RESUMO
OBJECTIVE To establish an in vitro test method and to evaluate the genotoxicity of chemicals using primary cultured mouse hepatocytes and the changes in phosphorylated histone H2AX(γH2AX)expression levels to provide a more reliable marker of the identification of genotoxicity. METHODS Hepatocytes were isolated from BALB/c mice by an improved two-step collagenase diges?tion method and then cultured in sandwich configuration. The primary cultured hepatocytes were treat?ed with various concentrations of four known genotoxic agents bleomycin(BLM),benzo(a)pyrene〔B (a)p〕,styrene and styrene-7,8-oxide(SO)within the range of 40 μmol · L-1 and two non-genotoxic agents azathioprine(Aza)and ciclosporin A(CsA)at different time points within 24 h. The cytotoxicity induced by these toxicants was assessed by CCK-8 assay. Then,the changes in γH2AX expression levels in treated cells were determined by flow cytometry. RESULTS The four genotoxic agents could be detected and two non-genotoxic agents could not be detected by this method. The γH2AX expression level was the highest when hepatocytes were exposed to BLM and SO for 3 h,or B(a)p and styrene for 6 h(P<0.01). The production of γH2AX was 25.67,18.36,12.43 and 14.25 for the four types of genotoxic agents,respectively,and was approximately 19,13,9 and 11 times that of the vehicle control group(P<0.01)at the optimum time point and concentration. There was a significant positive corre?lation between the indicated concentrations of genotoxic chemicals and γH2AX expression levels(P<0.01). In addition,the production ofγH2AX indicated no marked increase in two non-genotoxic agents such as Aza and CsA in comparison with the control group. CONCLUSION This test method can effec?tively distinguish genotoxic agents from non-genotoxic agents,and direct genotoxic agents from indirect genotoxic agents in the absence of S9. γH2AX might be a reliable marker for the identification of the potential genotoxicity of chemicals.
RESUMO
Ethanol is a principle ingredient of alcoholic beverages with potential neuronal-genotoxicity, and associated neuronal oxidative DNA damage, neurodegeneration in the CNS and neurocognitive deficits is well documented. Chronic consumption of alcohol is associated with disturbances of mnemonic functions and behavioural deficit. DNA-damaging molecules such as reactive oxygen species (ROS), lipidperoxidation product malondialdehyde (MDA) and acetaldehyde are potent genotoxic agents.Combined application of Quercetin and Coenzyme Q10 ameliorated the neurotoxicity by significantly reducing the potential biomarkers of oxidative stress, augmenting neurotransmitter, and cellular DNA and ATP contents. These results suggest that combined application of Quercetin and Coenzyme Q10 will be beneficial in prevention of neurodegeneration and cognitive deficits associated with alcoholism and these therapeutic interventions could have a clinical implication associated with alcoholism. The combined neuroprotective treatment of Quercetin and Coenzyme Q10 has been proven to ameliorate the neurotoxicity induced by ethanol.