Alterations of Mitochondrial DNA in CEM Cells Selected for Resistance Toward DDC Toxicity

2 ′,3 ′-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.     

The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and shows beneficial effects in AIDS therapy. The compound inhibits mitochondrial DNA (mtDNA) synthesis at a clinically relevant concentration, which could be responsible for the side effects of ddC observed in the clinic. Thymidine (dThd), one of the substrates of mitochondrial deoxypyrimidine kinase (dPyd kinase), was not able to reverse the mitochondrial toxicity of ddC in CEM cells. Furthermore, the cytoplasmic deoxycytidine kinase (dCyd kinase)-deficient CEM cells were highly resistant to the mitochondrial toxicity of ddC. These data suggest a critical role for cytoplasmic dCyd kinase in the mitochondrial toxicity of ddC. The metabolites of ddC, but not ddC itself, were able to inhibit mtDNA synthesis in isolated mitochondria. The potency of the inhibitory effect was in the order of ddCTP greater than ddCDP greater than ddCMP greater than ddC. The lack of inhibition by ddC of mtDNA synthesis could be due to the inefficient ddC phosphorylation in mitochondria. Although the mitochondrial dPyd kinase was reported to phosphorylate ddC, the phosphorylation of ddC in isolated mitochondria was not detectable. The data suggest that ddC is phosphorylated to ddCTP in the cytoplasm and then transported into mitochondria to exert its inhibitory effect on mtDNA synthesis.     

Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol gamma), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2',3'-dideoxycytidine (ddC)) and Lamivudine (beta-d-(+)-2',3'-dideoxy-3'-thiacytidine ((-)3TC])). (-)3TC is the only analog containing an unnatural l(-) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (-)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (-)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol gamma. This analysis reveals that Pol gamma incorporates (-)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3'-end of the DNA primer by the 3'-5'-exonuclease activity of Pol gamma were similar (0.01 s(-)1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.     

5-Fluoro-2'-deoxyuridine has effects on mitochondria in CEM T-lymphoblast cells

Fluoropyrimidines are useful anticancer agents and the compound 5-fluoro-2'-deoxyuridine (FdUrd) plays an important role in chemotherapy of colon cancers. Several nucleoside analogs, such as 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), can be incorporated into and cause depletion of mitochondrial DNA (mtDNA). These drugs are known to cause mitochondrial toxicity after prolonged treatment in patients. In this study we demonstrate that FdUrd reduces the mtDNA content and the expression level of the mtDNA encoded cytochrome c oxidase (COX II) in a CEM T-lymphoblastic cell line.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase

Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) > lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.     

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) > (+)3TC > (-)3TC > PMPA > azidothymidine (AZT) > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T > ddA (ddI) > ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.     

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs'' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.     

5‐Fluoro‐2′‐Deoxyuridine Has Effects on Mitochondria in CEM T‐Lymphoblast Cells

Fluoropyrimidines are useful anticancer agents and the compound 5‐fluoro‐2′‐deoxyuridine (FdUrd) plays an important role in chemotherapy of colon cancers. Several nucleoside analogs, such as 3′‐azido‐2′,3′‐dideoxythymidine (AZT) and 2′,3′‐dideoxycytidine (ddC), can be incorporated into and cause depletion of mitochondrial DNA (mtDNA). These drugs are known to cause mitochondrial toxicity after prolonged treatment in patients. In this study we demonstrate that FdUrd reduces the mtDNA content and the expression level of the mtDNA encoded cytochrome c oxidase (COX II) in a CEM T‐lymphoblastic cell line.     

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1^−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1^−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.     

Insulin-Like Growth Factor-1 Prevents Dorsal Root Ganglion Neuronal Tyrosine Kinase Receptor Expression Alterations Induced by Dideoxycytidine In Vitro

Dideoxycytidine (zalcitabine, ddC) produces neurotoxic effects. It is particularly important to understand the toxic effects of ddC on different subpopulations of dorsal root ganglion (DRG) neurons which express distinct tyrosine kinase receptor (Trk) and to find therapeutic factors for prevention and therapy for ddC-induced peripheral sensory neuropathy. Insulin-like growth factor-1 (IGF-1) has been shown to have neurotrophic effects on DRG sensory neurons. However, little is known about the effects of ddC on distinct Trk (TrkA, TrkB, and TrkC) expression in DRG neurons and the neuroprotective effects of IGF-1 on ddC-induced neurotoxicity. Here, we have tested the extent to which the expression of TrkA, TrkB, and TrkC receptors in primary cultured DRG neurons is affected by ddC in the presence or absence of IGF-1. In this experiment, we found that exposure of 5, 25, and 50 μmol/L ddC caused a dose-dependent decrease of the mRNA, protein, and the proportion of TrkA-, TrkB-, and TrkC-expressing neurons. IGF-1 (20 nmol/L) could partially reverse the decrease of TrkA and TrkB, but not TrkC, expression with ddC exposure. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μmol/L) blocked the effects of IGF-1. These results suggested that the subpopulations of DRG neurons which express distinct TrkA, TrkB, and TrkC receptors were affected by ddC exposure. IGF-1 might relieve the ddC-induced toxicity of TrkA- and TrkB-, but not TrkC-expressing DRG neurons. These data offer new clues for a better understanding of the association of ddC with distinct Trk receptor expression and provide new evidence of the potential therapeutic role of IGF-1 on ddC-induced neurotoxicity.     

Assessment of developmental toxicity of antiretroviral drugs using a rat whole embryo culture system

BACKGROUND: Previous guidelines for HIV-infected pregnant women have recommended zidovudine (ZDV) monotherapy during the second and third trimesters of pregnancy to prevent fetal HIV infection. New guidelines suggest that women should continue or be offered combination antiretroviral therapy (including protease inhibitors) during pregnancy. Nevertheless, little animal or human toxicity data underlie these recommendations. METHODS: We used an in vitro rat whole embryo culture system to assess the embryo toxicity of various nucleoside analogues, namely, ZDV, dideoxyinosine (ddI), and 2', 3'-dideoxycytidine (ddC), and the HIV-1 protease inhibitor, indinavir, both alone and in combination. RESULTS: Although human fetal concentrations of these compounds are unknown, no gross abnormalities were detected after incubation with these agents, either alone or in combination at concentrations that would be expected to be achievable in human maternal serum (1-50 microM). ZDV in combination with ddC at >100 microM, resulted in severe growth retardation and morphologic abnormalities not seen with either agent singly. CONCLUSIONS: We conclude that the combination of ZDV/ddC results in severe concentration-dependent embryo toxicity. No growth retardation or gross morphologic abnormalities were found for any of the agents, either singly or in combination, at clinically relevant concentrations.     

Does mitochondrial dysfunction during antiretroviral therapy in human immunodeficiency virus infection suggest antioxidant supplementation as a beneficial option?

Over the last few years, a relative decline of the morbidity and mortality of human immunodeficiency virus (HIV) infection in industrialised countries has been observed due to the use of a potent combined therapy known as high active antiretroviral therapies (HAARTs). It has led to a decrease of viral load and a quantitative and qualitative improvement of immune function in patients, especially CD4+ T-lymphocyte count, having as a consequence a decrease of infectious complications and a global clinical improvement. Besides the positive effects of HAARTs on immune and metabolic alterations during HIV infection, it has been reported that the commonly used drugs AZT, ddI, and ddC are toxic to hepatocytes. Recent reports continue to point to the mitochondria as targets for toxicity. The prevalence of these symptoms is continued during acquired immunodeficiency syndrome (AIDS). The effects of oxidative stress occurring as a consequence of mitochondrial toxicity may amplify some of the pathophysiological and phenotypic events during infection. Mitochondrial stabilisation and antioxidative strategies are possible new therapeutic aims since the antiretroviral treatment is prolonged with increased longevity from AIDS, which has become a more manageable chronic illness. The aim of the present review article is to summarize the current knowledge about mitochondrial dysfunction during HAART and its consequence for patients with chronic treatment. Oxidative stress may serve as one pathway for cellular damage in AIDS and its treatment. One important future goal is to prevent or attenuate the side effects of HAART so that improved disease management can be achieved.     

Effect of exercise on plasma paraoxonase1 activity in rugby players: Dependance on training experience

Abstract

Over the last few years, a relative decline of the morbidity and mortality of human immunodeficiency virus (HIV) infection in industrialised countries has been observed due to the use of a potent combined therapy known as high active antiretroviral therapies (HAARTs). It has led to a decrease of viral load and a quantitative and qualitative improvement of immune function in patients, especially CD4⁺ T-lymphocyte count, having as a consequence a decrease of infectious complications and a global clinical improvement. Besides the positive effects of HAARTs on immune and metabolic alterations during HIV infection, it has been reported that the commonly used drugs AZT, ddI, and ddC are toxic to hepatocytes. Recent reports continue to point to the mitochondria as targets for toxicity. The prevalence of these symptoms is continued during acquired immunodeficiency syndrome (AIDS). The effects of oxidative stress occurring as a consequence of mitochondrial toxicity may amplify some of the pathophysiological and phenotypic events during infection. Mitochondrial stabilisation and antioxidative strategies are possible new therapeutic aims since the antiretroviral treatment is prolonged with increased longevity from AIDS, which has become a more manageable chronic illness. The aim of the present review article is to summarize the current knowledge about mitochondrial dysfunction during HAART and its consequence for patients with chronic treatment. Oxidative stress may serve as one pathway for cellular damage in AIDS and its treatment. One important future goal is to prevent or attenuate the side effects of HAART so that improved disease management can be achieved.     

Mitochondrial dysfunction enhances the migration of vascular smooth muscles cells via suppression of Akt phosphorylation

Background

Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues.

Methods

We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC).

Results

The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC.

Conclusions

Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease.

General significance

Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis.     

Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.     

Effect of indinavir used alone or in double or triple combination with AZT and ddC on human immune functions

Indinavir (IDV) is a potent and selective human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) widely used in antiretroviral therapy, but its effects on the immune system are relatively unknown. In this study we have investigated the in vitro effect of IDV on normal human peripheral blood mononuclear cells (PBMC). We used the drug alone or in double and triple combination with AZT and ddC to assess whether IDV interferes with the previously observed immunomodulatory effects induced by AZT and ddC. We found that proliferative response, induction of immunoglobulins (Ig) production and cytokine production was not modulated by IDV. More importantly, IDV used in double or triple combination with AZT and ddC, does not further strenghten the inhibition of proliferative response induced by AZT and is able to abrogate the inhibitory effect induced by ddC on proliferative response. Similarly, IDV/AZT, IDV/ddC and IDV/AZT/ddC combinations does not strenghten the modulation of TNF-alpha, IFN-gamma and IL-4 induced by AZT, ddC and AZT/ddC. On the other hand, IDV neutralizes the up-regulating effects of AZT on IL-2 production while the up-regulating effects of ddC on IL-2 production is not affected. These data suggest that IDV used in combination with AZT and ddC did not add any further immunotoxicity.