[Influence of lactoferrin and enzymatiс hydrolysates of cow's and mare's milk proteins on anaphylactic sensitivity and cytokine profile of rats].

The development of innovative ingredients of specialized formula for children with intolerance to cow's milk proteins requires accounting the influence of the protein component on the allergic sensitivity. The aim of the research was to study the effect of lactoferrin (LF) from cow colostrum, cow's milk protein hydrolysate (CMPH) and mare's milk protein hydrolysate (MMPH) on the severity of the systemic anaphylaxis reaction, the levels of specific IgG antibodies and cytokines in rats parenterally sensitized with ovalbumin (OVA). Material and methods. The experiment was carried out on 4 groups of 26 male Wistar rats, which were sensitized intraperitoneally with chicken egg OVA and a systemic anaphylaxis reaction was induced on the day 29 by intravenous administration of a challenge dose of the antigen (6 mg per kg body weight). LF, CMPH and MMPH were introduced into the diet in doses of 1.4-2 g/kg body weight per day (on an average 1.59±0.04, 1.53±0.05 and 1.48±0.05 g per kg body weight respectively). The content of IgG antibodies in the blood serum was determined by an indirect ELISA; the levels of cytokines IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, GM-CSF, IFN-γ and TNF-α were detected by multiplex immunoassay. Results. Dietary LF did not have a significant effect on the severity of active anaphylactic shock (AAS), concentrations of antibodies and cytokines in the blood of animals. As a result of CMPH consumption, there were no significant changes in AAS severity and IgG antibodies to OVA but significant increase in TNF-α level was observed as well as a significant decrease in IL-1α (p<0.05). In animals receiving MMPH, there were also no significant changes in the severity of AAS, but a 1.9-fold decrease in the level of IgG antibodies to OVA (p<0.001) was noticed along with a significant increase in IL-12(p70) (p<0.05) and IL-10 (p<0.10) level. Conclusion. Cosumption of LF by sensitized rats didn't significantly affect their anaphylactic sensitivity and cytokine profile, while CMPH intake induced some signs of pro-inflammatory processes. Consumption of MMPH was accompanied by the formation of an anti-inflammatory cytokine profile, which corresponds to a decrease in the intensity of the humoral immune response to the model allergen. Differences in the effects of two hydrolisates, which have similar degrees of hydrolysis, may be associated with the specific composition of glycopeptides formed during the enzymatic cleavage of milk protein produced by these two species of dairy animals.

[Alternative protein sources: bacteria Methylococcus capsulatus concentrate, characteristic of composition and biological value].

A promising growth vector of food protein production in the context of the Russian Federation's food sovereignty security is the use of microbial synthesis. Taking into consideration the proven promising use of biotechnological processes in the production of alternative protein sources, modern scientific research is focused, among other issues, on improving the technology of obtaining food microbial protein using a variety of substrates and strains-producers, as well as evaluating the consumer properties, food, biological value and safety of such products. The purpose of the research was to study and comparatively evaluate protein concentrate (PC) from bacteria Methylococcus capsulatus and basic food of animal and plant origin within the development of the technology of optimal in nutritional and biological value PC production. Material and methods. Analysis of the nutritional and biological value of PC obtained from denucleinized and purified from cell walls biomass of methanoxidizing bacteria Methylococcus capsulatus (strain GSB-15) was carried out on 46 indicators, including estimation of protein content and amino acid composition, fat content and fatty acid composition, ash and moisture. Biological studies based on measuring of net protein ratio / net protein utilization were performed on 28 growing (between 25-50 days of life) male Wistar rats. Rats in the control group (n=14) received a semi-synthetic casein diet with a protein content of ~12% in calories, the test group (n=14) received a diet including an equivalent amount of PC protein. Body weight, feed intake, and fecal and urine nitrogen losses were measured during the experiment. The biological value and digestibility of protein were judged by coefficients of protein efficiency ratio, net protein ratio, true protein digestibility, true protein biological value, true net protein utilization. Results. The nutritional value study of PC showed high protein content - 69.0%, the share of fat, moisture and ash, accounted for 0.17, 9.5 and 14.4%, respectively. The carbohydrate content was 7.0% (of which mono- and disaccharides were <0.1%). The results of a comparative assessment of Methylococcus capsulatus protein amino acid profile and basic food of animal and plant origin showed a balanced content of the most amino acids, the level of which is comparable with the protein of chicken egg, which is traditionally a standard of quality of complete protein. At the same time, the content of the essential amino acid tryptophan in PC was an order of magnitude lower than in chicken egg protein; the content of this amino acid in PC is comparable with incomplete plant proteins (sunflower, flax, rapeseed). The results of the biological value evaluation of the Methylococcus capsulatus protein in the experiment on rats indicate a relatively low biological value of the microbial synthesis protein, that is caused, most likely, by tryptophan deficiency. Rats of the test group had a significant decrease in body weight gain, feed/protein intake, coefficient of protein efficiency ratio, coefficient of net protein ratio, true protein biological value, true net protein utilization. Conclusion. The results of a comparative evaluation of PC from methanotrophic bacteria Methylococcus capsulatus denucleinized biomass and basic food of animal and plant origin indicate its relatively high nutritional value. However, the characteristics of this PC sample were not optimal in regard of protein biological value by reason of tryptophan deficiency. A single amino acid deficiency is not a valid argument for not using microbially synthesized protein in human nutrition, considering the capabilities of the modern food industry, including ways to enrich foodstuffs with missing components. In addition, there is every cause to believe that adjusting the hydrolysis technology used in the production of PC will allow to eliminate the essential amino acid loss, thereby increasing the biological value of this product.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

[Nutritional and biological value of Hermetia illucens larvae biomass].

Recent years a worldwide interest in the use of alternative sources of protein, in particular, protein from insects, has increased. Edible insects for thousands of years have been a part of the human diet in Asian-Pacific region and South America, while in the European Union, the USA and Canada the use of insects for food purposes is a modern trend that is determined by the care of the environment, global warming combating, etc. Thus, the legal rules governing the food use of insects have significant differences among countries. In the Eurasian Economic Union requirements to food are regulated by the Customs Union Technical Regulations. Since none of the Technical Regulations contains the name of such food as "products obtained with the use of insects", these products may be classified as "food products of novel type" which are subjected to state registration. Fundamental and applied research should be conducted as a part of this novel food safety assessment system, that include the determination of nutritional and biological value of food raw materials derived from insects, and toxicological, reprotoxicological, allergological experiments in vivo on several generations of laboratory animals. The aim of the research was studying and comparing the nutritional and biological values of Hermetia illucens larvae dry biomass and basic foodstuffs of animal and plant origin. Material and methods. Nutritional and biological value analysis of H. illucens minced dry larvae biomass, dried at 110-120 °C, was carried out on 83 indicators, which included determination of protein content and amino acid composition, determination of fat level and fatty acid composition, determination of the content of carbohydrates, vitamins, minerals and trace elements, ash and moisture. Results. A study of the nutritional value of dry larvae biomass showed high levels of protein and fat (39 and 38%, respectively), while ash, dietary fiber and carbohydrate accounted for less than 20%. The amino acid profile had a balanced content of essential amino acids and was comparable to the protein of a hen's egg, as well as other animal products. The fatty acid composition of the biomass was characterized by a relatively high content of lauric acid (39.9% of the total fatty acid content), also found in some fruits and seeds of tropical plants; the ratio of other acids was more consistent with the fatty acid profile of fish oil. The dry larvae biomass contained carotenoids (0.23 mg/100 g), tocopherol (3.1 mg/100 g) and thiamine (53 µg/100 g) in amounts significantly lower to those of foods, which traditionally are sources of these vitamins. Based on the analysis of the mineral composition, the H. illucens biomass can be attributed to the sources of calcium, iron, copper and chromium. In terms of the content of the above elements, as well as magnesium and zinc, dry biomass significantly exceed the main food products of animal origin (beef, eggs, fish and seafood), and in terms of the content of potassium and phosphorus, it was comparable to them. Conclusion. The results of dry larvae biomass comparative evaluation with the basic foodstuffs of animal and plant origin evidence to its high nutritional and biological value, allowing to consider H. illucens as a promising source of complete protein, lauric acid, minerals and trace elements.

[Influence of the L-carnitine and resveratrol complex on physiological, biochemical and morphological indicators of normal and obese rats].

Specialized products and dietary supplements, enriched with complexes of minor biologically active substances (BAS), are often offered as components of therapeutic diets in the treatment of obesity and metabolic syndrome. At the same time, the possible effects of the interactions of BAS when consuming a multicomponent product have not been studied enough. The aim - to study the action on rats' organism of a complex supplement (RС), containing resveratrol (Res) and L-carnitine (L-Car), when consumed with a standard balanced or hypercaloric diet. Material and methods. Male Wistar rats received for 63 days a standard balanced diet (SD) or a high-fat-high-carbohydrate diet (HFCD) with an excess of total fat (30%) and fructose (20% solution instead of drinking water), or the same diets supplemented with RС in a low (25 mg/kg body weight as Res and 300 mg/kg body weight as L-Car) or high (50 and 600 mg/kg body weight, respectively) doses. The muscle grip strength, behavioral reactions in tests of the conditioned passive avoidance reflex (CPAR) and elevated plus maze (EPM) were studied. At the end of the experiment, the mass of adipose tissue and internal organs was determined together with the activity of microsomal and cytosolic liver enzymes for specific substrates, plasma biochemical parameters, liver morphology by lightoptical microscopy, accumulation of lipofuscin-like granules (LLG) in the liver and kidneys by laser confocal microscopy. Results. In the rats fed HFCD, compared with SD, there was an increase in the mass index of liver, total inguinal and retroperitoneal white adipose tissue, in the levels of glucose and triglycerides, in the activity of hepatic CYP1A1 and CYP3A monooxygenases, UDPglucuronosyltransferase, heme oxygenase, and simultaneous decrease of high and low density lipoprotein cholesterol, and quinone oxidoreductase activity. The RС intake stimulated the locomotor activity of rats in EPM, however, this effect was less pronounced against the background of HFCD consumption. In rats consuming SD (but not HFCD), the addition of RС caused an increase in search activity and anxiety according to the EPM and CPAR data. The effect on short- and long-term memory retention was statistically insignificant. RС intake did not have hypolipidemic and hypoglycemic properties but caused in low dose an increase in the ratio of the activity of transaminases AST/ALT in animals fed HFCD. The liver CYP3A activity increased in rats supplemented with RС in high dose fed HFCD. In the kidneys of animals, the consumption of RС resulted in increased accumulation of LLG. Conclusion. When studying the effect of the complex supplement RС on normal and obese rats according to the studied physiological, morphological and biochemical indexes, no positive effects were revealed, that would not have manifested themselves for Res and L-Car separate intake. No evidence of synergistic action of L-Car and Res were found, and some of the effects of the complex supplement can be considered as adverse. This requires careful assessment when combined using these substances in complex diet therapy of metabolic disorders in humans.

[Influence of the DAT gene knockout on exchange of essential and toxic trace elements in rats].

The maintenance of energy homeostasis of the body according to modern data is carried out with the active participation of dopaminergic neurons of the central nervous system. The synthesis and metabolism of dopamine (DA) occurs both in the brain and in peripheral tissues. Violation of the synthesis and metabolism of DA is considered as a link in the vicious cycle which it formed during the development of diet-induced obesity. According to modern data, a number of essential and toxic trace elements, such as Cd, Al, As, Mn, Fe, Cu, Zn, are actively involved in the exchange of DA in the brain and peripheral organs and tissues. One way to assess this relationship is to compare changes in the microelement status of the organism when consuming hypercaloric diets in animals with normal and impaired DA transport. The latter can be animals with a knockout of the DAT transporter gene, which performs DA reabsorption with subsequent storage in the composition of secretory granules. The aim is a comparative study of the content of a number of essential and toxic elements in the brain, liver, and kidneys of rats that differ in the allelic variants of the DAT gene fed balanced diet and the diet with an excess of energy value. Material and methods. The study was carried out on 30 male rats of the DAT-KO knockout line (homozygotes DAT-/- and heterozygotes DAT+/-), 8-10 weeks old, and 13 males rats of the outbred Wistar line (DAT+/+) of the same age. For 62 days the animals (6 groups) received a semi-synthetic diet containing essential elements in the salt mixture or a similar high-fat-highcarbohydrate diet (HFCD) with 30% fat and 20% fructose solution instead of drinking water. The content of 16 trace elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, V, Cs, Ag, Al, Cd, As, Pb, Ni) were determined by inductively coupled plasma mass spectrometry in the liver, kidneys, and brain of rats with a knockout of the dopamine DAT transporter gene: homozygotes (DAT -/-) and heterozygotes (DAT+/-), as well as wild-type rats (DAT+/+) of the Wistar strain. Results and discussion. In the liver, DAT knockout led to an increase in the content of As, Cd, Co, and Cs and a decrease in Fe; in the kidneys - to an increase in the levels of Pb, As, Cd and Se, in the brain - an increase in the content of most of the studied trace elements, including Pb, As, Cs, Al and Cu. Conclusion. Against the background of consumption of HFCD, the effect of DAT knockout on the content of a number of elements was more pronounced compared with the consumption of the control diet. The revealed changes in the trace element content in DAT knockout rats are considered in terms of the effect of DA metabolism in the central nervous system and in peripheral tissues on the status of trace elements.

[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.

[The content of essential and toxic microelements in the organs of mice of various lines receiving a high-carb high-fat diet and supplemented with quercetin].

Quercetin (Q) is known to be a powerful chelating agent for metal ions. Due to this property, Q, when ingested, is able to intervene actively in microelement homeostasis. The assessment of the possible significance of these effects for the dietary use of Q in obesity is complicated by the fact that in obese patients there are numerous changes in mineral metabolism and microelement homeostasis due to the pathogenesis of the disease. Thus, it is of considerable interest to identify the systemic effects of biologically active substances, including Q, on mineral metabolism in biological in vivo models (due to the limited choice of biosubstrates in clinical observations - blood plasma, urine, hair). The aim was to study the possible effects of Q on the levels of essential, non-essential and toxic elements in mice of three lines: db/db with knockout of the leptin receptor gene, prone to spontaneous development of obesity, inbred line C57Bl/6J, relatively resistant to the development of nutritional obesity, and complex hybrid of the 2nd generation DBCB, genetically more prone to developing obesity and fatty hepatosis when consuming diets with an excess quota of fat and simple carbohydrates. Material and methods. The content of 16 chemical elements (Fe, Mg, Cu, Mn, Co, Se, Zn, Cr, Al, Cd, As, Pb, Ni, Ag, V, Cs) was determined by mass spectrometry with inductively coupled plasma in the liver, kidneys, brain of genetically obese db/db mice (8-10 weeks old) receiving standard semisynthetic ration (SSD) and Q for 47 days at doses of 25 and 100 mg/kg body weight, C57Bl/6J mice treated with SPR or a high-fat high-carb diet (HFCD - 30% fats and 20% fructose solution instead of water) and Q supplementation at the same doses, DBCB tetrahybrid mice treated with SSD, HFCD and HFCD supplemented with Q for 63 days (25 mg/kg body weight). The compounds of Fe, Zn, Cu, Mn, Mg, Cr, Ni, Se, and V were included in the salt mixture in the form of inorganic salts or oxides in amounts close to physiological needs; Al, Cd, As, Pb, Ag, Cs, Co - were present in the diets in background amounts. Results and discussion. In db/db mice, in comparison with C57Bl/6J, a decreased content of Pb in the liver and increased in the kidneys and brain, decreased Co in the kidneys, increased Cs and As in the brain, which could not be explained by differences in the consumption of these elements with feed. The consumption of Q decreased the content of Mn, Cs, V, Ni, As in the liver in db/db mice and increased Cu and did not significantly affect the level of trace elements in the liver in animals C57Bl/6J. In kidneys of C57Bl/ 6J receiving Q, the content of As and Al increased. In brain of db/db mice, Q supplementation caused a decrease in the content of As, Pb, Cs and Se. In tetrahybrids DBCB, Q increased Pb levels in liver and brain and decreased in kidneys; increased the level of V in liver and brain; decreased As content in kidneys and increased in liver. Multiple correlations were noted between the organ content of elements in various valence forms, as well as between the content of trace elements and biochemical indicators of the intensity of catabolic and anabolic processes. Conclusion. Quercetin has an effect on the homeostasis of microelements, depending both on the animal genotype and on the diet, and not having unambiguous physiological significance. Indicators of the status of essential and toxic trace elements are recommended to be included in the protocols of preclinical trials of the efficacy and safety of minor biologically active food substances.

Effect of Vanadium Complex with Enzymatic Hydrolysate of Soy Protein on Carbohydrate and Lipid Metabolism Disorders in Male Wistar Rats.

The effects of vanadium complex with enzymatic hydrolysate of soy protein (V-EHSPI) were studied in male Wistar rats with induced disorders of carbohydrate and lipid metabolism. The content of vanadium (IV) in the studied complex was 15.8 mg/g dry product. High-lipid high-carbohydrate diet was used to induce disorders of lipid and carbohydrate metabolism. Addition of vanadium in three different doses to the diet over 100-day experiment reduced body weight gain and the levels of glucose, insulin, leptin, and triglycerides. V-EHSPI produced beneficial effects on carbohydrate and lipid metabolism even in a dose 5 µg/kg body weight/day (calculated from the mean food consumption over the entire treatment period). Significant inhibition of growth and changes in the weight of organs in animals treated with V-EHSPI attested to toxicity of vanadium in the studied dose range.

[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.

[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.

[Toxicological assessment of nanostructured silica. I. Integral indices, adducts of DNA, tissue thiols and apoptosis in liver].

Nanostructured amorphous silica (SiO2) is widely used in food additives, pharmaceuticals and cosmetics. Available data on the oral toxicity of this nanomaterial (NM) in vivo, obtained in acute and subacute experiments are contradictory. The purpose of this study is evaluation of some parameters of toxicity of nanostructured SiO2 when orally administered to rats for 3 months. We used commercial SiO2 preparation, obtained by gas-phase hydrolysis of tetrachlorosilane with a size of the primary nanoparticles close to 5-30 nm, which was characterized as NM by several independent methods. SiO2 in the form of sonicated aqueous dispersion was administered to male rats with initial weight of 80 +/- 4 g for the first 30 days by intragastric gavage and then for 62 days with consumed diets in daily dose of 0,1; 1,0; 10 and 100 mg/kg body weight. The control animals received vehicle--deionized water. Weight gain, relative mass of internal organs, intestinal permeability to protein macromolecules (determination of ovalbumin level in blood serum by solid-phase bivalent immunoassay), urinary excretion of oxidative degradation product of DNA 8-oxo-2-deoxyguanosine (8-oxo-G) (by reversed phase HPLC), the level of thiol compounds in liver (spectrophotometrically), liver cell apoptosis (flow cytometer), fixing efficiency of passive avoidance (CRPA) have been measured. It has been shown that three-month administration of nanostructured SiO2 in all doses resulted in animal body weight decrease by 10-15%; a significant increase in adrenal weight was noticed under doses of 1 and 10 mg/kg and urinary 8-oxo-G excretion was significantly reduced at the dose 10 mg/kg. At the maximum dose of NM, 100 mg/kg, after 2 months of administration the number of animals decreased that entered the dark compartment of the experimental setup at initial testing of CRPA. The rest of the studied indices did not experience any significant changes depending on the dose of NM. It is concluded that no toxic effect were expressed in indices studied under the influence of nanostructured SiO2 in rats at daily doses up to 100 mg per kg body weight for 3 months.