RESUMEN
Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.
Asunto(s)
ADN Mitocondrial/análisis , Animales , Hígado/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Wistar , Eliminación de SecuenciaRESUMEN
Ethanol metabolism causes oxidative stress and lipid peroxidation not only in liver but also in extra-hepatic tissues. Ethanol administration has been shown to cause oxidative degradation and depletion of hepatic mitochondrial DNA (mtDNA) in rodents, but its in vivo effects on the mtDNA of extra-hepatic tissues have not been assessed. We studied the effects of an acute intragastric ethanol administration (5 g/kg) on brain, heart, skeletal muscle, and liver mtDNA in mice. Ethanol administration caused mtDNA depletion and replacement of its supercoiled form by linearized forms in all tissues examined. Maximal mtDNA depletion was about similar (ca. 50%) in all organs studied. It occurred 2 h after ethanol administration in heart, skeletal muscle, and liver but after 10 h in brain. This mtDNA depletion was followed by increased mtDNA synthesis. A secondary, transient increase in mtDNA levels occurred 24 h after ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by vitamin E, melatonin, or coenzyme Q, three antioxidants. In conclusion, our study shows for the first time that ethanol metabolism also causes oxidative degradation of the mitochondrial genome in brain, heart, and skeletal muscles. These effects could contribute to the development of (cardio)myopathy and brain injury in some alcoholic patients. Antioxidants prevent these effects in mice and could be useful in persevering drinkers.
Asunto(s)
Antioxidantes/farmacología , Depresores del Sistema Nervioso Central/toxicidad , ADN Mitocondrial/efectos de los fármacos , Etanol/toxicidad , Mitocondrias/efectos de los fármacos , Animales , Southern Blotting , Encéfalo/efectos de los fármacos , Fomepizol , Masculino , Melatonina/farmacología , Ratones , Ratones Endogámicos ICR , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Musculares/efectos de los fármacos , Oxidantes/toxicidad , Pirazoles/farmacología , Timidina/metabolismo , Ubiquinona/farmacología , Vitamina E/farmacologíaRESUMEN
Like other antihuman immunodeficiency virus dideoxynucleosides, stavudine may occasionally induce lactic acidosis and perhaps lipodystrophy in metabolically or genetically susceptible patients. We studied the effects of stavudine on mitochondrial DNA (mtDNA), fatty acid oxidation, and blood metabolites in lean and genetically obese (ob/ob) mice. In lean mice, mtDNA was depleted in liver and skeletal muscle, but not heart and brain, after 6 weeks of stavudine treatment (500 mg/kg/day). With 100 mg/kg/day, mtDNA transiently decreased in liver, but was unchanged at 6 weeks in all organs, including white adipose tissue (WAT). Despite unchanged mtDNA levels, lack of significant oxidative mtDNA lesions (as assessed by long polymerase chain reaction experiments), and normal blood lactate/pyruvate ratios, lean mice treated with stavudine for 6 weeks had increased fasting blood ketone bodies, due to both increased hepatic fatty acid beta-oxidation and decreased peripheral ketolysis. In obese mice, basal WAT mtDNA was low and was further decreased by stavudine. In conclusion, stavudine can decrease hepatic and muscle mtDNA in lean mice and can also cause ketoacidosis during fasting without altering mtDNA. Stavudine depletes WAT mtDNA only in obese mice. Fasting and ketoacidosis could trigger decompensation in patients with incipient lactic acidosis, whereas WAT mtDNA depletion could cause lipodystrophy in genetically susceptible patients.
Asunto(s)
Fármacos Anti-VIH/farmacología , ADN Mitocondrial/efectos de los fármacos , Ácidos Grasos/metabolismo , Mitocondrias/efectos de los fármacos , Obesidad/metabolismo , Estavudina/farmacología , Animales , Fármacos Anti-VIH/sangre , Ciclo del Ácido Cítrico/efectos de los fármacos , ADN/biosíntesis , ADN/aislamiento & purificación , Genoma , Immunoblotting , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos ICR , Hibridación de Ácido Nucleico , Obesidad/genética , Oxidación-Reducción , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estavudina/sangre , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.