The Study Of Paternal Preconceptional Nonylphenol Exposure Induced Liver Impairment In F1 Male Offspring And The Epigenetic Mechanism

Nonylphenol(NP)is a typical endocrine disrupting chemicals(EDCs)with strong toxicity and accumulation in organisms.NP widely presents in water,atmosphere,soil and other environmental media,and has been listed by the United Nations Environment Programme as one of the priority controls of 27 persistent toxic pollutants.The annual market demand for NP in China has always been more than 20%of the total global demand and is the second largest consumer market after the U.S.NP can enter the human body from food and drinking water through the amplification of the biological chain and can also be transferred into food through food packaging materials.Therefore,dietary intake is the main and unavoidable route of NP exposure to the population.Since NP has estrogen-like activity and interferes with the metabolism(production,release,transport,metabolism,binding,action or elimination)of natural hormones in the body by inhibiting estrogen binding to estrogen receptors(ER).Moreover,gametes are sensitive to estrogenic effects.Thus,NP can affect gamete quality,gamete viability and fertilization processes.Embryonic development is highly plastic and is a programming process in which genetic information and environmental factors interact to produce specific phenotypes.Changes in maternal diet and environment during this period can affect embryonic development to varying degrees,which in turn affects the phenotype and health of the offspring,and paternal sperm has been considered to have the role of transmitting only half of the paternal DNA genetic material.These ideas had limited further exploration of the effects of paternal environmental factors on offspring health.As epigenetic research techniques have evolved,it has been discovered that lifestyle or environmental factors,for example,can alter the epigenetic information of male gametes without altering their DNA sequences and pass it on to the next generation,thereby affecting their phenotype and even increasing adult disease susceptibility.NP can therefore be toxic to both female and male reproduction and is also known as the "sperm killer" due to its estrogen-like effects that make males more sensitive to NP.Some present studies have shown that maternal preconception,perinatal and lactation exposure to NP can cause abnormal glucose metabolism in offspring,reduce the ability of the liver to scavenge oxygen free radicals,and affect liver function,as well as damage the nervous system,respiratory system and immune system of offspring.However,relevant studies about the effects of paternal preconceptional NP exposure on offspring health have not been reported.In this study,we will establish a paternal preconceptional NP exposure model to observe the health status of male offspring and analyze the mechanism of damage at epigenetic level.The main contents and results of this study are as follows:1.Paternal preconceptional NP exposure induced liver injury in F1 generation male ratsFirstly,a paternal preconceptional NP exposure model was established.The NOAEL value of 15mg/kg·bw/d for NP recognized by international researchers was used as the exposure dose for the low dose group,and 3 times the NOAEL(45mg/kg·bw/d)and 9 times the NOAEL(15mg/kg·bw/d)were used for the medium and high dose groups to simulate the exposure of general storm general population,high exposure general population and occupational high exposure population,respectively.Adult Sprague-Dawley male rats weighing 180-220 g were randomly divided into 4 groups of 15 rats each,and after 1 week of acclimatization,they were gavaged orally for 28 ds(the gavage solution was a mixture of nonylphenol diluted with corn oil).At the end of treatment,male rats were combined with unproductive female rats in a 1:1 ratio.After delivery,the sex and number of each litter were adjusted,and the serum,liver,kidney and testes were taken for storage when the F1 generation rats were developed.The results of organ coefficients showed that liver organ coefficients increased with increasing dose of NP exposure in the parental,and kidney organ coefficients were inversely proportional to the dose of NP exposure,and there was no statistically significant difference in the low dose exposure group compared with the control group,and the difference was significant in the middle and high dose groups.The testicular and epididymal organ coefficients were inversely correlated with the NP exposure dose,but there was no statistically significant difference between the groups.The results of serum biochemical indexes showed that the levels of serum GLU,CHOL,TG,LDL,ALB,Cr and BUN decreased and the levels of TBA,AST,ALT,ALP,HDL and UA increased in male rats with the increase of paternal NP exposure dose.The results suggested that the F1 male offspring with paternal preconceptional NP exposure had a more damaged liver and a depleted organism with abnormal glucose metabolism.The results of pathological histological examination showed that with the increase of NP dose,hepatic lobule structure was gradually lost,hepatocyte arrangement was disturbed,inflammatory infiltration area was gradually enlarged,and extensive hepatocyte degeneration and punctate necrosis were observed.The above results indicated that the F1 male offspring had more damaged liver,showing large area of hepatocyte necrosis and inflammatory infiltration,but the pathological level lesions were not yet obvious when exposed to the NOAEL level in rats.2.Epigenetic alterations in the liver of F1 male offspring from paternal preconceptional NP exposureTo investigate the epigenetic mechanism of hepatocyte necrosis and inflammation in F1 male offspring based on paternal preconceptional NP exposure leading to liver injury.Whole-genome DNA methylation sequencing and lncRNA&mRNA transcriptome sequencing were performed on the liver of F1 male offspring to screen for differentially methylated sites and IncRNAs and to perform GO and KEGG pathway enrichment analysis.The results showed that paternal preconceptional NP exposure altered the DNA methylation expression profile and lncRNA expression profile in the liver of F1 male rats.And most of the differentially enriched pathways were related to immune-related pathways,such as TNF signaling pathway,Thl and Th2 cell differentiation,Th17 cell differentiation,T cell receptor signaling pathway,B cell receptor signaling pathway,and chemokine signaling pathway.These results suggested that immune abnormalities in the liver are associated with the occurrence of liver injury in F1 male offspring.3.Hypomethylation of the ZBP1 promoter region causes necroptosis in the liver of F1 male offspringAccording to the genome-wide DNA methylation sequencing results,the DNA methylation level of ZBP1 promoter region was significantly changed.ZBP1 is an innate immune sensor that can mediate necroptosis and trigger excessive inflammatory response due to cell rupture.Since liver is not only a metabolic organ but also an immune organ with a large number of immune cells,excessive inflammatory response may induce abnormal activation of the immune system,which in turn aggravates hepatocyte damage.Therefore,it is proposed that the mechanism of liver injury in F1 male offspring exposed to preconceptional NP may be related to ZBP1-induced necrotizing apoptosis.According to the experimental results,the expression level of ZBP1-RIPK3-MLKL pathway in the liver tissues of F1 male offspring showed activation in proportion to the dose of preconceptional NP exposure in the sire.In the extracted primary liver parenchyma cells of F1 male offspring from control and high-dose groups,the ZBP1-RIPK3-MLKL pathway showed the same activation status in the high-dose group,and the ZBP1-RIPK3-MLKL pathway showed high expression in the control group after the use of DNA methyltransferase inhibitor 5-Aza.These results suggested that F1 male rat liver undergoes necroptosis,which is associated with ZBP1 methylation levels.However,it is interesting to note that the expression of DNMT1 increased with increasing dose of preconceptional NP exposure in the sire,and the DNA methylation level of ZBP1 should be increased,but the actual results were the opposite.Binding of ZBP1 to DNMT1 was shown to be reduced by chromatin immunoprecipitation experiments,which could explain the reduced level of DNA methylation in the promoter region of ZBP1,but also suggested that there may be other factors preventing DNMT1 from binding to it.4.lncRNA PVT1 involved in modulating the methylation level of ZBP1 promoter region in the liver of F1 male offspringlncRNA can regulate the DNA methylation level of gene by regulating DNA methyltransferase,which in turn affects the expression of gene.According to lncRNA sequencing results,lncRNA PVT1 was a significant differentially expressed lncRNA,which showed a positive ratio to the paternal preconceptional NP staining dose in F1 male offspring liver and the same trend in F1 primary rat hepatocytes.To determine its function,subcellular localization of lncRNA PVT1 in primary hepatic parenchymal cells was performed using nucleoplasm separation and in situ fluorescence hybridization.The results revealed that lncRNA PVT1 was mainly expressed in the nucleus,thus suggesting that lncRNA PVT1 may be involved in pre-transcriptional regulation.Antisense nucleotide(ASO)was selected to knock down lncRNA PVT1 in the nucleus of primary liver parenchyma cells.When lncRNA PVT1 was knocked down,the ZBP1-MLKL-RIPK3 pathway was also silenced and the expression level of DNMT1 was reduced,but pyrophosphate sequencing revealed that ZBP1 showed hypermethylation.It was shown that lncRNA PVT1 could bind to EZH2 and recruit DNA methyltransferase to regulate gene expression by regulating methylation levels.RNA immunoprecipitation and immunoprecipitation experiments revealed that lncRNA PVT 1 can bind DNMT1 through EZH2.It was concluded that lncRNA PVT1 binds to DNMT1 through EZH2 to reduce the binding of DNMT1 to the promoter region of ZBP1,causing it to show hypomethylation and promoting ZBP1 transcription,which in turn activates liver parenchymal cells to undergo necroptosis,causing extensive necrosis and inflammatory infiltration in the liver of paternally preconception NP-exposed F1 male rats.