Effects Of Cadmium On P-glycoprotein Kinetics And Expression In Carassius Auratus

With the development of urbanizationin and dustrialization in China,heavy metal pollution in water has emerged an important issue.Of the heavy metals,cadmium(Cd)is regarded as the most toxic one,and is considered to be one of the main marine pollutants.The Crucian carp is an excellent variety of freshwater aquaculture in China and can accumulate extremely high levels of Cd in their tissues.Bioaccumulation of Cd in aquatic animals may be harmful to non-target animals and humans.Hence,heavy metal pollution of The Crucian carp has become a potential threat of fish food and human health.Therefore,it is urgent to develop effective measures to reduce heavy metal residues in fish.For eliminating the harm of Cd and other harmful heavy metals residues in crucian carp and ensuring food safety,the most effective way is to reduce the content of Cd and other harmful heavy metals from the source.Therefore,the accumulation of Cd in the Crucian carp can be inhibited by improving the self-defense mechanism of the body,and the pollution of Cd in the Crucian carp can be reduced.Aquatic organisms has developed an effective defensive system termed multi-xenobiotic resistance(MXR)that is similar to the multi-drug resistance(MDR)of cancer cells.MXR can pump various endogenous and exogenous toxins out of the cells to protect cells from damage.MXR consists of multiple transmembrane proteins,of which P-glycoprotein(P-gp)plays an important role in heavy metal expulsion.P-gp can directly remove toxins from the body,including Cd.Extensive researches have been performed on the functions of P-gp in aquatic organisms,but its role in the bioaccumulation of Cd in the Crucian carp has rarely reported.Therefore,the purpose of this study is to investigate whether P-gp plays any role in the bioaccumulation of Cd from the Crucian carp.The main researches and results are as follows: 1 Bioaccumulation of heavy metal Cd in the Crucian carpDuring the exposure of the Crucian carp to Cd,The liver and gill tissues had a rapid and strong bioaccumulation of Cd.Moreover,The accumulation order of Cd in tissues was gills > liver.The results also indicated that the different concentrations and the different treatment time of Cd had significant effects on the bioaccumulation of Cd(p<0.05).At the same time,as the concentration of Cd increasing,the bioaccumulation of Cd increased significantly in the liver and gill(p<0.05).The liver and gill after exposed to same concentration of Cd,for the low concentration treatment,the bioaccumulation of Cd increased at the early period,and then decreased at the later period.While for the middle and high concentration treatment,the bioaccumulation of Cd always increased significantly through the time.2 Effects of heavy metal Cd on P-gp activity in the Crucian carpP-gp activity was performed using the efflux version of the method with rhodamine B as the model P-gp substrate.The results showed that P-gp played an important role in the resistance of Cd in the Crucian carp.When Cd bioaccumulated in the liver and gill,P-gp activity increased and could pump out Cd in order to protect the body.The results also indicated that the different concentrations and the different treatment time of Cd had significant effects on the P-gp activity(p<0.05).At the same time,P-gp activity increased significantly in the liver and gill with the increase of the concentration of Cd(p<0.05).The liver and gill after exposed to same concentration of Cd,P-gp activity showed a tendency to rise first and then decrease with the exposure time.3 Effect of P-gp inhibitor Verapamil(Ver)on P-gp activityThe inhibitor of P-gp,verapamil(VRP),in the 66.7 ?mol/L,could inhibit the efflux rate of Rho B in the liver and gill,and the activity of P-gp was inhibited.It is indicated that Ver can effectively inhibit the P-gp activity,and further reflected the importance of P-gp in the resistance of Cd in the Crucian carp 4 Effects of heavy metal Cd on the mRNA expression of P-gp in the Crucian carpEffect of Cd on the mRNA expression of P-gp in the Crucian carp tissues was detected by fluorescence quantitative PCR.The results showed that under the experimental condition,the P-gp mRNA expression was up-regulated in the liver and gill of the Crucian carp.For the low concentration treatment at 24 h,compared to the control group,the P-gp mRNA expression in the liver increased but had no significant difference(p>0.05).However,there was no similar pattern in the gill.At the same time,as the concentration of Cd increasing,the P-gp mRNA expression increased in the liver and gill.The liver and gill after exposed to same concentration of Cd,the P-gp mRNA expression increased at the early period,and then decreased at the later period.The changes of P-gp mRNA expression and activity showed similar time-effect and dose-effect patterns during Cd exposure.5 Effects of heavy metal Cd on the protein expression of P-gp in the Crucian carpEffect of Cd on the protein expression of P-gp in the Crucian carp tissues was detected by immunohistochemistry.The results showed that under the experimental condition,the protein expression of P-gp was up-regulated in the liver and gill of the Crucian carp.In the liver,at the same time,the protein expression of P-gp increased with the increase of the concentration of Cd.Exposed to same concentration of Cd,the protein expression of P-gp increased and reached the maximum at 72 h respectively,and then decreased.However,there was no similar pattern in the gill.Overall,Cd up-regulated the protein expression of P-gp in the gill.