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Bull. Vet. Inst. Pulawy 47, 153-156, 2003 METALLOTHIONEINS IN RATS EXPOSED TO BARIUM CHLORIDE BOLESŁAW FLORIAŃCZYK* AND ELŻBIETA STAROSŁAWSKA** *Department of Biochemistry and Molecular Biology, **Department of Oncology,. Medical University of Lublin, 20-123 Lublin, Poland e-mail: [email protected] Recived for publication January 05, 2003. The aim of the investigation was to determine metallothioneins concentration in tissues of rats intoxicated chronically with barium chloride. It was found that the intoxication resulted in a significant increase in metallothioneins content, especially in the brain. Key words: rat, barium, intoxication, metallothioneins. Metallothioneins (MTs) are the common proteins in animal tissues. Several molecular forms of metallothioneins were found (11, 12, 13). All of them contain 6061 amino acid residues. Cysteine is the most abundant amino acid component. It constitutes above 30 per cent of total amino acid residues. Such a high amount of cysteine, containing the reactive sulfhydryl groups is important for biological functions of metallothioneins (1, 2, 4). They take a part in the homeostasis of metal (Zn, Cu) ions, in biosynthesis of zinc-containing proteins and in regulation of zinc-dependent enzymes activity. Furthermore, they reduce toxic effects of heavy metals, oxygen, free radicals, electrophilic pharmacological agents used in the cancer therapy and mutagens (5, 6, 9). The biosynthesis of metallothioneins is affected by many factors, like: heavy metals, inflammatory factors, free radicals, steroid hormones and some pharmacological agents (12). Barium belongs to the second group of the periodic table. After the acidification of the environment, barium is rapidly removed from the soil and cumulates in the tissues of plants and animals. Water soluble barium compounds (e.g. chloride) are more toxic than those less soluble (e.g. sulphate or carbonate). Barium has a tendency to accumulate in animal tissues, especially in the skeleton where it inhibits the bone mineralization. The symptoms of barium intoxication are the result of its action on, among other things, the nervous system. In the present study an attempt has been made to gain information whether an increase in metallothionein concentration occurs in selected tissues of the rats intoxicated with barium. 154 Material and Methods Animals. The experiments were carried out on 6-month old Wistar rats, 150200 g of body weight. The animals were divided into control and investigated groups. The animals of the control group were treated with standard dry diet and redistilled water, whereas the animals of the investigated group were fed the same diet and redistilled water supplemented with 0.01% concentration of barium chloride. After 6 weeks the animals were anaesthetized with kethamine. The liver, kidney, and brain were excised and submitted to further procedures. Tissues. The excised organs were weighed, washed with physiological saline and homogenized in 4-time volumes of 10 mM Tris-HCl buffer, pH 7.4, with a glass homogenizer. The homogenates were centrifuged at 10 000 x g for 10 min and supernatants were collected and heated in a boiling water bath for 2 min. Precipitated proteins were separated by centrifugation under the above described conditions. The supernatants were collected and stored in a freezer. Determination of metallothioneins. The concentration of metallothioneins was determined by the cadmium-haemoglobin affinity assay (3). Statistical analysis. The results were submitted to statistical analysis with the Cohran and Cox test, accepting P < 0.05, as significant. Results The concentration of metallothioneins in the brain, liver and kidney was presented in Table 1. As can be seen from this table, significant differences in metallothioneins contents in particular organs were found. In the control group the highest concentration of metallothioneins was demonstrated in the kidney and liver; 9.64 ± 2.36 µg/g wet tissue and 9.27 ± 3.12 µg/g wet tissue, respectively. Distinctly lower amounts were found in the brain (3.03 ± 0.36 µg/g wet tissue). In barium intoxicated animals an increase in metallothioneins content in all the examined tissues was observed: the brain - 9.12 ± 3.05 µg/g wet tissue, kidney - 12.23 ± 3.65 µg/g wet tissue, liver - 11.98 ± 2.86 µg/g wet tissue. Table 1 The concentration of metallothioneins (in µg/g wet tissue) in rats exposed to barium Control group Intoxicated group Tissue Mean ± SD mean ± SD Significance Brain 3.03 ± 0.36 9.12 ± 3.0 5 p < 0.05 Liver 9.27 ± 3.12 11.98 ± 2.86 Insignificant Kidney 9.64 ± 2.36 12.23 ± 3.65 Insignificant 155 Discussion Metallothioneins (MT) are intracellular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MT have unique structural characteristics to give potent metal-binding and redox capabilities. Their primary role has not been identified and remains elusive, as further functions continue to be discovered. The most widely expressed isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators. In the alimentary tract and pancreas, MT respond mainly to zinc status. A brain isoform, MT-3, has a specific neuronal growth inhibitory activity, while MT-1 and MT-2 have more diverse functions related to their thiolate cluster structure. These include involvement in zinc homeostasis, protection against heavy metals (especially cadmium) and oxidant damage, and metabolic regulation via zinc donation, sequestration and/or redox control. In our study in the control group of animals the highest level of the metallothioneins was ascertained in the kidneys and liver. The high amounts of the compounds in the kidneys and liver allow to conclude that these organs are the main producers of the proteins. These organs are responsible for trace elements homeostasis and detoxication processes. In the group of barium intoxicated animals an increase in MT content in all the examined tissues was noted and the differences in the intensity of the MT induction depended on the kind of tissue. MT synthesis was induced most strongly in the brain. Hamer (7) suggests that metallothioneins content depends on a tissue and time of metal exposure. Besides, the MT synthesis relies on the affinity of the tissue towards the appropriate metal. The metallothionein produced in the liver promotes the bioaccumulation of barium and prevents the toxic effect of this metal on other tissues. In a critical situation, when the metallothionein ability to uptake barium in the liver is exceeded, barium becomes dangerous for cells of many tissues (8, 10, 13). It seems that the kidneys play a similar role. The main function of the kidneys is the excretion of waste metabolites and toxic substances, including metals. Since a considerable amount of blood flows through the kidneys, the metals (including barium) contained in the blood may stimulate the biosynthesis of metallothioneins in this organ. Such a mechanism may protect the kidneys against the toxic effects of heavy metals (14). References 1. Bremner J., Beattie J.H.: Metallothionein and the trace metals. Ann. Rev. Nutr., 1990, 10, 63-83. 2. Cousins R.J., Leinart A.S.: Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 1988, 65, 238-309. 3. Eaton D.L., Cherian M.G.: Determination of metallothionein in tissue by cadmiumhemoglobin affinity assay. in Methods in Enzymology, 1991, pp. 205-208. 4. Floriańczyk B.: Toksyczne i kancerogenne własciwości kadmu. Now. Lek., 1995, 64, 737-745. 5. Floriańczyk B.: Funkcja metalotionein w ustroju. Post. Hig. Med. Dośw., 1996, 50, 375-382. 156 6. Floriańczyk B.: Detoksykacyjna funkcja metalotionein. Now. Lek., 1997, 66, 310318. 7. Hamer D.H.: Metallothioneins. Ann. Rev. Biochem., 1986, 55, 913-951. 8. Johnston S.W., Ozols R.F., Hamilton T.C.: Mechanism of drug resisat interacts with a constitutively active transcrition factor MTF-1. Proc. Natl. Acad. Sci. USA, 1994, 91, 1219-1225. 9. Kelly S.L. Basu A., Teicher B.A., Hacker M.P., Hamer D.H., Lazo L.S.: Overexpression of metallothionein confers resistance to anticancer drugs. Science, 1988, 24, 1813-1815. 10. Naganuma A., Satoh M., Imura N.: Specific reduction of toxic side effects of adriamycin by induction of metallthionein in mice. Jpn. J. Cancer Res., 1988, 79, 406-411. 11. Palmiter R.D.: Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor MTF-1 Proc. Natl. Acad. Sci. USA, 1994, 91, 1219-1223. 12. Roesijadi G.: Metallothionein and its role in toxic metal regulation. Comp. Biochem. Physiol., 1996, 113C, 117-123. 13. Sudo J., Hayashi T., Terui J., Soyama M., Fukata M., Kakuno K.: Kinetics of Cd2 in plasma, liver and kidneys after single intravenous injection of Cdmetallothionein-II. Europ. J. Pharmacol., 1994, 270, 229-235. 14. Vestergaard P., Shaikh Z.A.: The nephrotoxicity of intravenously administered cadmium-metallothionein: Effe3ct of dose, mode of administration, and prexisting renal cadmium burden. Toxicol. Appl. Pharmacol., 1994, 126, 240-247.