Antiretroviral nucleoside and nucleotide analogues and mitochondria

Introduction This review is intended to provide understanding of the function of mitochondria, the advantages and disadvantages of available techniques for assessing mitochondrial function and quantity and to discuss the main clinical toxicities thought to be associated with mitochondrial dysfunction and how they may be managed or their prevalence reduced. Mitochondria are the key organelles in energy production in all human cells except erythrocytes. Energy, in the form of ATP, is produced through the highly efficient oxidative phosphorylation pathway. Additionally, mitochondria perform a range of other biological functions and carry a number of factors involved in cell apoptosis. Both HIV infection and antiretroviral nucleoside analogues (nucleoside reverse transcriptase inhibitors; NRTI) are known to affect mitochondrial DNA content and other aspects of mitochondrial function. A number of important clinical events occurring in individuals with HIV infection and on antiretroviral therapy have been linked to mitochondrial injury and dysfunction. In vitro studies have demonstrated that NRTI may differ in their effects on mitochondria and may affect mitochondria in different cell lines in different ways. This is likely to influence the clinical syndromes associated with toxicity to these agents. Dideoxy-NRTI have the greatest affinity for mitochondrial DNA polymerase-γ, the enzyme responsible for mitochondrial DNA replication, whereas other nucleoside analogues may influence mitochondrial function also through other mechanisms. These differences may be important in choosing techniques to evaluate the impact of antiretroviral agents on mitochondria. Mitochondria: roles and regulation The mitochondrion, from the Greek mito, thread and khóndrion, granule, was identified at the end of the nineteenth century. It was subsequently established that the mitochondrion was responsible for the majority of cellular energy production (in form of ATP) through the process of oxidative phosphorylation, driven by mitochondrial membrane potential (chemiosmosis) [1,2]. Subsequently, other biochemical and biological contributions of mitochondria to eukaryotic cellular function have been described (summarized in Table 1). More recently, we have expanded our understanding of the genetic basis of human diseases associated with mitochondrial DNA (mtDNA) mutations and the mechanism(s) that regulate the numbers and mass of these organelles, as well as the duplication of the DNA that these organelles contain [3].Table 1: Main roles and characteristics of mitochondria.The number, mass and morphology of mitochondria is partially controlled by fusion and fission events regulated by a variety of protein messengers [4]. Intracellular mechanisms exist to regulate the distribution of mitochondria during the cell cycle and cell division, their morphology, the replication and the inheritance of mtDNA into daughter cells. The morphology of mitochondria shows variability across different cell types [5]. Cells of hemopoietic origin (such as lymphocytes, monocytes, or platelets) typically display cigar-like organelles, while in muscle cells mitochondria are seen in rows between sarcolemma, in sperm cells they form spirals in the tail region, in fibroblasts and preadipocytes mitochondria form a reticulum. They may fuse or increase in size to form giant mitochondria or megamitochondria, and tend to locate near the structures where energy is required. Additionally, mitochondrial appearance and number in a tissue may change in response to changing tissue needs, most evident in cold adaptation [6] or under the situation of high metabolic activity. Conversely, the number of mitochondria can be reduced by pyknosis, ballooning, or autophagolysosome formation. Recent data underline the importance of mitochondria morphology and its correlation with the organelle's functionality, as the pleomorphicity of such organelle is probably linked to cell cycle stage or to the metabolic state of the organelle itself [7]. The mitochondrial matrix contains circular DNA of 16 569 base pairs, in punctate structures called ‘nucleoids'. Each nucleoid may contain several copies of mtDNA. Replication of mtDNA is performed by DNA polymerase-γ, a nuclear DNA (nDNA) encoded enzyme. In humans, several factors are required for transcription initiation, including the human mitochondrial promoter, h-mtRNA polymerase, and the DNA binding mitochondrial transcription factors, h-mtTF-A and -B [8,9]. Additional regulatory factors are hypothesized. Little is known of the factors that regulate the production and neogenesis of mitochondria. Studies are complicated by the fact that their stability varies considerably from tissue to tissue (their half-life in liver is suggested to be 3–5 days, in brain 30 days [10,11]), and that their components have different turnover rates. More is known about the mitochondrial changes provoked by agents which cause functional impairment or dysruption. When altered by a stimulus that does not provoke the necrotic death of the cell, mitochondria generate or amplify signals that lead to programmed cell death/apoptosis. Mitochondrial apoptosis, mitoptosis [12], occurs following stimuli provided by reactive oxygen species or tumour necrosis factor (TNF)-α and can result in cellular apoptosis because of the loss of mitochondrial membrane potential (ΔΨm), and the release of apoptogenic proteins such as cytochrome c or the apoptosis-inducing factor [13–16]. Mitochondria also contain antiapoptotic proteins whose role is yet to be clarified [17–21]. Non-fatal insults that damage mtDNA can be repaired by a proficient base excision repair of oxidative DNA damage [22]. However, mtDNA damage can impair the capacity of the organelle to synthesize crucial enzymes of the oxidative phosphorylation, so provoking a greater dependence on lactate generating cytosolic metabolism of glucose and a reduced capacity to clear H+ formed from the hydrolysis of ATP. Main methodologies for studying mitochondrial function Mitochondrial function can be assessed in several ways, by using a large variety of techniques that include assays of enzyme function from isolated organelles, bioenergetic studies of the ΔΨm, and assays evaluating release of specific factors after standardized stimuli (see Fig. 1).Fig. 1.: Main techniques utilized to analyse mitochondrial parameters in HIV infection. In the last years, several mitochondrial parameters have been investigated by scientists working in the field of HIV research in order to understand the effects of the infection and of different antiretroviral drugs on the activity of this organelle. Specific functions (indicated by black arrows) have been determined by a large variety of biochemical and cytometric assays. More general functions (white arrows), reflecting the global status of mitochondria, were also investigated by the indicated techniques.These assays all require consideration of the quantity of mitochondrial mass present in the sample. Defects in mitochondrial enzymes may be encoded for both by errors in mtDNA but also in nDNA. Methods for purification and extraction of intact mitochondria from cells have enabled the developement of a wide range of biochemical assays to evaluate the enzymes contained in the organelle and their activity. For example, to measure the functionality of different complexes (I–IV) of the respiratory chain, as well as the capability of the organelle to consume oxygen under different conditions. These assays have been recently used to investigate the presence of alterations in muscle mitochondria from HIV-positive lipodystrophic patients [23]. The assays on isolated organelles provide important information, but, in order to obtain enough biological material from muscle or fat tissues, open surgical procedures under local anaesthesia are often required. Red blood cells do not contain mitochondria hence large volume blood draws are required to obtain a sufficient number of organelles from platelets and white blood cells, necessary to perform biochemical measurements. However, in these established assays mitochondria are not studied in their natural environment, i.e., the cell. Bioenergetics involves the evaluation of ΔΨm to analyse mitochondria functionality in intact cells. The energy released by the mitochondrial respiratory chain during oxidation reactions is stored as an electrochemical gradient consisting of two components: a ΔΨm of about −200 mV, and a proton gradient of about 1 unit. This energy gradient drives the synthesis of ATP. To analyse the mechanisms which regulate changes in membrane potential of organelles with a negative interior, several membrane-permeable, lipophilic cations have been used, to their to into such A variety of have been to measure These assays have disadvantages the required to distribution of a mitochondrial membrane of binding of to a membrane in a such as in the of or which several binding effects of on mitochondrial functional from changes and from changes of mitochondrial of large of biological More recently, by using the lipophilic a was to in ΔΨm at the cell which been at the organelle advantages and other as is of into the mitochondria of cells, and changes from to as ΔΨm Both can be using the in or is the for of ΔΨm and been used for several studies on HIV infection Mitochondria contain a with that is in the membrane of these organelles, and is involved in the of a number of cellular including the of mitochondrial enzymes and hence production of energy by oxidative phosphorylation The distribution of between the and of mitochondrial membrane is crucial for the activity of several enzymes of the respiratory chain, and can be in different as well as by different the of of the characteristics of is to the of present on the two of the mitochondrial and obtain on the functional status of the organelles distribution was studied in from HIV-positive with with other mitochondria was to be Mitochondrial to the number of organelles present in a cell, can be using of This can be also investigated with a Mitochondrial mass is a crucial that to be into to that the cells under are not and that the functional changes are not to that the number of organelles in the under of the mitochondrial Each mitochondrion contains several copies of circular which are to to in the DNA repair local of oxygen and to the of Cells and in the number of mitochondria they and in mtDNA on the metabolic activity of the cell. Mitochondria are high in and brain cells in and in erythrocytes. may the quantity of mtDNA or the or of mutations These assays are functional assays. However, of mtDNA content during NRTI therapy the therapy is the function of DNA polymerase-γ, and as such may be most to therapy with the agents which have the greatest affinity for this enzyme aspects of mitochondrial enzyme function may also be altered by NRTI (see the impact of these agents on the mitochondria be mtDNA content is can be studied with several available techniques including and of can be performed by assays that the of a specific mitochondrial to that of a nuclear after the of mtDNA content in several cells, including blood from with or in from HIV-positive with or provided an for mtDNA with this that mtDNA in from patients with HIV infection is that in and in mitochondrial DNA were therapy to increase in the mtDNA However, in blood to a correlation between mtDNA or and in patients with or or data that fat but not blood cells from HIV-positive individuals contain mtDNA A in mtDNA copies cell been in and blood from HIV-positive on NRTI in from HIV-positive patients with or such drugs are associated with mtDNA and of mitochondria such as mtDNA content can be in all human cells, but, because of the number of organelles present in different cell to be to the of the biological material under For example, cells from blood using (such as on or are be In blood and platelets are considerably to contain mitochondria with mtDNA so platelets are not and mtDNA is in cell changes in mtDNA content be to several including in quantity and their mitochondrial which may be not by antiretroviral therapy but also by or is performed and cells of lymphocytes, can be by Additionally, as injury to mitochondria by antiretroviral agents may be tissue specific to of NRTI and the of changes in mtDNA in tissue or cell may not be of events which are occurring in other NRTI toxicities with a mitochondrial A wide range of events occurring in with HIV infection therapy have been suggested to be to mitochondrial function. 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