[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.

[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 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.

[Effect of vitamin sufficiency on adaptation syndrome in growing rats].

The influence of vitamin supply of growing male -Wistar rats (n=21) with an initial body weight 53,5±0,9 g on their resistance to a single distress induced by the electric shock has been investigated. Control rats within 21 days received a complete semisynthetic diet,providingadequate amounts of vitamins. Combined vitamin deficiency in experimental rats was caused by 5-fold decrease of vitamin mixture amount in the feed and the total vitamin E exclusion from the mixture. On the 21st day, one day before the end of the experiment, both groups of rats were subjected to stress impact (electrocutaneous irritation on paws, 0,4 mA for 8 sec) and then animals were placed in metabolic cages to collect urine. By the end of the experiment, the animals with the combined vitamin deficiency lag behind in growth. Vitamin B2, A, B1 and E liver content decreased in experimental rats by 1,6, 2,3, 4,4 and 15 fold accordingly. Retinol plasma concentration was significantly reduced by 18%, α-tocopherol level - by 5 fold, urinary excretionof riboflavin and 4-pyridoxic acid (vitamin B6 metabolite) was significantly reduced by 6,5 and 2,46 times accordingly. MDA blood plasma concentration and the urinary ratio of oxidized and not oxidized form of 8-hydroxy-2'-deoxy-guanosine did not differ in both groups of rats. Urinary excretion of stress biomarker corticosterone in rats with combined vitamin deficit was 2,5-fold higher than in control rats. Thus, reducing of vitamins supply resulted in an increase of urine corticosterone in stressed rats, that characterized the intensity of general adaptation syndrome. This fact shows the importance of optimal sufficiency with vitamins in nonspecific (general) resistance to stress.

[Studies of effects of aluminum oxide nanoparticles after intragastric administration].

Growing Wistar rats received intragastrically nanoparticles (NPs) of aluminum oxide (Al2O3) daily during 28 days at doses of 1 or 100 mg per kg body mass. There were studied body mass of animals, relative mass of internals, rate of protein macromolecules absorption in the gut, oxidative damage of DNA, pool of tissue thiols, activity of hepatic enzymes of xenobiotic detoxication system, biochemical and hematological blood indices, stability of lysosome membranes, condition of antioxidant defense system, apoptosis of hepatocytes. Conducted experiments didn't reveal any marked toxic action of Al2O3 NPs on rats after 28 days of administration both in high and low dose. Among effects probably related to NPs influence on animals there were lowering of relative liver and lung masses, decrease of hepatic thiol pool, activity of CYP1A1 isoform in liver and glutathione reductase in erythrocytes, increase of diene conjugates of fatty acids in blood plasma. Said shifts were small in magnitude, didn't come out of margins of physiological norm and didn't show any distinct relation to NPs dose. However considering great importance of this nanomaterial as probable environmental contaminant the studies of it's toxicity must be continued in conditions of low doses (less than 1 mg per kg body mass) for long period of time.

[Characterization of fullerene C60 peroral toxicity to rats in the 92-day experiment].

Toxicity of fullerene C60 in male Wistar rats under conditions of a daily intragastric administration at doses of 0.1; 1 and 10 mg/kg body weight for 92 days was studied. Integral, biochemical, physiological, hematological, immunological indicators were determined that characterized the body condition of animals together with fullerene C60 biodistribution in organs and tissues of rats. Majority of the results didn't show any influence of fullerene C60 on animals treated with it in the whole range of doses studied. On the other hand, some of the data not related to the action of the components used in carrier solution, demonstrated dose-dependent variation: found increased activity on 21-35% in the CYP2B1 at doses of 1 mg/kg and 10 mg/kg body weight, and reduced concentration of uric acid and increasing concentrations of urea at 10 mg/kg body weight. Nevertheless, these changes were within the physiological range of variability. However, it is worth noting an important significant (p = 0.02) increase of the absorption for antigenic proteins in the digestive tract in animals treated with fullerene C60 at a dose of 10 mg/kg, suggesting a marked effect on the intestinal wall.

[Toxicological sanitary characterization of silver nanoparticles introduced in gastrointestinal tract of rats].

Water suspensions of silver nanoparticles were introduced intragastrically to growing male Wistar rats daily for 28 days. There were studied animals mass gain, relative masses of viscera, intestinal barrier resistance against protein macromolecules, DNA oxidative damage, tissue non-protein thiol levels, first and second phase of xenobiotic detoxication system activity, lysosomal membranes stability in liver together with routine blood biochemical and hematological indices and caecal's microbiocenose state. The data testifying to possible toxic risks, connected with reaction of silver's nanoparticles is obtained.

[Toxicological and hygiene characterization of titanium dioxide nanoparticles introduced to the gastrointestinal tract of rats. Communication 1. Integral, biochemical and hematologic indices, small intestinal macromolecule absorption, DNA damage].

Water suspensions of titanium dioxide nanoparticles were introduced intragastrically to growing male Wistar rats daily for 28 days. The data obtained revealed the presence of toxic risks related to said nanomaterials action and demonstrate the necessity of sanitary regulation of titanium dioxide nanoparticles used in food contact materials, cosmetics and other commodity.

[Application of immunochemical and chromatographic techniques for monitoring of trichothecene mycotoxins type A in grains].

A sensitive HPLC-MS technique after immunoaffinity clean-up has been developed for the simultaneous determination of T-2 and HT-2 toxins of in the food grains. The limit of detection of method was 0,002 mg/kg for T-2 toxin and 0,005 mg/kg for HT-2 toxin. Comparison of existing immunochemical and chromatographic techniques of trichothecenes type A determination in grains was made. New combined scheme of monitoring of grain contamination by trichothecenes type A was proposed. It was based on screening by ELISAs followed by confirmation of positive samples by developed HPLC-MS technique with preliminary immune affinity purification of extracts. Proposed modification of immunochemical and chromatographic techniques allowed to lower the influence of grain matrix on the analytical results and to reduce of the probability obtaining of false-positive determination. Developed approach was used for determination of trichothecenes type A in natural contaminated grain samples.

[Fusariotoxins in the food production in the Russian Federation: situation 2006-2008 years].

The results of monitoring of the fusarium mycotoxins (deoxynivalenole (DON), zearalenone (ZL), T-2 and HT-2 toxins, fumonisins B1 and B2) contents in the food wheat, barley, rye, oats and corn (2006-2008 crops) in different regions of Russia were presented. DON occurrence in the food wheat, rye and barley were 9, 4 and 0,8% accordingly. DON wasn't detected in the food oats and maize in these years. ZL occurrence was relatively high and varied between 4 and 40%. The estimated daily intake of DON per 1 kg of body weight in Russia on average varied from 0,066 to 0,096 mg. The daily intake of DON in the North-Caucasian region was twice higher than in Russia on average and it didn't exceed tolerable daily intake (TDI) of DON - 1 mg/kg of body. HPLC-MC analyses results indicated that 14 and 16% of all investigated grain samples were contaminated by T-2 and HT-2 toxins accordingly. T-2 toxin content in grains was lower then MTL (0,1 mg/kg). High frequency and levels of fumonisins contamination were detected in the food corn.