Ecotoxicological Effects And Transcriptomic Regulation Of Diethyl Phthalate On Caenorhabditis Elegans

Diethyl Phthalate(DEP)is widely used as a plasticizer in various plastic products.However,it is easy to penetrate into the environment from plastic products due to the lack of covalent bonds with plastic molecules.Currently,DEP has been detected in various environmental media,and its environmental accumulation has been consistently increased with the rapid development of industrialization.Simultaneously,food was contaminated with varying degrees of DEP.Similarly,long-term exposure to the DEP-contaminated environment leads to the enrichment of DEP in organisms.As an environmental hormone,DEP is of the ability to disrupt the endocrine system,damage the nervous and reproductive systems,and further affect developmental process of organism.Currently,most studies on ecotoxicity of DEP mainly focused on the disruption of endocrine system,whereas comprehensive assessment on the ecotoxicological effects of DEP is still lacking.Moreover,studies on intrinsic toxicological mechanisms of DEP exposure are quite limited.Caenorhabditis elegans has been used for toxicological assessment of various target pollutants due to its well-established bioinformatics background and mature evaluation system.In this thesis,Caenorhabditis elegans was applied as a model animal to systematically investigate the toxic effects of DEP,including developmental,reproductive and neural indictors.Simultaneously,the developmental and neural indictors of F1-F3 generation was also evaluated through the maternal exposure manner.Based on transcriptomic second-sequencing technology,the regulatory effects of DEP exposure on the whole gene transcription level of nematodes were also investigated.(1)Developmental and reproductive toxic effects of DEP exposure on Caenorhabditis elegans at environmental concentrations:the effects of DEP on the body length were investigated under three toxicological exposure conditions,including acute,subacute and chronic exposure.Our results revealed that acute DEP exposure had no significant change to body length.Under the subacute exposure,the body length decreased drastically at the concentration of 2 mg·L-1 and the growth rate was slowed.In the chronic exposure experiment,all nematodes in the exposed groups showed an obvious decrease on body length,indicating that higher exposure concentration and longer duration would exacerbate the toxic effects.It can be figured out that the developmental toxicity of parental DEP exposure would pass on to the unexposed offspring,while the offspring(F3)would return to normal developmental levels gradually.In addition,the reduction of offspring number performed a dose-dependent relationship under DEP exposure concentration.Specially,the offspring number showed obvious difference at high DEP concentration of 2 mg·L-1 and spawning peak period.In comparison,low levels of DEP exposure would not affect the longevity of nematodes.When the DEP concentration increased to 0.02 mg·L-1,the longevity was drastically reduced.It should be noticed that such reduction on longevity was more significant at higher concentrations.Moreover,DEP was also proved to enable the increase of the accumulation on reactive oxygen radicals and lipofuscin in nematodes,causing apoptosis and oxidative stress damage in nematodes.(2)Neurotoxic effects of DEP exposure on Caenorhabditis elegans at environmental concentrations:The locomotor and feeding behaviors of nematodes showed different toxic effects under three exposure scenarios.Specifically,the head thrashes frequency under acute DEP exposure showed significant changes at low concentration(0.002 mg·L-1),while no obvious changes were observed in the pharyngeal pumping frequency.After subacute exposure to DEP,the head thrashes frequency showed an irritating increase,which induced an over-compensation mechanism.The head thrashes frequency decreased at 2 mg·L-1 and the overall change trend was an inverted U-shape.A significant decrease in the pharyngeal pumping frequency was also observed at 0.2 and 2 mg·L-1,indicating that the DEP exposure inhibited the feeding behavior of nematodes.After chronic exposure to DEP,the head thrashes frequency in each exposure concentration group did not change significantly compared with the control group.Nevertheless,a significant decrease in the pharyngeal pumping frequency was also observed at 0.2 and 2 mg·L-1,indicating that the DEP exposure inhibited the feeding behavior of nematodes.Furthermore,cross-generational results indicated that the neurotoxicity caused by DEP exposure could be passed on from parent to offspring,and the toxic effects would continuously exist until the third generation.In terms of biochemical indicators,up-regulation of the expression levels of two stress-related genes,namely,sod-3 and hsp-16.2,indicated that DEP exposure could activate the nematode's antioxidant activity and heat stress response.Simultaneously,the intensity of dopaminergic neural signals performed a reduction in expression.The damage of dopaminergic neuronal further proved that DEP exposure had a neurotoxic effect on nematodes.(3)Effect of DEP exposure on the regulation of whole gene transcript levels in Caenorhabditis elegans:DEP exposure(2 mg·L-1)resulted in changes in transcriptional responses compared with the control group.354 differentially expressed genes were obtained by differentially expressed genes analysis,including 241 up-regulated genes and 113 down-regulated genes.By the method of GO enrichment analysis,these differential genes were mainly related to biological processes,involving in changes at multiple physiological levels such as reproduction,growth and lipid regulation.By using KEGG metabolic pathways analysis,lifespan regulation was labelled as the most significant affected pathway.In addition,changes in the cytochrome P450 metabolic pathway,fatty acid degradation metabolism,2-oxocarboxylic acid metabolism,alanine,aspartate and glutamate metabolism,steroid biosynthesis,peroxisome,retinol metabolism,arginine and proline metabolism,and tyrosine metabolic pathways,were also found to play an important role in the toxic effects of DEP mechanisms.