Systematic Toxicity Study Of A New Disinfection By-product Haloactamides By Multi-models

As one of the most important public health advances of the twenty century,water disinfection not only kills the harmful microorganisms in drinking water but also simultaneously brings adverse health effects via disinfection by-products(DBPs),which are the reaction products of disinfectants(chlorine,ozone,chlorine dioxide,or chloramines)with naturally occurring organic and inorganic substances(including bromide and iodide)in source water.The class and content of DBPs in drinking water vary depending on the disinfectant used in the disinfection process and the contamination status of source water.To reduce the adverse health effects of DBPs,many countries or organizations have established regulations and guidelines to control selected DBPs,including trihalomethanes(THMs),haloacetic acids(HAAs),and so on.The implementation of these regulations brings new challenges to water utilities to improve water treatment process.Many water utilities alternatively use chloramines rather than chlorine as the final disinfectant to minimize the formation of these regulated DBPs.Nevertheless,Source water was more or less contaminated by production and domestic sewage or algal,which will increase the content of dissolved organic nitrogen,together with chloramines or other disinfectant,could increase the formation of a series of high concentration of nitrogenous DBPs(N-DBPs).One of the typical classes of N-DBPs is Haloacetamides(HAc Ams).It includes 13 members which are formed by the substitution of hydrogen of acetamide by halogen.HAc Ams were detected in drinking water from the United States,Australian,Japan and England with the maximum sum concentrations ranged from 3.80 ?g/L to 8.18 ?g/L and also detected from drinking water of our country with the concentration ranged from 0.1?g/L to 3.1 ?g/L.Limited toxicity data shows HAc Ams have higher cytotoxicity and genotoxicity than THMs,HAAs,Haloacetonitriles(HANs)and Halonitromethanes(HNMs).Overall,people exposure to HAc Ams,but the known toxicity data cannot demonstrate the possible health risk.It is necessary to carry out toxicity study about HAc Ams to clarify the adverse health effect and mechanism of action.The study will evaluate the toxicity and explore related mechanisms by the use of in vitro cell model,animal model and occupational exposure model.Firstly,two exposure pathway-related cell lines,human gastric epithelial cell line GES-1 and immortalized human keratinocyte cell line Ha Ca T,were utilized to investigate the cytotoxicity of HAc Ams.Then the developmental toxicity of three representative HAc Ams,including CAc Am,BAc Am,IAc Am,was analyzed by the use of animal model of zebrafish embryos and related toxicity mechanism and internal exposure biomarkers were explored and screened by the joint use of transcriptomics and metabonomics.Finally,the effect of DBPs represented by HAc Ams exposure on human metabolism was analyzed by metabonomics using DBPs occupational exposure population such as swimming pool coaches and lifeguards.Part ? Cytotoxicity of HAc Ams on GES-1 and Ha Ca T cellsObjectiveAs an emerging class of N-DBPs,HAc Ams have been found to have significantly higher cytotoxicity than regulated DBPs.Humans are exposed to DBPs mainly through drinking water ingestion and dermal contact.Studies using exposure pathway-related cell lines would be helpful to better recognize the toxicity of DBPs.MethodsAfter the identification of experiment parameters including cell density,exposure time,etc.,the cytotoxicity of HAc Ams on GES-1 and Ha Ca T cells was detected by Cell Counting Kit-8(CCK-8).The toxicity comparation between HAc Ams and chloroform(CHCl3)was also conducted under the same experiment condition.Cell-health parameters,including nuclear size,mitochondrial membrane potential changes,cytochrome c release,and changes in cell permeability,were analyzed by cell-based high-content screening(HCS)analysis.ResultsThe 50% inhibiting concentration(IC50)ranged from 7.29×10-7 mol/L to 8.93×10-3 mol/L for GES-1 cells and from 1.61×10-6 mol/L to 2.36×10-2 mol/L for Ha Ca T cells.The IC50 for Ha Ca T cells was 1.01-4.72 times that for GES-1 cells.Based on the IC50 values,the rank order of cytotoxicity of HAc Ams was DIAc Am>IAc Am>TBAc Am>BAc Am>DBCAc Am>BIAc Am> BDCAc Am> CIAc Am>BCAc Am?DBAc Am> CAc Am >TCAc Am>DCAc Am.IC50 of CHCl3 to GES-1 cells was 0.052 mol/L,which is 5.83-7.13×104 times the IC50 of 13 HAc Ams to GES-1 cells.HAc Am exposure more or less affected the cell-health parameters for both GES-1 and Ha Ca T cells.HAc Ams with Br showed a more significant effect.ConclusionThe gastric mucosa derived cell line GES-1 was more sensitive than the skin epithelial derived cell line Ha Ca T,indicating that drinking water brings higher health risk.Apoptosis may play an important role in the mechanism of cytotoxicity caused by HAc Ams.Part ? Developmental toxicity and related mechanisms of mono HAc Ams on zebrafish embryosSection ? Developmental toxicity of mono HAc Ams on zebrafish embryosObjectiveDevelopmental toxicity analysis of mono HAc Ams to zebrafish embryos using a zebrafish model.MethodsZebrafish embryos were exposed to CAc Am,BAc Am and IAc Am under the concentration of 2.50,5.00,10.0,20.0,40.0,80.0 mg/L from 4 hours post fertilization(hpf)to 120 hpf,respectively.The development status of the embryos,including hatching,deformity,death,etc.,was observed.The locomotor activity of zebrafish larvae exposed to 2.50,5.00,10.0 mg/L was assessed at 120 hpf by Nodus zebrafish locomotor behavior analysis system.ResultsAt 72 and 96 hpf,the embryo hatching rates of the highest concentration of CAc Am,BAc Am and IAc Am were all significantly lower than that of the control group,that of BAc Am exposure group decreased as the increasing of BAc Am concentration.The hatching rate of IAc Am exposure group also decreased as the increasing of IAc Am concentration at 72 hpf and was significantly lower than that of 96 hpf under the concentration of 10.0,20.0,40.0 mg/L.The embryo deformity rate increased with the increase of exposure concentration and time.The half maximal effective concentration(EC50)of 96 hpf were 21.10,9.77 and 16.60 mg/L for CAc Am,BAc Am and IAc Am,respectively.The embryo mortality started to increase with the increase exposure concentration and time from 48 hpf.The median lethal dose(LD50)of 96 hpf were 28.20?11.89 and 17.25 mg/L for CAc Am,BAc Am and IAc Am,respectively.Comparing to the control group,the locomotor activity,including activity,velocity and distance of relative movement,of zebrafish larvae was different between the exposure and control groups.The cycle change between bright and dark was disappeared in the larvae exposed to 10.0 mg/L BAc Am.ConclusionHAc Ams have developmental toxicity manifesting as the delayed hatching,malformations,increased mortality and altered locomotor activity of zebrafish embryos/ larvae.BAc Am has higher toxicity than that of CAc Am and IAc Am.Section ? Mechanisms of developmental toxicity in zebrafish embryos induced by BAc AmObjectiveBase on the discovery of developmental toxicity of mono HAc Ams in section ?,related toxicity mechanisms and internal exposure biomarkers will be explored and screened in this section.MethodsZebrafish embryos were exposed to BAc Am under the concentration of 6.00 ×10-4,6.00 ×10-2 and 6.00 mg/L from 4 hours post fertilization(hpf)to 120 hpf.Development status and locomotor activity was observed.Zebrafish larvae were collected at 120 hpf.Larvae under 6.00 ×10-2 mg/L BAc Am exposure were using for transcriptomics analysis by RNA-sequencing(RNA-seq)and under 6.00 ×10-4,6.00 ×10-2 and 6.00 mg/L BAc Am exposure were using for metabolomics analysis by Liquid chromatography-mass spectrometry(LC-MS).After the screen of differentially expressed genes and differential metabolite and functional analysis,the results from transcriptomics and metabolomics analysis were combined for confirming the target gene,metabolite and pathway and the screening of internal exposure biomarkers.Results6.00×10-4~6.00 mg/L BAc Am still shown a certain adverse effect on zebrafish embryo development,including the decreased hatching rate,increased deformity rate and change of locomotor activity in exposure groups,especially the high dose group.3751 differentially expressed genes were detected by transcriptomics analysis and 2084 ones were significantly differentially expressed genes with 1163 being upregulated and 921 being down-regulated.The enrichment pathway included glutathione metabolism,salivary secretion,phototransduction,etc.The involved differentially expressed genes include glutathione peroxidase family genes(Gpx2,Gpx1b),glutathione S-transferase superfamily gene(Gstm.2,Gstp1,Gstp2,Gsto1,Gsto2,Mgst1.2,Mgst3b),glutathione reductase gene(Gsr),glutamate-cysteine ligase catalytic subunit and modifier subunit gene(Gclc,Gclm).151 metabolites were detected by metabolomics analysis and 17 ones were in significantly different level,which were enriched in two pathways,glutathione metabolism and taurine and hypotaurine metabolism.The involved significant altered metabolites include LCysteine,Oxidized glutathione,Glycine and Taurine.The combined analysis revealed that BAc Am acts on the glutathione metabolic pathway and causing serious damage to the organisms through massive consumption of glutathione(GSH).Six potential internal exposure biomarkers,named BM1-BM6,were identified by SIEVE.ConclusionBAc Am generate developmental toxicity through the disturbing of glutathione metabolic pathway and the identified potential internal exposure biomarkers could serve as a basis for researches about the internal exposure of BAc Am and other DBPs.Part ? Metabonomics analysis of DBPs exposure populationObjectiveTo investigate the metabonomics change of a specific DBPs(represented by BAc Ams)exposure population,coach and lifeguard in swimming pool.MethodsAfter questionnaire,37 subjects donated 5 mL blood with or without anticoagulant respectively for blood routine,biochemical detection and metabolomics analysis.The metabolomics of whole blood and serum were detected by LC/MS and corresponding biomarkers were extracted according to information of the internal exposure biomarker obtained in the third part.Differential metabolites were screened and related pathways were enriched under different grouping model,including the differences of swimming pool water exposure and different expression level of internal exposure biomarker.Differences in blood routine and biochemical index were also analyzed under different grouping model.ResultsInternal exposure biomarker BM4 was extracted from serum and BM5 was extracted from whole blood.Sixteen and six pathways were enriched by differential metabolites of whole blood and serum under different swimming pool water exposure,respectively.Twenty-one pathways were enriched by differential metabolites of serum under different expression level of BM4.Fourteen pathways were enriched by differential metabolites of whole blood under different expression level of BM5.Some shared pathways were enriched under different grouping model,including cysteine and methionine metabolism,pantothenate and Co A biosynthesis,glutathione metabolism.Lymphocyte number,mean corpuscular hemoglobin(MCH)and mean corpuscular hemoglobin concentration(MCHC)of subjects were significantly different under different grouping model.ConclusionDBPs(represented by BAc Ams)exposure resulted in the metabonomics change of coach and lifeguard.The affected metabolic pathways included cysteine and methionine metabolism,pantothenate and Co A biosynthesis,glutathione metabolism.