Establishment And Application Of The Method For Environmental Genotoxicity Detection

Nowadays, water pollution caused by genotoxins is one of the most seriouspublic health problems in China and has aroused wide concern since chronic exposureto these genotoxins may increase the risk of cancer development. To defy this issueand implement an effective environmental management, it is important to identify thecausative chemicals in water. Therefore, in the present study we put emphasis on theestablishment and application of the sensitive method for detecting genotoxicity ofwater contamitated with toxic chemicals. The main results are as follows:We firstly presented the procedure of the SOS/umu test, as well as the strainpreservation and subculture. Then we obtained highly sensitive stainsTA1535/pSK1002and NM2009by strain screening. The maximum β-galactosidaseactivity of TA1535/pSK1002has improved by1.5times and the sensitivity ofNM2009has enhanced by20times after the strain screening. In addition, the effectsof the amount of S9fraction have been evaluated and the optimum amount of S9was25μL in300μL incubation mixture.In this work, river water and the adjacent underground water samples from ahigh cancer incidence area were sampled to detect the genotoxicity, and HPLCfractionation directed SOS/umu assay was performed. To get the structureinformation of genotoxins, three strains TA1535/pSK1002, NM2009and NM3009were adopted together. The results showed that: the genotoxic activities of the riverwater sample occured at F9and F10in both TA1535/pSK1002and NM2009strains.Nevertheless, the genotoxic activities based on NM2009are higher than that ofTA1535/pSK1002, indicating that the genotoxins in these fractions are possibly aromatic amines or nitroarenes. While NM3009, a strain can over-produce O-AT andNR and show specific response to nitroarenes, was applied, only Fraction10showedweak genotoxicity, demonstrating that the genotoxins in F9and F10should not benitroarenes, but is possibly aromatic amines. When the underground water fractionswere tested by NM2009strain, the experimental results of all fractions are negative,suggesting that the underground water has not been contaminated by the genotoxins insuface water.On the basis of studies of the previous chaptor, we used bioassay-directedchemical analysis, TIE (Toxicity Identification Evaluation), to identify keycompounds. In this study, SOS/umu assay in conjunction with UPLC-MS/MSmeasurements was performed to identify the potential genotoxins in river water. Theresults showed that: HPLC fractionation directed SOS/umu assay was performed toidentify the chemicals causing genotoxicity of river water sample. While genotoxicactivity was observed only in Fraction10upon using TA1535/pSK1002strainwithout S9addition, genotoxic activities were observed in both Fraction10and15byusing same strain with S9addition, indicating that some genotoxins in water sampleelicited their genotoxicity under metabolic activation. When using NM2009strainwhich can over-express O-AT and show specific response to aromatic amines andnitro-aromatic compounds, the genotoxic activities were detected in Fractions8,9and10; especially, the genotoxic activity of F10was greater than that when usingTA1535/pSK1002strain. These results indicated that the major causative agents of F8,F9, and F10should be aromatic amines or nitroarenes.In order to identify the key genotoxins, we established the UPLC-MS/MSmethod for22primary aromatic amines which have high toxicity and are suspected tobe carcinogenic compounds. The UPLC-MS/MS analysis results showed that: wesuccessfully detected4-COT (0.38ng/L) in F8,3,3’-DiMe-o (0.15ng/L) in F9, ando-An (0.127ng/L) in F10. However, according to the bioassays, none of the threearomatic amines is the causative agent, which indicated that the key compounds are probably nitroarenes or some other aromatic amines, and futher efforts are needed toidentify the exact compounds causing the genotoxic activity.