High-Carbohydrate Diets Affect Accumulation of Lipofuscin-Like Pigment in the Kidneys of Mice and Rats: Autofluorescence Confocal Microscopy Analysis.

The accumulation of lipofuscin-like granules in liver, kidneys, and spleen cells in mice and rats of different lines receiving 30% sugar solutions (fructose, glucose, their mixture, and sucrose) in addition to balanced semisynthetic diet for 62 or 122 days was studied by the method of laser scanning confocal microscopy. The granules were detected by their autofluorescence at maximum λex =570-600 nm and λex=488 nm. In the kidneys of rats receiving glucose and, especially, the mixture of glucose and fructose, significant accumulation of lipofuscin-like granules was found that was absent in the control group animals receiving water. Intensive accumulation of the granules was observed in the kidneys of all groups of mice receiving sugars (except for glucose). Lipofuscin-like granules were located in the cytoplasm of epithelial cells of the distal and proximal convoluted tubules. In the liver of rats and mice, the signs of accumulation of lipofuscin-like granules were absent or minimal. In rat spleen, lipofuscinlike granules were found in the red pulp in all groups, but their accumulation significantly increased in animals receiving the diet enriched with glucose and sucrose.

Comparative Analysis of JNK1 Expression in Liver Cells in Rats of Different Lines Receiving Excess of Easily Digested Carbohydrates: Confocal Microscopy.

The expression of JNK1 isoform of cJun-N-terminal kinase in hepatocytes of rats receiving excess of simple carbohydrates dissolved in drinking water was studied by immunohisto-chemical staining and confocal microscopy. In experiment I (60 days), the highest expression of JNK1 was observed in female Wistar rats receiving fructose (the difference with the group receiving a mixture of glucose and fructose was significant, p<0.05, the difference with the control group at the trend level, p=0.077; Mann-Whitney U test). In experiment II (120 days), an increase in JNK1 expression was observed in Wistar rats (males and females) receiving 30% fructose. In Dark Aguti rats (females and males) of comparable age, increased basal level of JNK1 expression was observed in comparison with Wistar rats. Three-way ANOVA showed that JNK1 expression was significantly (p<0.05) associated with consumption of fructose and animal line, but not sex. The level of JNK1 expression corresponded to previously identified changes in the biochemical markers of the metabolic syndrome.

Biochemical and Morphological Parameters of Inbred/Outbred Lines and DBCB Tetrahybrid Mouse in High-Sugar In Vivo Model of Metabolic Syndrome.

Integral, biochemical, and morphological parameters and concentrations of vitamins, particularly lipid soluble vitamins, were analyzed in female mice of inbred DBA/2J line, outbred ICR-1 (CD-1) line, and DBCB tetrahybrid mice on the in vivo model of metabolic syndrome induced by consumption of 30% sucrose for 2 days. In contrast to inbred and outbred lines, DBCB tetrahybrid mice developed abdominal obesity, hypercholesterolemia, and pronounced morphological picture of fatty liver disease. The lipid-coupled transport of vitamin E to the liver is also enhanced in these animals, which compensated decreased supply of vitamin E to the liver under conditions of high-sugar ration. The observed interstrain differences can be related to genetic features of the used mouse lines and DBCB tetrahybrid mice and require further genomic, transcriptomic, proteomic, and morphological studies. The results of the study based on the in vivo model of metabolic syndrome allow identifying the key biomarkers for complex diagnostics and prognosis of metabolic syndrome complications, such as nonalcoholic steatosis of the liver.

Effect of Silver Nanoparticles on Protein Composition of Rat Liver Microsomal Fraction.

We studied the effect of oral administration of metallic silver nanoparticles to rats on the proteome of the liver microsomal fraction. Nanoparticles (5-80 nm) were administered daily to growing Wistar male rats over 92 days. Controls received pure water. To control the effect of the carrier, the rats were administered aqueous solution of a stabilizer polyvinylpyrrolidone. The protein composition (proteome) of the liver microsomal fraction was analyzed by 2D-electrophoresis with identification of variable protein spots using the high-resolution nanoHPLC-MS/MS. Eight, 6, and 8 proteins absent in the control groups appeared in the microsomal fraction under the action of nanoparticles in doses of 0.1, 1, and 10 mg/kg body weight, among these, proteasome activator complex subunit 1 (Psme1 gene), and the heat shock protein HSP60 (Hspd1 gene) were reliably identified. The consumption of silver nanoparticles led to disappearance of protein of ß2a tubulin chain (Tuba1b gene) from the microsomal fraction. The expression of catalase, present in the proteome of the liver microsomal fraction in animals of all groups was significantly decreased after consumption of silver nanoparticles in doses of 0.1 and 10 mg/kg. The observed changes in the proteome are considered as manifestations of hepatotoxicity of silver nanoparticles and can be related to the antagonistic effect of silver on the status of the essential trace element selenium.

Interspecific Differences in Behavioral Responses and Neuromotorics between Laboratory Rodents Receiving Rations with Easily Digested Carbohydrates.

We assessed the effect of intake of easily digested carbohydrates for 133 days on quantitative parameters of neuromotorics and cognitive function in Wistar rats and C57Bl/6J mice. Neuromotorics (muscle tone) was assessed in rats and mice by the forelimb muscle force (grip strength) over 4 months. Anxiety was assessed in the elevated plus-maze test and cognitive function (short-term and long-term memory) was evaluated by conditioned passive avoidance response (CPAR) test over 3 months. The mice, in contrast to rats, receiving the diet with easily digested sugars demonstrated suppression of neuromotorics. Anxiety increased with age in female mice, but not in rats, irrespective of the diet. Cognitive function in rats receiving experimental rations did not change significantly in comparison with the control. In mice, consumption of equimolar mixture of fructose and glucose impared short-term, but not long-term memory, in comparison with the group receiving glucose alone. We revealed a small (by 14-17%), but statistically significant increase in the brain weight in mice receiving fructose and sucrose. The study demonstrates sufficient interspecies differences in the influence of carbohydrate rations on neuromotorics and behavioral responses in the in vivo metabolic syndrome model.

Detection of Gold Nanoparticles in Rat Organs by Transmission Electron Microscopy.

The effects of water-dispersed gold nanoparticles (8.0±0.9 nm in diameter) on the rat small intestinal mucosa and Peyer plaques, liver, and spleen were studied by electron microscopy. Water-dispersed gold nanoparticles injected into isolated intestinal loop not only accumulated in the small intestinal mucosa and Peyer plaques, but also penetrated into other organs, e.g. liver and spleen. Ultrastructural changes in the cells (hyperplasia of endoplasmic reticulum) were detected in the studied organs.

[Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles].

The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

Analysis of toxicity biomarkers of fullerene C60 nanoparticles by confocal fluorescent microscopy.

The methods of laser confocal microscopy were employed to study the changes in rat target organs (iliac mucosa and liver) provoked by peroral administration of dispersion of nanosized (31 nm) multimolecular fullerene C60 particles in doses of 0.1, 1.0, and 10 mg/kg body weight over 92 days. The micropreparations were selectively stained with fluorescent dyes to mark the cell nuclei (DAPI), actin microfilaments (fluorescently labeled phalloidin), and the membrane proteins CD106, CD31, and claudins in tight junctions (fluorescently labeled monoclonal antibodies). In rats treated with fullerene in the examined doses, the iliac mucosa demonstrated normal morphology of the villi. There were no signs of inflammation and no alterations in the actin fi laments of cytoskeleton and in enterocytic tight junctions. The count of CD106(+) and CD31(+) cells did not change. The highest examined doses of fullerene (1 and 10 mg/kg body weight) increased population and modified distribution of hepatic CD106(+) cells. They also resulted in accumulation of cytoplasmic granules presumably identified as Kupffer macrophages without any signs of visible inflammation or necrotic areas. This phenomenon can reflect the early stages of toxic reaction being a sensitive bioindicator of the damage produced by administered fullerene C60 in the hepatic tissue.

[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.

[Toxicological assessment of nanostructured silica. The acute oral toxicity].

Nanostructured amorphous silica (SiO2) is one of the priorities of nanomaterials, exposing human to the ever-increasing scale as a component of food additives, drugs and cosmetic products. According to numerous publications SiO2 nanoparticles (NPs) possess deleterious effects on animal and human cells in vitro and also exhibit inhalation toxicity. However, the biological effects in vivo of silica NPs taken orally are studied insufficiently. This article represents the first section of this study which aim is identification of silica preparation as nanomaterial and estimating of acute toxicity after oral administration in the form of aqueous suspension. Studies of size and shape of the particles in aqueous suspension of silica used in the study by electron and atomic force microscopy, spectroaqustic analysis and dynamic laser light scattering showed that the test substance is a nanomaterial. Estimation of acute toxicity of an aqueous suspension of nanostructured silica with a single intragastric gavage to male BALB/C mice allowed to conclude that the test material has LD50 by the oral route greater than 10 000 mg/kg and consequently belongs to class IV (low danger agents). Pathological changes in jejunum and colon of mice in the acute experiment (at a dose of 10 000 mg/kg) haven't been identified. Thus SiO2 NPs possess low toxicity when administered in the gastrointestinal tract. The available data, however, do not exclude possible presence of adverse effects under their long-term administration by oral way.

[Influence of titanium dioxide and silica nanoparticles on accumulation and toxicity of lead in experiments with intragastric co-administration].

The aim of this work was experimental verification of assumptions about the possibility of potentiation of accumulation and toxicity of lead (Pb) after its joint intragastric administration with nanoparticles (NPs) of titanium dioxide (TiO2) and silica (SiO2). Lead acetate was administered intragastrically to rats at a dose of 20 mg/kg body weight of lead over 21-23 days as a solution in water or in aqueous slurry of TiO2 or SiO2 NPs taken at 1 and 100 mg/kg body weight. The data was obtained that co-administration of Pb with NPs of SiO2 and TiO2 led to changes in a number of indicators that can be interpreted as a slight increase in the toxic effect of the tested substances. However, the size and direction of identified effects depended on the type and the dose of NPs of both kinds. In coadministration of Pb with NPs of TiO2 at both doses (rats with initial body mass 80 +/- 8 g) there was a decrease in hemoglobin concentration on 24% (p < 0.05), number of lymphocytes on 13% (p < 0.05), and platelets on 10% (p < 0.05) in the blood, together with the activation of apoptosis in hepatocytes. Introduction of Pb with SiO2 NPs (rats with initial body mass 140 +/- 4 g) contrary resulted in increased concentration of hemoglobin on 24% (p < 0.05) and significant decrease of urinary excretion of 5-aminolevulinic acid. Accumulation of Pb coadministered with TiO2 was not influenced in liver and decreased in spleen on 50% (p < 0.05), testis on 79% (p < 0.05) and brain on 38% (p < 0.05). SiO2 had no influence on these indices. It is concluded, that the hypothesis about Pb toxicity facilitation due to its transport across the intestinal wall in the form adsorbed on the NPs, does not receive experimental verification, and the observed effects were most likely due to both the toxicity of the Pb, and toxicity (in the studied doses) of NPs studied.

[Toxicological assessment of nanostructured silica. II. Enzymatic, biochemical indices, state of antioxidative defence].

Nanostructured silica (SiO2) "Aerosil" with the size of the primary nanoparticles (NPs) of 5-30 nm, in the form of ultrasound treated water suspension was administered to rats of 80 ± 4 g initial body weight for the first 30 days by intragastric gavage and then for 62 days with diets consumed in doses of 0.1; 1.0; 10 and 100 mg/kg body weight per day. The control group received vehicle of nanomaterial (NM)--deionized water. There were measured in liver of ani- mals the content of total cytochromes P450 and b5 in the microsomal fraction of liver, activity (Vmax) of microsomal monooxygenases with the mixed func- tion of isoforms CYP1A1, 1A2 and 2B1 on their specific substrates, the activity of conjugating liver enzymes glutathione-S-transferase and UDP-glucuronosyl-transferase in microsomal fraction and cytosol, the total and non sedimentable activity of lysosomal hydrolases (ß-glucuronidase, ß-galactozydase, arylsulphatase A, B). The content of PUFA's diene conjugates and TBA-reactive substances in the blood plasma and the activity of antioxidative enzymes (glutathionperoxidase, superoxidedismutase, glutathionreductase, katalase) in erytrocytes were estimated. A set of standard biochemical parameters of blood serum was also examined (total protein, albumin, glucose, creatinine, urea, uric acid, activities of hepatic transaminases). The studies revealed changes of a number of molecu lar markers that could be interpreted as unfavorable. These include isoforms of CYP2B1 activity decrease at a dose HM 1-10 mg/kg of body weight, decrease in the serum content of total protein, albumin and glucose levels in a dose range of 0.1-10 mg/kg. These changes were absent at the maximum dose of NM, which did not allow to clearly establish the dose-response. The remaining studied fig ures resided in the normal range or experienced changes that could not be interpreted as toxic.

[Bioaccumulation of silver and gold nanoparticles in organs and tissues of rats by neutron activation analysis].

Bioaccumulation of silver (Ag) and gold (Au) nanoparticles (NPs) with mean sizes of 6 nm and 35 nm, respectively, has been studied after their intragastric administration to rats at a dose of 100 µg/kg of body weight for 28 or 14 days. The organs and tissues (liver, kidney, spleen, heart, gonads brain, and blood) were subjected to thermal neutron activation, and, then, the activity of the 110mAg and 198Au isotopes generated was measured. The NPs of both metals were detected in all biological samples studied, the highest specific weight and content of Ag NP being found in the liver, and those of Au being found in kidneys of animals. The content of Ag NPs detected in the brain was 66.4 ± 5.6 ng (36 ng/g tissue), no more than 7% ofthese NPs being localized in the lumen of brain blood vessels. The content of Ag and Au NPs found in organs and tissues of rats could be regarded as nonhazardous (nontoxic) in accordance with the known literature data.

[About the human health safety estimation of ractopamine intake together with the food].

The analysis of scientific data including American and European scientific communities concerning use of ractopamine as a growth factor in food animal production and the argumentation of the maximum permitted levels of ractopamine and levels of ractopamine in meat and byproducts (offal) is carried out. The position of the Russian side stated at the Codex Alimentarius commission 35th session that acceptable ractopamine daily intake is insufficiently validated and cannot be used for the determination of maximum permitted levels of ractopamine in meat and byproducts (offal) is confirmed. It is represented that residual ractopamine intake together with food on the levels which are recommended by the Codex Alimentarius commission and by taking into account the levels of animal products consumption in Russian Federation will lead to unacceptable human health risk level that will promote increasing heart diseases and life expectancy reduction. In this connection Russia states against of acceptance of maximum permitted levels of ractopamine in food.

[Comparative analysis of modern approaches to risk estimation from artificially created nanoparticles and nanomaterials].

The article represents a review of modern approaches to problem of nanotechnologies and nanomaterials risk estimation for human health and environment that were elaborated in EU, USA and some international authorities. Submitted data suggests that there is a significant coincidence with approaches being developed and introduced in Russian Federation under the guidance of Rospotrebnadzor. Particularly criteria being used in Russian Federation and EU for classification of nanotechnologies and nanoindustry production are mainly similar. They include a) identification of nanomaterials in production, b) establishment of production disintegration possibility with concomitant migration of free nanoparticles, c) possibility of nanoparticles emission/migration both in normal conditions of utilization and in possible emergency conditions, d) degree of proximity of particular kind of production to it's consumer that means the possibility of exposition being from closely to zero (in conditions of unhabitated exploitation) up to approximately 100% (in case of medicine, food and cosmetics), e) biological examination of potential danger of nanomaterials according to current volume of scientific information. As applied to nanotechnology plants there are such criteria in use as a) nanomaterial identification, b) personnel exhibiting possibility, c) potential toxicity of stuff in aerosol nano-form, d) characteristics of biological action. Thus applied in Russia principles for nanomaterials safety estimation do not contradict to concepts of foreign authorities that builds up a possibility of said approaches harmonization to internationally recognized norms.

[Toxicological and sanitary characterization of bentonite nanoclay].

Intragastric administration of nanoclay to rats during 28 days led to reductions in the relative weight of the liver, the activity of its conjugating enzymes, the antagonistic activity of bifidoflora, and the hyperproduction of colonic yeast microflora. The findings lead to the conclusion that nanoclays that may be present in foods must be the object of sanitary regulation.

[Control of nanoparticles in food and biological objects. Report 2. Filtration, centrifugation, spectral methods and electrophoresis].

The large number of the analysis methods of engineered nanoparticles and nanoobjects as a part of disperse systems on the basis of principles of a membrane filtration (micro, ultra- and a nanofiltration) ultracentrifugation, spectral methods, including dynamic and static laser light scattering, Raman light scattering, low-angle X-ray scattering, x-ray techniques, laser decomposition spectroscopy, and other methods are developed. Mass spectrometry with inductively coupled plasma can be successfully used in studying of nanomaterials chemical composition in conditions when there is additional independent information on presence of analyzed substance in a nanoscale form. Methods of electrophoresis in a supportive environment and capillary electrophoresis are beginning to be successfully applied in the study of artificial nanomaterials. However, in terms of the identification of engineered nanoparticles and nanoobjects in complex, multicomponent, heterophase systems, that the objects of the environment and, in particular, food products are, all these methods currently can't compete transmission electron microscopy and atomic force microscopy, specified for purpose of certain particular applications, features of which been described in a previous eport in detail.

[Methods of nanoparticles control in food and biological objects. Report 1. Use of microscopic and chromatography investigation methods].

One of the promising applications of modern nanotechnology are food productions, which includes the improvement of food packaging, creation of new forms of nutrients that are characterized by improved assimilation and technological characteristics, quality control through the creation of compact and cheap test kits. All these applications of nanomaterials related to the risks of the possibility of receipt of potentially toxic nanoparticles in the diet. The task of regulation and hygienic standardization requires developing of the methods, their qualitative and quantitative analysis for such complex, multicomponent systems, which are the agricultural commodities and food products. The best hope in this plan are assigned to a group of approaches related to the microscopic visualization of artificial nanoparticles in the biological objects. While the typical size of nanoparticles (<100 nm) are below the theoretical maximum-resolution light optical methods, transmission electron microscopy often allows not only to identify nanoparticles on their size and shape, but also a qualitative and quantitative analysis their chemical composition with the use of additional analytical options. Another group of elaborate methods used in solving the problems of qualitative and quantitative analysis of nanoparticles are chromatographic methods, in particular, the exclusion, hydrodynamic, high-performance liquid chromatography, and the flow-field fractionation. Limitation of chromatographic approaches related with the need of complex sample preparation, as well as specific difficulties in nanoparticles detecting in chromatographic fractions. Transmission electron microscopy and high-performance liquid chromatography methods are officially recommended in Russia for the analysis of artificial nanoparticles in natural biological systems, including food products.

[Effect of titanium dioxide nanoparticles on protein expression profiles in rat liver microsomes].

There was studied an influence of intragastric administration of titanium dioxide (anatase form) nanoparticles (NP) on protein expression profiles in rat's liver microsomes by methods of proteomics. Animals received water suspension of NP in doses from 0,1 to 10 mg per kg body weight intragastrically daily during 28 days. Microsomes were isolated from liver by means of preparative ultracentrifugation. Proteins composition was studied by 2D-electrophoresis in acrylamide gel. Protein spots were identified by MALDI-TOF analysis. The results demonstrated appearance of 53 new protein spots and disappearance of 19 spots in animals subjected to NP irrespective of their dose. In addition 25 new spots appeared and 3 disappeared in higher doses of NP when compared to low dose group and control animals. Mass-spectrum analysis showed presence of few polypeptides registered in international database among proteins expressed under influence of NP. One of this dominant expressed proteins corresponded to enzyme glutathione transpherase Mu 2 isoform (M=41,55 kD, pI=8,0). The conclusion was made of well advances of proteomic analysis in artificial NP influences on biosynthetic processes estimation.

[Characterization of silver nanoparticles migration from package materials destined for contact with foods].

There was studied migration of artificial silver nanoparticles from polyethylene films destined for package of such foods as bread and poultry into model media reproducing physico-chemical properties and composition of said products. Modification of films was performed by 5- or 10-fold spraying of silver nanoparticles on the surface of package material. Model media were composed from water, alcohol and plant oil according to US FDA and Russian Federal Service for Surveillance of Consumer Rights Protection and Human Well-Being official recommendation. Nanoparticles were detected in model media by means of transmission electron and atomic force microscopy. Quantification of silver in nanoparticles migrating from films was performed by mass-spectrometry with inductively coupled plasma. The results obtained showed that silver migrated from films into test media in form of nanoparticles with mean diameter close to 10-20 nm. Migrated particles were partially aggregated to complexes with dimension about 50 nm with degree of aggregation depending on media composition. Quantification showed that amounts of silver nanoparticles migrating in foods did not exceed save level of this nanomaterial consumption even in aggravated conditions when almost all volume of product was consumed in form packaged in films modified with nanosilver.