Effects Of Benzo[a]Pyrene And Phenanthrene On Accumulation,Metabolism And Tissue HSP70 Gene Expression In Mytilus Coruscus

Polycyclic aromatic hydrocarbons(PAHs)are a kind of widespread persistent organic pollutants in the environment.Benzo[a]pyrene(BaP)is a typical representative of highly carcinogenic high molecular weight PAHs.Phenanthrene(Phe)is a simple model compound of tricyclic PAHs.BaP and Phe have high octanol water partition coefficients,which are easy to be enriched by marine organisms,transferred and amplified through the food chain,and have a variety of toxic effects on marine ecological environment and organisms.In this study,Mytilus coruscus was taken as the research object.Through enrichment and release experiments,high performance liquid chromatography,real-time quantitative PCR technology and other experimental methods,the accumulation law and release law of BaP(0 μg/L、0.2 μg/L、1.0 μg/L and3.0 μg/L)and Phe(0 μg/L、4 μg/L、20 μg/L and 100 μg/L)in three tissues(visceral mass,outer membrane and closed shell muscle)of Mytilus coruscus during the 1st day,the 3rd day,the 6th day and the 10 th day of exposure stage and the 12 th day and the15 th day of water release stage were studied,and the dose time effect relationship between metabolite 3-OH BaP and BaP,the effects of BaP and Phe on HSP70 mRNA expression in three tissues of Mytilus coruscus were also investigated.The results are as follows.(1)The content of BaP in the three tissues of Mytilus coruscus exposed to different concentrations of BaP solution increased significantly compared with the seawater control group at the enrichment stage and water release stage(P < 0.05).In the enrichment stage,the concentration and absorption rate of BaP in three tissues were as follows: the visceral mass > the outer membrane > the closed shell muscle,and the concentration of BaP increased with the increase of exposure concentration,which was related to the physical properties of BaP,life characteristics and tissue specificity of Mytilus coruscus.In the stage of water release,the release rate and release rate of BaP from the three tissues of Mytilus coruscus in different concentration groups were as follows: the visceral mass > the outer membrane > the closed shell muscle,which was related to the metabolic activity,excretion of Mytilus coruscus and diffusion of BaP in the solution.(2)3-OH BaP is the main hydroxyl metabolite of BaP in Mytilus coruscus.The production of 3-OH BaP is related to the metabolism transformation and tissue specificity of BaP.Under different concentrations of BaP solution,the content of 3-OH BaP in the visceral mass,outer membrane and closed shell muscle of mussel increased with the increase of BaP exposure time.The content of 3-OH BaP in the three tissues was significantly increased compared with that of the sea water control group(P < 0.05),that is,the content of 3-OH BaP in the visceral mass,outer membrane and closed shell muscle of Mytilus coruscus was significantly related to the time of BaP exposure.In addition,the relationship between the concentration of 3-OH BaP and BaP in Mytilus coruscus tissues was analyzed.The results showed that the content of 3-OH BaP in visceral mass,outer membrane and closed shell muscle was positively correlated with the concentration of BaP in each tissue,showing a significant dose-effect relationship.(3)The expression of HSP70 mRNA in visceral mass,outer membrane and closed shell muscle of Mytilus coruscus exposed to different concentrations of BaP was induced to varying degrees.HSP70 could protect cells from BaP stress in three tissues of Mytilus coruscus,of which 0.2 μg/L and 1.0 μg/L group: the expression of HSP70 mRNA in three kinds of tissues was induced to increase in BaP exposure stage,and there was no significant difference in the expression of HSP70 mRNA between the three groups and the control group in the release stage(P > 0.05).3.0 μg/L group: in the whole enrichment and release stage,the expression of HSP70 mRNA in three kinds of tissues was significantly increased compared with the control group(P < 0.05).(4)When Mytilus coruscus was exposed to Phe solution,the accumulation of Phe in three tissues of Mytilus coruscus was visceral mass > outer membrane > closed shell muscle at the same time and concentration.The content of Phe in the three tissues of Mytilus coruscus exposed to different concentrations of Phe solution increased significantly compared with the seawater control group in the enrichment stage and water release stage(P < 0.05).At the same time,it can be seen that the accumulation of Phe in three kinds of tissues increased with time,and showed a dose effect relationship.In the stage of water release,the release rate of Phe from visceral mass ranged from50% to 60%.The release rate of Phe from outer membrane gradually decreased with the increase of exposure concentration,and that from closed shell muscle gradually increased with the increase of exposure concentration,which was related to tissue specificity.(5)Under different concentrations of Phe stress,HSP70 mRNA expression in visceral mass,outer membrane and adductor muscle of Mytilus edulis was induced in different degrees.The order of HSP70 mRNA expression in visceral mass,outer membrane and closed shell muscle was outer membrane > visceral mass > closed shell muscle.The expression of HSP70 mRNA in visceral mass and outer membrane of Mytilus coruscus was significantly higher in enrichment stage than in water release stage;The expression of HSP70 mRNA in closed shell muscle increased significantly in the early stage of enrichment,and was significantly inhibited in the late stage of enrichment.However,the expression of HSP70 mRNA in closed shell muscle was induced again in the stage of water release.In this study,the enrichment of BaP and Phe in visceral mass,outer membrane and closed shell muscle of Mytilus coruscus was determined;The effects of BaP and Phe on HSP70 mRNA expression in three tissues of Mytilus coruscus were analyzed;The time dose effect relationship between 3-OH BaP and BaP was expounded,and the metabolism and transformation of BaP in Mytilus coruscus was preliminarily mastered.The results of this experiment can provide reference for the accumulation kinetics,metabolic transformation mechanism and toxic mechanism of Phe and BaP in shellfish.