Expression Of HMLH1,hMSH2 And P53 In Chromate Toxicity On Human Lung A549 Cells

Chromium (Cr) is used industrially in paints, dyes, inks, for chrome plateing, leather tanning; and wood preserving, which can induce occupational lung cancer. Research has shown that 90% of the ascorbic acid (Asc) in living cells is involved in the reductive metabolism of Cr(Ⅵ), and Asc is considered to be one of the main determining factors in Cr(Ⅵ) reducing. In vitro the dose of Asc was very low and could be increased by preincubated with dehydroascorbate (dehydroascrobate acid, DHA) close to normal physiological concentration. Reynolds found that H460 and IMR90 cells were exhibited higher cytotoxicity before treatment with chromate pre-incubated with DHA for 90 min. Takahashi found that hMLH1 and hMSH2 were repressed at a higher rate in chromate lung cancer than non-chromate lung cancer.Objective:The reserch of DHA on Cr(Ⅵ)-induced damage of A549 cells might provide the theoretical basis and prevention for carcinogenesis.Methods:1. The concentration of DHA is 0.6 mmol/L in serum-free medium, according to previous results and references. DHA is dissolved in DMSO firstly, and DMSO in serum-free culture medium is of 0.2%(v/v). Cells were exposed to K2Cr2O7.This study was established for the negative group, solvent group, control group and experimental group. Negative group was the complete control group and solvent group was incubated with DHA only for 90 min. The experimental group A was treated with Cr(Ⅵ), the experimental group B was preincubation for 90 min with DHA in erum-free medium before treated by Cr(Ⅵ).2. Effection of DHA on growth curve of A549 cells. These experiments were established for the negative group, solvent group, experimental group A and experimental group B and observed for 6 d. Cells had been exposed to 10μmol/ L Cr(Ⅵ) for 3 h.3. The influence of DHA on proliferation of A549 cells. Cells were exposed to 0.00, 1.25,2.50,5.00μmol/L of Cr(Ⅵ) for 24 h. Difference of cell proliferation between experimental group A and experimental group B were detected.4. The influence of DHA on the expression of hMLH1, hMSH2, p53. Cells were exposed to 0.00,1.25,2.50,5.00μmol/L of Cr(Ⅵ) for 24 h. Then total RNA was extracted and reverse transcription and the gene expression of hMLH1, hMSH2, p53 was examined by real time quantitative PCR (real time PCR) with GADPH as an internal reference.5.β-actin protein as a reference, cells were exposed to Cr(Ⅵ) for 24 h and the protein expression of hMSH2 was detected by Western blot. The images were scanned by Bioimaging System and protein expression was analyzed by Gene Tool software.6. Statistical analysis. The data was tested by Kolmogorov-Smirnov normality test, Levene test of homogeneity of variance, Independent Samples Test and ANOVA. The correlation of two varilables was analysed by Spearman. Statistical software is SPSS 12.0 (test level a=0.05).Results:1. The growth of A549 cells in negative group and solvent group was in good condition. And the cell curve reached the peak on the fourth day and followed proliferation inhibition on the fifth day. The A549 cells in experimental group A and experimental group B were inhibited with Cr(Ⅵ). Compared to negative group, their differences were significantly (P<0.05); the curve of experimental group Bwas significantly lower than experimental group A (P<0.05).2. The repression rates of A549 cells in experimental group A and experimental group B were significantly different (P<0.05); the IC50 in the experimental group A and experimental group B were 13.04μmol/L and 3.34μmol/L, the acute toxicity ratio of the two groups'was about 1:4.3. The relative expression of genes in the experimental group B was significantly lower than in the experimental group A (P<0.05).4. The relative expression of hMSH2 protein in the experimental group B was lower than in the experimental group A (P<0.05); the expression of hMSH2 protein in experimental group B was lower with increasing dose of Cr(Ⅵ); the expression of hMSH2 protein in experimental group A was higher in the dose of 1.25μmol/ L by compared with 0.00μmol/L (P<0.05); at 2.50～5.00μmol/L, the expressions were significantly lower (P<0.05).Conclusion:1. DHA played a catalytic role in the cytotoxicity induced by Cr (Ⅵ).2. The expressions of hMLH1, hMSH2, p53 and of hMSH2 protein DHA were higher with low dose of Cr (Ⅵ) and lower with high dose of Cr (Ⅵ).