[Peroral toxicological assessment of bentonite nanoclay used in the food industry].

Bentonite nanoclay (NC) manufactured from the natural sedimentary mineral bentonite contains more than 90% montmorillonite. Currently, it is widely used in food industry as processed aids - adsorbents for the purification of vegetable oils and beverages. Clay minerals have also applications as food additives and components in composite package materials. In vitro studies have shown that various forms of NC exerted cytotoxicity in many cell lines, whereas in vivo evidence of NC oral toxicity is contradictory. Therefore, this study aimed to assess the acute oral toxicity of NC and to evaluate its toxicological characteristics in a subacute 92-day experiment on Wistar rats with a daily oral administration in doses of 1, 10, and 100 mg/kg body weight (bw). Material and methods. The NC acute toxicity was evaluated in 8 male and 8 female rats with the initial bw 236±10 and 203± 10 g, respectively. NC was administered as an aqueous dispersion intragastrically at a dose of 5 g/kg bw. On the 14th day (end of the experiment), an autopsy of the chest and abdominal organs was performed. The subacute experiment was carried out on 64 male rats with an average initial bw of 117±7 g. During the experiment the levels of anxiety and memory function were evaluated using the test "Conditional reflex of passive avoidance". On the 90th day of the experiment, diurnal urinary excretion of creatinine and selenium was evaluated. At the end of the experiment, the integral parameters, the state of the intestinal wall permeability were assessed. Hematological and biochemical parameters were examined in blood, the content of non-protein thiols and the number of cells in apoptosis were determined in liver, and the state of cultivated microbiome populations was studied in cecum. Results. The results of the determination of NC acute toxicity showed the absence of rat's mortality and specific pathological changes in the internal organs at a dose as large as 5000 mg/kg bw, which allowed attributing NC to the V hazard class. Nevertheless, under the conditions of the 92-day experiment, NC caused some adverse biological effects on rat's organism. So, even at an NC dose of 1 mg/kg bw, there was a sharp inhibition of the symbiotic bifidobacterium growth, an increase in platelet count, in LDL and the LDL/HDL ratio, together with the presence of hypertriglyceridemia. At a dose of 10 mg/kg bw, an increase in spleen mass and a decrease in the de Ritis coefficient (AsAT/AlAT) were established. At a dose of 100 mg/kg bw there were shifts in the leukocyte blood count, an excessive enterococci growth in the cecum, significantly increased animal bw, along with the decrease of AsAT/AlAT and the level of serum nitrogen metabolites, indirectly indicating inhibition of catabolic processes. However, at the highest dose of NC, intestinal absorption of the protein antigen - ovalbumin, was apparently completely blocked. Conclusion. The data obtained have shown that NC has potentially adverse effects on the rats mainly at a dose of 100 mg/kg bw, nevertheless, its NOAEL in the 92-day daily oral exposure experiment is probably less than 1 mg/kg bw.

[Sex and line differences in biochemical indices and fat soluble vitamins sufficiency in rats on in vivo model of metabolic syndrome].

Consumption of diets that are inadequate in energy value to the actual energy expenditure can lead to the development of metabolic syndrome (MS), which has consequences such as type 2 diabetes mellitus, non-alcoholic steatohepatosis, atherosclerosis, gout, allergic diseases. Experimental models of MS are needed to develop new approaches to its dietary and drug correction. The aim of the work was a comparative analysis of functional, biochemical and vitamin markers characterizing the effect of a diet with a high content of fructose (F) on males and females of various rat lines and the selection on this basis of an optimal in vivo MS model. Male and female rats of the outbred Wistar line (W) and the inbred Dark Agouti line (DA) were used in the work number of 16 individuals of each sex and line. The animals of the 1st (control) groups of each sex and line received a balanced semi-synthetic diet according to AIN93, and the animals of the 2nd (experimental) groups - the same diet and 30% solution of F instead of water in the regime of free access. Within 121 days, energy value of diets consumed, the increase in body weight and blood pressure were determined; relative mass of internal organs, biochemical parameters of blood plasma, content of fat-soluble vitamins A and E in blood plasma and liver were determined at withdrawal of animals from experiment. It was shown that, in spite of the increased energy value of the diet in the experimental groups throughout experiment, DA males and females practically did not respond to this by an increase in body weight gain, in contrast to W rats (in particular, females). Consumption of diets with F led to an increase in glucose level irrespective of gender and line, whereas triglyceride level (TG) significantly increased only in the case of W female. Addition of F caused in DA rats of both sexes an increase in the mass of the kidneys, as well as more pronounced, in comparison with W rats manifestation of markers of toxic effects on the liver (increases alanine aminotransferase and γ-glutamyltransferase activity, elevated urea and bilirubin level in blood plasma). In rats of both lines intake of F suppressed the accumulation of retinol palmitate in the liver in terms of its specific content. The total content of α-tocopherol in liver was significantly higher in W compared with DA. At the same time, α-tocopherol levels in blood plasma correlated with TG, and the α-tocopherol/TG ratio significantly decreased in female W receiving F, which were characterized by hyperlipidemia. Thus, the effect of F on W males and, in particular, females, basically corresponded to the classical picture of MS with body weight increasing, elevated blood pressure, glycemia and TG increase, whereas the toxic effect of F prevailed in DA liver and, possibly, kidneys without development of marked dyslipidemia and obesity.

[Effect of B-vitamin deficiency on biochemical, immunologic markers and trace element status of rats and mice of various lines].

Biochemical, vitamin, trace element and immunological changes were searched for the combined nutritional deficiency of vitamins B1, B2, B6 on in vivo models in rats and mice. Female rats of Wistar (W) strain and hybrids of the 1st generation of Dark Aguti and Wistar (DA x W) strains, female mice of BALB/c strain and DBCB tetrahybrids were used in experiment. Animals received for 35 days a balanced diet (control) according to AIN-93 or a similar diet with the exception of vitamins B1, B2, B6 (experimental groups). The content of vitamins B1, B2 in liver, riboflavin blood plasma level and urinary excretion of thiamine, riboflavin and 4-pyridoxic acid were determined, as well as in rats: blood and liver content of α-tocopherol and retinol, blood biochemical indices of lipid and nitrogen metabolism, activity of cytochrome P isoforms-450 (CYP) in liver; in mice: the circulating levels of pro- and anti-inflammatory cytokines of blood plasma, in animals of both species - the content of essential and toxic elements in the kidneys. DAxW rats compared to W and DBCB mice compared to BALB/c were more sensitive to the development of B-vitamin deficiency judging by the B-vitamin status indicators. In the rats of the experimental groups, there were signs of a deterioration in blood and liver levels of vitamin E, multidirectional shifts in vitamin A sufficiency, increased activity of the CYP3A isoform (6ß-TG), a decrease in triglycerides, total protein and albumin fraction levels with an increase in urea level. Manifestation degree of these effects depended on the choice of the animal's line. In mice, the B-vitamin deficiency was characterized by an increase in the levels of proinflammatory cytokines TNF-α, IL-10, IL-Ιß, IL-6 and a decrease in IFN-γ and IL-17A. The content of magnesium, copper, zinc, chromium and silver was lowered, of cesium - was increased in the kidneys of the rats of the experimental groups. In mice, B-vitamin deficiency resulted in diminishment of magnesium, copper, zinc, chromium, selenium, cadmium and lead content, excess accumulation of cobalt and cesium. Some of these biomarkers are supposed to be used in pre-clinical evaluation of the effectiveness of new vitamin complexes, specialized foods and dietary supplements, as well as studies of interactions of various vitamins.

[Subacute oral toxicity in vivo of multi-walled carbon nanotubes].

Multiwall carbon nanotubes (MWCNTs) are regarded as environmental pollutants with increased risk. Recently MWCNTs have attracted attention as a promising component of packaging materials for food products, as carriers for agricultural plant growth stimulants, agrochemicals components and advanced pesticides, which creates the possibility of their exposure through the gastrointestinal tract. Objective of the research is assessment of sub-acute oral toxicity to rats of MWСNTs in an experiment lasting 100 days. MWCNTs preparation «Taunit-M®¼ was preliminary characterized by electron microscopy and Raman light scattering. Nanomaterial was administered to animals as sonicated dispersion in water with 1% by weight of nonionic surfactant Tween 20. The experiment was performed on 80 growing male Wistar rats with initial body weight (b.w.) 86±2 g. Rats of experimental groups (from 2nd to 5th) received MWCNTs dispersion instead of drinking water, the animals of the 1st control group - a carrier solution (Tween 20). Doses of MWCNTs consumed were, respectively, in groups 1-5: 0; 0.01; 0.1; 1.0 and 10 mg/kg b.w. Hematological and biochemical indices of blood were determined together with the activity of glutathione peroxidase of erythrocytes, the content of non-protein thiols in the liver, excretion of 8-hydroxy-2'-deoxyguanosine (8-oxo-G) and content of the main and transient components of gastrointestinal microbiocenosis in the cecum contents. Apoptosis of hepatocytes was studied by flow cytometry. As a result MWCNTs led to increase of blood glucose and creatinine in rats in group 2, a significant decrease in the number of neutrophils and monocytes by increasing the number of lymphocytes, decreased platelet volume, the most pronounced also in group 2, receiving the lowest dose of MWCNTs. There were no signs of oxidative DNA damage identified. At the lowest dose MWCNTs caused a significant decrease in the number of bifidobacteria, increase - citrate-assimilating Enterobacteriaceae, hemolytic aerobic microorganisms and yeast. These changes in the microbiota should be considered as adverse, apparently leading to disturbances in the immune function.

[Comparative evaluation of vitamin status and biochemical markers of metabolic syndrome on the models of rodents receiving rations with high quotas of different sugars].

Metabolic syndrome (MS) is one of the leading causes of non-infectious pathology among the population of developed countries. It is necessary to have experimental in vivo models of MS for pre-clinical testing of new approaches to its dietary therapy. The purpose of the study was a comparative analysis of functional, biochemical and vitamin markers that characterize the effect of diets with different composition of simple carbohydrates (sugars) on female Wistar rats and female C57Black/6J mice. Animals of each species (n=80) were divided into 5 groups of equal numbers. The animals of the 1st (control) group received a balanced semi-synthetic diet, and the animals of groups from the 2nd to the 5th - the same diet and 30% solutions of sugars - glucose (Gl), fructose (Fr), equimolar mixture Gl and Fr and sucrose instead of water, in the regime of free access for up to 133 days. Measured values included blood pressure, mass of internals, biochemical parameters of blood plasma, the activity of CYP1A1, CYP1A2, CYP2B1, CYP3A and glutathione transferase (GT) in liver, glutathione peroxidase (GP) in erythrocytes, the content of vitamins A and E in blood plasma and in liver, the level of vitamins B1 and B2 and nicotinamide coenzymes in liver. Interspecific differences in the response to sugars manifested in a decrease in the solid diet consumption in mice (in contrast to rats), so that the total consumed energy value in experimental groups of mice did not differ systematically from control, and the weight gain was reduced. Liver was the most sensitive organ to addition of sugars in both rats and mice with mass significantly increasing by the 2nd and the 4th months of the experiment. Hyperglycemia and triglyceridemia were the most noticeable in rats receiving Fr. The concentration of phosphorus increased significantly in blood plasma of all rats groups that received sugars. In rats there was a decrease in the activity of CYP1A1 and CYP1A2 in groups 3 and 5, the activity of CYP2B1 in groups 2 and 5, the increase in HT activity in groups 2, 4 and 5, and GP in group 3 at 56th day of experiment. There was a significant decrease in this index in group 3 at the 56th and the 133rd days of the experiment, and in groups 4 and 5 - at the 56th day. Plasma tocopherol to triglycerides ratio decreased in rats of group 3 at the 56th and 133rd days, groups 4 и 5 - at 56th day, which indicated the decrease of vitamin E safety. Sugars consumption suppressed retinol palmitate accumulation in the liver of rats and mice, and alpha-tocopherol in mice. It was concluded that Fr had the greatest effect on the studied indicators of the organism, and the rats showed the most significant similarity with the clinical picture of MS.

[Indicators of vitamins safety in experimental alimentary hyperlipidemia in rodents].

Rats and mice of different strains are used as a model of metabolic disturbances, caused by the consumption of diets with unbalanced content of macro-nutrients (fat, carbohydrate), as well as having elevated cholesterol quota. The aim of this study was to determine the magnitude and direction in change of vitamins status indices produced in rats and mice with experimental-mental hyperlipidemia, developing under consumption of high fat diet (HFD), fructose (Fr) and cholesterol (Cho). The experiment was conducted on 48 female growing Wistar rats with initial body weight 122±12 g, and 48 female growing C57Black/6 mice with initial body weight 18±1 g, which were divided into 12 groups of 8 animals per group. Within 63 days the rats and mice of the first (control) group received a balanced semi-synthetic (BD), 2nd groups - HFD with 30% of the total fat by weight of dry feed, 3rd groups - BD and Fr solution instead of water, 4th groups - HFD+Fr, 5th groups - BD supplemented with 0.5% Cho by weight of dry food, 6th groups - the same ration and Fr. After removal of animals from the experiment there were determined the content of vitamin A (retinol and retinol palmitate) and E (α-tocopherol) in blood plasma and liver by HPLC, 25-hydroxycholecalciferol [25(OH)D] in blood plasma by HPLC-MS, vitamins B1, B2 and oxidized NAD coenzymes in liver by fluorimetric methods. Consumption of HFD resulted in marked increase in the concentration of vitamin A by 32% and by 45% in rat blood plasma and in the mice liver respectively, elevation of vitamin E level by 46% in the rat liver. Unlike rats, vitamin E in the liver of mice treated with HFD was lower by 32% compared with the control. Cho additive resulted in increased vitamin E accumulation in rat and mice liver (α-tocopherol level was 2.5 и 1.5 fold higher than in control respectively). Convincing evidence wasn't revealed of the impact of the additional Fr on vitamins A and E safety in rats and mice. Consumption of Fr on background of HFD in rats significantly reduced the level of 25(OH)D compared with HFD without Fr. Fr reception in combination with the addition of Cho significantly reduced stores of vitamin A and increased - of vitamin E in the liver of rats and mice. 25(OH)D level for this type of diet was significantly reduced. Cho consumption in rats significantly decreased the content of NAD+NADP in the liver by 12%; the introduction of fructose into the diet neutralized this impact. Feeding rats with HFD resulted in a significant improvement, and uptake of Cho in reduce of vitamin B2 levels in the liver by 12.8 and 28%, respectively. Fr partially neutralized these effects. Thus, changes in the ratio of macronutrients and Cho in the diet of rats and mice may lead to a partially species-specific vitamin sufficiency variations, including in some cases the development of functional deficiency of vitamins А, B2, D and NAD coenzymes.

[Carbon nanotubes: mechanisms of the action, biological markers and evaluation of the (review of literature)].

The article contains the review and analysis of data on the mechanisms of the toxic action of carbon nanotubes (CNTs) on the body and available results of CNT toxicological evaluation after inhalation and oral routes of the action. Identification of cellular and molecular mechanisms of CNTs toxicity allows to justify the list of the most sensitive biochemical toxicity markers that could be used for monitoring the occupational effects of CNTs and serve as a promising target for the corresponding pharmacological and immunopharmacological interventions aimed on specific prophylaxis and therapy of diseases caused by CNT. A considerable amount of experimental data obtained in vivo on inhalation animal models allows to establish the hygienic standardfor CNT in the air of the working area. As to safe levels of the oral route of CNT it needs further study for their reliable assessment. The search and selection of sources for the review was executed with the use of public databases, including (in order of relevance) PubMed, Scopus, Google Scholar, and RISC, for the period from 2004 to 2017.

[Toxicological evaluation of colloidal nano-sized silver stabilized polyvinylpyrrolidone. III. Enzymological, biochemical markers, state of antioxidant defense system].

Nanosized colloidal silver (NCS) with primary nanoparticles (NPs) size in the range of 10-80 nm in aqueous suspension was administered to rats with initial weight 80±10 gfor the first 30 day intragastrically and for lasting 62 days with the diet consumed in doses of 0.1; 1.0 and 10 mg/kg of body weight b.w) per day based on silver (Ag). The control animals received deionized water and carrier of NPs - aqueous solution of stabilizer polyvinylpyrrolidone. Activity (Vmax) was determined in liver of microsomal mixed function monooxygenase isoforms CYP 1A1, 1A2 and 2B1 against their specific substrates, the activity of liver conjugating enzymes (glutathione-S-transferase and UDP-glucuronosyltransferase) in the microsomal fraction and a cytosol, and the overall and non-sedimentable activities of lysosomal hydrolases. In blood plasma there were evaluated malonic dialdehyde, PUFA diene conjugates, in erythrocytes - the activity of antioxidant enzymes. A set of standard biochemical indicators of blood serum was also determined. The studies revealed changes in a number of molecular markers of toxic action. Among them - the increase in the activity of key enzymes I and II stages of detoxification of xenobiotics, indicating its functional overvoltage; reducing the activity of glutathione peroxidase (GP), the total arylsulfatase A and B, ß-galactosidase (in the absence of changes in their non-sedimentable activity), levels of uric acid, increased alkaline phosphatase activity. These changes occurred mainly at the dose Ag of 10 mg/kg b.w., except for the GP to which the threshold dose was 1 mg/kg b.w. No significant changes in the studied markers in a dose Ag 0,1 mg/kg b.w. were identified. Possible mechanisms of the toxic action of silver NPs are discussed.

[Toxicological evaluation of nanosized colloidal silver, stabilized with polyvinylpyrrolidone, in 92-day experiment on rats. II. Internal organs morphology].

The aim of the study was to evaluate the safe doses of commercially available nanosized colloidal silver (NCS), stabilized with polyvinilpirrolidone (PVP, food additive E1201) when administered in gastrointestinal tract of rats in the 92-day experiment in terms of the morphological changes in the internals of animals. The sample studied contained non-aggregated nanoparticles (NPs) of silver belonging to size fractions with a diameter of less than 5 nm, 10-20 nm or 50-80 nm. 80% of NPs were inside the range of hydrodynamic diameters 10.6-61.8 nm. The preparation of NCS was administered to growing male Wistar rats. (initial body weight 80 ± 10 g) for 1 month by intragastric gavage and then consumed with food at doses of 0.1, 1.0 and 10 mg/kg of body weight based on silver. The control animals received water or vehicle of nanomaterial--water solution of PVP. After withdrawal of animals from the experiment by exsanguination under ether anesthesia organs (liver, spleen, kidney, ileum) were isolated and their slides were prepared by standard methods following 'by staining with hematoxylin-eosin. Analysis was performed in light optical microscope equipped with a digital camera at a magnification from 1 x 100 to 1 x 1000. It was shown that the experimental animals treated with the NCS developed series of morphological changes in the tissues of the internal organs (liver, spleen and kidney) with the elevation of the range and severity of structural changes with increasing doses of silver. The most sensitive target of NCS action was apparently liver, which has already shown at a dose of 0.1 mg of silver NP/kg of body weight marked eosinophilic infiltration of portal tracts, which was accompanied at doses of 1.0 and 10.0 mg/kg by the emergence of medium and large-drop fat vacuoles in the cytoplasm of hepatocytes, swelling and lympho-macrophage. infiltration of the portal tracts. Detectable changes can be regarded as symptoms of inflammation of hepatocytes, at least, at a dose nanomaterial of 1.0 mg/kg body weight or more. Relative intensity of morphological changes in the internal organs correlated with published data on the biodistribution of silver NP administered to the gastrointestinal tract. It is concluded that the threshold dose corresponding to the minimum adverse effect of NCS is, according to the study of the above, no more than 1.0 mg/kg of body weight based on silver.

[Toxicological evaluation of colloidal nano-sized silver stabilized polyvinylpyrrolidone. IV. Influence on intestinal microbiota, immune indexes].

Nano-sized colloidal silver (NCS) stabilized with polyvinylpyrrolidone (PVP) containing nanoparticles (NPs) of silver with a diameter of 10-80 nm was administered to growing male rats (body weight 80±10 g) during the first 30 days by intragastric gavage and then for 62 days with diet consumed in doses of 0.1, 1.0 and 10 mg/kg of body weight per day based on silver (Ag). The control animals received deionized water and PVP. The composition of microbiota from the cecum was studied using standard microbiological methods with determination of the main and transient components, together with antagonistic activity of symbiotic bifidobacteria. Expression of antigens CD45RA, CD3, CD4, CD8, CD161a on lymphocytes (Ly) of peripheral blood was determined by flow cytometry; blood serum levels of cytokines IL10, IL13, TNFα were examined by ELISA. It was shown that subacute administration of colloidal Ag in all studied doses did not lead to significant changes in the composition of the main components of normal microbiota, providing, however, the inhibitory effect on the growth of some transitory components probably including opportunistic species of microorganisms. Among the studied immunological parameters decreased amount of B-Ly was noticed at the highest dose of the NCS, while changes in the other parameters of the immune system were depended ambiguously on the dose of the product. The results were analyzed in conjunction with the data of previous publications concerning the impact on the NCS on integrated, morphological, hematological, biochemical and enzymological indexes of animals in the 92-day experiment. It was concluded that significant symptoms of NCS sub-acute oral toxicity manifested starting from a dose of 1 mg/kg body weight of Ag, and the maximum not observed adverse effect dose (NOAEL) can be estimated as 0.1 mg/kg body weight.

Influence of nanosized amorphous silica on assimilation of vitamins B1, B2 and B6 in rats.

Amorphous silica (SiO2) in the form of nanoparticles (NPs) is widely used as a food additive E551 in many enriched foods and food supplements. The aim of this study was to evaluate the effect of oral administration of SiO2 NPs on assimilation and metabolism of vitamins B1, B2 and B6 in laboratory rats. Amorphous SiO2 «Orisil-300 ®¼ was used with the size of the primary NPs 20-60 nm according to the electronic, atomic force microscopy and dynamic light scattering. The experiment was conducted on 8 groups of growing male Wistar rats (with initial body weight 70-80g) number, respectively, 7, 7, 10, 10, 12, 12, 14 and 16 animals. Animals of the 1st, 3rd, 4th and 5th groups received through­out the experiment balanced semi-synthetic diet. Animals of the 2nd group received a diet depleted of vitamins B1, B2 and B6 until day 21; animals of the 6th, 7th and 8th groups -the same diet from the 1st to the 21th day, and then, before the closure of the experiment, the diet provided with the indicated B vitamins at 100% of normal level. From day 22 of experiment and until the end at day 29 the animals of the 3rd and 6th groups received deionized water (placebo) through intragastric gavage; rat of the 4th and 7th groups -aqueous suspension of SiO2 dose of 1 mg/kg body weight /day, and the 5th and 8th group -100 mg/kg/day. Urinary excretion of thiamine, riboflavin, 4-pyridoxilic acid and liver and brain content of vitamins B1 and B2 (after acid and enzyme hydrolysis) were deter­mined by fluorimetric methods. It was found that rats in group 2 lagged in weight gain at day 21 significantly compared to group 1, and developed a marked deficiency of vitamins B1, B2 and B6 according to studied safety parameters. In groups from 6 to 8 at day 29 par­tial recovery was achieved in vitamin status. Administration of SiO2 to animal of groups 4 and 5, with normal consumption of B vitamins, had no significant effect on any param­eters of vitamin status in comparison to group 3. However, intragastric administration of SiO2 led in animals of groups 7 and 8 to an increase in the urinary excretion of vitamins B1 and B2 and lowering of their content in liver as compared to group 6. Administration of SiO2 had no effect on indices of vitamin B6 sufficiency. Possible reasons are discussed for the adverse lowering impact of SiO2 NPs on the availability of vitamins B1 and B2 and their increased clearance from the body.

[Toxicological assessment of nanostructured silica. IV. Immunological and allergological indices in animals sensitized with food allergen and final discussioin].

This paper is the final in a series of publications on the assessment of subacute oral toxicity of nanostructured silica (SiO2). Preparation studied was a commercial nanopowder of SiO2, obtained by hydrolysis of tetrachlorosilane in the gaseous phase with the size of primary nanoparticles (NPs) of 5­30 nm. The experiment was conducted in 95 male Wistar rats weighing 150­180 g, divided into 6 groups numbering 25 (group 1), 26 (group 2), 11 (groups 3­6) of animals. The aqueous dispersion of SiO2 after sonication was administered to animals of groups 2, 4 and 6 for 28 days by intragastric gavage at a dose of 100 mg/kg of body weight per day. Animals of groups 1, 3, and 5 were treated with deionized water. On the 1st, 3d, 5th and 21st day of experiment the rats of groups 1, 2, 3 and 4 were sensitized intraperitoneally with hen's egg ovalbumin (OVA) adsorbed to aluminum hydroxide. Intravenous administration of the challenge dose OVA to rats in groups 1 and 2 was carried out on the 29th day. In the same period animals of groups 3­6 were bled for analysis of cellular immunity. There were evaluated the severity of systemic anaphylaxis reaction, the level of specific IgG antibodies to OVA in sensitized animals, state of erythrocytes, platelets and leukocytes of peripheral blood using standard methods. Using flow cytometry there were measured contents of lymphocyte populations of B-lymphocytes (CD45RA+), total T-lymphocytes (CD3+), T-helper cells (CD4+), T-cytotoxic cells (CD8+), NKcells (CD161a+), phagocytic activity of polymorphonuclear leukocytes in respect of latex particles. Serum levels of TNFα and IL-10 cytokines were determined by ELISA. The result showed that NPs SiO2, at dose of 100 mg/kg body weight had no any marked effect on severity of active anaphylactic shock and level of specific antibodies. The changes in cellular immunity under the influence of nanomaterial had similar direction in sensitized and non-sensitized animals and were more pronounced in the latter. Based on the discussion of the results, together with data from previous publications it was concluded that oral maximum level without observable adverse effect (NOAEL) of nanostructured SiO2 is located below 100 mg/kg body weight.

[Toxicological evaluation of nanosized colloidal silver, stabilized with polyvinylpyrrolidone. I. Characterization of nanomaterial, integral, hematological parameters, level of thiol compounds and liver cell apoptosis].

Nano-sized colloidal silver (NCS) is currently one of the most widely used nanomaterials in medicine and consumer's products. Nanoparticles (NPs) of silver, in addition to the direct exposition through products may expose human via various environmental objects. The aim of the study is to assess the safe doses of silver NP received orally. The investigated NCS contained silver NPs with diameter of 10­60 nm, predominantly with a nearly spherical form stabilized with polyvinylpyrrolidone (PVP). The experiment was performed during 92 days in 5 groups of male Wistar rats (n=15 in each group), receiving a balanced semisynthetic diet. Animal of group 1 (control) received vehicle (deionized water) intragastrically for 30 days and then with food, groups from 2nd to 4th ­ PVP and groups from 3rd to 5th NCS, in doses respectively, 0.1; 1.0 and 10 mg/kg body weight (b.w.) in terms of silver. The dose of PVP in groups from 2nd to 5th did not differ, amounting to 200 mg/kg b.w. During the experiment, the weight gain, skin condition, activity, stool, cognitive function were assessed. At the end of the feeding period weight of internal organs, intestinal wall permeability to protein macromolecules, liver thiols, standard values of blood erythrocytes, leukocytes and platelets, hepatocyte apoptosis by flow cytometry were studied. These results suggest that in terms of weight gain, lung relative mass, average erythrocyte volume, hemoglobin content and concentration in erythrocytes, the relative proportion of lymphocytes and neutrophils adverse changes have been observed at a dose of 10 mg NPs per kg of b.w. At lower levels of exposure (0.1 and 1.0 mg/kg b.w.) some specific changes were also observed (in terms of thiols pool in liver, cognitive function, relative abundance of monocytes, the number of dead hepatocytes), which, however, did not possess an unambiguous dependence on the dose. Possible mechanisms of the toxic action of the NCS have been discussed.

[Lead toxicity at its joint administration with nanostructured silica].

Nanoparticles (NPs) are present in the composition of the objects in the environment and foods together with contaminants of conventional nature, and may enhance their toxicity in certain conditions. The aim of this work was to study the effect of nanostructured silicon dioxide (SiO2), widely used as a food additive, on the accumulation and manifestation of the toxic effect of lead (Pb) when co-administered to rats in the subacute experiment. Rats were divided into 6 groups. Animals of the 1st group (control) received distilled water intragatrically by gavage. Rats of the 2nd and 3rd groups received dispersion of SiO2 NPs with primary particle size of 5-30 nm in the doses of 1 and 100 mg/kg body weight, respectively, of Group 4--lead acetate in the dose of 20 mg of lead/kg body weight, group 5 and 6--Pb acetate and SiO2 NPs dispersion in doses, respectively 1 and 100 mg/kg body weight, for 22 days by intragastric gavage. The weight of the body and organs, standard haematological parameters, serum protein, creatinine, uric acid, albumin, glucose level, and -alkiline phosphatase, ALT and AST activity, urinary 5-aminolevulinic acid (ALA) were determined. Hepatocyte apoptosis was studied by flow-cytometry. The content of Pb, cadmium (Cd), arsenic (As), selenium (Se), iron (Fe), zinc (Zn), copper (Cu), chromium (Cr), manganese (Mg), aluminum (Al) and cobalt (Co) in organs (liver, kidney, brain) was determined by ICP-MS. The result showed that the introduction of Pb, but not NPs resulted in a significant increase in the mass of the kidneys and testes. Urinary excretion of 5-ALA in animals from groups 4-6 significantly (p < 0.001) increased, compared with animals 1-3 groups, not influenced by SiO2 NPs administration (p > 0.05). Introduction of SiO2 NPs resulted in a dose-dependent increase in blood hemoglobin, the effect of lead on this index had the opposite direction. Marked changes were noticed in the levels of glucose and serum albumin in combined exposure of Pb and NPs. No additional adverse effect was noticed in Pb co-administration with NPs on haematological parameters and hepatocyte apoptosis. Pb accumulation was revealed in the liver, kidneys and brain in animals of groups 4-6, which was not enhanced by the combined effects of NPs (p > 0.05). The average biosubstrates content of Cd, As, Se, Zn, Cu, Al, Cr, Mn, Co was similar in animals treated and untreated with Pb, and demonstrated no dependence on NPs dose. The only alleged adverse impact on the trace element profile was almost twofold significant increase in the content of Al in the liver in animals treated with lead and SiO2 NPs in the highest dose (0.859 ± 0.085 mg/kg vs 0.468 ± 0.065 mg/kg in the 4th group, p < 0.05). In the absence of lead exposure, this effect was not observed. Thus, there wasn't obtained any unequivocal evidence of lead adverse effects potentiation in the case of a joint income with SiO2 NPs. Moreover, for some indicators, such as weight gain, serum AST activity, hemoglobin concentration, erythrocyte parameters, the introduction of the SiO2 NPs "abolished" to a certain extent some of the adverse effects of lead, which may be due to the effects of its enterosorption. Investigation of the influence of different types of NPs on the intestinal absorption and toxicity of various chemical contaminants should be continued.

[Lead toxicity in its joint administration with the aluminium oxide nanoparticles to rats].

In this paper we studied the effect of aluminum oxide (Al2O3) nanoparticles (NPs) on the accumulation and biomarkers of toxic action of lead (Pb) when co-administered to rats in subacute experiment. 36 Wistar rats with initial body weight 120-140 g were divided into 4 groups. Animal of group 1 (control group) were given distilled water by gavage. Rats in group 2 received Pb acetate solution in a dose of 20 mg/kg body weight (based on Pb), animal in the 3rd and 4th groups additionally to this received suspension of Al2O3 NPs in doses of 1 and 100 mg/kg body weight, respectively. The experiment lasted 22 days. Body and organ weight, standard haematologic parameters, protein, creatinine, uric acid level, aminotransferase activity (ALT and AST) in serum, urinary 5-aminolevulinic acid (5-ALA) concentration were measured. Apoptosis of hepatocytes was studied by flow cytometry. Pb content was determined by atomic absorption spectrometry. It has been shown that the administration of Al2O3 NPs together with Pb resulted in a significant dose-dependent increase in the relative weight of the kidneys (0.88 ± 0.03% and 0.94 ± 0.06% vs. 0.74 ± 0.02% and 0.85 ± 0.01% in control and group 1). The excretion of 5-ALA in the urine of animals treated with lead acetate significantly (p < 0.001) increased compared to the animals of group 1 (0.80 ± 0.08/µmol/l); while any dependence of this parameter on the dose of Al2O3 NPs was absent (p > 0.05) (group 2: 4.54 ± 0.56 µmol/l; group 3: 7.34 ± 1.35 µmol/l; group 4: 5.71 ± 1.74 µmol/l). The hemoglobin content was significantly reduced in animals of groups 2-4 (134.0 ± 2.9; 133.6 ± 1.8 and 129.9 ± 2.9 g/l) compared to the animals of the control group (144.6 ± 1.5 g/l), the dependence of this parameter on the dose of Al2O3 NPs was also absent. A marked and significant increase of the level of glucose has been noticed on the background of the Al2O3 and NPs (7.46 ± 0.49 u 8.24 ± 0.80 vs. 6.28 ± 0.34 mmol/l in group 2), and its level went beyond physiological norms in the 4th group and ANOVA indicated the influence of Al2O3 NPs administration. The toxic effects of lead on hematological parameters of blood on the background of the Al2O3 and NPs weren't weakened, and in the case of hematocrit even enhanced, as evidenced by one-way analysis of variance (p < 0.05). When administered together with the Al2O3 Pd accumulated in increased amounts in the liver [up to 1.96 ± 0.25 (group 3) and 2.16 ± 0.23 (group 4) vs. 1.17 ± 0.19 (group 2) mg/kg] (p < 0.05). Thus, Al2O3 NPs possibly presented as a contaminant in food can enhance the bioavailability of lead and some indices of its toxic action.

[Toxicological assessment of nanostructured silica. III. Microecological, hematological indices, state of cellular immunity].

This paper is the third in a series of publications on the experimental study of subacute oral toxicity of nanostructured silicon dioxide (SiO2). We used commercial nanostructured SiO2, obtained by hydrolysis of tetrachlorosilane in the gaseous phase, with the size of the primary nanoparticles (NPs) of 5-30 nm. The aqueous dispersion of SiO2 after treatment with ultrasound was administered to rats with initial weight of 80 +/- 5 g for the first 30 days by intragastric gavage and further for 60 days with diets in doses of 0.1; 1.0; 10 and 100 mg/kg body weight per day. Animals of the control group were treated with deionized water. The amount of basic and transient populations of gut microbiocenosis, hematological indexes were measured using standard methods. Specific content of the B-lymphocytes (CD45RA+), total T-lymphocytes (CD3+), T-helper cells (CD4+), T-cytotoxic cells (CD8+), NK-cells (CD161a+) in general population of lymphocytes was evaluated byflow cytometry; serum cytokine levels of TNF-alpha, IL-6, IL-10 were determined by ELISA. No significant changes in the qualitative and quantitative composition of the intestinal microbiota populations regardless of the dose of administered nanomaterial have been found. This gave reason to believe that the postulated mechanism of the toxic effects of the NPs of SiO2, mediated by modification of the composition of the intestinal microflora and the corresponding changes in its functional activity, apparently, is not realized. The main target of nanostructured SiO2 was the T-cellular system of the immune system of animals, that was manifested in the significant decrease of the number of leukocytes (33%), number of T-helper cells (13%), CD4/CD8 ratio (27%) and increasing the number of cytotoxic lymphocytes (19%) and the level of TNF-alpha (590%). The value of the maximum dose (NOAEL) of nanostructured SiO2, has no effect on T-cell immunity was not more than 100 mg/kg body weight per day.

[Development of the system for nanomaterials and nanotechnology safety in Russian Federation].

The article discloses a system of guidelines establishing unified order of detection, identification of nanoparticles and nanomaterials, estimation of their safety, sanitary regulation, control and supervision in environmental objects and production of nanoindustry, evaluation and management of risk produced by nanoparticles and nanomaterials. Recent development of nanosafety system in Russian Federation serves the aim of balanced achievement of unconditional ensuring welfare of present and future human population together with promotion of advanced technologies promising lot of useful applications.