Toxicity And Carcinogenic Mechanism Of Microcystins

Objective To study the relationship between microcystins and human health, and to explore in depth the role and molecular mechanism of mocrocystins in hepatocarcinogenesis and anti-TMV activities of mocrocystins in vitro.Methods (1) We carried out an investigation on the microcystins water pollution and cause of death in Fuqing and Yongtai, and conducted a case-control study including newly diagnosed 388 hepatocellular carcinoma (HCC) and 388 hospital controls. These controls without history of malignant tumor were selected during the same period and matched to each case by gender, age, nationality and inhabited areas. The genotypes of XRCC3 and ERCC1 were analyzed with PCR-RFLP. General Relative Risk Regression Models were applied to analyze the interaction between drinking and other environmental factors, and the interaction between drinking and genes in hepatocarcinogenesis. (2) The two-stage-medium-term theory was applied to the establishment of the animal model. The promoting effect of MC-LR on liver tumor was evaluated with the Albertγ-GT methods and micronucleus test. During the tumor-promoting course, the effects of MC-LR on the regulation and expression of the oncogenes and suppressor genes were studied with immunohistochemical technique, RT-PCR, PCR-SSCP, Molecular Cloning and Sequencing. (3) anti-TMV activities of mocrocystins was detected by leaf-disc method.Results (1) The positive reaction rate of microcystins in Fuqing and Yuntai was 28.4%. The positive reaction rate of microcystins in pond-ditch, river, shallow, and well were 41.7%, 29.4%, 13.6%, 8.0%, respectively; The positive reaction rate of microcystins in Fuqing was 46.5%, which was significantly higer than that in Yongtai 11.1%(P<0.05); The standard cancer mortality in Fuqing was double higher than that in Yongtai. The standard mortality rate of the esophagus cancer, the stomach cancer, the liver cancer, the intestines cancer, the lung cancer, and the urinary bladder cancer in Fuqing were higher than in Yongtai. (2) Logistic regression analysis showed that drinking pond-ditch, eating moldy food and hepatitis B were the risk factors of hepatoma cancer in fuzhou; The analysis of interaction showed that drinking pond-ditch appeared super-additive interaction with hepatitis B, super-multiplicative interaction with eating moldy food, and super-additive interaction with hepatitis B and eating moldy food. The point mutation in ERCC1-8092 increased the risk of hepatoma cancer in Fuzhou. Super-additive interaction was observed between genetic polymorphism of ERCC1-4533 and drinking pond-ditch. (3) MCLR can enhance the positive reaction ofγ-GT which is a preneoplasm marker. The positive reaction rate ofγ-GT in DEN+MC group is 100%, significantly higher than that of the DEN control group (22.22%, P<0.05). MCLR can significantly enhance the micronucleus ratio, which indicates the condition of chromosome aberration. The micronucleus ratio in DEN+MC group is 13‰, significantly higher than that in the other groups (P<0.05). (4) MCLR can increase the expression of Bcl-2 and PCNA and decrease the protein expression of Bax and p21WAF1. The intension of Bcl-2 and PCNA protein expression in DEN+MC group are 0.0377 and 0.0088, respectively, significantly higher than those in DEN control group (P<0.05). The intensions of Bax and p21WAF1 protein expression in DEN+MC group are significantly lower than those in DEN control group (P<0.05). The intension values of the two genes in DEN+MC group are 0.0073 and 0.0058, respectively(P<0.05)。(5) The mRNA expression of Bcl-2 and p53 are significantly increased by MCLR. The intension of Bcl-2 and p53 mRNA expression in DEN+MC group are 2.244 and 2.5719, respectively, significantly higher than those in the other groups (P<0.05). The mRNA expression of Bax, p21WAF1 and p16 are not significantly different between DEN+MC group and the other groups (P>0.05). (6) The point mutation in exon 8 of p53 gene was detected in the promotion of liver tumor by MCLR. p53 gene mutations were found in 26.6 % of cases in DEN+MC group(4/15). There was no p53 gene mutation found in other groups. The results of cloning and sequencing indicated that three cases showed 268th condon T→C missense mutation, and this mutation made Phe change to Leu in p53 protein. There was one case of mutation with a G→A tranvertion, located at condon283, and this mutation made no change of p53 protein. No Bax gene mutation was detected during the tumor-promoting course. (7) MCLR inhibited TMV multiplication in N. tabacum var.K326 weakly. The highest inhibition rate against Tobacco Mosaic Virus infection in Nicotiana glutinosa was only 46.03%.Conclutions (1) The high mortality rate of cancer in Fuqing is positively correlated with the microcystins pollution. (2) Drinking pond-ditch was a risk factor of hepatoma cancer in Fuzhou, and act synergistically with other risk factors in hepatocelluar carcinogenesis. (3) MCLR can strongly promote liver tumor and affect the regulation and expression of the oncogenes and suppressor genes in different levels. The expression change of oncogenes and suppressor genes possibly plays an important role in the promotion of liver tumor by MCLR. (4) MCLR inhibited TMV multiplication in N. tabacum var.K326 weakly.