A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line.

Ethidium bromide (EtBr) is widely used to deplete mitochondrial DNA (mtDNA) and produce mitochondrial DNA-less cell lines. However, it frequently fails to deplete mtDNA in mouse cells. In this study we show by using a highly sensitive real-time PCR, that low doses of EtBr (10 microM) did lead to a three-fold increase of the total amount of mitochondrial DNA in a human neuronal cell line (Ntera 2). A higher dose of EtBr (25 microM) led to the expected decrease of mtDNA until day 22 when the cells almost died. Cell growth and mtDNA content could be restored after additional 22 days of non-EtBr treatment. The highest concentration of 50 microM also led to a significant increase of mtDNA. The cells died when they had only about 10% of mtDNA left, indicating a mtDNA threshold for cell survival. Additionally, the so-called common 4977 bp deletion could be induced by prolonged exposure to ethidium bromide. Whereas the higher doses led to significant higher amounts of deleted mtDNA.

Quantification of human mitochondrial DNA in a real time PCR.

Recently, a moderately priced machine for real-time quantitative PCR has become available, the Perkin Elmer 5700. The rapid and quantitative assay of mitochondrial DNA (mtDNA) copy number is potentially useful in a variety of molecular, evolutionary and forensic fields. Using this new tool, we have evaluated the precision and reliability of the real time PCR to quantify undeleted mitochondrial genome copy number, and to determine the frequency of an age-associated deletion of 4977 base pairs in length, in 42 human iliopsoas muscle DNA samples from persons of known age. We have evaluated the accuracy with which age can be predicted, knowing only the frequency of this common 4977 bp deletion, and derived a statistical formula which describes the confidence with which the 4977 bp frequency predicts age. The results indicate that the mutation frequency could be used to distinguish between tissue from young and old individuals. However in this data set, while there was considerable agreement of 4977 bp frequency among replicates from the same individual sample, there was substantial diversity of mean mutation frequency between individuals of the same or similar ages. The simplest interpretation of these results is that there are biological modifiers of 4977 bp frequency that are age-independent, which are potentially interesting but may limit the usefulness of this deletion frequency alone as a "molecular forensic clock."

Frataxin expression rescues mitochondrial dysfunctions in FRDA cells.

Friedreich's ataxia (FRDA) is the result of mutations in the nuclear-encoded frataxin gene, which is expressed in mitochondria. Several lines of evidence have suggested that frataxin is involved in mitochondrial iron homeostasis. We have transfected the frataxin gene into lymphoblasts of FRDA compound heterozygotes (FRDA-CH) with deficient frataxin expression to produce FRDA-CH-t cells in which message and protein are rescued to near-physiological levels. FRDA-CH cells were more sensitive to oxidative stress by challenge with free iron, hydrogen peroxide and the combination, consistent with a Fenton chemical mechanism of pathophysiology, and this sensitivity was rescued to control levels in FRDA-CH-t cells. Iron challenge caused increased mitochondrial iron levels in FRDA-CH cells, and a decreased mitochondrial membrane potential (MMP), both of which were rescued in FRDA-CH-t cells. The rescue of the low MMP, and high mitochondrial iron concentration by frataxin overexpression suggests that these cellular phenotypes are relevant to the central pathophysiological process in FRDA which is aggravated by exposure to free iron. However, even at physiological iron concentrations, FRDA-CH cells had decreased MMP as well as lower activities of aconitase and ICDH (two enzymes supporting MMP), and twice the level of filtrable mitochondrial iron (but no increase in total mitochondrial iron), and the observed phenotypes were either fully or partially rescued in FRDA-CH-t cells. Free iron is known to be toxic. The observation that frataxin deficiency (either directly or indirectly) causes an increase in filtrable mitochondrial iron provides a new hypothesis for the mechanism of cell death in this disease, and could be a target for therapy.

Cellular characterization of leukotoxin diol-induced mitochondrial dysfunction.

Leukotoxin, a cytochrome P450-derived epoxide of linoleic acid, has been implicated as a causative factor in acute respiratory distress syndrome. Conversion of this fatty acid epoxide to leukotoxin diol by epoxide hydrolase has been hypothesized as the critical activation step in leukotoxin-induced cellular toxicity. In both human and insect cells, we observed that leukotoxin diol causes acute cellular toxicity and that cyclosporin A, an inhibitor of the mitochondrial permeability transition, ameliorates leukotoxin diol-associated toxicity. To evaluate mitochondria as a target of leukotoxin diol, multiple aspects of mitochondrial integrity were evaluated in both cell- and organelle-based assays. Leukotoxin diol specifically activated the mitochondrial permeability transition, resulting in release of cytochrome c and subsequent cell death. Pretreatment with cyclosporin A inhibited these effects and, furthermore, limited in vivo toxicity. While the mechanisms underlying leukotoxin-mediated toxicity remain to be fully elucidated, the observation that leukotoxin diol disrupts mitochondrial function specifically through activation of the mitochondrial permeability transition suggests at least one mechanism through which leukotoxin diol may exert its activity in physiological contexts.

Genetic basis for susceptibility to noise-induced hearing loss in mice.

The C57BL/6J (B6) and DBA/2J (D2) inbred strains of mice exhibit an age-related hearing loss (AHL) due to a recessive gene (Ahl) that maps to Chromosome 10. The Ahl gene is also implicated in the susceptibility to noise-induced hearing loss (NIHL). The B6 mice (Ahl/Ahl) are more susceptible to NIHL than the CBA/CaJ (CB) mice (+(Ahl)). The B6xD2.F(1) hybrid mice (Ahl/Ahl) are more susceptible to NIHL than the CBxB6.F(1) mice (+/Ahl) [Erway et al., 1996. Hear. Res. 93, 181-187]. These genetic effects implicate the Ahl gene as contributing to NIHL susceptibility. The present study demonstrates segregation for the putative Ahl gene and mapping of such a gene to Chromosome 10, consistent with other independent mapping of Ahl for AHL in 10 strains of mice [Johnson et al., 2000. Genomics 70, 171-180]. The present study was based on a conventional cross between two inbred strains, CBxB6.F(1) backcrossed to B6 with segregation for the putative +/Ahl:Ahl/Ahl. These backcross progeny were exposed to 110 dB SPL noise for 8 h. All of the progeny were tested for auditory evoked brainstem responses and analyzed for any significant permanent threshold shift of NIHL. Cluster analyses were used to distinguish the two putative genotypes, the least affected with NIHL (+/Ahl) and most affected with PTS (Ahl/Ahl). Approximately 1/2 of the backcross progeny exhibited PTS, particularly at 16 kHz. These mice were genotyped for two D10Mit markers. Quantitative trait loci analyses (log of the odds=15) indicated association of the genetic factor within a few centiMorgan of the best evidence for Ahl [Johnson et al., 2000. Genomics 70, 171-180]. All of the available evidence supports a role for the Ahl gene in both AHL and NIHL among these strains of mice.

Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria.

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca(2+)), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed.

Sensitivity of FRDA lymphoblasts to salts of transition metal ions.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease resulting from decreased expression of the nuclear-encoded mitochondrial protein, frataxin. FRDA patients have characteristic iron deposits and dysfunction of mitochondrial enzymes in the heart. Inactivation of the frataxin homologue in yeast causes dysregulation of both mitochondrial iron levels and iron export. Previously, we have observed sensitivity of FRDA fibroblasts to FeCl3 and hydrogen peroxide, results consistent with the hypothesis that FRDA cells may experience increased Fenton chemistry. To determine whether the sensitivity of FRDA cells to transition metal ions is a general or specific property, we have compared the sensitivity of lymphoblasts from FRDA patients and healthy controls to the transition metal salts CoCl2, CuSO4 FeCl3 FeSO4, MnCl2, and ZnCl2. FRDA lymphoblasts were significantly more sensitive to FeCl3 and MnCl2 than control cells. However, there were no significant differences observed in sensitivity to CoCl2, CuSO4, FeSO4 and ZnCl2 in the concentration ranges studied. Thus, the sensitivity of FRDA lymphoblasts exposed to transition metals appears to be specific, and could be relevant to the pathophysiological mechanism, which is discussed.

Mitochondrial defects and hearing loss.

The techniques of human molecular genetics have been rapidly applied to the study of hearing loss. These studies have implicated more than 60 loci as causes of nonsyndromic hearing loss. Mutations at more than a dozen nuclear genes have been demonstrated to cause hearing loss, and these have been covered in recent reviews. However, a perhaps unexpected feature of the molecular characterization of human hearing loss has been the occurrence of mutations in the mitochondrial DNA (mtDNA). The importance of mitochondrial function in hearing is emphasized by the recent discovery of mutations in a nuclear-encoded mitochondrial protein which results in hearing loss. This article reviews the current status of our knowledge of mtDNA mutations that have been shown to cause hearing loss, and the suggestion of potential molecular, cellular and tissue-specific pathophysiological mechanisms by which dysfunction of mitochondria may lead to a loss of hearing.

Frequencies of hprt(-) mutations and bcl-2 translocations in circulating human lymphocytes are correlated with United Kingdom sunlight records.

Between 1983 and 1995 we have monitored human populations for evidence of exposure to environmental mutagens, taking blood samples to measure hprt(-) mutant frequency in T cells and more recently bcl-2 t(14:18) translocation frequency in B cells. We have now analysed data from 785 assays on 448 blood samples from 308 normal subjects and find that there is a highly significant statistical correlation between hprt(-) mutant frequency and the sunlight record for the 3 weeks prior to taking the blood sample. We discuss the weaknesses in retrospective studies of this nature and the possibility of spurious epidemiological correlations that may result. More controlled experiments can be envisaged that would give a firmer basis to the statistical associations observed. hprt(-) mutations in T cells show little evidence of a UV fingerprint, so that the correlation may be due to immunomodulation rather than mutation. We also find a correlation between the sunlight record and bcl-2 translocation. This translocation is found at a low frequency in the B cells of many normal subjects and is the commonest translocation observed in non-Hodgkin's lymphoma. Our results strengthen the case for a link between sunlight and this increasingly common cancer.

Mitochondria in organismal aging and degeneration.

Several lines of experimentation support the view that the genetic, biochemical and bioenergetic functions of somatic mitochondria deteriorate during normal aging. Deletion mutations of the mitochondrial genome accumulate exponentially with age in nerve and muscle tissue of humans and multiple other species. In muscle, a tissue that undergoes age-related fiber loss and atrophy in humans, there is an exponential rise in the number of cytochrome-oxidase-deficient fibers, which is first detectable in the fourth decile of age. Most biochemical studies of animal mitochondrial activity indicate a decline in electron transport activity with age, as well as decreased bioenergetic capacity with age, as measured by mitochondrial membrane potential. Mitochondrial mutations may be both the result of mitochondrial oxidative stress, and cells bearing pure populations of pathogenic mitochondrial mutations are sensitized to oxidant stress. Oxidant stress to mitochondria is known to induce the mitochondrial permeability transition, which has recently been implicated in the release of cytochrome c and the initiation of apoptosis. Thus several lines of evidence support a contribution of mitochondrial dysfunction to the phenotypic changes associated with aging.

The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis.

Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease. Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency. These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients. FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls. The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40%. Treatment of cells with the intracellular Ca2+chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death. Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine-induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts. These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.

dATP causes specific release of cytochrome C from mitochondria.

The induction of the Mitochondrial Permeability Transition (MPT) has recently been associated with the release of apoptogenic cytochrome c, which could come about in a swelling-dependent or swelling-independent manner. We observed that canonical inducers of MPT (Ca2+, t-butyl hydroperoxide, atractyloside) induce a swelling-dependent release of cytochrome c, and that osmotic support of mitochondria with PEG-1000 abolishes mitochondrial swelling, protein release, and cytochrome c release by these inducers. By contrast, it was observed that dATP is a potent inducer that caused release of cytochrome c in a swelling independent manner, i.e. even in the presence of osmotic support by PEG-1000; in addition this release of cytochrome c is inhibitable by cyclosporin A. The dATP-dependent and swelling-independent release of cytochrome c from mitochondria is not inhibitable by the protease inhibitor z-VAD, suggesting that it is not mediated by upstream caspases. This is the first report to our knowledge that a chemical compound may directly cause release of cytochrome c from mitochondria, and could explain the toxicity of dATP in the context of the genetic immunodeficiency diseases Adenosine Deaminase deficiency and Purine Nucleotide Phosphorylase deficiency.

Analysis of oxygen consumption and mitochondrial permeability with age in mice.

Biochemical and physiological parameters have been investigated in purified liver mitochondria from C57BL/6J mice of relatively young and old age, 1 month vs. 36 months. Under identical purification conditions, mitochondria from old animals consumed significantly less O2 under state 3 conditions (i.e. with saturating ADP stimulation), consistent with a lower activity of the electron transport chain. In the absence of ADP (i.e. state 4 conditions), old mitochondria consumed significantly more O2 than young mitochondria; one possible explanation was increased mitochondrial permeability as a result of induction of the mitochondrial permeability transition (MPT), and this was investigated by the mitochondrial swelling assay. In response to induction by 20 microM Ca2+, MPT rates were observed to be variable, but significantly faster in old mitochondria (t1/2 = 105 s) than in young mitochondria (t1/2 = 155 s), and in all cases MPT was inhibitable by cyclosporin A (CsA). The implications of lower state 3 respiration, higher state 4 respiration and increased rate of MPT in old mitochondria are discussed.

Induction of the mitochondrial permeability transition causes release of the apoptogenic factor cytochrome c.

It was recently reported that the mitochondrial protein cytochrome c is required for the induction of apoptosis, and that the overexpression of Bcl-2 caused increased retention of this apoptogenic factor by mitochondria. Several cellular toxins, including H2O2, tBOOH and Ca++, induce the Mitochondrial Permeability Transition (MPT); we tested the possibility that MPT is an intracellular sensor of toxicity that results in the release of cytochrome c. We observe that the release of cytochrome c from purified mitochondria is stimulated by the classical inducers of MPT, and is inhibited by the classical inhibitor of MPT, cyclosporin A (CsA). After induction of MPT, mitochondrial supernatants gained the activity to induce cleavage of caspase 3 (CPP32) in cytosolic extracts, and this gain of activity was inhibited by CsA pretreatment of mitochondria, and was cancelled by immunodepletion of cytochrome c from the supernatants. After induction of MPT, mitochondrial supernatants mixed with or without cytosolic extract gained the activity to ladder nuclei, and this gain of activity was inhibited by CsA pretreatment of mitochondria, and cancelled by immunodepletion of cytochrome c from the supernatants. These results demonstrate that the induction of MPT causes release of cytochrome c from mitochondria, which is required for the hallmarks of cytosolic and nuclear apoptosis, caspase 3 activation and nuclear laddering, and identify the MPT as a potential intracellular sensor of oxidants and other toxins, and as a target for the pharmacological inhibition of apoptosis.

Familial streptomycin ototoxicity in a South African family: a mitochondrial disorder.

The vestibular and ototoxic effects of the aminoglycoside antibiotics (streptomycin, gentamycin, kanamycin, tobramycin, neomycin) are well known; streptomycin, in particular, has been found to cause irreversible, profound, high frequency sensorineural deafness in hypersensitive persons. Aminoglycoside ototoxicity occurs both sporadically and within families and has been associated with a mitochondrial DNA (mtDNA) 1555A to G point mutation in the 12S ribosomal RNA gene. We report on the molecular analysis of a South African family with streptomycin induced sensorineural deafness in which we have found transmission of this same predisposing mutation. It is now possible to identify people who are at risk of hearing loss if treated with aminoglycosides in the future and to counsel them accordingly. In view of the fact that aminoglycoside antibiotics remain in widespread use for the treatment of infections, in particular for tuberculosis, which is currently of epidemic proportions in South Africa, this finding has important implications for the family concerned. In addition, other South African families may potentially be at risk if they carry the same mutation.

mtDNA mutations confer cellular sensitivity to oxidant stress that is partially rescued by calcium depletion and cyclosporin A.

The complete mechanism by which pathogenic mtDNA mutations cause cellular pathophysiology and in some cases cell death is unclear. Oxidant stress is especially toxic to excitable nerve and muscle cells, cells that are often affected in mitochondrial disease. The sensitivity of cells bearing the LHON, MELAS, and MERRF mutations to oxidant stress was determined. All were significantly more sensitive to H2O2 exposure than their nonmutant cybrid controls, the order of sensitivity was MELAS > LHON > MERRF > controls. Depletion of Ca2+ from the medium protected all cell lines from oxidant stress, consistent with the hypothesis that death induced by oxidant stress is Ca(2+)-dependent. A potential downstream target of Ca2+ is the mitochondrial permeability transition, MPT, which is inhibited by cyclosporin A. Treatment of MELAS, LHON, and MERRF cells with cyclosporin A caused significant rescue from oxidant exposure, and in each case significantly greater rescue of mutant than control cells. The pronounced oxidant-sensitivity of mutant cells, and their protection by Ca2+ depletion and CsA, has potential implications for both the pathophysiological mechanism and therapy of these mitochondrial genetic diseases.

Mitochondria-mediated cell injury. Symposium overview.

Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.

The rate of mitochondrial mutagenesis is faster in mice than humans.

We have investigated mitochondrial DNA (mtDNA) mutagenesis in the laboratory mouse. Using a nested PCR method for quantification, the absolute frequency, tissue distribution and rate of increase of mitochondrial deletion mutations was determined. Multiple deletions arise in brain, cardiac muscle and kidney tissues: deletions occur most frequently at regions of directly repeated mtDNA homology. Deletion frequencies rose by 2.5 x 10(5), 6300- and 4000-fold in heart, brain and kidney, respectively, between young and old mice. The rates of mtDNA mutation accumulation in mouse and human hearts are modeled well by exponential equations, with r-values of 0.96 and 0.97, and mutations rose much faster in mouse than human mtDNA per unit time. Thus, maintenance of the human mitochondrial genome is much better than that of mice, consistent with the higher rate and final extent of total DNA repair in humans than mice, that has been observed by others and consistent with the predictions of the disposable soma model of aging. A comparison of mtDNA mutagenesis from cardiocytes vs. whole heart tissue was undertaken. Deletion mutations were observed to be 100-fold lower in DNA prepared from isolated cardiocytes than from whole heart homogenates, consistent with a model of uneven mtDNA mutation accumulation.

Multiple origins of a mitochondrial mutation conferring deafness.

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation.