Mitochondrial DNA Replication, NucleosideReverse-Transcriptase Inhibitors, and AIDSCardiomyopathy

William Lewis

Nucleoside reverse-transcriptase inhibitors (NRTIs)in combination with other antiretrovirals (HAART)are the cornerstones of current AIDS therapy, butextensive use brought mitochondrial side effects tolight. Clinical experience, pharmacological, cell,and molecular biological evidence links altered mi-tochondrial (mt-) DNA replication to the toxicity ofNRTIs in many tissues, and conversely, mtDNAreplication defects and mtDNA depletion in targettissues are observed. Organ-specific pathologicalchanges or diverse systemic effects result from andare frequently attributed to HAART in which NRTIsare included. The shared features of mtDNA deple-tion and energy depletion became key observa-tions and related the clinical and in vivo experimen-tal findings to inhibition of mtDNA replication byNRTI triphosphates in vitro. Subsequent to thosefindings, other observations suggested that mito-chondrial energy deprivation is concomitant with orthe result of mitochondrial oxidative stress in AIDS(from HIV, for example) or from NRTI therapy itself.Copyright 2003, Elsevier Science (USA). All rightsreserved.

W ith increased clinical experience as the ac-quired immune deficiency syndrome

(AIDS) epidemic continues, clinical, pharmaco-logic, cell, and molecular biologic evidence linksaltered mitochondrial (mt-) DNA replication tothe toxicity of nucleoside reverse-transcriptase in-hibitors (NRTI)1-7 in cardiac and skeletal muscle,liver, and peripheral nerve. Defective mitochon-drial (mt-) DNA replication depletes mtDNA intissue targets. Ultimately, mtDNA depletion re-sults in organ-specific pathologic changes or sys-temic effects that are diverse. These may includemuscle weakness, cardiac failure, liver failure, pe-ripheral neuropathic changes, and elevatedplasma lactate levels. The latter finding is being

observed more commonly with combined antiret-roviral therapy that includes NRTIs (highly activeantiretroviral therapy [HAART]).8-18

Mitochondrial toxicity of NRTIs was estab-lished by clinical, in vitro, and in vivo investi-gations that related mtDNA depletion with orwithout lactic acidemia to treatment with NR-TIs in HAART combinations or in NRTI mono-therapy.2,3,6,10,19-25 In many cases, the clinical im-pact of NRTI toxicity in patients with AIDSremains controversial. Long-term side effects ofNRTIs may be more common because of increasedAIDS survival and increased treatment withHAART. Heightened clinical awareness raises theindex of suspicion, which in turn may raise thetrue incidence. Nonetheless, the risk/reward ratiois weighted heavily toward treatment withHAART.

Our group and others suggested that interre-lated events may be mechanistically operative inNRTI toxicity from AIDS therapeutics. The firstevents that suggested NRTI mitochondrial toxic-ity was observed in parallel clinical and experi-mental systems.19,26,27 The shared features ofmtDNA depletion and energy depletion becamekey observations and related the clinical and invivo experimental findings to inhibition of

From the Department of Pathology, Emory UniversityAtlanta, GA.

Supported by National Heart, Lung, and Blood Institute(RO1 HL59798).

Address reprint requests to William Lewis, MD, Depart-ment of Pathology, Emory University, 1639 Pierce Dr,Room 7117, Atlanta, GA 30322; e-mail: wlewis@emory.edu.

Copyright 2003, Elsevier Science (USA). All rights reserved.0033-0620/03/4504-0003$30.00doi:10.1053/pcad.2003.3

305Progress in Cardiovascular Diseases, Vol. 45, No. 4, (January/February) 2003: pp 305-318

mtDNA replication by NRTI triphosphates invitro.28,29 Another series of observations sug-gested that mitochondrial energy deprivation isconcomitant with or the result of mitochondrialoxidative stress in AIDS (eg, from human immu-nodeficiency virus [HIV]) or from NRTI therapyitself. In vivo studies with NRTI treatment of in-bred mice30,31 support this hypothesis and datafrom our group and others using AIDS transgenicmice (TG) revealed that oxidative stress resultsfrom transgenic expression of HIV Tat in the heartand liver.32-34 Last, mtDNA mutations may resultfrom oxidative mtDNA damage, aberrant mtDNAreplication, and altered mtRNA transcription. To-gether, these interlinked events are the corner-stones of the mitochondrial dysfunction hypothe-sis7 that applies to cardiomyopathy (CM) fromAIDS therapeutics. The hypothesis (detailed later)underpins pathophysiologic events that are im-portant in NRTI toxicity. In some ways, analysis ofmechanisms of NRTI-induced mitochondrial tox-icity is analogous to approaches that examine de-fects in genetic mitochondrial illnesses in whichthe defective mitochondrial gene product, oxida-tive stress, and the environment contribute to dis-ease pathogenesis.35

mtDNA, DNA Polymerase-�, andNRTI Toxicity

Nuclear DNA encodes 80% of the oxidative phos-phorylation genes (OXPHOS; the principal sourceof myocardial energy), but 13 OXPHOS geneproducts are encoded by mtDNA.36 Although mi-tochondrial genetic diseases result from point mu-tations or deletions of mtDNA, acquired defects inmtDNA replication resulting from NRTI’s inhibi-tion of mtDNA replication may yield phenotypicOXPHOS defects that mimic the genetic illnesses,but may relate to increased mtDNA mutations,oxidative stress, or mtDNA depletion.

For emphasis, mtDNA replication is driven bypolypeptides that are encoded by the nuclear ge-nome, thus, DNA polymerase-� (DNA pol-�, themitochondrial DNA polymerase) is the nuclearencoded mtDNA replication enzyme in eukary-otic cells. DNA pol-� extracted from flies, frogs,and humans reveals significant sequence homol-ogy. DNA pol-� contains a subunit of 125-140 kdwith polymerase and exonuclease catalytic activ-ity. A smaller accessory subunit of 41-55 kd is

required for processive synthesis.37-42 Mutationsin DNA pol-� active site result in genetic mito-chondrial diseases including chronic progressiveophthalmoplegia (CPEO).43 Mutations in the ac-cessory subunit also result in defective mtDNAreplication.44 The accessory subunit providestighter DNA binding of the complex, thus allow-ing highly processive DNA synthesis.42 mtDNAreplication defects are becoming an important as-pect of mitochondrial pathobiology.45 Con-versely, defects in mtDNA replication that relateto DNA pol-� likely will be better understood withincreased understanding of biochemical mecha-nisms of mtDNA replication and the polypeptidesthat are involved critically in mtDNA replication.

The polymerase function of DNA pol-� is fun-damental to the first proposed mechanism ofNRTI toxicity of the mitochondrial dysfunctionhypothesis7: decreased energy production sec-ondary to decreased mtDNA abundance. WhenDNA pol-� activity is inhibited by NRTI triphos-phates, mtDNA synthesis is inhibited and mtDNAdepletion results. This also suggests that NRTItoxicity may be cumulative and toxic manifesta-tions increase with duration of exposure owing inpart to the kinetics of mtDNA depletion in thismodel.

The 2-subunit DNA pol-� is highly processivebecause of its accessory subunit. This high proces-sivity allows the DNA pol-� complex to replicatethe entire mitochondrial genome in one bindingevent.42 Processivity of DNA pol-� may relate inpart to heteroplasmy (an intracellular or intrami-tochondrial mix of normal and mutant mitochon-drial DNA molecules that ultimately may reflect aphenotype).39,40 Because of high DNA pol-� pro-cessivity, deletion mutants (truncated mtDNAtemplates) may be replicated more quickly andefficiently than native mtDNA counterparts.40,42

Abundance of defective mtDNA may increase to apoint at which energy depletion may occur withsymptomatic manifestations. This threshold isanalogous to that seen with heritable mitochon-drial illnesses, including those that includemtDNA depletion.36,46,47 One potential defenseagainst NRTI toxicity exists in the 3�3 5� exonu-clease within the enzyme. This exonucleolyticfunction48,49 is inhibited by nucleoside 5�-mono-phosphates.50

306 WILLIAM LEWIS

Nucleoside Reverse-TranscriptaseInhibitors and the Relationship

to CM in AIDS

At present there are at least 5 NRTIs used in thetreatment of HIV infection. Zidovudine (AZT 3�-azido-2�,3�-deoxythymidine), zalcitibine (ddC2�,3�-dideoxycytidine), didanosine (ddI 2�,3�-dideoxyinosine), stavudine (d4T 2�,3�-didehy-dro-3�-dideoxythymidine), and lamivadine(3TC; 3 thiacytidine; cis-1-[2�-hydroxymethyl-5�-(1,3oxathiolanyl)]cytosine) are formidableNRTIs that also serve as tools in vitro and invivo in biomedical and cell biologic models ofinhibition of DNA pol-�.

Other agents were of promise in AIDS ther-apy, but ultimately were found to be toxic. Onesuch agent, fluoro-dideoxyadenosine (FDDA; 2�-fluoro-2�,3�-dideoxyadenosine) was discontinuedbecause of adverse events in clinical trials. OtherNRTIs are used to treat common co-infectionsseen with HIV. Chronic hepatitis B infection wasand is considered a serious and common co-infec-tion in AIDS that may increase morbidity andmortality. Treatment of chronic hepatitis B infec-tion would be beneficial to patients who are co-infected. Accordingly, the pyrimidine nucleosideanalog fialuridine (FIAU; 1-[2-deoxy-2-fluoro-�-D-arabinofuranosyl]-5-iodouracil) was used inclinical trials with some promising early results.However, in further trials, FIAU was found to beextremely toxic to liver, skeletal and cardiac mus-cle, pancreas, and peripheral nerve. The extent ofmitochondrial toxicity was so profound that pre-mature death and hepatic failure in some patientsrequired early termination of clinical trials andabandonment of these pharmacologic agents ow-ing to mitochondrial toxicity.

The mitochondrial dysfunction hypothesis7 in-corporates the DNA pol-� hypothesis,2 oxidativestress, and acquired mtDNA mutations in a con-tinuum with an end point of energy depletion andtissue dysfunction in the target. In essence, thispotentially common side effect (NRTI mitochon-drial toxicity3) becomes an acquired mitochon-drial disease because it affects mtDNA replicationin target tissues and yields a phenotype. As a ther-apeutic side effect, however, presently there areno known genetic predispositions or somatic mu-tations that enhance pharmacologic events criticalto NRTI intracellular metabolism (such as compe-

tition by NRTIs and native nucleotide pools, orbetween NRTIs and and native nucleotides at thenucleotide binding site of DNA pol-�). Ulti-mately, depletion of mtDNA in affected tissues isthe subcellular correlate to target tissue dysfunc-tion or elevated plasma lactate levels, an indicatorof inefficient oxidative phosphorylation. The phe-notype of NRTI toxicity is variable, similar to thatof the mitochondrial diseases that it mimics, butthe phenotype has been identified in blood sam-ples from patients treated with NRTI combina-tions.24 Deleterious effects on mitochondrialstructure and function in selected targets havebeen documented.2 Overall, depletion of mtDNAappears to be an important marker of the toxicprocess, and may serve as a diagnostic hall-mark,20,51 a way to monitor successful HAARTtherapy,24 and to tailor changes in HAART regi-mens.

The DNA pol-� hypothesis2 is the working hy-pothesis used in our group that relates the clinicalobservations, biochemical, pharmacologic, andpathologic data. Its premises are based on phar-macologic and biologic reasoning. First, the intra-cellular and intramitochondrial abundance of theNRTI must be sufficient to impact on the intrami-tochondrial pool of nucleotides and compete forincorporation into nascent mtDNA. Mitoticallyquiescent tissues such as myocardium and liverpossess intramitochondrial nucleoside kinases fornucleoside salvage (including thymidine kinase 2[TK2], the mammalian mitochondrial isoform).52

These kinases must be able to phosphorylate ade-quately the NRTI to provide sufficent substrate fordownstream phosphorylation to NRTI triphos-phate, the pharmacologically active moiety. Fromthat point, the NRTI triphosphate must be an ef-fective inhibitor of DNA pol-�. This depends onthe NRTI triphosphate’s ability to compete withthe native nucleotide at DNA pol-�’s nucleotidebinding site and its ability to adulterate the nas-cent mtDNA on its incorporation and cause chaintermination of mtDNA. Last, the metabolic re-quirements of target tissues may be significantlyaffected by energy deprivation in the face of de-pleted mtDNA.

Each aspect of the DNA pol-� hypothesis isfounded in and has a clinical analogy in mitochon-drial medicine. If the intramitochondrial pool ofnucleosides is disrupted, altered energetics canoccur as seen in the neurogastric syndrome

307mtDNA, ANTIRETROVIRALS, AND AIDS CARDIOMYOPATHY

(NGIE).53 Inefficient monophosphorylation ofthymidine (by TK2) results in genetic illnesseswith lactic acidosis and muscle weakness.54 Katz55

explained the role of mitochondrial alterations inthe development of low-output congestive heartfailure by using similar reasoning. The inability ofmitochondria to function normally in that lattersetting related to decreased cardiac performance.It generally is considered axiomatic that many ge-netic mitochondrial illnesses manifest with athreshold based at least in part on the heteroplas-mic effects of the associated mtDNA mutation asstated in the OXPHOS paradigm.56 Energy depri-vation, possibly the initiating step of NRTI toxic-ity based on mtDNA depletion, relates decreasedenergy abundance in tissues (eg, heart) to de-creased functional mitochondria. To that extent,the threshold phenomenon is involved intimatelywith phenotypic change.

Oxidative Stress and Its Relationshipto mtDNA Alterations and HIV

Infection

Although energy depletion from altered mtDNAreplication in NRTI toxicity is a logical conse-quence,2,19,20,23,28,57-59 related events of oxidativestress also impact on energetics in striated mus-cle30,60 and mtDNA replication, on heart failure ingeneral, and on HIV infection and AIDS. In thecontext of NRTI toxicity, oxidative stress is animbalance between the production of reactive ox-ygen species (such as superoxide, hydrogen per-oxide, lipid peroxides, hydroxyl radical, and per-oxynitrite) and the antioxidant defenses thatprevent damage to cells.61 Mitochondria serve asboth a logical target for the stress and as a sourceof the biochemical moieties that contribute to orcause it. The proximity of mtDNA, mtRNA, mito-chondrially and nuclear-encoded proteins, andlipids to the highest gradient of oxidants (near thesource) is a crucial factor as well. Thus, it is rea-sonable to implicate mitochondria and oxidativestress in some aspects of the toxicity of NRTIs.Conversely, it may be reasonable to use therapeu-tic strategies that are focused on the prevention ofoxidative stress as a means to prevent, attenuate,or ameliorate NRTI toxicity. Some studies in ourlaboratories focus on this important issue.

As a brief review, reactive oxygen species arephysiologic products of mitochondria during

OXPHOS in which approximately 2% to 4% ofelectron flux results in reduction of oxygen tosuperoxide. Mitochondria generate superoxideand hydrogen peroxide.62-64 If electron flowthrough the electron transport chain may be dis-rupted, production of reactive oxygen species isincreased. Both the NADH dehydrogenase and theubquinone Q-cytochrome b complex produce su-peroxide. Mitochondrial hydrogen peroxide isformed rapidly from superoxide by spontaneousdismutation (105 M�1 s�1) or by mitochondrialmanganese superoxide dismutase (109 M�1 s�1).Hydroxyl radicals have been detected at siteswhere superoxide and hydrogen peroxide areformed in the mitochondria65 and are produced inliver mitochondria in vivo in disease states.66 Mi-tochondria concentrate iron for use in cyto-chromes and non–heme-iron proteins67 in whichiron serves in Fe-S-containing enzymes, such asaconitase. Reactive oxygen species can releaseFe(II) from aconitase.68 Fe (II) can then bindmtDNA, providing oxidants near this critical bio-logic target. Importantly, DNA pol-� particularlyis susceptible to oxidation and its inactivity couldfurther enhance mtDNA mutations or mtDNA de-pletion.69 It is logical to expect that oxidativestress impacts on mtDNA replication by alteringmtDNA templates through oxidation, but proof ofconcept is difficult.

Nitric oxide is another reactive oxygen speciesthat is generated by both a calcium-calmodulin–dependent (constitutive) and calcium-indepen-dent (inducible) form of nitric oxide synthase.Nitric oxide can affect energy production becauseit binds tightly to and inhibits cytochrome oxi-dase.70 This modest direct toxicity is enhancedgreatly by its reaction with superoxide to formperoxynitrite.71 Protonation and decompositionof peroxynitrite to more reactive species leads toDNA damage.72

Mitochondria contain antioxidants to protectagainst damage from reactive oxygen species.Manganese superoxide dismutase (MnSOD) cata-lyzes the dismutation of superoxide into hydrogenperoxide plus oxygen at a rate that approaches thediffusion limit. MnSOD eliminates superoxide,but generates injurious hydrogen peroxide.73 Hy-drogen peroxide in mitochondria is eliminatedprimarily by glutathione peroxidase by which hy-drogen peroxide is converted to water and oxi-dized glutathione.74,75 Glutathione reductase and

308 WILLIAM LEWIS

the reduced form of nicotinamide-adenine dinu-cleotide phosphate (NADPH) (from the pentoseshunt) recycle oxidized glutathione. The cyclecontinues under physiologic conditions and theseenzyme systems prevent hydrogen peroxide accu-mulation and limit the formation of more reactivespecies (such as hydroxyl radical).76 Recent evi-dence suggests that both HIV polypeptides (Tat34)and AZT in vitro and in vivo60,77 may depleteglutathione in selected tissues, increase oxidation,and could impact on the oxidative balance withintissue targets.

Mutations of mtDNA and CM in AIDS

In addition to NRTI-induced energy deprivation,oxidative damage to mtDNA by respiration-linkedreactive oxygen species may relate to damage ofcardiac myocytes and development of CM.78,79

Reactive oxygen species, including hydrogen per-oxide and hydroxyl radical, are generated close tothe inner mitochondrial membrane. They can re-act with and oxidize mtDNA.80

mtDNA from rat liver has more than 100-foldoxidative DNA damage compared with nuclearDNA. Differences in oxidative damage betweennuclear DNA and mtDNA may relate to (1) a lackof known repair enzymes for mtDNA error exci-sion; (2) a lack of histones protecting mtDNA; and(3) a subcellular proximity of mtDNA to theseoxidants. Exposure of DNA to superoxide gener-ating systems causes extensive strand breakageand degradation of deoxyribose.81 Additionally,peroxynitrite is a potent initiator of DNA strandbreaks82 and causes DNA base modifications.83

On a mass action basis, random mtDNA muta-tions would likely inactivate complex I, owing tothe significant contribution from mtDNA-en-coded elements. Moreover, deficiency of complexI proteins could amplify superoxide formationand increase oxidative stress.84

mtDNA oxidation by hydroxyl radicals yields 8-hydroxydeoxyguanosine (8-OHdG). This oxi-dized base is present in hepatic mtDNA at 16-fold higher levels than corresponding nuclearDNA.85,86 In human hearts, similar observationswere made.87 Base modification can lead to mis-pairing and point mutation.88 It follows stochasti-cally that during any given oxidative event,mtDNA will sustain more damage than nuclearDNA.89,90 The number of oxidative events in rat

DNA is estimated at about 100,000 per cell perday.

With regard to repair, enzymes for repair ofnuclear DNA efficiently remove most, but not all,of the adducts in nuclear DNA.90 Although mostcomponents of a mitochondrial base excision re-pair system have been identified,45,91 it is unclearhow efficiently this repair removes the wide spec-trum of adducts that may occur from oxidativedamage. The co-existence of malondialdehydeon (or near) the inner mitochondrial mem-brane92 is an additional factor that may impactmtDNA oxidation. Malonaldehyde interactionwith mtDNA could lead to cross-linking, dele-tion errors in transcription, or mtDNA polymer-ization.

In the presence of oxidative stress, 8-OHdGabundance in mtDNA is higher than that in nu-clear DNA.93 This observation may relate to theabundance of mtDNA deletions.87,94 With respectto heart tissue, a random accumulation of mtDNAdefects may result in myocytes with an array ofoxidative capacity from normal to severely im-paired. This would effectively produce a myocar-dial bioenergy mosaic in NRTI-treated cells in theaging heart.95 Such a mosaic may be absent histo-chemically, in which a spectrum of activity may beseen in a given tissue (such as myocardial cyto-chrome c oxidase activity as a function of aging).96

Pathophysiologic events would not occur untilthe threshold of damage was severe enough toimpact on organ function.36 Although the AIDSepidemic is approximately 20 years old, treatmentwith NRTIs in combined HAART have been un-dertaken for less than a decade. The long-termside effects of HAART combinations are not wellunderstood.

NRTI Pharmacology and theDevelopment of Mitochondrial

Toxicity

NRTIs may be divided into mtDNA replicationinhibitors based on the importance of DNA chaintermination, NRTI incorporation into nascentmtDNA, and substitution for the natural base.1,5,97,98

In one model system, the drug inhibits mtDNAreplication in ways that resemble the action ofFIAU in which NRTI monophosphate incorpora-tion into mtDNA is crucial. Among NRTIs thatmay share this characteristic are FIAU, FIAC

309mtDNA, ANTIRETROVIRALS, AND AIDS CARDIOMYOPATHY

(1-[2-deoxy-2-fluoro-�-D-arabinofuranosyl]-5-iodocytosine), FMAU (1-[2-deoxy-2-fluoro-�-D-arabinofuranosyl]-5-methyluracil), and FEAU(1-[2-deoxy-2-fluoro-�-D-arabinofuranosyl]-5-ethyluracil). Each showed virucidal efficacy indisease models.99 Many of their triphosphates in-hibit mammalian DNA pol-� in vitro.57 With theseNRTIs, competition with the native nucleotideand NRTI at the nucleotide binding site of DNApol-� appears to be a critical event and competi-tive inhibition kinetics may be predicted.

The second type of NRTI is represented bysome dideoxynucleosides such as AZT, ddC, andD4T. With these NRTIs, 5�-triphosphates serve assubstrates for mtDNA synthesis by DNA pol-�,but the focus of toxic mechanisms is slightly dif-ferent. Once they are fully phosphorylated toNRTI triphosphates, NRTI triphosphates competewith the natural nucleotides (as described earlier).However, once incorporated into nascent mtDNAthey terminate nascent mtDNA chains becausethey lack 3�-hydroxyl groups (3�-OH) for contin-ued mtDNA polymerization. In many ways, suchNRTIs (such as AZT) have toxic mechanisms thatresemble their respective pharmacologic mecha-nisms of action. However, because incorporationinto mtDNA is not complete, mtDNA replicationdefects from NRTI toxicity may be reversible. Thismay be possible because the 3�3 5� exonucleaseof DNA pol-� may excise the inserted NRTImonophosphate at the time of its insertion intonascent mtDNA (if it is recognized as an errone-ous moiety). If the NRTI is not recognized as er-roneous, it remains in the chain. mtDNA synthesisceases because the 3�-OH necessary for DNA rep-lication is absent in dideoxy NRTIs.

To support evidence that mitochondrial toxic-ity may be relatively long term, terminally incor-porated AZT monophosphate is not removed bythe 3�3 5� exonuclease of Saccharomyces cerevi-siae DNA pol-�.100 Similar results were found us-ing porcine DNA pol-� against dideoxynucleotidetermini.101 Human DNA pol-� removes chain ter-minators poorly as compared with normal nucle-otides. AZT monophosphate is the most persistentchain terminator to DNA pol-� exonuclease activ-ity.69 It is possible that this could be significantmechanistically in the observed mitochondrialtoxicity of DNA by AZT and related NRTIs. Withnon–dideoxy-NRTIs, molecular similarities to thebiologic condition exist. Accordingly, use of these

compounds has potentially more hazardous con-sequences, some of which have been observed inhumans. Postreplicational repair mechanismsthat remove the internally incorporated NRTImonophosphate are understood incompletely anddamage to mtDNA is more likely to be more diffi-cult to reverse.

Biochemical Evidence for NRTIToxicity

In studies that suggested mtDNA replication wasaffected, kinetics were performed. Incubation ofAZT triphosphate with DNA pol-� in vitro re-sulted in mixed kinetics with a competitive Ki of1.8 � 0.2 �mol/L and a noncompetitive Ki of6.8 � 1.7 �mol/L. In studies by others, AZTtriphosphate inhibited DNA pol-� activity ap-proximately 30% at 4 �mol/L, compared with80% inhibition of reverse-transcriptase activity atthe same concentration in vitro.29 A mixed inhibi-tion pattern also was determined for inhibition ofcardiac DNA pol-� by D4T triphosphate, but witha much lower Kis.102

Human DNA pol-� is inhibited by 50% with 20�mol/L AZT triphosphate in a reverse-transcrip-tase assay.69 Gray et al103 showed that Hela cellDNA pol-� incorporated 3TC triphosphate nearlyas well as ddC triphosphate into DNA. Martin etal104 compared the inhibition kinetics of humanDNA polymerase �, � and found DNA pol-� to behighly inhibited by dideoxynucleotide triphos-phates, D4T triphosphate, and flouro-substitutedanalogs.104 AZT triphosphate, 3TC triphosphate,and carbovir triphosphate were moderate inhibi-tors of DNA pol-� in vitro. They also showed thatddC, D4T, and FIAU inhibited mtDNA synthesisin vivo below known physiologic levels.104

Recent studies have corraberated the findingsand extended them to and have shown incorpora-tion of NRTI triphosphates into mtDNA with dif-ferent efficiencies based on their biochemical69 orisomeric structure98 and support the observationsmade earlier in vivo.

Dideoxy NRTI Toxicity: AZT as theModel System

As a bona fide complication of AZT therapy,a cummulative mitochondrial skeletal myop-athy occurred in AZT-treated, adult AIDS pa-

310 WILLIAM LEWIS

tients.26,105,106 Characteristic microscopic raggedred fibers107 and ultrastructural paracrystalline in-clusions26 were observed in muscle samples fromthese patients. The features resulted from subsar-colemmal accumulation of mitochondria in theskeletal muscle with long-term, high-dose treat-ment in adult AIDS patients. Mitochondria wereenlarged and swollen ultrastructurally and con-tain disrupted cristae and occasional paracrystal-line inclusions.2,19,27,108 Extracts of muscle biopsyspecimens of AZT-treated patients revealed de-creased skeletal muscle mtDNA. Mitochondrialdysfunction in AZT-induced myopathy resultedin inefficient use of long-chain fatty acids for�-oxidation. Fat droplets accumulated. AZT my-opathy developed after at least 6 months of ther-apy and occured in up to 17% of treated pa-tients.109,110 Jay and Dalakas111 showed that itoccured with high-dose therapy and with current,low-dose regimens. In pediatric populations withAIDS, AZT skeletal myopathy is observed less fre-quently and may be masked by coexisting enceph-alopathy.111

Clinical features of AZT muscle toxicity in-cluded fatigue, myalgia, muscle weakness, wast-ing, and elevation of serum creatine kinase lev-els,51,109 features of mitochondrial myopathies ofgenetic etiology. Clinical improvement accompa-nies histologic improvement and reversal ofzidovudine-induced mtDNA changes.51,109 Patho-logic changes in AZT myopathy appear to be re-versible after discontinuation of drug. Serum ana-lyte levels that are elevated include lactatedehydrogenase, creatine kinase, and serum glu-tamic-oxaloacetic transaminase. These occurredafter prolonged therapy.112 Exercise decreasedmuscle phosphocreatine (detected by 31P nuclearmagnetic resonance) in AZT-treated patients.113

Abnormal mitochondrial respiratory function wasfound. Enzyme histochemical analysis of musclebiopsy examinations showed partial deficiency ofcytochrome c oxidase activity.109,114 A high lac-tate/pyruvate ratio (consistent with abnormal mi-tochondrial function) is seen in the blood of pa-tients with AZT myopathy.115 Assessment ofmuscle metabolism in vivo using magnetic reso-nance spectroscopy showed marked phosphocre-atine depletion with slow recovery only in AZT-treated, HIV-positive patients.113

In the case of CM from NRTIs in patients withAIDS, findings are less clear. CM related to AZT

and/or other antiretroviral therapy has been re-ported. Interestingly, discontinuation of NRTIsresulted in improved left ventricular function,116

perhaps the earliest report of planned therapeuticinterruption of antiretroviral therapy.

Clinical features of AZT CM resemble some ofthose described for CM of other etiologies, butwith the addition of AIDS or HIV infection. Datasuggest that cardiomyopathy in the setting ofAIDS has an ominous prognosis,117 but the directrelationship to NRTI or HAART therapy has notbeen made.

AZT CM occurs after prolonged treatment.Clinical features include congestive heart failure,left ventricle dilatation, and reduced ejection frac-tion. In general, biopsy examination data in AZTCM is incomplete. One small study showed ultra-structural changes of intramyocytic vacuoles,myofibrillar loss, dilated sarcoplasmic reticulum,and disruption of mitochondrial cristae.118

The role of NRTI CM in children is controver-sial because clinical studies are inconclusive. Inlarge-scale studies of pediatric patients with AIDSand of neonates treated with AZT both in uteroand perinatally, Lipshultz et al119,120 reported thatimpaired cardiac function was not attributed toAZT. Importantly, myocardial biopsy findingswere absent. Additionally, because AZT skeletalmyopathy is uncommon in children with AIDS,111

it may be possible to infer that the pediatric stri-ated and cardiac muscle tissue responds differ-ently to NRTI-related toxicity.

Contrasting evidence in other reports suggeststhat AZT CM in pediatric patients may be moreprevalent than previously reported.121 It furthersuggests a causal relationship between NRTI ther-apy and cardiac dysfunction. Perhaps the mostinteresting data to date come from in vivo studiesin primates using pregnant Erythrocebus patas.Neonatal E. patas treated with AZT in utero revealmitchondrial toxicity to heart and skeletal mus-cle77,122 that resembles those features described inexperimental systems with rodents

Cell biologically, AZT decreased the abundanceof mtDNA in human lymphoblastoid cells.123 Invitro, AZT (25 �mol/L) inhibited the incorpora-tion of [3H] thymidine by 90%123 and 1 �mol/LAZT inhibited it by 25% to 38%.124 Selective lossof mtDNA occurred in MOLT-4F lymphoblastsexposed to ddC, AZT, and other dideoxynucleo-sides in vitro.123,125,126 In various human and ro-

311mtDNA, ANTIRETROVIRALS, AND AIDS CARDIOMYOPATHY

dent muscle cell lines, exposure to AZT causedabnormal mitochondria with extensive lipid accu-mulation.

Oral AZT decreased rat cardiac mtRNA and al-tered mitochondrial ultrastructure.19,20,27,127,128

In similarly treated rats, AZT administration de-creased mtDNA, mtRNA, and mitochondrialpolypeptide expression, and altered mitochon-drial ultrastructure in skeletal muscle.20 Mito-chondrial changes we observed in the AZT-treatedrat heart and muscle were also found in striatedmuscle of hamsters treated with AZT intraperito-neally.129 Rats treated with AZT developed ultra-structural abnormalities in skeletal and cardiacmuscle mitochondria associated with depressionof muscle mtDNA and mitochondrial polypeptidesynthesis, impaired cytochrome c reductase, andan uncoupling effect.27,127

FIAU: Toxicity of a Nondideoxy NRTI

Although the DNA pol-� hypothesis2 and its sub-sequent refinements7 is based on biochemical rea-soning, the point was substantiated in a tragicclinical trial using FIAU in patients with chronichepatitis B virus infection with and without con-commitant HIV infection. Patients who weretreated with FIAU revealed hepatic, muscle, andcardiac mtDNA replication that was disturbedprofoundly. Patient deaths occurred. In studiesperformed in vitro and in vivo using animalmodels, we showed that adenosine tracts wereanalogous to template-related hot spots, that wereparticularly sensitive to mtDNA replication inhi-bition and resembled those found in some herita-ble mitochondrial diseases36,47,57,58 because theyaltered mtDNA synthesis. Adenosine tracts inDNA templates inhibit the incorporation of fialu-ridine monophosphate into DNA in vitro.57

Although FIAU exhibited thorough virucidalactivity, serious toxicity included liver failure (re-quiring liver transplantation).3,130-132 Clinicalmanifestations included profound lactic acidosis,hepatic failure, skeletal and cardiac myopathy,pancreatitis, and neuropathy. Microvesicular he-patic steatosis was prominent. Previously, weshowed that FIAU triphosphate inhibited DNApol-� in vitro (competitively with a nanomolar Ki

and that FIAU monophosphate was incorporatedinto DNA).57,58 FIAU and FMAU triphosphateshowed nanomolar, competitive Ki values.

HepG2 cells treated with FIAU and FMAU re-sulted in each NRTI to be found in nuclear andmtDNA. Ultrastructural defects were found in mi-tochondria.133 mtDNA decreased in abundance inHepG2 cells after 14 days of exposure to FIAU andFMAU.58 FIAU and FMAU (but not FAU) causedmitochondrial structural defects in vitro after atleast 2 weeks of treatment. Changes were visibleon Oil-red-O–stained HepG2 monolayers. Mor-phologic changes correlated with lactate abun-dance in the medium.134 In U937 or MOLT-4 cellstreated with FIAU, a higher Inhibitory concentra-tion was found with 1% to 2% replacement ofcellular thymidine by fialuridine.135

Oil-red-O–stained heart samples from FIAU-treated woodchucks revealed neutral lipid drop-lets in cardiac myocyte cytoplasm. Ultrastructuralevidence of mitochondrial destruction was seen.The steady-state abundance of mtDNA in theliver, myocardium, skeletal muscle, and kidneywas decreased significantly in tissue samples fromfialuridine-treated woodchucks. The magnitudeof the decreases varied among the tissues exam-ined.23,136

Toxicity From NRTIs: DifferingTissue Targets

On the basis of the OXPHOS paradigm56 and ourworking hypothesis,2,7 it is logical to expect mito-chondrial events to impact on other tissues. He-patic toxicity from AZT, ddI, and ddC was re-ported.137-139 It is presumed to relate to toxicity toliver mitochondria. Fatal hepatomegaly with se-vere steatosis,137 severe lactic acidosis,138 andadult Reye’s syndrome139 in AZT-treated HIV-se-ropositive patients were all linked pathogeneti-cally to AZT-induced hepatotoxicity. Clinical fea-tures resembled some of those seen in FIAUtoxicity. The prevalence of metabolic abnormali-ties is increasing in AIDS patients treated withNRTI analogs and the relationship to a variety ofmetabolic and cardiovascular changes in AIDS arebeing investigated more closely.

Treatment with certain NRTIs (d4T/3TC) re-sults in anion gap acidosis.140 Moreover, thelactic acidosis/hepatic steatosis syndrome may bemore common than previously appreciated inadults8,13,14 and children10 treated with NRTIs.d4T treatment caused lipodystrophy.142 Mecha-nisms may involve altered mitochondrial biogen-

312 WILLIAM LEWIS

esis and/or oxidative changes, and possibly adipo-cyte apoptosis.2,143 Recently, we showed arterialdysfunction in FVB/n mice treated with AZT,144

which may be another important target of toxicity.NRTIs have peripheral neuropathies as side ef-fects.2,145

Summary

NRTI toxicity now is an important clinical prob-lem with long-term significance to AIDS patientsand CM from NRTIs may be an important clinicalevent. Mechanisms likely relate to energy deple-tion, oxidative stress, and mtDNA mutations.Analogous to treatment of other serious infectiousagents, combinations of multiple anti–HIV-1drugs are used to target different viral proteins orpoints in the virus-host life cycle146-148 and maycreate combined toxicities to mitochondria. Be-cause current clinical guidelines recommendcombined therapy usually containing NRTI,149

such regimens may be important to the develop-ment of mitochondrial toxicity in new tissue tar-gets. Future studies will pinpoint susceptible pa-tient populations, genetic predispositions, andpharmacologic mechanisms for NRTI toxicity andits tissue selectivity.

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