| Phoxim,as an efficient and low toxic organophosphorus pesticide,is widely used in agricultural production,pond cleaning and pest control.It has a good killing effect on fish diseases and pests.Phoxim is low toxic to mammals,but high toxic to fish.When entering the water environment,it may cause potential harm to fish.Nowadays,there are few studies on the effects of phoxim in fish,especially in Carassius auratus gibebio,which is widely cultivated in China.Therefore,the effect of phoxim on the activity of major subtypes of cytochrome P450 in liver microsomes of Carassius auratus gibebio was studied in this study.The effects of phoxim on the expression of CYP1 A and CYP3 A protein and mRNA in Carassius auratus gibebio liver microsomes were investigated by Western blot and real-time quantitative PCR,respectively.Furthermore,the effect of phoxim on the phosphorylation of CYP450 protein in hepatic microsomes was studied by SDS-PAGE electrophoresis and Pro-Q Diamond fluorescence staining.The main results are as follows:1 Effects of phoxim on the activity of main cytochrome P450 subtypes in the liver of Carassius auratus gibebioThe effects of phoxim exposure at different concentrations and times on total CYP450 content,CYP2 E,CYP1A and CYP3 A activity in the liver of Carassius auratus gibebio were determined.In general,phoxim could reduce the production of CYP450 content.The influence of phoxim on the activity of CYP450 was different at different time points.The effect of phoxim on the activity of CYP450 was significantly different in different concentration groups after 96 h(P < 0.01),but there was no significant time-dependent manner in different concentration groups.The effect of phoxim on the activity of CYP2 E was not entirely inhibition or induction.It showed induced effect in low and medium concentration groups at the early stage of exposure(24,48 h),the others showed inhibition.There was a significant dose-dependent manner after 24 h and 96 h exposure,and time-dependent manner existed in both low and medium concentration groups,while CYP2 E activity increased in high concentration group after 96 h exposure.On the whole,phoxim inhibited the activity of CYP1 A.Except for the obvious dose-dependent manner after 24 h,with the increase of phoxim concentration,the more obvious inhibitory effect was.The dose-dependent manner was not significant in other groups.But there was significant time-dependent manner among low,medium and high concentration groups.The longer the exposure time,the lower the activity of CYP1 A.The activity of EROD in the low,medium and high concentration groups after 96 h was significantly different from that in the control group(P < 0.01);Phoxim inhibited the activity of CYP3 A as a whole and showed a dose-dependent relationship after 24 h and 72 hours.Compared with the control group,the activity of CYP1 A in the high concentration group after 24 h was significantly different(P < 0.01).After being poisoned,the difference of enzyme activity between medium and high concentration groups was significantly different(P < 0.01).After 96 hours exposure,there was only significant difference in the high concentration group,but no significant difference in the low and medium concentration group compared with the control group(P > 0.05).2 Effects of phoxim on mRNA expression of CYP1 A and CYP3 A in the liver of Carassius auratus gibebioThe effects of phoxim exposure at different concentrations and times on the mRNA expression of CYP1 A and CYP3 A in the liver of Carassius auratus gibebio were determined by real-time quantitative PCR.The results of the effect of phoxim on the mRNA expression of CYP1 A in the liver of Carassius auratus gibebio showed that it was significantly down-regulated on the whole,and there was a strong dose-dependent relationship among the groups exposed at different time points,but the expression level was positively correlated with the concentration after 48 h and negatively correlated with the other time groups.After 48 h and 72 h,the differences between the groups were significant compared with the control group(P < 0.01).Low and medium concentration groups showed a low expression level after 48 h exposure,and then increased gradually with the increase of exposure time,while the expression level in high concentration groups did not differ significantly at each time point(P > 0.05).The results of the effect of phoxim on the mRNA expression of CYP3 A in Carassius auratus gibebio liver showed that it was significantly down-regulated on the whole.Except that the mRNA expression of CYP3 A increased with the increase of concentration after 48 h exposure,there was no significant dose-dependent relationship among other time groups.After 72 h and 96 h exposure,there were significant differences among the concentration groups compared with the control group.The expression level in low concentration group was low after 72 h,and then increased slightly.In medium concentration grounps,there was a significant time-dependent manner.With the increase of exposure time,the expression level decreased.3 Effects of phoxim on protein expression of CYP1 A and CYP3 A in the liver of Carassius auratus gibebioThe effects of phoxim exposure at different concentrations and times on protein expression of CYP1 A and CYP3 A in the liver of Carassius auratus gibebio were determined by Western blot.The results of the effect of phoxim on protein expression of CYP1 A in the liver of Carassius auratus gibebio showed that it was significantly different from that of the control group(P < 0.01).The protein expression of CYP1 A in different concentration groups at different time showed the same dose-dependent manner.Compared with the control group,the protein expression of CYP1 A in low,medium and high concentration groups was different after 96 h exposure.The results showed that phoxim could decrease the expression of CYP3 A protein in the liver of Carassius auratus gibebio as a whole.The protein expression of CYP3 A showed the same dose-dependent manner in different concentration groups at different time points.After 24 h exposure,the expression of CYP3 A protein in each concentration group was significantly different from that in the control group(P < 0.01).There were significant differences in high concentration group(P < 0.05).And there were significant differences between medium and high concentration groups at 72 and 96 h(P < 0.05).The protein expression of CYP3 A only showed significant time-dependent manner at low concentration.The protein expression of CYP3 A increased slightly at 48 h in medium and high concentration groups,and then decreased continuously.4 Effects of phoxim on the phosphorylation of CYP450 in the liver of Carassius auratus gibebioThe effects of phoxim on the phosphorylation of CYP450 protein in hepatic microsomes were studied by SDS-PAGE electrophoresis and Pro-Q Diamond,which is the specific phosphorylation protein dye.Electrophoresis results showed that there were four bands those molecular weights ranged from 45 to 60 kDa.The molecular weights of bands 1,2,3 and 4 after being analyzed by image lab software were about 59,56,51 and 45 kDa,respectively.Except for the significant difference between the medium and high concentration groups exposed to phoxim after 72 h,there was no significant difference in the phosphorylation of CYP450 protein among the groups of each band(P > 0.05).The P/T values of phosphorylation protein in each band were positively correlated with CYP1 A and CYP3 A activities.Except band 2,Pearson correlation coefficients of phosphorylation protein level and CYP1 A activity in the other bands were all greater than 0.6(P < 0.01),showing strong correlation;Pearson correlation coefficients of phosphorylation protein level and CYP3 A activity in band 2 and band 4 were both greater than 0.6,and it showed the strongest correlation in band 4 of which Pearson correlation coefficient was 0.727(P = 0.001). |
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