Abstract Book - Pathology and Laboratory Medicine - University of ...

oral presentationGRADUATE STUDENTBASIC SCIENCESABSTRACT #19Measuring mitochondrial DNA oxidative damage by HIVnucleoside reverse transcriptase inhibitors in cultured cell modelsGurmeet K Sohi 1 , Kyle Hukezalie 2 , Judy Wong 2 , Martine Caron 3 , Jacqueline Capeau 3 , Hélène Côté 11Department of Pathology and Laboratory Medicine, UBC; 2 Department of PharmaceuticalSciences, UBCBackground /objectivesThere are 34 million people currently living with HIV worldwide. Treatment with lifelong highly activeantiretroviral therapy (HAART) has greatly decreased mortality and morbidity in HIV+ individuals. Nucleosidereverse transcriptase inhibitors (NRTIs) form the backbone of HAART and have been implicated in mitochondrialdysfunction. Aging and age-associated diseases such as cardiovascular disease and cancer are reported to occur earlierin the HIV infected population. While the mechanisms for this is not fully understood, it is possibly linked tomitochondrial dysfunction. We hypothesize that NRTIs may contribute to mitochondrial toxicity by increasingoxidative damage to mitochondrial DNA (mtDNA). A qPCR-based assay that quantifies mtDNA oxidative damageby exploiting the fact that oxidative damage slows down the progression of PCR polymerase was optimized. Usingthis assay, the amplification of study mtDNA relative to an undamaged control mtDNA is used to quantify relativelevels of mtDNA oxidative damage. We measured mtDNA oxidative damage in cultured human cells exposed toNRTIs.MethodsHuman coronary artery endothelial (hCAE) cells were exposed to the following NRTIs, TDF, 3TC, AZT, andd4T, for a period of ~30 days at clinically relevant concentrations. Human colorectal adenocarcinoma (HT29)cells were exposed to the following NRTIs, TDF, 3TC, AZT, d4T, ddI, and ABC at various concentrations (withthe exception of 3TC) for a period of ~30 days. Cells were collected longitudinally at various time points andDNA was extracted. The DNA was then quantified by mtDNA copy number, amplified by long template PCRand products of this PCR were quantified also by qPCR mtDNA copy number. The mtDNA oxidative damage ofNRTI-exposed cells was expressed relative to that of unexposed control cells. The longitudinal mtDNA content wasalso quantified using qPCR.ResultsIn hCAE cells, mtDNA oxidative damage was detected with d4T and TDF exposure but not 3TC or AZT. NomtDNA oxidative damage was detected with any of the NRTIs in the HT29 culture. Depletion of mtDNAcontent was demonstrated in both cultures with d4T exposure, and in a dose dependent manner with HT29 cells,along with ddI. No changes were seen in mtDNA content with the other NRTIs in both cultures.ConclusionsDespite lower NRTI concentrations used, greater mtDNA oxidative damage was seen with primary hCAE cells,compared to immortalized HT29 cells. Of particular interest was evidence of mtDNA oxidative damage seen withTDF which is currently recommended in first line HAART. Differences seen in mtDNA oxidative damage in thetwo cultures suggest that the two cell lines are affected differently by NRTI exposure despite the fact that drugssuch as d4T are active in both as reflected by the observed decrease in mtDNA content. For example, HT29 cellsmay have undergone adaptations that protect them in unfavorable conditions conditions such as oxidative stress.Primary cells such as hCAE cells may therefore offer a more appropriate model for the study of NRTI-inducedtoxicity including mtDNA oxidative damage.Oral/Poster Presentations * 2012 31