【作者】 王林；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2009， 博士

【摘要】 铅、镉是环境中两种常见的重金属污染物;近年来,由于工业生产中铅、镉使用量的增加及相关工业废物带来的污染加重,环境中的铅、镉含量呈快速上升趋势。由于铅、镉常从许多天然和人工污染源同时进入环境而引起复合污染,因此铅镉联合暴露给公众健康带来的危害引起了广泛关注。肾脏是慢性铅、镉毒性损伤的靶器官和蓄积部位,国内外研究者从职业性暴露、环境污染及动物试验多方面对铅镉所致肾毒性机理进行了广泛研究;但铅、镉单独肾毒性作用的报道较多,而对两者的联合肾毒性研究则相对较少。本研究以SD大鼠为试验动物,通过体内和体外试验相结合的方法较系统地探讨了铅、镉及其联合对大鼠肾脏的毒性损伤效应及可能的作用机理,为进一步认识铅镉及其联合肾毒性作用提供了理论依据。一、体内试验1月龄雌性SD大鼠24只随机分为4组,每组6只,分别为对照组、铅组（300mgPbAc2/L）、镉组（50mgCdAc2/L）、铅镉联合组（300mgPbAc2/L+ 50mgCdAc2/L）。对照组大鼠自由饮用超纯水,其余各组自由饮用配置毒液,每天称量大鼠体重与饮水量,连续染毒8周,进行如下试验:①分别于染毒前一天、染毒后2、4、6、8周收集24h尿液,测定尿酶UALP、UNAG、UGGT、ULDH活性和尿蛋白UTP、Uα₁-MG、Uβ₂-MG、UmAlb含量的动态变化;同时测定尿液中Zn、Cu、Mn、Fe、Se排泄量的动态变化;②染毒结束,测定血清与肾皮质中Zn、Cu、Mn、Fe、Se、GSH、MDA含量和SOD、CAT、GSH-Px活性;③采用光学显微镜、透射电子显微镜观察染毒后大鼠肾皮质组织病理学及超微结构的变化;④采用荧光定量PCR法检测染毒后大鼠肾皮质中线粒体细胞色素氧化酶亚基COX-I、COX-II、COX-III mRNA表达量的变化;免疫组织化学法与荧光定量PCR法检测金属硫蛋白MT-1、MT-2在大鼠肾皮质中的表达。结果表明:①除染毒2周时Pb、Cd组Uα₁-MG含量与Pb组UALP活性外,Pb、Cd组其余各项指标（UNAG、UGGT、ULDH、UTP、Uβ₂-MG、UmAlb）均从染毒2周开始即显著或极显著高于对照组（P<0.05或P<0.01）,且升高幅度与染毒时间呈正相关;铅镉联合组从2周开始,所测各项指标均显著高于对照组（P<0.05）。在整个试验过程中,联合组各项指标均高于Pb组、Cd组,且在不同染毒时间有显著差异;②染毒结束,各染毒组血清与肾皮质中SOD、CAT、GSH-Px活性与GSH、Zn、Cu、Mn、Fe、Se含量均显著低于对照组（P<0.05）,但MDA含量均显著高于对照组（P<0.05）;③除染毒2周时尿Zn含量外,Pb、Cd组其余4种微量元素（Cu、Mn、Fe、Se）均从染毒4周开始尿中排泄量显著增多（P<0.05）;联合染毒除2周时尿Fe含量外,其它元素（Zn、Cu、Mn、Se）均从染毒2周开始尿中排泄量显著增多（P<0.05）;④各染毒组肾皮质部肾小管和肾小球有明显组织学病理变化,同时超微结构变化明显,表现核染色质分布不均、染色质边聚、近端小管刷状缘微绒毛脱落、线粒体肿胀、嵴断裂、部分或完全消失,铅镉联合暴露的病理学损伤较铅、镉单独染毒严重;⑤各染毒组大鼠肾皮质COX-I、COX-II、COX-III基因表达量均显著低于对照组（P<0.05）,以铅镉联合组降低幅度最大;⑥Pb组MT-1、MT-2表达量与对照组无显著差异（P>0.05）,但Cd组和铅镉联合组MT-1、MT-2表达均显著高于对照组（P<0.05）。结合上述试验结果,可以得出以下结论:①铅镉单独或联合暴露可损伤大鼠肾小管的重吸收功能和肾小球的滤过功能,其肾损伤程度与染毒时间呈正相关;②铅镉染毒可导致肾组织抗氧化功能降低,抗氧化微量元素含量降低加剧了氧化应激介导的肾组织损伤,染毒组大鼠体内微量元素含量降低与其尿液中排泄量显著增加直接相关;③铅镉暴露可引起肾皮质显著病理学损伤和多种细胞器损伤,其中线粒体损伤较显著;④染毒组肾皮质COX-I、COX-II、COX-III基因表达量显著下降可能与铅镉暴露导致的线粒体脂质过氧化损伤有关,金属硫蛋白MT-1、MT-2基因表达量显著升高在铅镉联合肾毒性过程中发挥重要作用。总之,铅镉联合肾毒性呈协同效应。二、体外试验采用机械筛网结合酶消化法建立大鼠原代肾小管上皮细胞（rPTCs）培养模型,在传一代细胞增殖活性最强时间段进行铅（0.5μmol/L、1μmol/L）、镉（2.5μmol/L、5μmol/L）单独或联合染毒。主要进行以下试验:①cck-8还原法测定不同组合的铅镉在不同染毒时间（3、6、12、24h）对rPTCs存活率的影响;②测定铅镉单独或联合染毒12h对rPTCs凋亡率、坏死率、乳酸脱氢酶释放率及凋亡形态学的影响,同时添加N-乙酰半胱氨酸（NAC）,观察其对铅镉所致细胞毒性损伤的保护效果;③测定铅镉染毒12h对rPTCs胞内SOD、CAT、GSH-Px活性及GSH、MDA含量的影响;④测定铅镉染毒12h对rPTCs膜ATP酶(Ca²⁺-ATPase、Na+/K+-ATPase)活性、胞内pH、线粒体膜电位、活性氧及钙离子水平的影响。结果表明,①铅镉单独染毒高剂量组从3h开始、低剂量组从6h开始,其细胞存活率显著低于对照组（P<0.05）;铅镉联合组从3h开始,细胞存活率极显著低于对照组（P<0.01）,且存活率降低幅度与染毒剂量、染毒时间呈正相关;②染毒12h,各染毒组细胞凋亡率、坏死率、乳酸脱氢酶释放率均极显著高于对照组（P<0.01）,且铅镉联合组各项指标均高于各相关单独染毒组;各染毒组细胞表现核皱缩、呈新月形、染色质致密浓染、核碎裂等典型凋亡特征;NAC对铅镉所致细胞凋亡有显著保护效应,但对细胞坏死率和乳酸脱氢酶释放率无明显影响;③与对照组比较,染毒组SOD、CAT、GSH-Px活性和GSH含量均显著或极显著降低（P<0.05或P<0.01）,而MDA含量均极显著升高（P<0.01）;④染毒各组细胞内活性氧和钙离子水平均极显著高于对照组（P<0.01）,线粒体膜电位水平、胞内pH、Ca²⁺-ATPase与Na⁺/K⁺-ATPase活性均显著或极显著低于对照组（P<0.05或P<0.01）。上述试验结果可以得出以下结论:①铅镉暴露对rPTCs的毒性损伤呈浓度依赖性和时间依赖性,联合暴露呈协同毒性损伤;②细胞凋亡与细胞坏死是铅镉所致rPTCs死亡的2种死亡类型,其中凋亡性死亡在铅镉该剂量组合所致细胞损伤过程中发挥主导作用。氧化应激在铅镉染毒所致细胞凋亡性死亡过程中发挥重要作用,细胞内抗氧化酶活性降低进一步加剧铅镉对rPTCs的氧化损伤;抗氧化剂NAC对铅镉所致的肾小管上皮细胞毒性损伤有显著保护效应;③铅镉暴露导致rPTCs线粒体膜电位降低而促进细胞凋亡,同时细胞内酸化、钙离子超载、氧化还原平衡状态失调等一系列细胞内环境稳态失衡促进了细胞凋亡。总之,铅镉联合暴露对大鼠肾小管上皮细胞的损伤程度重于单独染毒,呈协同效应。更多还原

【Abstract】 Lead （Pb） and cadmium （Cd） are now recognized to be two of most important heavy metal contaminants in the environment. Due to their increased industrial uses and environmental pollution with the related waste products, concentrations of lead and cadmium are increasing rapidly in the environment in recent years. Since the two elements are often released simultaneously in the environment from a number of natural and man made sources, adverse health effects caused by combined exposure to lead and cadmium has provoked a significant public health concern. The kidney is the target organ and the primary accumulation site of chronic lead and cadmium exposure. The nephrotoxicity induced by lead and/or cadmium have been extensively studied and widely reported in occupationally and environmentally exposed human subjects, as well as in various experimental models. Most studies were implicated in the single exposure of lead/cadmium on the kidney. However, systemic studies of toxic damage on the combination of lead and cadmium were little referred. In this study, the toxic effects of lead and/or cadmium on the kidney of Sprague-Dawley （SD） rats were investigated in vitro and in vivo, which will offer some theoretic evidences for further exploring the mechanism in nephrotoxicity of lead and/or cadmium.1. In vivo studies The study was carried out on female one-month-old SD rats. Twenty- four rats were allocated randomly to four groups of six animals each. The experimental period was eight weeks. （1） Control: rats consumed distilled water as drinking water. （2） Lead treated group: rats consumed a solution of PbAc2 （300mg/L） as drinking water. （3） Cadmium treated group: rats consumed a solution of CdAc2 （50mg/L） as drinking water. （4） Pb+Cd treated group: these rats received both Pb and Cd at the doses, periods and ways of administration described above. During the experimental period, water consumption and weight gain were measured every day. A series of tests were carried out:①On the day before the experiment and at the end of 2, 4, 6 and 8 weeks of treatment, rats were kept for 24h urine collection. Activities of alkaline phosphatase （ALP）, N-acetyl-β-D-glucosaminidase （NAG）,γ-glutamyl-transpeptidase （GGT）, lacticacid dehydrogenase （LDH） and contents of total protein （TP）,α₁-microglobulin （α₁-MG）,β₂-microglobulin （β₂-MG）, microalbumen （mAlb） in urine were determined. Also, concentrations of Zn, Cu, Mn, Fe and Se in the urine were detected during the experiment.②At the end of treatment, the levels of Zn, Cu, Mn, Fe, Se, glutathione （GSH）, malondialdehyde （MDA） and activities of total superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GSH-Px） in the renal cortex and serum were measured.③Changes in histopathology and ultrastructure of renal cortex were detected by light microscope and transmission electron-microscope, respectively.④The relative gene expression levels of cytochrome oxidase submits （COX-I, COX-II, COX-III） in the renal cortex were quantified by Fluorescent Quantitative-PCR （FQ-PCR）. The expressions of metallothionein submits （MT-1, MT-2） in the renal cortex were detected by immunohistochemistry method and FQ-PCR, respectively. The results are as follows:①Compared with the control group, all of the indices（NAG, GGT, LDH, TP,β₂-MG, mAlb） except the contents ofα₁-MG in the Pb, Cd group and activities of ALP in the Pb group at the time of 2-week exposure increased significantly from the beginning of exposed for two weeks （P<0.05 or P<0.01）. Especially the changes in the （Pb+Cd） group were the greatest （P<0.05）. Positive correlation lied in the process between the degree of increase in activities of urinary enzymes/contents of urinary proteins in the exposed groups and the exposure time. During the experiment, the levels of indices in the （Pb+Cd） group were all higher than those in the Pb, Cd group. There is significant difference between the （Pb+Cd） group and Pb or Cd group in different times.②In comparison with the control group, activities of SOD, CAT, GSH-Px and contents of GSH, Zn, Cu, Mn, Fe, Se in the renal cortex and serum of the three exposed groups decreased significantly（P<0.05）. However, the contents of MDA in these exposed groups were significantly increased than that of the control group （P<0.05）.③As far as the Pb group and the Cd group is concerned, concentrations of trace elements （Cu, Mn, Fe, Se） in the urine except the concentrations of urinary Zn at the time of 2-week exposure increased significantly from the beginning of exposed for four weeks（P<0.05）. Regarding the （Pb+Cd） group, the excretion of trace elements （Zn, Cu, Mn, Se） in the urine except that of urinary Fe increased significantly from the beginning of exposed for two weeks （P<0.05）.④Obvious pathological changes in the renal tubule and glomeruli renis were observed in the renal cortex of these three exposed groups. Also, the changes in ultrastructure of renal cortex is obvious, which pyknosis of nucleus, chromatin assemble, partial loss of brush border microvilli in the proximal tubular cells, mitochondrial swelling, disappearance and fragmentation of carina were seen under transmission electronic microscope. The degree of pathological damage in the （Pb+Cd） group was more serious than that in the Pb/Cd group. Compared with the Pb or Cd group, more severe pathological damage was found in the （Pb+Cd） group.⑤The relative expression levels of cytochrome oxidase submits （COX-I, COX-II, COX-III） in the renal cortex of these three exposed groups were significantly lower than those in the control group （P<0.05）. The most significant change occurred in the （Pb+Cd） group.⑥There is no significant difference in the expression level of MT-1 and MT-2 gene in the kidneys between the lead group and control group （P>0.05）, whereas those in the cadmium group and （Pb+Cd） group were significantly higher than that in the control group （P<0.05）. Based on these results, the conclusions are as following:①Tubular reabsorptive function and glomerular filtration function were damaged after exposed to lead and/or cadmium. The degree of renal injury is positively correlated with the increase of exposure time.②Exposure to lead and/or cadmium can induce the decrease of the anti-oxidative function in the kidneys of rats. Also the decreased contents of trace elements related to antioxidative function made the renal damage induced by oxidative stress worse. The decreased levels of these trace elements in the tissues exposed to lead and/or cadmium were due to an increase of their excretion in the urine.③Obvious pathological changes and damage of many organelles in the renal cortex were medicated by lead and/or cadmium. Among these organelles, mitochondria underwent the greatest changes.④The decreased expression levels of COX-I, COX-II, COX-III in the renal cortex exposed to lead and/or cadmium may be related to the mitochondrial lipid peroxidation. The increased expression level of MT-1 and MT-2 gene played an important role in the nephrotoxicity induced by lead and cadmium. In summary, there was an obvious synergistic effect of lead combined with cadmium on the kidney of rats.2. In vitro studies The primary cultures of rat proximal tubular cells （rTECs） were cultured by mechanical grinding, filtering and chemical digestive methods. The first passage was used to perform the experimental design when it was in its highest cell viability. Effects of lead （0.5μmol/L, 1μmol/L） and/or cadmium （2.5μmol/L, 5μmol/L） on the rTECs were investigated in the following assays.①Effects of different doses of lead and/or cadmium on the survival rates in rPTCs for a time range of 3, 6, 12 and 24h were detected by using the cck-8 reduction method.②Effects of lead and/or cadmium on the apoptotic rates, necrotic rates, LDH release and apoptotic morphological changes in rPTCs over a 12 h period were investigated. In addition, the protective effect of N-acetyl-L-cysteine （NAC） against lead and/or cadmium induced cellular damage was investigated.③Activities of SOD, CAT, GSH-Px and contents of GSH, MDA in rPTCs were measured when exposed to lead and/or cadmium over a 12h period.④Activities of Ca²⁺-ATPase and Na⁺/K⁺-ATPase, intracellular pH, levels of mitochondrial membrane potential （ΔΨ）, reactive oxygen species （ROS） and intracellular [Ca²⁺]i in rPTCs were detected after exposed to lead and/or cadmium for 12h. The results are as follows:①The cell survival rates in the single lead and cadmium groups were significantly lower than those of control groups since these cells were exposed to high-dose and low-dose groups for three and six hours, respectively （P<0.05）. The cell survival rates in the combined groups were significantly lower than those of control groups after a 3-h exposure （P<0.01）. Furthermore, the degree of decrease in the cell survival rate was positively correlated with the dose and the exposure time.②After exposure to lead and/or cadmium for 12h, the apoptotic rates, necrotic rates, LDH release in these exposed groups were significantly higher than those in the control group （P<0.01）. Also, the above indices induced by （Pb+Cd） were always higher than those in the related Pb or Cd group in the same exposure time. After a 12-h exposure time, it showed morphological changes typical of apoptosis in the lead and/or cadmium groups, i.e., nuclear chromatin condensed and fragmented chromatin was characterized by a scattered, drop-like structure. Apoptosis induced by lead and/or cadmium can be efficiently prevented by NAC, but the necrotic rates and LDH release were not affected by NAC.③Compared with the control group, activities of SOD, CAT, GSH-Px and the GSH level in the exposed groups decreased significantly （P<0.05 or P<0.01）; But the content of MDA increased significantly （P<0.01）.④After exposed to lead and/or cadmium for 12h, intracellular ROS and [Ca²⁺]_i in rPTCs increased significantly （P<0.01）, while the mitochondrialΔΨ, intracellular pH, activities of Ca²⁺-ATPase and Na⁺/K⁺-ATPase decreased significantly （P<0.05 or P<0.01）. Based on these results, the conclusions are as follows:①Lead and/or cadmium exposure induced cellular death in rPTCs, depending on both the concentration and the exposure time. Synergistic effect lies in the administration of lead combined with cadmium.②Cellular death induced by lead and/or cadmium is medicated by two mechanisms, necrotic and apoptotic. The apoptotic mechanism played a chief role in the cellular death induced by lead and/or cadmium at these doses. Moreover, oxidative stress could be implicated in the apoptotic mechanism mediated by lead and/or cadmium. Decreased activities of anti-oxidative enzymes further enhanced oxidative damage in rPTCs caused by lead and/or cadmium. In addition, the cellular damage induced by lead and/or cadmium can be significantly prevented by NAC.③Depletion of mitochondrialΔΨand a disorder of intracellular homeostasis, i.e. intracellular acidification, calcium overload, disturbance in the prooxidant–antioxidant balance, promoted the development of apoptosis in rPTCs. In a word, there was an obvious synergistic effect of lead combined with cadmium on the cellular damage in rPTCs.更多还原