MX-Induced Ras Gene Activation and Oxidative Stress in Human Derived Fetal Hepatocytes
|School||Huazhong University of Science and Technology|
|Course||Occupational and Environmental Health|
|Keywords||MX Carcinogenesis ras gene Gene mutation P21ras Oxidative Stress Malondialdehyde Reduced glutathione 8 - OHdG|
3 - chloro-4 - dichloro-methyl-5 - hydroxy-2 (5 hydrogen) - furanone (3-chloro-4-(dichloromethyl)-5-hydroxy-2 [5H]-furanone, MX), the case of chlorine of furan ketones, mainly produced in the paper industry chlorinated bleach and drinking water chlorination process, a new member of drinking water chlorination byproducts. The International Agency for Research on Cancer (IARC) has listed it as a probable carcinogen mankind. MX concentration in drinking water is only nanogram, but its extensive genetic toxicity, multiple organ carcinogenic effects on animals, coupled with the detection level of the rising in recent years, caused by the international attention. Numerous laboratories in the world in the Salmonella typhimurium reverse mutation assay (Ames test) confirmed the MX extremely mutagenic activity; vivo and in vitro mammalian cell tests also show a wide range of genetic toxicity, such as caused by genetic material base mutation occurs DNA damage, chromosomal aberrations (CAs), sister chromatid exchange (SCEs); the overall animal tests MX experimental rats can lead to multiple organ tumors. However, very little MX genotoxic effects of human-derived cells, the carcinogenic mechanism is not clear. The laboratory in vitro cultures of normal human source of embryonic liver cells (L-02 cells) to target cells by in situ hybridization experiments found that the MX can induce the expression of L-02 cells ras-mRNA inferred ras gene may be involved in the MX cancer process. To further clarify the ras gene involved in the MX induced malignant transformation of human cells mechanism, this study used PCR-cloned and sequenced and immunocytochemistry MX role ras gene mutations in the L-02 cells, the detection of the ras protein levels. Furthermore, oxidative stress plays an important role in tumorigenesis, chemical mutagens to produce one of the genotoxic mechanism; tumorigenesis is the complex process of interleaving by multiple mechanisms. MX carcinogenic process involving oxidative stress, MX can lead to L-02 cells from oxidative injury? Research and focus on the three oxidative stress biomarkers - lipid oxidative damage markers malondialdehyde (malondialdehyde MDA), antioxidant molecules of reduced glutathione (reduced glutathione, GSH), DNA oxidative damage markers 8 - hydroxy-deoxyguanosine (8-hydroxydeoxyguanosine ,8-OHdG), the use of the chemical colorimetric, high performance liquid chromatography - Electrochemical - Ultraviolet (high of performance liquid chromatography, electrochemistry-ultraviolet HPLC-EC-UV) detection technology MX L-02 cells oxidative stress levels, to investigate the carcinogenic mechanisms of MX from the other side. This article consists of three parts: the first part: MX-induced human embryonic stem cells ras gene mutation study set dose of MX 300μmol / L, dimethyl sulfoxide (DMSO, 10ml / L) solvent control, L-02 cells cultured for 12 days continuous exposure, cells were harvested and extracted genomic DNA by PCR-cloning and sequencing assay ras gene (K-ras, H-ras, N-ras) 12,13,61 the codon and around base exists mutations. The results showed: the MX group of L-02 cells exposed to 57 H-ras gene codon exists by GAT to GGT replacement, does not detect the cells of K-ras, N-ras and H-ras12, 13,61 codon of mutation; DMSO solvent control group target fragment ras gene mutation was not detected. The test results show that the MX L-02 cells may induce ras gene mutations, ras gene mutations may be involved in the MX induced human cancer process. Second part: MX-induced human embryonic liver cells ras protein expression MX exposure concentration is set to 10, 30, 100, 300 micromol / L 4 doses, dimethyl sulfoxide (DMSO, 10ml / L) as solvent control L-02 cells exposed to 24 hours, using the the immunocytochemical techniques detect intracellular P21ras protein expression level. The results show: the highest dose group (MX300μmol / L) visible cytoplasm presents obvious brown-positive cells, the cytoplasmic average luminosity compared with the solvent control group, the difference was significant (P lt; 0.001); compared to the average luminosity of the rest of the cytoplasm of each dose group with the control group were not significantly increased. Thus think The MX can induce L-02 cells P21ras protein expression increased, but the existence of threshold dose. Part III: MX-induced oxidative stress in fetal liver cells MX exposure concentration set 10,30,100,300 μmol / L 4 doses, dimethyl sulfoxide (DMSO, 10ml / L) as solvent control, L-02 cells exposed to 24 hours after the detection of the L-02 cells lipid oxidation products MDA, antioxidant molecule GSH and oxidative DNA damage marker 8-OHdG content. Found that: compared with the solvent control 30,100,300 μmol / L MX significantly increased lipid oxidation products MDA content (P lt; 0.05 L-02 cells, P lt; 0.001, P lt; 0.001) , 100,300 μmol / L MX make the L-02 cells in anti-oxidative molecules GSH level significantly significantly decreased (P lt; 0.001, P lt; 0.001), oxidative DNA damage marker 8-OHdG generated amount significantly with sex increase (P lt ; 0.05, P lt; 0.05). MX0 ~ well plates, and treated by 300 μmol / L concentration range, L-02 cells MDA content of 8-OHdG generated amount was being related (r = 0.767, P lt; 0.01); GSH level and 8-OHdG generated amount was negatively correlated (r = 0.761, P lt; 0.01). The experimental results show that the MX can induce L-02 cells from oxidative stress and its lipid peroxidation, reduced antioxidant effects, increased DNA oxidative damage; MX-induced lipid peroxidation and decreased antioxidant capacity may be oxidative DNA injury factors. MX via oxidative stress damage to biological macromolecules may be one of the mechanisms of its carcinogenic.