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 tissue, and formalin-fixed placenta and umbilical cord. There are three experimental groups: HIV-1-uninfected mothers not given 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 umbilical cord and have analyzed all the samples for mitochondrial DNA (mtDNA) quantity using the HC-CA. We found that the mtDNA quantity is variable for the unexposed infants (11-173% of the blood bank control), but the ARV-exposed infants all had smaller amounts of mtDNA (20-80% of the blood bank control). 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: 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 and progressive depletion of mtDNA in patas fetal brain up to 1 yr. Currently we are investigating further persistence of NRTI-induced damage in 3 yr old patas (equivalent to a 15 year old human) exposed 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 morphological damage in heart and brain of 3 yr old offspring. Currently pregnant patas dams are being 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 used both before and during pregnancy for HIV-1 prophylaxis. In addition we are examining the stable free radical Temopl as a potential chemopreventive agent for NRTI-induced mitochondrial toxicity. Genotoxicity in Cultured Cells: Mechanisms underlying genomic instability induced by the antiretroviral NRTI AZT are not entirely understood. Our exploratory studies revealed that the microRNA hsa-miR-770-5p was down-regulated in the mammary epithelial cell line MCF10A, as a result of exposure to 100 or 200 micromolar AZT for 24hr. We therefore chose to study the hsa-miR-770-5p target gene Stathmin1 (STMN1), because the concomitant upregulation of this gene would cause microtubule erosion and mitotic spindle destabilization, and we previously showed that 23% of normal human mammary epithelial cells exposed to 200 micromolar AZT for 24 hr lacked the ability to polymerize microtubules. In these experiments we performed reverse transfections to introduce overexpression of hsa-miR-770-5p (defined as mimic) and inhibition of hsa-miR-770 (defined as inhibitor) in MCF10A cells. Cells, analyzed for STMN1 by RT-PCR, showed high levels of hsa-miR-770-5p in the mimics. Untreated mimic transfected cells were 70.1% positive for STMN1 by immunochistochemical (IHC) staining, and the untreated cells transfected with the inhibitor were 82.9% positive for STMN1 by IHC, confirming STMN1 expression increased when hsa-miR-770-5p levels were very low. AZT-exposed mimic transfected cells showed 54.9% of cells positive for STMN1, while AZT-exposed inhibitor transfected cells showed 49.5% of cells positive for STMN1. Western blot confirmed downregulation of STMN1 protein levels, by hsa-miR-770-5p overexpression in mimic transfected cells. In summary, down-regulation of hsa-miR-770-5p caused an increase in cells expressing STMN1, and AZT-induced genomic instability may occur through dysregulation of STMN1 and tubulin erosion. Mitochondrial Toxicity in Cultured Cells: The NRTIs compromise mitochondria in cardiomyocytes and other host cells, limiting the clinical use of these drugs. To explore underlying mechanisms we overexpressed PGC-1 alpha, a master regulator of mitochondrial biogenesis, two-fold in H9c2 rat cardiomyocyte cultures, hypothesizing that this might protect the mitochondria from damage induced by the NRTI combination zidovudine (AZT) and didanosine (ddI). The experimental groups, evaluated during 16 passages (P) of drug exposure, included: PGC-1 alpha-overexpressing cells with no exposure, or exposure to 50 micromolar AZT plus 50 micromolar ddI;and control cells with no exposure or exposure to the same doses of AZT and ddI. The AZT/ddI combination caused a growth inhibition of 15-20% in control cells, but none in PGC-1 alpha cells. Apoptosis was highest in AZT/ddI-exposed control cells, and PGC-1 alpha overexpression protected cells from AZT/ddI-induced apoptosis. At P3, P6, P8 and P12, uncoupled mitochondrial oxygen consumption rate (OCR), determined by Seahorse 24 XF Analyzer, as higher in AZT/ddI-exposed PGC-1 alpha cells, compared to AZT/ddI-exposed control cells (p0.05 at all P). Complex I activity was higher in AZT/ddI-exposed PGC-1 alpha-overexpressing cells than in AZT/ddI-exposed control cells (p0.05), and reactive oxygen species (ROS) levels were lower in PGC-1 alpha-overexpressing cells than in control cells (p0.05) when both were exposed to AZT/ddI. Taken together, these experiments show proof of concept that overexpression of PGC-1 alpha protects cardiomyocytes from NRTI-induced toxicity, and suggest that a pharmaceutical agent with similar activity may protect against NRTI-induced mitochondrial toxicity.
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