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PubMed2001

Relationship between various surrogate indices of insulin resistance and mitochondrial DNA content in the peripheral blood of 18 healthy volunteers.

Mutations or deletions of mitochondrial DNA (mtDNA) are associated with diabetes mellitus. In this study, we investigated the relationships between the mtDNA content in peripheral blood and surrogate indices of insulin resistance in 18 healthy young women (mean age 20.8 +/- 1.5 years). The mtDNA content was significantly correlated with the area under the curve of insulin during an oral glucose tolerance test (r = -0.622), the homeostasis model assessment for insulin resistance (r = -0.616), the ratio of fasting glucose to insulin concentration (r = 0.586) and the fasting insulin level (r = -0.552). Further study is warranted to elucidate the mechanism by which the mtDNA content is associated with insulin resistance.

Journal Article↗
PubMed1999

Biochemical and molecular consequences of ethidium bromide treatment on Drosophila cells.

KC167 Drosophila cells were incubated with low concentrations of ethidium bromide (200 ng/ml), causing changes in mitochondrial DNA (mtDNA) content (2-184% of that of controls). SSCP (single strand conformational polymorphism) analysis of mtDNA indicated that the incubation with ethidium bromide also generated mutations. Compared with controls, there were marked reductions in the activities of respiratory complexes III and IV measured in these cells, and in respiration and ATP synthesis capacities measured in isolated mitochondria. These reductions matched that in mtDNA content. In contrast, no link could be demonstrated between mtDNA content and steady-state concentrations of the transcripts of genes COIII and Cyt b.

Adenosine Triphosphate↗
PubMed2003

Evaluation of mitochondrial DNA content and enzyme levels in tenofovir DF-treated rats, rhesus monkeys and woodchucks.

The antiviral compound tenofovir DF (Gilead Sciences) was evaluated for possible mitochondrial toxicity in rats, rhesus monkeys and woodchucks. Animals were treated by oral gavage with tenofovir DF, and the levels of mitochondrial enzymes cytochrome c oxidase and citrate synthase were assayed. In rats (6/group) treated daily for 28 days with 300 mg/kg tenofovir DF the enzyme levels were unchanged versus control in liver, kidney, and skeletal muscle. In a parallel study, rats (6/group) were treated with 40 mg/kg of the antiviral adefovir dipivoxil (Gilead Sciences) and enzyme levels were also unchanged versus control. In rhesus monkeys (6/group) treated daily with 30 mg/kg or 250 mg/kg tenofovir DF for 56 days, and in woodchucks (6/group) treated daily with 15 mg/kg or 50mg/kg tenofovir DF for 90 days, the enzyme levels were unchanged in liver, kidney, skeletal muscle and cardiac muscle. Mitochondrial DNA (mtDNA) content was determined in tissue from treated versus control animals by utilizing a quantitative real-time PCR (QPCR) technique, where the relative ratios of mitochondrial cytochrome b gene to the genomic actin gene were measured. The relative mtDNA content from rats, rhesus monkeys and woodchucks were unchanged in the various treatment groups. Variations in mtDNA content between animals in the same treatment group were noted. The actual species-dependent mitochondria/genomic ratios were estimated from the QPCR assay. In summary, treatment with tenofovir DF, or with adefovir dipivoxil, did not affect mtDNA content or level of mitochondrial enzymes, and no liver, muscle or renal microscopic abnormalities were observed in tenofovir-treated animals.

Adenine↗
PubMed2005

Stability and association with the cytomatrix of mitochondrial DNA in spontaneously immortalized mouse embryo fibroblasts containing or lacking the intermediate filament protein vimentin.

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer.

BACKGROUND: The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. METHODS: The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma) of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE), followed by direct DNA sequencing to identify the mutations. RESULTS: Fourteen somatic mtDNA mutations were identified in 55% (11/20) of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64%) were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. CONCLUSION: Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations.

Adenocarcinoma↗
PubMed2005

Ethanol feeding enhances age-related deterioration of the rat hepatic mitochondrion.

Chronic ethanol feeding damages the hepatic mitochondrion by increasing mitochondrial DNA (mtDNA) oxidation, lowering mtDNA yields and impairing mitochondrial respiration. These effects are also seen during aging. By employing a 21-day chronic feeding regimen, we investigated the effects of ethanol consumption on mtDNA content and mitochondrial respiration in 2-, 12-, and 24-mo-old male rats. Aging resulted in decreased mtDNA content, increased mtDNA damage (as indicated by inhibition of Taq polymerase progression), and a decline in state 3 respiration; effects that were further exacerbated by ethanol feeding. Additionally, ethanol consumption caused an increase in the levels of citrate synthase while not impacting mitochondrial protein content. In conclusion, ethanol and aging combine to cause deterioration in the structural and functional integrity of the hepatic mitochondrion. The additive effects of aging and ethanol feeding may have serious consequences for hepatic energy metabolism in aged animals, and their detrimental combination may serve as one of the molecular mechanisms underlying the progression of alcoholic liver disease.

Aging↗
PubMed1997

Insensitivity of cultured rat cortical neurons to mitochondrial DNA synthesis inhibitors: evidence for a slow turnover of mitochondrial DNA.

Mitochondrial dysfunction is a major contributor to aging and neurodegeneration. Defects in mitochondrial DNA (mtDNA) have been identified in several neuromuscular diseases. Even though there is a high rate of phenotypic expression of mtDNA mutations in the central nervous system and replication of DNA introduces errors, little is known about the replicative activity of mtDNA in the brain. In this study, we investigated the sensitivity of cultured rat cortical neurons to mtDNA synthesis inhibitors as a means to assess the turnover rate of mtDNA. Four-day treatment with dideoxycytidine (ddC) (0.2 microM) or ethidium bromide (EtB) (0.25 microg/mL) reduced the mtDNA content approximately 80% in the human lymphoblastoid cell line, CEM. Concentrations of ddC ranging from 0.2 to 10 microM did not reduce mtDNA content in primary cultures of rat cortical neurons. Similarly, treatment with EtB (0.1, 0.25, and 0.5 microg/mL) did not affect significantly neuronal mtDNA. EtB (0.25 microg/mL) was effective in reducing mtDNA content in the undifferentiated embryonic carcinoma cell line, P 19. However, once P 19 cells were differentiated into a neuronal phenotype, they became insensitive to inhibition of mtDNA synthesis by EtB. Thus, cultured rat cortical neurons were less sensitive to mtDNA synthesis inhibitors than cell lines, suggesting that the turnover of mtDNA in central neurons is very slow. This may protect central neurons from accumulating mutations during the replication of mtDNA.

Animals↗
PubMed2005

The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: a quantitative study using laser-captured microdissected tissues.

OBJECTIVE: Microsatellite instability (MSI) is a frequent genetic event in the D-loop region (which controls mitochondrial DNA (mtDNA) replication) of mitochondrial genome of endometrial cancer. We therefore investigated the relationship between mtMSI and mtDNA content in endometrial cancer. METHODS: Tumor tissues from 65 cancer patients and normal tissues from 41 non-cancer patients were used in this study. Pure endometrial adenocarcinoma cells and normal endometrial glandular epithelial cells were collected by laser capture microdissection, and analyzed for levels of mtDNA copy number by real-time quantitative PCR. RESULTS: Our data show that mtDNA copy number was not related with age in both endometrial cancer and normal endometrium cells. Great inter-individual variations in mtDNA copy number in endometrial cancer group were found; and mtDNA content was significantly larger than that in normal endometrium group. About 2-fold increase of mtDNA copy number was found in endometrial adenocarcinoma compared with normal endometrial glandular epithelium (P = 0.001). In particular, the analysis also shows that the copy number of mtDNA in the cases that carried the mtMSI at nucleotide position 303 was significantly higher than that of the negative cases (P = 0.048). CONCLUSIONS: Our data indicate that mtDNA copy number increased during endometrial cancer development. There is also a correlation between the mtDNA instability and mtDNA content in endometrial cancer cells. Role of mitochondrial genome changes in carcinogenesis warrants further investigation.

Adenocarcinoma↗
PubMed2005

Age-related changes of mitochondrial DNA content and mitochondrial genotypic and phenotypic alterations in rat hind-limb skeletal muscles.

Mitochondrial DNA (mtDNA) content relative to nuclear DNA content as well as mitochondrial transcription factor A (TFAM) content was measured in four hind-limb skeletal muscles, namely soleus (S), tibialis anterior (TA), gastrocnemius (G), and extensor digitorum longus (EDL) of adult rats. Content of mtDNA in 6-month-old rats is in the rank order of S > TA > G > EDL, and TFAM content is higher in S than in the other studied muscles. After the rat is 6 months of age, the mtDNA content decreases only in S and TA, whereas the TFAM content increases only in S. Deletions in mtDNA appear quite early in life in S and later on in the other muscles. Fibers defective for mitochondrial respiratory enzymes appear in rats at 15 months of age. In the oldest animals, the highest frequencies of occurrence of mtDNA deletions as well as of mitochondrial phenotypic alterations are found in S according to its highest mtDNA content and oxidative potential.

Aging↗
PubMed2003

Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblasts.

Deoxyguanosine kinase is a constitutively expressed, mitochondrial enzyme of the deoxyribonucleoside salvage pathway. Deficiency of deoxyguanosine kinase causes early-onset, hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome. To clarify the molecular mechanism of the disease, a skin fibroblast culture was studied from a patient carrying a homozygous nonsense mutation in the gene for deoxyguanosine kinase. In situ examination of DNA synthesis demonstrated that, although mtDNA synthesis is cell cycle independent in control fibroblasts, mtDNA synthesis occurs mainly during the S-phase in deoxyguanosine kinase-deficient cells. Consistent with this observation, it was found that the mtDNA content of exponentially growing, deoxyguanosine kinase-deficient cells is only mildly affected. When cycling is inhibited by serum-deprivation and cells are in a resting state, however, the mtDNA content drops considerably in deoxyguanosine kinase-deficient cells, yet remains stable in control fibroblasts. The decline in mtDNA content in resting, deoxyguanosine kinase-deficient cells can be prevented by dGMP and dAMP supplementation, providing conclusive evidence that substrate limitation triggers mtDNA depletion in deoxyguanosine kinase-deficient cells.

Cell Cycle↗
PubMed2005

13C-methionine breath test detects distinct hepatic mitochondrial dysfunction in HIV-infected patients with normal serum lactate.

OBJECTIVE: To assess mitochondrial respiratory chain dysfunction in different treatment groups of HIV-infected patients with normal serum lactate by measuring hepatic mitochondrial decarboxylation capacity by the C-methionine breath test (MeBT) and to correlate MeBT results with mitochondrial DNA (mtDNA) content in peripheral blood mononuclear cells (PBMCs). METHODS: Four groups were studied: HIV-negative controls (n = 10), treatment-naive patients (n = 15), antiretroviral therapy (ART)-treated patients with asymptomatic disease (n = 15), and patients with long-term treatment and clinical evidence of lipoatrophy (n = 15). After oral administration of C-methionine, CO2 exhalation was determined by infrared spectroscopy. MtDNA content in PBMCs was assessed by real-time polymerase chain reaction quantification. RESULTS: CO2 exhalation in lipoatrophic patients and therapy-naive patients was distinctly decreased when compared with that in healthy controls and asymptomatic patients (P < 0.001). The functional mitochondrial impairment in lipoatrophic patients was associated with a 47% decline in mtDNA content. MeBT results and mtDNA were significantly correlated in ART-treated patients (r = 0.77, P < 0.0001). CONCLUSIONS: MeBT is a simple noninvasive method to detect mitochondrial dysfunction in HIV-infected patients that correlates with mtDNA depletion in PBMCs of ART-treated individuals. Decreased hepatic methionine metabolism in therapy-naive patients may reflect the functional relevance of viral-mediated mitochondrial toxicity.

Adult↗
PubMed2005

Mechanisms of HIV and nucleoside reverse transcriptase inhibitor injury to mitochondria.

Available evidence suggests that a number of important clinical events in individuals with HIV infection are related to mt dysfunction. Several factors may contribute to the development of these events and the tissue(s) in which the event occurs. Some individuals are likely to have important genetic predispositions for mt disease, which may be unmasked by the presence of HIV infection or the introduction of NRTI antiretrovirals. HIV infection per se is associated with reduction in mtDNA content and changes in mt morphology and function, which in some cases leads to clinical events such as myopathy or peripheral neuropathy. NRTI antiretrovirals may impact mtDNA content and function through a number of different mechanisms and have been demonstrated to be causative of a number of clinical toxicities. In in vitro and in clinical studies, newer nucleoside and nucleotides agents such as lamivudine, emtricitabine, abacavir and tenofovir appear to be much weaker inhibitors of mtDNA polymerase-gamma or other mt functions, and appear to be associated with a lower risk of events thought to be related to mt toxicity. Simple, non-invasive tests for mt function are not available at present in the clinical routine, and assays of mtDNA content in blood cells may miss key aspects of mt function, require careful sample handling and may not reflect events occurring in other tissues. There remains a need for the development of rapid, cheap and clinically applicable assays that would enable the prediction of increased likelihood of mt events.

Anti-HIV Agents↗
PubMed2005

Hearing loss in mitochondrial disorders.

Hearing loss is a common clinical feature in mitochondria-syndrome disorders. The underlining molecular etiology of hearing loss has not been fully investigated. In this study, 83 patients with mitochondrial syndromic hearing loss were evaluated clinically and their blood and tissue samples were examined molecularly. Using modified Walker's criteria, 31, 31, 14, and 7 patients had been classified as having definite, probable, possible, and unlikely diagnosis of mitochondrial disease, respectively. Deleterious mtDNA point mutations and/or abnormal mtDNA content or multiple deletions were identified in 20 patients with definite diagnosis and 2 patients with probable diagnosis. In addition to known, undisputed pathogenic mutations, several novel mutations believed to be clinically significant were found. Furthermore, abnormal mtDNA content and mtDNA deletions were found in some of the cases. Evaluation of clinical and diagnostic features associated with hearing loss revealed that cardiomyopathy, lactic acidosis, deficient respiratory chain enzyme complex activities, histochemical and ultrastructural abnormalities in mitochondria, and abnormal brain imaging results occurred significantly more frequently in patients with mtDNA alterations than in those without. This study revealed that the majority of the mtDNA defects in patients with mitochondrial syndromic hearing loss affect the overall mitochondrial gene expression.

DNA, Mitochondrial↗
PubMed2005

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors.

BACKGROUND: Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1), and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1), and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA). RESULTS: Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. CONCLUSION: Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

Animals↗
PubMed2003

Mitochondrial DNA content of human spermatozoa.

Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

Case-Control Studies↗
PubMed1995

Mitochondrial deoxyribonucleic acid content is specifically decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of noninsulin-dependent diabetes.

Considerable interest has recently been focused on the putative role of mutations in the mitochondrial genome for the development of noninsulin-dependent diabetes. The Goto-Kakizaki (GK) rat, a genetic model of defective insulin secretion and hyperglycemia, is characterized by partial maternal inheritance. Because the mitochondrial genome is known to be maternally transmitted, the aim of this study was to investigate whether the GK syndrome can be explained in terms of alterations of the mitochondrial DNA (mtDNA). For this purpose, pancreatic islets were isolated from adult and fetal control Wistar and diabetic GK rats. Using electron microscopy, the ultrastructural morphology of beta-cell mitochondria was analyzed in control and GK islets. It was found that the beta-cells of adult GK rats had a significantly smaller mitochondrial volume and an increased number of mitochondria per unit tissue volume as compared with the beta-cells of corresponding control islets. Moreover, mtDNA and mtRNA were isolated from the islets and, as a control tissue, from liver, and subsequently analyzed using Southern and Northern blot techniques. No major deletions or restriction fragment polymorphism could be detected in mtDNA from both GK liver and GK islets. The mtDNA sequence of the transfer RNAleu(UUS) gene was identical in both strains of rats. mtDNA contents of fetal GK islets and fetal GK liver were not different from those of fetal Wistar rats. However, adult GK islets contained markedly less mtDNA than the corresponding control islets, contrary to the mtDNA contents of adult liver, which were similar in the two strains. The lower islet mtDNA contents were paralleled by a decreased content of islet mtRNA (12S ribosomal RNA and cytochrome b messenger RNA). Islet insulin messenger RNA contents were similar in GK and Wistar rats. In conclusion, our results do not support a role of a genetic defect in mtDNA as a cause of the GK syndrome. Instead, mtDNA damage may occur specifically in islet cells as a consequence of the disturbed metabolic environment of the adult GK rat. It is speculated that a long-lasting metabolic dysfunction may induce mtDNA damage and/or inhibition of mtDNA replication leading to a gradual and late decrease in the mitochondrial volume fraction and subsequently an impaired capacity for oxidative metabolism.

Animals↗
PubMed2005

In vivo effects of highly active antiretroviral therapies containing the protease inhibitor nelfinavir on mitochondrially driven apoptosis.

BACKGROUND: In vitro studies have reported controversial effects of protease inhibitors (PIs) on mitochondrially driven apoptosis. Additionally, since PIs in the clinical setting are almost always given in combination with nucleoside analogues, which may have negative effects on mitochondrial DNA (mtDNA), the impact of PI-containing highly active antiretroviral therapy (HAART) on apoptosis and mtDNA content is unclear. PATIENTS AND METHODS: A cross-sectional study was performed including 20 HIV-negative (HIV-) patients, 16 HIV-positive, antiretroviral-naive (HIV+) patients and 17 HIV-positive patients receiving the PI nelfinavir (NFV) plus zidovudine and lamivudine (AZT+3TC) or didanosine and stavudine (ddl+d4T)--collectively known as HIV+PI--as first-line antiretroviral treatment for at least 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated. BCL2 expression (anti-apoptotic) and the levels of the cleaved, active form of caspase-9 (pro-apoptotic) were determined by western blot. An index of mitochondrially driven apoptotic activation was estimated calculating the ratio caspase-9:BCL2. Mitochorldrial DNA content was measured by real-time PCR. RESULTS: BCL2 expression was lower in HIV+ than in HIV-patients (P < 0.01), whereas levels of caspase-9 were higher (P = 0.001). The caspase-9:BCL2 ratio was significantly increased in HIV+ compared with HIV-individuals (P < 0.001). Mitochondrial DNA content was also decreased in HIV+ compared with HIV-patients (P < 0.001). The HIV+PI group exhibited a trend to normalization for BCL2 expression and caspase-9 compared with the HIV+ group, whereas the caspase-9:BCL2 ratio significantly improved (decreased, P < 0.05 compared with HIV+ group). The mtDNA content in the HIV+PI group was similar to that of the HIV+ group, although the results of mtDNA content differed depending on whether NFV was combined with AZT+3TC (preserved) or with ddl+d4T (depleted). Conversely, no differences were found in apoptotic markers between the two subgroups of HIV+PI. CONCLUSIONS: NFV-based PI-containing HAART regimens may exert some beneficial effects counteracting the increased mitochondrially driven apoptosis present in HIV-infected people.

Adult↗
PubMed1997

Effect of nucleoside analogs on neurite regeneration and mitochondrial DNA synthesis in PC-12 cells.

The effects of several anti-human immunodeficiency virus nucleoside analogs were examined on neurite regeneration and mitochondrial DNA (mtDNA) synthesis in nerve growth factor-primed PC-12 cells. Under pharmacologically relevant concentrations, the exposure of cells to 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC) and 2',3'-didehydro-3'-deoxythymidine (d4T) led to a marked dose-dependent inhibition of neurite regeneration with a 50% inhibitory concentration approximating 1, 5 and 15 microM, respectively. In contrast, 3'-azido-3'-deoxythymidine (AZT) and beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) had no effect on neurite regeneration. Inhibition of mtDNA synthesis by ddI was dose dependent, and ddC at a concentration of 10 microM strongly reduced mtDNA content by >75%. However, no inhibition of mtDNA synthesis was detected in cells exposed to 10 microM 3TC or d4T and to 25 microM AZT, suggesting a lack of definite correlation between mtDNA depletion and blockage of neurite regeneration. High performance liquid chromatographic analysis demonstrated that AZT, ddC, 3TC and d4T were anabolized to their respective monophosphate, diphosphate and triphosphate derivatives in the PC-12 cells. In addition, d4T was phosphorylated to form its monophosphate, diphosphate and triphosphate derivatives in isolated mitochondria, whereas ddC was metabolized only to its monophosphate form and no phosphorylated metabolites of 3TC were detected under the same conditions. In summary, the peripheral neuropathy induced by ddC and ddI in patients with acquired immune deficiency syndrome may be accounted for by the depletion of mtDNA content in the neurons. As for d4T, some other mechanism(s) may be involved in its clinical neurotoxicity. Both AZT and 3TC lacked any substantial toxicity in our in vitro model, which is in agreement with the clinical action of these drugs.

Animals↗