected bleeding with the Truvada-exposed dams and offspring. This appears to be something unique to Truvada and will continue to be investigated. We are interested in examining agents that might protect heart and skeletal muscle from the toxic effects of NRTI therapy, with a particular focus on mitochondrial damage. This is an important question since HIV-1-uninfected infants born to HIV-1-infected mothers show evidence of mitochondrial, particularly cardiac, compromise (Lipshultz et al., J.Am.College of Cardiology, 2010). Studies in NRTI-exposed rat cardiomyocytes (see above), showed a loss in mitochondrial function in cells exposed to AZT and ddI, and a restoration of mitochochondrial function when the stable free radical Tempol was added, along with AZT/ddI. Therefore, we are currently dosing pregnant patas dams with Tempol and studying their infants taken at birth. To date there is no evidence of toxicity in the patas pregnancies. In order to evaluate genotoxocity in infants, born to HIV-1 infected women and exposed in utero to ARV drugs during the last 6 months of development and the first 6 wk after birth, we have grown bone marrow cells from patas infants taken at birth, 1 year and 3 years of age. In these studies, patas dams (n=3/group) were given human-equivalent daily dosing with AZT/3TC, AZT/3TC/ABC, or AZT/3TC/NVP, for the last half (10 wk) of gestation. Their offspring were given the same drug combination for the first 6 wk of life. Fibroblast cells cultured from the offspring were examined, with no further treatment, for % of cells with CA, MN and MN+C. In patas offspring exposed in utero to AZT/3TC and taken at birth there were significant (p<0.05) increases in %CA, %MN, and %MN+C, compared to the unexposed controls. Similar increases (p<0.05) in the same end points were observed in 1 yr old infants exposed in utero to AZT/3TC/ABC, and in 3 yr old infants exposed to AZT/3TC/NVP, by comparison with age-matched controls. Significant (p<0.05) increases were also observed in % of cells with CA and MN+C, but not MN, at 3 yr in patas infants exposed in utero to AZT/3TC/ABC. Therefore, genotoxicity, including abnormal chromosomal segregation (CA and MN) and aneuploidy (MN+C), initiated by in utero exposures to human-equivalent ARV protocols, persisted in patas offspring bone marrow (possibly in stem cells) for up to 3 yr of age. The data suggest a mechanism for AZT-induced transplacental tumorigenesis in mice, and imply that human infants exposed in utero to ARV therapy may also be at risk.
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