A Study On The Injury And Mechanism Of DEHP To Male Reproductive Health From The Perspective Of Occupational Safety

Di-(2-ethylhexyl) phthalate(DEHP) is a widely used plasticizer with significant male reproductive toxicity,which can cause decreased serum testosterone,sperm motility and sperm count in people with occupational exposure.However,there are no comprehensive occupational exposure limits and occupational hazard protection measures for DEHP worldwide.In order to promote the research on the occupational exposure limit of DEHP,strengthen the prevention and control of DEHP on male reproductive toxicity,and promote the health and safety of people exposed to DEHP,animal experiments were used to confirm the effect of DEHP on male reproductive toxicity and the regulatory pathways of DEHP-induced male reproductive toxicity were predicted based on the differentially expressed pi RNA and PIWI proteins in testes.Then the role of the predictive pathways in vivo and in vitro models was explored,and eventually the sensitive effect index and possible mechanism of DEHP-induced male reproductive toxicity were preliminarily proposed.The possible pathways of DEHP-induced male reproductive toxicity were screened out,which provided a basic research direction for the screening of sensitive effect markers and the study of occupational exposure limit.DEHP exposed rat model was established,and the expression levels of pi RNA and PIWI proteins in testes were detected by pi RNA microarray and Western blot respectively.The results showed that1351 pi RNA were up-regulated and 944 pi RNA were down-regulated in rat testes exposed to DEHP.Ten differentially expressed pi RNA verified by real-time quantitative PCR were selected,and bioinformatics method was used to predict target genes and for the enrichment of GO and KEGG pathway.Literature study was conducted to explore the regulatory pathways of differentially expressed Piwil2 protein.The Piwil2/STAT3/p53,Piwil2/PI3K/Akt,INSR/IRS1/PI3K/Akt/Fox O1 and AMPK/Fox O1 pathways were screened and verified to be involved in the regulation of DEHPinduced male reproductive toxicity initially through comprehensive analysis of the pathways regulated by differentially expressed pi RNA and Piwil2.The role of the selected pathways in DEHP-induced reproductive toxicity in male rats were studied.Male rats were administrated with 250,500 and 1000 mg/kg DEHP by oral for 28 days.Serum testosterone,epididymal sperm concentration,the changes of testicular histological structure,testicular cells apoptosis and autophagy were detected to evaluate male reproductive toxicity.The expression levels of proteins related to screening pathways in testes were examined to explore their roles in DEHPinduced male reproductive toxicity.Serum testosterone decreased in all DEHP exposure groups and was a sensitive indicator of DEHP exposure.In the 250 mg/kg·day DEHP group,autophagy was induced by DEHP to avoid cell apoptosis,while in the 500 and1000 mg/kg·day DEHP groups,the level of autophagy decreased and DEHP activated mitochondrial apoptosis pathway,leading to increased apoptosis of testicular cells and testis damage,which may be related to the STAT3/p53 pathway.INSR,IRS1,Piwil2 and STAT3 are sensitive and stable indicators of DEHP exposure,and their expression levels are significantly decreased in all exposure groups,which can be used as biomarkers for early exposure of DEHP for further study.To investigate the toxicity effects of different pathways regulated by pi RNA/PIWI on the mouse primary spermatocytes(GC-2spd)induced by MEHP,a metabolite of DEHP.GC-2spd cells were treated with 0,1,10,100 ?M MEHP and 100 ?M NAC+100?M MEHP,respectively.The levels of oxidative stress,apoptosis and autophagy,as well as the expression levels of proteins related to pi RNA/PIWI regulatory pathways were detected in GC-2spd cells.The results showed that MEHP induced apoptosis of GC-2spd cells through STAT3/p53 pathway mediated by oxidative stress in 10 and 100?M MEHP groups.MEHP also promotes autophagy in GC-2spd cells by affecting PI3K/Akt/m TOR and AMPK/m TOR pathways regulated by oxidative stress.The current sensitive effect index of male reproductive toxicity of DEHP is testosterone,and INSR,IRS1,PIWIL2 and STAT3 can be used as new effector indicators for further screening.DEHP affects male reproductive toxicity by regulating STAT3/p53 and PI3K/Akt/m TOR pathways mediated by oxidative stress.This study provides a possible target for the prevention and control of male reproductive toxicity of DEHP,and provides basic data for the study of occupational exposure limits of DEHP,which is of great significance for the promotion of occupational health and safety of DEHP.