The Risk Of Esophageal Cancer And Combined Effect Of Nitrosamines And Microcystin In Drinking Water

Objective:In recent years, due to a wide range of industrial pollution, and excessive use of fertilizers and pesticides, which result in nitrogenous compounds' increasing and water eutrophication, and algal excessive reproduction. After the water containing high nitrogen compounds by centralized and chlorinated treatment, which can produce nitrosamines(NAms, reasearch shows that NAms can be detected in the raw water, the factory water and the tap water pipe network, and its concentration is gradually increasing. Microcystin also can be detected in the raw water and it can't be completely eliminated after conventional purification treatment, and can be detected after the raw water treatment. As a result, people can exposure to NAms and microcystin for a long time. At the low concentration, long-term and combined effect of NAms and microcystins in drinking water, may increase the carcinogenic risk of people in conventional purification treatment.This study aim to investigate the combined effects of methyl-benzyl-nitrosamin (NMBzA) and microystin (MC-LR) exposure of F-344 rat s' esophageal carcinogenesis; for further study this combined effects on the development of esophageal carcinoma, we choose normal esophageal epithelial cells HET-1A and esophageal cancer EC 109 cells treated with NMBzA and MC-LR. The combined effects of NMBzA and MC-LR make normal esophageal epithelial cells malignant transformation involved in the regulation of signal transduction pathways were preliminarily known. Methods and Results:1. Carcinogenic experiment of rats induced by combined treatment of NMBzA and MC-LRIn this study, male F-344 rats were divided into four groups, i e, control group, NMBzA group, MC-LR group and combined group. Three times per week and last for five weeks. We killed three combined group rats at 19,22,25 and 28 weeks, respectively. All rats were killed at 31 weeks. Take the middle part of esophageal tissue form the rats and fixed in 10% buffered formalin for 4 hour, then embedded by routine paraffin wax for observation of histological change by H-E staining. The results showed that:25 weeks later, the esophagus tissue of rats were occured hyperplasia and aggravated with time. No lesions were observed in the control group and group and MC-LR group at 31 weeks; The ratio of NMBzA groupproliferation in NMBzA group was 60%, of which 40% were mild hyperplasia,20% were moderate hyperplasia; The ratio of combined group was 100%,25% were mild hyperplasia,62.5% were moderate hyperplasia and 12.5% were severe dysplasia. These results showed that only exposed NMBzA could induce precancerous lesion, MC-LR could not induce precancerous lesion, however, MC-LR could combined with NMBA induce the occurrence of cancer as a accelerator.2. The combined effects of NMBzA and MC-LR on the occurrence and development of esophageal carcimoma2.1 The malignant transformation of normal esophageal epithelial HET-1A cells induced by combination of NMBzA and MC-LR2.1.1 Study on the combined effects of NMBzA and MC-LR(1) The HET-1A cells in logarithmic growth phase were gathered and divided into four groups, negative control group, NMBzA (1 nmol/L,10 nmol/L,100 nmol/L,1?mol/L, 10?mol/L,100?mol/L and 1 mmol/L) single exposure group, MC-LR (1 nmol/L,10 nmol/L, 100 nmol/L,1 ?mol/L and 10?mol/L) single expose group and the concentration of joint exposure to formaldehyde and composition of combined exposure group, after exposured for 24h, The cell inhibition rate of NMBzA group, MC-LR and combined group were detected by MTT.The results showed that the inhibition rate was significantly increased with the concentration in the NMBzA single group (P<0.05); In the MC-LR single group, the cell inhibition rate were decreased firstly and then increased (P<0.05); The cell inhibition rate with the increase of concentration of NMBzA and MC-LR decreased first and then increased in the combined group (P<0.05).(2) The results by anglysis of vanance of factorial design shows that, different doses of MC-LR and NMBzA were all have significant cell inhibition rate (P<0.05), and NMBzA with a dose increasing inhibition rate also increases; MC-LR and NMBzA two toxicants have interaction effect (P<0.05) and NMBzA 1 mmol/L and MC-LR 10?mol/L inhibition rate was the highest.(3)The results by the principle of efficiency to determine the type of two toxic joint action shows that, when fa<0.23, CI>1, the combined effects of this two poison was antagonistic effect; when CI<1, fa>0.23, it shows the synergistic effect.2.1.2 The effects of MC-LR and NMBzA alone and combination on the cell clonality in HET-1A cells of 20 generationThe het-la cells of 20 generations were gathered and count after cultured into 6-well plates for plate-clone formation assay and soft agar colony formation assay. The results show that NMBzA and MC-LR exist interaction and NMBzA and MC-LR could make cell clone formation rates increased and have significant difference and with the clone formation rates in the combined group is higher than the NMBzA and MC-LR single group, this investigate that NMBzA and MC-LR could synergistically promote cell malignant transformation.2.1.3 The effects of MC-LR, NMBzA and combined on the biological function of HET-1A cells in 20 generationsThe HET-1A cells were collected after 20 generations, and analysis of the changes of cell cycle, apoptosis, migration and invasion ability of MC-LR and NMBzA and combination(1) Flow cytometry (PI staining) detected the changes of cell cycle after 24h and CO-exposure to MC-LR and NMBzA. The results showed that MC-LR and NMBzA had interaction, MC-LR,NMBzA single group and combined exposure group G1 shorter, S/G2 phaseprolonged, the difference was statistically significant.(2) The apoptosis rate of MC-LR, NMBzA group and combined group were detected by flow cytometry (V-FITC Annexin labeling) at 24h. The results showed that MC-LR and NMBzA could not be considered to have interaction, the NMBzA single group can promote apoptosis, and the difference was statistically significant.(3) Using the Trans well assay to detected the cell migration and invasion, the results show that MC-LR and NMBzA exited the interaction, MC-LR can promote cell migration, NMBzA and MC-LR can promote cell invasion, the difference was statistically significant, these results suggested that MC-LR and NMBzA can promote cell invasion.2.2 The effect of combined exposure NMBzA and MC-LR on the occurrence and development of esophageal cancer EC109 cellsAccording to the results of the combined effects of HET-1A cells, established low, middle and high dose group of MC-LR, NMBzA and combined exposure Ee109 cells for 24h, analysis of the changes of cell cycle, apoptosis, migration and invasion ability of the two poisons MC-LR and NMBzA and combination were analyzed.(1) use flow cytometry (PI staining) to detect cell cycle when NMBzA, MC-LR and CO exposure after 24h. The results showed that, compared with the control group, the cell G1 phase of each exposed group was shortened, and the S phase was prolonged (P<0.05), indicating that the cell block in the S phase, there was a certain degree of proliferation out of control.(2) the apoptosis rate of NMBzA, MC-LR and combination were detected by flow cytometry (V-FITC Annexin labeling) after 24h. There was no significant change in cell apoptosis in low dose group, the combination of NMBzA and MC-LR in middle dose group inhibited apoptosis (P<0.05), high dose group NMBzA and MC-LR two poison combined to promote apoptosis (P<0.05).(3) cell migration and invasion ability were detected by Transwell assays, the cell migration and invasion were not significantly changed in low dose group, MC-LR and NMBzA co promoted cell migration and invasion (P<0.05) in the middle and high dose groups.3. The mechanism of the malignant transformation of HET-1A cells by combined exposure of NMBzA and MC-LR3.1 The expression of OATP1B1 and OATP1B3 in different esophageal cellsHET-1A cells were collected from 10 and 20 generations, and the expression of OATP1B1 and mRNA in different esophageal cells was measured by real-time fluorescence quantitative PCR. Experimental results show that compared with normal esophageal epithelium het-1a cells, OATP1B1 in esophageal carcinoma cells C17, EC 109 and EC9706 expression were increased (P<0.05); OATP1B3 in esophageal carcinoma cell line EC 109 and EC9706 expression were increased (P<0.05).3.2 Expression of PP2A and Ras genes in the regulatory pathway of MC-LR, NMBzA and combinationmRNA expression levels of PP2A and Ras genes in 10 and 20 generations of HET-1A cells were detected by real-time fluorescence quantitative PCR.(1) for PP2A gene, exposure for 10 generations have not NMBzA and MC-LR interaction and NMBzA single exposure promotes the expression of PP2A, the difference is statistically significant. exposed for 20 generations, NMBzA and MC-LR exist interaction, NMBzA single exposure promotes the expression of PP2A, MC-LR inhibits the expression of individual exposure PP2A, the differences were statistically significant.(2) for Ras gene, exposure for 10 generations have NMBzA and MC-LR interaction and NMBzA single exposure promotes the expression of Ras, the difference is statistically significant, exposed to 20 generations, NMBzA and MC-LR exist interaction, NMBzA and MC-LR alone exposure increased ras gene expression, difference had statistical significance, expression of Ras in the combined exposure group was significantly higher than that of the expression of poisons, suggesting that poisons can cooperate to promote the expression of ras gene.3.3 Analysis of the signal pathway of MC-LR, NMBzA and combinationUse Western blotting to determine have been exposed to 10 and 20th passages of het-la cells, MAPK family members ERK, JNK and p38 MAPK, MAPK downstream transcription factor ELK1 and c-Myc, P53 pathway in cell cycle related protein P53, Cdc25C, Cdc2 and apoptosis related proteins Bcl-2 and Bax protein expression and phosphorylation expression.(1) the phosphorylation protein expression levels of ERK are significantly increased in MC-LR group, and with the algebra of MC-LR exposure increases, NMBzA group and the combined exposure group decreased first and then increased, and in the combined exposure group expressed the highest, and the difference was statistically significant; JNK phosphorylation level with the poisoning algebraic increase increased, in the 20 generations of combined exposure group expressed the highest, and the difference was statistically significant; the phosphorylation level of p38 MAPK protein with the poisoning algebraic increase showed a tendency to reduce; prompts combined exposure of NMBzA and MC-LR can be activated through phosphorylation of ERK and JNK activation of the MAPK pathway.(2) phosphorylation of ELK1 protein with the poisoning algebraic increased first decreased and then increased, expression in the 20 generations of combined exposure group is the highest, and the difference was statistically significant; c-Myc in MC-LR group and the combined exposure group phosphate level was with the poisoning algebraic increase increased, the difference was statistically significant. It showed that the combined exposure group could activate the MAPK pathway and its downstream transcription factors to promote the malignant transformation of cells.(3) in P53 gene mRNA expression, exposure for 10 generations fashion no NMBzA and MC-LR interaction, exposed for 20 generations NMBzA and MC-LR exist interaction, NMBzA and MC-LR group could promote the expression of P53, differences were statistically significant, but combined exposure P53 expression was significantly decreased, suggesting that two toxic synergistic effects in inhibiting the expression of P53 gene, P53 protein (ser37) expression level with algebraic exposure decreases with increasing also confirmed this point.The phosphorylation level of Cdc25C protein in the combined exposure group with the poisoning algebraic increase and increase, in 20 generations of combined exposure group expressed the highest, and the difference was statistically significant; phosphorylation of CDC2 protein (ser37) expression level increases as the exposure algebra decreased, the difference is statistically significant. The combination of NMBzA and MC-LR could block the cell cycle, DNA synthesis increased, and the proliferation out of control.(4) the expression of Bcl-2 protein, Bax protein expression and Bcl-2/Bax ratio increased with the increase of exposure algebra, the difference was statistically significant. It suggested that the trend of cell apoptosis was gradually decreased, which promoted the malignant transformation of cells.Conclusion:1. NMBzA exposure alone can induce F344 rats with precancerous lesions, MC-LR exposure alone cannot induce cancer precancerous lesions, but can play a cancer promoting agent to promote the carcinogenic effects of NMBzA in the joint exposure to promote NMBzA induce cancer precancerous lesions.2 The combination of the low dose of NMBzA and the MC-LR has the antagonistic effect, and the high dose has the synergistic effect. NMBzA and MC-LR can promote the malignant transformation of HET-1A cells. NMBzA and MC-LR collaboration will block the cell in S phase, proliferation out of control; NMBzA promotes the apoptosis; MC-LR can promote cell migration; NMBzA and MC-LR could synergistically promote cell invasion.3. Different doses of NMBzA and MC-LR exposure of esophageal cancer cells, collaboration will block the cell in S phase, proliferation out of control; the middle dose group combined inhibition of apoptosis, high dose group NMBzA and MC-LR jointly promote cell apoptosis; NMBzA and MC-LR could synergistically promote cell migration and invasion.4 Organic anion transporter OATP1B1 and OATP1B3 are both expressed in normal esophageal epithelial cells and esophageal cancer cells, compared with normal esophageal epithelial cells HET-1 A, the expression of OATP1B1 in esophageal cancer cells C17, EC 109, EC9706 were increased, OATP1B3 in esophageal cancer cells EC109, EC9706 expression were increased.5 compared with the control group, the expression of PP2A was decreased, the expression of Ras was increased, and the expression of the combined group was increased significantly compared with the control group. It showed that NMBzA and MC-LR could inhibit the expression of PP2A and promote the activation of Ras expression by activating downstream signal transduction pathways to promote the malignant transformation of cells.6 Combined exposure of NMBzA and MC-LR can through the activation of MAPK pathway, promote MAPK downstream transcription factor ELK1 and c-Myc expression; inhibition of P53 expression and promote Cdc25C protein expression and inhibition of CDC2 protein expression to the cell cycle arrest of the cell cycle, the increase in DNA synthesis, cell proliferation out of control. Bcl-2 protein expression, Bax protein expression and Bcl-2/Bax ratio increased with the increase of exposure algebra, the trend of apoptosis was gradually weakened, which promoted the malignant transformation of cells.