Study On The Toxicity Mechanism Induced By 2-ethylhexyl Diphenyl Phosphate In Human And Mouse Hepatocytes And Its Biotransformation Products

With the phase-out of brominated flame retardants,the organophosphorus flame retardants(OPFRs)which serve as their alternatives,have gradually been used widely throughout the world.However,more and more scientific evidence suggests that OPFRs have harmful effects on living organisms.In this study,a toxicity screening of 15 OPFRs was performed on human normal hepatocytes L02 and AML-12(alpha mouse liver 12)cell line to obtain the target compound.The 15 OPFRs can be divided into three categories including alkyl-,aryl-,and halogenated-OPFRs.And then toxicity mechanism of the target compound based on transcriptomics and its biotransformation profile in vitro were also studied.The cell viability results showed that 2-ethylhexyl diphenyl phosphate(EHDPP)was one of the most toxic OPFRs,and the 48 h median inhibiting concentrations on L02 and AML-12 cells were 22.10 and 26.65 mg/L,respectively.Transcriptome sequencing results showed that there were 365 differential expression genes(DEGs)in L02 cells treated with 10 ppm of EHDPP for 48 h and 151 DEGs in AML-12 cells exposed to 15 ppm of EHDPP.Gene ontology(GO)analysis found that the function of DEGs in L02 cells was mainly referred to the biological processes which were related to glycolysis/gluconeogenesis and endoplasmic reticulum(ER)stress.As for AML-12 cells,DEGs were mainly involved in the biological processes that related to steroid biosynthesis/metabolism and cell proliferation/apoptosis.Further Kyoto Encyclopedia of Genes and Genomes(KEGG)function enrichment found that DEGs of L02 cells were mainly enriched in glycolysis/gluconeogenesis,amino acid metabolism,p53 and AMPK signaling pathways,while DEGs of AML-12 cells were mainly enriched in cholesterol biosynthesis pathway.The hub genes were screened and analyzed by Cytoscape software.It was found that the hub genes of L02 cells were mainly enriched in glycolysis/gluconeogenesis and pyruvate metabolism,while hub genes of AML-12 cells were mainly enriched in cholesterol biosynthesis.Fluorescence quantitative PCR results verified the concentration-dependent effects on the expression of hub genes after cells were exposed to EHDPP.Apoptosis results showed that significant apoptosis rates of L02 and AML-12 cells were observed exposed to 10 ppm and 15 ppm of EHDPP for 48 h,respectively.In vitro biotransformation results of 1 ppm of EHDPP showed that the metabolic rates in L02 and AML-12 cells changed significantly at 48 h with metabolic rate of 6.1% and 11.2%,respectively.Its biotransformation pathway mainly involved phase I metabolism including O-dealkylation,diarylation,redox reaction and so on,as well as phase II metabolism such as glucosylation,sulfation and phosphorylation of metabolites generated in phase I metabolism.In summary,this study preliminarily revealed the hepatotoxicity mechanism induced by EHDPP and analyzed the potential biotransformation pathways of EHDPP in vitro.These results provided a strong data support for ecological risk assessment.