Analysis of Experimental Data on Changes in Various Structures and Functions of the Rat Brain following Intranasal Administration of Fe2O3 Nanoparticles.

Particulate matter, including iron nanoparticles, is one of the constituents of ambient air pollution. We assessed the effect of iron oxide (Fe2O3) nanoparticles on the structure and function of the brain of rats. Electron microscopy showed Fe2O3 nanoparticles in the tissues of olfactory bulbs but not in the basal ganglia of the brain after their subchronic intranasal administration. We observed an increase in the number of axons with damaged myelin sheaths and in the proportion of pathologically altered mitochondria in the brains of the exposed animals against the background of almost stable blood parameters. We conclude that the central nervous system can be a target for toxicity of low-dose exposure to Fe2O3 nanoparticles.

Comparative Evaluation of the Cytotoxic Effects of Metal Oxide and Metalloid Oxide Nanoparticles: An Experimental Study.

Industrial production generates aerosols of complex composition, including an ultrafine fraction. This is typical for mining and metallurgical industries, welding processes, and the production and recycling of electronics, batteries, etc. Since nano-sized particles are the most dangerous component of inhaled air, in this study we aimed to establish the impact of the chemical nature and dose of nanoparticles on their cytotoxicity. Suspensions of CuO, PbO, CdO, Fe2O3, NiO, SiO2, Mn3O4, and SeO nanoparticles were obtained by laser ablation. The experiments were conducted on outbred female albino rats. We carried out four series of a single intratracheal instillation of nanoparticles of different chemical natures at doses ranging from 0.2 to 0.5 mg per animal. Bronchoalveolar lavage was taken 24 h after the injection to assess its cytological and biochemical parameters. At a dose of 0.5 mg per animal, cytotoxicity in the series of nanoparticles changed as follows (in decreasing order): CuO NPs > PbO NPs > CdO NPs > NiO NPs > SiO2 NPs > Fe2O3 NPs. At a lower dose of 0.25 mg per animal, we observed a different pattern of cytotoxicity of the element oxides under study: NiO NPs > Mn3O4 NPs > CuO NPs > SeO NPs. We established that the cytotoxicity increased non-linearly with the increase in the dose of nanoparticles of the same chemical element (from 0 to 0.5 mg per animal). An increase in the levels of intracellular enzymes (amylase, AST, ALT, LDH) in the supernatant of the bronchoalveolar lavage fluid indicated a cytotoxic effect of nanoparticles. Thus, alterations in the cytological parameters of the bronchoalveolar lavage and the biochemical characteristics of the supernatant can be used to predict the danger of new nanomaterials based on their comparative assessment with the available tested samples of nanoparticles.

Experimental Testing of an Approach to Establishing Combined Toxicity of Ternary Nanoparticle Mixtures.

Our studies of exposure to binary mixtures of nanoparticles (TiO2 + SiO2; TiO2 + Al2O3 and SiO2 + Al2O3) based on mathematical modelling show that their combined subchronic toxicity can either be of an additive type or deviate from it depending on the outcome, dose ratio, and levels of effect. To characterize the type of toxicity of ternary mixtures of nanoparticles, we successfully tested a previously developed approach for assessing the combined toxicity of metal ions. In this approach, the effects are classified by a null, positive, or negative change in the toxicity of binary nanoparticle mixtures when modeled against the toxicity of the third agent added.

Changes in the Cardiotoxic Effects of Lead Intoxication in Rats Induced by Muscular Exercise.

Exposure to lead is associated with an increased risk of cardiovascular diseases. Outbred white male rats were injected with lead acetate intraperitoneally three times a week and/or were forced to run at a speed of 25 m/min for 10 min 5 days a week. We performed noninvasive recording of arterial pressure, electrocardiogram and breathing parameters, and assessed some biochemical characteristics. Electrophoresis in polyacrylamide gel was used to determine the ratio of myosin heavy chains. An in vitro motility assay was employed to measure the sliding velocity of regulated thin filaments on myosin. Isolated multicellular preparations of the right ventricle myocardium were used to study contractility in isometric and physiological modes of contraction. Exercise under lead intoxication normalized the level of calcium and activity of the angiotensin-converting enzyme in the blood serum, normalized the isoelectric line voltage and T-wave amplitude on the electrocardiogram, increased the level of creatine kinase-MB and reduced the inspiratory rate. Additionally, the maximum sliding velocity and the myosin heavy chain ratio were partly normalized. The effect of exercise under lead intoxication on myocardial contractility was found to be variable. In toto, muscular loading was found to attenuate the effects of lead intoxication, as judged by the indicators of the cardiovascular system.

Looking for the LOAEL or NOAEL Concentration of Nickel-Oxide Nanoparticles in a Long-Term Inhalation Exposure of Rats.

Rats were exposed to nickel oxide nano-aerosol at a concentration of 2.4 ± 0.4 µg/m3 in a "nose only" inhalation setup for 4 h at a time, 5 times a week, during an overall period of 2 weeks to 6 months. Based on the majority of the effects assessed, this kind of exposure may be considered as close to LOAEL (lowest observed adverse effect level), or even to NOAEL (no observed adverse effect level). At the same time, the experiment revealed genotoxic and allergic effects as early as in the first weeks of exposure, suggesting that these effects may have no threshold at all.

Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles.

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO-NP and PbO-NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling ß-MHC. The type of CdO-NP + PbO-NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb-NP and CdO-NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

Manifestation of Systemic Toxicity in Rats after a Short-Time Inhalation of Lead Oxide Nanoparticles.

Outbred female rats were exposed to inhalation of lead oxide nanoparticle aerosol produced right then and there at a concentration of 1.30 ± 0.10 mg/m3 during 5 days for 4 h a day in a nose-only setup. A control group of rats were sham-exposed in parallel under similar conditions. Even this short-time exposure of a relatively low level was associated with nanoparticles retention demonstrable by transmission electron microscopy in the lungs and the olfactory brain. Some impairments were found in the organism's status in the exposed group, some of which might be considered lead-specific toxicological outcomes (in particular, increase in reticulocytes proportion, in δ-aminolevulinic acid (δ-ALA) urine excretion, and the arterial hypertension's development).

Toxic Effects of Low-Level Long-Term Inhalation Exposures of Rats to Nickel Oxide Nanoparticles.

Rats were exposed to nickel oxide nanoparticles (NiO-NP) inhalation at 0.23 ± 0.01 mg/m³ for 4 h a day 5 times a week for up to 10 months. The rat organism responded to this impact with changes in cytological and some biochemical characteristics of the bronchoalveolar lavage fluid along with a paradoxically little pronounced pulmonary pathology associated with a rather low chronic retention of nanoparticles in the lungs. There were various manifestations of systemic toxicity, including damage to the liver and kidneys; a likely allergic syndrome as indicated by some cytological signs; transient stimulation of erythropoiesis; and penetration of nickel into the brain from the nasal mucous membrane along the olfactory pathway. Against a picture of mild to moderate chronic toxicity of nickel, its in vivo genotoxic effect assessed by the degree of DNA fragmentation in nucleated blood cells (the RAPD test) was pronounced, tending to increasing with the length of the exposure period. When rats were given orally, in parallel with the toxic exposure, a set of innocuous substances with differing mechanisms of expected bioprotective action, the genotoxic effect of NiO-NPs was found to be substantially attenuated.

Combined Subchronic Toxicity of Aluminum (III), Titanium (IV) and Silicon (IV) Oxide Nanoparticles and Its Alleviation with a Complex of Bioprotectors.

Stable suspensions of metal/metalloid oxide nanoparticles (MeO-NPs) obtained by laser ablation of 99.99% pure elemental aluminum, titanium or silicon under a layer of deionized water were used separately, or in three binary combinations, or in a ternary combination to induce subchronic intoxications in rats. To this end, the MeO-NPs were repeatedly injected intraperitoneally (i.p.) 18 times during 6 weeks before measuring a large number of functional, biochemical, morphological and cytological indices for the organism's status. In many respects, the Al2O3-NP was found to be the most toxic species alone and the most dangerous component of the combinations studied. Mathematical modeling with the help of the Response Surface Methodology showed that, as well as in the case of any other binary toxic combinations previously investigated by us, the organism's response to a simultaneous exposure to any two of the MeO-NP species under study was characterized by a complex interaction between all possible types of combined toxicity (additivity, subadditivity or superadditivity of unidirectional action and different variants of opposite effects) depending on which outcome this type was estimated for and on effect and dose levels. With any third MeO-NP species acting in the background, the type of combined toxicity displayed by the other two remained virtually the same or changed significantly, becoming either more or less unfavorable. Various harmful effects produced by the (Al2O3-NP + TiO2-NP + SiO2-NP)-combination, including its genotoxicity, were substantially attenuated by giving the rats per os during the entire exposure period a complex of innocuous bioactive substances expected to increase the organism's antitoxic resistance.

Attenuation of Combined Nickel(II) Oxide and Manganese(II, III) Oxide Nanoparticles' Adverse Effects with a Complex of Bioprotectors.

Stable suspensions of NiO and Mn3O4 nanoparticles (NPs) with a mean (±s.d.) diameter of 16.7±8.2 and 18.4±5.4 nm, respectively, purposefully prepared by laser ablation of 99.99% pure nickel or manganese in de-ionized water, were repeatedly injected intraperitoneally (IP) to rats at a dose of 2.5 mg/kg 3 times a week up to 18 injections, either alone or in combination. A group of rats was injected with this combination with the background oral administration of a "bio-protective complex" (BPC) comprising pectin, vitamins A, C, E, glutamate, glycine, N-acetylcysteine, selenium, iodide and omega-3 PUFA, this composition having been chosen based on mechanistic considerations and previous experience. After the termination of injections, many functional and biochemical indices and histopathological features (with morphometric assessment) of the liver, spleen, kidneys and brain were evaluated for signs of toxicity. The Ni and Mn content of these organs was measured with the help of the atomic emission and electron paramagnetic resonance spectroscopies. We obtained blood leukocytes for performing the RAPD (Random Amplified Polymorphic DNA) test. Although both metallic NPs proved adversely bio-active in many respects considered in this study, Mn3O4-NPs were somewhat more noxious than NiO-NPs as concerns most of the non-specific toxicity manifestations and they induced more marked damage to neurons in the striatum and the hippocampus, which may be considered an experimental correlate of the manganese-induced Parkinsonism. The comparative solubility of the Mn3O4-NPs and NiO-NPs in a biological medium is discussed as one of the factors underlying the difference in their toxicokinetics and toxicities. The BPC has attenuated both the organ-systemic toxicity and the genotoxicity of Mn3O4-NPs in combination with NiO-NPs.

Toxicodynamic and toxicokinetic descriptors of combined chromium (VI) and nickel toxicity.

After repeated intraperitoneal injections of nickel and chromium (VI) salts to rats, we found, and confirmed by mathematical modeling, that their combined subchronic toxicity can either be of additive type or depart from it (predominantly toward subadditivity) depending on the effect assessed. Against the background of moderate systemic toxicity, the combination under study proved to possess a marked additive genotoxicity assessed by means of the random amplification of polymorphic DNA test. We also demonstrated that chromium and nickel reciprocally influenced the retention of these metals in some organs (especially in the spleen) but not their urinary excretion in this study.

Some characteristics of free cell population in the airways of rats after intratracheal instillation of copper-containing nano-scale particles.

We used stable water suspensions of copper oxide particles with mean diameter 20 nm and of particles containing copper oxide and element copper with mean diameter 340 nm to assess the pulmonary phagocytosis response of rats to a single intratracheal instillation of these suspensions using optical, transmission electron, and semi-contact atomic force microscopy and biochemical indices measured in the bronchoalveolar lavage fluid. Although both nano and submicron ultrafine particles were adversely bioactive, the former were found to be more toxic for lungs as compared with the latter while evoking more pronounced defense recruitment of alveolar macrophages and especially of neutrophil leukocytes and more active phagocytosis. Based on our results and literature data, we consider both copper solubilization and direct contact with cellular organelles (mainly, mitochondria) of persistent particles internalized by phagocytes as probable mechanisms of their cytotoxicity.

Subchronic toxicity of copper oxide nanoparticles and its attenuation with the help of a combination of bioprotectors.

In the copper metallurgy workplace air is polluted with condensation aerosols, which a significant fraction of is presented by copper oxide particles<100 nm. In the scientific literature, there is a lack of their in vivo toxicity characterization and virtually no attempts of enhancing organism's resistance to their impact. A stable suspension of copper oxide particles with mean (±SD) diameter 20±10 nm was prepared by laser ablation of pure copper in water. It was being injected intraperitoneally to rats at a dose of 10 mg/kg (0.5 mg per mL of deionized water) three times a week up to 19 injections. In parallel, another group of rats was so injected with the same suspension against the background of oral administration of a "bio-protective complex" (BPC) comprising pectin, a multivitamin-multimineral preparation, some amino acids and fish oil rich in ω-3 PUFA. After the termination of injections, many functional and biochemical indices for the organism's status, as well as pathological changes of liver, spleen, kidneys, and brain microscopic structure were evaluated for signs of toxicity. In the same organs we have measured accumulation of copper while their cells were used for performing the Random Amplification of Polymorphic DNA (RAPD) test for DNA fragmentation. The same features were assessed in control rats infected intraperitoneally with water with or without administration of the BPC. The copper oxide nanoparticles proved adversely bio-active in all respects considered in this study, their active in vivo solubilization in biological fluids playing presumably an important role in both toxicokinetics and toxicodynamics. The BPC proposed and tested by us attenuated systemic and target organs toxicity, as well as genotoxicity of this substance. Judging by experimental data obtained in this investigation, occupational exposures to nano-scale copper oxide particles can present a significant health risk while the further search for its management with the help of innocuous bioprotectors seems to be justified.

Comparative in vivo assessment of some adverse bioeffects of equidimensional gold and silver nanoparticles and the attenuation of nanosilver's effects with a complex of innocuous bioprotectors.

Stable suspensions of nanogold (NG) and nanosilver (NS) with mean particle diameter 50 and 49 nm, respectively, were prepared by laser ablation of metals in water. To assess rat's pulmonary phagocytosis response to a single intratracheal instillation of these suspensions, we used optical, transmission electron, and semi-contact atomic force microscopy. NG and NS were also repeatedly injected intraperitoneally into rats at a dose of 10 mg/kg (0.5 mg per mL of deionized water) three times a week, up to 20 injections. A group of rats was thus injected with NS after oral administration of a "bioprotective complex" (BPC) comprised of pectin, multivitamins, some amino acids, calcium, selenium, and omega-3 PUFA. After the termination of the injections, many functional and biochemical indices and histopathological features of the spleen, kidneys and liver were evaluated for signs of toxicity, and accumulation of NG or NS in these organs was measured. From the same rats, we obtained cell suspensions of different tissues for performing the RAPD test. It was demonstrated that, although both nanometals were adversely bioactive in all respects considered in this study, NS was more noxious as compared with NG, and that the BPC tested by us attenuated both the toxicity and genotoxicity of NS.

Toxic Kidney Damage in Rats Following Subchronic Intraperitoneal Exposure to Element Oxide Nanoparticles.

Chronic diseases of the urogenital tract, such as bladder cancer, prostate cancer, reproductive disorders, and nephropathies, can develop under the effects of chemical hazards in the working environment. In this respect, nanosized particles generated as by-products in many industrial processes seem to be particularly dangerous to organs such as the testes and the kidneys. Nephrotoxicity of element oxide particles has been studied in animal experiments with repeated intraperitoneal injections of Al2O3, TiO2, SiO2, PbO, CdO, CuO, and SeO nanoparticles (NPs) in total doses ranging from 4.5 to 45 mg/kg body weight of rats. NPs were synthesized by laser ablation. After cessation of exposure, we measured kidney weight and analyzed selected biochemical parameters in blood and urine, characterizing the state of the excretory system. We also examined histological sections of kidneys and estimated proportions of different cells in imprint smears of this organ. All element oxide NPs under investigation demonstrated a nephrotoxic effect following subchronic exposure. Following the exposure to SeO and SiO2 NPs, we observed a decrease in serum creatinine and urea, respectively. Exposure to Al2O3 NPs caused an increase in urinary creatinine and urea, while changes in total protein were controversial, as it increased under the effect of Al2O3 NPs and was reduced after exposure to CuO NPs. Histomorphological changes in kidneys are associated with desquamation of the epithelium (following the exposure to all NPs except those of Al2O3 and SiO2) and loss of the brush border (following the exposure to all NPs, except those of Al2O3, TiO2, and SiO2). The cytomorphological evaluation showed greater destruction of proximal sections of renal tubules. Compared to the controls, we observed statistically significant alterations in 42.1% (8 of 19) of parameters following the exposure to PbO, CuO, and SeO NPs in 21.1% (4 of 19)-following that, to CdO and Al2O3 NPs-and in 15.8% (3 of 19) and 10.5% (2 of 19) of indicators, following the exposure to TiO2 and SiO2 nanoparticles, respectively. Histomorphological changes in kidneys are associated with desquamation of epithelium and loss of the brush border. The cytomorphological evaluation showed greater destruction of proximal sections of renal tubules. The severity of cyto- and histological structural changes in kidneys depends on the chemical nature of NPs. These alterations are not always consistent with biochemical ones, thus impeding early clinical diagnosis of renal damage. Unambiguous ranking of the NPs examined by the degree of their nephrotoxicity is difficult. Additional studies are necessary to establish key indicators of the nephrotoxic effect, which can facilitate early diagnosis of occupational and nonoccupational nephropathies.

PM10, and children's respiratory symptoms and lung function in the PATY study.

Studies of the impact of long-term exposure to outdoor air pollution on the prevalence of respiratory symptoms and lung function in children have yielded mixed results, partly related to differences in study design, exposure assessment, confounder selection and data analysis. We assembled respiratory health and exposure data for >45,000 children from comparable cross-sectional studies in 12 countries. 11 respiratory symptoms were selected, for which comparable questions were asked. Spirometry was performed in about half of the children. Exposure to air pollution was mainly characterised by annual average concentrations of particulate matter with a 50% cut-off aerodynamic diameter of 10 µm (PM(10)) measured at fixed sites within the study areas. Positive associations were found between the average PM(10) concentration and the prevalence of phlegm (OR per 10 µg · m(-3) 1.15, 95% CI 1.02-1.30), hay fever (OR 1.20, 95% CI 0.99-1.46), bronchitis (OR 1.08, 95% CI 0.98-1.19), morning cough (OR 1.15, 95% CI 1.02-1.29) and nocturnal cough (OR 1.13, 95% CI 0.98-1.29). There were no associations with diagnosed asthma or asthma symptoms. PM(10) was not associated with lung function across all studies combined. Our study adds to the evidence that long-term exposure to outdoor air pollution, characterised by the concentration of PM(10), is associated with increased respiratory symptoms.

Electron microscopy study on the transport of lead oxide nanoparticles into brain structures following their subchronic intranasal administration in rats.

White outbred female rats were exposed intranasally to 50-µL of suspension of lead oxide nanoparticles (PbO NPs) at a concentration of 0.5 mg/mL thrice a week during six weeks. A control group of rats was administered deionized water in similar volumes and conditions. The developed intoxication was manifested by altered biochemical and cytochemical parameters, as well as behavioral reactions of animals. Using electron microscopy and energy-dispersive X-ray spectroscopy techniques, we revealed deposition of PbO NPs in the olfactory bulb, but not in basal ganglia, and an increase in the number of axons with damage to the myelin sheath in the tissues of olfactory bulb and basal ganglia, changes in the ultrastructure of mitochondria of neurons in the tissues of olfactory bulb and basal ganglia of the brain, and differences in the mitochondrial profile of neurons in different regions of the rat brain. Our results collectively suggest that the central nervous system may be a target of low-level toxicity of lead oxide nanoparticles.

Subchronic systemic toxicity and bioaccumulation of Fe3O4 nano- and microparticles following repeated intraperitoneal administration to rats.

Aqueous suspensions of 10 nm, 50 nm, or 1 µm Fe(3)O(4) particles were injected intraperitoneally (ip) to rats at a dose of 500 mg/kg in 4 mL of sterile deionized water 3 times a week for 5 weeks. Following exposure, functional and biochemical indices and histopathological examinations of spleen and liver tissues of exposed rats were evaluated for signs of toxicity. The iron content of the blood was measured photometrically, and that of the liver and the spleen by atomic adsorption spectroscopy (AAS) and electron paramagnetic resonance (EPR) methods. It was found that, given equal mass doses, Fe(3)O(4) nanoparticles possess considerably higher systemic toxicity than microparticles, but within the nanometric range the relationship between particle size and resorptive toxicity is intricate and nonunique. The latter fact may be attributed to differences in different nanoparticles' toxicokinetics, which are controlled by both more or less substantial direct penetration of nanoparticles through biological barriers and their unequal solubility.

New Data on Variously Directed Dose-Response Relationships and the Combined Action Types for Different Outcomes of in Vitro Nanoparticle Cytotoxicity.

Spherical selenium-oxide and copper-oxide nanoparticles (SeO-NP with mean diameter 51 ± 14 nm and CuO-NP with mean diameter 21 ± 4 nm) were found to be cytotoxic for human fibroblast-like cells in vitro, as judged by decreased ATP-dependent luminescence. Compared with SeO-NP, CuO-NP produced a somewhat stronger effect of this kind. Along with cell hypertrophy developing in response to certain doses of SeO-NP and CuO-NP, our experiment also revealed doses causing a decrease in cell and cell-nucleus sizes. We observed both monotonic and different variants of nonmonotonic dose-response relationship. For the latter, we have succeeded in constructing adequate mathematical expressions based on the generalized hormesis paradigm that we had considered previously in respect of CdS-NP and PbS-NP cytotoxicity for cardiomyocites. It was demonstrated as well that combined toxicity of SeO-NP and CuO-NP is of different types depending on the outcome.

On an extended understanding of the term "hormesis" for denoting alternating directions of the organism's response to increasing adverse exposures.

The authors propose to consider as hormesis phenomenon not only a realization of the Arndt-Schulze rule but any non-monotonic dose-response relationship for a certain outcome that is characterized by changing direction of a response between adjacent ranges of doses of an initiator of this response, the number of such ranges being two or more. This approach is illustrated with results of several in vitro experiments on different established cell lines exposed to CdS or PbS nanoparticles.