Metabolic Mechanism Of 8:2 Polyfluoroalkyl Phosphoric Diesters In Ruditapes Philippinarum

Poly-and Perfluoroalkyl Substances(PFAS)are widely used in consumer products and industrial application due to the unique stability accompanied with hydrophobic and lipophobic properties.However,the strong persistence,long-range transport and bioaccumulation,and toxic effects render the phase-out of some PFAS.In recent years,more alternatives to PFAS are introduced,among them,8:2 polyfluoroalkyl phosphoric diesters(8:2 diPAP)is one of the most frequently detected alternatives.Studies have revealed that 8:2 diPAP have a high potential to accumulate in marine bivalves and can biotransformed to perfluoroalkyl carboxylic acids(PFCAs)with longer half-time and more toxicity.At present,the mechanism of the transformation behavior and metabolic mechanism in response to 8:2 diPAP is still unclear.This study was done on Ruditapes philippinarum with greater residual risk of PFCAs to explore the bioaccumulation,biotransformation and physiological stress to 8:2 diPAP.Then physiological response mechanishasm has been studied through analyzing a combination of metabolomics and transcriptomics data.(1)In order to explore the bioaccumulation,transformation behavior and physiological stress response of 8:2 diPAP in R.philippinarum,the metabolic transformation characteristics and physiological stress response of 8:2 diPAP were studied.The results suggested that five metabolites of 8:2 diPAP were identified in R.philippinarum,inclubng 7:3 fluorotelomer carboxylic acid(7:3 FTCA),perfluorooctanoic acid(PFOA),perfluorononanoic acid(PFNA),perfluoroheptanoic acid(PFHp A)and perfluorohexanoic acid(PFHx A),with a biotransformation rate of3.54 mol%.The hepatopancreas was found to be the target organ where bioaccumulation and biotransformation primarily occur,and 8:2 diPAP tended to be excreted in feces.Superoxide dismutase(SOD)was significantly inhibited throughout the whole experimental stage;catalase(CAT)and glutathione peroxidase(GSH-Px)activity displayed similar patterns: significant stress during the uptake phase,and basically returned to pre-exposure levels after 7 days of metabolism;glutathione Stransferase(GST)activity was significantly inhibited in the elimination phase;malondialdehyde(MDA)contents significantly increased with time.Pathological section of the hepatopancreas also showed some pathological damage.(2)Using untargeted metabolomic techniques,we analyzed the effect of 8:2 diPAP on metabolites in the hepatopancreas of R.philippinarum.The results showed that the differential metabolites in the hepatopancreas exposed to 8:2 diPAP were mainly enriched in metabolic pathways such as cholinergic synapses,glyoxylate and dicarboxylate metabolism,purine metabolism,and amino acid metabolism.Further screening resulted in 6 significantly different metabolites including 5’-AMP,glycerol-3-phosphate,α-ketoglutarate,choline,aspartate and methionine.From the differential metabolites and enriched metabolic pathways,it can be seen that 8:2 diPAP stress can lead to stress response and energy regulation disorder,and the oxidative stress response was a primary candidate for the stress response.(3)Using transcriptome technology,the effect of 8:2 diPAP on gene expression in the hepatopancreas of R.philippinarum was analyzed.The results showed that a total of4503 differentially expressed genes were screened out,and a large number of differentially expressed genes were enriched in the endocrine system,digestive system and immune system,mainly related to energy regulation,apoptosis and stress response.Differentially expressed genes were enriched into 20 trends,of which 4 trends appeared significant,The overall gene expression trend was characterized by more of the downward trend than the upward trend,which mainly affected the pathways of apoptosis,phagosome,pyruvate metabolism and amino acid metabolism.Therefore,R.philippinarum mainly activate the programmed cell death through the regulation of immune stress and energy metabolism,thereby exerting biological functions.(4)Building prediction models based on transcriptomics and metabolomics datasets to reveal the metabolic mechanism of 8:2 diPAP in R.philippinarum.Results showed that 8:2 diPAP exposure in the hepatopancreas mainly affected the genes and metabolites related to energy metabolism,osmotic regulation and stress response,reflected in AMPK and PI3K-Akt signaling pathway,pyruvate metabolism and amino acid metabolism.In addition,this study also enriched the key enzyme GST in glutathione metabolism.