Download METALLOTHIONEINS IN RATS EXPOSED TO BARIUM CHLORIDE

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.
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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
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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).
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