Mitochondria analysis for investigating toxicity of antiretroviral drugs.

Mitochondria are the key organelles in intracellular energy production, but performseveral other biologic functions and carry factors involved in cell apoptosis.Both HIV infection and antiretroviral nucleoside analogues (NRTI) can affectmitochondrial (mt) function and DNA content. Several side effects occurring inindividuals with HIV infection who are on antiretroviral therapy have been linkedto mitochondrial injury and dysfunction. Clinical studies of NRTIs, while collectingadverse event data, have not specifically evaluated mitochondrial toxicityin a systematic way. In vitro studies have demonstrated that NRTIs may differ intheir effects on mitochondria activities and may affect mtDNA content in differentcell lines in different ways, and several models are actually employed to betterunderstand not only the mechanism(s) of action of antiretroviral compounds,but also their possible side effects. Accordingly, it has been hypothesized thatclinical syndromes associated with toxicity to these agents are likely caused, atleast in part, by drug-related mitochondria alterations. Dideoxy-NRTIs have thegreatest affinity for mtDNA polymerase-γ, the enzyme responsible for mtDNAreplication, whereas other nucleoside analogues may influence mitochondrialfunction also through other mechanisms. By using cytometric assays to analyzemt or cell functionality (mt membrane potential by JC-1, mt mass by Mitotrackergreen, cell viability and apoptosis by classical stainings) along with originalmolecular biology techniques based upon real time PCR for determining mtDNAcontent, we have investigated the effects of different drugs commonly employedin anti-HIV therapy. We have used several human cell lines of different origin(lymphocytic such as CEM; monocytic, U937 or hepatocytic, HepG2). We foundthat the aforementioned parameters can be altered by antiretroviral drugs, with adifferent extent in the cell lines we studied. In general, stavudine seemed morepotent than zidovudine or didanosine in inducing damages to mt function ormtDNA. Antiretroviral drugs of the protease inhibitor category (such as indinavir)had no effects on any mt parameter we analyzed. From our experience, it can beconcluded that the choice of adequate molecular or cellular techniques and modelsis important in evaluating differences in drug activity that are related to theimpact of antiretroviral agents on mitochondria, as well as in understanding theside effects of such drugs on different cells, organs or systems.