Adverse effects of reverse transcriptase inhibitors

Introduction After zidovudine (ZDV), a 3′-azido analogue of thymidine, was found to be an effective antiretroviral drug against HIV [1,2], other nucleoside analogues inhibiting reverse transcriptase (RT) soon followed: didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (D4T), and recently abacavir (1592U89) [3–7]. These drugs have demonstrated efficacy in reduction of morbidity and mortality, especially in combination therapy [8–10]. A special feature of some of these drugs is the protection against AIDS dementia complex, which appears to be related to good penetration of the blood–brain barrier [11–13]. Although the introduction of protease inhibitors has changed the management of HIV infection drastically, this cerebro-protective property will assert the role of these nucleoside RT inhibitors (NRTI) as a cornerstone of antiretroviral therapy [9,10]. More than 10 years of experience with NRTI therapy has revealed important adverse effects ranging from mild (myopathy) to fatal in some cases (pancreatitis, liver failure and lactic acidosis). Behind most of these side-effects there appears to be a common mechanism: a decreased mitochondrial energy-generating capacity. In this review we will summarize the literature in which this mechanism is analysed and will emphasize the importance of acquired mitochondrial dysfunction that will accumulate during long-term treatment with antiretroviral nucleoside analogues. Nucleoside analogues and DNA polymerases During the synthesis of DNA, the DNA duplex is unwound by a helicase and each DNA strand directs the synthesis of a complementary DNA strand to generate two DNA duplexes. New nucleotides (triphosphorylated nucleosides: dATP, dCTP, dGTP and dTTP) are added to a pre-existing polynucleotide strand (primer) by an enzymatically catalysed formation of a phosphate ester between the 3′-hydroxyl group of the sugar residue of the nucleotide of the primer and the 5′-phosphate group of the nucleotide to be added. An original DNA strand serves as a template during this process. The enzymes that catalyse this formation of new DNA strands on a template are called DNA polymerases. In eukaryotic cells five types of DNA polymerase are active (DNA polymerase α, β, γ, δ and ε), which all utilize a DNA strand as template. HIV encodes a DNA polymerase (RT) that uses RNA as template. All DNA polymerases have in common the utilization of dNTP as substrate [14–16]. Modification of dNTP can affect the functioning of DNA polymerases: 2′,3′-dideoxy analogues of dNTP (so-called ddNTP) can serve both as inhibitors and substrates of certain DNA polymerases. Since these ddNTP lack the hydroxyl group in the 3′-position, incorporation of a ddNTP will terminate primer elongation (Fig. 1). This mechanism forms the basis of dideoxy sequencing of DNA [17,18] and it was found that ddNTP also inhibited the proliferation of HIV by inhibiting RT [19,20]. All currently used NRTI, such as ZDV, ddC, ddI, 3TC, D4T and abacavir, are dideoxynucleosides, which are phosphorylated intracellularly by host kinases to ddNTP. Since every NRTI (as a ddNTP) might not only inhibit viral RT but also human DNA polymerases, serious toxicity can be expected.Fig. 1: . Scheme of DNA replication. The arrow marks the lacking hydroxyl (OH) group in the 3′-position of a dideoxynucleoside triphosphate (ddNTP), which is responsible for chain termination.For all currently used NRTI, the interaction with human DNA polymerases has been studied (Table 1): in general, DNA polymerase α, δ and ε are insensitive to inhibition by ddNTP, but both DNA polymerases β and γ can be inhibited in vitro by these compounds [15,21–28]. Fortunately, during the cell cycle DNA polymerase α and δ are responsible for the necessary DNA duplication and NRTI apparently do not interfere in this process. DNA polymerases β and ε are involved in DNA repair mechanisms [14] and so far little is known whether the inhibitory effect of NRTI on DNA polymerase β has any pathophysiological importance. However, since DNA polymerase γ is the only DNA polymerase involved in mitochondrial DNA (mtDNA) replication, the inhibitory action of NRTI on this enzyme can easily interfere in mitochondrial replication and function [16,24,28–30]. Interestingly, as an exception to the other NRTI, 3TC is both an inhibitor of the polymerase activity and a substrate of the integral 3′–5′ exonuclease activity of DNA polymerase γ, which makes incorporation less feasible.Table 1: . Kinetic interactions of reverse transcriptase inhibitors with human DNA polymerases.Other RT inhibitors (RTI) are also used as HIV inhibitors, including non-nucleoside analogues such as nevirapine, delavirdine, efavirenz [31–33], and nucleotide analogues such as adefovir [9-(2-phosphomethoxyethyl)adenine (PMEA)] [23,34]. Although data on the affinity for human DNA polymerases are less abundant for these compounds, it appears that nonnucleoside analogues do not interfere with any of these polymerases, whereas the currently available nucleotide analogues appear to have a strong affinity for DNA polymerases β and γ in particular [23,34] (Table 1). Mitochondrial function and replication Mitochondria, subcellular organelles present in all cells except erythrocytes, contain the enzymes, enzyme complexes and proteins necessary for the intramitochondrial generation of ATP and its exportation to the cytoplasm. The oxidative phosphorylation system The most important function of mitochondria is oxidative phosphorylation: the oxidation of fuel molecules by oxygen and the concomitant energy transduction into ATP [35]. The synthesized ATP is used for energy-requiring reactions in the matrix or exported to the cytosol by the adenine nucleotide translocator in exchange for cytosolic ADP [35]. The oxidative phosphorylation system consists of the four multisubunit enzyme complexes of the mitochondrial respiratory chain (complexes I–IV) and the F1–F0 ATP synthetase complex (complex V). All are embedded in the lipid bilayer of the inner mitochondrial membrane (Fig. 2). Besides ATP production via the oxidative phosphorylation system in mitochondria, the process of anaerobic glycolysis (i.e., the conversion of glucose to lactate) in the cytoplasm delivers energy. However, glycolysis produces little ATP compared with the oxidative phosphorylation.Fig. 2: . Diagram of the oxidative phosphorylation system, containing four respiratory chain complexes (I-IV), F1-F0 ATP synthetase (complex V) plus the adenine nucleotide translocator (ANT), located in the mitochondrial inner membrane. Each complex is composed of several subunits, each of which is encoded by either nuclear or mitochondrial DNA (nDNA or mtDNA). Adapted from Wallace [38].With the exception of complex II, which is encoded entirely by nuclear DNA (nDNA), the other respiratory chain complexes I, II and IV and complex V are encoded both by nDNA and extrachromosomal mtDNA (Fig. 2). mtDNA consists of a double-stranded circular DNA molecule composed of 16 569 base pairs, coding for 22 transfer RNA, two ribosomal RNA, and 13 subunits of the oxidative phosphorylation system. mtDNA can be replicated, transcribed and translated independently of nDNA metabolism. However, cell function and mitochondrial function are interdependent [36,37]: for replication of mtDNA the nuclear-encoded DNA polymerase γ is needed (see above). nDNA controls the synthesis of 90–95% of all mitochondrial proteins [38,39]; mtDNA is therefore semiautonomous. There are several differences in structure and function between nDNA and mtDNA. At first, mtDNA is predominantly maternally inherited. The DNA of mitochondria is directly inherited from the cytoplasm of mainly the oocyte; less than 0.1% of the mtDNA is contributed by the sperm [36,38,40]. Second, mtDNA does not recombine and undergoes replicative segregation during both mitosis and meiosis. Each human cell contains hundreds of mitochondria and each mitochondrion contains two to 10 mtDNA molecules. When a cell divides, both mutated and non-mutated forms of mtDNA are randomly segregated into the daughter cells, resulting in mixtures of mutant and wild-type mtDNA in cells and human lineages [37–39,41]. Due to this coexistence of mutant and wild-type mtDNA, called heteroplasmy, otherwise lethal mutations can persist [36]. The severity of a defect due to mtDNA mutation depends on the nature of the mtDNA mutation and on the proportion of mutant mtDNA within the cell; mtDNA mutations will result in cellular malfunction when a certain threshold is reached, a phenomenon called threshold expression [37–39]. This expression depends on the severity of the oxidative phosphorylation defect and the relative reliance of each organ system on mitochondrial energy production. Mitochondria replicate more often than nuclei, and therefore the relative proportion of mutant and wild-type mtDNA may change within a cell cycle [38,39]. More replications indicate a larger chance to develop replication abnormalities. Because mtDNA has no introns, a random mutation will usually strike a coding DNA sequence. Furthermore, mutations and defects can easily occur because mtDNA has neither an effective repair mechanism nor protective histones, and it is exposed to oxygen radicals generated by the respiratory chain [16,36,38,40,42]. Altogether, mtDNA appears to be extremely vulnerable to genetically and exogeneously acquired mutations. Since DNA polymerase γ appears to be the only regulating enzyme of mtDNA replication, inhibition of this enzyme with RTI might easily downregulate this replication resulting in decreased mitochondrial energy generation. Oxidative phosphorylation disorders Genetically inherited defects in mtDNA or nuclear genes encoding the oxidative phosphorylation system, leading to an impaired oxidative phosphorylation, give rise to a variety of clinical diseases due to failure in ATP synthesis [36,37,39,41,43–45]. This failure can affect virtually all organ systems, but tissues with the highest energy demand are most susceptible [38,46]. Disease symptoms will appear when the mitochondrial energy-generating capacity will fall below the energetic threshold of an organ [36,37,41,44]. Many organ systems have been described to be possibly affected (Table 2): liver, pancreas, heart, skeletal muscle, nervous system, haematopoietic system, inner ear, kidney (renal failure is rare), and eye. Liver cells and pancreatic β cells are highly dependent on oxidative metabolism and are therefore easily vulnerable to energy depletion, leading to liver disease and diabetes. Other (genetically inherited) clinical manifestations encountered in mytochondriocytopathies are blindness, deafness, dementia, movement disorders, weakness, cardiac failure, and renal dysfunction [36,43,44,47].Table 2: . Clinical manifestations of oxidative phosphorylation disorders [36,37,43,45].The amount of mtDNA defects is one of the principal factors that determines whether a defect is expressed clinically. Usually the highest levels of mtDNA defects are in post-mitotic tissues such as skeletal muscle. Lower levels are seen in rapidly dividing tissues such as blood. Tissues with a slow turnover of mtDNA accumulate the largest number of mtDNA defects [36,44]. When a certain threshold is reached after accumulation of mtDNA defects, a deficient production of ATP with its consequences for a specific tissue will emerge. As deficient oxidative phosphorylation increases with mitochondrial damage, mitochondrial ATP production declines until it falls below the minimum energy levels (threshold expression) necessary for oxidative tissues and organs to function [16,37]. A disturbed function of the oxidative phosphorylation system will give rise to an altered oxidoreduction status (Fig. 3): a disturbed redox state (increased NADH/NAD+ ratio) shifts the pyruvate/lactate equilibrium in the direction of lactate and leads to a functional impairment of the Krebs cycle. Consequently, both lactate, leading to lactic acidaemia or even lactic acidosis, as well as the lactate/pyruvate ratio increase. This is particularly true in the post-absorptive period, when more NAD+ is required for the adequate metabolism of glycolytic substrates [45]. Similarly, a postprandial increase of ketone bodies synthesis can be observed, related to the channelling of acetylcoenzyme A towards ketogenesis [48]. Fat (triglycerides and free fatty acids) will accumulate intracellularly, which can be demonstrated histologically (macrovesicular hepatic steatosis).Fig. 3: . Schematic presentation of pyruvate oxidation pathway leading to ATP production. When oxidative phosphorylation function is interrupted, ATP production will decline and the NADH/NAD+ ratio will rise, followed by (i) impairment of the flux through the Krebs cycle, (ii) channelling of acetyl-coenzyme A (CoA) towards ketogenesis, (iii) lactic acidaemia, and (iv) an increased lactate/pyruvate ratio. OMM, Outer mitochondrial membrane; IMM, inner mitochondrial membrane; LDH, lactate dehydrogenase; PDHc, pyruvate dehydrogenase complex; FADH2, reduced form of flavin adenine dinucleotide; ANT, adenine nucleotide translocator.In electron microscopy, histological damage can be demonstrated as swollen enlarged mitochondria, with or without loss of cristae, matrix dissolution, lipid droplets, paracristalline and scattered vesicular inclusions [16,46,47,49,50]. Nucleoside analogues and mitochondrial toxicity Apart from the inheritable route, mtDNA defects can also be acquired exogeneously by toxic agents such as alcohol, tobacco and drugs [16,36,47]. In the latter group, drugs that have been shown to induce mitochondrial toxicity are nucleoside analogues used in chemotherapy and antiretroviral therapy, such as HIV RTI (as discussed in this review), but also cytarabine, vidarabine, aciclovir, and ribavirin. Since these nucleoside analogues elicit complete mtDNA replication deficits, clinical features can be regarded as a compilation of those seen in the genetic mitochondriocytopathies. These features include myopathy, cardiomyopathy, neuropathy, lactic acidosis, exocrine pancreas failure, liver failure and bone-marrow failure [16,44,46,51–54]. A classical example of a drug with this mitochondrial toxicity is fialuridine. A trial with this nucleoside analogue in patients with chronic hepatitis B infection ended after 13 weeks due to severe adverse events. Hepatic failure, lactic acidosis, pancreatitis, neuropathy and myopathy due to mitochondrial toxicity were found and even persisted after discontinuation of the of the patients due to one or more of these serious adverse decreased the of mtDNA in which was also seen in the in the The decline in mtDNA was in heart, in in liver and in tissue is not a dideoxynucleoside analogue and has a of action from RTI Although it is the of this this drug the consequences of mtDNA replication by nucleoside analogues In this it is not that RTI have also been demonstrated both in vitro and in to induce mitochondrial In vitro Mitochondrial toxicity of antiretroviral nucleoside analogues was in in vitro with nucleoside analogues in human cell and demonstrated that within a the antiretroviral drugs decreased the mtDNA of the cells (Fig. There was a compared with Furthermore, in mitochondria were such as of the mitochondria and of the lactic production was as a result of this damage (Fig. Although mitochondrial damage will result in an increase of energy production the capacity of the drugs to lactic production was not directly with inhibition of mitochondrial synthesis The to inhibit the mtDNA between the several the required to the mtDNA of cells by after a drug treatment was for ddC, for for and for the of ZDV, and D4T is and of of a pancreatic cell to 10 for both impaired cell with lactic production and increased and of lipid with mitochondria In a cell both ddC, D4T and inhibited in a whereas and 3TC no this not be to a of mtDNA for all drugs ZDV, and mitochondrial toxicity in human cells, with decreased cell proliferation and increased lactic lipid accumulation and impaired activity of respiratory chain enzymes data of in vitro mitochondrial toxicity have been to for abacavir or . The effects of of zalcitabine (ddC), stavudine and zidovudine on the mitochondrial DNA (mtDNA) of cells after of Adapted from of lactic production in cells after of with ddC, or Adapted from in the severity of toxicity in the tissues be related to the in cell and production. studied the effect of cell and to mitochondrial found an increase in mtDNA after of cells into cells A effect by antiretroviral nucleoside analogues on mtDNA in cells is on the turnover of mtDNA compared with nDNA These demonstrated that there is a in the drug necessary for cell inhibition and inhibition of mtDNA are required to inhibit mtDNA synthesis Furthermore, it be demonstrated that mtDNA is a more of nucleoside analogue toxicity than cell and mitochondrial In Many adverse effects of RTI have been (Table and some of these include by mitochondrial 3: . of reverse transcriptase in long-term therapy with due to mitochondrial damage has been described by several features of proliferation of have been demonstrated This mitochondrial myopathy appears to be since some patients have shown a reduction in and a concomitant increase in mtDNA after discontinuation of myopathy from myopathy, and electron features of were The were found in the of the patients with and not in the Furthermore, histological was seen in the of patients in myopathy to discontinuation of A with patients exposed and neuropathy as the most important clinical of this The of these symptoms after several weeks of was after weeks of therapy and neuropathy was after and haematopoietic toxicity with was found and pre-existing even after was of neuropathy or has also been seen in and D4T therapy with Mitochondrial were demonstrated in cells of and and and the complex of mitochondria was to be an to the mitochondrial dysfunction Many clinical have the of neuropathy during NRTI treatment and in one the neuropathy when treatment was to ddI, a interaction of the two agents Hepatic and hepatitis are of severe toxicity in long-term antiretroviral due to this hepatic with severe lactic has been for several NRTI and after several of (Fig. and without features of or agents This clinical often with of weakness, and and may and to fatal may In one the of this in a of antiretroviral was to be of . hepatic in a with zidovudine and lamivudine toxicity has been demonstrated for ZDV, but ddC, and D4T also in vitro toxicity to haematopoietic cells The mechanism of this toxicity appears to be in which both ddNTP and to be involved to inhibit the mitochondrial most through the inhibition of DNA polymerase γ and it has been that the of might this toxicity in every and can induce and in to of on the Furthermore, it can a loss of in in a and has been in patients and Although is a feature in an with the of RTI has not been Apart from mitochondrial toxicity in patients with of 13 RTI therapy with ZDV, or in four patients after discontinuation of therapy, but of cardiac function was seen after was to either or found a in but were to this for toxicity has not been for any NRTI or but during the AIDS in the most of after weeks of treatment a of patients some of renal acidosis, in cases by Although the of this toxicity has to be in this the dysfunction seen in oxidative phosphorylation disorders (Table 2). As shown there is that mitochondrial toxicity is the in the adverse effects of NRTI A with this toxicity is its and therefore side-effects are seen in long-term therapy with nucleoside analogues. In some of symptoms was after of the and in toxicity persisted drug with a fatal Since NRTI are to the cornerstone of combination antiretroviral therapy, this toxicity might the of this to damage but to factors that to the of these side-effects have been In one it was that and HIV status of of are factors but not this and that both and and patients in and of HIV infection to be susceptible to adverse However, it that only some appear to be susceptible to the of even after a of which is by a certain genetic of DNA polymerase γ with for NRTI might a but this also for the of mtDNA Although a tissue metabolism might appear to function the threshold expression of energy might be due to a oxidative phosphorylation system. These will be more susceptible to the of mtDNA by This mechanism was demonstrated in vitro in from patients with a by a in mtDNA. In a of cells, nucleoside analogues increased the levels of mutated mtDNA the levels of wild-type mtDNA, by inhibiting the proliferation of cells with little or no mtDNA mutations Since mtDNA is highly with a strong between and mtDNA may a role in the for NRTI As with the expression of inherited mtDNA during for NRTI toxicity might increase in no data are available on these Apart from these is the that some patients develop certain adverse whereas other patients the drug develop adverse events. Furthermore, do patients not develop mild adverse first, followed by more severe all nucleoside analogues the side-effects (Table and there appears to be a tissue in nucleoside analogue toxicity This tissue may be related to phosphorylation of nucleoside analogues or of cellular kinases for phosphorylation of nucleoside analogues in tissues In most tissues have cell enzyme This in metabolism may a role in the differences in which the nucleoside analogues are to active there may be a in mitochondrial between and can to the tissue also of adverse events. All these factors have been in the γ so because of the role of DNA polymerase γ in this process. that the toxicity of antiretroviral nucleoside analogues depends on (i) the subcellular and of the antiretroviral nucleoside analogues in the (ii) the of cellular nucleoside to the antiretroviral nucleoside analogue as a substrate resulting in and of the antiretroviral nucleoside (iii) the of the triphosphate of the antiretroviral nucleoside analogue to inhibit DNA polymerase γ either by as a substrate or by chain of the mtDNA strand and (iv) the reliance on oxidative phosphorylation in the tissues Mitochondrial toxicity is a adverse effect of The clinical features of this which can be both and and between several In some cases fatal can be to which factors a role in the to develop this in the of this is lacking and that can the of this toxicity an are not be of this toxicity in the management of long-term treatment with NRTI since the toxicity of these drugs might easily the of antiretroviral The of the by and was highly