Cadmium Poisoning And Effect Of Zinc Supplement On Cadmium Cytotoxicity

Cadmium(Cd) is a toxic heavy metal that can cause toxic damage to a variety of organs and systems both in people and animals. Liver and kidney are the primary targets of Cd poisoning. High concentration or acute Cd treatment mainly induce hepatotoxicity; while low dose or chronic Cd poisoning preferentially lead to kidney damage. Various ways could contribute to the Cd-induced cell apoptosis, among which, ROS over-accumulation in cells by Cd is the pivotal one. Cd could induce ROS production in cells through either direct or indirect way, consuming peroxiredoxins such as MT, GSH and SOD etc, ultimately resulting in lipid peroxide reaction and oxidative damage in cells. A series of consequences following cell oxidative damage which include cytoskeleton damage, mitochondrial dysfunction, chromosome breakage and protein inactivation would happen, and cells undergo non-recoverable damage even death at last.Zinc(Zn) is an important trace element indispensable for our body, involving in the biosynthesis and functional regulation of over 120 and 300 types of enzymes in body respectively. Zn deficiency could lead to fetal anomaly, weakened immunity, nervous disorder, incidence and mortality rates of disease increase. In recent years, it was found that Zn can against cytotoxicity caused by a variety of stimulus including heavy metals, and be used as an antidote. Zn protection to cells is related to a variety of aspects. Among them, contribution of Zn to cellular antioxidant capacity by promoting the expression of antioxidant enzymes, improving the efficiency of ROS scavenging in cell and countervailing cell oxidative damage are widely studied. MT, with low molecular weight(6-7 KDa) rich in cysteine, acts as a free radical scavenger to eliminate surplus ROS and alleviate Cd intoxication because of its high capacity to bind Cd2+ which can transfer toxic Cd2+ to nontoxic MT-Cd. Expression of MT relies on the mediation of MTF-1, most of which are normally located in cytoplasm but rapidly translocate to the nucleus under different stress conditions. MTF-1 contains six zinc fingers of the Cys-2-His type, and in nucleus it mediates DNA-binding to MRE which presents in the promoter of MT genes and initiates the gene expression.From the 1960 s, people established various kinds of cadmium poisoning models including rat, rabbit, swine, monkey and fish to elucidate the underlying mechanism about Cd damage and cytotoxicity. Recently, scientists devoted themselves on effect of Zn on Cd toxicity by adding Zn2+ to the Cd-treated cells. Although some advances have been gained, it is still controversial involving the mechanism of Zn on Cd poisoning. Based on references from previous researches, as well as using some metallomics method, we carried out the present experiment according to the items below, aiming to understand more about the mechanism of cadmium poisoning and effect of zinc supplement on cadmium poisoning in cell. 1. Effect of chronic Cd poisoning on MT expression in liver and kidney of ratsKunming rats were given intraperitoneal injection with a sterile solution of 1.0 mg/kg body mass Cd2+ in 0.9% Na Cl for 21 days. Livers and kidneys were isolated for the MT gene and protein analysis. According to the results, expression levels of both MT-1 and MT-2 m RNA in Cd treated livers was considerably higher than those from control group(P<0.01), rising to 7.7 times and 10 times respectively. In kidney, there was also a significant increase on MT-1/2 m RNA after Cd exposure. In contrast to the liver, MT-1 m RNA expression level was higher than MT-2 gene in kidney. From the immunohistochemistry analysis, MT protein evenly distribute in the cytoplasm of liver cells; and can be found also in the cytoplasm of renal proximal as well as distal tubule cells. At the third week after Cd treatment, MT protein synthesis in liver was considerably higher than the control group(P<0.01). MT protein concentration in Cd-treated rat kidney was similar to control group(P>0.05). However, MT protein concentration in kidneys from both the Cd treatment group and control group were higher than those in livers. 2. Effect of chronic Cd poisoning on metal metabolism in liver and kidney of ratsAs expected, Cd2+ content in Cd-treated liver and kidney were both higher than that in control group. Cd concentration in liver of Cd treatment group was about twice higher than that in kidney. Zn2+ had a significant increase in liver and kidney after Cd exposure, reaching 66.04 μg/g(P<0.01) and 35.33 μg/g(P<0.05) respectively. Contrary to Zn2+ distribution, Ca2+ content had high increase in kidney(reach to 700 μg/g), while a significant reduction in liver. In accordance with Ca2+ tendency,Fe2+ content in liver was lower than that in control group. But in kidney, the situation was opposite. Fe2+ concentration in control group was unexpected higher than that in Cd treatment group. As to Cu2+, a slight rise both in liver and kidney was examined before and after experiment, but not so considerable as other metals. 3. Cytotoxicity of Cd against MDBK cellIn order to further uncover the mechanism of Cd poisoning and the role of Zn in Cd poisoning, we exposed Madin-Darby Bovine Kidney(MDBK) Cells with Cd2+ alone or in combination with Zn2+. It can be shown from the results that Cd cause MDBK cell apoptosis in a time-concentration way. Upon Cd attack, cells underwent a series of damage including structure breakdown, skeleton protein degradation, ROS over-production and mitochondrial membrane potential decline. 10 μM Cd treatment caused moderate cytotoxicity; while 50 μM Cd attack leads to irretrievable damage on cell. Upon high concentration of Cd, ROS accumulated sharply in cell within a short time which subsequently aggravates mitochondria damage, resulting in cell apoptosis and necrosis rate increase ultimately. 4. Effect of Zn supplement on cytotoxicity caused by CdResults from the present study indicated that Zn supplement exhibits significant inhibition effect on Cd-induced cytotoxicty, under certain constraints however. When cells were exposed to 10 μM Cd Cl2, supplementation with 10 μM or 50 μM Zn2+ produced comparable improvements in cell survival, ROS elimination and MMP normalization up to 24 h after treatment. In contrast, Zn2+-mediated protection could only be detected at earlier time points(3 h and 6 h) when 50 μM Cd Cl2 was present. After 12-h and 24-h exposure to 50 μM Cd2+, addition of 50 μM Zn2+ even intensified Cd2+-induced cytotoxicity, ROS production and mitochondria depolarization. These results suggested that Zn2+ prevented mild Cd2+-induced cell damage. However, exposure to high levels of Cd2+ could result in irreversible damage to bovine renal cells, which would not be alleviated by Zn2+ administration. 5. Effect of Zn on MTF-1 and MT expression in Cd-exposed MDBK cellsAs shown in our study, 6-h exposure to Cd2+ or Zn2+ alone led to comparable up-regulation of MT-1 and MT-2 m RNA levels. Moreover, incubation with both Zn2+ and Cd2+ substantially enhanced transcription of these MTs. Co-treatment with both Zn2+ and Cd2+ significantly enhanced MTF-1 m RNA levels in MDBK cells. To study the kinetics of Zn2+- and Cd2+-mediated up-regulation of MT-1, MT-2 and MTF-1, MDBK cells were exposed to the same conditions as planed at a range of time points. Results showed that levels of the MTs and MTF-1 proteins peaked dose-independently after 24 h and 12 h Zn2+ treatment, respectively. The addition of Zn2+ to Cd2+-exposed cells enhanced the MTs and MTF-1 protein levels in a dose- and time-dependent manner. These findings indicated that Cd2+ and Zn2+ stimulated expression of MTs and MTF-1 in a synergistic manner. We also observed increased levels of MT and MTF-1 proteins in MDBK cells following 6-h exposure to Cd2+ and Zn2+ using fluorescence imaging. 6. Effects of Zn2+ administration on Cd2+ accumulation in MDBK cellsIntracellular levels of Zn2+ and Cd2+ were determined over time using ICP-MS. In cells exposed to 10 μM Cd2+, addition of Zn2+ resulted in a dose-dependent reduction in the intracellular Cd2+ level, which was associated with increased levels of intracellular Zn2+. In contrast, the inhibitory impact of Zn2+ supplementation on Cd2+ uptake was not obvious in cells exposed to a high concentration of Cd2+(50 μM). Moreover, exposure to a high concentration of Zn2+(50 μM) even tended to increase intracellular Cd2+ levels. Notably, the intracellular Zn2+ level under these circumstances was comparable to that of control samples after 24 h treatment, even though transient Zn2+ absorption was detected. These results indicated that Zn2+ supplementation was able to prevent Cd2+ uptake into MDBK cells, but its efficacy was dependent on the level of Cd2+ exposure.