of Work: There are more than 40 million people infected by the HIV virus worldwide where 5 million new infections occurred during 2003. Worldwide, approximately 1 in every 100 adults aged 15 to 49 is infected with HIV. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 3 million people died from AIDS last year. Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side effects by inducing mitochondrial toxicity. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 20% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease and D4T and ddC cause neuropathy in 15-20% of patients. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2'-3'-dideoxy-CTP (ddCTP), 2'-3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-dGTP (CBV-TP). Human pol gamma readily incorporated all five analogs into DNA but with varying efficiencies. Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) = ddI(didanosine) = D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol g utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. With the exception of terminally incorporated 3TC, the pol gamma 3'-5' exonuclease was inefficient at removing these five analogs from DNA and removal required enzyme levels exceeding substrate concentrations. Even though discrimination against inserting AZT and CBV makes them only moderate inhibitors in vitro, their inefficient excision suggest AZT and CBV may persist in vivo once incorporated into mtDNA by pol gamma. Finally, we found that the exonuclease activity is inhibited by AZT-monophosphate at concentrations known to occur in cells. Thus, although these analogs exert their greatest effect by insertion and chain termination of DNA synthesis, the persistence in DNA and inhibition of proofreading activity may also contribute to mitochondrial toxicity. Increasing evidence now shows that children exposed in utero to NRTI treatment used to prevent vertical transmission from mother to child, are prone to mitochondrial damage and possible future heart problems later in life. The long-term risk for these children is unknown. The goal of this project should address the human health risks from these iatrogenic agents. Antiretroviral therapies based on NRTIs, like AZT and 3TC, dramatically reduce mother-to-child transmission of HIV. However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mtDNA damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To characterize the effects of AZT and 3TC on mtDNA after perinatal exposure, we treated pregnant CD-1 mice throughout gestation and their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg AZT, 75 mg/kg 3TC, 125/62.5 mg/kg AZT/3TC, or the vehicle control. Half the pups were sacrificed on PND 28; the remaining pups continued without further dosing until sacrifice at week 10. At sacrifice, heart tissue was collected, total DNA extracted, and mtDNA assays performed using PCR-based methods. Analyses revealed an increase in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC; but not 10-week-old mice, suggesting the global effect disappeared after treatment ceased. Point mutations were elevated in 10 week-old females treated with AZT but not 3TC. Ten-week-old females treated with AZT/3TC had significant increases in mtDNA damage, and one had a point mutation. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and female mice appear to be more sensitive than males to AZT/3TC-induced mtDNA damage.
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