Estimation of the infl uence of cytochrome P450 induction on carbon tetrachloride genotoxicity in vitro

Abstract

Introduction. One of the stages of the pathogenesis of the toxic eff ect of carbon tetrachloride is the formation of compounds of reactive oxygen species with DNA, leading to the modifi cation of nitrogenous bases. The frequency of formation of nucleotides modifi ed by nitrogenous bases correlates with the number of single-and double-chain breaks of deoxyribonucleic acid (DNA) molecules. The initiator of the formation of active forms of oxygen and lipid peroxidation in liver cells upon receipt of carbon tetrachloride is a trichloromethyl radical formed during biotransformation by microsomal enzymes of cytochrome P450.The aim of the study was to analyze the changes in the genotoxicity of carbon tetrachloride in hepatocytes at normal and increased activity of cytochrome P450 caused by the infl uence of an inductor (sovol).Materials and methods. Evaluation of genotoxicity is performed by the method of DNA-comets aft er gavage with carbon tetrachloride culture of mouse hepatocytes МН22а in 96-well microplates without the induction of cytochrome P450 and chemical induction of cytochrome P450 by sovol. Determination of DNA content in comet tail (%), comet tail length (μm) and tail moment was performed in ImageJ 1.48. Statistical analysis of the results was performed in the program SPSS Statistics 21.Results: Experimental data on the genotoxic eff ect of carbon tetrachloride on hepatocytes of the MN–22A cell line without induction of cytochrome P450 and chemical induction of cytochrome P450 by sovol are presented. It was found that 0.5 mm solution of carbon tetrachloride in 1 hour aft er addition to the culture medium is genotoxic for hepatocytes MH–22a without the use of sovol (p<0.001). Th e lack of determined using the method of DNA-comet signs of genotoxicity of 5 mm carbon tetrachloride (p>0.05) in the culture medium, probably due to the transition of the cells into a state of parametros. Genotoxic eff ect is not detected by DNA comet aft er 3 and 24 hours of incubation of hepatocytes MN–22A with 0.5 and 5 mm solutions of carbon tetrachloride without preincubation with sovol (p>0.05), which may indicate repair of the damage. Aft er 72 hours of preliminary incubation of hepatocytes with sovol and the followin g four-hour cell priming with 2.5 mm tetrachloromethane solution, higher values of the parameters of DNA comets are observed than when seeding with tetrachloromethane without incubation with sovol (p<0.05).Conclusions: According to the results of the study 72 hours of cytochrome P450 induction by sovol increases the genotoxicity of carbon tetrachloride in vitro, compared with 24 h of inductor exposure, which may indirectly indicate a higher level of formed reactive oxygen species caused by increased activity of cytochrome P450 enzymes.