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There has been a study conducted to determine the possibility of using Na-thiosulphate to neutralize forages with an increased content of Cd, Pb and other toxic elements. It has been discovered that rats fed for two months with a diet including special fodder mixed with compounds of Cd (0.91 mg/kg), Pb (18.3 mg/kg) and Na-thiosulphate (2-10 g/kg) reduced by two times the accumulation of metals as compared to animals receiving only compounds of Cd and Pb. Animals fed with Cd and Pb compounds accumulated these metals in different organs, and had increased levels of γ-glutamiltransferase and metallothioneines in liver and kidneys. There was also observed a decrease of SH-concentrations in blood serum and the activity of porfibilinogensynthase, and a minor depression of levels of haemoglobin as compared to the control group. Na-thiosulphate also caused a remarkable gain of rat and sheep biomass and reduced the assimilation of Hg by sheep in conditions of mercury- enrichment biogeochemical provinces. It was not discovered some effect of Na-thiosulphate on level As and Sb in animal organism.

Introduction

It is known that some groups of mines, those located in the vicinity of complex ore extraction and processing facilities, as well as those near electrochemical and metallurgical plants face problems with selling fodder and live-stock products due to the high content of heavy metals including cadmium in them [1, 2, 4, 7]. Under natural conditions Cd and Pb are often accumulated in coarse fodder together with Hg and other toxic element compounds which occurs in the vicinity of metallurgical plants, exploitation regions of Pb and Zn deposits and tail-depositories which makes it unhealthy to develop animal breeding in the areas [3, 6, 8, 9]. This fact necessitates detoxication of fodder having a high Cd, Pb and Hg contents. This article describes the data on the experiments on detoxication of Cd, Pb, Hg in animals.

Materials and Methods

We have tested sodium thiosulphate to value Cd and Pb detoxication in diet and in animal organism. For the experiment we used mongrel white rats originally weighing 180 g. They were fed for 60 days with        combined fodder especially produced for rats. The experiment included two phases. During the first phase (30 days) 75 female rats were divided into five groups (Table 1). Group 1 was used as a control and was fed during the entire experiment with combined fodder having natural content of Hg, Cd, Pb, Cu, and Zn: 0.052, 0.031, 0.24, 6.98, and 56.8 mg/kg, respectively.

Table 1

Group 2 was fed with a combined fodder mixed with lead acetate and cadmium chloride introduced by spraying. Hg, Cu, and Zn concentrations in the treated fodder were 0.042, 6.00, and 46.8 mg/kg; Cd and Pb concentrations were 0.91 and 18.3 mg/kg which were 2.5 to 3.7 times above the maximum permissible level of Cd and Pb for cattle and sheep [11]. The rats of groups 3, 4, and 5 were fed with combined fodder mixed with 0.91 mg/kg of Cd and 18.3 mg/kg of Pb that was treated with  a 20%  solution of sodium thiosulphate: 2.5 and 10 g of salt per kg, respectively. In 30 days some of the animals were killed and the remaining rats, with the exception of those of group 2, were kept on the same diet for 30 days more. The rats of group 2 were divided into three sub-groups (2-1, 2-2, and

2-3). The animals of subgroup 2-1 were fed with the same combined fodder mixed with Cd and Pb as those of the original group 2 for a month. The rats of sub-group 2-2 were fed with metal-containing fodder modified with sodium thiosulphate (10 g/kg). Metal was not introduced in the fodder for the animals of group 2-3 but it was amended with sodium thiosulphate (10 g/kg). The animals were observed and periodically weighed. In 30 and 60 days five rats in all the groups were killed, samples of blood, brain, muscular tissue, liver, and kidneys were taken for analyses. Analogical experiments were carried out on effect  of Na-thiosulphate on detoxication of Hg, As, Sb (0.2, 1.0 and 1.0 mg/kg of diet, respectively) in rats and sheep inhabited in the conditions of Hg-enrichment biogeochemical provinces of South Fergana [6].

The amount of heavy metals in fodder, organs, and tissues of the animals was deter- mined using the flameless and flame atomic absorption technique with  the Kortec spectrophotometer, model “Kvant-AFA” and AAC- Z.2A with Zeeman correct after mineralizing the  material  with  peroxide  hydrogen  and nitric acids with  addition of perchloric acid. Nitric acid concentration in sampled solution was 2 per cent. Atomizing conditions were chosen in compliance with  the Service Manual for these instruments. In order to validate the technique, some reference samples of human hair, serum of blood, soil and mixture of plants were analyzed.

Biochemical analyses were conducted using the unified and known procedures. The liver and kidneys were tested for metallothioneines (MT) in the form of MT-like acid-resistant protein [5] and for the activity of porfibilino- gensynthase (ICI 4.2.1.24,  PBS) using  the procedure of L. Semenova [10]. Most  data were processed using method of variation statistics. The neutralizing action of Na-thiosulphate was evaluated by the clinical biochemical characteristics and basing on the accumulation of Cd, Pb and other elements in the muscular tissue, liver, and kidneys of the rats and sheep.

Results and Discussion

1. Accumulation of cadmium and lead in muscle tissues, liver and kidneys of the experimental rats

Table 2 shows the results of tests for the accumulation of Pb and Cd in liver, and kidneys for  rats  of  different  experimental  groups. Both metals are primarily accumulated in the kidneys and liver of rats (group 2) fed with metal-containing  fodder  for  30  days  was 5.00 and 0.80 mg/kg (30 days) while  after 60 days it amounted to 7.20 and 2.02 mg/kg. We should note that the Pb content in the kidneys and liver of group 2 animals was 1.3-3.7 and 5.0-7.2 times, respectively, above its maximum permissible level for food products [11]. Cd concentration in the organs of rats of group 2 increased by 6-15 times after 30 days and 8-15 times after 60 days compared with metal concentration in the kidneys and liver of the control group of animals, but it did not exceed the maximum permissible level of elements (0.3 mg/kg for the liver and 1.0 mg/ kg for the kidneys).

Introducing Na-thiosulphate in the amount of 2, 5, and 10 g/kg reduced the accumulation of Pb and Cd in the organs both in 30 and 60 days. We have noted that the concentrations of metals in the liver and kidneys were reduced in average two-fold under the action of Na-thiosulphate. We  have also noted that the reduction in the concentration of both Pb and Cd in the organs was reduced with an increase in the amount of Na-thiosulphate introduced. However, while the detoxication effect in the reduction the amount of Pb in the animal liver was attained after 30 and 60 days of the experiment, full reduction of Pb, accumulation in the kidneys, did not occur (it was 2.3-3.6 times above the  maximum  permissible  level even in group 5 at the maximal dose of Na- thiosulphate).

With the tested dose of Cd, we have not noted metal accumulation in the organs to a level exceeding maximum permissible Cd concentration in the liver and kidneys after 30 and 60 days of the experiment. At the same time, the detoxication effect  of  Na-thiosulphate is evident even with the mentioned dose of Cd (Table 2). The antidote, especially in the proportion of 10 g/kg of fodder, provides in average a two-fold reduction of Cd concentrations in the kidneys and liver.

We should note that for the tried doses neither Cd nor Pb were  noticeably accumulated in muscle tissue. The concentrations of lead, cadmium and mercury in muscle tissue of animals were low and changed as follows  (on all groups): 10-35 µg/kg (Pb), 25-59 µg/ kg (Hg) 4-60 µg/kg (Cd). The maximum content of Cd was revealed in muscle tissue of rats from in group 1-1 after the month period of feeding. Their concentrations in muscles did not exceed metal content of the tissues of the control- group rats and amounted to 15-28, 4-10 and 2-5 µg/kg for Pb, Cd, and Hg, respectively. This phenomenon is consistent with the data of L. Zaharova [12]. Feeding rats with  2 mg/kg of Cd and

25 mg/kg of Pb for 90 days caused respective concentrations as high as 0.73 and 1.72 mg/kg in the liver and 3.58 and 4.04 mg/kg in the kidneys. In this case, both metals were not accumulated in tissue compared with the control-group animals. The concentration of metals in muscle was lowest for animal, receiving sodium thiosulphate.

Table 2

A transfer of the animals fed for a month with fodder mixed with  these metals (group 2) to the diet of the control group modified with 10 g/kg of antidote also gives a neutralizing effect. This exhibited an approximate two-fold reduction of Pb and Cd concentrations in the organs compared with metal contents in those of the control-group animals. Na-thiosulphate was efficient in particular when rats received fodder with a low background concentration of metals and a maximal dose of antidote (10 g/kg) for a month. When this is the case, the Pb concentration, even in the kidneys, does not exceed the maximum permissible level and amounts in average to 1.13 mg/kg.

Image 1

The Cu concentration in blood serum, brain, muscular tissues, liver, and kidneys of all the animals was in the range of 0.7-1.1, 1.2-1.8,

0.5-0.8, 1.8-3.5, and 3.7-8.6 mg/kg, respectively. Zinc content of the mentioned organs and tissues varied between 1.8 and 4.3 mg/l for blood serum, 7.2 and 12.1 mg/kg for bra- in, 7.1 and 12.1 mg/kg for muscular tissue, 28.0 and 70.0 mg/kg for liver, 30.0 and 50.0 mg/kg for kidneys. Na-thiosulphate does not usually produce an evident impact on Cu and Zn levels in the organs and tissues of rats. In some cases Cur and Zn concentrations varied both under the action of Cd and Pb and under the influence of Na-thiosulphate, but it was kept within the physiological limits.

As a whole,  Na-thiosulphate evidently possesses a neutralizing effect at various stages of metal concentrations for those rats who- se fodder was modified with only Cd and Pb salts, the metals accumulated in the organs. This fact enables us to assume that Na-thiosulphate functions as a detoxicating agent both at the feeding stage and in the course of the delivery of these metals to the organism, which increased the release of these metals.

2. Clinical  biochemical changes  in the organism of rats due to the introduction of Cd, Pb, and Na-thiosulphate in fodder.

In order to estimate the detoxication action of Na-thiosulphate, we have chosen some specific  clinical  biochemical tests whose results for different groups of animals are given in Table 3-4. During the experiment, an increase in the mass of the rats of different groups varied within a month from 16+-3g. to 41+-3 g. and within two months, from 40+-8g. to 60+-8g. The smallest gain in the mass of group 2 animals, whose diet included fodder mixed up with Cd and Pb was obtained at the end of the first month (16±3 g) and only slightly differed  from  that of the control group; by the end of the second phase it amounted to 45±7 and 40±8 g. With an introduction of Na-thiosulphate there is a remarkable gain in biomass appreciably exceeding that  of  the control group (32-41 g in 30 days and 58-60 g in 60 days). We have also noted growth- stimulating  properties of Na-thiosulphate in mercury detoxication [6].

Table 3

A slightly lower level of hemoglobin was noted in group 2 animals especially after 30 days of the experiment. Then hemoglobin of all the rats was gradually equalized and approached 14-gram percent. Hematocrite and urea content  in the blood serum of all the animals regardless of their group was within the physiological standards (Table 3-4). The concentration of thiogroups in blood serum of group 2 rats was 1.2 to 1.5 times lower in 30 days and 1.5 times lower in 60 days com- pared with the content of sulfhydryl groups in blood serum of the control group of animals. Various amounts of Na-thiosulphate introduced in the diet have abruptly increased the concentration of thiogroups in serum up to or higher than the control values which we no- ted earlier in our experiments with mercury compounds but not as to As and Sb [6].

Keeping group 2 animals on a long diet of fodder mixed with Cd and Pb evidently inhibits PBS activity in blood (2.2-3.1 times compared with  the control group). The activity of this enzyme increased when the rats received Na- thiosulphate but still remained below the activity of the control group. On the other hand, the activity of γ-glutamiltransferase increased to about twice that in the blood serum of group 2 rats compared with the control group and was brought to a normal level with introduction of Na-thiosulphate.

A combination of Cd and Pb in the diet caused a 2.3-2.6 fold activation of MT synthesis in the liver and kidneys of group 2 animals compared with the control group. Introduction of Na-thiosulphate inhibits the synthesis of metalloproteide but its level  in  the  organs  is still 1.6-1.9 times higher than metallothioneine content in the liver and kidneys of group 1 rats (control group).

Table 4

Conclusion

Thus, in spite of the fact that Cd, Pb and Hg are not accumulated in muscular tissue of the rats, changes in the clinical biochemical characteristics (gain in biomass, reduced level of hemoglobin, activity of PBS, serum thiogroups, increased activity of γ-glutamiltransferase and MT synthesis) point to a certain intoxication with Cd and Pb compounds in the doses used (0.91 and 18.3 mg/kg of fodder).

In most cases Na-thiosulphate normalizes the mentioned characteristics.

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