RU2530639C1 - Method for prevention of adverse effect of general toxic and genotoxic action of nanosilver on human body - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: complex of biologically active preparations containing glutamic acid, glycine, cysteine, a pectin enterosorbent, a selenium polyvitamin-polymineral complex, a calcium preparation, as well as a cod liver oil preparation rich in nonesterified fatty acids omega 3 is administered. This complex is prescribed as a course of treatment once or twice a year for 4-6 weeks daily in the doses to provide the amounts of glycine of 300 mg, cysteine - 600 mg, glutamic acid - 4 g, selenium - 100 mcg, calcium - 4 g, cod liver oil - 25 ml with 12-15% nonesterified fatty acids omega 3, pectin - 4-5 g, as well as other microelements and vitamins in the doses to fill the normal physiological needs of the body.

EFFECT: lower adverse effects of the general toxic and genotoxic action of nanosilver on the body by normalising lipid peroxidation and the morphofunctional state of the liver and spleen, a lower degree of the DNA fragmentation.

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Description

The invention relates to medicine, in particular to the toxicology of nanomaterials (nanotoxicology), and can be used to reduce the general toxic and genotoxic effects of nanosilver on the body in risk groups, covering individuals who are exposed to this nanomaterial in the conditions of its production and use.

The high practical importance of nanosilver in various fields of technical and medical use is the reason for studying the toxic effects of this metal and finding means to reduce its general toxic and genotoxic effects on the body.

Works have been published in which various tests and on various cellular objects demonstrate the genotoxic effect of nanosilver, although predominantly in vitro (for example, Ahamed M., Karns M., Goodson M. et al. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. // Toxicol, and Applied Pharm. - 2008. - Vol.233, No. 3. - P.404-410) [1]. Some studies not only show that nanosilver significantly increases the formation of oxygen radicals, but also significantly blocks the genotoxic effects of nanosilver in the presence of antioxidants and scavengers of free radicals (for example, Choi JE, Kim S, Ahn JH et al. Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish. // Aquatic Toxicology. - 2010. - Vol.100, No. 2. - P.151-159) [2]. However, information retrieval has not found examples of the use of this protective effect with prolonged exposure of the nanosilver to the whole organism of mammals.

From the practical standpoint of prophylactic toxicology and health risk assessment due to exposure to nanoparticles, it is essential that the in vivo nanosilver genotoxicity is shown at such a low level of exposure at which the general toxic effect is still not very pronounced. Apparently, for some nanomaterials, not so much general toxicity as genotoxicity and thus likely carcinogenicity should be considered as a limiting risk. Judging by the results of studies conducted by the authors, nanosilver refers specifically to such nanomaterials (Katsnelson V.A., Privalova LI, Gurvich VB et al. Comparative in vivo assessment of some adverse bioeffects of equidimensional gold and silver nanoparticles and the attenuation of nanosilver's effects with a complex of innocuous bioprotector // Int. J. Mol. Sci. - 2013. - V.14. - P.2449-2483) [3]. Therefore, the objective of the invention is to provide means of protecting the body not only from the general toxic, but also from the genotoxic effect of nanosilver.

Based on the proposed mechanisms of the toxic and genotoxic effects of nanosilver on the body, a method has been developed for the prevention of the harmful effects of the general toxic and genotoxic effects of nanosilver on the body, which consists in the fact that people belonging to the risk group of this action are prescribed a complex of biologically active drugs, including glutamate, glycine, cysteine, pectin enterosorbent, selenium-containing multivitamin-polymineral complex, calcium-containing preparation, as well as a fish oil preparation rich in non-esterified fatty acids of the omega-3 class, and people at risk take complex preparations by repeated courses 1-2 times a year for 4-6 weeks daily in doses that provide 300 mg of glycine, 600 mg of cysteine, 4 g of glutamic acid, 100 mcg of selenium, 4 g of calcium, 25 ml of fish oil with 12-15% content of unesterified omega-3 fatty acids, 4-5 g of pectin, as well as other trace elements and vitamins in doses that ensure normal physiological needs of the body.

The claimed complex of biologically active preparations includes amino acids, namely glutamate, glycine and cysteine as precursors for the biosynthesis of reduced glutathione, which is a systemic protector against oxidative and free radical damage, while glutamate is also a powerful stabilizer of cell membranes. Pectin enterosorbent in the claimed complex is intended to serve as a blocker of intestinal reabsorption of metals isolated from bile. Calcium contained in a calcium-containing preparation acts as a silver antagonist. The selenium-containing multivitamin-polymineral complex contains selenium, which acts as a component of the antioxidant system and as an antagonist of silver, while the multivitamin-polymineral complex also contains other trace elements and vitamins in doses that ensure normal physiological needs of the body. The claimed complex also contains a fish oil preparation rich in non-esterified omega-3 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 scheme of use of the complex by people at risk, namely: in repeated courses 1-2 times a year for 4-6 weeks daily in doses that provide 300 mg of glycine, 600 mg of cysteine, 4 g of glutamic acid, 100 μg of selenium, 4 g of calcium, 25 ml of fish oil with a 12-15% content of unesterified omega-3 fatty acids, 4-5 g of pectin, as well as other trace elements (in particular copper) and vitamins in doses that ensure normal physiological needs of the body, justified by recounting the content of the listed active factors (bio protectors) in a bioprophylactic complex (BOD), the protective effectiveness of which was proved in an experiment conducted on laboratory white rats, in doses for human use on the basis of the ratio of the rat and human basal metabolic rates, taking into account also reference and published data on the daily human need for these substances .

The protective mechanisms of the bioprotectors included in the complex are complex and, apparently, mutually potentiate each other. The following may be important: (a) antioxidant action 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 the mitochondria by silver nanoparticles and thereby oxidative stress; (c) antagonism of a number of trace elements (selenium, copper, partly calcium) and silver. It has been shown for the first time that against the background of an oral combination of bioprotectors: pectin, vitamins, glutamate, glycine, cysteine, calcium, selenium and non-esterified omega-3 fatty acids, subchronic toxicity and, in particular, genotoxicity of nanosilver can be significantly reduced. 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. A new technical result achieved by the claimed invention is to reduce the harmful effects of the general toxic and genotoxic effects of nanosilver on the body.

The claimed method was experimentally tested on white outbred female rats with an initial body weight of 150-220 g. The animals were kept in a specially organized vivarium, corresponding to veterinary requirements. In drinking, they received artesian water, cleaned up to the first quality category, for food - full-feed mixed fodder Labkormorm LLC. Nanoserber suspensions (colloidal solutions) were prepared by laser ablation of a metal in a liquid using a Fmark-20 RL laser system for processing materials (CLT, Russia). 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 average particle diameter of silver was 49 ± 5 nm and remained stable for a week after preparation. However, freshly prepared suspensions were used for each animal administration.

The resulting nanosuspensions with a concentration of 0.5 mg / ml were administered intraperitoneally to rats at a dose of 10 mg / kg of body weight for metal 3 times a week. Control animals were similarly injected with an appropriate volume of the same sterile deionized water on which suspensions were prepared. Each rat received a total of 20 injections over an incomplete 7 weeks.

The introduction of nanosilver or water and the killing of animals of different groups were carried out in parallel. A group of animals was also conducted in parallel through the experiment, in which a similar effect of nanosilver was carried out against the background of oral administration of the bioprevention complex.

Rats were given sodium glutamate as a 1.5% solution in unlimited drink instead of water. Apple pectin production (Promavtomatika enterprise, Belgorod, Russia) was given as an additive in food in an amount corresponding to an average dose consumed of about 1000 mg / kg body weight. Glycine, cysteine in a more accessible and metabolically active form of acetylcestein, Complivit-selenium as a source of vitamins, selenium and calcium, Complivit-calcium as a source of vitamins and calcium were used in tablets, which were crushed and added to another portion of feed based on average dose consumed in accordance with the recommended needs of the rat. As a preparation of fish oil, rich in non-esterified omega-3 fatty acids, the drug “Eicosavitol” (Farmavit, Tyumen, Russia) was used, which was given intragastrically through a probe at a dose of 1 ml per rat.

In 4 animals of each group, organs were removed from the body within 2-3 minutes after the rats were sacrificed by decapitation in the sequence: liver - spleen - kidney - thigh bone - and immediately, like the blood leaked during decapitation, were placed in chilled to -80 ° C special vessels that were urgently delivered to the genetic laboratory in cryo-containers for research.

To isolate genomic DNA, 96 tissue samples were analyzed, each in triplicate. Samples of solid organs were ground with a scalpel. Then, in order to obtain a homogeneous mass, the samples were subjected to freezing four times in liquid nitrogen and thawing in a water ultrasonic bath (38 ° C) in a PBS solution (Ca ⁺⁺ , Mg ⁺⁺ free, Sigma), followed by passing through a syringe needle of decreasing diameter. Bone marrow cells were washed with the same solution from the femur after cutting off the epiphyseal and diaphyseal sites, followed by passing through a needle of decreasing diameter. Nucleated peripheral blood cells were isolated from whole blood by centrifugation in a percol gradient (Sigma). To isolate DNA from cell cultures, a GenElute reagent kit (Sigma) was used according to the manufacturer's recommended protocol. The amount of DNA (Ultraspec 1100 pro) was determined spectrophotometrically (Ultraspec 1100 pro) in the obtained samples, frozen and stored at -84 ° C in a kelvinator (Sanyo) until the start of the study.

PDAF analysis (PDAF - polymorphism of the lengths of amplified DNA fragments) was performed as follows. Randomized oligonucleotide and NotI-STS primers were designed on line using the Oligo version 3.0 service (http://www.oligo.net) and synthesized on an ASM 800 automatic synthesizer (Biosset company).

The choice of the optimal amplification mode was carried out based on the length of the amplified fragment after a trial cycle of modeling the temperature gradient. The stages of initial denaturation (2 min at 94 ° C) and final polymerization (3 min at 72 ° C) were mandatory in each trial amplification cycle. The number of cycles was selected empirically for the highest yield of a specific amplicon with a minimum content of non-specific fragments. The mixture in a volume of 25 μl was placed in a thermocycler with a given program of temperature-time cycles: T _den 940 ° C - 2 min; (T _den 95 ° C - 0.2 min, T _anne 64 ° C, T _elon 72 ° C - 0.5 min) - 29 cycles, T _elon 72 ° C - 3 min.

In order to increase the sensitivity, specific primers and nucleotides (dCTP, dATP, methyl dTTP, Amersham) labeled with tritium were used in the reaction. The resulting amplification was separated during horizontal agarose gel electrophoresis in TAE buffer at 100 V for 15 min. At the end of electrophoresis, the gel plates were divided into tracks, each of which was cut into sections 5 mm long. The resulting gel fragments were placed in vials containing 3.0 ml of absolute isopropanol. The vials were heated to 80 ° C for 2 hours. After extraction of the amplified fragments containing the isotopic tag from the gel, a simple toluene scintillator (6.0 ml) was added to the bottles. The results were recorded on a Beta-2 automatic liquid scintillation counter.

This technique allows you to quantify the degree of DNA fragmentation, as an indicator of the genotoxicity of the estimated damaging agents and the effect of the studied complex of bioprotectors. The basis of the method is that, unlike fragmented DNA, which forms the so-called “comet tail” during electrophoresis in an agarose gel, non-fragmented DNA has an extremely low degree of migration and remains practically in place (“comet nucleus”), and the degree of migration is directly proportional degree of DNA fragmentation. To characterize the degree of DNA damage, a “fragmentation coefficient” was used, that is, the ratio of the total radioactivity of all fractions of the “tail” to the radioactivity of the “core”.

The state of the organism of rats in all groups was evaluated by a large number (over 40) of universally recognized functional, biochemical and morphological criteria for toxic action. As an example of the protective effectiveness of the applied BOD for the effects of the general toxic effect of nanosilver, the following are given: normalization of lipid peroxidation (one of the most important free radical processes in the body), enhanced by the action of nanosilver, as well as some morphometric indicators of the effect on the liver and spleen as organs rich in cells reticulo-endothelial system (RES) and therefore serving as one of the main stores of any nanoparticles that enter the bloodstream.

The statistically significant increase in lipid peroxidation (LP) during subchronic intoxication by nanosilver particles without biological protection and the absence of this effect with the same intoxication while taking BOD are illustrated by the data in Table 1, which shows the concentration of the final LPO product - malonyl dialdehyde (MDA) in rat serum .

Table 2 shows morphometric indicators characterizing the state of the liver and spleen of rats under the action of nanosilver while protecting the body with and without BOD. It was shown that although the main indicator of liver damage (% of nuclear-free hepatocytes) was only slightly increased under the influence of nanosilver, however, against the background of BOD, it was statistically significantly lower than not only this, but also the control indicator, which indicates a non-specific hepatoprotective effect of the applied complex. Along with this, judging by the percentage of binuclear cells, regenerative mitosis of hepatocytes was significantly enhanced, which is a positive effect of the action of BOD. Activated by the influence of BOD, the release of the liver from nanoparticles resulted in a statistically significant decrease in both the percentage of Kupffer cells (RES cells) and their particle loading. The normalization of the morphological and functional state of the spleen under the influence of BOD is indicated by the absence of the statistically significant decrease in the ratio of white pulp to red, which was observed during intoxication of nanoserbr without the BOD.

For the same two organs, Table 3 shows the fragmentation coefficients (Kfr) of the genomic DNA of rats that were seeded with nanosilver particles without protection and while receiving the claimed complex. It was shown that during subchronic intoxication with NS particles, significant DNA damage occurs in the cells of the liver, spleen, kidneys, bone marrow and peripheral blood, which is noticeably weakened if the same effect of nanosilver occurred while taking BOD.

A comparison of the obtained data with the literature indicates that the applicants for the first time convincingly demonstrated the multi-organotoxicity of nanosilver with prolonged exposure to the whole organism, and in a dosage that showed only moderate systemic toxicity, and it was shown for the first time that, against the background of the exposure of the proposed complex of bioprotectors to the genotoxicity of nanosilver and its general toxic effect can be significantly attenuated.

Table 1 The effect of subchronic intoxication of rats with nanosilver on the level of lipid peroxidation without protection and while taking BOD (by the concentration of malonyl dialdehyde in blood serum, X ± S _x ) The concentration of MDA in serum, nmol / l Control group (water injection) The group receiving nanosilver (NS) The group receiving NS on the background of BOD 5.84 ± 0.12 6.23 ± 0.14 * 5.72 ± 0.25 * - The difference from the control indicator is statistically significant at P <0.05 (according to the Student t-test). table 2 Some morphometric indicators of the cellular structure of the liver and spleen of rats during intoxication with nanosilver without protection and while taking BOD (x ± s.e.) Indicators The control Nanosilver Nanosilver on the background of BOD The number of nuclear-free hepatocytes per 100 liver cells 17.6 ± 0.6 18.5 ± 1.3 13.0 ± 1.0 * ^• The number of binuclear hepatocytes per 100 liver cells 5.9 ± 0.8 7.8 ± 0.6 12.0 ± 1.5 * ^• Kupffer cells per 100 liver cells 16.5 ± 0.5 25.0 ± 0.8 * 20.0 ± 0.6 * ^• Weighted average particle loading score for Kupffer cells 0 0.91 ± 0.7 0.51 ± 0.09 ^• The ratio of white to red pulp of the spleen 0.59 ± 0.036 0.37 ± 0.035 * 0.59 ± 0.086 ^• * - statistically significant difference with the control group; • - a statistically significant difference with the nanosilver group (P <0.05; according to Student t-test).

Table 3 Fragmentation coefficients (Kfr) of the genomic DNA of rats subjected to subchronic inoculation with nanosilver particles without protection and while taking BOD (according to the results of the PDAF test), X ± S _x A group of rats treated with: Fabrics Liver Bone marrow Spleen Bud Nuclear cells of peripheral blood Water (control) 0.399 ± 0.001 0.385 ± 0.003 0.379 ± 0.002 0.385 ± 0.003 0.383 ± 0.001 Nano Silver (NS) 0.461 ± 0.002 * 0.455 ± 0.032 * 0.462 ± 0.001 * 0.423 ± 0.008 * 0.413 ± 0.012 * NA + BOD 0.408 ± 0.011 ⁺ 0.373 ± 0.003 * ⁺ 0.419 ± 0.003 * ⁺ 0.407 ± 0.006 * ⁺ 0.390 ± 0.007 * statistically significant (P <0.05 by t-student test) difference from the control group. ⁺ statistically significant (P <0.05 by Student t-test) difference between the group receiving NS and BOD from the group receiving only NS,

Claims (1)

A method for the prevention of the harmful effects of the general toxic and genotoxic effects of nanosilver on the body, which consists 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, pectin enterosorbent, selenium-containing multivitamin-polymineral complex, calcium-containing a drug, as well as a fish oil preparation, rich in non-esterified omega-3 fatty acids, and people at risk take drugs to of the complex by repeated courses 1-2 times a year for 4-6 weeks daily in doses providing 300 mg of glycine, 600 mg of cysteine, 4 g of glutamic acid, 100 μg of selenium, 4 g of calcium, 25 ml of fish oil from 12-15 % of non-esterified fatty acids of the omega-3 class, 4-5 g of pectin, as well as other trace elements and vitamins in doses that ensure normal physiological needs of the body.