Background: The pathogenesis of osteoarthritis (OA) has been linked with the elaboration of increased amounts of nitrotyrosine in cartilage as a stable marker of tyrosine nitration by nitric oxide. Reactive oxygen species (ROS) are known to damage mitochondrial DNA (mtDNA); this process is associated with aging of non-cartilaginous tissues. This study aimed to link intraarticular nitrotyrosine content, with mtDNA lesions and OA severity in order to gain insights into the contribution of nitrotyrosine and mitochondria to cartilage aging and the development of OA. Methods: Femoral heads were prospectively obtained from individuals undergoing hip arthroplasty or postmortem autopsy (22 male, 26 female, age range 16-93 years). OA severity was graded radiographically, and histologically by means of the Mankin score. Articular chondrocytes were isolated, and the ‘common’ mtDNA deletion quantified with a Polymerase Chain Reaction (PCR) technique. mtDNA copy numbers per chondrocyte were determined by quantitative PCR. Articular nitrotyrosine content in chondrocytes was quantified by ELISA. Multivariate associations between parameters were computed by linear regression analysis or Spearman rank correlations, as appropriate. Results: Articular nitrotyrosine concentrations were independently correlated with both, the age of the subjects (r=0.39, p=0.01) and the presence of the common deletion (r=0.48, p=0.004), but not with the Mankin Score (p=0.84) or wild type mtDNA copy numbers. The severity of hip OA (Mankin score) however was only correlated with patient age, but not with articular nitrotyrosine, mtDNA deletions or wild type mtDNA copy numbers. Conclusions: mtDNA lesions are correlated with articular nitrotyrosine content in hip cartilage, but a link of mitochondrial mutagenesis and NO-mediated ROS formation with the age of the subjects, or with OA severity cannot be demonstrated in this study.
Some nucleoside analogue reverse transcriptase inhibitors (NRTI) may cause depletion of mitochondrial (mt) DNA in liver by inhibiting polymerase-gamma. mtDNA depletion may contribute to lactic acidosis, steatohepatitis and liver failure.To evaluate the long-term mitochondrial toxicity of NRTI combinations.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 (C ), and also in one-third and 10 times C. 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.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.The data indicate additive or synergistic long-term mitochondrial toxicity in some NRTI combinations.
Appearance of nitrate reductase (NR, EC 1.6.6.1–3), nitrite reductase (NiR, EC 1.7.7.1) and glutamine synthetase (GS, EC 6.3.1.2) under the control of nitrate, ammonium and light was studied in roots, hypocotyls and needles (cotyledonary whorl) of the Scots pine ( Pinus sylvestris L.) seedling. It was found that appearance of NiR was mainly controlled by nitrate whereas appearance of GS was strongly controlled by light. In principle, the NR activity level showed the same dependency on nitrate and light as that of NiR. In the root, both nitrate and ammonium had a stimulatory effect on GS activity whereas in the whorl the induction was minor. The level of NiR (NR) activity is high in the root and hypocotyl and low in the cotyledonary whorl, whereas the GS activity level per organ increases strongly from the root to the whorl. Thus, in any particular organ the operation of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is not closely connected to the operation of the nitrate reduction pathway. The strong control of GS/GOGAT by light and the minor sensitivity to induction by nitrate or ammonium indicate a major role of the GS/GOGAT cycle in reassimilation of endogeniously generated ammonium.
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.)
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.)
Some nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) combinations cause additive or synergistic interactions in vitro and in vivo.We evaluated the mitochondrial toxicity of tenofovir (TFV), emtricitabine (FTC) and abacavir as carbovir (CBV) alone, with each other, and in combination with additional NRTIs. HepG2 human hepatoma cells were incubated with TFV, FTC, CBV, didanosine (ddl), stavudine (d4T), lamivudine (3TC) and zidovudine (AZT) at concentrations equivalent to 1 and 10x clinical steady-state peak plasma levels (C(max)). NRTIs were also used in double and triple combinations. Cell growth, lactate production, intracellular lipids, mtDNA and the mtDNA-encoded respiratory chain subunit II of cytochrome c oxidase (COXII) were monitored for 25 days.TFV and 3TC had no or minimal toxicity. FTC moderately reduced hepatocyte proliferation independent of effects on mtDNA. ddl and d4T induced a time- and dose-dependent loss of mtDNA and COXII, decreased cell growth and increased levels of lactate and intracellular lipids. CBV and AZT strongly impaired hepatocyte proliferation and increased lactate and lipid production, but did not induce mtDNA depletion. The dual combination of TFV plus 3TC had only minimal toxicity; TFV plus FTC slightly reduced cell proliferation without affecting mitochondrial parameters. All other combinations exhibited more pronounced adverse effects on mitochondrial endpoints. Toxic effects on mitochondrial parameters were observed in all combinations with ddI, d4T, AZT or CBV. TFV and 3TC both attenuated ddI-related cytotoxicity, but worsened the effects of CBV and AZT.The data demonstrate unpredicted interactions between NRTIs with respect to toxicological endpoints and provide an argument against the liberal use of NRTI cocktails without first obtaining data from clinical trials.
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.
