Study On The Multi-generation Exposure Effects And Toxicity Mechanism Of 2-bromo-4,6-dinitroaniline Based On Adverse Outcome Pathway

An adverse outcome pathway(AOP)is a conceptual framework for summarizing existing knowledge about linkages between a direct molecular-level initiating event(MIE)and an adverse outcome(AO)at a level of biological organization relevant to ecological risk assessment.So far,a lot of short-term acute toxicity screening of zebrafish have been conducted to get more toxicological data to improve the development of AOP.We have also used the in silico and in vivo methods to screen the acute toxicity of toxic substances to zebrafish embryos/lavae,but we have not been able to proposed the potential AOPs because of the inconsistency between adverse outcome at higher exposed concentration and molecular pathway at lower exposed concentration.In this paper,we proposed the multi-generation chronic experiments of zebrafish at environment relevant concentrations.On the one hand,multi-generation chronic experiments can accurately simulate the actual environment in which toxic substances may elicit adverse outcome on parents at any life stages and even its offprings of organisms.This will provide more direct and effective evidence for cological risk assessment.On the other hand,based on the adverse outcomes(AO)in individual level and population level that concluded from multi-generation experiments,we delved down to progressively lower levels of biological organization in an effort to connect that outcomes with a specific MIE(or multiple MIEs)which would contribute to toxicity prediction.Disperse Blue 79,one of the largest azo dyes for production and use,is mainly degraded to BDNA in the environment due to its instability,and BDNA is the main raw material of disperse blue 79.BDNA is abundant in the environment,but the environmental toxicology reseach about it is still lacking.Therefore,we choosed BDNA as the target compound of zebrafish multi-generation experiment.Based on the above research purposes,the research steps of this paper are as follows:(1)Firstly,we select 2-bromo-4,6-dinitroaniline(BDNA)as our target compound,and then conduct the acute experiment about BDNA to obtain its acute toxicity data and then derived chronic toxicity data.Finally,a series of appropriate exposure concentrations of multi-generation chronic experiments were seted according to the chronic toxicity data and the actual concentration of BDNA.(2)Secondly,the multi-generation exposure experiments(including the 21-days chronic exposure test of F0 generation,full-life cycle exposure test of F1 generation and early life stage exposure and recovery test of F2 generation)of BDNA were conducted,thus we get a lots of AOs about parental and offspring zebrafish.(3)Finally,we designed the experiment in molecular level and protein level to explore the possible toxic mechanism that caused those AOs,then combined with the existing study and the concluded AOs to explore possible AOP that based on the multi-generation zebrafish experiments through the "top-down" strategy.The experimental results are as follows:(1)The 5-day acute exposure test showed that the LC50 of BDNA at 24 h,48 h,72 h and 96 h was 623107,16102,8016,1568 and 1023 μg/L,respectively.And the predicted no observed effected concentration(PNOEC)of BDNA was 394 μg/L.According to the PNOEC and environmental concentration of BDNA,the exposure concentrations of the multi-generation experiment were determined to be 0.5 μg/L,5 μg/L,50 μg/L and 500 μg/L.(2)Exposure to BDNA elicited the adverse effects on reproductive development of zebrafish,for example,it reduced the fecundity of F0-generation zebrafish,reduced the hatching rate of F1 larvae,skewed sex ratio towards males and then decreased fecundity and weight of F1-generation female zebrafish significantly.In addition,the effects on early life development of F2 generation including decreased fertilization rate,reduced hatching rate,malformations and mortality were caused through mother and child delivery.(3)The study about BDNA reproductive toxicity mechanism have shown that BDNA affected the gene expression associated to hypothalamus-pituitary-gonad(HPG)axis and estrogen receptor(ER),thus interfered with the balance of hormone levels and ultimately lead to the effects on sex ratio,fecundity and other reproductive process.The Vtgs was down-regulated significantly,and the protein level of vitellogenin(VTG)was also decreased in fish serum in this study.Moreover,it has been reported that VTG gene may reduce egg quality and retard the development of the ovary,thus the reduced fertility rates,increased malformations and increased mortality in F2-generation zebrafish maybe resulted from the down-regulation of vtg.(4)An AOP about multi-generation experimental results were constructed based on a series of AOs in this study and existing AOPs related to the reproductive development of fish.The MIE of this AOP was that BDNA had antagonistic activity on ER.