Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy

We have evaluated mitochondrial (mt) DNA content in CD4 and CD8 peripheral blood lymphocytes (PBLs) from HIV-infected patients taking highly active antiretroviral therapy (HAART) who display different types of adipose tissue alterations. A cross-sectional study was performed in a total of 23 patients with lipodystrophy (LD): nine patients with fat accumulation, six patients with fat loss, eight patients with combined form, who were compared to 11 individuals infected by HIV without LD (HIV-positive) and 10 seronegative controls (CTRL). PBLs were obtained by standard methods, that is, gradient density centrifugation on Ficoll, and CD4 or CD8 cells were positively isolated by magnetic sorting to eliminate the contamination of platelets. mtDNA content was then measured by an original assay based upon real-time PCR. mtDNA content was significantly increased in CD4 T cells from patients with LD, while no differences were present between CD4 and CD8 cells from HIV-positive and CTRL individuals. Nor were any differences found when comparing LD or HIV-positive patients treated with stavudine or zidovudine, or taking D-drugs or non D-drugs. Patients with fat accumulation had significantly higher mtDNA content compared to HIV-positive and CTRL, this phenomenon regarding both CD4 and CD8 PBLs. Considering all HIV-positive patients (including LD), mtDNA content showed a significant, positive correlation with cholesterolaemia but not with triglyceridaemia and glycaemia. Relatively high mtDNA content in LD patients, as well as the correlation between mtDNA content and cholesterol in all HIV-positive subjects, suggest the involvement of mitochondria in such a pathology. However, further studies are needed to confirm these initial observations and ascertain whether the quantification of mtDNA in PBL is a useful and reliable marker to investigate and monitor HAART-related changes in fat distribution.     

Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy.

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A-->G3260 transition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho degrees). The presence and amount of the mutant G3260 vs the wild-type A3260 were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I- and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A-->G3260 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations.     

Quantification of mitochondrial DNA deletion,depletion, and overreplication: application to diagnosis

BACKGROUND: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. METHODS: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. RESULTS: Both methods quantified 5-76% of deleted mtDNA in muscle, 59-97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r(2)) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. CONCLUSIONS: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.     

Cellular and molecular events leading to mitochondrial toxicity of 1-(2-deoxy-2-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil in human liver cells.

We have explored the mechanism(s) related to FIAU-induced liver toxicity, particularly focusing on its effect on mitochondrial function in a human hepatoma cell line-HepG2. The potential role of FMAU and FAU, metabolites detected in FIAU-treated patients were also ascertained. FIAU and FMAU inhibited cell growth and were effectively phosphorylated. A substantial increase in lactic acid production in medium of cells incubated with 1-10 microM FIAU or FMAU was consistent with mitochondrial dysfunction. Slot blot analysis demonstrated that a two week exposure to 10 microM FIAU or FMAU was not associated with a decrease in total mitochondrial (mt) DNA content. However, FIAU and FMAU were incorporated into nuclear and mtDNA and relative values suggest that both compounds incorporate at a much higher rate into mtDNA. Electron micrographs of cells incubated with 10 microM FIAU or FMAU revealed the presence of enlarged mitochondria with higher cristae density and lipid vesicles. In conclusion, these data suggest that despite the lack of inhibition of mtDNA content, incorporation of FIAU and FMAU into mtDNA of HepG2 cells leads to marked mitochondrial dysfunction as evidenced by disturbance in cellular energy metabolism and detection of micro- and macrovesicular steatosis.     

Drosophila melanogaster mitochondrial DNA: completion of the nucleotide sequence and evolutionary comparisons

The nucleotide sequence of the regions flanking the A + T region of Drosophila melanogaster mitochondrial DNA (mtDNA) has been determined. Included are the genes encoding the transfer RNAs for valine, isoleucine, glutamine and methionine, the small ribosomal RNA and the 5'-coding sequences of the large ribosomal RNA and NADH dehydrogenase subunit II. This completes the nucleotide sequence of the D. melanogaster mitochondrial genome. The circular mtDNA of D. melanogaster varies in size among different populations largely due to length differences in the control region (Fauron & Wolstenholme, 1976; Fauron & Wolstenholme, 1980a, b); the mtDNA region we have sequenced, combined with those sequenced by others, yields a composite genome that is 19,517 bp in length as compared to 16,019 bp for the mtDNA of D. yakuba. D. melanogaster mtDNA exhibits an extreme bias in base composition; it comprises 82.2% deoxyadenylate and thymidylate residues as compared to 78.6% in D. yakuba mtDNA. All genes encoded in the mtDNA of both species are in identical locations and orientations. Nucleotide substitution analysis reveals that tRNA and rRNA genes evolve at less than half the rate of protein coding genes.     

The mitochondrial genome of the primary screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae)

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16,022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT-rich, reflected in the predominance of AT-rich codons in protein-coding genes. Non-optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.     

A 6-month interruption of antiretroviral therapy improves adipose tissue function in HIV-infected patients: the ANRS EP29 Lipostop Study

OBJECTIVE: To examine the reversibility of adipose tissue alterations in HIV-infected patients after a 6-month interruption of antiretroviral therapy (ART). METHODS: Forty HIV-infected patients on stable effective ART were enrolled, 33 of them completed the study. Patients underwent a physical examination, laboratory tests and needle biopsy of subcutaneous abdominal adipose tissue both at inclusion and at month 6. Changes in fat morphology, mitochondrial DNA (mtDNA) content and gene expression were examined in 29, 23 and 20 patients, respectively. RESULTS: Body fat distribution was not clearly modified at month 6. Adipose tissue inflammation improved markedly, with fewer infiltrating macrophages and fewer tumour necrosis factor alpha (TNFalpha)- and interleukin 6 (IL6)-expressing cells. Expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma) and of markers of mitochondrial function and biogenesis (cytochrome oxidase 2 and PPAR-gamma coreceptor 1alpha) improved after protease inhibitor (PI) withdrawal. In patients who stopped taking stavudine or zidovudine, the number of TNFalpha- and IL6-expressing cells was lower at month 6 than at month 0, and so was CD68 expression, a macrophage marker. Adipocyte mitochondrial status also improved, with lower mitochondrial density and cytochrome oxidase 4 mRNA levels, and higher mtDNA content. Sterol regulatory element binding protein 1 mRNA levels increased, reflecting better adipocyte differentiation. CONCLUSIONS: A 6-month ART interruption markedly improved adipose tissue functions, although fat distribution did not visibly change. Stavudine and zidovudine were associated with marked inflammation, which improved when these drugs were withdrawn; they also had a negative effect on differentiation and mitochondrial status. PIs were also associated with altered adipocyte differentiation and mitochondrial status. These data clearly show the detrimental effect of antiretroviral drugs, in particular thymidine analogues, on adipose tissue and argue for switch strategies sparing these drugs.     

Induction and properties of cytoplasmic factor(s) which enhance nuclear nonhistone protein phosphorylation in lymphocytes stimulated by anti-Ig

An increased in vitro phosphorylation of nonhistone nuclear proteins (NHP) was observed in the nuclei isolated from rabbit lymphocytes which had been stimulated with anti-Ig for 4 h. No concomitant increase of phosphorylation in histones or 0.14 M NaCl-soluble proteins was observed. The increase of in vitro phosphorylation of NHP was also observed in the nuclei isolated from nonstimulated cells when these nuclei were preincubated for 2 h with cell-free extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was maximally induced when lymphocytes were stimulated with anti-Ig for 2 h. The induction of an increased phosphorylation of NHP in nonstimulated nuclei with the cell-free extracts was not due to decrease of the adenosine triphosphate pool in the extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was not NHP-protein kinase itself, but it probably activated NHP-protein kinase in quiescent nuclei. The active substance was nondialyzable and probably protein. It was resistant against heating at 56 degrees C for 30 min, but the activity was completely destroyed by heating at 90 degrees C for 30 min. The active substance may be responsible for the transduction of the membrane-mediated signals given through Ig receptors to nuclei.     

Molecular mechanisms of mitochondrial diabetes (MIDD)

Mitochondria provide cells with most of the energy in the form of adenosine triphosphate (ATP). Mitochondria are complex organelles encoded both by nuclear and mtDNA. Only a few mitochondrial components are encoded by mtDNA, most of the mt-proteins are nuclear DNA encoded. Remarkably, the majority of the known mutations leading to a mitochondrial disease have been identified in mtDNA rather than in nuclear DNA. In general, the idea is that these pathogenic mutations in mtDNA affect energy supply leading to a disease state. Remarkably, different mtDNA mutations can associate with distinct disease states, a situation that is difficult to reconcile with the idea that a reduced ATP production is the sole pathogenic factor. This review deals with emerging insight into the mechanism by which the A3243G mutation in the mitochondrial tRNA (Leu, UUR) gene associates with diabetes as major clinical expression. A decrease in glucose-induced insulin secretion by pancreatic beta-cells and a premature aging of these cells seem to be the main process by which this mutation causes diabetes. The underlying mechanisms and variability in clinical presentation are discussed.     

Mitoepigenetics and drug addiction

Being the center of energy production in eukaryotic cells, mitochondria are also crucial for various cellular processes including intracellular Ca²⁺ signaling and generation of reactive oxygen species (ROS). Mitochondria contain their own circular DNA which encodes not only proteins, transfer RNA and ribosomal RNAs but also non-coding RNAs. The most recent line of evidence indicates the presence of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (mtDNA); thus, the level of gene expression – in a way similar to nuclear DNA – can be regulated by direct epigenetic modifications. Up to now, very little data shows the possibility of epigenetic regulation of mtDNA. Mitochondria and mtDNA are particularly important in the nervous system and may participate in the initiation of drug addiction. In fact, some addictive drugs enhance ROS production and generate oxidative stress that in turn alters mitochondrial and nuclear gene expression. This review summarizes recent findings on mitochondrial function, mtDNA copy number and epigenetics in drug addiction.     

Molecular mechanisms of mitochondrial diabetes (MIDD)

Mitochondria provide cells with most of the energy in the form of adenosine triphosphate (ATP). Mitochondria are complex organelles encoded both by nuclear and mtDNA. Only a few mitochondrial components are encoded by mtDNA, most of the mt‐proteins are nuclear DNA encoded. Remarkably, the majority of the known mutations leading to a mitochondrial disease have been identified in mtDNA rather than in nuclear DNA. In general, the idea is that these pathogenic mutations in mtDNA affect energy supply leading to a disease state. Remarkably, different mtDNA mutations can associate with distinct disease states, a situation that is difficult to reconcile with the idea that a reduced ATP production is the sole pathogenic factor. This review deals with emerging insight into the mechanism by which the A3243G mutation in the mitochondrial tRNA (Leu, UUR) gene associates with diabetes as major clinical expression. A decrease in glucose‐induced insulin secretion by pancreatic beta‐cells and a premature aging of these cells seem to be the main process by which this mutation causes diabetes. The underlying mechanisms and variability in clinical presentation are discussed.     

Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.     

Mitochondrial effects of antiretroviral therapies in asymptomatic patients

BACKGROUND: A decrease in the mitochondrial (mt) DNA to nuclear DNA ratio has gained acceptance as a marker of mitochondrial toxicity in treated HIV-infected patients, but the functional meaning of this alteration is unclear. METHODS: We assessed mtDNA content, mitochondrial content and function in peripheral blood mononuclear cells (PBMCs) of consecutive asymptomatic HIV-infected patients. Patients selected had been receiving a first-line highly active antiretroviral therapy (HAART) regimen for at least 6 months, consisting of zidovudine plus lamivudine or stavudine plus didanosine plus either nelfinavir or nevirapine, or were antiretroviral-naive. The mtDNA content was assessed by quantitative real-time PCR, mitochondrial content by citrate synthase activity, enzyme activity of complexes III and IV (both partially encoded by mtDNA) of the electron transport chain by spectrophotometry, oxygen consumption by polarography, and oxidative damage in cell membranes by monitoring cis-parinaric acid fluorescence. RESULTS: Mitochondrial content was significantly lower in all treated groups. Patients receiving stavudine plus didanosine had mtDNA depletion and a decrease in complex IV activity. However, oxygen consumption capacity and lipid peroxidation were unaffected in all groups. CONCLUSION: Long-term HAART may induce mitochondrial abnormalities in PBMC mitochondria, which do not necessarily translate into functional abnormalities, at least in asymptomatic patients.     

Simultaneous detection and quantification of mitochondrial DNA deletion(s), depletion, and over-replication in patients with mitochondrial disease

Heterogeneous clinical expression of mitochondrial DNA (mtDNA) disorders depends on both qualitative and quantitative changes in mtDNA. We developed a sensitive and effective method that simultaneously detects mtDNA deletion(s) and quantifies total mtDNA content. The percentage of deletions and mtDNA content of 19 patients with single or multiple deletions were analyzed by real-time quantitative polymerase chain reaction (real-time qPCR) using TaqMan probes specific for mtDNA (tRNA leu(UUR), ND4, ATPase8, and D-loop regions) and nuclear DNA (AIB1, beta-2-microglobulin, and beta-actin). The proportion of deletion mutants determined by real-time qPCR was consistent with that determined by Southern analysis. Most patients with mtDNA deletions also demonstrated compensatory mtDNA over-replication. Multiple mtDNA deletions that were not detectable by Southern analysis due to low percentage of each deletion molecule were readily detected and quantified by real-time qPCR. Furthermore, 12 patients with clinical features and abnormal biochemical/histopathological results consistent with mitochondrial respiratory chain disorders without identified mtDNA mutations had either substantially depleted or significantly over-replicated mtDNA content, supporting the diagnosis of mitochondrial disease. Our results demonstrate that both qualitative and quantitative analyses are important in molecular diagnosis of mitochondrial diseases. The presence of deletion(s) and mtDNA depletion or compensatory over-replication can be determined simultaneously by real-time qPCR.     

Evaluation of mitochondrial toxicity in Marmota himalayana treated with metacavir, a novel 2',3'-dideoxyguanosine prodrug for treatment of hepatitis B Virus

Metacavir (PNA) is a novel synthetic nucleoside analogue for the treatment of hepatitis B virus (HBV). Our recent studies showed that PNA, a prodrug of 2',3'-dideoxyguanosine (ddG), exhibited lower mitochondrial toxicity in long-term cultures of HepG2 cells. In the current study, we examined the long-term effects of PNA on mitochondrial toxicity in Marmota himalayana (Himalayan marmot). Himalayan marmots were treated daily with oral PNA (50 or 100 mg/kg), ziduvidine (AZT) (100 mg/kg), or water (control) for 90 days. PNA treatment did not alter the body weight or plasma lactate acid level. In livers from the animals treated with PNA at 100 mg/kg/day, histopathology showed mild steatosis or small focal liver cell necrosis. Electron microscopy also showed minor proliferation and partial mitochondrial swelling with crista reduction. Measurement of respiratory chain complex enzyme activity and mitochondrial DNA (mtDNA) content revealed no significant differences in skeletal muscle, liver, and kidney tissues between animals treated with PNA and controls. In contrast, in Himalayan marmots treated with AZT we observed delayed toxicity, including lactic acidosis, severe hepatic steatosis, obvious mitochondrial damage, and significant decreases in respiratory chain complex enzyme activity and mtDNA content. This is similar to the delayed toxicity syndrome observed previously in animals and humans. In summary, PNA treatment did not alter mitochondrial enzyme activity or mtDNA content. This suggests that PNA could pose a very low risk for adverse mitochondrion-related effects. However, long-term hepatotoxic effects of PNA were observed, and this indicates a need for continued monitoring of PNA-associated hepatotoxicity in clinical trials.