Effects Of Single And Joint Subacute Exposure Of Copper And Cadmium On Heat Shock Proteins In Common Carp(cyprinus Carpio)

In recent years, heavy metal pollution incidents happened frequently. The effects of heavy metals on human health draw great attention of the public. Copper and Cadmium, widely found in industry waste water,dometic sewage. are two harmful heavy metals to organsims. Fish, the representative of aquatic organisms, receives great harm of heavy metals. Common carp(Cyprinus carpio) plays an important role in aquaculture, but emerging "cadmium rice", water pollution and other events that brought the fish farming environment is worrying.Heat shock protein(heat shock protein, HSP) family is a class of stress proteins. When the body is in stress, the expression of many members of this family increased in response to stress-induced protein denaturation, misfolded and to help the nascent polypeptide chain to form a correct conformation. Meanwhile, many members of the HSP family has also been widely applied to the study of ecotoxicology, the expression level of the body which can reflect the stress state.In this paper, we select common carp as test animals to study the effects of Cu, Cd single and joint exposure on HSP60 HSP70 and HSP90 m RNA levels and protein expression, and their relation to oxidative stress. Furthermore, the research is designed to investigate the mechanisms of heavy metal toxicity to fish and provides evidence to the HSP family in toxicology studies function as a biomarker.420 healthy carp juveniles(average length 13.5087 ± 1.5465 cm, average weight 45.3694 ± 6.7102 g) were randomly selected and divided into the following seven groups: Cu2+ low dose group(0.05 mg/L) and Cu2+ high dose group(0.1 mg/L), Cd2+ low dose group(0.63 mg/L), Cd2+ high dose group(1.26 mg/L), mixed low dose group(0.045 mg/L) and mixed high dose group(0.09 mg/L), and a control group. Each treatment group has 30 fish and two parallel groups. The liver and brain were collected at 24 h, 48 h, 72 h and 96 h. SOD activity, CAT activity are determined. The m RNA levels of HSP60, HSP70 and HSP90 were detected by real-time quantitative PCR. The protein levels were detected by Western blot. The results indicated that:(1) Compared with the control group, the SOD activity of liver in the Cu2+ high dose group was significantly increased at 24h(p<0.05); the other groups’ SOD activity were higher than the level of the control group but no significant change was observed(P>0.05); significant decrease was observed in the mixed high dose group at 48h(p<0.05); significant decrease was also observed in each treatment group(p<0.05), except for the Cd2+ low dose group and the mixed low dose group at 72h; at 96 h, there was significant reduce in each treatment group(p<0.05). In brain tissue, there was no significant difference in SOD activity between the treatment groups and the control group at 24h(P> 0.05); the SOD activity of the Cu2+ high dose group and the mixed treatment groups were significantly lower than the control group(P <0.05); the SOD activity of each group were decreased significantly at 72 h and 96h(P <0.05).(2) Compared with the control group, the CAT activity of liver in each treatment group at 24 h was higher than the level of the control group, but there is no significant change except for the mixed high dose group; at 48 h, each group showed a decrease, but only changed significantly in the Cu2+ high dose group, the Cd2+ low dose group and the mixed low dose group(P <0.05); the CAT activity of each treatment group were decreased significantly at 72 h and 96 h. The changing pattern in brain was similar with that in liver.(3) Compared with the control group, the HSP60 m RNA levels in the Cu2+ low dose group and the mixed low dose group didn’t show significant increase throughout the 96 h exposure(P>0.05); the other groups’ HSP60 m RNA levels increased significantly at 48 h, 72 h and 96h(P <0.05) and reached the peak at 96h; The HSP60 m RNA level of each treatment group showed a significant increase in brain at 48h(P <0.05). The HSP70 m RNA level of each treatment group showed a significant increase in liver at 72h(P <0.05); the significant increase of HSP70 m RNA level in brain appeared at 48 h in liver(P <0.05). In liver, except for the Cu2+ low dose group, the HSP90 m RNA level of each exposure group showed significant increase at 24h(P <0.05); the HSP90 m RNA level of brain increased significantly at 24h(P <0.05).(4) The protein levels of HSP60 in liver showed decrease during the test but the decrease was not significant(P >0.05); the protein levels of each high dose group increased significantly in brain at 48h(P <0.05). In liver, the protein levels of HSP70 decreased significantly at the beginning of the exposure(P <0.05) and increased significantly at 96h(P <0.05); the protein levels of HSP70 in brain didn’t show significant changes until 72 h. In liver, the protein levels of HSP90 didn’t show significant changes all over the exposure; there was significant increase in Cu2+ high dose group, Cd2+ high dose group and two mixed groups at 72hIn summary, single and joint exposure of Cu2+ and Cd2+ could cause oxidative stress in juvenile carps with their SOD and CAT activity significantly decreased and the m RNA levels of HSP60, HSP70 and HSP90 increased significantly in liver and brain. The increase of the protein levels of HSP60, HSP70 and HSP90 indicated that the heavy metal exposure could cause the overexpression of heat shock proteins. Meanwhile, more research is needed to reveal the mechanism of the inconsistent phenomenon between m RNA level and protein level.