【作者】 邓睿；

【导师】 林道辉；

【作者基本信息】 浙江大学 ， 环境科学， 2020， 博士

【摘要】 碳纳米管（CNTs）是目前使用最广泛的纳米材料之一,经排放进入环境后可与共存污染物产生复合生物效应,危害生态环境。目前对CNTs与有机污染物（OCs）复合毒性的案例研究以及相关机理的认识还很欠缺,有必要研究CNTs-OCs的细菌毒性以及OCs性质和细菌种类的影响。五氯酚（PCP）属于优先控制污染物和人类致癌物,是一种典型的传统OCs,而环丙沙星（CIP）作为氟喹诺酮类抗生素的代表,已被确定为环境中常见的新型OCs;PCP和CIP分别是疏水性和亲水性物质,与CNTs的相互作用和复合毒性可能不同。革兰氏阴性菌大肠杆菌（E.coli）和阳性菌枯草芽孢杆菌（B.subtilis）具有不同的理化性质和生理特征,对同一暴露体系的响应可能不同。本文通过显微观察、生物化学指标测定、生物累积实验、转录组学和代谢组学分析等方法,探讨了一种氧化多壁碳纳米管（OCNTs）与PCP或CIP对E.coli和B.subtilis的复合毒性及致毒机理。取得如下主要研究结果:（1）OCNTs和PCP均能通过破坏细胞膜、诱导氧化应激和干扰基因表达来抑制E.coli的生长繁殖,OCNTs和PCP暴露3 h的半数抑制浓度(IC₅₀)分别为14.4±1.8和11.5±1.6 mg/L,且两者的复合毒性为拮抗。PCP能通过增加静电斥力和降低细胞表面疏水性（CSH）而减少OCNTs的生物累积及其诱导产生的胞内活性氧;OCNTs能缓解PCP对生物合成、蛋白质和小分子代谢以及细胞器相关基因表达的干扰作用。（2）CIP对E.coli生长的3-h IC₅₀为244±14 mg/L,与OCNTs复合为拮抗毒性,细胞膜破损和氧化应激有所缓解。CIP能通过降低CSH减少OCNTs的生物累积,从而降低OCNTs的毒性。OCNTs可减轻CIP对DNA回旋酶活性的抑制作用,从而减少CIP对基因表达的干扰,尤其是与含氮化合物代谢、氧化还原酶活性和铁硫蛋白成熟相关的基因。OCNTs还可通过调节不饱和脂肪酸的合成和一些氨基酸及谷胱甘肽的代谢来降低CIP对代谢过程的影响。（3）OCNTs、PCP、CIP对B.subtilis生长的3-h IC₅₀分别为12.5±2.6、3.5±0.5、0.46±0.03 mg/L,OCNTs与PCP或CIP联合暴露分别为相加和协同毒性,PCP或CIP优先暴露后再与OCNTs共暴露复合毒性会增强。三种污染物均能破坏细胞膜,加剧氧化应激,显著改变代谢产物脂肪酸类、氨基酸、甘油、糖胺和小分子酸的含量。PCP能降低OCNTs的生物累积,CIP则没有显著影响。OCNTs可以升高PCP和CIP的生物累积,并可能通过“木马效应”增强CIP的毒性。OCNTs的共存会增大CIP对拓扑异构酶Ⅳ活性的抑制作用,并干扰与ABC转运蛋白和赖氨酸合成相关基因的表达。更多还原

【Abstract】 Carbon nanotubes（CNTs）is one of the most extensively used nanomaterials.After being discharged,CNTs has the possibility of interacting with co-existing contaminants,endangering the ecological environment.At present,the case study of the joint toxicity of CNTs and organic contaminants（OCs）and the understanding of relative mechanisms are very insufficient.It is necessary to study the joint toxicity of CNTs and OCs to bacteria and the influence of contaminant properties and bacterial species.Pentachlorophenol（PCP）,listed as priority pollutant and human carcinogen,is a typical traditional OCs.Ciprofloxacin（CIP）,as a representative of fluoroquinolones,has been identified as a common new OCs in the environment.PCP and CIP are hydrophobic and hydrophilic substances,respectively,and they may have differences in the interaction and joint toxicity with CNTs.Gram-negative Escherichia coli and positive Bacillus subtilis possess different physicochemical properties and physiological characteristics,and their responses to a same exposure system may be different.Based on microscopic observation,determination of biochemical indexes and bioaccumulation,and the analyses of transcriptomics and metabonomics,the involved interactions and toxic mechanisms of oxidized multi-walled carbon nanotubes（OCNTs）with PCP or CIP to E.coli and B.subtilis were investigated.The main results of the work are as follows:（1）Both OCNTs and PCP significantly inhibited the growth and reproduction of E.coli through membrane disruption,oxidative stress,and genetic damage.The half inhibitory concentration(IC₅₀)values for OCNTs and PCP after 3-h exposure were 14.4±1.8 and 11.5±1.6 mg/L,respectively.Their co-exposure exhibited antagonistic toxicity.PCP increased the electrostatic repulsion between bacteria and OCNTs and decreased cell-surface hydrophobicity（CSH）.The bioaccumulation of OCNTs and subsequent OCNTs-induced intracellular reactive oxygen species accumulation were therefore reduced.OCNTs attenuated the PCP-induced disturbances to gene expressions in biosynthetic,protein metabolic,and small molecule metabolic processes,as well as for organelles.（2）The 3-h IC₅₀ of CIP to E.coli growth was 244±14 mg/L,and the co-exposure of CIP and OCNTs revealed an antagonistic toxicity.Mitigations in cell membrane disruption and oxidative stress were involved in the antagonistic action.CIP decreased the bioaccumulation of OCNTs via reducing CSH,which attenuated the OCNTs toxicity.OCNTs alleviated the disturbance of CIP to gene expressions,especially related to nitrogen compound metabolism,oxidoreductase activity,and iron-sulfur protein maturation,through relieving the CIP-induced inhibition of DNA gyrase activity.Further,OCNTs reduced the impact of CIP on bacterial metabolome via the regulation of biosynthesis of unsaturated fatty acids and metabolisms of some amino acids and glutathione.（3）The 3-h IC₅₀ values of OCNTs,PCP,and CIP to B.subtilis were 12.5±2.6,3.5±0.5,and 0.46±0.03 mg/L,respectively.In the co-exposure of OCNTs with PCP or CIP,there were additive toxicity or synergistic toxicity,respectively.The pre-exposure of PCP or CIP before its co-exposure with OCNTs had significantly increased the joint toxicity.All of the three pollutants caused cell membrane damage,aggravation of oxidative stress,and changes in the contents of fatty acids,amino acid,glycerol,osamine,and small molecular acid.PCP reduced the bioaccumulation of OCNTs,while CIP had no significant influence on the OCNTs bioaccumulation.OCNTs increased the bioaccumulation of PCP and CIP,and may enhance the toxicity of CIP through the‘Trojan horse effect’.The co-existence of OCNTs increased the inhibition effect of CIP on the activity of topoisomeraseⅣ,and interfered with the expression of genes related to ABC transporters and lysine biosynthesis.更多还原