| PAHs(polycyclic aromatic hydrocarbons)are a type of persistent organic pollutants,which are widely present in the environment.They can enter water through various means,such as atmospheric sedimentation,surface runoff,waste water discharge,and sea oil spill.Because PAHs are fat-soluble,difficult to degrade in the environment,and get easily accumulated in aquatic organisms,they induce a variety of toxic effects.Benzo[a]pyrene(BaP)is a PAH with a five-ring structure.Numerous studies have proven that it is highly toxic to aquatic organisms.Due to the sessile lifestyle,filter feeding,and low metabolic rate,pollutants can easily bioaccumulate in marine bivalves.In this study,we focus on the pearl oyster Pinctada martensii,a vital bivalve species for producing artificial pearls,which is widely distributed in subtropical and tropical oceans.The toxic effects of BaP exposure on P.martensii were studied at individual,cellular,and molecular levels.Herein,we aimed to elucidate the toxic mechanisms associated with growth and development,stress response,immunization,and metabolic pathways of P.martensii from the perspectives of molecular biology,ecotoxicology,proteomics and metabolomics.Further,we aimed to provide some important theoretical data for predictive monitoring of marine environmental pollution as well as ecological risk assessment of coastal marine environmental pollution.The main findings are shown as follows:(1)On morphological observation of the early developmental stages of P.martensii exposed to BaP,it was found to have significant effects on the morphological changes of the D-shaped veliger larvae.There was a positive correlation between teratogenicity and BaP concentration.In the same exposure time,the abnormality of the D-shaped veliger larvae showed a dose-response relationship with the concentration of BaP exposure.In the time range of 12-72 h,the abnormality showed a certain time-response relationship with the duration of BaP exposure.BaP exposure significantly affected the mortality of P.martensii.The mortality of the D-shaped veliger larvae increased with increasing concentrations and durations of exposure.The dose-response and time-response relationships were evident.Taken together,the adverse effects of BaP exposure on the early development of P.martensii could change the population structure.(2)The effects of BaP on the activities of antioxidant enzymes in the digestive gland and gill tissues of P.martensii were studied by assaying antioxidant enzyme activity.In the digestive glands,the lowest-observed-effect concentration(LOEC)values of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx),and glutathione-S-transferase(GST)were 1 ?g/L,whereas in the gills,the LOEC values of SOD,CAT,and GPx were 1 ?g/L and that of GST was 10 ?g/L.The results indicated that digestive gland tissues are more sensitive to BaP exposure than gill tissue.The concentration of MDA was significantly increased in the digestive glands and significantly decreased in the gill tissues.The results indicated that BaP induced relatively more severe lipid peroxidation damage to digestive glands.BaP exposure caused different degrees of oxidative stress damage to the digestive gland tissues and gill tissues of P.martensii and also affected its antioxidant system differently.Flow cytometry was used to analyze the effects of BaP exposure on the immune system of P.martensii.The results showed that BaP exposure induced respiratory burst and apoptosis.With the increase of BaP exposure concentration,the production of reactive oxygen species and the degree of apoptosis showed a certain dose-response relationship.The results indicated that oxidative stress and severe immunotoxicity in hemolymph of P.martensii were induced by BaP exposure.(3)The mRNA expression of key genes(AhR,CYP4,and HSP90)related to the the aryl hydrocarbon receptor(AhR)pathway of P.martensii was analyzed by real-time quantitative PCR.The mRNA expression of these genes showed tissue-specific responses after BaP exposure.The mRNA expressions of these genes were significantly inhibited in the digestive glands(LOEC = 1 ?g/L),whereas the mRNA expressions of CYP4 and HSP90 were induced in the gill tissues(LOEC>10 ?g/L).However,in the gill tissues,the mRNA expression of AhR was significantly induced at high concentrations of BaP(10 ?g/L).The results indicated that BaP exposure caused affected the expression of the related genes in different tissues of P.martensii.The response of genes of the AhR pathway of digestive glands to BaP is more sensitive than gills.(4)Two-dimensional electrophoresis combined with mass spectrometry-based proteomics was applied to analyze the differential proteomic responses in the BaP-exposed digestive glands and gill tissues of P.martensii.The MALDI-Biotyper system was used to establish the protein fingerprint.The differential proteomic responses were induced by different BaP exposure concentrations in both tissues.Moreover,the protein responses were tissue-specific.Twenty-eight differentially expressed proteins were successfully identified in the digestive glands;nineteen proteins were up-regulated and nine were down-regulated.These proteins were mainly involved in the cytoskeleton,material and energy metabolism,stress response,and signal transduction.Thirteen differentially expressed proteins were successfully identified in the gills;eight proteins were up-regulated and five were down-regulated.These proteins were principally involved in signal transduction,material and energy metabolism,and growth and development.The effects of BaP exposure on small molecule protein compounds of P.martensii were analyzed by protein fingerprinting.The characteristic responses of these proteins to BaP exposure were studied.The results showed that protein fingerprints of the different tissues of P.martensii were tissue-specific,and the molecular weight of the protein was 8573 and 13325 Dalton in the digestive glands.The molecular weight of the protein was 8570,9714,and 13542 Dalton in the gills.The response trends of protein fingerprints suggested that both tissues show higher sensitivity to low concentration of BaP(1 ?g/L)exposure.(5)Nuclear magnetic resonance-based metabolomics was applied to analyze the BaP exposure-induced production of differential metabolites of the digestive glands and gill tissues of P.martensii.The results showed that a low BaP concentration(1 ?g/L)caused no significant change in the metabolites of both tissues,whereas a high BaP concentration(10?g/L)caused differences in the metabolic responses of both tissue types.Differential metabolites are mainly involved in energy metabolism,osmotic regulation,and neural signaling pathway.In the digestive glands,the levels of major osmolytes(hypotaurine and dimethylglycine)and the branched-chain amino acids involved in osmotic regulation changed significantly.The levels of branched amino acids changed significantly in the gills,while the major osmotic modulators did not.Glycine induced neurotoxicity in the digestive glands.Glutamine was involved in neural signaling but did not induce significant neurotoxicity in the gills.The results indicated that tissue-specific metabolic responses of P.martensii were induced by BaP exposure.(6)The combined analysis of the response pathways of BaP exposure in the digestive glands and gill tissues showed that BaP exposure mainly changed the heat shock protein HSP60,78 kDa glucose-regulated protein,copper/zinc-SOD involved in oxidative stress,proteasome subunit beta type-4,and alpha type-7-like protein associated with the proteolytic pathway had different responses in digestive glands.N-terminal EF-hand calcium-binding protein 1,calmodulin,and calumenin were involved in Ca+ signaling pathway in the gills.The primary metabolites involved in tricarboxylic acid cycle,urea cycle,and glycolysis had changed.Malonate,glycine,and phosphocholine significantly increased in digestive glands,whereas acetoacetate,glutamine,threonine,and others significantly decreased.Arginine,malonate,acetoacetate,and phosphocholine significantly increased in the gills,while the branched chain amino acids,glutamate and glucose significantly decreased.In digestive glands,aspartate aminotransferase,methylmalonate-semialdehyde dehydrogenase,and glutamine synthetase were involved in amino acid metabolism,and aldehyde dehydrogenase was involved in ethanol metabolism.In gills,mitochondrial malate dehydrogenase and fructose-bisphosphate aldolase were involved in malonate and glycolysis metabolism,respectively. |
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