Abstract. The purpose of the study was a comparative assessment of the integral indicators of the functional state of experimental animals under conditions of prolonged exposure to cadmium chloride and nanoparticles (NP) of cadmium sulfide of different sizes.
Materials and Methods. The studies were carried out on male rats of the mature age of the Wistar line weighing 160–180 grams, which were injected intraperitoneally with cadmium chloride (CdCl) and cadmium sulfide (CdS) nanoparticles with a size 4–6 nm and 9–11 nm in a dose of 0,08 mg/kg/day in terms of cadmium. The body weight was measured weekly. The weight of the internal organs and the muscular endurance measured by the duration of retention time on the pole was evaluated after 30 injections (1,5 months), after 60 injections (3 months), and after cessation of introduction.
Results. CdCl2 and NP CdS caused a decrease in the average body weight and body weight gain in comparison with control animals. NP CdS caused a mentioned decrease earlier than CdCl2, but under the influence of CdCl2, these changes were more pronounced. In animals, after the introduction of cadmium compounds, were observed an increase in the relative mass of the brain, liver, spleen, thymus, a decrease in lung mass. There were also observed an increase in the relative mass of the heart and kidney after 30 and 60 injections and their decrease in the post-exposure period. The greatest changes in the weight of the organs where caused by CdCl2, while the smallest ones where caused by NP CdS with the size of 9–11 nm. Reduction of muscular endurance of rats after 30 injections was most pronounced when exposed to NP CdS in size of 4–6 nm. But, in a period after 60 injections and in the post-exposure period, the reduction of muscular endurance was most pronounced with CdCl2 exposure.
Key words: cadmium, nanoparticles, cadmium sulfide, cadmium chloride, integral indices, body weight, weight of internal organs, muscular endurance.
Cadmium compounds play an important role among the global environmental pollutants [1]. The effect of cadmium on humans occurs mainly when inhaled aerosols containing it, or when they enter the digestive tract with food. In case of excessive administration into the body under acute exposure, cadmium causes damage to the lungs, kidneys, liver, reproductive organs, and under chronic exposure, it exhibits predominantly nephrotoxic, immunotoxic and osteotoxic effects [2, 3]. In general, cadmium compounds are highly toxic substances, characterized by mutagenic and genotoxic effects, having high cumulative properties. Cadmium is classified as carcinogens [4, 5].
The development of nanotechnologies promoted scientific studies of the synthesis and action of nanoparticles (NPs) of heavy metal compounds, in particular of cadmium compounds, their usage in various industries [6]. Cadmium-containing nanomaterials are widely used in optical and electronic devices [7]. Cadmium sulphide as a p-type semiconductor is used in electronics, in particular, it is an active medium in semiconductor lasers, a material for the manufacture of photocells, solar cells, photodiodes and LEDs. Quantum dots based on cadmium compounds may be used as biological labels (markers), combining them with certain proteins. Thanks to its optical properties, quantum dots based on cadmium chalcogenides are promising for ultra-sensitive multicolour registration of biological objects, medical diagnostics, cytometry, and optical and electron microscopy and fluorescence analysis [8]. The introduction of cadmium compounds NPs causes the need to study the mechanism of their action, both at the cellular level and at the level of organs and systems, as well as the determination of biomarkers of their effects, especially when compared to the ionic form.
Purpose of the study. Comparative evaluation of integral parameters of the functional state of experimental animals in the setting of prolonged exposure to cadmium chloride and cadmium sulphide NPs of different sizes.
Materials and methods of the study. In this work, nanoforms of cadmium compounds were used: Cadmium sulphide NPs (CdS NPs) with an average size of 4–6 nm and 9–11 nm and ionic forms: Cadmium chloride (CdCl2), which is well soluble in water. CdS NPs were obtained by chemical synthesis using a sodium polyphosphate stabilizer (NaPO3)n. NPs sizes were determined by electron microscopy.
The study was carried out on sexually mature male Wistar rats weighing 160–180 g. The animals were kept in a vivarium on a standardized diet with free access to drinking water. The rats were divided into 3 experimental groups and the control one. The 1st experimental group received CdS NPs of 4–6 nm in size, the 2nd group – CdS NPs of 9–11 nm in size, the 3rd group — CdCl2; the control group received a normal saline solution. The study substances were administered intraperitoneally daily 5 times a week (working week simulation) at a dose of 0.08 mg/kg expressed in terms of cadmium. Toxic effects were evaluated after 30 injections (1.5 months), 60 injections (3 months), and 1.5 months after discontinuation of the exposure. The animals were weighed once a week under fasting conditions during the experiment. Muscle strength was estimated evaluated based on hanging time in the wire hang test in rats at a height of 1.5 m above the support. At the end of the experimental period, the animals were decapitated, observing the requirements for humane treatment. Immediately after drawing the blood, internal organs were removed and weighed. All manipulations with animals were conducted in accordance with the provisions of the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 1985).
Statistical processing of primary data was performed using Microsoft Excel 2003 and SPSS 21.0. The significance of differences between the parameters was evaluated by the Mann-Whitney U-test.
The study was carried out within the framework of the research work of Institute of Occupational Medicine of the National Academy of Medical Sciences of Ukraine, State Enterprise, “Scientific Substantiation of Principles, Methods and Parameters of Experimental Evaluation of Toxicity of Nanoparticles and Nanomaterials (Based on Metal Nanoparticles)” (research No. 0113U001447) and “Investigation of the Peculiarities of Toxic Effects of Heavy Metal Nanoparticles, Search and Justification of Prophylactic Methods” (research No. 0116U00497).
Study results. The dynamics of changes in the body weight of experimental animals during exposure reflects the state of the body and the general (non-specific) reaction to intoxication. This allows a rapid and non-invasive evaluation of the animal’s condition, level of intoxication development as well as adjustment of the experiment, if necessary in the process of the test. Due to its simplicity and non-invasiveness, this method is extremely attractive and important especially for toxicological experiments.
The results of the conducted studies indicate that the average body weight and its growth in rats of the first and second experimental groups exposed to CdS NPs were significantly lower than those of the control group of animals starting from Week 3 of the experiment. The average body weight and its growth in rats exposed to CdCl2 were significantly lower than those of the control group of animals starting from Week 4 (Table 1). There are no significant differences in the valuesof the average body weight of animals exposed to CdS NPs of 4–6 nm and 9–11 nm in size. However, the increase in the body weight of animals in the first experimental group compared to that in the second experimental group was significantly lower at Weeks 5 and 6 of the exposure, while at Weeks 7 and 8 it was significantly higher. This suggests that in the animals exposed to smaller CdS NPs the weight gain began to reduce earlier than that of the animals exposed to larger cadmium sulphide NPs.
The average body weight of the animals of the first experimental group (CdS4–6 nm NPs) was lower than the body weight of the animals of the third experimental group (CdCl2), starting from Weeks 3 to 7 of the experiment, and the average weight of animals in the second experimental group (CdS9–11nm NP) was lower than that of the animals of the third experimental group (CdCl2) starting from Weeks 4 to 9. The increase in body weight of rats exposed to cadmium chloride decreased in comparison with that of animals in the first and second experimental groups, starting from Weeks 8 to 12 of the experiment.
Thus, the weight of the animal body of all three experimental groups and body weight gain were significantly lower than those of the control group of animals. Reduction in the average body weight of rats exposed to both sizes of CdS NPs occurred earlier compared to those of the animals exposed to cadmium chloride, but these changes were more pronounced under the influence of cadmium chloride. This may indicate that the development of intoxication in animals following administration of CdS NPs occurs earlier, compared to the ionic form of cadmium. Fluctuations in body mass gain were more pronounced in animals exposed to CdS NPs of a larger size.
The relative weight of internal organs of the experimental animals, exposed to CdS NPs and CdCl2, significantly differed from those of the animals of the control group (Table 2). In rats exposed to CdS NPs of 4–6 nm in size, there was a significant increase in the relative weight of the heart after 30 and 60 injections, and in the post-exposure period, there was a decrease compared to that of the control group. In animals exposed to CdS NPs of 9–11 nm in size, a significant increase in the relative weight of the heart was recorded after 60 injections and in the post-exposure period. CdCl2 caused an increase in the relative weight of the heart after 30 injections and its decrease in the post-exposure period.
The relative weight of the lungs in animals exposed to CdS NPs varied insignificantly: Only after 30 injections, there was a statistically significant increase. Administration of CdS NPs of 9–11 nm in size resulted in a decrease in the relative weight of the lungs after 60 injections and in the post-exposure period. In animals exposed to CdCl2, a statistically significant decrease in the relative weight of the lungs was reported in all the study periods, especially one month after discontinuation of the exposure.
A significant increase in the relative weight of the kidneys after 30 and 60 injections was reported in rats of all three experimental groups. In the post-exposure period, a decrease in the relative weight of the kidneys was reported in animals exposed to CdS NPs of 9–11 nm in size and cadmium chloride compared to the control group of animals.
Table 1
Average body weight and weight gain of experimental animals (M ± m, n = 10)
Notes: * – statistically significant difference of the parameters of the experimental groups animals compared to those of the control group; # — statistically significant difference between the parameters of the groups of animals exposed to CdS NPs of different sizes; ~ — statistically significant differences between the parameters of the groups of animals exposed to CdS NPs and CdCl2; p < 0.05.
The relative weight of the thymus was significantly increased in the group of animals exposed to CdS NPs of 4–6 nm in size and cadmium chloride, after 60 injections and in the post-exposure period. In animals exposed to CdS NPs of 9–11 nm in size, there was an increase in the relative weight of the thymus after 30 and 60 injections and its decrease in the post-exposure period.
The relative weight of the spleen was significantly increased in the animals of all three study groups after 30 and 60 injections of cadmium compounds compared to those of the animals of the control group. In the post-exposure period, it was statistically increased in rats exposed to CdS NPs of 9–11 nm in size and cadmium chloride.
The relative weight of the brain was significantly increased in rats of all study groups in all periods of the study, especially significant changes were reported after 60 injections of cadmium chloride and CdS NPs of 9–11 nm in size.
Thus, summing up the above changes in the relative weight of the internal organs, the following can be concluded. In animals of the experimental groups, there was a significant increase in the relative weight of the brain, liver and spleen. In general, CdS NPs of 9–11 nm in size caused larger changes than CdS NPs of 4–6 nm in size. The relative weight of the internal organs is a rather sensitive parameter, which reflects morphofunctional changes in the internal organs and characterizes the degree of development of intoxication.
Table 2
Relative weight of internal organs of experimental animals (M ± m)
Notes: * – statistically significant difference of the parameters of the experimental groups animals compared to those of the control group; # — statistically significant difference between the parameters of the groups of animals exposed to CdS NPs of different sizes; ~ — statistically significant differences between the parameters of the groups of animals exposed to CdS NPs and CdCl2; p < 0.05.
The parameter of muscle strength based on hanging time in the wire hang test in rats is an integral parameter indicating the general state of the body and level of the development of intoxication. The conducted studies showed a significant decrease in the hanging time in the wire hang test in rats exposed to cadmium compounds, both in the first and in the second period of the study (Fig. 1). Moreover, in the first period there was a decrease in the hanging time in the wire hang test by 2.8 times in animals exposed to CdS NPs of 4–6 nm in size, by 2.3 times — exposed to CdS NPs of 9–11 nm in size, by 1.9 times — exposed to CdCl2.
Fig. 1. Changes in muscle strength of rats exposed to cadmium compounds (based on hanging time in the wire hang test in rats, seconds).
Notes: * – statistically significant difference of the parameters of the experimental groups animals compared to those of the control group; # — statistically significant difference between the parameters of the groups of animals exposed to CdS NPs of different sizes; ~ — statistically significant differences between the parameters of the groups of animals exposed to CdS NPs and CdCl2; p < 0.05.
In the second study period, the greatest reduction in muscle strength was observed in animals exposed to cadmium chloride — 3.7 times. Reducing hanging time in the wire hang test in animals exposed to CdS NPs of cadmium of a lower size — by 2.6 times, and exposed to CdS NPs of a larger size — 2.5 times. In the post-exposure period, the increase in the duration of the hanging time in the wire hang test in rats was observed compared to the second period, but it was statistically significant only in animals exposed to CdS NPs of a larger size.
Thus, the data presented above show that in animals exposed to cadmium compounds, there is a significant decrease in the muscular strength, which is most pronounced in the first period under the influence of CdS NPs of 4–6 nm in size, and in the second and in the post-exposure period — under the influence of CdCl2.
Summarizing the above results of the study, the following can be concluded:
1. Integral parameters such as the general condition of animals, body weight, body weight gain, relative mass of internal organs, muscular strength, reflect the general functional state of experimental animals, allow assessing the presence and degree of intoxication, its onset time, and so on.
2. CdCl2 and CdS NPs caused a decrease in the average body weight of animals and its gain compared to control animals in the long-term administration. Reduction in the average body weight of rats exposed to both sizes of CdS NPs occurred earlier compared to those of animals exposed to CdCl2, but these changes were more pronounced under the influence of CdCl2. This may indicate that the development of intoxication in animals following administration of CdS NPs occurs earlier, compared to the ionic form of cadmium.
3. The relative weight of the internal organs is a rather sensitive parameter, which reflects morphofunctional changes in the internal organs and characterizes the degree of development of intoxication. In animals of the experimental groups, there was a significant increase in the relative weight of the brain, liver and spleen. In general, CdS NPs of 9–11 nm in size caused larger changes than CdS NPs of 4–6 nm in size.
4. In animals exposed to cadmium compounds, there is a significant decrease in the muscular strength, which is most pronounced in the first period under the influence of CdS NPs of 4–6 nm in size, and in the second and in the post-exposure period — under the influence of CdCl2.
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Надійшла до редакції 01.08.2017 р.