The Detection Of Environmental Disrupting Chemicals In Fish Bile And Their Inductive Effects On Apoptosis In Zebrafish Embryos

With the development of industry and improvement of human life, a large number ofchemicals have been discharged into the environment. Endocrine disrupting chemicals (EDCs), asa global problem, have attracted extensive attention because they could interfer with the hormonalbalance of organisms. In recent years, the EDCs have been widely detected in the environment andthey have potential impacts on the nervous system, immune system and reproduction system inorganisms even at relatively low concentrations. Bisphenol A (BPA), alkylphenols (APs) andnorethindrone (NET), which are used in daily life, are ubiquitous in water, but their lowconcentrations makes them difficult to be detected by chemical analysis. Fish are very closelyrelated to human life, and their bile has strong bioaccumulating capacity to store contaminantsreaching high concentrations. In addition, apoptosis is an autonomous cell death program that canprotect bodies by removing unnecessary cells and it is particularly important in the embryonicdevelopment of vertebrates. The presence of EDCs in water may affect the apoptosis process offish that will further affect their normal growth and finally have a threat to human.The present study was carried out from two aspects. On the one hand, using the LC–MS/MS,we detected the concentrations of bisphenol A, alkylphenols and norethindrone in Xin’an Riverwater and fish bile that collected from Xin’an River and fish markets, then analyzed theiraccumulation effects in fish biles and compared the difference between fish species. On the otherhand, we assessed the effects of embryonic exposure to BPA (0.1,1,10,100,1000μg/L), NP (0.1,1,10,100μg/L) and their mixtures (1+1,10+10μg/L) on the apoptosis of zebrafish. The resultsare shown as follows:1. The estrogenic activity, calculated as estradiol equivalents (EEQs), in Xin’an River islower. The presence of BPA, NP,4-t-OP,4-t-BP and NET was detected in the surface water andtheir mean concentrations were38.77,7.90,1.98,2.66and0.12ng/L, respectively. The ecologicalrisk of Xin’an River to aquatic organisms is relatively low by calculating the hazard quotient ofBPA and NP.2. BPA, NP,4-t-OP,4-t-BP and NET were present in the fish bile which collected fromXin’an River and the mean concentrations were38.68,159.64,2.07,5.86and0.96μg/L, atconcentrations with a magnitude higher than the water. Base on the calculated bioaccumulation factors and EEQ value, it was shown that fish bile had a strong ability to accumulate these fivechemicals, and thus, it was an ideal indicator to assess their contamination in water.3. The analysis of fish bile which collected from Shanghai markets showed that the meanconcentrations of BPA, NP,4-t-OP,4-t-BP and NET were240.45、527.76、76.54、12.75and5.26μg/L, respectively. Different fish species have different abilities to accumulate these contaminants,and the order for accumulating ability for BPA is: Hypophthalmichthys nobilis> Parabramispekinensis> Mylopharyngodon piceus> Carassius carassius, and for NP is: Hypophthalmichthysnobilis> Parabramis pekinensis> Carassius carassius> Mylopharyngodon piceus. Fish absorbpollutants mainly through water and food, so the differences in bioaccumulation depend on thefeeding patterns, life habits and their trophic levels in the food chain.4. Following exposure of zebrafish embryos to BPA, NP and their mixtures, the resultsshowed that the activities of caspase3were significantly induced at10,100μg/L of BPA and themixtures (10μg/L+10μg/L) compared to the control. The DNA fragmentation also appeared inthe10,100μg/L of BPA exposure group. Furthermore, apoptosis-related genes (caspase3, Bax,p53, C-Jun) and oxidative stress-related genes (NF-kB, Ucp-2) were up-regulated after BPAexposure. Therefore, those preliminary data indicated that BPA exposure might induce theapoptosis in zebrafish embryos. In contrast, the impact of NP on apoptosis in zebrafish embryoswas not significant. The apoptosis pathways include intrinsic and extrinsic pathways, and thesetwo pathways eventually lead to the activation of caspase3. Our study showed that the activity ofcaspase3and the expression of related genes in apoptosis pathways were all induced in zebrafishembryos exposed to BPA, and, further studies should be needed to explore the mechanism forthose effects.In conclusion, our results indicated that fish bile could serve as an ideal indicator to assessthe xenoestrogeic pollution in water owing to its ability to accumulate EDCs. In addition, BPAexposure might induce the apoptosis in zebrafish embryos, which threatened the development andsurvival of fish.