Bernhard Setzer

Increased long-term mitochondrial toxicity in combinations of nucleoside analogue reverse-transcriptase inhibitors

Background: Some nucleoside analogue reverse transcriptase inhibitors (NRTI) may cause depletion of mitochondrial (mt) DNA in liver by inhibiting polymerase-γ. mtDNA depletion may contribute to lactic acidosis, steatohepatitis and liver failure. Objective: To evaluate the long-term mitochondrial toxicity of NRTI combinations. Methods: The HepG2 human hepatoma cell line was cultivated in the presence of zalcitabine (ddC), didanosine (ddI), stavudine (d4T), lamivudine (3TC), zidovudine (ZDV) and efavirenz at concentrations equivalent to steady-state peak plasma levels (Cmax), and also in one-third and 10 times Cmax. The NRTI were added to the medium alone or in combination. Control cells were incubated without any NRTI or with efavirenz. Cell growth, lactate production, intracellular lipid droplets, mtDNA and the mtDNA-encoded respiratory chain subunit COX II were monitored over a period of up to 30 days. Results: Time- and dose-dependent mtDNA depletion was observed with ddC > ddI > d4T and mtDNA depletion preceded or coincided with a decline in COX II expression, a decrease in cell growth, increased lactate production and increased intracellular lipids. 3TC and efavirenz did not affect any measurement. ZDV increased lactate moderately and cell growth was inhibited, despite normal mtDNA and COX II levels. The negative effects on some measurements were more pronounced in the 3TC–ZDV and ddC–d4T combinations, than in the single-NRTI incubations. The combination of ddI–d4T was not more toxic than ddI alone. Mitochondrial damage by ZDV, d4T, ddI, and ddC did not reach steady-state by day 25. Using a Southern blot technique, mtDNA deletions were never observed. Conclusion: The data indicate additive or synergistic long-term mitochondrial toxicity in some NRTI combinations.

Evidence of nucleoside analogue reverse transcriptase inhibitor--associated genetic and structural defects of mitochondria in adipose tissue of HIV-infected patients.

&NA; To investigate if possible mitochondrial injury can be found in adipose tissue of nucleoside analogue reverse transcriptase inhibitor (NRTI)‐treated patients, subcutaneous fat was taken from the buttocks of 24 HIV‐positive patients and 8 HIV‐negative controls. The content of mitochondrial DNA (mtDNA) was quantified using a Southern blot technique. Fat biopsies were examined by electron microscopy and screened by restriction fragment length polymorphism analysis for the presence of the nt 8344 and 3243 mtDNA point mutations. Age, sex, and body mass index did not differ between the HIV‐negative controls, the HIV‐positive patients currently treated with NRTIs (NRTI group, n = 19), and the HIV‐positive patients without NRTIs (no‐NRTI group, n = 5). The mean mtDNA content was 44% lower in the NRTI group compared with the no‐NRTI group (p = .01) but did not differ between the control group and the no‐NRTI group. When the HIV‐infected patients were stratified to a group with clinical signs of lipoatrophy at the biopsy site (LA group, n = 11) and a group without lipoatrophy (no‐LA group, n = 13), the mean mtDNA content in the LA group was 39% lower than that in the no‐LA group (p = .02). No point mutations or deletions were observed. The adipocytes of patients with lipoatrophy contained multiple small lipid vacuoles, and the mitochondria harbored inclusions reminiscent of mtDNA cytopathies. mtDNA depletion and ultrastructural abnormalities of adipocytes suggest a link between mitochondrial damage, the use of NRTIs, and lipoatrophy in HIV‐infected patients.

Time-Dependent and Tissue-Specific Accumulation of mtDNA and Respiratory Chain Defects in Chronic Doxorubicin Cardiomyopathy

Background—Doxorubicin causes a chronic cardiomyopathy of unknown pathogenesis. We investigated whether acquired defects in mitochondrial DNA (mtDNA) and interconnected respiratory chain dysfunction may represent a molecular mechanism for its late onset. Methods and Results—Rats were treated weekly with intravenous doxorubicin (1 mg/kg) for 7 weeks, starting at 11 weeks of age (group B). Controls received saline. Group C received doxorubicin identically to group B, but the course was started at 41 weeks of age. All rats were killed at week 48. Doxorubicin was also injected once, either 6 days (group D) or 2 hours (group E) before euthanasia. Heart and skeletal muscle were examined. Only group B rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Group B hearts had the lowest cytochrome c oxidase (COX) activity (24% of controls; P =0.003), the highest citrate synthase activity (135% of controls; P =0.005), and the highest production of superoxide. In group B, the respiratory subunit COXI, which is encoded by mtDNA, was reduced (P <0.001), as was mtDNA (49% of controls, P <0.001). Group C hearts differed from group B in their lower cardiomyopathy score (P =0.006), higher COX activity (P =0.02), and higher mtDNA content (P =0.04). Group B and to a lesser extent group C hearts contained deleted mtDNA. There was no detectable mitochondrial toxicity in group D and E hearts or in skeletal muscle. Conclusions—In doxorubicin cardiomyopathy, mtDNA alterations, superoxide, and respiratory chain dysfunction accumulate long-term in the absence of the drug and are associated with a late onset.

Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine

The “D drug” HIV reverse‐transcriptase inhibitors zalcitabine, didanosine, and stavudine are relatively strong inhibitors of polymerase‐gamma compared with the “non–D drugs” zidovudine, lamivudine, and abacavir. D drugs deplete mitochondrial DNA (mtDNA) in cultured hepatocytes. This mtDNA depletion is associated with an increased in vitro production of lactate. To investigate the origin of hyperlactatemia in HIV‐infected patients and the effects of antiretroviral therapy on liver mtDNA, we biopsied liver tissue from 94 individuals with chronic hepatitis C virus (HCV) infection. Eighty subjects were coinfected with HIV. Serum lactate was measured at the time of biopsy. Hepatic mtDNA and liver histology were centrally assessed. Liver mtDNA content of HIV‐infected patients receiving D drugs at the time of biopsy (n = 34) was decreased by 47% (P<.0001) compared with those without D drugs (n = 35). Aside from a possible association between HCV genotype I status and mtDNA depletion in multivariate analysis, there were no other virologic, immunologic, histologic, demographic or treatment‐related variables that could explain the mtDNA depletion. Lactate was above the upper limit of normal in only three patients, all of whom were treated with D drugs. The mtDNA in each of them was lower than in any non–D drug patient and significantly (P = .017) depleted compared with D drug patients with normal lactate. In conclusion, D drug treatment is associated with decreased hepatic mtDNA in HIV‐infected patients with chronic HCV infection. Moderate mtDNA depletion in liver does not necessarily lead to hyperlactatemia, but more pronounced decreases in hepatic mtDNA may be an important contributor to lactate elevation. (HEPATOLOGY 2004;39:311–317.)

Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin.

Doxorubicin causes a chronic cardiomyopathy. Although the exact pathogenesis is unknown, recent animal data suggest that somatically acquired alterations of mitochondrial DNA (mtDNA) and concomitant mitochondrial dysfunction play an important role in its onset. In this study, skeletal and myocardial muscles were examined from human autopsies. Compared to controls (n = 8), doxorubicin‐exposed hearts (n = 6) showed low absolute enzyme activity of mtDNA‐encoded nicotinamide adenine dinucleotide hydrogen dehydrogenase (NADH DH, 79% residual activity, p = 0.03) and cytochrome c oxidase (COX, 59% residual activity, p < 0.001), but not of succinate dehydrogenase (SDH), which is encoded exclusively by nuclear DNA. NADH DH/SDH and COX/SDH ratios were 37% (p < 0.001) and 27% (p < 0.001) of controls. Expression of the mtDNA‐encoded subunit II of COX was reduced (82%, p = 0.04), compared to its unchanged nucleus‐encoded subunit IV. MtDNA‐content was diminished (56%, p = 0.02), but the ‘common’ mtDNA‐deletion was increased (9.2‐fold, p = 0.004). Doxorubicin‐exposed hearts harboured numerous additional mtDNA rearrangements lacking direct repeats. They contained elevated levels of malondialdehyde (MDA) (p = 0.006, compared to controls), which correlated inversely with the COX/SDH ratio (r = −0.45, p = 0.02) and the mtDNA‐content (r = −0.75, p = 0.002), and correlated positively with the levels of the ‘common’ deletion (r = 0.80, p < 0.001). Doxorubicin‐exposed hearts also contained the highest levels of superoxide (p < 0.001, compared to controls), which correlated negatively with the mtDNA‐encoded respiratory chain activities, such as the COX/SDH ratio (r = −0.57, p = 0.02) and the NADH/SDH ratio (r = −0.52, p = 0.04), as well as with the mtDNA content (r = −0.69, p = 0.003), and correlated positively with the frequency of the ‘common’ deletion (r = 0.76, p < 0.001) and the MDA levels (r = 0.86, p < 0.001). Doxorubicin‐exposed hearts contained electron‐dense deposits within mitochondria. Hearts exposed to other anthracyclines (n = 6) or skeletal muscle (all groups) had no mitochondrial dysfunction. Doxorubicin, unlike other anthracyclines, augments lipid peroxidation, induces mtDNA mutations and decreases mtDNA content in human hearts. These lesions have an impact on mitochondrial function and could be of importance in the pathogenesis of clinical cardiomyopathy. Copyright

The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage.

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long‐term and explain in part the delayed onset of heart dysfunction. DOXO‐EMCH a 6‐maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO‐EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO‐EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO‐EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c‐oxidase (COX, 26% of controls), reduced expression of the mtDNA‐encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO‐EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high‐dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin‐exposed hearts and to a lesser extent the myocardia of both DOXO‐EMCH groups contained mtDNA‐deletions. In summary both DOXO‐EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX‐activity, COX II expression, mtDNA‐content, mtDNA mutation loads and superoxide production in rats.

Effects of Didanosine-Related Depletion of mtDNA in Human T Lymphocytes

The normal metabolism of mitochondria in T lymphocytes is unknown, as are the effects from nucleoside-analogue reverse-transcriptase inhibitors that impair mitochondrial polymerase- gamma . We isolated peripheral-blood CD4 and CD8 T lymphocytes from 6 healthy men and stimulated them with anti-CD3 and anti-CD28 antibodies, in the presence and in the absence of didanosine (ddI). In the absence of ddI, mitosis of T lymphocytes was paralleled by a transient up-regulation of both mtDNA and production of lactate. In CD4 lymphocytes, 10-day incubation with ddI at concentrations of 11.8 mu mol/L, 35.4 mu mol/L, 59.0 mu mol/L, and 118.0 mu mol/L induced (1) a concentration-dependent reduction of both mtDNA (to 73%, 29%, 24%, and 23%, respectively, of the levels in control samples) and subunit II of mtDNA-encoded cytochrome c oxidase (to 86%, 81%, 55%, and 31%, respectively, of the levels in control samples) and (2) a concentration-dependent increase in production of lactate (to 139%, 222%, 276%, and 312%, respectively, of the levels in control samples). Activation of lymphocytes (which was measured in terms of expression of CD25) was unaffected. Mitochondrial depolarization (assessed by staining with JC-1) was observed as early as day 7 of incubation. All changes were time dependent and also were observed in isolated CD8 lymphocytes. Electron microscopy revealed enlarged mitochondria with vacuoles, inclusions, and reduced electron density. ddI at a concentration of 11.8 mu mol/L induced changes that bordered statistical significance. After stimulation, there was a wide range in the change of mtDNA content in lymphocytes. Therefore, mtDNA measurements in blood are not necessarily a marker for the mitochondrial toxicity of ddI. Nevertheless, ddI does lead to depletion of mtDNA in lymphocytes and to functional impairment.

Pyrimidine Nucleoside Depletion Sensitizes to the Mitochondrial Hepatotoxicity of the Reverse Transcriptase Inhibitor Stavudine

Stavudine is a hepatotoxic antiretroviral nucleoside analogue that also inhibits the replication of mitochondrial DNA (mtDNA). To elucidate the mechanism and consequences of mtDNA depletion, we treated HepG2 cells with stavudine and either redoxal, an inhibitor of de novo pyrimidine synthesis, or uridine, from which pyrimidine pools are salvaged. Compared with treatment with stavudine alone, co-treatment with redoxal accelerated mtDNA depletion, impaired cell division, and activated caspase 3. These adverse effects were completely abrogated by uridine. Intracellular ATP levels were unaffected. Transcriptosome profiling demonstrated that redoxal and stavudine acted synergistically to induce CDKN2A and p21, indicating cell cycle arrest in G1, as well as genes involved in intrinsic and extrinsic apoptosis. Moreover, redoxal and stavudine showed synergistic interaction in the up-regulation of transcripts encoded by mtDNA and the induction of nuclear transcripts participating in energy metabolism, mitochondrial biogenesis, oxidative stress, and DNA repair. Genes involved in nucleotide metabolism were also synergistically up-regulated by both agents; this effect was completely antagonized by uridine. Thus, pyrimidine depletion sensitizes cells to stavudine-mediated mtDNA depletion and enhances secondary cell toxicity. Our results indicate that drugs that diminish pyrimidine pools should be avoided in stavudine-treated human immunodeficiency virus patients. Uridine supplementation reverses this toxicity and, because of its good tolerability, has potential clinical value for the treatment of side effects associated with pyrimidine depletion.

Uridine supplementation antagonizes zalcitabine-induced microvesicular steatohepatitis in mice†

Zalcitabine is an antiretroviral nucleoside analogue that exhibits long‐term toxicity to hepatocytes by interfering with the replication of mitochondrial DNA (mtDNA). Uridine antagonizes this effect in vitro. In the present study we investigate the mechanisms of zalcitabine‐induced hepatotoxicity in mice and explore therapeutic outcomes with oral uridine supplementation. BalbC mice (7 weeks of age, 9 mice in each group) were fed 0.36 mg/kg/d of zalcitabine (corresponding to human dosing adapted for body surface), or 13 mg/kg/d of zalcitabine. Both zalcitabine groups were treated with or without Mitocnol (0.34 g/kg/d), a dietary supplement with high bioavailability of uridine. Liver histology and mitochondrial functions were assessed after 15 weeks. One mouse exposed to high dose zalcitabine died at 19 weeks of age. Zalcitabine induced a dose dependent microvesicular steatohepatitis with abundant mitochondria. The organelles were enlarged and contained disrupted cristae. Terminal transferase dUTP nick end labeling (TUNEL) assays showed frequent hepatocyte apoptosis. mtDNA was depleted in liver tissue, cytochrome c‐oxidase but not succinate dehydrogenase activities were decreased, superoxide and malondialdehyde were elevated. The expression of COX I, an mtDNA‐encoded respiratory chain subunit was reduced, whereas COX IV, a nucleus‐encoded subunit was preserved. Uridine supplementation normalized or attenuated all toxic abnormalities in both zalcitabine groups, but had no effects when given without zalcitabine. Uridine supplementation was without apparent side effects. Conclusion: Zalcitabine induces mtDNA‐depletion in murine liver with consequent respiratory chain dysfunction, up‐regulated synthesis of reactive oxygen species and microvesicular steatohepatitis. Uridine supplementation attenuates this mitochondrial hepatotoxicity without apparent intrinsic effects. (HEPATOLOGY 2007;45:72–79.)

Skeletal muscle mitochondrial DNA content and aerobic metabolism in patients with antiretroviral therapy-associated lipoatrophy

OBJECTIVES To assess whether mitochondrial dysfunction in skeletal muscle characterizes antiretroviral therapy (ART)-associated lipoatrophy (LA). METHODS A cross-sectional study comparing HIV-infected, antiretroviral-treated patients with LA (n = 5; LA+) and without LA (n = 5; non-LA) was conducted. Positron emission tomography was used to measure blood flow, oxygen extraction and oxygen consumption in quadriceps femoris muscle during rest and aerobic exercise. Mitochondrial DNA (mtDNA) was quantified by PCR. Body composition was measured by dual-energy X-ray absorptiometry and magnetic resonance imaging. All data are given as means +/- SEM. RESULTS Compared with the non-LA group, the LA+ group had significantly less limb fat and more intra-abdominal fat, but similar leg muscle mass. The LA+ group versus the non-LA group had reduced mtDNA content per nucleus in adipose tissue (173 +/- 38 versus 328 +/- 62; P = 0.067), but not in skeletal muscle (2606 +/- 375 versus 2842 +/- 309; P = 0.64). Perfusion in resting muscle (34 +/- 7 versus 28 +/- 6 mL/kg/min in the LA+ group versus the non-LA group; P = 0.5), and the mean absolute (277 +/- 30 versus 274 +/- 43 mL/kg/min, respectively; P = 0.95) and relative (10.6 +/- 2.5- versus 11.9 +/- 1.5-fold change, respectively; P = 0.67) increases in perfusion during exercise were similar between the groups. Oxygen consumption at rest (2.2 +/- 0.7 versus 2.1 +/- 0.3 mL/kg/min in the LA+ group versus the non-LA group; P = 0.9), and the mean absolute (14.6 +/- 1.7 versus 24.3 +/- 8.8 mL/kg/min, respectively; P = 0.3) and relative (10.3 +/- 2.8- versus 11.7 +/- 2.4-fold change, respectively; P = 0.73) exercise-induced increases in oxygen consumption were similar between the groups. The oxygen extraction fraction was comparable between the groups, both at rest and during exercise. Plasma lactate concentrations remained unchanged in both groups during exercise. CONCLUSIONS HIV-infected patients with ART-associated LA have similar mtDNA content in skeletal muscle and comparable skeletal muscle aerobic exercise metabolism to antiretroviral-treated non-lipoatrophic patients.