神经抑制污染物连续暴露下大型溞（Daphnia magna）外在行为响应及内在机制研究
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摘要
水体污染严重影响水生生态系统稳定及威胁人类的健康安全。利用水生生物行为变化监测水体环境可以实现对水体环境质量的实时、快速的监测。水生生物在环境污染物暴露下，对水生生物产生的毒性效应会表现在五个水平上，分别是分子水平、细胞水平、组织水平、个体水平和群体水平。通常水生动物在个体水平上表现为游泳能力、回避行为、条件反射能力等各种行为的变化。个体水平上生物的行为强度与暴露污染物的相对效应浓度和暴露的时间有着明显的相关性。这种行为变化与环境压力大小直接相关，其在一定环境压力下的行为变化与环境压力阈模型(Stepwise Stress Model, SSM)所述的生物行为环境模型相一致。环境毒物对生物个体影响，首先作用于生物机体的相关生物指示物(酶或蛋白)。因此，生物行为强度变化的同时，生物体内相应的标志物必然也做出了相应的反应。
本实验研究了体内抑制和体外抑制试验下，有机磷类农药敌敌畏、氨基甲酸酯类农药灭多威以及拟除虫菊酯类农药溴氰菊酯分别在2TU和5TU浓度下的指示生物大型溞(Daphnia magna)的生物标志物乙酰胆碱酯酶(AChE)活性的变化趋势。结合生物的环境压力阈模型，分析大型溞的乙酰胆碱酯酶活性的变化规律，探索大型溞对受试污染物的内在响应机制。
通过实验发现大型溞的AChE活性与暴露农药的浓度和暴露时间有明显的相关性，其中体外抑制试验中，AChE活性还与农药的毒理机制有关：
1. 24 h急性毒性试验结果表明：不同污染物对大型溞的毒力大小有所差异，敌敌畏、灭多威和溴氰菊酯对24~48 h大型溞的24 h-LC50分别为0.746μg/L、9.829μg/L和6.680μg/L。大型溞的死亡率与污染物的浓度呈正相关性，具有明显的剂量－效应关系。大型溞在受到不同种类环境污染物的刺激时，均会表现出明显的行为变化，出现回避行为，这与污染物的致毒机理无直接关系。
2.敌敌畏、灭多威以及溴氰菊酯在体内抑制试验中对大型溞AChE活性具有明显的抑制作用。整体上，随着暴露时间的增加，实验组大型溞的AChE活性呈下降趋势明显低于空白对照组，且高浓度组(5 TU)污染物对酶活性的抑制作用明显高于低浓度组(2 TU)；在一定浓度污染物暴露中，AChE活性的抑制率随时间增加呈上升趋势。体内抑制试验由于受生物体内代谢的影响，AChE活性在某些时间会出现回升现象。
3.体外抑制实验结果显示，有机磷类与氨基甲酸酯类农药对AChE活性的抑制作用明显。敌敌畏、灭多威的作用下，AChE活性的抑制率在一定时间范围内随着作用时间的增加而上升且5 TU浓度组的对酶活性的抑制强度要高于2 TU浓度组。由于溴氰菊酯的作用靶位点非AChE，因此对AChE活性的影响较小。
在体内抑制试验中，污染物连续暴露下，不同时间酶活性的相对活性不一定，可能会出现升高或降低的情况，但总的变化趋势是下降。这与生物的体外行为强度变化规律基本吻合，符合环境压力阈模型(SSM)。通过比较污染物在体内和体外抑制试验中对AChE活性的影响，发现同种污染物对生物的AChE活性的抑制率存在差异。
Recently, water pollution has been a serious influence on the stability ofaquatic ecosystem and human security. Using the behavior change of aquaticorganism, can carry out real-time and rapid water quality monitoring. Aquaticorganisms exposed to the environmental pollution would produce the toxiceffects of performance in five levels, respectively molecular level, cellular level,organizational level, the individual level and the group level. Usually aquaticanimals in individual performance level display various kinds of behaviorchange such as swimming ability, avoidance behavior, reflex ability and so on.The behavior change of aquatic organism which exposed to pollutants has beenrelativity with contaminant concentration and exposure time on the individuallevel. This kind of behavior change is directly related to environmentalpressure, under certain environmental pressure, the trend of behavior changecorresponds with Stepwise Stress Model. Contaminants have a primordiallyeffect on biological indicator of organisms like enzymes or proteins. Therefore,with the biological indicator change, biological behaviors will also make acorresponding reaction.
In this study, the inhibitory effects of three insecticides, dichlorvos,methomyl and deltamethrin, on acetylcholinesterase of Daphnia magna wereinvestigated in the laboratory. The concentrations of insecticides are 2 TU and5 TU, and 1 TU is defined as the 24 h-LC50 of insecticides. Combined withStepwise Stress Model, to analyze the trend on cholinesterase activity ofDaphnia magna exposed to pollutants and explore the response mechanism.
The results show that the AChE activity has obviously relationship withthe relative concentration of insecticides based on 24 h-LC50 and the exposure time, hereinto, there is obviously relationship between AChE activity andtoxicological mechanism of pesticides in vitro.
Firstly, the results of acute toxicity indicated that different pollutants havedifferent virulence to Daphnia magna and the 24h-LC50 of insecticides, DDVP,methomyl and deltamethrin, were 0.746μg/L, 9.829μg/L and 6.680μg/L.Meanwhile, an obvious positive relationship was observed between themortality rate of Daphnia magna and the concentration of insecticides. Underthe stimulation of different types of environmental pollutants, Daphnia magnawere showing significant behavioral changes.
Secondly, in vivo, dichlorvos, methomyl and deltamethrin had obviousinhibition on AChE activity of Daphnia magna. Overall, along with theincrease of the exposure time, AChE activities of experimental groups dropsignificantly lower than blank control groups. The inhibition rates of threeinsecticides on AChE were increasing gradually with time, and the 5 TUgroups were more serious influence on AChE activity than the 2 TU groups.The result indicated that metabolism maybe had an effect on the insecticidesinhibit AChE activity, sometimes; the back sampling's AChE activity maybeappears higher than the previous one with time.
Finally, in vitro, just dichlorvos and methomyl had important influence onAChE activity of Daphnia magna. Within the scope of a certain time, theinhibition on AChE activity of Daphnia magna would go up with the increaseof exposure time exposed to dichlorvos or methomyl. However, deltamethrinhad less impact in AChE activity, because AChE was not the target position.
In conclusion, AChE activity was indeterminacy at different time, mayappear raise or lower, but total trend is downward trend with time in pollutantscontinued exposure. The trend is consistent with the rule of biologicalbehaviors, which accorded with Stepwise Stress Model (SSM). Comparing theresults between the inhibition on AChE activity of Daphnia magna in vitro andin vivo, concluded that the same insecticides caused different inhibition rate onthe activity of AChE.
本实验研究了体内抑制和体外抑制试验下，有机磷类农药敌敌畏、氨基甲酸酯类农药灭多威以及拟除虫菊酯类农药溴氰菊酯分别在2TU和5TU浓度下的指示生物大型溞(Daphnia magna)的生物标志物乙酰胆碱酯酶(AChE)活性的变化趋势。结合生物的环境压力阈模型，分析大型溞的乙酰胆碱酯酶活性的变化规律，探索大型溞对受试污染物的内在响应机制。
通过实验发现大型溞的AChE活性与暴露农药的浓度和暴露时间有明显的相关性，其中体外抑制试验中，AChE活性还与农药的毒理机制有关：
1. 24 h急性毒性试验结果表明：不同污染物对大型溞的毒力大小有所差异，敌敌畏、灭多威和溴氰菊酯对24~48 h大型溞的24 h-LC50分别为0.746μg/L、9.829μg/L和6.680μg/L。大型溞的死亡率与污染物的浓度呈正相关性，具有明显的剂量－效应关系。大型溞在受到不同种类环境污染物的刺激时，均会表现出明显的行为变化，出现回避行为，这与污染物的致毒机理无直接关系。
2.敌敌畏、灭多威以及溴氰菊酯在体内抑制试验中对大型溞AChE活性具有明显的抑制作用。整体上，随着暴露时间的增加，实验组大型溞的AChE活性呈下降趋势明显低于空白对照组，且高浓度组(5 TU)污染物对酶活性的抑制作用明显高于低浓度组(2 TU)；在一定浓度污染物暴露中，AChE活性的抑制率随时间增加呈上升趋势。体内抑制试验由于受生物体内代谢的影响，AChE活性在某些时间会出现回升现象。
3.体外抑制实验结果显示，有机磷类与氨基甲酸酯类农药对AChE活性的抑制作用明显。敌敌畏、灭多威的作用下，AChE活性的抑制率在一定时间范围内随着作用时间的增加而上升且5 TU浓度组的对酶活性的抑制强度要高于2 TU浓度组。由于溴氰菊酯的作用靶位点非AChE，因此对AChE活性的影响较小。
在体内抑制试验中，污染物连续暴露下，不同时间酶活性的相对活性不一定，可能会出现升高或降低的情况，但总的变化趋势是下降。这与生物的体外行为强度变化规律基本吻合，符合环境压力阈模型(SSM)。通过比较污染物在体内和体外抑制试验中对AChE活性的影响，发现同种污染物对生物的AChE活性的抑制率存在差异。
Recently, water pollution has been a serious influence on the stability ofaquatic ecosystem and human security. Using the behavior change of aquaticorganism, can carry out real-time and rapid water quality monitoring. Aquaticorganisms exposed to the environmental pollution would produce the toxiceffects of performance in five levels, respectively molecular level, cellular level,organizational level, the individual level and the group level. Usually aquaticanimals in individual performance level display various kinds of behaviorchange such as swimming ability, avoidance behavior, reflex ability and so on.The behavior change of aquatic organism which exposed to pollutants has beenrelativity with contaminant concentration and exposure time on the individuallevel. This kind of behavior change is directly related to environmentalpressure, under certain environmental pressure, the trend of behavior changecorresponds with Stepwise Stress Model. Contaminants have a primordiallyeffect on biological indicator of organisms like enzymes or proteins. Therefore,with the biological indicator change, biological behaviors will also make acorresponding reaction.
In this study, the inhibitory effects of three insecticides, dichlorvos,methomyl and deltamethrin, on acetylcholinesterase of Daphnia magna wereinvestigated in the laboratory. The concentrations of insecticides are 2 TU and5 TU, and 1 TU is defined as the 24 h-LC50 of insecticides. Combined withStepwise Stress Model, to analyze the trend on cholinesterase activity ofDaphnia magna exposed to pollutants and explore the response mechanism.
The results show that the AChE activity has obviously relationship withthe relative concentration of insecticides based on 24 h-LC50 and the exposure time, hereinto, there is obviously relationship between AChE activity andtoxicological mechanism of pesticides in vitro.
Firstly, the results of acute toxicity indicated that different pollutants havedifferent virulence to Daphnia magna and the 24h-LC50 of insecticides, DDVP,methomyl and deltamethrin, were 0.746μg/L, 9.829μg/L and 6.680μg/L.Meanwhile, an obvious positive relationship was observed between themortality rate of Daphnia magna and the concentration of insecticides. Underthe stimulation of different types of environmental pollutants, Daphnia magnawere showing significant behavioral changes.
Secondly, in vivo, dichlorvos, methomyl and deltamethrin had obviousinhibition on AChE activity of Daphnia magna. Overall, along with theincrease of the exposure time, AChE activities of experimental groups dropsignificantly lower than blank control groups. The inhibition rates of threeinsecticides on AChE were increasing gradually with time, and the 5 TUgroups were more serious influence on AChE activity than the 2 TU groups.The result indicated that metabolism maybe had an effect on the insecticidesinhibit AChE activity, sometimes; the back sampling's AChE activity maybeappears higher than the previous one with time.
Finally, in vitro, just dichlorvos and methomyl had important influence onAChE activity of Daphnia magna. Within the scope of a certain time, theinhibition on AChE activity of Daphnia magna would go up with the increaseof exposure time exposed to dichlorvos or methomyl. However, deltamethrinhad less impact in AChE activity, because AChE was not the target position.
In conclusion, AChE activity was indeterminacy at different time, mayappear raise or lower, but total trend is downward trend with time in pollutantscontinued exposure. The trend is consistent with the rule of biologicalbehaviors, which accorded with Stepwise Stress Model (SSM). Comparing theresults between the inhibition on AChE activity of Daphnia magna in vitro andin vivo, concluded that the same insecticides caused different inhibition rate onthe activity of AChE.
引文
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