【作者】 梁艺怀；

【导师】 金泰廙； Gunnar Nordberg； Monica Nordberg； Ingvar Bergdahl； Eva Lundin；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2010， 博士

【摘要】 长期的职业或环境镉暴露可引起多器官损伤,肾脏是镉慢性毒作用的主要靶器官,肾小管为靶部位。镉慢性毒作用以低分子量蛋白尿为特征,尿金属硫蛋白(UMT)、β2-微球蛋白(UB2M)等是常被用于反映肾小管损伤的生物标志。镉在人体内的半衰期很长,即使接触停止,大部分既往蓄积的镉仍继续停留在人体内。停止或减少镉接触后,镉致肾功能损害的预后引起了国际学术界的普遍关注。对华东某镉污染区的环境监测和流行病学调查显示,食用当地自产大米是当地居民最主要的镉接触来源,自1996年起,当地居民停止食用镉污染的自产大米。本课题组分别于1995年、1998年、2006年先后对该地区居民进行了三次健康调查,通过观察和比较镉接触降低后肾功能预后情况,探讨相关的影响因素。对先后参加过1995年和1998年调查的148名居民的1995年尿镉水平与UB2M、尿白蛋白(UALB)变化情况分析后发现,接触水平下降前的尿镉水平是影响肾功能预后的重要因素,而10μg/g Cr是一重要的阈值,尿镉水平低于此值时肾功能损伤是可逆的,否则不可逆、甚至进一步恶化。2006年底,本课题组对参加过上述1998年调查的居民再次进行调查,随访到475名受访者。研究发现,时隔八年后,随着环境镉接触的降低,居民血镉水平下降明显,而尿镉变化并不显著。通过比较前后两次调查中尿N-乙酰-β-D-氨基葡萄糖苷酶(UNAG)和UB2M的平均值显示,居民总体的肾小管功能状况并无显著恢复、甚至出现恶化趋势,特别是,既往过量的UB2M排泄是肾功能不良预后的危险因素；而UALB水平却明显降低,意味着肾小球功能的改善。除了既往镉接触水平,还发现镉致肾小管损伤仅在特定的性别或年龄组出现恢复的迹象。人群研究有很多不确定因素,比如接触剂量、时间及接触途径,需要受到严格控制的动物试验加以佐证。为阐明金属硫蛋白(MT)在镉暴露致肾功能损伤发生和预后中的作用,建立了两个动物模型。第一个模型将雄性Wistar大鼠分别进行锌、铜预处理,皮下注射镉-金属硫蛋白复合物(CdMT)造成急性镉中毒,观察到不同剂量镉染毒后,锌或铜诱导动物肝MT中金属含量比例存在差异,铜／镉高于锌／镉,即与镉相比,铜更易将MT结合锌取代,并认为肝脏中非MT结合锌也是影响与MT结合的金属离子稳态的重要因素。第二个模型将Wistar大鼠经饮用水染镉12周,之后停止染毒、继续观察16周。该试验选取的染毒途径、时间是为了模拟生活在镉污染区普通居民的镉接触情况,旨在通过MT免疫组化法测定和比较终止镉染毒前后大鼠肝、肾组织MT表达情况,探讨其与以UNAG、UMT为指标的肾功能损伤预后的关系。研究发现,亚慢性经口染镉后大鼠肝脏和肾脏MT增加；当染毒终止后,肝脏镉负荷下降而肝脏MT水平上升。到28周后,低剂量组雄性大鼠UNAG降低和所有剂量组UMT下降,可能预示着由肝脏转运至肾脏的CdMT减少和肾小管功能的部分恢复。这些发现有助于揭示人类停止镉接触后机体功能的变化过程,并提示个体间合成MT能力的差异可能是镉致肾毒性发生的重要因素。蛋白质组学技术的出现为镉致肾功能损害及其预后研究提供了新的思路。为了解镉致肾损伤尿蛋白质表达情况,采用增强激光解析电离-飞行时间质谱(SELDI-TOF MS)蛋白质芯片技术,对饮水染镉试验中雄鼠染毒前(0周)和染毒后第2、4、8、12周尿样进行分析。结果显示,镉处理组大鼠在不同时点的尿液蛋白质峰数目与对照组基本相同,主要集中在2000-5000Da、8000-12000Da、17000-19000 Da 3个分子量区域内,但是对照组的蛋白质峰强度比镉处理组低很多。镉处理组大鼠尿液平均有11个蛋白质峰,与对照组相比都表达上调。此外,对照组大鼠尿液中蛋白排泄量基本保持恒定,而镉处理组大鼠的蛋白峰强度呈现出时间-效应趋势。据此,初步了解镉接触早期尿蛋白峰的强度和分布模式,并揭示染毒剂量高低与差异蛋白峰出现时间先后的关系,有助于筛选出一组能反映肾功能损伤的敏感的生物标志,为镉肾毒性的早期防护和预后研究提供依据。SELDI分析提供的信息有限,为获取镉致肾损伤及其预后的尿蛋白质表达谱,利用双向凝胶电泳(2DE)技术分析大鼠饮水染镉试验尿样和华东某地镉污染区居民尿样,获取银染的双向电泳图像,经比较发现：镉性肾损害尿蛋白质组(大鼠)及其不同预后尿蛋白质组(人)的差异主要出现在低分子量区域,表明慢性镉中毒的损伤与预后均以低分子量蛋白尿为特征；同性别的各剂量组大鼠尿蛋白分布模式基本上是一致的,而不同个人之间的蛋白分布模式差异却很大。进一步利用双向差异凝胶电泳结合基质辅助激光解析电离-飞行时间质谱(2DE DIGE with MALDI-TOF MS)技术分析差异表达,鉴定出常用的镉肾毒性效应指标血清白蛋白(ALB)、NAG,还发现了潜在的可用于镉肾毒性及预后研究的蛋白质人α1-微球蛋白/bikunin前体、IgG kappa chain。更多还原

【Abstract】 Occupational or environmental exposure to cadmium (Cd) can give rise to adverse health effects, particularly kidney damage, as a result of Cd accumulation in the kidneys. Cd-induced kidney damage is characterized by proximal tubular reabsorptive dysfunction, the earliest manifestations of which are increased urinary excretion of low-molecular-weight proteins such as urinary metallothionein (UMT) andβ2-microglobulin (UB2M), etc. An important toxicological feature of cadmium is its extremely long biological half-time in the organism. Once absorbed, cadmium is efficiently retained in the organism and accumulates throughout life. The prognosis of Cd-induced kidney dysfunction after the cessation of or reduction in Cd-exposure is concerned by researchers all over the world.In late 2006 we followed up the residents in Cd-polluted areas in Zhejiang Province, China, after the 1998 survey. In the pilot study conducted in 1995, the Cd content of rice produced in the highly-polluted area was found as high as 3.7 mg Cd/kg, and thus, the consumption of rice grown locally was identified as the most important determinant of Cd exposure among the residents. Since the year 1996 the residents living in the Cd-contaminated areas stopped to eat rice in their own fields and turned to rice from non-polluted areas. Data of a number of 148 individuals who were examined both in the 1995 and 1998 surveys were picked up to investigate the evolution of kidney function after reduction in Cd-exposure. Based on defined cut-off points of specific kidney markers, the prevalence of adverse renal effects in 1995 and 1998 were compared to each other, indicating that the Cd-induced renal dysfunction might be reversible if UCd concentration was low-level before exposure decreasing, otherwise it might be irreversible or aggravated. Even so, it was still unknown what would happen in a longer time perspective. Therefore, a number of 475 residents who participated in the 1998 survey were followed up in 2006. In order to evaluate the changes in renal function among the population after reduction in Cd-exposure for years, the cut-off values of kidney markers were redefined considering the aging effect and the prevalence of kidney dysfunction in 1998 and 2006 was compared to each other. To find out how the prevalence of impaired kidney function develops after cessation of exposure, and how that development relates to the initial exposure level, three different biomarkers of kidney effects were assessed:urinary N-acetyl-β-D-glucosaminidase (UNAG), UB2M and albumin (UALB). For these biomarkers we also investigated how predicitive an impaired level was for future impairment in the same individual. This study over eight years shows clearly that albuminuria from cadmium exposure (at the here observed exposure levels) is a reversible effect that recovers after cessation of exposure. For the markers of tubular effects, a tendency towards improvement, but not complete recovery, was observed for UNAG, but only among women. Maybe there is also some improvement for UB2M, but then only in relatively young individuals. The pattern previously indicated in a smaller three-year study, with improvements for individuals with low exposure levels and impairment for those with high exposure levels did not repeat itself. Based on the individual level, the study demonstrates that an impaired UNAG value is very little prognostic for the result of a future sampling for UNAG. In contrast, an impaired UB2M value is a strong predictor of impaired values also in the future.Animal studies with well-defined exposure doses, duration and route, can help understand the process of evolution of renal function after changes of Cd-exposure. In one experiment, Wistar male rats pre-treated with zinc (Zn) or copper (Cu), were administered with CdMT, to observe the ratios of Zn/Cd, Cu/Cd in liver metallothionein (MT) induced by zinc or copper. It was concluded that Zn bound to MT was more easily replaced by Cu than Cd bound to MT. The non-MT-bound Zn in liver tissues may be one of the explanations for the difference. In another one, to investigate the role of MT in tissues after cessation of Cd-exposure, Wistar rats of both genders were exposed to Cd via drinking water for 12 weeks, and then exposure was stopped; the animals were monitored for the following 16 weeks, i.e., until 28 weeks after start of the experiment. We observed that the levels of immunohistochemically determined MT in rat liver and kidneys were responsive to chronic oral Cd exposure. Once the exposure ceased, the present study confirmed a decreased Cd burden in the liver tissue and found increased hepatic MT levels. The decreased UNAG levels in the lowest-dose group and the decreased UMT levels in the urine in all of the dose groups after cessation of exposure might reflect a partial recovery of renal tubular function and decreased transport of CdMT from the liver. These findings should also contribute to our understanding of the events taking place in tissues after cessation of Cd exposure in humans.Recent developments in proteomic technologies have provided tools for discovering and identifying disease-associated biomarkers. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) analysis was performed on urine samples for Wistar male rats orally exposed to Cd via drink water. The samples collected at different time points of the exposure, demonstrated a time-dependant increase in the density of protein/peptides peaks at the range of 2000-5000 Da,8000-12000 Da and 17000-19000 Da. The dose-effect relationship was also found between dose groups and the density of those peaks. This experiment revealed the time-related and dose-related changes of urinary protein patterns under Cd-exposure, which might be a good start to investigate the Cd nephrotoxicity and its prognosis from a proteomic perspective. Because of the limited information provided by SELDI analysis, two-dimensional gel electrophoresis (2DE) was performed to profile urinary proteins from animals (rats exposed to Cd via drinking water) and humans (residents living in the Cd-contaminated areas in southeast China). The silver-staining images showed a similar pattern of protein spots for the urine from rats of the same gender at the same dose, but varied patterns for individuals of human. In spite of that, both urinary proteome of Cd-induced renal dysfunction (rats) and its prognosis (human) indicated increasing spots located in the low-molecular-weight area. Furthermore, by labeling urinary proteins with different fluorescent dyes, the 2D difference gel electrophoresis (2D-DIGE) analysis revealed 95 protein spots for rats and 78 for humans, the expression levels of which were altered during cadmium exposure.36 protein spots up-regulated in rat urines and 21 in human urines (>1.49-fold changes and P<0.05) were picked up for subsequent identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. Some of them were identified as Serum albumin, NAG, Ig kappa chain C region, human alpha-1-microglobulin/bikunin precursor (Protein AMBP) and rat alpha-2u-globulin. The elevated excretion of urinary NAG and albumin (both of which are widely used in the study of Cd nephrotoxicity), and probably Protein AMBP, was a sign of impaired renal function. Ig Kappa chain may increase when the capacity of tubular reabsorption decrease. Although not all urinary biomarkers related to cadmium toxicity were covered by our findings, the present study should be regarded as exploratory in an attempt to profile cadmium-induced proteinuria with proteomic techniques.更多还原