The Establishment And Application Of An In Vivo Screening Method Of Environmental Estrogens Using Caenorhabditis Elegans Strain RT130as A Model Organism

Environmental estrogens are exogenous chemicals which can interfere with themetabolism of hormones, thus affecting growth, development, reproduction and otherphysiological functions. Reproduction distortion, reproductive abnormalities andreduced sperm count caused by environmental estrogens had attracted wide attentionaround the world since1996. Caenorhabditis elegans is an internationally recognizedmodel organism and has been widely used in environmental toxicology, since it iseasy to train and exerts strong resistance to environmental stress. The strain RT130isa kind of gene recombinant C. elegans whose reporter gene green fluorescent proteingene (gfp) is regulated by the vitellogenin gene (vit-2). Green fluorescence could beemitted at gut of the nematode RT130strain after UV or blue light excitation. Thegreen fluorescence can be observed by fluorescence microscopy directly, and thesignal strength can characterize estrogen levels. In this study, we established adose-response standard curve regression equation about relationship between the17β-estradiol concentration and the fluorescence signal strength, constructing ascreening method for environmental estrogens in vivo initially. This screening methodcould be applied to the measure of estrogen equilalent of single target pollutant in thelaboratory and integrate real-time monitoring of estrogen effects in differentenvironments.(1) After exposure to different concentrations of E2solutions, reporter gene gfpof this transgenic nematode expressed in gut, which issued the fluorescence signal.The gfp expression and the fluorescence signal intensity were positively correlatedwith estrogen levels, and the fluorescence signal intensity has dependency withestrogens. According to the relationship between E2exposure concentration andfluorescence signal intensity, we established the standard curve regression equation: y=4.8165e0.22(In(1E12x)/2.3026)(R2=0.9871)This in vivo screening method shows high sensitivity and could to applied toscreen estrogen substances at10-10g/L level.(2) We evaluated the estrogenic effects of single target pollutant using the abovementioned equation. Monocrotophos, bisphenol A, polychlorinated biphenyl andmercury chloride showed significant estrogenic effects, while lindane, semicarbazideand tributyltin showed no estregenic effects. This result is consistent with domesticand international researches, verifying the accuracy of the established environmentalestrogen screening method. According to the E2standard curve regression equation,the equivalents of four pollutants were: mercuric chloride> bisphenol A>polychlorinated biphenyl> monocrotophos.(3) The composition of hormone-like substances in different environments iscomplex, and there is a variety of effects between different pollutants such assynergism, antagonism and adduction. Our screening method could be applied tomonitoring estrogen levels in different environmental waters by using transgenicnematode RT130strain as a model organism. The fluorescence intensity of thenematode could characterize integrated estrogenic effects of complex environmentalcontaminants:The estrogen equivalent of water after the process inlet, grit chamber, primarysetting tank, MSBR(Membrane Sequencing Batch Reactor) treatment, rotating biodisc andfinal effluent in Haibo River sewage treatment plant were:3.48×10-7g/L,9.36×10-7g/L,3.30×10-7g/L,1.30×10-6g/L,3.67×10-7g/L and3.60×10-6g/L,respectively.The estrogen equivalent of water after the process inlet, grit chamber, settingtank and final effluent in Maidao sewage treatment plant were:3.29×10-7g/L,2.73×10-6g/L,2.28×10-5g/L and9.35×10-7g/L, respectively.The estrogen equivalent of leachate (0.5%), MBR(Membrane Biological Processor)effluent (0.5%) and final effluent in Xiaojianxi municipal waste landfill were:3.89×10-10g/L,1.95×10-8g/L and3.66×10-8g/L, respectively.The estrogen equivalent of the Second Beach in Qingdao was5.98×10-12g/L; the estrogen equivalent of the estuary of the Haibo River, Moshui River and LoushanRiver were:2.16×10-6g/L,2.83×10-6g/L and9.03×10-9g/L, respectively.In summary, we constructed a screening method for environmental estrogens invivo initially. This screening method could be applied to measure estrogen equilalentof single target pollutant in the laboratory and integrate real-time monitoring ofestrogen effects in different environments.