Human studies In a collaboration with Drs. K. Oursler and W. Blattner (Institute of Human Virology and Veterans Administration Hospital, Baltimore, MD), we have examined the content of mtDNA in leukocytes taken from HIV-1-infected patients before and after participating in a planned exercise program (the Get Fit Study). We hypothesized that a consistent program of aerobic exercise might improve the mitochondrial status of HIV-1-infected patients, despite the effects of antiretroviral therapy. Using the HC-CIA to measure mtDNA levels in 60 patient samples and 9 blood bank controls, we found that mtDNA levels were significantly higher in the controls compared to the patients. This was not unexpected based on previous studies, however, there was no correlation between mtDNA levels and oxidative capacity, and no increase in mtDNA after a program of exercise, among the exercising HIV-1-infected patients. (Manuscript submitted.) Monkey studies We 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). The antiretroviral drugs were given to pregnant patas dams (n=3-4/group) during the last half (10 weeks) of gestation and to their neonates for the first 6 weeks after birth. This is an ongoing investigation, and data have been published for heart, skeletal muscle, brain and liver for the at birth and 1 year old patas. The 3 year study for the AZT/3TC/NVP and AZT/3TC/ABC infants is complete and samples are being analyzed. Pregnancies have been successful throughout 2011, and dams are now being dosed with the NRTI combination Truvada (Tenofovir and Emtricitabine). 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 toxicity. 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). We hypothesized that the radioprotective antioxidant amifostine (AMF) might reduce NRTI-induced mitochondrial toxicity. We examined patas offspring taken at birth from pregnant patas dams given either AMF alone, AZT/3TC/AMF, AZT/3TC or no drug during the last half of gestation. Based on mitochondrial morphology, there was no protection for the hearts of infants exposed to AZT/3TC/AMF, compared to AZT/3TC alone, but there was protection in the brain. However, serum cardiac troponin (an indicator of heart toxicity) levels were very high in offspring of dams given AMF, with or without AZT/3TC, and because troponins are transient indicators of toxicity, AMF will not likely be a good choice for use in human pregnancy. The troponin studies also showed clearly that damage levels were high at birth, and for a week or two in the perinatal period, in patas infants born to NRTI-exposed dams, but that levels returned to normal by 4 weeks of age despite continuing antiretroviral drug exposure during the first 6 weeks of life. 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 (80-95%) cell survival in the H9c2 rat and HL-1 mouse cardiomyocytes, and followed mitochondrial integrity for 36 passages. Mitochondrial function, as measured by Seahorse, is expressed as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The Seahorse studies showed that OCR was impaired in cells treated with AZT or AZT/ddI at most passages, 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. When the AMF metabolite WR1065 was added, along with the NRTIs, substantial toxicity was encountered, further indicating that WR1065 is not a suitable mitochondrial protective agent. An additional candidate is the stable free radical Tempol, a cyclic nitroxide with antioxidant properties. When H9c2 cells were cultured for 15 passages in the presence of 150 microM AZT/150 micro M ddI, with or without 100 microM Tempol, OCR, which was impaired by the AZT/ddI, was restored in the presence of AZT/ddI/Tempol to the level of the unexposed controls. We will continue to evaluate underlying mechanisms related to the effect of Tempol as a mitochondrial protective agent, and we will evaluate Tempol in the offspring of pregnant patas dams given NRTIs during gestation. To-date we have administered Tempol to 2 adult female patas and encountered no toxicity. In cultured cells, exposure to AZT produces genomic instability, including cell cycle arrest, micronuclei (MN), sister chromatid exchanges, shortened telomeres and centrosomal amplification (CA, defined as >2 centrosomes /cell). In this study we examined CA and MN in bone marrow cells from WT C57BL/6J mice and their counterparts having genotypes of Xpa(-/-)p53 (+/+), Xpa(-/-)p53 (+/-), and Xpa(-/-)p53 (-/-), which were cultured to obtain mesenchymal-derived fibroblasts and exposed to AZT for 24 hr. Immunohistochemical staining with an anti-pericentrin antibody revealed CA in 12, 15 and 20% of WT cells exposed to 0, 10 or 100 microM AZT, respectively. CA values were about 10% higher for Xpa(-/-)p53 (+/+), Xpa(-/-) p53 (+/-), and Xpa(-/-)p53 (-/-) cells after exposure to the same doses of AZT, compared to WT cells (ANOVA, p= 0.0025 for WT cells vs. each transgenic group). MN formation was revealed by DAPI staining, and the presence of whole chromosomes in MN was revealed using anti-CREST staining. In WT cells exposed to 0, 10 or 100 microM AZT, the percentage of total MN, including those containing whole and partial chromosomes, was 3.4, 3.6, and 5.3%, respectively, compared to 15.2, 16.5, and 15.0%, respectively, for the Xpa(-/-)p53(-/-)cells. Therefore, both CA and aneuploidy (whole chromosomal loss) occur in mouse fibroblasts cultured in the presence of AZT. These studies indicate that the presence of an intact NER system protects cells against genoxicity induced by AZT exposure.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010770-05
Application #
8349147
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$1,275,318
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Liu, Yongmin; Shim Park, Eunwoo; Gibbons, Alexander T et al. (2016) Mitochondrial compromise in 3-year old patas monkeys exposed in utero to human-equivalent antiretroviral therapies. Environ Mol Mutagen 57:526-34
Liu, Yongmin; Shim, Eunwoo; Crespo-Mejias, Yasmin et al. (2015) Cardiomyocytes are Protected from Antiretroviral Nucleoside Analog-Induced Mitochondrial Toxicity by Overexpression of PGC-1?. Cardiovasc Toxicol 15:224-31
Poirier, Miriam C; Gibbons, Alexander T; Rugeles, Maria T et al. (2015) Fetal consequences of maternal antiretroviral nucleoside reverse transcriptase inhibitor use in human and nonhuman primate pregnancy. Curr Opin Pediatr 27:233-9
Olivero, Ofelia A; Ongele, Michael O; Braun, Hannan M et al. (2014) Selective protection of zidovudine-induced DNA-damage by the antioxidants WR-1065 and tempol. Environ Mol Mutagen 55:566-72
Herman, Eugene H; Knapton, Alan; Liu, Yongmin et al. (2014) The influence of age on serum concentrations of cardiac troponin I: results in rats, monkeys, and commercial sera. Toxicol Pathol 42:888-96
Onwuamah, Chika K; Ekama, Sabdat O; Audu, Rosemary A et al. (2014) Exposure of Allium cepa root cells to zidovudine or nevirapine induces cytogenotoxic changes. PLoS One 9:e90296
Liu, Yongmin; Shim, Eunwoo; Nguyen, Phuonggiang et al. (2014) Tempol protects cardiomyocytes from nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity. Toxicol Sci 139:133-41
Olivero, Ofelia A; Torres, Lorangelly Rivera; Gorjifard, Sayeh et al. (2013) Perinatal exposure of patas monkeys to antiretroviral nucleoside reverse-transcriptase inhibitors induces genotoxicity persistent for up to 3 years of age. J Infect Dis 208:244-8
Liu, Yongmin; Nguyen, Phuonggiang; Baris, Tara Z et al. (2012) Molecular analysis of mitochondrial compromise in rodent cardiomyocytes exposed long term to nucleoside reverse transcriptase inhibitors (NRTIs). Cardiovasc Toxicol 12:123-34
Torres, Salina M; March, Thomas H; Carter, Meghan M et al. (2010) In utero exposure of female CD-1 Mice to AZT and/or 3TC: I. Persistence of microscopic lesions in cardiac tissue. Cardiovasc Toxicol 10:37-50

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