Identification And Characterization DEHP-dependent Markers In Clam (Venerupis Philippinarum)

Di(2-ethylhexyl) phthalate (DEHP) is an environmental hormone which has beenwidely used in all kinds of plasticizer industries. With the mass production andwidespread application of plastic products, DEHP is not only the culprit behind the"white pollution" of terrestrial environment, but also a major source of pollution ofmarine environment. DEHP exposure can be harmful for the marine environment aswell as the marine lives, for it can be accumulated by some ocean species ordeposited in the sediment and suspended solids, which. Although the negative impactof DEHP on environment and organisms has been extensively studied, identificationof molecular markers for monitoring this pollutants still need to be investigated.In this paper, GS-MS was employed to determine the accumulated levels of DEHP inclamVenerupis philippinarum foot and the whole body under two exposure doses of0.4mg/Land4mg/L. Real-time PCR and two-dimensional gel electrophoresis were used toexplore the promising molecular markers.The research results are as follows:(1) DEHP concentration assay by GC-MS: We found that DEHP was significantlyaccumulated in the clam foot and whole body in the first24h, then declined at96h. In thefoot, DEHP concentration decrease from0.203±0.022μg g-1to0.104±0.011μg g-1at0.4mgL exposure dose and from1.689±0.018μg g-1to1.172±0.012μg g-1at4mg L-1exposure in thetwo examined times. In the whole body, the corresponding vales were0.166±0.062μg/g and0.071±0.004μg/g at lower exposure dose, and2.300±0.024μg/g and1.047±0.011μg/gat higher exposure dose, respectively.(2) Identification of immune-related molecular markers by qPCR: DEHPmediates the immune system mainly by triggering the production of reactive oxygenspecies (ROS) and nitric oxide (NO) in higher animals. In the present study, spatialvariation in the expression of immune-related genes in clam (Venerupis philippinarum) under acute short-term DEHP treatment was assessed by qPCR. The expression of sixgenes including glutamine synthetase (GS), IkB (IK), transcription factor activatorprotein-1(AP-1), cyclophilin A-1(CypA-1), heat shock protein90(HSP90) andsuperoxide dismutase (SOD) was dose-dependent. A negative correlation betweenexpression and DEHP treatment was observed for big defensin (BD), glutathioneS-transferase (GST), and thioredoxin peroxidase (TP). Surprisingly, lysozyme (LYZ)exhibited two distinct expression patterns at two DEHP doses. Significant differencesbetween the experimental and control groups were observed for all tested genes at thevarious time points. Overall, our results revealed that DEHP mediates immuneresponses in clams by various means, and certain genes are promising candidate forbiomarkers in DEHP monitoring.(3) Identification of protein molecular markers in clams haemocytes by2-DE:In order to obtain the molecular marker of instructed DEHP contamination on theprotein level, we present a comparative proteomic analysis of the global proteinexpression changes in clam Venerupis philippinarum haemocytes after twoconcentrations of DEHP exposures. Twenty-seven proteins with significantdifferences in abundance were identified and characterized, in which six proteinsrelated to glycolysis pathway and five members in TCA cycles were induced. ThemRNA expression levels analysis of malate dehydrogenase (MDH) were furtherassessed by qPCR, and MDH mRNA transcripts were elevated compared to that inethanol control group.(4) Identification of protein molecular markers in clams foot by2-DE: Duringthe experiment commenced, we also observed a strong decrease in clams movementcompared to that in the control group. Therefore, clam foot were selected to analyzethe proteomic expression map under different concentration of DEHP exposure.Twenty-eight proteins with significant differences in abundance were identified andcharacterized, in which six enzymes related to glycolysis pathway were suppressedand two members in TCA cycle were induced. The activity and mRNA expressionlevels analysis of malate dehydrogenase were further assessed by qPCR andenzymatic assay. Malate dehydrogenase (MDH) activity and mRNA transcripts wereboth elevated compared to that in ethanol control group.