Epigenetic Factors in Cancer Risk: Effect of Chemical Carcinogens on Global DNA Methylation Pattern in Human TK6 Cells

In the current study, we assessed the global DNA methylation changes in human lymphoblastoid (TK6) cells in vitro in response to 5 direct and 10 indirect-acting genotoxic agents. TK6 cells were exposed to the selected agents for 24 h in the presence and/or absence of S9 metabolic mix. Liquid chromatography-mass spectrometry was used for quantitative profiling of 5-methyl-2′-deoxycytidine. The effect of exposure on 5-methyl-2′-deoxycytidine between control and exposed cultures was assessed by applying the marginal model with correlated residuals on % global DNA methylation data. We reported the induction of global DNA hypomethylation in TK6 cells in response to S9 metabolic mix, under the current experimental settings. Benzene, hydroquinone, styrene, carbon tetrachloride and trichloroethylene induced global DNA hypomethylation in TK6 cells. Furthermore, we showed that dose did not have an effect on global DNA methylation in TK6 cells. In conclusion we report changes in global DNA methylation as an early event in response to agents traditionally considered as genotoxic

Toxicity of styrene and its metabolites styrene oxide and 4-vinylphenol on mouse Clara cells

Styrene has been found to be both hepato- and pneumotoxic in mice, in addition to causing lung tumors. The mode of action of styrene’s carcinogencity in mice is still unknown; however, it is likely that oxidative stress may play a role. The hypothesis of this study is that oxidative stress plays a significant role in acute styrene toxicity and may provide insight into the lung tumor formation observed in mice, but not rats, following styrene exposure. Significant decreases in glutathione levels in isolated Clara cells in vitro and in vivo were observed 3 hours following the administration of styrene, R-, S-, or racemic styrene oxide. At this same time point, increases in reactive oxygen species (ROS) and increases in superoxide dismutase were found. Increased 8-OHdG levels were observed following styrene or R-SO administration, likely due to increased ROS. CC10 is an antioxidant and anti-inflammatory protein secreted by Clara cells, the main target for styrene toxicity. Following administration of styrene or its metabolites, a general downward trend through 24 hours was observed in CC10 mRNA levels. R-SO administration caused CC10 protein levels to remain decreased 5 days following a single dose and 10 days following 5 consecutive doses. CC10 is thought to have a protective effect against pulmonary carcinogenesis, and these decreases observed in CC10 may be an early indicator of an increased susceptibility to tumorigenesis. Decreases in CC10 may also be causing an increase in oxidative stress in the lung. While decreased glutathione levels cause increases in apoptosis, our findings of apoptosis were minimal. TUNEL staining of whole lung tissue revealed increased fluorescence 12 hours following R-SO. However, the increase in caspase 3 at this time was small. The bax/bcl-2 mRNA ratio increased steadily through 240 hours following a single dose of styrene and through 24 hours following a single dose of R-SO. However, increases in the ratio of bax/bcl-2 protein levels were not observed until 240 hours following administration of both compounds. Our findings of limited apoptosis in Clara cells following acute exposure to styrene or styrene oxide are in agreement with others and may reflect the minimal extent to which apoptosis plays a role in acute styrene toxicity. Increased oxidative stress is clearly occurring following acute styrene or styrene oxide exposure, and these changes may be involved in the chronic lung tumorigenesis in mice