Cell Culture Studies At mitosis the centrosome directs chromosomal migration, and normal kinetochore material is required for spindle formation. In Chinese Hamster Ovary (CHO) and normal human mammary epithelial cells (NHMECs), exposure to NRTIs induced centrosomal fragmentation with multipolar mitotic figures. In addition, loss of whole chromosomes from the nucleus was evidenced by kinetochore positive micronuclei and the formation of nuclear buds. In experiments designed to evaluate functional viability of cells containing amplified centrosomes, microtubule nucleation was arrested with nocodazole and immediately continued in the abnormal centrosomes when nocodazole was removed. In addition, we demonstrated the occurrence of aneuploidy, in the form of NRTI-induced micronuclei containing whole chromosomes. Overall the study shows that centrosome amplification occurs as a result of exposure to all NRTIs, and that cells with centrosome amplification are able to accomplish cell division, but that the same cells can sustain loss of a whole chromosome in micronuclei. In subsequent studies we added amifostine and its metabolite WR-1065, antioxidants with cytoprotective properties, to evaluate their ability to modulate NRTI-induced centrosomal damage. These studies showed that addition of WR-1065 to the cell culture media reduced the NRTI-induced centrosomal amplification by about 50%. Therefore, WR-1065 provided some protection against NRTI-induced genotoxicity in these cells. We hypothesized that amifostine might protect host organs from mitochondrial toxicity induced by NRTI therapy, and we first exposed human HIV-1-infected phytohemagglutinin (PHA)-stimulated T-cell blasts with WR1065 to observe any alteration in the antiretroviral capacity of AZT. Unexpectedly we found that WR1065 has antiretroviral properties [Patent NIH332.001VPC Organic Thiophosphate Antiretroviral Agents]. Subsequent extensive WR1065 dosimetry studies in human T-cell blasts and in T-cell blasts taken from macaques infected long-term with Simian Immunodeficiency virus (SIV) showed substantial inhibition of both HIV-1 and SIV replication. Additive antiviral activity was observed with the combination of AZT plus WR1065. Therefore, amifostine, may be useful, given either alone or in combination with NRTIs, as adjuvant antiretroviral therapy. To determine mechanisms of NRTI toxicity, and possibly design therapeutic interventions, we established cardiomyocyte cultures of rat H9c2 cells and mouse HL-1 cells, and exposed them to AZT and ddI. Short-term growth inhibition assays revealed drug doses that would sustain growth in the absence of substantial toxicity (survival at 70-100% of unexposed controls), and the cells were exposed for 32 passages. Mitochondrial function, in the form of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), were measured by Seahorse XF24 analyzer at various times during exposure. The Seahorse data revealed significant depletion in maximal oxidative capacity for most of the passages (P) between P2 and P32 in the H9c2 cells and about half of the passages in the HL-1 cells. In addition, compared to unexposed controls, ECAR, a measure of lactic acid production, was elevated at some passages in both cell strains. Therefore, long-term NRTI exposures in cultured rodent cardiomyocytes impair maximal mitochondrial capacity and induce glycolytic compensation at doses that support cell viability. We plan to use this system to evaluate a series of potentially protective intervention strategies Studies in animal models We are performing ongoing investigations of mitochondrial toxicity in fetal Erythrocebus patas monkeys, taken at birth, 1 year and 3 years of age, from dams exposed to human-equivalent protocols containing 3TC, AZT, AZT/3TC, AZT/ddI, 3TC/d4T, AZT/3TC/ABC or AZT/3TC/NVP. To model human clinical use, pregnant patas dams (n=3-4/group) are dosed during the last half of gestation, and the neonates for 6 weeks after birth. Biomarkers include: clinical chemistry;electron microscopy (EM) for mitochondrial morphology;scoring of damage in EM photos;quantitation of mitochondrial (mt)DNA using hybrid capture-chemiluminescence assay (HC-CA);and oxidative phosphorylation (OXPHOS) enzyme assays. Dosing with 1-2 NRTIs has been completed. A manuscript describing analysis of liver and brain mitochondria from NRTI-exposed patas, both at birth and 1 year of age, were recently accepted for publication. Similar to the heart and skeletal muscle (data published previously), the livers and brains of NRTI-exposed fetuses sustained mitochondrial morphological damage at birth that was not improved by 1 year of age. In addition, the HC-CA showed that brain and liver mtDNA levels were largely normal at birth but significantly depleted (p less than 0.05) in all NRTI-exposed groups at 1 year of age, suggesting progressive damage in both organs. It was particularly notable that in the 1 year old monkeys exposed perinatally to 2-NRTI combinations (AZT/3TC, AZT/ddI or d4T/3TC) the brain mtDNA levels were only about 50% of those found in unexposed controls. Because of anecdotal reports indicating cognitive problems in some children exposed in utero to NRTIs we will examine cognitive function and personality parameters in the NRTI-exposed patas and controls. A preliminary set of experiments in which amifostine was given to pregnant patas monkeys was performed in order to examine toxicity and evaluate whether or not amifostine could protect fetal mitochondria from NRTI-induced toxicities. Daily doses of amifostine were administered simultaneously with a human-equivalent course of AZT/3TC for the last half of the patas gestation. The experimental groups included no drug, amifostine alone, AZT/3TC alone and AZT/3TC/amifostine. In the heart mitochondria there was no apparent protection, but the brain mitochondria did appear to be protected by amifostine, and analysis of those studies is in progress.
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