INVESTIGATIONS OF OXIDATIVE STRESS EFFECTS AND THEIR MECHANISMS IN RAT BRAIN AFTER SYSTEMIC ADMINISTRATION OF CERIA ENGINEERED NANOMATERIALS

Advancing applications of engineered nanomaterials (ENM) in various fields create the opportunity for intended (e.g. drug and gene delivery) or unintended (e.g. occupational and environmental) exposure to ENM. However, the knowledge of ENM-toxicity is lagging behind their application development. Understanding the ENM hazard can help us to avoid potential human health problems associated with ENM applications as well as to increase their public acceptance. Ceria (cerium [Ce] oxide) ENM have many current and potential commercial applications. Beyond the traditional use of ceria as an abrasive, the scope of ceria ENM applications now extends into fuel cell manufacturing, diesel fuel additives and for therapeutic intervention as a putative antioxidant. However, the biological effects of ceria ENM exposure have yet to be fully defined. Both pro-and anti-oxidative effects of ceria ENM exposure are repeatedly reported in literature. EPA, NIEHS and OECD organizations have nominated ceria for its toxicological evaluation. All these together gave us the impetus to examine the oxidative stress effects of ceria ENM after systemic administration. Induction of oxidative stress is one of the primary mechanisms of ENM toxicity. Oxidative stress plays an important role in maintaining the redox homeostasis in the biological system. Increased oxidative stress, due to depletion of antioxidant enzymes or molecules and / or due to increased production of reactive oxygen (ROS) or nitrogen (RNS) species may lead to protein oxidation, lipid peroxidation and/or DNA damage. Increased protein oxidation or lipid peroxidation together with antioxidant protein levels and activity can serve as markers of oxidative stress. To investigate the oxidative stress effects and the mechanisms of ceria-ENM toxicity, fully characterized ceria ENM of different sizes (~ 5nm, 15nm, 30nm, 55nm and nanorods) were systematically injected into rats intravenously in separate experiments. Three brain regions (hippocampus, cortex and cerebellum) were harvested from control and ceria treated rats after various exposure periods for oxidative stress assessment. The levels of oxidative stress markers viz. protein carbonyl (PC), 3-nitrotyrosine (3NT), and protein bound 4-hydroxy-2-trans-nonenal (HNE) were evaluated for each treatment in each control and treated rat organ. Further, the levels and activities of antioxidant proteins, such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), were measured together with levels of heat shock proteins heme oxygenase -1 and 70 (HO-1 and Hsp-70). In addition, the levels of pro-inflammatory cytokines IL-1β, TNF-α, pro-caspase-3, and autophagy marker LC-3A/B were measured by Western blot technique. In agreement with the literature-proposed model of oxidative stress hierarchy mechanism of ENM-toxicity, the statistical analysis of all the results revealed that the ceria ENM-induced oxidative stress mediated biological response strongly depends on the exposure period and to some extent on the size of ceria ENM. More specifically, a single intravenous injection of ceria ENM induced tier-1 (phase-II antioxidant) response after shorter exposure periods (1 h and 20 h) in rat brain. Upon failure of tier-1 response after longer exposure periods (1 d to 30 d), escalated oxidative stress consequently induced tier-2 and tier-3 oxidative stress responses. Based on our observations made at chronic exposure period (90 d) after the single i.v. injection of ceria ENM, we could extend the model of oxidative stress hierarchy mechanisms for ceria-ENM-induced toxicity. Considering the evaluation of all the oxidative stress indices measured in 3-brain regions, oxidative stress effects were more prominent in hippocampus and the least in cerebellum, but no specific pattern or any significant difference was deduced

Plasma and Serum Proteins Bound to Nanoceria: Insights into Pathways by which Nanoceria May Exert Its Beneficial and Deleterious Effects \u3cem\u3eIn Vivo\u3c/em\u3e

Nanoceria (CeO2, cerium oxide nanoparticles) is proposed as a therapeutic for multiple disorders. In blood, nanoceria becomes protein-coated, changing its surface properties to yield a different presentation to cells. There is little information on the interaction of nanoceria with blood proteins. The current study is the first to report the proteomics identification of plasma and serum proteins adsorbed to nanoceria. The results identify a number of plasma and serum proteins interacting with nanoceria, proteins whose normal activities regulate numerous cell functions: antioxidant/detoxification, energy regulation, lipoproteins, signaling, complement, immune function, coagulation, iron homeostasis, proteolysis, inflammation, protein folding, protease inhibition, adhesion, protein/RNA degradation, and hormonal. The principal implications of this study are: 1) The protein corona may positively or negatively affect nanoceria cellular uptake, subsequent organ bioprocessing, and effects; and 2) Nanoceria adsorption may alter protein structure and function, including pro- and inflammatory effects. Consequently, prior to their use as therapeutic agents, better understanding of the effects of nanoceria protein coating is warranted

Rat Hippocampal Responses up to 90 Days After a Single Nanoceria Dose Extends a Hierarchical Oxidative Stress Model for Nanoparticle Toxicity

Ceria engineered nanomaterials (ENMs) have very promising commercial and therapeutic applications. Few reports address the effects of nanoceria in intact mammals, let alone long term exposure. This knowledge is essential to understand potential therapeutic applications of nanoceria in relation to its hazard assessment. The current study elucidates oxidative stress responses in the rat hippocampus 1 and 20 h, and 1, 7, 30 and 90 days following a single systemic infusion of 30 nm nanoceria. The results are incorporated into a previously described hierarchical oxidative stress (HOS) model. During the 1-20 h period, increases of the GSSG: GSH ratio and cytoprotective phase-II antioxidants were observed. During the 1-7 d period, cytoprotective phase-II antioxidants activities were inhibited with concomitant elevation of protein carbonyl (PC), 3-nitrotyrosine (3NT), heme oxygenase-1 (HO-1), cytokine IL-1β and the autophagy marker LC-3AB. At 30 day post ceria infusion, oxidative stress had its major impact. Phase-II enzyme activities were inhibited; concurrently PC, 3NT, HO-1 and Hsp70 levels were elevated along with augmentation of IL-1β, pro-apoptotic pro-caspase-3 and LC-3AB levels. This progress of escalating oxidative stress was reversed at 90 days when phase-II enzyme levels and activities were restored to normal levels, PC and 3NT levels were reduced to baseline, cytokine and pro-caspase-3 levels were suppressed, and cellular redox balance was restored in the rat hippocampus. This study demonstrates that a single administration of nanoceria induced oxidative stress that escalates to 30 days then terminates, in spite of the previously reported continued presence of nanoceria in peripheral organs. These results for the first time confirm in vivo the HOS model of response to ENM previously posited based on in vitro studies and extends this prior hierarchical oxidative stress model that described three tiers to a 4th tier, characterized by resolution of the oxidative stress and return to normal conditions

In Vivo Processing of Ceria Nanoparticles inside Liver: Impact on Free-Radical Scavenging Activity and Oxidative Stress

The cytotoxicity of ceria ultimately lies in its electronic structure, which is defined by the crystal structure, composition, and size. Despite previous studies focused on ceria uptake, distribution, biopersistance, and cellular effects, little is known about its chemical and structural stability and solubility once sequestered inside the liver. Mechanisms will be presented that elucidate the in vivo transformation in the liver. In vivo processed ceria reveals a particle-size effect towards the formation of ultrafines, which represent a second generation of ceria. A measurable change in the valence reduction of the second-generation ceria can be linked to an increased free-radical scavenging potential. The in vivo processing of the ceria nanoparticles in the liver occurs in temporal relation to the brain cellular and protein clearance responses that stem from the ceria uptake. This information is critical to establish a possible link between cellular processes and the observed in vivo transformation of ceria. The temporal linkage between the reversal of the pro-oxidant effect (brain) and ceria transformation (liver) suggests a cause-effect relationship

Rat Brain Pro-Oxidant Effects of Peripherally Administered 5 nm ceria 30 Days After Exposure

The objective of this study was to determine the residual pro-or anti-oxidant effects in rat brain 30 days after systemic administration of a 5 nm citrate-stabilized ceria dispersion. A ∼4% aqueous ceria dispersion was iv-infused (0 or 85 mg/kg) into rats which were terminated 30 days later. Ceria concentration, localization, and chemical speciation in the brain was assessed by inductively coupled plasma mass spectrometry (ICP-MS), light and electron microscopy (EM), and electron energy loss spectroscopy (EELS), respectively. Pro- or anti-oxidant effects were evaluated by measuring levels of protein carbonyls (PC), 3-nitrotyrosine (3NT), and protein-bound-4-hydroxy-2-trans-nonenal (HNE) in the hippocampus, cortex, and cerebellum. Glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase levels and activity were measured in addition to levels of inducible nitric oxide (iNOS), and heat shock protein-70 (Hsp70). The blood brain barrier (BBB) was visibly intact and no ceria was seen in the brain cells. Ceria elevated PC and Hsp70 levels in hippocampus and cerebellum, while 3NT and iNOS levels were elevated in the cortex. Whereas glutathione peroxidase and catalase activity were decreased in the hippocampus, GR levels were decreased in the cortex, and GPx and catalase levels were decreased in the cerebellum. The GSH:GSSG ratio, an index of cellular redox status, was decreased in the hippocampus and cerebellum. The results are in accordance with the observation that this nanoscale material remains in this mammal model up to 30 days after its administration and the hypothesis that it exerts pro-oxidant effects on the brain without crossing the BBB. These results have important implications on the potential use of ceria ENM as therapeutic agents

Distribution, Elimination, and Biopersistence to 90 Days of a Systemically Introduced 30 nm Ceria-Engineered Nanomaterial in Rats

Nanoceria is used as a catalyst in diesel fuel, as an abrasive in printed circuit manufacture, and is being pursued as an antioxidant therapeutic. Our objective is to extend previous findings showing that there were no reductions of cerium in organs of the mononuclear phagocyte (reticuloendothelial) system up to 30 days after a single nanoscale ceria administration. An ~5% aqueous dispersion of citrate-stabilized 30 nm ceria, synthesized and characterized in-house, or vehicle, was iv infused into rats terminated 1, 7, 30, or 90 days later. Cageside observations were obtained daily, body weight weekly. Daily urinary and fecal cerium outputs were quantified for 2 weeks. Nine organs were weighed and samples collected from 14 tissues/organs/systems, blood and cerebrospinal fluid for cerium determination. Histology and oxidative stress were assessed. Less than 1% of the nanoceria was excreted in the first 2 weeks, 98% in feces. Body weight gain was initially impaired. Spleen weight was significantly increased in some ceria-treated groups, associated with abnormalities. Ceria was primarily retained in the spleen, liver, and bone marrow. There was little decrease of ceria in any tissue over the 90 days. Granulomas were observed in the liver. Time-dependent oxidative stress changes were seen in the liver and spleen. Nanoscale ceria was persistently retained by organs of the mononuclear phagocyte system, associated with adverse changes. The results support concern about the long-term fate and adverse effects of inert nanoscale metal oxides that distribute throughout the body, are persistently retained, and produce adverse changes

Alteration of hepatic structure and oxidative stress induced by intravenous nanoceria.

Beyond the traditional use of ceria as an abrasive, the scope of nanoceria applications now extends into fuel cell manufacturing, diesel fuel additives, and for therapeutic intervention as a putative antioxidant. However, the biological effects of nanoceria exposure have yet to be fully defined, which gave us the impetus to examine its systemic biodistribution and biological responses. An extensively characterized nanoceria (5 nm) dispersion was vascularly infused into rats, which were terminated 1 h, 20 h or 30 days later. Light and electron microscopic tissue characterization was conducted and hepatic oxidative stress parameters determined. We observed acute ceria nanoparticle sequestration by Kupffer cells with subsequent bioretention in parenchymal cells as well. The internalized ceria nanoparticles appeared as spherical agglomerates of varying dimension without specific organelle penetration. In hepatocytes, the agglomerated nanoceria frequently localized to the plasma membrane facing bile canaliculi. Hepatic stellate cells also sequestered nanoceria. Within the sinusoids, sustained nanoceria bioretention was associated with granuloma formations comprised of Kupffer cells and intermingling CD3 + T cells. A statistically significant elevation of serum aspartate aminotransferase (AST) level was seen at 1 and 20 h, but subsided by 30 days after ceria administration. Further, elevated apoptosis was observed on day 30. These findings, together with increased hepatic protein carbonyl levels on day 30, indicate ceria-induced hepatic injury and oxidative stress, respectively. Such observations suggest a single vascular infusion of nanoceria can lead to persistent hepatic retention of particles with possible implications for occupational and therapeutic exposures

Rat hippocampal responses up to 90 days after a single nanoceria dose extends a hierarchical oxidative stress model for nanoparticle toxicity

Abstract Ceria engineered nanomaterials (ENMs) have very promising commercial and therapeutic applications. Few reports address the effects of nanoceria in intact mammals, let alone long term exposure. This knowledge is essential to understand potential therapeutic applications of nanoceria in relation to its hazard assessment. The current study elucidates oxidative stress responses in the rat hippocampus 1 and 20 h, and 1, 7, 30 and 90 days following a single systemic infusion of 30 nm nanoceria. The results are incorporated into a previously described hierarchical oxidative stress (HOS) model. During the 1-20 h period, increases of the GSSG: GSH ratio and cytoprotective phase-II antioxidants were observed. During the 1-7 d period, cytoprotective phase-II antioxidants activities were inhibited with concomitant elevation of protein carbonyl (PC), 3-nitrotyrosine (3NT), heme oxygenase-1 (HO-1), cytokine IL-1b and the autophagy marker LC-3AB. At 30 day post ceria infusion, oxidative stress had its major impact. Phase-II enzyme activities were inhibited; concurrently PC, 3NT, HO-1 and Hsp70 levels were elevated along with augmentation of IL-1b, pro-apoptotic pro-caspase-3 and LC-3AB levels. This progress of escalating oxidative stress was reversed at 90 days when phase-II enzyme levels and activities were restored to normal levels, PC and 3NT levels were reduced to baseline, cytokine and pro-caspase-3 levels were suppressed, and cellular redox balance was restored in the rat hippocampus. This study demonstrates that a single administration of nanoceria induced oxidative stress that escalates to 30 days then terminates, in spite of the previously reported continued presence of nanoceria in peripheral organs. These results for the first time confirm in vivo the HOS model of response to ENM previously posited based on in vitro studies and extends this prior hierarchical oxidative stress model that described three tiers to a 4th tier, characterized by resolution of the oxidative stress and return to normal conditions

Oxidative modification of lipoic acid by HNE in Alzheimer disease brain

AbstractAlzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder

PLEASE SCROLL DOWN FOR ARTICLE Nanotoxicology Biodistribution and oxidative stress effects of a systemically-introduced commercial ceria engineered nanomaterial

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Biodistribution and oxidative stress effects of a systemically-introduced commercial ceria engineered nanomaterial Abstract The objective was to characterize the biodistribution of nanoscale ceria from blood and its effects on oxidative stress endpoints. A commercial 5% crystalline ceria dispersion in water (average particle size Â3194 nm) was infused intravenously into rats (0, 50, 250 and 750 mg/kg), which were terminated 1 or 20 h later. Biodistribution in rat tissues was assessed by microscopy and ICP-AES/MS. Oxidative stress effects were assessed by protein-bound 4-hydroxy 2-transnonenal (HNE), protein-bound 3-nitrotyrosine (3-NT), and protein carbonyls. Evans blue (EB)-albumin and Na fluorescein (Na 2 F) were given intravenously as blood-brain barrier (BBB) integrity markers. The initial ceria t ½ in blood was Â7 min. Brain EB and Na 2 F increased some at 20 h. Microscopy revealed peripheral organ ceria agglomerations but little in the brain. Spleen Ce concentration was !liver !blood !brain. Reticuloendothelial tissues cleared ceria. HNE was significantly increased in the hippocampus at 20 h. Protein carbonyl and 3-NT changes were small. The nanoparticle characterizations before and after biodistribution, linked with the physiological responses, provide a foundation for evaluating the effects of engineered nanomaterial physico-chemical properties on peripheral organ distribution, brain entry and resultant toxicity