| GFP fusion yeast genotoxicity assay has many advantages and some limitation,including the single DNA damaging agent test,single pathway response,and specific repair pathway related to complicated indicator proteins.This study aimed to establish a short-term toxicity method based on DNA damage response network for five different DNA damage inducing agents.This method is simplified and covers multiple DNA damage repair pathways.It was applied to investigate the toxicity mechanism of haloacetaldehyde disinfection by-products and to evaluate the toxicity level of surface water and suspected pollution point sources in typical villages and towns located in the lower Yellow River.The main research results are as follows:1)Eight key genes,namely CHK1,RAD9,MSH2,RAD1,RAD30,RAD52,NTG2,and XRS2,were selected as the targets for genotoxicity assessment.To evaluate the feasibility of the method,genotoxicity assays were conducted on five known model toxins and four negative controls.The results showed that the four negative controls,including BPA,aspirin,erythromycin and tetracycline hydrochloride,didn’t show any toxicity.The five model toxins,including MMC,MMS,ETO,CDDP,and MNNG were all detected as toxic,with corresponding toxic effect endpoints of 1.196×10-3,3.632×10-4,6.796×10-5,1.342×10-3and 2.719×10-3m M,respectively.The maximum toxic effect observed were1.97,2.05,2.03,1.68,and 1.91,respectively.In addition,the toxicity response patterns of the nine chemicals exhibited clear chemical specificity and concentration dependence,and their potential molecular mechanisms of DNA damage were consistent with the previously reported toxicity mechanisms.Clustering analysis showed that samples with similar DNA damage response patterns tended to cluster together,and a significant correlation was observed between the toxic effect endpoint PELIalland the traditional phenotypic endpoints Tail DNA%（rp=0.853,P<0.001）and IR（rp=0.812,P<0.01）.2)When the method was applied for the toxicity test of haloacetaldehyde disinfection by-products,it was found that the haloacetaldehydes with similar structures had no similar DNA damage properties in general,but rather exhibited unique and conserved DNA molecular damage mechanisms.Their temporal protein expression profiles also showed chemical specificity and concentration dependence to some extent.In addition,the toxic effect endpoints of CAL and TCAL were detected as 6.370×10-3m M and 3.392×10-3m M,respectively,with the maximum toxic effect PELImaxof 1.62 and 1.60,respectively.Moreover,a significant correlation was observed between the toxic effect endpoint PELIalland the traditional phenotpyic endpoint Tail DNA%（rp=0.865,P<0.01）as well as IR（rp=0.773,P<0.05）.3)The method was applied to evaluate the toxicity levels of 13 surface waters and 5suspected pollution point sources in a county and town in Shandong Province.The potential toxicity risks were found at points situated in suspected pollution sources,points flowing through suspected pollution sources,and points in complex surrounding environments,when the water samples were concentrated 100 times.However,when the water samples were concentrated 10 times,only one of the above potential risk points showed toxic,while other points were normal. |
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