Nucleoside reverse transcriptase inhibitors (NRTIs) are an important component of long-term AIDS therapy and are associated with a host of mild to lethal side effects that are caused by mitochondrial toxicity of the drugs, often localized to adult non-replicating tissues. These drugs must be converted by cellular enzymes to the triphosphate to be active as inhibitors of the viral reverse transcriptase. Two mechanisms of toxicity have been proposed. The analog triphosphates inhibit the mitochondrial DNA polymerase leading to mtDNA depletion;or for at least one NRTI, AZT, toxicity may be caused by the inhibition of thymidine phosphorylation with alterations of the dNTP precursor pools supporting mtDNA replication. While the rates and levels of phosphorylated NRTI analogs have been measured in dividing cells in culture, the degree to which these drugs are modified or phosphorylated in adult non-replicating tissues in which the expression of salvage enzymes are expected to be different has not been studied. The goal of this proposal is to obtain a better understanding of the toxicities of the recently released and actively used NRTIs by studying their metabolism in non-replicating tissues and isolated mitochondria. This goal will be accomplished by: 1) Determining the conversion of radioactive NRTIs to their tri-phosphates in mitochondria isolated from a variety of rat tissues, in the perfused heart, and in vivo in a variety of rat tissues;2) Using NRTIs and tissues identified in (1) that have high levels of NRTI-triphosphate, perform long term treatment studies to determine if NRTI triphosphates are associated with mtDNA depletion, and if so;3) study the effect of exogenous addition of competing deoxynucleosides and/or uridine on NRTI phosphorylation and mtDNA depletion;4) Determine if any of NRTIs inhibit phosphorylation of the typical deoxynucleosides in mitochondria, the perfused heart, or in vivo in other non-replicating rat tissues;5) Using NRTIs identified in (4), determine if dNTP pools are disrupted and if this disruption leads to decreases in mtDNA level, and if so;6) Study the effect of addition of the naturally occurring competing deoxynucleosides and/or uridine on correcting the dNTP defect. To broaden this analysis, mRNA levels of enzymes of the deoxynucleoside salvage and synthesis pathways will be measured by RT-PCR to capture changes in gene expression that may be caused by long-term drug therapy. Further, microarray and proteomic analysis will be included to correlate with the RT-PCR results and to capture genome wide gene expression changes that may provide important information relative to toxicity. Finally, as NRTI toxicities are reported to be different between males and females in both humans and rodents, and because these differences may be related to sex specific differences in metabolism, all of the above studies will be done in male versus female cohorts. The successful completion of the aims of this proposal will not only provide significant essential data in understanding NRTI toxicity, but will also address sex specific differences and potential treatments including uridine supplementation, which can only improve rational therapy to limit toxicity.
The major aim of this grant is to obtain a better understanding of the toxicities of the more recently released nucleoside reverse transcriptase inhibitors (NRTIs) in non-replicating rat tissues and compare their metabolism and toxicities in both male and female cohorts. As NRTIs are identified with toxicities, we propose to test the effect of the competing exogenous deoxynucleoside and/or uridine in relieving the toxicity in long-term studies in a rat model. The successful completion of the aims of this proposal will not only provide essential data in understanding NRTI toxicity, including sex differences, but will also address potential treatments including uridine supplementation (now in Phase III trials), which can only improve rational therapy to limit toxicity.
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