【作者】 杨光；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2005， 硕士

【摘要】 前言:镉作为重要的工业原料,随着工业生产的迅速发展,它对环境污染日益严重,而且它在环境中非常稳定,不易降解。镉不仅是公知公认的肾脏毒物,镉还是被确认的致癌物。国际癌症研究机构(IARC)早在1993 年就将其归类为第一类致癌物。近年来,对于镉肾毒性的作用机制的研究有了很大发展,认为金属硫蛋白(MT)可对体内镉的运输、储存及毒性起作用;还有人认为镉可通过干扰酶的活性而引起肾脏功能的损害;但越来越多的研究表明,作为一种强脂质过氧化物作用的诱导剂, 镉所致的肾毒性主要与氧化应激有关[5]。N-乙酰半胱氨酸(N-acetylcysteine,NAC)是一种含巯基化合物,近年来的研究发现,NAC 分子中的半胱氨酸,作为体内活性氧类(reactive oxygen species, ROS)的清除剂和还原性谷胱甘肽的供给体,在体内抗氧化反应中有重要作用,具有干扰自由基生成,预防DNA 的损伤,对抗细胞凋亡等作用。本研究以肾小管细胞系及大鼠整体动物为试验系统,用NAC 为干预物,探讨氯化镉(CdCl2)引起肾脏毒性的氧化性损害机制,为防制镉危害提供实验依据。方法:以猪肾小管细胞系LLC-PK1 细胞作为体外试验系统。噻唑蓝(MTT)法检测CdCl2 的细胞毒性,通过单细胞凝胶电泳(SCGE)试验检测引起的DNA 损伤,荧光法测定氧自由基水平。同时,以NAC为干预物,观察其对上述效应终点的影响。体内试验以成年健康雄性Sprague—Dawley (SD)大鼠为试验对象,按体重随机分为4 组,每组6 只。各组及其处理如下:(1) 对照更多还原

【Abstract】 Introduction: As an important industrial material, cadmium has polluted environment more and more seriously with the quickly development of industrial production. Further more, it is difficult to decompose in environment and organism. Cadmium is toxic to kidney, and in 1993, International Agency For Research on Cancer named cadmium as the first carcinogen. In recent years, it has attained a great development on the study of nephrotoxicity and mechanism of cadmium. Rencent studies have suggested that it relate to the oxidative stress. N-acetylcysteine(NAC) is a thiol-containing compound. It has been known that cysteine in the NAC molecule, as a scavenger of reactive oxygen species (ROS) and provider of reductive glutathione, is very important during the antioxidant reactions. Including directly reducing the level of reactive oxygen species, preventing DNA damage, affecting the gene regulation, and so on. In this study, we will investigate the possible mechanism of DNA damage of kidney by cadmium, and we will also study the preventive effect of NAC on cadmium-induced nephrotoxicity and provide scientific evidence against cadmium-induced genotoxicity in kidney cells. Method:LLC-PK1 cells were used as in vitro experimental system. Cytotoxicity was determined by MTT color assay. DNA damage was determined by single cell gel electrophresis assay (SCGE), and ROS were measured by fluorescence method. With the help of the method described above, NAC as an intervenor was added into cultures together with CdCl2 to explore its preventive effects on oxidative damage and DNA damage induced by CdCl2. Male Sprague-Dawley rats were randomly divided into 4 groups according to their weights (six rats in each). Each group was treated as follows: (1) the control, (2) high dose of CdCl2 (5.00mg/kg) group, (2) middle dose of CdCl2 (2.50mg/kg) group, (3) low dose of CdCl2 (1.25mg/kg) group. Rats were treated intraperitoneally by injection (ip) Once a day for 3 consecutive days, and killed on day 4. Nephrotoxicity was assessed by the measurement of body weight, kidney index, contents of blood urea nitrogen (BUN), changes in malondialdehyde (MDA) formation, the kidney cortex and histological changes. And 8-hydroxydeoxyguanosine (8-OhdG) was investigated by immunohistochemical method. Result: After LLC-PK1 cells were treated by different concentrations of CdCl2 for 24 h, growth of the cells was significantly inhibited and inhibitory concentration 50%(IC50 ) was 7.79±0.6mg/L, the cytotoxocity was significantly decreased by NAC and IC50 was 28±5.56mg/L. Single cell gel electrophresis assay showed that CdCl2 at concentrations of above 3μg/ml induced DNA strand-breaks, and the length of the tail of DNA was significantly decreased by 1mM NAC. The production of ROS was significantly increased after LLC-PK1 cells were treated by CdCl2 for 1 h, and 1mM NAC can decrease the production of ROS. In vivo studies, on day 3 after administration of CdCl2 , significantly nephrotoxicity appeared in rats, including the decrease of body weight, increase of kidney index, and contents of BUN. The formation of MDA in kidney cortex were significantly higher than that of the control (P<0.05). Histologically, acute renal damages in structure were observed. The 8-OHdG were positive in the kidney specimens of rats treated with CdCl2, and the percentage of adduct labeling cell (PALC%) at doses of 5.00, 2.50,and 1.25 mg/kg were 79±7.59 %, 69±10.69%, and 47±10.81%, respectively. Whereas the normal kidney tissues were weak positive with 8-OHdG, and PALC% in this group was 6±2.08%. PALC% of rats in groups treated with CdCl2 was significantly higher than that of the control (P<0.01). Conclusion: CdCl2 significantly inhibited the growth of LLC-PK1 cells, induced DNA strand-breaks, and increased production of ROS. NAC can attenuated the cytotoxicity produced by CdCl2. CdCl2 (1.25mg/kg, 2.5 mg/kg, 5 mg/kg) once a day, for 3 days can result in noticeable kidney damage in rats. The 8-OHdG was positive in the kidney specimens of rats treated with CdCl2, which may due更多还原