RU2642674C1 - Method for increase of organism resistance to combined toxic action of nanoparticles of copper, zinc and lead oxides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for reduction of the adverse effects of combined effects of copper (CuO), zinc (ZnO) and lead (PbO) oxides nanoparticles on organism in risk groups covering individuals exposed to such effects under production conditions. Method includes prescription of a complex of biologically active drugs: glutaminic acid, glycine, N-acetylcysteine, pectin enterosorbent, fish oil preparation rich in unesterified omega-3 fatty acids, Vitamins A, C, D3, E, selenium, iron and iodized preparations. This complex is taken by repeated courses 1-2 times a year for 4-6 weeks daily at doses providing daily intake of 300 mg of glycine, 600 mg of cysteine, 4 g of glutaminic acid, 25 ml of fish oil with 12-15% content of polyunsaturated omega-3 fatty acids, 4-5 grams of pectin, as well as selenium, iron, iodine and these vitamins in doses that provide the normal physiological needs of the organism.

EFFECT: reduction of all three metals in the blood, improved elimination function of the liver and kidneys, reduced integral signs of chronic intoxication, including signs of neurotoxicity, and genotoxic combined action of copper, zinc and lead oxides nanoparticles on the body.

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Description

The invention relates to medicine, in particular to the toxicology of nanoparticles (nanotoxicology), and can be used to reduce the adverse effects of the combined action of nanoparticles (NPs) of copper oxides (CuO), zinc (ZnO) and lead (PbO) on the body at risk, covering persons who are exposed to such effects in a production environment, in particular in the smelting industry.

Works have been published in which various tests and on various biological objects demonstrate that the cytotoxic, systemic toxic and genotoxic effects of metal and metal oxide particles of the nanometer range are significantly higher than the corresponding action of chemically similar particles with sizes> 100 nm (Boris A Katsnelson, Larisa I Privalova, Marina P Sutunkova, Vladimir B. Gurvich, Nadezhda V Loginova, Ilzira A Minigalieva et al. (2015)). Some inferences rom in vivo experiments with metal and metal oxide nanoparticles: the pulmonary phagocytosis response, subchronic systemic toxicity and genotoxicity, regulatory proposals, searching for bioprotectors (a self-overview). International Journal of Nanomedicine, 10: 3013-309).

In particular, there are many experimental data on the high toxicity and danger of CuO nanoparticles (for example, Chen Z, Meng H, Xing G et al (2006) Acute toxicological effects of copper nanoparticles in vivo. J Toxicol Lett 25: 109-120; Karlsson H , Cronholm P, Gustafsson J et al (2008) Copper oxide nanoparticles are highly toxic: A comparison between metal oxide nanoparticles and carbon nanotubes. J Chem Res Toxicol 21: 1726-1732; Studer AM, Limbach LK, Van Duc L et al ( 2010) Nanoparticle cytotoxicity depends on intracellular solubility: comparison of stabilized copper metal and degradable copper oxide nanoparticles. J Toxicol Lett 1: 169-174; Magaye R, Zhao J, Bowman L et al (2012) Genotoxicity and carcinogenicity of cobalt-, nickel - and copper-based nanoparticles. J Exp Ther Med 4: 551-561; Alarifi S, Ali D, Verma A et al (2013) Cytotoxicity and genotoxicity of copper oxide nanoparticles in hum an skin keratinocytes cells. Int J Toxicol 32: 296-307; Xu J, Li Z, Xu P et al (2013) Nanosized copper oxide induces apoptosis through oxidative stress in podocytes. J Arch Toxicol 87: 1067-1073; Privalova L.I., Katsnelson, B.A., Loginova, N.V., Gurvich, V.B., et al. (2014). Subchronic toxicity of copper oxide nanoparticles and its attenuation with the help of a combination of bioprotectors. International Journal of Molecular Sciences 15, 12379-12406); ZnO (Wang B, Feng WY, Wang TC et al (2006) Acute toxicity of nano- and micro-scale zinc powder in healthy adult mice. J Toxicol Lett 161 (2): 115-123; Cho WS, Duffin R, Howie S et al (2011) Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2 + dissolution inside lysosomes. J Part Fib Toxicol 8:27; Adamcakova-Dodd A, Stebounova LV, Kim JS et al (2014) Toxicity assessment of zinc oxide nanoparticles using sub-acute and sub-chronic murine inhalation models. J Part Fib Toxicol 11:15; Jacobsen NR, Stoeger T, van den Brule S et al (2015) Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories. Food Chem Toxicol 85: 84-95; Gao F, Ma NJ, Zhou H et al (2016) Zinc oxide nanoparticles induced epigenetic change and G2 / M arrest are associated with apoptosis in human epidermal keratinocytes. Int J of Nanomedicine 11: 3859-3874) and, to a lesser extent, PbO (Shaikh SM, Shyama SK, Desai P V (2015) Absorption, LD50 and effects of CoO, MgO and PbO nanoparticles on mice "Mus musculus". IOSR-JESTFT 9 (2): 32-38; Amiri A, Mohammadi M, Shabani M (2016) Synthesis and Toxicity Evaluation of lead oxide (PbO) nanoparticles in rats. Electronic J Biol 12: 2).

However, neither an example of studying the chronic systemic toxicity of the triple combination of the oxides under consideration, nor testing or even theoretical justification of biological protection agents (bioprotectors) from the harmful effects of prolonged combined exposure of these nanoparticles to the body, an information search was found. As for the isolated action of the components of the combination in question, it was only with respect to the toxicity of copper oxide nanoparticles that an effective complex of bioprotectors was previously proposed (patent of the Russian Federation No. 2560682). However, the use of the same complex for the purpose of biological protection against the harmful effect of a combination of nanoparticles is not sufficiently substantiated, given that the nanoparticles of copper, zinc and lead oxides have, along with common to many nanoparticles, also metal-specific ones (in particular, characteristic of lead itself dangerous component of this combination) harmful mechanisms that, when combined exposure, enter into complex interactions (I.A. Minigalieva (2016) Some patterns of combined toxicity of metal oxide Tox Herald's nanoparticles, №6:. 18-24).

The objective of the invention is to provide a protection method based on increasing the body's resistance to the harmful effects of the combined action of nanoparticles of copper, zinc and lead oxides.

The technical result consists in reducing the harmful effects of the toxic and genotoxic combined effects of nanoparticles of copper, zinc and lead oxides on the body.

A method for preventing the harmful effects of the organo-systemic level due to the combined chronic general toxic and genotoxic effects of these nanoparticles on the body is claimed. It lies in the fact that people belonging to the risk group of this action are prescribed a complex of biologically active drugs, including glutamic acid, glycine, cysteine in the metabolically active form of N-acetylcysteine. pectin enterosorbent, selenium, iodine, iron, calcium, a fish oil preparation rich in omega-3 polyunsaturated fatty acids, as well as vitamins A, D3, E and C, and people at risk take complex preparations with repeated courses 1-2 times a year for 4-6 weeks daily in doses providing 300 mg of glycine per day, 600 mg of cysteine, 4 g of glutamic acid, 25 ml of fish oil with 12-15% content of omega-3 polyunsaturated fatty acids, 4-5 g pectin, as well as trace elements and vitamins in doses that ensure normal phi iologicheskie needs of the body.

The amino acids included in the complex, namely glutamate, glycine and cysteine, are included as precursors for the biosynthesis of reduced glutathione, which is a systemic protector from oxidative and free radical damage to cells and subcellular structures that characterizes the primary mechanisms of cytotoxicity and genotoxicity of various metal-containing nanoparticles, that glutamate is also a powerful stabilizer of cell membranes, as well as the most important neurotransmitter of the central nervous system, specifically and lead damage. Selenium and vitamins A, E, and C also have antioxidant effects. The introduction of iodine into the bioprotective complex is due to the fact that lead intoxication is associated with impaired thyroid function, and the introduction of iron is due to the inhibition of heme synthesis under the influence of lead, including at the final stage of inclusion of this metal, the protoporphyrin molecule IX. Calcium in combination with vitamin D3 is used as a well-known antagonist of many toxicokinetic and toxicodynamic mechanisms of action on the body of the same lead.

The pectin enterosorbent in the claimed complex is designed to block intestinal absorption of metal ions generated by the dissolution of nanoparticles transferred to the gastrointestinal tract after deposition in the respiratory tract, as well as reabsorption of ions excreted by the liver with bile.

The claimed complex also contains a fish oil preparation rich in not only the aforementioned vitamins A and D3, but also omega-3 polyunsaturated fatty acids, the intracellular derivatives of which are eicosanoids that activate DNA replication, thereby playing an important role in the repair of its damage.

The fact that people at risk are recommended to take complex preparations with repeated courses 1-2 times a year for 4-6 weeks daily in doses that ensure that 300 mg of glycine, 600 mg of cysteine, 4 g of glutamic acid, 25 ml of fish oil are received per day with a 12-15% content of polyunsaturated fatty acids of the omega-3 class, 4-5 g of pectin, as well as selenium, calcium, iron, iodine and vitamins in doses that ensure normal physiological needs of the body, it is justified by recalculating the content of the listed active factors in bioprotective complex ( PC) protective efficacy has been proved in an experiment carried out on laboratory white rats. Recalculation to the indicated doses for human use was carried out on the basis of the ratio of the levels of the main metabolism of the rat and the person, taking into account also reference and literature data on the daily human need for these factors. Only doses of biomicroelements and vitamins are caused not by such a conversion, but by normal physiological needs of the body, including compensation for endogenous vitamin deficiency and microelement imbalance arising from intoxication.

, Е. Cellular targets and mechanisms in the cytotoxic action of non-biodegradable engineered nanoparticles. J. Curr. Drug. Metab. 2013, 14, 976-988).; (в) компенсация некоторых функциональных и биохимических нарушений, связанных с токсикодинамическими механизмами как общего характера, так и специфичных для конкретного вида наночастиц.The protective mechanisms of the bioprotectors included in the complex are complex and, apparently, mutually potentiate each other. The following may be important: (a) the anti-radical (including antioxidant) effect, different in molecular mechanisms, to one degree or another inherent in a number of bioprotectors of the claimed complex (antioxidant synergism); (b) the membrane-stabilizing effect of glutamate, since it can prevent damage to mitochondria by nanoparticles of metal oxides and thereby oxidative stress, which, according to modern concepts, is one of the main mechanisms of the cytotoxic and genotoxic effects of metal nanoparticles (for example,

, E. Cellular targets and mechanisms in the cytotoxic action of non-biodegradable engineered nanoparticles. J. Curr. Drug. Metab. 2013 , 14, 976-988) .; (c) compensation of certain functional and biochemical disturbances associated with toxicodynamic mechanisms of both a general nature and specific for a particular type of nanoparticles.

A characteristic feature of the claimed method is the integrated use of all of the above mechanisms. It has been shown for the first time that, against the background of oral administration of the proposed combination of bioprotectors, chronic systemic toxicity and genotoxicity of copper, zinc and lead oxide nanoparticles acting on the body together can be significantly attenuated. As a result of a search by sources of scientific, technical and patent literature, no means were identified that aimed at solving such a problem.

The inventive method was experimentally tested on outbred white male rats with an initial body weight of 150-220 g. Animals were kept in a specially organized vivarium, corresponding to veterinary requirements. In drinking, they received artesian water, purified to the first quality category, and for food - full-feed compound feed of LLC Labkorm. Suspensions of nanoparticles were prepared by laser ablation of the corresponding pure (99.99%) metals in deionized water using a Fmark-20 RL laser system for processing materials (CLT, Russia). The chemical composition of the nanoparticles was determined by Raman spectroscopy and was identified as CuO, ZnO and PbO. The size distribution of nanoparticles was characterized by direct measurement using scanning electron microscopy and dynamic light scattering using a Zetasizer Nano ZS analyzer (Malvern Instruments, UK). The CuO and PbO particles had a spherical shape with an average diameter (± σ) of 24.5 ± 4.8 nm and 47.0 ± 16.0 nm, respectively. ZnO particles had a rod-shaped (close to cylindrical) shape with average sizes of 83.0 ± 20.0 × 30.0 ± 11.0 nm. For each administration to animals, freshly prepared suspensions were used.

Aqueous suspensions of nanoparticles at a dose of 0.5 mg per rat (each type of nanoparticles in 1 ml of the corresponding suspension) were administered to rats intraperitoneally at a dose of 1 mg per rat 18 times 3 times a week. In order to avoid direct interaction of oxides, accelerating the agglomeration of nanoparticles, suspensions were collected in separate syringes and injected separately with an interval of about 1 minute. 3.0 ml of the same sterile deionized water on which suspensions were prepared was similarly added to control animals. Separate groups of animals received the same injections against the background of oral administration of a bioprotective complex (BOD). The introduction of nanoparticles and the killing of animals of different groups were carried out in parallel.

Preparations containing the aforementioned bioprotectors were given in the same manner and at the dosages shown in Table 1.

The state of the organism of rats in all groups was evaluated by a large number (over 50) of generally recognized functional, biochemical and morphological (with morphometry under optical and electron microscopy) criteria for toxic action. To assess the genotoxic effect of nanoparticles in vivo, we used the PDAF analysis (PDAF is the polymorphism of the lengths of amplified DNA fragments), and to characterize the degree of DNA damage, we used the “fragmentation coefficient”, that is, the ratio of the total radioactivity of all tail fractions to the “core” radioactivity. The accumulation of metals in the body, estimated by their content in the central toxicokinetic pool - in the blood, and their content in the secretions was evaluated using inductively coupled plasma atomic emission spectroscopy on an ICAP 6500 DUO instrument (Thermo scientific, USA).

As can be seen from the data presented in Table 2, the most important toxicokinetic result of exposure to BOD was a statistically significant decrease in the content of all three metals in the blood compared to the corresponding indicator when seeds were coated with nanoparticles without BOD (1.3 times for copper, 3.3 times for zinc, 3.7 times lead). Since, within the framework of the laws of first-order toxicokinetics, the concentration of metal in the secretions is in dynamic equilibrium with the concentration in the blood, it is not surprising that in both urine and feces at the time of the study (end of the seed period), the content of all three metals was also reduced by BOD. However, in itself, a decrease in the delay of these metals in the body, evaluated by their concentrations in the central toxicokinetic pool, which is blood, indicates that. that over a significant part of the seed period, the elimination function of the kidneys and liver (which plays a special role in the excretion of copper) was improved. In turn, this improvement is due to the protection of these excretory organs from toxic damage provided by the influence of BOD (Table 3).

The general tendency revealed in the analysis of the functional indicators of the state of the body is that all those for which, when exposed to a combination of nanoparticles without BOD, revealed one or another shift in relation to the control value, especially if it is statistically significant, like this occurs for almost all indicators shown in Table 4. With the same effect against the background of BOD, it is weakened - often until the significance of the difference from the control is lost, while the difference from the group exposed to nanoparticles without BOD, on the contrary, is becomes statistically significant. In this regard, special attention should be paid to the weakening of the action of BOD:

- changes in the content of delta-aminolevulinic acid in urine and the content of reticulocytes in the blood, characteristic of lead intoxication;

- inhibition of research behavior and general motor activity, which, on the one hand, is one of the integral indicators of most intoxications, but at the same time, a probable sign of the specific neurotoxic effect of lead (mainly on the hypocampus) and excess copper (mainly on the basal nuclei).

The statistically significant attenuation shown in Table 5 is also of paramount importance against the background of BOD taking the genotoxic (prognostically - mutagenic and carcinogenic) effect inherent in all metal and metal oxide nanoparticles studied so far.

In this experiment, both the central neurotoxicity of the combination of nanoparticles of copper, zinc and lead oxides, and the attenuation of this effect while taking BOD are confirmed by histological examination of the brain with morphometry of damage to the neurons of the basal nuclei and the hippocampus (Table 6).

The general weakening of redox processes in the body under the influence of a toxic factor, reflected by a decrease in the activity of LDH, which loses its statistical significance when seeds are taken while taking BOD (Table 4), also has a morphological correlate - noted in all examined organs (liver, spleen, kidneys) , myocardium, brain, thymus, testicles) homogeneous mitochondrial damage with more or less complete destruction of cristae. With a 3-point assessment of the prevalence of such damage, a total score of 2 in the control group was obtained (for all organs as a whole), 14 - when exposed to nanoparticles without BOD, and only 6 - with the same effect while taking BOD. Based on nonparametric criteria (the Friedman rank test and the Kruskal-Wallis test), it was found that the difference between both LF-exposed groups from the control, as well as LF-exposed while taking BOD from LF-exposed, is statistically significant (P <0.05) without BOD.

A comparison of the obtained data with the literature indicates that for the first time in a toxicological experiment on a whole organism, hepatotoxicity, nephrotoxicity, and specific neurotoxicity of a combination of copper, zinc and lead oxide nanoparticles, as well as its genotoxicity, were convincingly demonstrated for the first time in a toxicological experiment on a whole organism. It was shown that when using the claimed method, these harmful effects are significantly attenuated.

A statistically significant difference from the control index is indicated by the index *, and from the index of the group of rats receiving a combination of NPs without BOD, by the index ⁺ (P <0.05 by student t).

A statistically significant difference from the control index is indicated by the index *, and from the index of the group of rats receiving a combination of NPs without BOD, by the index ⁺ (P <0.05 by student t).

A statistically significant difference from the control index is indicated by the index *, and from the index of the group of rats receiving a combination of NPs without BOD, by the index ⁺ (P <0.05 by student t).

A statistically significant difference from the control index is indicated by the index *, and from the index of the group of rats receiving a combination of NPs without BOD, by the index ⁺ (P <0.05 by student t).

A statistically significant difference from the control index is indicated by the index *, and from the index of the group of rats receiving a combination of NPs without BOD, by the index ⁺ (P <0.05 by student t).

Claims (1)

A way to increase the body's resistance to the development of the harmful effects of the combined action of copper, zinc and lead oxide nanoparticles on it, namely, to persons at risk of this action, a complex of biologically active drugs is prescribed, including glutamic acid, glycine, N- acetylcysteine, pectin enterosorbent, a fish oil preparation rich in non-esterified omega-3 fatty acids, as well as vitamins A, C, D3, E, selenium, iron and iodine preparations, and those at risk take this complex in repeated courses 1-2 times a year for 4-6 weeks daily in doses that provide 300 mg of glycine per day, 600 mg of cysteine, 4 g of glutamic acid, 25 ml of fish oil with 12-15% the content of polyunsaturated fatty acids of the omega-3 class, 4-5 g of pectin, as well as selenium, iron, iodine and these vitamins in doses that ensure normal physiological needs of the body.