Citotoxicidade do corante dinitrofenilazo CI DB291: biotransformação, estresse oxidativo e alteração epigenética em células HepG2 / Cytotoxicity of the CI DB291 dinitrofenilazo dye: biotransformation, oxidative stress and epigenetic change in HepG2 cells

O produto comercial CI Disperse Blue 291 (CI DB291) é amplamente utilizado pela industria têxtil. Estudos mostram que corantes dinitrofenilazo são genotóxicos no ensaio de Ames/Salmonella, mas há poucos estudos sobre seus efeitos em células eucariontes. Este trabalho teve como objetivo investigar a genotoxicidade e citotoxicidade do corante DB291 em células humanas em cultura, bem como vias pelas quais o corante atua levando aos efeitos tóxicos. O corante comercial foi purificado por HPLC-DAD e utilizado para incubação com células HepG2 (5-100 µM por 3-72 h). A citotoxicidade foi avaliada pelos ensaios de XTT, corante cristal violeta, lactato desidrogenase extracelular e consumo de glicose. A geração de ROS intracelular foi verificada pela emissão de fluorescência da 2',7'-diclorofluoresceína. Análises de ciclo celular, fragmentação do DNA, potencial de membrana mitocondrial (Ψ) e cálcio intracelular foram realizadas por citometria de fluxo. A produção de ATP foi mensurada por quimiluminescência. Níveis de 8-oxodG e 5-mdC foram avaliados por HPLC-ESI-MS/MS e HPLC-UV, respectivamente. A expressão da enzima DNMT1 foi avaliada por western blot. Um produto de biotransformação foi identificado e caracterizado estruturalmente por MS/MS e 1H-RMN. A exposição ao corante DB291 diminuiu significativamente a sobrevivência das células em um modo tempo- e dose-dependente (IC50 = 74 µM), com a concomitante formação de um produto de biotransformação reduzido. A morte celular ocorreu sem lise da membrana plasmática. Nas células expostas, foi observado aumento da atividade enzimática mitocondrial, acompanhado por um aumento da taxa de consumo de glicose. Índices elevados de ATP, Ψ e cálcio foram verificados após a incubação das células com concentrações crescentes do corante. Alterações mitocondriais e o processo de biotransformação impeliram a aumento na produção de ROS intracelular, 8-oxodG e fragmentação do DNA. O dano ao DNA induziu a parada no ciclo...

The commercial CI Disperse Blue 291 (CI DB291) is widely used by textile industry. It is mutagenic in the Ames/S. typhimurium assay, but there are few studies showing its effects in eukaryotic cells. We evaluated here the toxicity of CI DB291 in the human HepG2 cell line. The commercial dye was purified by HPLC-DAD and used for incubation with HepG2 cells (5-100 µM for 3-72 h). Cytotoxicity was assessed by the XTT, crystal violet dye, extracellular lactate dehydrogenase and glucose consumption assays. ROS formation was assessed by 2',7'-dichlorofluorescein fluorescence. Analyses of cell cycle, DNA fragmentation, mitochondrial membrane potential (Ψ) and intracellular calcium were analyzed by flow cytometry. ATP level was measured by chemiluminescence. Levels of 8-oxodG and 5-mdC were evaluated by HPLCESI-MS/MS and HPLC-UV, respectively. DNMT1 expression was assessed by western blot. A biotransformation product was identified and structurally characterized by MS/MS and 1H-NMR. DB291 significantly decreased cell survival in a time- and dose-dependent manner (IC50 = 74 µM), with concomitant formation of a reduced biotransformation product. Plasma membrane lysis did not occur. Increased mitochondrial enzymatic activity, accompanied by increase in glucose consumption rate, was observed in cells incubated with DB291. Elevated ATP, Ψ, and intracellular calcium was verified after cell incubation with increasing dye concentrations. Mitochondrial changes and the biotransformation process accounted for the observed raise in intracellular ROS, 8-oxodG, and DNA fragmentation. DNA damage induced cell cycle arrest and DNMT1 overexpression, followed by DNA hypomethylation in the subsequent cell cycle. Results point for the first time to toxicity of the dye through biotransformation, mitochondrial changes, and oxidative stress.

Association between polymorphism in the promoter region (G/C-915) of PAX9 gene and third molar agenesis

PURPOSE: Hypodontia is the congenital absence of one or more (up to six) permanent and/or deciduous teeth, being one of the most common alterations of the human dentition. Genetic polymorphisms are variations of DNA sequences occurring in a population. This study investigated whether G-915C single nucleotide polymorphism (SNPs) in the PAX9 gene promoter is associated with hypodontia in humans. MATERIAL AND METHODS: The polymorphism in region G/C-915 of PAX9 gene (NCBI ref SNP ID: rs 2073247) of 240 patients was analyzed, being 110 controls and 130 individuals with third molar agenesis. After DNA extraction, the region of interest was amplified by PCR technique using two different primers. The significance of the differences in observed frequencies of polymorphisms in both groups was assessed by odds-ratio and chi-squared test with 95 percent confidence interval. RESULTS: Genotype CC was more frequent in patients with agenesis (11.5 percent) compared to the control (1.8 percent), while GG was more prevalent in the control group (39.1 percent) compared to the individuals with agenesis (26.2 percent). CONCLUSION: These data showed that the allele C could be associated with the third molar agenesis.