Human studies. Human placenta was obtained from uninfected mothers and HIV-1-infected mothers receiving antiretroviral drugs during pregnancy. We extracted DNA from the placentas and analyzed all the samples for mitochondrial DNA (mtDNA) quantity using HCCA. For the unexposed infants (n=48) the placental mtDNA values were 11-173% of the blood bank control, but the antiretroviral drug-exposed infants (n=41) all had less placental mtDNA (20-80% of the blood bank control). By dichotomizing values above and below the blood bank control, there was a statistically-significant difference between the two groups (p below 0.01). Monke Monkey studies. We are investigating the effects of human-equivalent transplacental NRTI protocols on Erythrocebus patas monkey offspring taken at birth, 1 and 3 yrs of age. The current drug combinations of interest include zidovudine (AZT)/lamivudine (3TC)/abacavir (ABC) and AZT/3TC/Nevirapine (NVP), which are given to the pregnant dams for the last 50% (01 wk) of gestation and to their neonates for the first 6 weeks of life. The studies have revealed persistent (up to 3 yr) genotoxicity in bone marrow mesenchymal cells, as well as mitochondrial morphological damage in hearts and brains of 1 and 3 year old offspring. There is persistent loss in quantity of mtDNA, at birth, 1 year and 3 years of age, in hearts of offspring exposed to AZT/3TC/NVP. In addition there is a functional loss in mitochondrial OXPHOS capacity in bone marrow cells taken at 3 years of age from patas offspring exposed to AZT/3TC/ABC. Because a 3 year old patas is developmentally-equivalent to a 15 year old human, these studies indicate that perinatal antiretroviral drug exposure may result in adverse events that persist into puberty and beyond. Attenu Attenuation of NRTI toxicity with Tempol in cultured cells and patas. We are interested in examining agents that might protect heart and brain from toxicities induced by NRTI therapy. In 2014 we published studies showing that Tempol and Tempol-H protect mitochondria of cultured cardiomyocytes from NRTI-induced damage, and protect human lymphoblastoid cells from formation of the genotoxic end points micronuclei and nuclear buds. Armed with these favorable data in cultured cells, we are now analyzing newborn offspring of patas dams given no drugs, AZT/3TC alone, AZT/3TC/Tempol or Tempol alone during the last 10 weeks of gestation. Scoring of heart and brain electron microscopy photomicrographs has demonstrated that infants given AZT/3TC/Tempol are protected from mitochondrial damage induced by AZT/3TC. Analysis of other end points is in progress. Mechanisms of AZT-induced genomic instability 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 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. Previously we showed a substantial loss in the ability of AZT-exposed normal human mammary epithelial cells 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 immuno-histochemical (IHC) staining, and the untreated cells transfected with the inhibitor were 82.9% positive for STMN1 by IHC, confirming that STMN1 expression increased when hsa-miR-770-5p levels were low. Western blot confirmed downregulation of STMN1 protein levels, by hsa-miR-770-5p overexpression, in mimic transfected cells. In summary, because down-regulation of hsa-miR-770-5p caused an increase in cells expressing STMN1, AZT-induced genomic instability may occur through dysregulation of STMN1 and tubulin erosion. DNA repair mechanisms induced by AZT exposure has been explored in cultured human fibroblasts from normal or DNA repair defective donors having Bloom's (BLM) or Werner's syndromes, or xeroderma pigmentosum (XP) with defects in proteins A, B, C D, E, F and G. We evaluated the genotoxic end points micronuclei (MN) and centrosomal amplification (CA, 2 centromeres/cell). Only BLM cells showed a dose-response for MN with exposure from 0 to 200 micromolar AZT (p=0.02). Elevated CA levels were found in AZT-exposed XPB and XPD cells (p=0.08 and p=0.03, respectively). All three cell lines, BLM, XPB and XPD, are deficient in helicases required for normal DNA replication. Therefore this study suggests that removal of AZT at the DNA replication fork requires normal helicase activity.
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