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PubMed1997

Quantification of wild-type mitochondrial DNA and its 4.8-kb deletion in rat organs.

Oxidative damage to mitochondrial DNA (mtDNA) is considered a major contributor in aging. An age-dependent increase of oxidative damage and of the quantity of partially deleted mtDNA was reported for several rat and human organs. Here, a systematic investigation of ten different tissues and organs of 20-months-old rats was performed. The amount of mtDNA and age-dependent 4.8 kb deletion (delta mtDNA4834) was determined by competitive polymerase chain reaction, along with the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx). The data were related to the corresponding metabolic rates. MtDNA content was highest in heart and lowest in spleen. delta mtDNA4834 was detected in all ten tissues and organs, and its amount was highest in liver and lowest in intestine. In heart, lung, muscle, and bone-marrow the deletion could not be quantified because of a point mutation, an A-->T transition at position 8107. Activities of SOD and GSHPx were highest in liver and lowest in intestinal mucosa. A negative correlation between mtDNA content and delta mtDNA4834, and a positive correlation between metabolic rate, GSHPx, and the deletion was found. These results suggest that the occurrence of delta mtDNA4834 in rat is related to oxidative stress.

Animals↗
PubMed2006

Mitochondrial abnormalities in patients with LHON-like optic neuropathies.

PURPOSE: To investigate certain biochemical and molecular characteristics of mitochondria in patients with Leber hereditary optic neuropathy (LHON)-like optic neuropathies. METHODS: Patients who had LHON-like optic neuropathies in both eyes were selected from neuro-ophthalmology clinics. Evaluation included clinical examination, neuroimaging, and assessment of several mitochondrial parameters in the blood, including sequencing the entire mitochondrial (mt)DNA coding region, measuring relative mtDNA content, studying mitochondrial respiratory function in some patients, and sequencing the OPA1 and OPA3 genes. RESULTS: Thirty-five patients (21 men and 14 women; average age at onset 19.0 +/- 8.7 years) met inclusion and exclusion criteria for LHON-like optic neuropathies with median visual acuity approximately 20/200. Other hereditary retinopathies and optic neuropathies were unlikely because of inclusion and exclusion criteria, because ERGs were normal, and because no patient had pathogenic sequence changes in the OPA1 or OPA3 genes. Compared with control subjects, these patients had more potentially pathogenic nonsynonymous mtDNA changes, greater relative mtDNA content (P < 0.001), and less mitochondrial respiratory activity (P < 0.001). Only six patients (17%) had primary LHON mutations; however, even the 29 patients without primary LHON mutations had significant evidence of mitochondrial abnormalities. Mitochondrial haplogroup distribution was similar in patients and control subjects. CONCLUSIONS: Primary LHON mutations are less common in patients with LHON-like optic neuropathy selected from a clinical setting than in patients with LHON from multigenerational families. The results suggest that mitochondrial dysfunction plays a role in this type of optic neuropathy whether or not primary LHON mutations are present. This information has implications for diagnostic testing and for future investigations into mechanisms of disease.

Adolescent↗
PubMed1993

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells.

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin are potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA.

Animals↗
PubMed2000

Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells.

Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H(2)O(2) at concentrations of 90-360 microM. After the treatment for 24-72 h, we found that cells were arrested at G(0) and G(1) phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H(2)O(2) treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.

Cell Cycle↗
PubMed2005

Site specific alterations of adipose tissue mitochondria in 3'-azido-3'-deoxythymidine (AZT)-treated rats: an early stage in lipodystrophy?

Although it is well accepted that treatment with nucleoside reverse transcriptase inhibitors (NRTIs) modifies fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examines the effects of chronic oral administration of 3'-azido-3'-deoxythymidine (AZT) on the mitochondrial metabolism and the redox status management of rat white adipose tissues originating from two anatomical sites, as well as of the rat liver. Results showed that AZT treatment induced differential effects on the mitochondrial functions depending on the anatomical localisation. Indeed, in inguinal adipose tissue, a significant decrease in the cytochrome c oxidase activity and in the mitochondrial DNA (mtDNA) content was observed, whereas the activity of citrate synthase, a mitochondrial protein exclusively encoded by the nucleus, was not affected. In contrast, no significant change in these parameters could be detected for epididymal tissue and for liver. In parallel, no oxidative stress could be detected after treatment, for both white adipose tissues and for liver, even though treated liver exhibited several modifications in redox management. Taken together, these data demonstrate differential mitochondrial effects of AZT on subcutaneous versus visceral white adipose tissue. Moreover, the decrease in mitochondrial oxidative capacity of inguinal adipocyte consecutive to AZT treatment is not primarily due to an oxidative stress per se, but rather to a depletion of the mtDNA content per cell.

Adipose Tissue↗
PubMed1996

Effect of beta-enantiomeric and racemic nucleoside analogues on mitochondrial functions in HepG2 cells. Implications for predicting drug hepatotoxicity.

A group of enantiomeric nucleoside analogues with beta-D or beta-L configuration, which represent potential candidates for the treatment of hepatitis B virus (HBV) infection, were incubated in human hepatoblastoma HepG2 cells at concentrations between 0.1 and 10 microM for 4-14 days. Then the effect on mitochondrial DNA (mtDNA) content, lactic acid production, lipid droplet formation, and mitochondrial morphology were evaluated. No effect on lactic acid production was detected in cells treated with beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-L-FTC), beta-D-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-D-FTC), racemic cis 2',3'-dideoxy-5-fluoro-3'thiacytidine [(+/-)-FTC], and 2,4-diamino-7-(2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2',3'-d]pyrimidine (T70178), whereas a slight increase was associated with beta-D-2-hydroxymethyl-5-(2,6-diaminopurin-9-yl)-1,3-dixolane++ + (beta-D-DAPD) and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimi dine -5-thiocarboxamide (T70182) at 10 microM. A concentration-dependent increase in lactic acid production was observed in cells exposed to beta-D-2',3'-dideoxy-3'-thiacytidine [(+)-BCH-189], racemic cis 2',3'-dideoxy-3'-thiacytidine [(+/-)-BCH-189], beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC), beta-L-2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), beta-D-2-hydroxymethyl-5-(5-fluorocytosin-I-yl)-1,3,-dioxolane (beta-D-FDOC), 2,4-diamino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2,3-d]pyrimidine (T70080), and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo [2,3-d]pyrimidine (T70179). Inhibition on mtDNA content was demonstrated to be concentration-dependent with (+)-BCH-189, beta-D-FddC, and T70080, whereas 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-D-DAPD, T70178, T70179, and T70182 had no effect. beta-D-FDOC resulted in a marked inhibition of mtDNA synthesis at 10 microM but not at lower concentrations. Cells treated with 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-D-DAPD, T70178, T70179, and T70182 did not show morphological changes compared with the control. In contrast, increased cytoplasmic lipid droplets associated with a loss of cristae in mitochondria were detected in cells treated with either beta-D-FDOC, beta-D-FddC, or T70080, (+)-BCH-189 treatment resulted in loss of cristae in mitochondria. In summary, 3TC, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-D-DAPD, T70178, and T70182 exhibited a relatively safe profile, supporting their further development.

Antiviral Agents↗
PubMed2004

Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions.

PURPOSE: Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions. EXPERIMENTAL DESIGN: DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and beta-actin genes. RESULTS: Mean CoxI/beta-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P = 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. CONCLUSIONS: Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.

Adult↗
PubMed2000

Vertebrate mitochondrial DNA-a circle of surprises.

Evidence for the existence of a vertebrate mitochondrial genome first arose over 30 years ago. Application of emerging techniques of molecular biology established the structure of vertebrate mitochondrial DNA (mtDNA) as a small closed-circular species. The ability to purify these mtDNAs to a high degree facilitated studies on the overall replication and expression pattern of the genome. With the acquisition of the genomic sequences of human and mouse mtDNAs, it was possible to infer the genetic organization and some of the genes contained therein, as well as providing a basis for developing strategies to assign important regulatory elements involved in mtDNA replication and transcription. This, in turn, presented the opportunity to identify nucleus-encoded proteins that target to mtDNA and, in doing so, determine the replication and expression modes of the genome. Vertebrate cells, in general, need mtDNA due to the requirements for maintaining a functional oxidative phosphorylation pathway. Depression of mtDNA content or mutations in mtDNA can result in metabolic dysfunction severe enough, in some cases, to result in human lethality. The emergence of mouse models for human mitochondrial diseases should provide the experimental context to understand the full role of mtDNA in different cells, tissues, and organs; the control of organelle biogenesis; and the development of therapeutic strategies for treatment of mitochondrial disorders.

Animals↗
PubMed2005

Effects of 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.

Adult↗
PubMed2005

Mitochondrial toxicity of nucleoside analogues in primary human lymphocytes.

OBJECTIVE: To evaluate if nucleoside analogue reverse transcriptase inhibitors (NRTIs) and polymerase-gamma inhibitors deplete mitochondrial DNA (mtDNA) in cultured primary lymphocytes and if such depletion might be associated with functional defects. METHODS: Primary peripheral blood CD4 and CD8 lymphocytes were purified from six healthy humans (three male and three female), stimulated mitotically (CD3/CD28) and cultured for 10 days in the presence or absence of NRTIs. Lymphocyte proliferation, mtDNA content, the expression of mtDNA-encoded cytochrome c-oxidase II (COXII) and lactate production were assessed. RESULTS: In CD4 lymphocytes, 10-day exposure to zalcitabine (1.77 microM), didanosine (118 microM) and stavudine (361 microM) induced a time-dependent decline of mtDNA. Compared with controls, residual mtDNA levels were 25%, 21% and 40%, respectively. COXII was reduced to 55%, 35% and 70% of control values. Lactic acid production was increased (by 214%, 294% and 175%, respectively). At day 10, lymphocyte counts were reduced (to 60%, 51%, and 41%, respectively). Zidovudine (71 microM) also reduced lymphocyte counts to 34% and increased lactic acid production by 170%, but did not induce mtDNA and COXII depletion. All these changes were highly significant. Lower NRTI concentrations (0.177 microM of zalcitabine, 11.8 microM of didanosine, 3.6 microM of stavudine and 7.1 microM of zidovudine) had effects at the border of significance. Similar observations were made in CD8 lymphocytes. CONCLUSIONS: In human lymphocytes, zalcitabine, didanosine and stavudine induce dose- and time-dependent mtDNA depletion, which is associated with decreased cell proliferation and increased lactate production. Zidovudine impairs lymphocyte division without inducing mtDNA depletion.

CD4-Positive T-Lymphocytes↗
PubMed2002

Bioenergetic remodeling of heart during treatment of spontaneously hypertensive rats with enalapril.

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in <10 days). Despite changes in LV mass, specific activities of bioenergetic (cytochrome-c oxidase, citrate synthase, lactate dehydrogenase) and reactive oxygen species (ROS) (total cellular superoxide dismutase) enzymes were actively maintained within relatively narrow ranges regardless of treatment duration, organismal age, or transmural region. Although enalapril led to parallel declines in mitochondrial enzyme content and ventricular mass, total ventricular mtDNA content was unaffected. Altered enzymatic content occurred without significant changes in relevant mRNA and protein levels. Transcript levels of gene products involved in mtDNA maintenance (Tfam), mitochondrial protein degradation (LON protease), fusion (fuzzy onion homolog), and fission (dynamin-like protein, synaptojanin-2alpha) were also unchanged. In contrast, enalapril-mediated ventricular and mitochondrial remodeling was accompanied by a twofold increase in specific activity of catalase, an indicator of oxidative stress, suggesting that rapid cardiac adaptation is accompanied by tight regulation of mitochondrial enzyme activities and increased ROS production.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Pgp-positive leukaemic cells have increased mtDNA but no increased rate of proliferation.

Cells of solid tumours tend to rely on glycolysis for energy. On the other hand, increased glycolysis in solid tumour cells expressing the multidrug resistance protein MDR-1 has been associated with increased malignancy in tumours. We have previously shown that cells of the MDR-1-positive CEM/VLB100 leukaemic cell line have increased mitochondrial electron transport chain (mtETC) activity compared with parental CEM cells. In the present study we used infrared (IR) spectroscopy to demonstrate that the mitochondrial DNA (mtDNA) content in the CEM/VLB100 cell line was significantly increased compared to that in the parental CEM cells. The increase in mtDNA was not accompanied by an increase in mitochondrial protein as both lipid and protein levels were decreased in CEM/VLB100 mitochondria. The ATP content was similar in these two cell lines. However, the ATP-dependent membrane efflux pump function in CEM/VLB100 cells was significantly reduced when mitochondrial ATP synthesis was inhibited by oligomycin, a specific inhibitor of mitochondrial F0F1-ATPase. Proliferation of CEM/VLB100 cells was significantly decreased compared to parental CEM cells, and was independent of p53 expression. Thus, we conclude that: (1) IR spectroscopy is a potential powerful technique for detecting mtDNA, protein and lipid contents simultaneously; (2) leukaemic cells mainly rely on mtDNA for energy; (3) increased expression of an ATP-dependent membrane efflux pump such as Pgp may up-regulate ATP generation and mtDNA content. These metabolic perturbations may exist merely to serve the efflux pump and do not result in an increase in leukaemic cell proliferation. In addition, the associated reduction in mitochondrial lipid and protein may contribute to sensitize the cells to cytochrome c release.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle.

To have a clearer picture of how mitochondrial damages are associated to aging, a comprehensive study of phenotypic and genotypic alterations was carried out, analyzing with histochemical and molecular biology techniques the same skeletal muscle specimens of a large number of healthy subjects from 13 to 92 years old. Histochemical data showed that ragged red fibers (RRF) appear at about 40 years of age and are mostly cytochrome c oxidase (COX)-positive, whereas they are almost all COX-negative thereafter. Molecular analyses showed that the 4977 bp deletion of mitochondrial DNA (mtDNA(4977)) and the 7436 bp deletion of mtDNA (mtDNA(7436)) are already present in individuals younger than 40 years of age, but their occurrence does not change with age. After 40 years of age the number of mtDNA deleted species, as revealed by Long Extension PCR (LX-PCR), increases, the 10422 bp deletion of mtDNA (mtDNA(10422)) appears, although with a very low frequency of occurrence, and mtDNA content is more than doubled. Furthermore, mtDNA(4977) level directly correlates with that of COX-negative fibers in the same analyzed subjects. These data clearly show that, after 40 years of age, the phenotypic and genotypic mitochondrial alterations here studied appear in human skeletal muscle and that they are closely related.

Adolescent↗
PubMed2006

The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription.

DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT-PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (-)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.

Animals↗
PubMed1998

Creation and characterization of a mitochondrial DNA-depleted pancreatic beta-cell line: impaired insulin secretion induced by glucose, leucine, and sulfonylureas.

It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.

Adenosine Triphosphate↗
PubMed2004

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.

Antimetabolites, Antineoplastic↗
PubMed1994

Effects of 2',3'-dideoxynucleosides on proliferation and differentiation of human pluripotent progenitors in liquid culture and their effects on mitochondrial DNA synthesis.

2',3'-Dideoxynucleosides (ddNs) including 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-3'-deoxythymidine (FLT), 3'-amino-3'-deoxythymidine (AMT), 2',3'-dideoxycytidine (ddC), and 2',3'-didehydro-3'-deoxythymidine (D4T) were tested for their effects on proliferation and differentiation of pluripotent progenitor cells (CD34+) purified from human bone marrow cells grown in liquid cultures. These highly purified progenitor cells undergo extensive proliferation during 14 days, with a marked differentiation during the last 7 days. These differentiated cells exhibit normal morphological features in response to specific hematopoietic growth factors of both erythroid and granulocyte-macrophage lineages, as demonstrated by flow cytometry cell phenotyping. The potencies of these ddNs in inhibiting proliferation of granulocyte-macrophage lineage cells were in the order FLT > AMT = ddC > AZT >> D4T, and the potencies in inhibiting proliferation of erythroid lineage cultures were in the order FLT > AMT > AZT > ddC >> D4T. The toxic effects of ddNs assessed in these liquid cultures were in agreement with data obtained by using semisolid cultures, demonstrating the consistency of these two in vitro hematopoietic systems toward ddN toxicity. ddC was toxic to CD34+ progenitor cells and/or cells in the early stages of differentiation, whereas the inhibitory effect of AZT on the erythroid lineage was predominantly observed on a more mature population of erythroid progenitors during the differentiation process. Slot blot analysis of granulocyte-macrophage cultures demonstrated that exposure to ddC and FLT was associated with a decrease in total mitochondrial DNA (mtDNA) content, suggesting that these two ddNs inhibit mtDNA synthesis. In contrast, no difference in the ratio of nuclear DNA to mtDNA was observed in cells exposed to toxic concentrations of AZT and AMT is not associated with an inhibition of mtDNA synthesis. This human pluripotent progenitor liquid culture system should permit detailed investigations of the cellular and molecular events involved in ddN-induced hematological toxicity.

Antibody Specificity↗
PubMed1995

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.

Adenosine Triphosphate↗