Effects Of Combined Pollution In Hun River On Gene Expression Profiles In Zebrafish And Human Stem Cells

Combined pollution is one of the most typical characteristics of water pollution,as complex mixtures of hazardous pollutants coexist at low concentrations.It is of great significance to understand their effects on aquatic ecosystem and human health.Human bone marrow mesenchymal stem cells(hBMSCs)are multipotent cells and self-renewable,which represent highly correlation with human health in toxicity evaluation of chemicals.Zebrafish has a fully mapped genome,which has significant homology with the human genome.Therefore,evaluating toxicity caused by combined pollution in zebrafish and hBMSCs simultaneously are beneficial to predict and evaluate toxicity crossing species,and to clarify the association between adverse effects in ecosystem and human health.Compared to bioassays based on specific endpoints,transcriptomic approaches were effective to predict early toxic responses,to reveal toxicity mechanisms of chemicals,and to evaluate toxicity crossing species.Toxicgenomic researches concerning complex mixtures are still on the beginning.The relationship between composition and concentrations of contaminates and gene expression profiles are not well-understood.In the present study,combined pollution in Hun River was concerned as a case study.Surface water samples were collected from key monitoring sections,which represent different pollution gradients,as well as receiving different types of wastewater discharges.Effects of surface water on gene expression profiles of zebrafish and human stem cells were measured to analyze potential toxicity of surface water,and to illustrate the relationship between gene expression profiles and pollution gradients and characteristics of contaminant.Furthermore,the differentially expressed orthologs of zebrafish and human stem cells were identified to predict common toxicity of surface water in zebrafish and human stem cells.The main research contents and results of present study are as follows,(1)Effects of surface water from key monitoring sections of the Hun River on the transcriptomic profiles in zebrafish were studied.Changes in the transcriptomic profiles in liver of zebrafish exposed to seven water samples from the Hun River for 96 h was analyzed using zebrafish microarray.The numbers of differentially expressed genes(DEGs)with fold changes(FC?2)in each treatment ranged from 728 to 3292,which were positively correlated(r=0.788,P=0.038)with chemical oxygen demand(COD).Predominant transcriptomic responses affected included peroxisome proliferator-activated receptors(PPAR)signaling and steroid biosynthesis,which were identified by pathways enrichment analysis based of DAVID.The up-regulations of genes related to growth arrest and DNA-damage-inducible(gadd45)and cell division cycle(cdc6)and the disturbance of cell cycle pathways indicated genotoxicity of surface water.In addition,the interference of intestinal immune network infers potential immunotoxicity of surface water on zebrafish.Notably,two human diseases related pathways,insulin resistance and salmonella infection were enriched.Clustering analysis and principle component analysis with DEGs differentiated the upstream and downstream site of Shenyang City,and the mainstream and the tributary sites near the junction.Results suggested that the transcriptomic profiles of zebrafish liver revealing several potential toxic effects of surface water,and differentiating surface water with pollution gradient and different discharges.(2)The effects of surface water extracts from the Hun River on transcriptomic profiles of hBMSCs were evaluated.Organic extracts of surface water collected from Hun River inhibited 9.7-52%of cell viability at relative enrichment factor 5-20 in a dose-dependent manner.Results from human gene expression microarray found that surface water extracts at noncytotoxic concentrations induced 533 to 1055 DEGs in hBMSCs after 48 h of exposure.Total of 370 genes were commonly affected by surface water from different sites.Pathways related to genetic information processing,human diseases and organismal systems were enriched based on DEGs analyzed by DAVID.Interleukins(IL1B,IL6 and IL8)were affected and involved in most human diseases related pathways.The significantly downregulation of COL1A1 and the variation of rheumatoid arthritis pathway suggested that surface water potentially inhibited osteogenic differentiation of hBMSCs.Clustering analysis and principle component analysis with DEGs distinguish the surface water from tributary and mainstream.Results indicated that the transcriptomic profiles of hBMSCs revealed potential adverse effects of surface water on human health,and differentiated pollution source of surface water.(3)The association of transcriptome response between zebrafish and hBMSCs exposed to surface water from the Hun River was analyzed.NCBI HomoloGene system was used to identify orthologous genes shared between zebrafish and human.DEGs identified in both species were matched to HomoloGene ID for identification of differentially expressed orthologs(DEOs).The results showed that the exposure of surface water induced 311-1477 DEOs in zebrafish and 402-768 DEOs in hBMSCs.202 DEOs were commonly changed in zebrafish and hBMSCs.Pathways enrichment analysis of DEOs based on DAVID indicated focal adhesion and ECM-receptor interaction pathways were commonly enriched in zebrafish and hBMSCs.Further GO enrichment analysis found nucleoside metabolism was altered in both species.Results suggested that exposure to surface water commonly induced potential genotoxicity in zebrafish and hBMSCs.(4)Genotoxicity of surface water on zebrafish and hBMSCs were investigated.Comet assay found DNA damage in the hepatocyte of zebrafish exposed to surface water at 60-100%concentrations were 88-1072%higher than control.Oxidative stress analysis resulted 36-242%increase of MDA concentrations in the hepatocyte of zebrafish.Surface water extracts from all sites induced genotoxicity in hBMSCs,and the surface water from downstream exhibiting stronger genotoxicity than surface water from upstream.qPCR analysis presented the upregulation of RNA23A,HSP90AA1 and STIP1 which are related to DNA repair,and the downregulation of GADD45A which maintains DNA stability.Non-polar fraction(NPF)resulted in stronger DNA damage,oxidative stress and differentiation toxicity than mid-polar fraction(MPF)and polar fraction(PF)indicated that the key toxicants exist in the non-polar fractions.Genotoxicity assay were consistent with that of microarray analysis,suggesting that gene profiles in zebrafish and hBMSCs are beneficial to predict genotoxicity crossing species.In summary,the changes of transcriptomic profiles of zebrafish and hBMSCs induced by surface water from key monitoring sections of the Hun River indicated the potential genotoxicity,endocrine disrupting effects and immunotoxicity.Transcriptomic profiles were capable of differentiating surface water with pollution gradients and different discharges,and to predict genotoxicity crossing species.The present study revealed the transcriptomic associations between the toxic responses in zebrafish and human cells exposed to surface water,suggesting that abnormal changes in the gene profiles of zebrafish and hBMSCs is beneficial to comprehensively and sensitively predict the adverse effects in ecosystem and humans caused by combined water pollution.