Developmental Toxicity And Gene Response To Mixture Of TCDD,Chlorpyrifos,Estrogen And Heavy Metals In The Zebrafish Embryo(Danio Rerio)

With the continuous development of society and the advancement of science and technology,a large amount of toxicants and chemical pollutants have been released into the environment.Among them,organic substances and heavy metals are the majority of pollutants and exist ubiquitously in the environment.Organic pollutants generally have high toxicity,persistence and bioaccumulation.Heavy metals such as mercury,lead,and cadmium belong to non-degradable materials in the environment.Due to the complexity of the joint toxic effects of chemicals and the theory of joint toxicology of substances is not well understood,the current policies managing the environmental pollutant are almost based on the health effects of a single substance.Moreover,there is no acceptable methods to assess the health risk involving in exposure to mixture of environmental pollutants.Existing research evidence supports the notion that a mixture of multiple pollutants with a concentration which is below the single pollutant effect threshold still produces visible toxic effects,indicating that ignoring the combined toxic effects of environmental pollutants will seriously underestimate the environmental impact and health damage caused by the exposure of environmental pollutant mixtures.It is urgent to develop a risk assessment method based on the combined toxic effects of pollutants.Investigating the combined toxic effects and the mechanism of toxicity is prerequisite and basis for establishing methods on the joint toxicity effect of environmental pollutants.In this study,zebrafish were used as a model.Mercury,cadmium,lead,arsenic,carcinogenic chemicals named 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）,chlorpyrifos,an acetylcholinesterase inhibitor,and estrogen were selected as the typical pollutants.The concentration effect relationship of exposure of zebrafish embryos to single and combined pollutants was analyzed.The marker genes response to single and mixture of pollutants were screened.The effects of exposure concentration and exposure time on the expression of marker genes were studied.Objective to compare the relationship between changes in gene expression and toxic effects induced by single and mixtures exposure and to elucidate the molecular mechanism,so as to establish a method for assessing the combined toxic effects of environmental pollutants.The main results are as follows:（1）Toxic effects associated with exposure of zebrafish embryos to TCDD,chlorpyrifos,estrogen,mercury,cadmium,lead and arsenic separately and mixtures.The single LC₅₀ and EC₅₀of 120 hpf zebrafish embryos exposed to three organic substances and four heavy metals were determined by following the method of acute toxicity test.The ranges of LC₅₀ and EC₅₀in the 7 groups of combined exposure groups were determined.（2）Screening of marker genes induced by single and combined exposure.Based on the previous results of our laboratory,18 marker genes with different molecular functions were selected.（3）The changes in expression of marker genes response to single and mixture exposure.The results of q PCR showed that there is a certain relationship between exposure concentration and the relative expression of marker gene under the same exposure conditions.When the exposure concentration of pollutants is certain,the the relative expression of genes is dependent on the exposure time.In addition,the expression levels of the same gene are different during single exposure and mixture exposure.The different heavy metals have different effects on the expression of corresponding genes when exposed to organic mixtures.In conclusion,exposure of zebrafish embryos to different mixtures and different signle chemicals induce the difference of toxic effects.The difference of toxic effects between different mixtures in heavy metals and organic compounds were observed.The results laid a foundation for establishing a method to assess health risk of mixture of pollutants by using quantitative analysis of marker gene expression.