| As a very popular nanopesticide formulation on the market,copper hydroxide nanopesticide is widely used in the prevention and control of bacterial and fungal pathogens in filed crops.In recent years,it is gradually replacing the traditional pesticide formulation copper sulfate.Although a lot of studies have found that the nanopesticide formulations are superior to the traditional formulations in terms of improving effective utilization and alleviating environmental pollution,however,the specific toxic effects of the nanopesticide formulations is scarcely known.Therefore,it is of great practical significance to understand their environmental and ecological risks for safe,effective and rational use of the nanopesticides.This nanopesticide is one of the few commercial nanopesticides that can reach the agricultural watersheds with rainwater and irrigation water,posing a threat to aquatic ecosystems.However,the current toxicological studies of copper hydroxide nanopesticide only paid attention to its acute toxicity to aquatic organisms,antioxidant system interference and developmental toxicity.In view of the mitochondrial toxicity of copper reported in the literatures,this paper paid attention to the ecological risk of the copper hydroxide nanopesticide,using zebrafish as the testing organism.The interfering effect of the nanopesticide on zebrafish energy metabolism was comprehensively investigated.In the meantime,this study revealed the molecular regulation mechanism of the nanopesticide interfering energy metabolism based upon AMPK signaling pathway.The specific results of the paper are as follows:(1)The release rate of copper ions from the nanopesticide was about 70 to 75 %at exposure concentrations.The mortality rates of zebrafish larvae were 2.83,3.50 and21.58 %,respectively,and the malformation rates were 1.08,30.52 and 91.83 %,respectively,at copper concentration of 1,50 and 100 μg/L.The main manifestations were yolk sac edema,spinal curvature and hatching failure.Compared with the copper ions alone at the same copper concentration,exposure of copper ions caused a higher level of copper accumulation in zebrafish than nanopesticide,thus resulting in a higher level of metallothionein and ABC transporters in zebrafish.However,the lethality and teratogenicity of zebrafish exposed to the nanopesticide were significantly higher than those of copper ions at copper concentration of 100 μg/L.The growth and development of zebrafish larvae are closely related to mitochondrial respiration.Exposure to 150 and200 μg/L copper showed that exposure to nanopesticides and copper ions resulted in a significant decrease in mitochondrial respiration in zebrafish embryos.Therefore,the developmental toxicity of new copper hydroxide nanopesticide to zebrafish larvae is not lower than that of copper ions.(2)The reduced mitochondrial oxygen consumptions in zebrafish embryos is one of the important indicators of disturbed energy metabolism in zebrafish larvae.Using metabolomics of energy-related metabolites,activities of key enzymes,and expression levels of related genes,in aspects of glycolysis(GLY),tricarboxylic acid cycle(TCA)and oxidative phosphorylation(OXPHOS),the interfering effect of copper hydroxide nanopesticide on energy metabolism of zebrafish larvae was investigated.The results showed that exposures of the nanopesticides and copper ions alone decreased the levels of a diverse of GLY intermediate metabolites in zebrafish,and the inhibitory effect of the nanopesticide on GLY was more prominent.Compared with copper ions exposure,the nanopesticide also upregulated the levels of several TCA intermediate metabolites in zebrafish,and the activities of TCA rate-limiting enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase were also increased,indicating an increased TCA rate to compensate for the energetic pressure caused by glycolytic inhibition.In addition,the activities of several complex enzymes in the respiratory chain and expressions of their genes were not changed,indicating no effect on OXPHOS by the nanopesticide,but zebrafish attempted to enhance the mitochondrial respiratory chain to alleviate the energetic pressure induced by copper ions alone.We also found that AMP-dependent protein kinase(AMPK)plays an important role in promoting TCA cycle and relieving decreased GLY.(3)Zebrafish adjusted the ratio of different energy substances as changes of energy metabolism.The nanopesticide at 100 μg/L caused a big increase of α-ketoglutarate in TCA cycle in fish.As glutamate and glutamine can be converted into α-ketoglutarate,we measured concentrations of glutamate and glutamine,and found that both of them were significantly decreased in zebrafish.The expressions of glutamate dehydrogenase and glutaminase was also upregulated,indicating that glutamate and glutamine were largely converted into α-ketoglutarate,which entered TCA cycle in mitochondria,as an energy substance.The nanopesticide also upregulated the expressions of Acetyl-Co A carboxylase and fatty acid synthase in zebrafish,thus the concentration of fatty acids was significantly increased,and the Nile Red staining was deepened.However,copper ions at 100 μg/L did not cause this large consumption of glutamate and glutamine and fat accumulation in zebrafish.The dramatic increase of α-ketoglutarate induced by the nanopesticide returned to the control level,and fat accumulation in zebrafish was also alleviated when zebrafish exposed to a combination of AMPK inhibitor Compound C and copper hydroxide nanopesticide.According to western blotting,the nanopesticide activated AMPK in zebrafish larvae and inhibited the synthesis of downstream proteins through AMPK-m TOR signaling pathway,prompting more amino acids to enter the TCA cycle to provide energy,and a part of them were converted into fatty acids and synthesizes fat accumulation in fish.In summary,the copper hydroxide nanopesticide induced an imbalance of energy metabolism,and modulated the ratio of energy substances in zebrafish larvae through AMPK signaling pathway,to relieve the energetic pressure at environmentally relevant concentrations,thus leading to abnormal growth and development.The results of this paper can provide valuable data for the ecological risk assessment of copper hydroxide nanopesticide,and have certain theoretical guiding significance for the development,application and promotion of new nanopesticide formulations. |
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