为探明铜离子(Cu2+)对两栖动物肝脏线粒体中三羧酸(Tricarboxyl acid, TCA)循环及自由基代谢的毒理作用,采用静水暴露实验,研究了Cu2+不同浓度和不同暴露时间对虎纹蛙(Hoplobatrachus chinensis)肝脏线粒体中异柠檬酸脱氢酶(ICDHm)活性、α-酮戊二酸脱氢酶(α-KGDH)活性、抗超氧阴离子(anti-·O2–)活性、过氧化氢(H2O2)含量、抑制羟自由基(inhabit-·OH)活性、一氧化氮(NO)含量以及一氧化氮合成酶(NOS)活性的影响。暴露实验共设置6个Cu2+浓度组(0.0、2.0、4.0、6.0、8.0和10.0 mg/L),分5个暴露时间(0、24h、48h、72h和96h)取材,对每个浓度的不同暴露时间分别取6个样本,测定TCA循环及自由基代谢的相关指标。结果显示,在TCA循环中随着Cu2+浓度的增加和暴露时间的延长,时间和浓度因素对ICDHm活性影响无显著性交互作用(P>0.05),暴露时间的延长对 ICDHm 活性无显著性影响(P>0.05),但随着 Cu2+浓度的增加ICDHm 活性逐渐减小；而时间和浓度因素对α-KGDH 活性影响有显著交互作用(P<0.05),暴露处理后α-KGDH活性下降,分别在24h和96h的4.0、6.0 mg/L时活性最低。在自由基代谢中,时间和浓度因素对抗·O2–活性、H2O2含量影响有显著交互作用(P<0.05),而对抑制·OH 活性、NO 含量、NOS 活性的影响无显著性交互作用(P>0.05)。不同时间随着 Cu2+浓度的增加,抗·O2–活性均呈现出逐渐下降的趋势；实验处理后H2O2含量升高,在24h的6.0 mg/L时含量最大；随着暴露时间的延长和Cu2+浓度的增加抑制·OH活性均逐渐降低；而NO 含量和NOS 活性的变化趋势基本相同,即随着Cu2+浓度的增加先增加后减少并趋近0浓度组,且都在6.0 mg/L时达到最大。研究结果表明急性Cu2+暴露对虎纹蛙肝脏线粒体中TCA循环及自由基代谢有显著的毒性作用。%In this study, we applied acute toxicity test to investigate the toxic effects of copper ion (Cu2+) on tricarbo-xylic acid cycle (TCA) and free radical metabolism in the mitochondria of the liver ofHoplobatrachus chinensis. We treated the animals with Cu2+at different concentrations and for different exposure time, and tested the activities of TCA-related enzymes and free radicals. We found that although Cu2+ at high concentrations could reduce the activity of ICDHm, prolonged exposure time had no significant effect on ICDHm (P > 0.05). We did not observe cross effects be-tween the concentration of Cu2+ and the exposure time on the activity of ICDHm (P > 0.05). However there were sig-nificant cross effects between the exposure time and the concentration of Cu2+ on the activity ofα-KGDH (P < 0.05). The activity ofα-KGDH decreased when exposed to Cu2+, and the lowest activities were detected when the exposure times were 24h and 96h and concentrations were 4.0 and 6.0 mg/L. In terms of free radical metabolism, the exposure time and copper concentration had strong cross effects on the activities of anti-·O2– and H2O2 (P < 0.05), but not on the activity of inhibit-·OH, the content of NO, and the activity of NOS. The activities of anti-·O2– decreased along with the increase in the concentration of Cu2+. The content of H2O2 increased after the treatments and perked at 24h and 6.0 mg/L. There was a negative correlation between the activity of inhibit-·OH and the exposure time and the concentration of Cu2+. Along with the increase in the concentration of Cu2+, the content of NO and the activity of NOS first increased and then decreased to the values of the control group, and the maximum values appeared in the 6.0 mg/L group. Our study demonstrated that the acute exposure of Cu2+ could have significant toxic effects on TCA cycle and free radical metabo-lism in the mitochondria of the liver ofH. chinensis.
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