| 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. |
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