MX-Induced Ras Gene Activation And Oxidative Stress In Human Derived Fetal Hepatocytes

3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX), the most important representative of the family of chlorinated furanones, which is mainly produced in process of wood pulp bleaching and chlorinated waters, is a new byproduct in chlorine-disinfected drinking water. International Agency for Research on Cancer (IARC) has placed MX on the list of possibly human carcinogen. Though MX occurs in drinking water at the concentration of ng/L level, its strong and extensive toxic properties, its carcinogenesis on the multiple organs in experimental animals, together with its elevated detection level recently, captivate the interest of many researchers.Up to now, MX has been confirmed to have an extremely high mutagenic activity measured by the Salmonella typhimurium Ames assay in many laboratories of the world. Most studies have showed that MX can induce some genotoxic damage such as base mutation, DNA damage, chromosome aberrations (CAs), sister chromatid exchanges (SCEs) in mammalian cells in vitro and in vivo, and have exhibited carcinogenicity in experimental rats at multiple sites. But few studies are reported on MX's genotoxicity in human cells. The mechanism of MX-induced carcinogenicity still remains unclear. During our former work, we founded that MX enhanced ras-mRNA transcription in human derived fetal hepatocytes (L-02 cells) in vitro using in situ hybridization. It is thus evident that ras gene probably involves with the carcinogenesis of MX. For the further elucidation of the malignant transformation mechanism of MX on human cells that ras gene referred, in the present study, we investigated ras genes'mutation and ras protein's expression of L-02 cells after MX treatment using the PCR-clone sequencing and immunocytochemistry. In addition, oxidative stress plays an important role in the development of many cancers and is always used to explain the genotoxicity mechanism produced by chemical mutagen. The tumor's development presents a multiplicity procedure with several mechanisms interlaced. Quibus, we draw off the questions whether MX-induced carcinogenesis relates to oxidative stress and whether MX can initiate L-02 cells'oxidative damage. Thus we focus on the three biomarkers of oxidative stress, malondialdehyde (MDA, biomarker of lipid peroxidation), reduced glutathione (GSH, biomarker of antioxidation), and 8-hydroxydeoxyguanosine (8-OHdG, biomarker of DNA oxidative damage), to study MX-induced oxidative stress in L-02 cells and explore the carcinogenesis mechanism of MX from another way. In this part chemical colorimetry and high performance liquid chromatography-electrochemistry-ultraviolet (HPLC-EC-UV) were applied. This paper is consisted of three parts:PartⅠ:MX-induced ras genes mutation in human derived fetal hepatocytesL-02 cells were treated consecutively with 300μmol/L of MX for 12 days and genome DNA was extracted after cell's harvesting. Dimethyl sulfoxide (DMSO, 10ml/L) was used as solvent control. The sequences of codons 12, 13, and 61 and their neighbors of the ras genes (K-ras, H-ras, and N-ras) were detected applying the PCR-clone sequencing with the genome DNA from the cells.The results showed that codon 57 of the H-ras gene existed substitution from GAT to GGT in the MX-treated group, but codons 12, 13 and 61 of K-ras, H-ras, N-ras were the same with normal sequence. There was no mutation in the solvent control group.Our study suggested that MX could induce ras gene mutation of L-02 cells probably and ras gene performs some role in the carcinogenesis of MX.PartⅡ:MX- induced ras protein's expression in human derived fetal hepatocytesL-02 cells were treated with 10, 30, 100, 300μmol/L of MX for 24 hours. The expression of P21ras protein in L-02 cells after MX treatment was tested by immunocytochemical technique. DMSO (10ml/L) was used as solvent control. The results showed that positive cells with obviously buffy cytoplasm were found in the 300μmol/L MX treated L-02 cells and the average OD of this group was significantly higher than that of solvent control group (P<0.001). No overpressions of p21ras were observed in L-02 cells treated with MX of 10, 30, 100μmol/L in this experiment. So the conclusion drawed from this study is that MX could cause increase in ras protein expression in L-02 cells, but there maybe exist a dose threshold of MX.PartⅢ:MX-induced oxidative stress in human derived fetal hepatocytes L-02 cells were treated with 10, 30, 100, 300μmol/L of MX for 24 hours. DMSO (10ml/L) was used as solvent control. MDA, GSH and 8-OHdG, which are the product of lipid peroxidation, the representative of antioxidative molecules and the marker of DNA oxidative damage respectively, were detected in L-02 cells after MX exposure.The results are reviewed as follow: (1) The content of MDA was significantly increased in L-02 cells exposed to MX at the concentration of 30, 100, 300μmol/L in comparison with solvent control (P<0.05, P<0.001, P<0.001). (2) The level of GSH was significantly decreased and 8-OHdG was significantly increased in L-02 cells exposed to MX at the concentration of 100, 300μmol/L in comparison with solvent control (GSH: P<0.001, P<0.001; 8-OHdG: P<0.05, P<0.05). (3) There was a striking positive association between MDA content and 8-OHdG level (r=0.767, P<0.01) and a negative association between GSH level and 8-OHdG level (r=0.761, P<0.01) in L-02 cells exposed to MX with concentrations from 0 to 300μmol/L.Our study indicated that MX could induce oxidative stress in L-02 cells including increase of lipid peroxidation and DNA oxidative damage, and decrease of antioxidative molecules. DNA oxidative damage in L-02 cells might be ascribe to MX-induced lipid peroxidation and the weakening of antioxidation ability.