水体中抗生素与铜绿微囊藻的复合污染机制和联合毒性研究

As an increasing concern for water quality assurance, combined pollution control depends on a comprehensive understanding of the interaction effects and mechanisms of combined pollutants. In a previous study of our laboratory, two typical antibiotic pollutants, amoxicillin and spiramycin, were found to interact separately with Microcystis aeruginosa. In the present study, the interaction effects between antibiotics and Microcystis aeruginosa were further investigated, including the combined effects of these two antibiotics on the growth and microcystin-production in Microcystis aeruginosa, as well as the simultaneous degradation of two antibiotics by Microcystis aeruginosa. The molecular and cellular mechanisms of the above interaction effects were then interpreted by examining the responses of microcystin synthetase gene B, antioxidant enzymes and other functional molecules in the algal cells under exposure to single and combined antibiotics. Further, the action pathways of single and combined antibiotics in Microcystis aeruginosa were explored on the protein level via comparative proteomic analysis between the antibiotic-exposed and unexposed algae, according to which the target proteins of antibiotics in the algae were screened. At last, the effects of single and combined antibiotics on the toxicity of microcystins/Microcystis aeruginosa were evaluated using the protein phosphatase inhibition test. The results in the present study could not only indicate the alterations in the pollution levels of antibiotics and Microcystis aeruginosa based on their interactions, but also contribute to the current knowledge on the interaction mechanisms and combined toxicity between them, according to which a combined pollution control method could be suggested.

揭示复合污染机制并建立水体复合污染控制方法，对保障水质安全具有重要意义。本项目通过前期研究证实了单一的抗生素类污染物可与微囊藻发生相互作用。本项目选定两种典型抗生素类污染物阿莫西林和螺旋霉素，进一步研究二者共存时与铜绿微囊藻之间的相互作用，探究混合抗生素对微囊藻增殖和产毒性能的联合生物效应，以及微囊藻对混合抗生素的降解效应。通过测定多肽合成酶基因、抗氧化酶系和多种胞内生物分子对抗生素的响应，分析产生上述相互作用效应的细胞学和分子生物学机制。并基于藻细胞全蛋白质组分析技术，深入探索抗生素与微囊藻之间的作用途径，筛查抗生素对微囊藻的作用靶蛋白。最后选取对微囊藻毒素具有特异响应的"蛋白磷酸酶抑制法"评价抗生素对微囊藻毒素/微囊藻的生物毒性的影响效应。研究结果可以阐明水体中抗生素与微囊藻复合污染水平和联合毒性的变化规律，揭示抗生素与微囊藻的复合污染机制，为抗生素与微囊藻的复合污染治理提供理论依据。

水环境中的抗生素类污染物可以与水华蓝藻发生复杂的相互作用，导致复合污染数量和毒性不再是简单的叠加，有必要深入揭示复合污染变化规律。由于天然水环境中多种抗生素类污染物经常同时存在，本项目以混合抗生素为研究对象。选定两种典型抗生素类污染物阿莫西林和螺旋霉素为目标物质，选定水华蓝藻模式种铜绿微囊藻为目标藻种，研究混合抗生素与铜绿微囊藻之间的相互作用效应、机制以及联合毒性变化规律。采用毒性单位（TU）法评价混合抗生素对微囊藻的联合效应。当目标抗生素分别按照等毒性和等比例混合时，混合抗生素对微囊藻的半数效应浓度分别为1.25 TU和1.83TU，表明二者之间可能存在拮抗效应。当目标抗生素以毒性反比混合时，半数效应浓度为0.86TU，表明二者之间可能存在协同效应。进一步采用浓度加和模型(CA)和独立作用模型(IA)来模拟螺旋霉素和阿莫西林单一毒性的简单加和结果。将模拟数据与实际实验数据进行对比，对比结果证实等毒性配比的两种抗生素确实产生拮抗效应；以毒性反比混合的两种抗生素确实产生协同效应。等比例混合的两种抗生素，随着暴露浓度的增加，由协同效应逐渐转变为拮抗效应。选定与现有水环境中抗生素污染浓度相近的暴露浓度(800ng/L)进行后续试验，发现随着混合抗生素中螺旋霉素的比例上升，对藻细胞生长的效应由刺激逐步转变为抑制，复合污染物的生物毒性也由高于微囊藻单一污染变为低于微囊藻单一污染。混合抗生素在各比例下均刺激藻毒素的合成，而阿莫西林是刺激微囊藻生长和藻毒素合成的主因。叶绿素a、psbA、psaB和rbcL基因的表达量变化规律显示抗生素可能经由光合作用调控微囊藻的生长。此外，ntcA和mcyB基因在混合抗生素调控下的表达量与藻毒素合成量之间存在正相关关系。抗氧化系统的响应表明，螺旋霉素的高毒性可以导致过氧化氢在藻细胞内累积从而产生细胞氧化损伤，这可以解释随着螺旋霉素比例上升，混合抗生素对微囊藻混合效应的变化规律。蛋白质组学研究表明，混合抗生素可以直接调控微囊藻的光合作用、能量和物质代谢、藻毒素合成。从微囊藻蛋白质组中，共筛查出6个与其他差异蛋白交联程度最高、参与蛋白质相互作用最多的靶蛋白。项目研究发现，共存的低浓度抗生素污染物会刺激微囊藻爆发，需要加以控制，尤其是阿莫西林，应将其在混合抗生素中的比例控制在50%以下。研究结果可为水环境中抗生素和微囊藻复合污染控制策略制定提供依据。