The Content of Metallothionein in Mice Liver After Acute Administration of Cadmium, Zinc and Selenite Ions

Abstract

Introduction Metallothionein is a low molecular mass (3500 to 14000 Da), cysteine-rich, metal-binding protein. It has the capacity to bind both physiological (Zn, Cu, Se) and xenobiotic (Cd, Hg) heavy metals through the thiol group of its cysteine residues, which represents nearly the 30 % of its amino acidic residues [1]. Metallothionein function is not clear, but experimental data suggest that it may provide protection against metal toxicity and oxidative stress, and may be involved in regulation of physiological metals [2]. It has been demonstrated that Cd-induced metallothionein have a protective function against hydroxyl radicals [3]. This heavy metal stimulates free radical production, resulting in oxidative deterioration of lipids, proteins and DNA, and initiating various conditions in humans and animals [4]. Antioxidants Zn and Se may suppress deleterious effects of Cd. The present study was conducted to investigate the effect of Cd2+, Zn2+ and SeO3 2- on the content of metallothionein in mouse liver. Materials and methods Outbreed mice weighing 20-25 g were used in the study. All experiments were performed according to the Republic of Lithuania Law on the Care, Keeping and Use of Animals (License of State Veterinary Service for working with laboratory animals No 0136). Mice were randomly assigned into six groups: five experimental and one control. Each group included 8-15 mice. Mice of the first experimental group were injected intraperitoneally with CdCl2 solution (1.6 mg Cd / kg body mass). Mice of the second experimental group received intraperitoneal injection of ZnSO4 solution at a dose level 1.56 mg Zn2+ / kg body mass. Mice of the third experimental group were injected intraperitoneally with ZnSO4 solution and after 20 min – with CdCl2 solution in aforementioned dose. Mice of the fourth experimental group received intraperitoneal injection of Na2SeO3 solution at a dose level 1.25 mg Se / kg body mass. Mice of the fifth experimental group were injected intraperitoneally with Na2SeO3 solution and after 20 min – with CdCl2 solution in aforementioned dose. Control animals (sixth group) received injection of the same volume of physiological solution. The content of metallothionein determination was assayed according to the method as described in [5]. Aliquots of 1 ml of supernatant were added with 1.05 ml of cold (-20 ºC) absolute ethanol and 80 μl of chloroform; the samples were then centrifuged at 6000 x g for 10 min. The collected supernatant was combined with three volumes of cold ethanol (-20 ºC), maintained at -20 ºC for 1 h and centrifuged at 6000 x g for 10 min. The metallothionein – containing pellets were then rinsed with 87 % ethanol and 1 % chloroform and centrifuged at 6000 x g for 10 min. The metallothionein content in the pellet was evaluated using the colorimetric method with Ellman’s reagent. The pellet was resuspendent in 150 μl 0.25 M NaCl and subsequently 150 μl 1 N HCl containing EDTA 4 mM was added to the sample. A volume of 4.2 ml 2 M NaCl containing 0.43 mM DTNB buffered with 0.2 M Na – phosphate, ph 8.0 was then added to the sample at room temperature. The sample was finally centrifuged at 3000 x g for 5 min; the supernatant absorbance was evaluated at 412 nm and metallothionein concentration was estimated utilizing cysteine as a reference standard and expressed as micrograms of SH per gram of wet weight. All samples were run in duplicate. Results were expressed as the mean standard error of mean. Statistical significance was set at p<0.05. Results The effects of Cd 2+, Zn2+ and SeO3 2- on the content of metallothionein in mice liver 2, 8 and 24 h after injection are shown in Figures 1-3. [...].