Abstract. Today there is a rapid development of nanomaterials and nanotechnology. Metal nanoparticles (NPs) are widely implemented in various spheres of economic activity, creating a potential risk for workers’ health and the environment. Especial attention deserves nanoparticles of lead since the possibility of their formation in the production process is quite large, and the nature of the effect on the body to these almost not studied.
The aim of the study. Morpho-functional assessment of internal organs of rats in application micro and nano of inorganic compounds of lead to intact skin.
Materials and Methods. A chronic experiment was conducted on 24 Wistar male rats, divided into 4 groups. 1 ml of colloids was applied to rats of groups II, III and IV on the previously sheared back skin of 2 cm area, daily (5 days per week) for 3 months for 4 hours. PbS NPs were of size of 12,5 nm and 100 nm, and aqueous solution of lead acetate, group I included control intact rats. A morphological study of internal organs (myocardium, liver and kidney) was carried out, using general histological methods.
Results of the Study. It is established that a prolonged exposure to undamaged rats skin of 12,5 nm and 100 nm PbS nanoparticles, unlike aqueous trihydrate lead acetate solution, the average particle size of which is 700 nm, is accompanied by resorption of nanoparticles through the intact skin, which ensures their absorption into the body and intake to target organs, where nanoparticles can exhibit toxic effects.
Conclusions. PbS nanoparticles of different sizes with long-term effects on the intact skin in contrast to the particles of the micrometer range cause a wide range of morphological changes, characterized by their cardiovasotoxic, hepatotoxic and nephrotoxic effects.
Key words: lead nanoparticles, skin, inner organs.
Introduction
Lead is one of the oldest and most thoroughly studied toxicants among environmental and occupational ones. This metal has rather large volumes of production and a wide scope of application, which results in its penetration into various objects of the environment [1-3].
Lead nanoparticles (NPs) are of particular attention, since the possibility of their formation in the production process is rather high, which increases the risk of contact of workers with them in the production of NPs, as well as in their application [4].
The source of penetration of ultrafine particles of less than 100 nm in size into the air can be production area, where there are conditions for the formation of condensation aerosols, in particular, during lead smelting, welding and cutting structures, and recuperation of lead batteries [5].
The danger of lead in the form of nanoparticles is due to their physical and chemical properties, such as small size, large specific surface area. This greatly increases the reactivity and biological activity of lead, opening up prospects for a wider application. At the same time, there is a potential danger to the environment and human beings [5]. One of the most dangerous manifestations of the general toxic effect of NPs is their translocation in the intracellular environment and distribution in the internal organs. A detailed study of the effect of lead NPs on the functioning of internal organs and systems is an important toxicological and hygienic problem. The solution of these tasks is possible only on the basis of new fundamental knowledge about the regularities and peculiarities of NPs ways to the living organism, their distribution in organs (tissues, cells), depositing, metabolic transformations and excretion.
It is believed that the main ways of NPs penetration to the human body are inhalation (with air), oral (with food and water) and transcutaneous (through the skin). It has been established that NPs are able to overcome biological barriers (hematoencephalic, placental), penetrate through the intact skin [6].
Therefore, the study of the peculiarities of the morphofunctional state of the internal organs under the exposure of lead NPs on the intact skin is very pressing, since they provide an opportunity to deepen understanding of the mechanism of NPs resorption through the skin, as well as mechanisms of toxic effects of the metal on the body.
The purpose of the study is a morphofunctional evaluation of internal organs of rats upon chronic exposure of micro and nanoparticles of inorganic lead compounds on their intact skin.
Materials and methods of the study. Experiments were carried out on 24 white, sexually mature male Wistar rats weighing 150–180 g, which were kept under standard vivarium conditions with an average luminous period of 11–12 hours, air temperature 21–240C and its relative humidity of 40–60 % on a standard diet with free access to water settled for at least 24 hours in accordance with the recommendations [7]. All manipulations with rats were carried out in compliance with the essential requirements of the European Convention for the Protection of Vertebrate Animals used in Experiments for Scientific Purposes [8].
The rats were divided into 4 groups (3 experimental, 1 control), with 6 animals in each group. In experimental rats, a model of chronic exposure on the intact skin of lead nanoparticles (NPs) was reproduced. For this purpose, the experiment involved colloids of lead sulphide NPs (PbS; molecular weight 239.3 g/mol) with different sizes of NPs and aqueous solution of lead acetate trihydrate (AC TH; Pb (C2H3O2) 2 • 3H2O; molecular weight 325.29 g/mol; solubility in water at 25°C – 55.2 g/L).
Colloids of PbS NPs were obtained by chemical synthesis (L. V. Pisarzhevskyi Institute of Physical Chemistry of the National Academy of Sciences of Ukraine) and stabilized with gelatin at a final concentration of 0.5 % and 1.0 %, which allowed obtaining NPs of greater and smaller size. The colloids of PbS NPs and AC TH solution (ChemLaborReaktiv) were standardized by mass fraction of the chemical, equal to 0.01 mmol/L.
To determine the physicochemical characteristics of PbS and AC TH particles (form and size) required to determine the average volume and average particle specific surface area, a raster electron microscopy (Tescan MIRA3 microscope, Tesla with the local elemental energy-dispersion microanalysis system Oxford Advanced Aztec Energy (IE350)/X-max 80 and the method of dynamic light scattering (DysaSizer laser spectrometer from Fritsch, Germany).
To reproduce in a toxicological experiment a model of chronic exposure of PbS NPs and AC TH on the intact skin, the fur on the back of the rats was cut off with scissors, releasing the space ofskin with an area of 2 cm2. The experimental studies used the working solutions of PbS colloids and AC TH aqueous solution, which was prepared ex tempore, by ten-fold dilution of the stock solution with deionized water. Under such conditions, the concentration of chemicals applied to the skin was 0.001 mol/L. The working solutions of PbS NPs and AC TG were applied daily (5 days a week), within 3 months. For this, the rats were placed in a special plastic chamber for skin applications, 1.0 mL of the working solution was pipetted onto a prepared skin surface and the animals were left in the chamber for 4 hours for exposure to chemicals. After exposure, the chemicals were washed off the surface of the skin with deionized water.
From the experiment, animals were withdrawn three months later, by decapitation after previous anaesthesia. Narcotization of rats was carried out by introducing 2.5 % solution of 2,2,2-tribromoethanol (Aldrich) into 2-methyl butanol (working dilution 1:50 in PBS) in the abdominal cavity at a rate of 300 mg/kg. After decapitation and bleeding in animals, the liver, kidneys and heart were taken, from which pieces of 0.5 cm in the thickness and no more than 1 cm2 were cut. The cut pieces were immersed for 72 h in a 10 % solution of neutral formalin or in Lily fixer. Individual pieces of liver and kidney before formalin fixation were kept for 6–12 hours in an aqueous 20 % solution of cadmium sulphate (CdSO4) [9]. After fixation, pieces of organs were washed in water, dehydrated in a series of ethanols, clarified in xylene and sealed in paraffin using a standard procedure [10]. Paraffin slices, 5–7 µm thick, were prepared using a microtome, mounted on the glass slide (at least 3 sections) and stained with hematoxylin and eosin, as well as by Mallory-Slinchenko’s method [10]. To detect catabolic ferritin, which is one of the markers of lead toxic effect on paraffin sections of the liver and kidneys, histochemical Perls’ reaction was carried, according to the recommendations [9].
Histological studies were performed with the help of a light microscope Olympus BX 54 with a digital camera Olympus C-5050 ZOOM and software Olympus DP-Soft. Microscopy determined the nature and frequency of morphological changes. To do this, according to the recommendations of H. H. Avtandilov [11], at least 3 histological preparations were used (9 sections of each organ from one rat), in which at least 5 fields of view were examined using a microscope lens with magnification of x 10 and, respectively, 10 fields of view using a microscope lens with magnification of x 40. The results of the evaluation of morphological changes were presented in relative units (%), which determined their frequency in the total volume of the test material. For statistic data processing, the non-parametric method of angular transformation φ* – Fisher’s[12] was used. The essence of the method is to convert the index of the frequency of morphological characters (P) to the value of the central angle φ, which is measured in radians and is determined by the formula:
(1)
where φ is the values of the central angle (in radians),
P is the value of the frequency of the detected morphological characters (%),
followed by a comparison of the calculated empirical value φ*emp with the critical value φ*cr. The calculation of the empirical value of the criterion φ*emp was carried out according to the formula:
(2)
where φ1 is the angle corresponding to a larger percentage,
φ2 is the angle corresponding to a smaller percentage
n1, n2 is the number of tests in the samples (groups)
The obtained empirical values of the criterion φ*emp were compared with its critical values φ*cr (tabular) [12, p. 332]. On the basis of the comparison of φ* empirical and critical values, we conclude that one of the accepted statistical hypotheses is consistent.
Critical values φ*cr corresponding to accepted in practice of morphological studies with levels of statistical significance, were:
(3)
Results and their discussion. The results of conducted electron microscopic studies showed that almost all of PbS NPs, obtained by the method of chemical synthesis, had the form of an elongated ellipse (Fig. 1 a, b). At the same time, the size of its long axis in almost 80 % of PbS NPs stabilized with 1 % gelatin solution ranged from 7 nm to 16 nm (average value 12.87 ± 0.57 nm), while the short axis ranged from 5.5 nm to 7.5 nm (average value 6.5 ± 0.23 nm). Moreover, the size of the longer axis in 65 % of PbS NPs stabilized with 0.5 % gelatin solution ranged from 75 nm to 130 nm (average 106.0 ± 6.0 nm) and short respectively from 19 nm to 31 nm (average value 24.0 ± 5.5 nm). The electron microscopic examination of AC TH particles showed that they had a crystalline structure with a monocline syngony and a shape in the form of a hexagonal flattened prism, about 20 nm in height and an average length between the sides of the hexagonal base of about 700 nm (Fig. 1 c).
Fig. 1. Electron microscopic characteristic of PbS NPs (a, b) and AC trihydrate crystals (c) by the results of raster electron microscopy and PbS NPs average size according to the dynamic light scattering method.
Table 1
Basic physical-chemical characteristics of PbS and AC TH particles used in the experiment
Based on the data obtained, it was found that when applied to the intact skin of rats, 1 mL of PbS colloids with the chemical concentration of 0.001 mol/L, the concentration of PbS NPs of 12.5 nm per 1 cm2 of skin surface area was 0,61*1013 particles/cm2, and PbS NPs of 100 nm in size – 1.2*1010 particles/cm2. At the same time, the concentration of AC TH particles of 700 nm in size at a similar concentration of the chemical substance (0.001 mmol/L) was 10.13*108 particles/cm2. Under these conditions, the average specific surface area of PbSNPsof 12.5 nm applied to the intact skin of rats was 1.28 *10-3 m2, PbS NPs, of 100 nm in size — 1.61*10-4 m2, and AC particles — 4.69 * 10-6 m2, respectively.
The results of microscopic studies of the myocardium, liver and kidneys of rats exposed to chronic AC TH exposure on the intact skin showed that the histological structure of organs did not differ significantly from the control ones. At the same time, the effects of PbS NPs of 12.5 nm in the myocardium of the experimental rats often resulted in the enlargement of the lumen and congestion of the small intramural arteries, veins and capillaries, which caused the oedema of the interstitial connective tissue (Fig. 2а). Such changes indicate a disorder in the myocardial microcirculation system, which determined the development of cardiomyocyte dystrophy in the form of swelling of their sarcoplasm, as well as fragmentation and lysis of myofibrils (Fig. 2b).
It should be noted that dystrophic changes in the myocardium were often associated with the development of compensatory and adaptive reactions in the form of compensatory alterations, which were aimed at supporting tissue homoeostasis and manifested by hypertrophy of cardiomyocytes and/or their nuclei. In such cases, in cardiomyocytes, when the size of the nucleus was increased, sinuosity of the nuclear membrane contour was often observed, which increased the contact area of the nucleus and the cytoplasm, as well as the exchange between them [14].
The effects on the intact skin of rats exposed to PbS NPs of 100 nm in size in the myocardium, as well as under the influence of NPs of a smaller size, there was an oedema of the interstitial connective tissue, which was accompanied with oedema, swelling and dissection of collagen fibres. In this case, compared to PbS NPs of a small size, there were small foci of subendothelial oedema in the intima of arteries and veins of small calibre (up to 50 µm in diameter), which caused the orientation of the nuclei of these cells i. e. perpendicular to the basal membrane which is not characteristic of this type of cells (Fig. 2c). In this case, in the blood vessels the development of vascular endothelial dystrophy in the form of swelling of the cytoplasm, basophilia of the nucleus, often with signs of their heterochromatization, as well as the accumulation of small crystalline inclusions, capable of basophilia were often observed (Fig. 2c).
In addition, the myocardium often showed dystrophic changes in cardiomyocytes, in which, together with swelling of the sarcoplasm, fragmentation and lysis of myofibrils, accumulation of small crystalline cytoplasmic inclusions, capable of basophilia were observed (Fig. 2d).
The results of the analysis of the frequency of detected changes in the myocardium of the experimental rats showed that the chronic exposure of 100-nm PbS NPs on the intact animal skin was characterized by a statistically significant increase in the frequency of oedema changes in the interstitial connective tissue, vascular endothelium dystrophy, and cardiomyocyte dystrophy, as compared with the action of 12.5-mm PbS NPs (p<0.05; Table 2). At the same time, the frequency of morphological changes that characterize the development of adaptive reactions in the myocardium with the action of 100-nm PbS NPs did not show statistically significant differences compared with the action of 12.5 nm PbS NPs (p>0.05; Table 2).
Fig. 2. Histological myocardial changes in rats under the exposure of PbS NPs of 12.5 nm (a, b) and 100 nm (c, d): a) – dilated lumens and congestion of the capillaries with the development of interstitial oedema; b) – dystrophic changes of cardiomyocytes in the form of swelling of sarcoplasm and lysis of myofibrils (►); c) – perivascular oedema, small foci of sub-endothelial oedema and endothelial cell dystrophy with small crystalline inclusions in their cytoplasm (◄); d) – small crystalline inclusions in the sarcoplasm of cardiomyocytes (▲). Hematoxylin and eosin.
In the histological examination of the liver of rats exposed to PbS NPs of different sizes chronically on the intact skin, in contrast to control rats and animals exposed to AC TH, they often demonstrated dilatation and congestion of central veins and sinusoid capillaries accompanied with hyperplasia and hypertrophy of stellate reticuloendothelial cells (SRE, Kupffer cells), in which the cytoplasm the accumulation of small crystalline inclusions were observed (Fig. 3 a, b). Together with this, unlike the effect of AC TH, the action of a 12.5 nm PbS NPs was accompanied with swelling of hepatocyte cytoplasm and nuclei of the 3rd zone of the liver acini, and often — with the development of cytoplasm granular degeneration. In this case, the parenchyma of the experimental rats often showed small foci of lymphoma-microphage infiltration, which, according to the literature, are usually formed in places of hepatocyte necrosis [15]. Dystrophic and necrotic changes in hepatocytes were accompanied by the development of compensatory and adaptive reorganization in the organ parenchyma, which was determined by an increase in the number of hepatocytes with obvious signs of hypertrophy of the cytoplasm and nucleus, as well as an increase in the number of binuclear hepatocytes. This corresponds to the literature data, which indicate that the hypertrophy of hepatocyte nuclei and the increase in the number of binuclear hepatocytes are one of the morphological manifestations of physiological regeneration of the organ [16,17,18].
In the histological examination of the liver of rats exposed to 100-mm PbS NPs chronically, the morphological changes in the organ were the same as those under the action of smaller-sized NPs. Differences in the action of PbS NPs of different sizes were also established. Thus, degenerative changes in hepatocytes under the action of larger PbS NPs, in contrast to smaller-sized NPs, were often manifested by swelling of hepatocyte cytoplasm of the 3rd zone of liver acini with the deposition of small crystalline inclusions in it, which often acquired a basophilic colouration (Fig. 3c). Under such conditions, in the dilated lumens of sinusoidal capillaries, apoptotic bodies were often found, represented by fragments of the densified cytoplasm and hyperchromic nucleus (Fig. 3d). At the same time, the foci of inflammatory infiltration were rare.
The results of the analysis showed that chronic exposure of 100-nm PbS NPs on the intact skin of rats was characterized, compared with the action of smaller PbS NPs, by a significant (p<0,05) increase in the incidence of hepatocyte dystrophy, with no significant differences in the development of venous congestion of the organ (p>0,05; Table 2). At the same time, the frequency of inflammatory changes in the parenchyma, associated with hepatocyte necrosis under the action of 100-nm PbS NPs, was significantly lower than under the action of smaller PbS NPs (p<0.05; Table 2). Together with this, the action of larger NPs was characterized by a significant increase in the frequency of apoptosis of hepatocytes, compared with the action of smaller PbS NPs (p<0.05; Table 2).
Table 2
Frequency (φ) of morphological changes under chronic exposure on intact skin of PbS NPs of 12.5 nm and 100 nm, radians
Fig. 3. Histological liver changes in rats under the exposure of PbS NPs of 12.5 nm (a, b) and 100 nm (c, d): a) – dilated lumens and congestion of sinusoid capillaries; SRE hepatocyte dystrophy and hypertrophy; b) – small crystalline inclusions in SRE cytoplasm (←); c) – small crystalline inclusions in the cytoplasm of hepatocytes with signs of swelling (◄); d) – heterochromatization of hepatocyte nuclei (↑) and apoptotic bodies in the lumens of dilated sinusoidal capillaries (◄). Hematoxylin and eosin.
In response to injuries in the liver parenchyma of rats exposed to 100-nm PbS NPs, there were often compensatory and adaptive alterations in the form of hypertrophy of hepatocytes or their nuclei, as well as an increase in the number of binuclear hepatocytes. According to the analysis, the frequency of such changes under the action of smaller and larger-sized PbS NPs did not differ significantly (p>0,05; Table 2).
In the kidney of rats under long-term exposure on intact skin, 12.5-nm PbS NPs, in contrast to the control and AC TH action, there was dilatation and congestion of peritubular capillaries, lumens of renal corpuscle capsules as well as of capillaries of renal glomerules, predominantly, of the juxtamedullary nephrons (Fig. 4 b), which indicates their greater functional activity, through which, in the first place, they react to changes in the internal environment. In this case, the endothelium of glomerular capillaries was often subjected to dystrophic changes in the form of swelling of their cytoplasm and nucleus. Under these conditions, epithelial proximal tubules often showed the development of protein degeneration. However, loose and dense acidophilic protein masses were often found in the dilated lumen of the distal tubules and the collecting tubules (Fig. 4 b), indicating disorders of protein reabsorption processes in the kidneys. Under the action of 100-mm PbS NPs, the morphological changes in the kidneys were the same as those of smaller PbS NPs. However, under the action of larger NPs, in contrast to smaller NPs, in the epithelium of the proximal tubule of the kidneys with signs of protein degeneration in their cytoplasm, along with protein inclusions, small crystalline inclusions were found that often exhibited the ability to basophilia (Fig. 4 c). In this case, histochemically, protein inclusions showed a number of characteristic features. First, a greater number of cytoplasmic protein inclusions were represented by acidic proteins, which were stained in red with chromotropium 2B by Mallory-Slinchenko’s method. Secondly, in the structure of protein cytoplasmic inclusions, iron polymorphic granular inclusions were often detected under the action of cadmium sulphate by Granin and Klochkova method (modified Perls’ method) (Fig. 4 e), which, according to literature, characterizes such inclusions as deposition of catabolic ferritin [9]. Thirdly, the accumulation in the cell cytoplasm of crystalline inclusions, capable of staining with basic nuclear stains, was accompanied with pronounced densification of the cell cytoplasm and nuclei, often with signs of their heterochromatization (Fig. 4 c), indicating the irreversible nature of such changes, the final of which may be necrosis and/or apoptosis.
Deposition of catabolic ferritin in the epithelium of proximal tubules of the kidneys under the action of PbS NPs may be due to the mechanisms of toxic action of lead compounds, in particular, via influence on processes of iron metabolism, the violation of which determines the development of inherited hemochromatosis [19] morphologically manifested by various forms of hemosiderosis of the internal organs [20].
The results of the analysis of the detected morphological changes have shown that their frequency in the kidneys of rats exposed to 100-nm PbS chronically was not significantly different from that of 12.5-nm PbS NPs (p>0.05; Table 1). 2).
Fig. 4. Histological renal changes in rats under the exposure of PbS NPs of 12.5 nm (a, b) and 100 nm (c, d): a) – dilated lumens and congestion of intratubular capillaries, dystrophic changes in the epithelium of the proximal tubules; b) – acidophilic protein masses in the dilated lumen of proximal and distal tubules; c) – small crystalline cytoplasmic inclusion in the epithelium of the proximal tubule (►); d) – protein degeneration of the epithelium of the proximal tubules with the inclusions of ferritin (←) Hematoxylin and eosin. (a, b, c); histochemical reaction to ferritin (d).
Thus, the results of the experimental studies have shown that PbS NPs obtained by chemical synthesis have the form of an elongated ellipse, the determined size of which allowed the calculation method to determine characteristics most important for assessing NPs biological and toxic effects, in particular, the concentration of PbS NPs in colloids, NPs average volume, as well as NPs average specific surface area.
It has been found that chronic exposure of 12.5- and 100-nm PbS NPs on the intact skin of rats when applied to the skin at a concentration of 0.001 mmol/L (for the chemical, PbS) is characterized by the fact that the concentration of 12.5-nm NPs applied on 1 cm2 of the skin surface is 3 orders of magnitude higher than that of 100-nm PbS NPs. At the same time, smaller PbS NPs are characterized by a greater, 2 orders of magnitude, average specific surface area, compared with smaller PbS NPs, which predetermines a much larger area oftheir interaction with different structural elements of the skin surface, compared with larger NPs.
In the course of the experimental studies it has been found that chronic application of 12.5-nm and 100-nm PbS NPs on the intact skin, in contrast to AC TH aqueous solution, with the average particle size of 700 nm, is accompanied with resorption of nanoparticles through intact skin, which ensures the process of their absorption into the body and penetration into target organs, where nanoparticles can exhibit toxic effects.
It should be noted that the spectrum of morphological changes in stroma and parenchyma of target organs characterized by circulatory disorders in the system of the microcirculatory bloodstream of the myocardium, liver and kidneys, as well as development of dystrophic changes in the mesenchyma and parenchyma of the organs, reflect the complex process of damage, which often acts as a trigger mechanism for the development of structural transformations, which are aimed at compensating for functions partly lost by target organs. At the same time, different morphological changes of the internal organs of rats, which develop during long-term exposure of PbS NPs of different sizes on intact skin, determine the specific features of biological and toxic action of NPs. Thus, the mechanisms of action of PbS NPs directly depend not so much on the concentration of the chemical substance acting on the organism (PbS), but on the physical and chemical characteristics of NPs, applied to the intact skin, in particular concentration, size, shape as well as average specific surface area of NPs. It is the physicochemical characteristics of PbS NPs that influence the processes of their interaction in the body with different proteins, including transport ones at the stages of absorption and transport of NPs. This is evidenced by changes in the walls of the blood vessels of the myocardium, capillaries of renal glomerules, as well as protein inclusions in the epithelium of renal proximal tubules. In this case, larger NPs at the stages of their interaction with proteins and penetration through the endothelium of the blood vessels can form aggregates and agglomerates that actively perceive basic nuclear stains, thus defining their basophilic character. This enables to detect these crystal-like inclusions by means of a light microscope. It is likely that agglomerates and aggregates may also form smaller PbS NPs, but it is possible that their very small sizes will not allow detecting deposition of such aggregates and agglomerates in cells using a light microscope.
It should be noted that physicochemical characteristics of NPs are also very important in assessing their effect on metabolic processes. The histochemical studies of the kidneys indicate these properties of PbS NPs of different sizes, which have found that the action of larger PbS NPs may affect the processes of iron metabolism, manifested by the accumulation of proximal tubules of acidic proteins and catabolic ferritin in the epithelium of the proximal tubules of the epithelium cytoplasm.
Conclusions
1. The results of the experimental studies have shown that lead inorganic compounds, represented by particles of the nanometric range, in contrast to micrometric particles, are able to penetrate through the intact skin and penetrate into the target organs under their chronic exposure, in which their toxic effects are manifested. It acquires not only theoretical but also practical value because it allows assessing lead harmful effects on the health of workers and the population, taking into account the possibility of resorption of various lead compounds through intact skin.
2. It has been established that under the action of similar concentrations of lead compounds (0.001 mmol/L), represented by particles of the nano- and micrometric range on the rat intact skin, smaller NPs are characterised by significantly higher NPs average specific surface area than larger NPs indicating a much larger area of interaction of smaller NPs with different structural elements of the skin.
3. The results obtained determine the most promising directions for further research, among which the study of ultrastructural changes of target organs under the chronic exposure on intact skin of PbS NPs of different sizes, as well as mechanisms of resorption of PbS NPs through intact skin, is very important.
REFERENCES
1. Lugovskyi, S.P. 2002, «Mechanisms of biological action of lead on the digestive system», Modern problems of toxicology, food and chemical safety, no. 2, pp. 45–50 (in Ukrainian).
2. Smolyar, V.I. 2007, «Lead in food and diets», Problems of nutrition. , no. 4, pp. 42–51 (in Ukrainian).
3. Gidlow, D.A. 2004, «Lead toxicity», Occupational medicine. no. 54, pp. 76–81 (in Ukrainian).
4. Dmytrukha, N.М., Lugovskyi, S.P., Lagutina, O.S. 2014, «Characteristics of immunotoxic effect of lead compounds with micro- and nanoparticles», Modern problems of toxicology, food and chemical safety, no. 1–2, pp. 59–66 (in Ukrainian).
5. Trakhtenberg, I.М., Dmytrukha, N.M., Chekman, I.S. Kypriy, V.O. 2015, «Lead-dangerous pollutant. The problem is old and new», Modern problems of toxicology, food and chemical safety, no. 3, pp. 14–24 (in Ukrainian).
6. Trakhtenberg, І.М., Dmytrukha, N.M. 2013, «Nanoparticles of metals, methods of obtaining, spheres of application, physico-chemical and toxic properties», Ukr. J. Occup. Health, no. 4, pp. 62–74 (in Ukrainian).
7. Kozhemiakin, Yu.М., Khromov, О.S, Filonenko, М.А., Saifetdinova, G.А. 2002, Scientific and practical recommendations on maintenance of laboratory animals and work with them. Кyiv: Аvicenna, 156 p. (in Ukrainian).
8. European Convention for the protection of vertebrate animal, used for experimental and other scientific purposes, Council of Europe, Strasburg, 1986, 53 p.
9. Medentsov, A.A., Kateneva, U.A. 2005, «Catabolic ferritin as an additional criterium of ethanol intoxication», Modern High Technologies, no. 10, pp. 51–52 (in Russian).
10. Sarkisov, D.S., Petrov, Yu.L. 1996, Microscopic technique. Manual for doctors and laboratory assistants. Moscow: Medicine, 427 p. (in Russian).
11. Avtandilov G.G. 1990, Medical morphometry. Guidance. Moscow: Medicine, 384 p. (in Russian).
12. Gubler E.V. 1973, Application of nonparametric statistical criteria in biomedical research. Moscow: Medicine, 144 p. (in Russian).
13. Leonenko, N.S., Demetska, O.V, Leonenko, O.B. 2016, «Features of physical and chemical properties and toxic effects of nanomaterials — to the problem of assessing their hazardous effects on living organisms (literature review)», Modern problems of toxicology, food and chemical safety, no. 1, pp. 64–76 (in Ukrainian).
14. Sarkisov, D.S. 1987, Clinical aspects of modern ideas about the structural basis of adaptation and compensation of disturbed functions, Structural basis of adaptation and compensation of disturbed functions. Moscow: Medicine, pp. 57–83 (in Russian).
15. Palydeva, M.A., Paukova, V.S., Ulumbskova, E.G. 2002, Pathology: Manual. Moscow: Gaoter-med, 960 p. (in Russian).
16. Uryvaeva, I.V. 2001, «Replication potential of hepatocytes and liver stem cells», Izvestiya AN. Ser. Boil, no. 6, pp. 728–737 (in Russian).
17. Garbuzenko, D.V. 2008, «Mechanism of indemnify cation of structure and liver function at its damage and their practical value», Ros. zhurn. gastroenterol., gepatol., koloproktol, no. 6, pp. 14–20 (in Russian).
18. Shalakhmetova, T.M., Umbayev, B.A., Kolumbayeva, S.Zh., Kudryavtsev, B.N. 2009, «About mechanisms of formation of multinuclear hepatocytes during toxic action of N-nitrosodimethylamine on rats», Tsitologiya, v. 51, no. 1, pp. 32–40 (in Russian).
19. Lubianova, I.P. 2010, «Modern conceptions about the metabolism of iron from the position of the occupational pathologist», Айші problems of transport medicine, no. 2, pp. 47–57(in Ukrainian).
20. Lugovskoy, S.P. 2009, «Morphofunctional characteristic of hemosiderosis at experimental lead intoxication», Аctual problems of transport medicine, no. 2, pp. 124–132 (in Ukrainian).
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