【作者】 贺超才；

【导师】 雷毅雄；

【作者基本信息】 广州医学院 ， 卫生毒理学， 2011， 硕士

【摘要】 1.目的镉(Cadmium,Cd)及其化合物是常见的工业毒物和环境污物,进入机体内的镉可引起多种组织器官的损害,甚至导致肿瘤的发生。根据国际癌症研究组织(IARC)的报告,镉及其化合物是确认的第一类致癌物,是人类危害最大的金属毒物之一。因此,镉暴露及其健康损害,特别是镉低水平长期毒作用已经成为毒理学研究的热点,其中关于DNA修复基因在亚慢性镉暴露动物体内的表达变化鲜见报道。本实验通过创建大鼠亚慢性镉暴露模型,对镉低水平较长期致大鼠的损伤及其与DNA修复基因ERCC1表达变化的关系进行研究,为镉化物对机体的亚慢性损伤及其毒作用机制补充新的实验数据。2.方法2.1在急性毒性试验的基础上,用SPF级别SD大鼠96只按体重随机分4组,3个CdCl2染毒组(高剂量1.225mg/kg、中剂量0.612mg/kg和低剂量0.306mg/kg)和1个生理盐水对照组(0.9% NaCl),每组24只大鼠,雌雄各半,腹腔注射,每天1次,每周5天,连续14周。然后对大鼠进行24小时尿液收集,穿刺心脏采血,剖杀切取内脏器官并对其进行称重。2.2用原子吸收分光光度法测定肝和肾组织的镉含量,计算肝脏和肾脏的脏器系数,同时从每组中随机抽出4只大鼠(雌雄各半),分别对其肝脏和肾脏进行病理切片以观察组织形态学变化。测定血清丙氨酸转氨酶(ALT)和谷草转氨酶(AST)作为肝功能生化指标,测定血中尿素氮(BUN)、血肌酐(SCR)、尿肌酐(CR)以及24小时尿蛋白(24hPro)作为肾功能生化指标。2.3在构建亚慢性镉暴露大鼠模型的基础上,探讨DNA修复基因ERCC1在肝和肾组织中的表达情况。根据ERCC1基因的mRNA序列,用Primer Express 2.0软件设计ERCC1上下游引物。应用Trizol试剂盒并按照其一步法提取大鼠肝组织和肾组织中总RNA,经逆转录(RT)后用实时荧光定量PCR(FQ-PCR)检测方法分析其ERCC1基因的表达情况。2.4分析雌雄大鼠的肝、肾脏器系数及其与镉含量的关系,进一步对大鼠肝脏和肾脏的ERCC1基因表达水平与肝、肾镉含量,以及肝、肾功能变化分别作关联性分析。数据均以x±s表示,运用t-检验、方差分析、Games-Howell检验、Student- Newman-Keulsa检验和相关分析等对数据进行处理,P<0.05有统计学意义。3.结果3.1大鼠经过不同镉浓度染毒14周后,不同镉暴露的大鼠肝、肾脏器中的镉含量均有不同程度升高,并随着镉的暴露水平的增加而升高,存在明显的剂量依赖关系(P<0.01)。实验组大鼠肝、肾组织的脏器系数均有不同程度增大,并随着镉的暴露水平的增加而增大,具有明显的剂量依赖关系(P<0.01)。实验组大鼠肝、肾组织的病理切片中与对照组相比也均观察到明显的病理学改变。提示亚慢性CdCl2染毒已经引起肝、肾靶器官不可逆的的严重损伤。3.2亚慢性染毒试验的结果显示,CdCl2染毒的大鼠中肝功能生化指标AST和ALT的活性以及肾功能生化指标Scr、BUN和24hPro的活性等,相对于对照组均有不同程度的升高,并随着镉的暴露水平的增加而升高,具有明显的剂量-反应关系(P<0.05)。这些数据显示镉亚慢性染毒已经引起肝、肾组织的不同程度的损害并出现了功能性的改变。以上检测结果显示雌雄大鼠的变化趋势相同,而且雄性大鼠的变化有大于雌性大鼠变化的趋势。3.3经FQ-PCR分析,肝脏和肾脏的ERCC1基因的表达量以β-actin为内参照进行标化计算,在镉高、中、低剂量染毒大鼠组中,肝脏ERCC1表达量分别是对照组的66.7%、70.9%和74.3%,而肾脏ERCC1表达量分别是对照组的69.4%、79.39%和85.2%,显示随着大鼠CdCl2暴露水平的升高而其肝、肾的ERCC1表达水平下调更明显,具有明显的剂量-反应关系(P<0.01),而且随着染镉剂量的升高雌性大鼠ERCC1表达量的降低程度比雄性大鼠更为明显。3.4经过统计学的相关性分析,发现大鼠肝、肾靶器官的ERCC1 mRNA表达量与大鼠肝、肾中的镉蓄积浓度呈负相关关系;大鼠肝、肾中的ERCC1 mRNA表达水平还分别与肝功能生化指标(AST,ALT)和肾功能生化指标(24hpro,BUN, Scr)异常存在负相关关系,并与肝、肾组织的病理学改变相关。结果表明,肝、肾靶器官的ERCC1基因表达水平与大鼠的镉暴露程度、肝和肾组织中的镉镉蓄积浓度及其引起的内脏器官异常效应呈现出良好的负相关性(P<0.01或P<0.05)。4.结论4.1构建了大鼠亚慢性镉暴露模型,发现镉可以引起肝、肾脏器系数的增大和不同程度的病理改变,同时引起肝、肾生化功能的改变,有明显的剂量-反应关系,表明镉对肝、肾等靶器官均产生明显的毒性损害并得到病理学证实,为镉化物对机体的亚慢性损伤及其毒作用机制补充新的实验数据。4.2在亚慢性镉中毒模型中观察到镉对大鼠内脏器官DNA修复基因ERCC1表达的诱导作用,揭示大鼠肝、肾ERCC1基因表达下调与肝、肾组织的镉蓄积量及其引起的内脏器官异常效应呈现出良好的剂量-反应关系,提示ERCC1基因的表达改变有可能成为一个有价值的镉亚慢性暴露或效应生物标志物。更多还原

【Abstract】 1. Objective:Cadmium (Cd) and its compounds are known occupational hazardous chemicals and environmental contaminants,and they can induce a wide variety of adverse health effects, even result in human tumors after absorption. According to IARC, Cd and its compounds are classified as human carcinogens, and they are the one of the most hazard metallic poisons in human. Therefor, Cd exposure and its health damage, especially toxic effects of long-term low level exposure to Cd has become a hotspot in toxicological study, in which the expression changes of DNA repair genes in rats exposed to Cd has been rarelly reported. The present objective is to investigate the the oxicity injuries and DNA repair gene ERCC1 expressions in rats exposed subchronically to Cd through establishing a model of long-term low level exposure to Cd in rats, supplement experimental data of Cd inducing subchronically damages and toxic mechanisms.2. Methods:2.1 Based on acute toxicity test, the 96 Sprague-Dawley rats (half male and half female) were culled by weight and randomly assigned to four experimental groups with 24 rats each group. The rats were respectively given i.p. injections of ultrapure water solution containing the CdCl2 in doses of 1.225 mg/kg,0.612 mg/kg,0.306 mg/kg body wt (5 times i.p. per week) for 14 weeks. After 14 weeks of treatment all rats were placed separately in glass metabolic cages for 24-h urine collection, as well as the blood and urine and internal organs were collected.2.2 Cd levels were detected by atomic absorption spectrometry in liver and kidney, and their organ coefficients were respectively calculated. At the same time we select ed4 rats from every group randomly (half male and half female), and the biopsies of liver and kidney were prepared to observe the histological changes. The activities of alanine aminotransferase (ALT) and asparate aminotransferase (AST) in serum were measured as indicators of the liver function. The blood urea nitrogen(BUN), serum creatinine(SCR), urine creatinine(CR), 24 hour protein (24hPro) were detected as parameters of the kidney function.2.3 Through establishing a model of subchronic exposure to Cd in rats, we could explore the expressions of ERCC1 in liver and kidney. According to mRNA sequence of ERCC1, upstream and downstream primers of ERCC1 were designed by Primer Express 2.0. Using TRIzol reagent. The total RNAs from liver and kidney were isolated and the ERCC1 expressions were analysed with reverse transcrispion (RT) and fluorescent quantitative-polymerase chain reaction (FQ-PCR).2.4 The relationships between liver, kidney coefficients and their Cd concentration, and the correlations between ERCC1 expression levels and Cd concentration in liver and kidney were analysed. Data were expressed as mean±standard deviation ( x±s), and statisticlly analysed with t-test, variance analysis, Games-Howell test, Student- Newman-Keulsa test and correlation analysis and so on. The level of significance was set at P<0.05.3. Results:3.1 After rats were treated by different Cd concentrations for 14 weeks, the levels of Cd concetration in liver, kidney were significantly increased compared to controls, and there were distinct dose-dependent relationships among different groups(P<0.01). The levels of organ/body coefficients in liver and kidney were significantly increased with compared to controls and there were dose-dependent relationships among different groups (P<0.01). The internal organs (liver and kidney) of rats showed pathological changes compared to control, which influenced in tissues by the administration of Cd.3.2 As the result of subchronic experiment shown,the biochemical indicators like ALT, AST for hepatic function and 24hpro, BUN, SCR for renal function induced by Cd displayed significantly increased with the levels of Cd-exposure compared to controls, and there were dose-response relationships among different groups (P<0.05).This data indicated series of damages as well as dysfunctions had been occured in the liver, kidney by subchronic Cd-exposed. The results above were almost the same between males and females, while the viarations showed in males were more obviously than that of females.3.3 After FQ-PCR analysis, under the inner standard ofβ-actin, showed primarily that the levels of ERCC1 expression were 66.7%、70.9%、74.3% down regulation in liver , and were 69.4%、79.39%、85.2% down regulation in the kidney of rats treated with CdCl2 in high-dose, med-dose, and low-dose, respectively (P<0.01). when compared with controls. The data above displayed a obviously dose-response relationship since the down regulations were along with Cd-exposure levels (P<0.01). Besides, the level of ERCC1 down regulation in females is a little more obviously than that in males with the Cd concentration increased (P<0.05).3.4 The statistical correlation analysis showed that, in liver and kidney respectively, there were the negative correlation between ERCC1 under-expression and Cd cumulation. Besides in liver and kidney respectively, there were the negative correlation between ERCC1 under-expression and the biochemical indicators such as ALT, AST for hepar function and 24hpro, BUN, Scr for renal function induced, as well as the pathological changes in the liver, kidney respectively. The results showed the levels of ERCC1 factor had good negative dose-response relationship along with the Cd exposure levels in liver and kidney tissue (P<0.01or P<0.05).4. Conclusions:4.1 A rat model of subchronic Cd exposure was established. Cd could induce the organ coefficient of liver and kidney increasing ,various pathological changes and biochemical function changes in liver and renal, with the dose-response relationship. The results above confirm the obviously toxicity in liver and kidney induced by Cd which will provide new scientific data for long-term toxicity of Cd compounds.4.2 Indution of DNA repair gene ERCC1 was observed in rat internal organs on the basis of the subchronic Cd-exposed model, revealing that there was a good negative dose-response relationship between the ERCC1 expression levels and Cd exposure in rats’liver and kidney, respectively. These findings indicate that expression damages of ERCC1 factor seem to be a significant biomarker of exposure or effect to Cd subchronic toxicity.更多还原