Toxic Effects Study Of New Disinfection Byproducts Halobenzoquinones In Zebrafish Embryos

The by-products of halobenzoquinones?HBQs?produced in drinking water disinfection process have been attracted great attention because of their low concentration and high toxicity.In vitro studies have shown that HBQs can induce oxidative stress effects in bladder cancer cells;it also inhibits the proliferation of human neural stem cells?h NSC?and exhibits potential neurotoxicity.However,the toxicological effects of these new disinfection by-products are still at the level of vitro cells,and their toxic effects in vivo need further study.In this paper,zebrafish embryos are used as model to explore the toxic effects of HBQs on zebrafish embryos,including developmental toxicity,oxidative stress,neurotoxicity and cardiovascular toxicity.First,the developmental toxicity and oxidative stress effects of HBQs on zebrafish embryos were studied.Five kinds of HBQs were exposed to zebrafish embryos at 4 hours after fertilization?4 hpf?at different concentrations,and the toxic effects were evaluated at different developmental stages.The main research contents include statistics on mortality,deformity rate and hatching rate at different exposure times,and calculated LC₅₀.The related factors in the redox system of the embryo were measured and the morphology of different developmental stages was observed.At the same time,the combined exposure of HBQs and antioxidant N-acetyl-L-cysteine?NAC?was carried out to observe whether the abnormal development caused by exposure to HBQs has been alleviated.The results showed that the mortality,hatching rate and deformity rate were positively correlated with the exposure concentration of HBQs.The mortality,deformity rate and hatching rate of the NAC composite exposure group were significantly decreased.When HBQs was exposed to96 hpf,the results of acridine orange staining?AO?showed that there were more apoptotic cells in larvae exposed to high dose of HBQs.When HBQs were exposed to 120 hpf,the related redox products including reactive oxygen species?ROS?,superoxide dismutase?SOD?,glutathione?GSH?,8-hydroxyuridine?8-OHd G?were determined in larvae.Embryo DNA Ladder assay and q RT-PCR of apoptosis-related genes were also performed.These results showed that the content of ROS was in a concentration-dependent manner;the content of SOD,GSH,8-OHd G decreased with the increase of exposure concentration;in the high concentration group,there were more fragments of DNA and the relative expression of apoptosis-related gene mdm2 was decreased and the relative expression of p53 gene was up-regulated.Finally,The traditional disinfection by-product haloacetic acid?HAAs?including:dibromoacetic acid?DBH?,dichloroacetic acid?DCH?,and iodoacetic acid?IAA?were used to expose zebrafish embryos.The results showed that the LC₅₀of the new disinfection by-product HBQs was more than 200 times smaller than LC₅₀of the HAAs.These studies have shown that HBQs have significant embryonic developmental toxicity and can induce a strong oxidative stress effect.Secondly,the neurotoxicity of HBQs exposure to zebrafish was studied.In this study,we selected three kinds of HBQ:2,5-DCBQ,2,5-DBBQ and 2,6-DCBQ.The concentrations of each exposed drug were 0.5,2.5,5.0?M respectively,in which the DMSO content was less than 0.07%,and the control group was 0.065%DMSO.Zebrafish embryos exposed from 4 hpf to 120 hpf,the motor nervous system of larvae was evaluated by zebrafish behavior analyzer and Ethovision software.The results showed that the motor behavior ability of the high-dose exposure group was weakened.In addition,the contents of related neurotransmitters,such as dopamine,?-aminobutyric acid,and the activity of acetylcholinase,were determined by molecular biological experiments.The results showed that the contents of these neurotransmitters were down-regulated.Besides,the relative expression levels of neuromodulation-related genes?gfap,mbp,syn-2a,and?1-tubulin?were determined by q RT-PCR,the in situ expression of gfap gene was observed by whole in situ hybridization experiments.The results showed that the expression of neuromodulation-related genes was abnormal.The active protein of the exposed group was analyzed by proteomics,then the results showed that more differentially expressed proteins were enriched in the pathways associated with neurodevelopment.The above results indicate that HBQs are neurotoxic to zebrafish embryos.Finally,2,6-dichloro-1,4-dibenzo?2,6-DCBQ?were selected to expose the embryos of zebrafish,and the cardiovascular toxicity effects of 2,6-DCBQ on zebrafish embryos were studied based on transcriptomics analysis.The exposure dose was optimized according to the results of the previous studies,and the four concentrations of 0,2,4,8?M were selected respectively.Significant differentially expressed genes were obtained by transcriptome analysis,and the functional clustering analysis and pathway enrichment were performed on these differential genes.The results show that there are more differential gene clusters in oxidative stress,neurodevelopmental systems and cardiovascular system-related functions.In addition,two transgenic zebrafish?Tg?kdrl:EGFP?blood vessels with fluorescent markers And Tg?zp3:fsta,myl7:EGFP?heart fluorescent label?were selected as the research object.The heart and vascular development of transgenic zebrafish were observed by fluorescence microscope.It was found that the heart development of zebrafish was abnormal,mainly manifested in pericardial edema and atrial ventricular separation.In the high concentration exposure group,the number of mandibular vascular tributaries of larvae decreased significantly,which confirmed that2,6-DCBQ has cardiac developmental toxicity to zebrafish.At the same time,Danioscope software was used to analyze the blood flow activity of larvae,the results showed that the blood flow activity of larvae decreased significantly at 8?M exposure dose.Finally,the in situ expression of cardiac specific marker gene myl7 was observed by in situ hybridization.The results showed that the expression of myl7 gene was inhibited with the increase of exposure concentration.These results indicate that HBQs are cardiovascularly toxic to zebrafish embryos.In summary,the in vivo toxicity of HBQs,a new disinfection by-product,shows that it has embryonic development toxicity,oxidative stress effect,neurotoxicity and cardiovascular toxicity.Results the environmental toxicological effects of HBQs were expounded at the molecular level and the overall level.At the same time,it also has application value,which can provide scientific basis for the drinking water safety management department of waterworks to formulate reasonable standard provisions,and scientifically evaluate the potential health hazards of this new disinfection by-products to humans.Finally,it provides a scientific basis for drinking water safety management.