RESUMO
To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.
Assuntos
DNA Mitocondrial/genética , Helicobacter pylori/fisiologia , Mutação , Espécies Reativas de Oxigênio/metabolismo , Antígenos de Bactérias/análise , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Sequência de Bases , Extratos Celulares/química , Extratos Celulares/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Citocromos b/genética , Análise Mutacional de DNA , DNA Mitocondrial/efeitos dos fármacos , Helicobacter pylori/metabolismo , Humanos , Dados de Sequência Molecular , Mutação/efeitos dos fármacos , Mutação/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the relationship between the helicobacter pylori (HP) infection and the genetic instability of mitochondrial DNA (mtDNA) in human gastric adenocarcinoma epithelial cells (AGS). METHODS: After treated with extracts of HP11638 (CagA+, VacA+) or Hp11638 mutant strain (CagA+, VacA-), AGS cells were collected, and mitochondrial DNA was extracted and Cox-I, Cox-II, Cox-III, ATPase6, ATPase8 and Cytb genes and the D-Loop region were amplified by PCR and then sequenced. RESULTS: The mutation rates of the mtDNA in AGS cells were correlated with the extracts of the two HP strains in a concentration- and time-dependent manner. But the mtDNA mutation rate in AGS cells treated with the HP11638 extract was higher than that treated with the Hp11638 mutant extract. Total of 616 mutations in D-Loop region were detected, including 489 point mutations, 81 insertions and 46 deletions. Among them, 70.9% (437/616) belonged to GC to AT and AT to GC transition. Seventeen out of 20 (85%) AGS cells treated with extract of HP had mutations in 303PolyC, 16184PolyC and 514CA regions of mtDNA D-Loop. No mutation was detected in Cox-I, Cox-II, Cox-III, ATPase6 and ATPase8 genes, three point mutations were found in the Cytb gene. CONCLUSION: HP can cause the accumulation of mutations in mtDNA, in particular, in the D-Loop region, and the VacA participated in the process.
