| Summary: | Background/objectives: HIV antiretroviral therapy, specifically nucleoside reverse transcriptase inhibitors (NRTIs) have been associated with mitochondrial DNA (mtDNA) alterations, possibly through mtDNA oxidative damage leading to mitochondrial dysfunction, which is associated with degenerative diseases and aging. A published assay exploits that oxidative damage can slow down/inhibit DNA polymerase progression, such that the PCR amplification of damaged mtDNA template yields less product compared to undamaged mtDNA. I sought to optimize this assay using in-house tools and to quantify apparent mtDNA oxidative damage in cultured cells exposed to NRTIs.
Methods: Three DNA quantification methods were compared: PicoGreen fluorescence quantification, UV spectrophotometry, and qPCR mtDNA copy number. Human hepatocellular carcinoma cells (HepG2) were exposed to hydrogen peroxide (H₂O₂) followed by recovery time to allow mtDNA repair. To determine whether NRTI exposure induces mtDNA damage, human coronary artery endothelial cells (hCAE) cells and human colorectal adenocarcinoma cells (HT29) cells that had been exposed to various NRTIs were subjected to the assay. To assess the assay’s future applicability to clinical samples, human skeletal muscle DNA samples were also assayed.
Results: Quantification of long PCR mtDNA product by UV (CV=6.5%) and qPCR (CV=7.0%) showed lowest variability while PicoGreen quantification was noticeably higher (CV=21%). DNA from H₂O₂-exposed cells showed decreased amplification of long PCR product that increased with repair time. MtDNA depletion occurred in both cultures treated with stavudine. While there was no apparent mtDNA oxidative damage in HT29 cells with any NRTI, both tenofovir and stavudine yielded increased mtDNA oxidative damage in hCAE cells. A wide degree of apparent mtDNA oxidative damage was observed in clinical samples.
Conclusions: The preferred method for DNA quantification is qPCR mtDNA copy number. The observed mtDNA depletion indicated that NRTIs were active in both cell lines. The primary hCAE cells incurred greater mtDNA oxidative damage than cancer-derived HT29 cells. Cancer cells may have enhanced anti-oxidant mechanisms, suggesting that primary cells may be better model for studying mtDNA damage. The broad range of mtDNA damage detected in clinical samples bodes well for the assay’s use with diverse samples.
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