【作者】 张甜；

【导师】 李曦；

【作者基本信息】 武汉理工大学 ， 应用化学， 2011， 硕士

【摘要】 本文采用微量热法研究了氯苯类化合物和重金属的单一及联合毒性,并用ICP-AES探讨细胞中金属离子的积累情况；用ATR-FTIR研究了细胞膜上官能团的变化；用SEM观察细胞受药物作用下表面的破损情况。并探讨了毒性机理。具体结果如下：(1)采用微量热法研究了五种氯苯类化合物对四膜虫的毒性效应,在五种氯苯类化合物的作用下,生长速率常数K随着化合物浓度的增加而减小,说明化合物对四膜虫的生长代谢产生了抑制作用。这五种化合物的毒性大小顺序为：1,2,4-三氯苯>邻二氯苯>对二氯苯>间二氯苯>氯苯。其半抑制浓度分别为21、30、37、38、45 mg/L。采用生物显微镜观察发现四膜虫在受到药物作用下,细胞膜慢慢破损,形貌发生明显变化,最终内部物质溢出,改变了细胞膜的渗透性。采用ATR-FTIR研究了四膜虫在氯苯类化合物作用下,细胞膜上的胺基峰和P02ˉ峰变化明显,部分峰消失,说明药物对细胞膜有一定的破坏程度。(2)采用微量热法研究了三种重金属离子对四膜虫的毒性效应,随着Cu2+、Cd2+、Cr3+浓度的增加,K值逐渐减小；金属离子不同抑制程度不同,其半抑制浓度分别为150、0.405、100 mg/L,因而这三种重金属离子对四膜虫的毒性大小顺序为：Cd2+>Cr3+>Cu2+。通过ICP-AES研究表明：随着加入金属离子量的增加,四膜虫细胞内金属离子的量也增多,但是达到一个最大值后反而减少,说明足够的药物破坏细胞膜的通透性,细胞内外的金属离子会产生交换。结合四膜虫细胞膜的分子结构,通过ATR-FTIR的研究细胞膜上官能团的变化情况,结果表明：金属离子的加入改变了细胞膜上的官能团,使有些官能团的峰值减少,有些官能团消失,引起了细胞膜的破坏。(3)采用微量热法研究了三种重金属离子和氯苯类化合物对四膜虫的联合毒性研究,联合作用下,K也随着药物浓度的增加而减少,抑制率I逐渐增大。不同的联合物质,抑制程度不同,相同的金属离子,不同的氯苯物质,抑制程度也不相同。采用相加指数法对联合毒性进行了评价,结果表明研究的体系都属于协同作用。通过ATR-FTIR的研究细胞膜上官能团的变化发现联合作用同样造成了细胞膜上最外层的亲水端上的胺基和P02ˉ的破坏,且破坏程度比单一毒性明显要强。采用FE-SEM法观察了四膜虫表面细胞膜的微观情况,发现受到毒物的作用后细胞出现明显空洞,足够时间后细胞会有很大程度的受损。(4)对氯苯类化合物的毒性进行了定量构效关系的研究,构建了QSAR模型：logIC50=-0.3781ogKow+0.476ELOMO-0.618△QnR-1.804(n=5, R=0.706, SE= 0.132),对毒性机理探讨的结果是氯苯类化合物的毒性作用主要发生在糖蛋白质分子中的—H,—NH—等亲核基团上,它们破坏细胞膜上的磷脂和糖蛋白,氯苯穿过细胞膜进入细胞,与大分子发生电荷转移,从而影响细胞的正常代谢；过量的铜和铬会使形成的金属蛋白,金属核酸络合物过量而引起毒性效应。联合毒性的作用类型为协同作用,可能是由于两者共同存在于生物体内,金属物质形成的络合物、螯合物,及某种金属蛋白酶,能促进氯苯物质进入靶点,从而快速的破坏细胞膜的通透性,造成毒性加强。更多还原

【Abstract】 In this paper, microcalorimetry was used to study the single toxicity and joint toxicity on Tetrahymena, the accumulation of intracellular heavy metals was studied by ICP-AES, damage of the cell membrane and change of function groups was studied by ATR-FTIR spectra, the cell surface damage on the control of the compounds was observed by SEM, and then, the mechanisms of toxicity was explored. The main results and conclusions are summarized as follows:1. The toxicity of five chlorobenzenes to Tetrahymena growth metabolism was studied by microcalorimetry. And the growth constant (k) decreases with the increasing of drug concentration, indicating that the growth metabolism had been inhibited. The results suggest that the order of toxicity is 1,2,4-trichlorobenzene> o-dichlorobenzene> p-dichlorobenzene> m-dichlorobenzene> chlorobenzene.These five compounds half-inhibition concentrations are 21,30,37,38,45 mg/L. Biological microscopy was used to observe the membrane damage slowly, morphology change significantly, until internal material spill. It reflected drugs changed the permeability of cell membrane. ATR-FTIR spectra showed that under the action of chlorobenzenes, the amine and PO2 peak change significantly, and some peaks disappeared, indicating that certain compounds can cause the cell membrane damage.2. The toxicity of three heavy metals to Tetrahymena growth metabolism was studied by microcalorimetry. And the growth constant (k) decreased with the increasing of metals concentrations, indicating that the growth metabolism had been inhibited. The results suggest that the order of toxicity is Cd2+>Cr3+>Cu2+.These three metals half-inhibition concentrations are 150、0.405、100 mg/L. By ICP-AES results showed that:with the increase in the amount of added metal ions, Tetrahymena cells also increased the amount of metal ions, but it has decreased after reaching a maximum value, indicating that the enough metal ions damaged the cell membrane permeability, both inside and outside cells metal ions happened the exchange. Tetrahymena with the molecular structure of cell membranes, ATR-FTIR spectra showed that under the action of chlorobenzenes,the amine and PO2peak changed significantly, and some peaks disappeared, indicating that certain metal ions damaged the cell membrane.3. The joint toxicity of five chlorobenzenes and three heavy metals to Tetrahymena growth metabolism was studied by microcalorimetry. Under the combined effect, k was also increased with the reduction of mixture concentrations, the inhibition rate I gradually increased. Different joint compounds, different degrees of inhibition, with the same metal ions, and different chlorobenzenes, were not the same degree of inhibition. The joint toxicity was evaluated using Additive Index method. The results showed that the studied system was all synergies. By ATR-FTIR study showed that under the action of joint drug, the amine and PO2-peak of the phospho-lipid phospho-diester, both in the hydrophobic end exposed to the outer layer were changed significantly, and some peaks disappeared, indicating that certain drugs on the cell membrane damage. And damage was significantly stronger than a single toxic. Surface of the cell membrane of Tetrahymena micro-situation was observed by FE-SEM, the role of drugs found to be significant after the empty cells; the cells have sufficient time to a large degree of damage.4. The toxicity of chlorobenzene compounds in a quantitative structure-activity relationship was studied, QSAR model is constructed:logIC50 =- 0.378logKOW +0.476 ELOMO-0.618△QπR-1.804 (n= 5, R= 0.706, SE= 0.132). Mechanism of toxicity of chlorobenzenes compounds, mainly happened in the sugar and protein molecules of the-SH,-NH-and other pro-nuclear groups, they damage the cell membrane phospholipids and glycoproteins, chlorobenzenes through the cell membrane into the cells, and then transfer charge with macromolecules, thus affecting the normal metabolism of cells; excess copper and chromium metal will form a protein, nucleic acid complexes, when these were excessive, metal caused toxic effects. synergy type of joint toxicity may be due to both co-exist in vivo, the formation of metals complex, chelate, and a metal protease, can promote the chlorobenzene substances into the target, so fast destruction the permeability of cell membranes, resulting in toxic strengthened.更多还原