Possible Involvement Of Oxidative Stress In Trichloroethylene-induced Genotoxicity In Human HepG2 Cells

Introduction: Trichloroethylene (TCE) is an important vapor degreaser for the cold cleaning of fabricated metal parts and a general solvent for fats, waxes, resins, oils, rubber, cellulose esters and ethers, paints, and varnishes. Owing to its widespread use, partial water solubility and volatility, TCE has been found extensively in the environmental medium, such as the air, soil, surface water, and groundwater. The data of epidemiological studies reveal that TCE could be detected in blood and urine of the residents in many countries.TCE is known an animal carcinogen in rodents. In 1995, the International Agency for Research on Cancer (IARC) classified it to be "probably carcinogenic in humans" (group 2A) both on the basis of limited evidence for carcinogenicity in humans and a sufficient evidence for carcinogenicity in experimental animals. Recently, some studies revealed that there are species differences in response to trichloroethylene. The toxicity of TCE is dependent on the metabolism by cytochrome P450 (P450)-dependent oxidation and GSH conjugation. TCE could increase the level of lipid peroxidation in the tested cells of experimental animals and TCE exposed population. Considering that liver is the potential tumor target site of TCE in human and P450 play an important role in the oxidation metabolism of TCE in this organ, we selected HepG2 cells as the experimental system in vitro in our study. The HepG2 cell line retained many of the functions of normal liver cells and expresses the activities of several phases I and II xenobiotic metabolizing enzymes that play key roles in the activation and/or detoxification of DNA-reactive carcinogens. It has been shown to be a suitable system for genotoxicity testing.The aim of this study was to assess the genotoxic effects of TCE and, moreover, to explore the role of oxidative stress in TCE-induced genotoxicity. We hope the results could give some useful information for the safety assessment to humans on TCE.Methods:HepG2 cells were selected as test system. We used the single cell gel electrophoresis assay (SCGE) in addition to the micronucleus test (MNT) to study the genotoxic effects of TCE. Since the molecular mechanism of DNA damage by TCE may involve the oxidative stress, we evaluated the level of lipid peroxidation by measurement of thiobarbituric acid–reactive substances (TBARS). In addition, Hydroxydeoxyguanosine (8-OHdG), which is a reliable marker for oxidative DNA damage, was also measured by immunoperoxidase staining analysis. To explore the role of GSH in TCE-induced DNA damage, the intracellular GSH level in HepG2 cells was monitored with o-phthalaldehyde (OPT) assay. DL-buthionine sulphoximine (BSO) and N-acetylcysteine (NAC) were used to modulate the level of GSH in HepG2 cells, and the effects of GSH on TCE-induced DNA damage were determined by the SCGE. Furthermore, the effect of GSH depletion on cytotoxicity of TCE in HepG2 cells was examined by the cell viability, using the methyl thiazol tetrazolium bromide (MTT) assay. The data were statistically analyzed by SPSS v 13.0 software.Results: In the SCGE and MNT, TCE increased the DNA migration and the MN frequencies in a dose-dependent manner at all tested concentrations (0.5-4 mM, P < 0.05 or 0.01), respectively. The formation of TBARS was observed in HepG2 cells exposed to TCE. With the increasing of TCE concentration, the staining intensity of 8-OHdG increased obviously. There was no significant decrease in viability of HepG2 cells over an 8 h exposure to TCE concentration ranging from 0.25 to 10 mM. However, HepG2 cells became more susceptible to TCE at 8 h after pretreated with BSO (150μM) for 20 h and produced remarkable cytotoxic effects at concentrations above 4 mM. In BSO-pretreated HepG2 cells, TCE-induced significantly more DNA strand breaks than that in corresponding control cells. Co-treatment with NAC almost completely prevented the formation of TCE-induced DNA strand breaks at all tested concentrations. Conclusion: TCE could be genotoxic to HepG2 cell. TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.