Response Of Detoxification Genes And Related Enzymes Activities Of Chinese Mitten Crab (Eriocheir Sinensis) To Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons(PAHs)are persistent organic pollutants that are carcinogenic,teratogenic and mutagenic.PAHs could be metabolized by Phase I and Phase II metabolizing enzyme.In order to study the response of Phase I and Phase II metabolic enzyme in Chinese Mitten Crab(Eriocheir sinensis)exposed to polycyclic aromatic hydrocarbons(PAHs),we choose Phe(Phenanthrene),Chr(Chrysene)and BaP(Benzo(a)pyrene)-three representatives of polycyclic aromatic hydrocarbons.1.Gene cloning and expression profileCYP2A of Chinese Mitten Crab(861bp),CYP2B and CYP4 open reading frames(1476bp and 1557bp)were cloned.Results of bioinformatics and phylogenetic tree confirmed the correctness of the gene.In this study,four genes were commonly expressed in Eriocheir sinensis.The liver,pancreas,gills,hemolymph and muscle are highly expressed.2.Effects of toxicant exposure on gene expression and enzyme activity.The hepatopancreas,gill,hemolymph and muscle with high expression of those four genes were selected to study the expression of four genes and the activities of related enzymes under the exposure of PAHs.(1)Effects of phenanthrene exposure on gene expression and enzyme activity Exposure concentration test:The phenanthrene concentration was set at 0,100,200,400,600 and 800μg/L,and the expression of target gene and the activity of related enzymes were detected after 24 hours exposure.The results showed that CYP2A and CYP4 gene increased firstly and then decreased,CYP2B gene firstly decreased and then increased and then decreased,GST gene is on the rise,CYP2A,CYP2B,CYP4 and GST four genes reached the highest value at 400,600 and 200 and 800 μg/L of phenanthrene,respectively.In contrast,CYP4 and GST genes are more sensitive to phenanthrene concentration,at 200 and 800μg/L they were significantly higher than other-groups(P<0.05).The results showed that the activity of APND,ERND and GST increased firstly and then decreased with the increase of phenanthrene concentration,but the activity of APND,ERND at 600μg/L reached the highest value;GST enzyme activity at 400μg/L reached the highest value.In conclusion,the activities of APND,ERND and GST of three enzymes were significantly higher than those of control group(P<0.05).Exposure time test:To carry out safety exposure experiments in the concentration range of 200μg/L,in 4h,6h,12h,1d,2d,4d,7d,14d,21d and 28d,the expression of the target gene and the activities of related enzymes were detected.The CYP genes may play an important role from the initial exposure stage of phenanthrene and the GST gene was induced at the later period of exposure.Detection of APND,ERND and GST enzyme activity in hepatopancreas,they are sensitive to phenanthrene exposure.In addition to the ERND enzyme in 6h,the activity of three enzymes are all in higher state during 28d,comparing to the control.(P<0.05).(2)Effects of chrysene exposure on gene expression and enzyme activityExposure concentration test:The exposure concentration of chrysene was set at 0μg/L,10μg/L and 100μg/L for 28 days.The expression and the activities of these genes and enzymes were detected in hepatopancreas at Id,2d,4d,7d,14d,21 d and 28d.Results showed that the high concentration groups significantly increases the expression of four genes.The expression of CYP2A and CYP4 at 1d in high concentration was significantly higher than that of control group(P<0.05),The GST gene in 1-21d and CYP2B gene in 1-14d were significantly higher than those in the control group(P<0.05);In addition,CYP2A,CYP2B and GST three genes were also induced by low concentration of chrysene and show significantly high expression in 2d(P<0.05);CYP2B exposed to low concentration shows significantly high expression in 1d(P<0.05),which are more sensitive than the other two genes.Three enzyme activity were significantly induced by high and low concentration of chrysene since 1d.At 1 and 2d,the activity of the three enzymes in the low concentration group was significantly induced than that in the high concentration group(P<0.05).The activity of APND enzyme in 4d,7d,21d and 28d,ERND enzyme in 4d,7d,14d and 21d,as well as GST enzyme in 14d,21d and 28d,showed a significant increasing trend with the increase of chrysene concentration.Exposure time test:Compared with 0d,the relationship between the expression of four genes and different times were studied.Results showed that the expression of four genes in hepatopancreas in both two exposure groups showed a significant time effect,especially in 2d.After 2d,the four genes expressed differently.Among the four genes,The response time of CYP2B gene were more shorter than the other three genes no matter high or low concentration(P<0.05).In addition,the response time of CYP4 gene in the low concentration group was more shorter than the high concentration group(P<0.05).The three enzymes were all induced by chrysene no matter exposed in high or low concentration,and the activity of three enzymes change when time are different(P<0.05).(3)Effects of BaP exposure on gene expression and enzyme activityExposure concentration test:To detect the expression of the target gene and the activity of related enzymes,we design exposure concentration of BaP:0.15μg/L,0.45μg/L,and we design different time to collect samples:0d,Id,2d,4d,7d,14d,21d,28d,and the dates are compared with that from solvent control at each time point.The results showed that four genes in hepatopancreas were significantly induced at high concentrations of BaP.However,the activity of three enzymes were significantly induced since 2d no matter high or low concentration,but at 1 and 2d,the activity of three enzymes in the low concentration group was more significantly induced compared with the high concentration group.However,APND at 21d and 28d,ERND at 4d and 28d,GST at 4-28d showed a significant dose effect.Exposure time test:We compared the date we detect with that in 0d,and the results showed that the expression of CYP2A,CYP4 and GST genes were significantly induced by high concentration of BaP group,and the CYP4 gene was induced more quickly than the other two genes,which was induced significantly since 1d in high concentration group(P<0.05).The CYP2B gene was more sensitive in low concentrations of BaP group compared with other genes.Three enzymes were all induced by BaP no matter in high or low concentration,and the change showed obvious time effect(P<0.05),three enzymes were all induced more quickly to low concentration of BaP group,compared to high concentration group.Only APND showed significantly change at 1d in high BaP concentration group.3.Effects of hepatopancreas-exposed to toxicant in the microstructure.Histology analysis was conducted to evaluate the morphology of hepatopancreas exposed to Phe,Chr,and BaP for four weeks.The results show that all of them have caused obvious damage.Based on the results above,three kinds of toxicants can all stimulate the expression of the target gene,and show a certain time effect and concentration effect.The toxin also had a significant time effect and concentration effect on the related enzymes,and caused damage to the hepatopancreas.