| Nonylphenol(NP)serves as fine chemical of raw materials and intermediates for a class of industrial productions.Due to its continuous environmental input and the stability of chemical structure,it is getting higher and higher of its universality and concentration which detected in various environmental medias.Meantime,various crops and commercial foods have also been contaminated by NP at differental degrees.Long-term exposure to NP leads to a large amount of accumulation in biological tissues,and even higher concentrations are detected in lactic and urine in human.As an endocrine disruptor,NP has been shown to interfere with the reproductive system,nervous system,and genetic processes.However,most of the current studies are focused on the unidirectional and high dose of toxicological effects for NP exposure,and there is a lack of overall assessment of NP at environmentally relevant concentrations.In addition,it is still currently unclear the comprehensive toxic mechanism of NP.Caenorhabditis elegans(C.elegans),as organism model which is extremly sensitive environmental change,has been widely used in the toxicological evaluation and mechanism research of chemicals in the environment,due to its good physiological structure and clear genetic background.In the present study,the organism C.elegans was used to evaluate the toxicological effects of reproductive,developmental and nervous system at low concentrations of NP.The high-throughput sequencing platform was applied to transcribe the genome,as well as metabolome for related molecular regulatory mechanisms,to explore the underlying molercular mechanism affected by NP exposure.Specifically,the study included the following levels:NP exposure to the reproductive and developmental processes together with lipid storage of C.elegans at the condition of subchronic exposure;The influence to neurobehavioral changes and molecular mechanism at chronic condition of NP exposure to the nematode;The negative effect to nematode's transcription level and gene regulation pathway under the situation of NP exposure;The adverse effect of NP exposure on the metabolic molecule level and metabolic pathway of C.elegans.The main conclusions of the preaent study were as follows:(1)The results revealed that developmental indictors of C.elegans were significantly inhibited,and the lipid stroge together with reproductive system were also disturbed under NP exposure,which further caused the negative change of population structure and intrinsic growth rate.The body length,width and the growth curves were adversely affected under 400 ?g L-1 NP exposure.The contrast between different life stages of L1 and L4 larva indicated that the disturbance of development caused by NP invasion mainly targeted to the later period of C.elegans.NP exposure also led to changes in the morphology of lipid droplets,the increase in the density of lipid droplets and the distribution of lipid ratio.In addition,abnormalities in reproductive systems were also observed under NP exposure.Such abnormalities displayed a dose-dependent relationship with NP concentrations.In the appearance of NP at concentration of 1 ?g L-1,the genital morphology was significantly affected,including ovipositor malformations,reduced fertilized eggs and delayed first-day spawning,which were closely related to the decreased reproductive rates.The population's parameters,including age composition and intrinsic growth rate(rm d-1).displayed significant changes.with a suppressed potentiality of population growth and the dynamic stability of the population.(2)NP exposure caused obvious effects to C.elegans,including decreased head thrashes,body bends and forging behaviour from a relatively low concentration(10 ?g L-1),accompanied with impaired plasticity of the learning and memory ability.The level of reactive oxygen species(ROS)in head was significantly elevated with the increase of NP concentrations from 10 to 200?g L-1.The oxidative damage caused by NP restored to some extent through antioxidant experiment.Meanwhile,significantly increased expression of stress-related genes,including SOD-related genes(sod-3 and sod-1),CTL-related genes(ctl-2 and ctl-3),CYP-related genes(cyp-35A2)was observed under 200 ?g L-1 NP exposure.It was confirmed by the strains of GA186 and VC710 that the overexpression of sod-3 gene played an important role in protecting the worm from oxidative stress.The expression of Tryptophan hydroxylase(TPH)in ADF and NSM neurons notably decreased in C.elegans when exposed to 10 to 200 ?g L-1 NP.The expression of TPH-related and serotonin-related genes were obviously suppressed in the present study,including tph-1,cat-1,cat-4,ser-1,and mod-5.Finally,those processes lead to a decrease in the level of serotonin in the C.elegans.Through the the addition of ascorbic acid,the neurotoxic mechanism of NP exposure to C.elegans could be explained from oxidative stress and serotonin-related progress.(3)An exploration of the response mechanism at the transcription level of C.elegans under NP exposure was proceeded with the assistance of the second-generation Illumina gene sequencing platform.Total 127 differential genes were identified based on the gene database of Caenorhabditis elegans(Fold change>1.5,FDR<0.05).According to the analysis results of Gene Ontology(GO),the enriched differential genes mainly involved in the regulation of developmental and reproductive processes.The biological processes in GO term,such as nematode larval development,hermaphrodite genitalia development,reproductive process,embryo development ending in birth or egg hatching,and growth processes,etc.,were related with the abnormally expressed genes,including anc-1,C14C6.5,atp-5,and nduf-7,which played an important role in the toxicological mechanism of NP.Those results provided a mechanism explanation for the aforementioned the inhibition of growth and the damage of reproductive system.Kyoto Encyclopedia of Genes and Genomes(KEGG)was used to analyzed the affected pathways and finally enriched in 10 pathways related to lipid anabolic metabolism,indicating that abnormal lipid storage occured on the level of gene regulation,in which stdh-2,gpx-3,K09H11.7,cpt-1,T01B11.2,cysl-2 genes were involved in those processes.According to the verification results of qRT-PCR experiment,the tendency of gene quantitative results was basically consistent with the results of transcriptome.(4)In order to further explore the changes of small molecule metabolites in the downstream of transcription regulation,UPLC-MS technology was applied to conduct a comparative analysis of metabolomics in the control group and the 400?g L-1 NP exposure treatment group of C.elegans.Results showed that a total of 431 metabolites were screened and 113 differential metabolites were effectively annotated.The functional annotation and enrichment analysis for differential metabolites were compared based on the KEGG(Kyoto Encyclopedia of Genes and Genomes)database and the Human Metabolome Database(HMDB).Those results involved in lipid metabolism,amino acid metabolism,and sugar metabolism,dopamine neurotransmitter metabolism and other processes.The upregulation of glycerophosphocholine and citramalic acid were closely related to the upregulation of intestinal lipid storage of nematode.Carboxylic acids and derivatives,especially the disorder of amino acid metabolism,participated in the anabolic process of four molecules through the tricarboxylic acid cycle(TCA),which perhaps caused an impact on life activities.The down-regulation of catecholamine neurotransmitters affected by NP exposure involved in the processes of abnormal neurobehaviors.Combined with the analysis results of transcriptome data,the metabolite changes caused by gene transcription regulation provided a basis for the negative effects of the physiological level.In conclusion,the present study found that NP exposure at environmental concentrations interfered with the regulatory changes of transcriptional and metabolic levels,causing damage to growth and development,reproductive development,and nervous system of C.elegans.The results from this study impied that NP exposure triggered a comprehensive toxicity to the organism,which posed a potential threat to the safety of ecological environment. |
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