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1
Rat brain pro-oxidant effects of peripherally administered 5 nm ceria 30 days after 1
exposure 2
3
Sarita S. Hardas*, Rukhsana Sultana*, Govind Warrier*, Mo Dan‡, Rebecca L. Florence‡, Peng 4
Wulll, Eric A. Grulkelll, Michael T. Tsengllll, Jason M. Unrine¶, Uschi M. Graham||, Robert A. 5
Yokel‡, § and, D. Allan Butterfield *, † 6
7
Sarita S. Hardas: *Department of Chemistry, University of Kentucky, Lexington, Kentucky 8
40506-0055. Email: Sarita.Hardas@uky.edu 9
Rukhsana Sultana: *Department of Chemistry, University of Kentucky, Lexington, Kentucky 10
40506-0055. Email: rsult2@uky.edu 11
Govind Warrier: *Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-12
0055. Email: govind.warrier@uky.edu 13
Mo Dan: ‡Department of Pharmaceutical Sciences, University of Kentucky Academic Medical 14
Center, University of Kentucky, Lexington, Kentucky 40536-0082. Email: mo.dan@uky.edu 15
Rebecca L. Florence: ‡Department of Pharmaceutical Sciences, University of Kentucky 16
Academic Medical Center, University of Kentucky, Lexington, Kentucky 40536-0082. Email: 17
rlstep2@uky.edu 18
Peng Wu: IIIChemical and Materials Engineering Department, University of Kentucky, 19
Lexington, Kentucky 40506-0503. Email: peng.wu@uky.edu 20
Eric Grulke: IIIChemical and Materials Engineering Department, University of Kentucky, 21
Lexington, Kentucky 40506-0503. Email: eric.grulke@uky.edu 22
Michael T. Tseng: IIIIDepartment of Anatomical Sciences & Neurobiology, University of 23
Louisville, Louisville, Kentucky 40202. Email: mttsen01@louisville.edu 24
Jason M. Unrine: ¶Department of Plant and Soil Sciences, University of Kentucky, Lexington, 25
Kentucky 40546-0091. Email: jason.unrine@uky.edu 26
*Manuscript
Click here to view linked References
2
Uschi M. Graham: IICenter for Applied Energy Research, University of Kentucky, Lexington, 1
Kentucky 40511. Email: uschi.graham@uky.edu 2
Robert A. Yokel: ‡Department of Pharmaceutical Sciences, University of Kentucky Academic 3
Medical Center, University of Kentucky, Lexington, Kentucky 40536-0082, § Graduate Center for 4
Toxicology, University of Kentucky Academic Medical Center, Lexington, Kentucky 40506-9983. 5
Email: ryokel@email.uky.edu 6
D. Allan Butterfield: *Department of Chemistry, University of Kentucky, Lexington, Kentucky 7
40506-0055, †Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 8
40506-0059. Email: dabcns@uky.edu 9
10
11
Corresponding author: 12
D. Allan Butterfield, Ph.D. 13
Department of Chemistry, 14
University of Kentucky, 15
Lexington, 16
Kentucky 40506-0055, 17
Phone: 859-257-3184 18
Fax: 859-323-1069 19
Email: dabcns@uky.edu 20
21
Short title: Nanoceria pro-oxidant effect on brain 22
23
24
25
26
3
Abbreviations: 1
3NT protein bound 3-nitrotyrosine 2
Ce cerium 3
Cat catalase 4
EELS electron energy loss spectroscopy 5
ENM engineered nanomaterial 6
GR glutathione reductase 7
GPx glutathione peroxidase 8
H2O2 Hydrogen peroxide 9
Hsp70 heat shock protein 70 10
HNE protein-bound 4-hydroxy-2-trans-nonenal 11
ICP-MS inductively coupled plasma mass spectrometry 12
iNOS inducible nitric oxide synthase 13
MDL method detection limit 14
PC protein carbonyl 15
ROS reactive oxygen species 16
RNS reactive nitrogen species 17
SOD superoxide dismutase 18
TEM transmission electron microscopy 19
20
21
22
4
Abstract: 1
The objective of this study was to determine the residual pro-or anti-oxidant effects in rat brain 2
30 days after systemic administration of a 5 nm citrate-stabilized ceria dispersion. A ~4% 3
aqueous ceria dispersion was iv-infused (0 or 85 mg/kg) into rats which were terminated 30 4
days later. Ceria concentration, localization, and chemical speciation in the brain was assessed 5
by inductively coupled plasma mass spectrometry (ICP-MS), light and electron microscopy 6
(EM), and electron energy loss spectroscopy (EELS), respectively. Pro- or anti-oxidant effects 7
were evaluated by measuring levels of protein carbonyls (PC), 3-nitrotyrosine (3NT), and 8
protein-bound-4-hydroxy-2-trans-nonenal (HNE) in the hippocampus, cortex, and cerebellum. 9
Glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), and 10
catalase levels and activity were measured in addition to levels of inducible nitric oxide (iNOS), 11
and heat shock protein-70 (Hsp70). The blood brain barrier (BBB) was visibly intact and no ceria 12
was seen in the brain cells. Ceria elevated PC and Hsp70 levels in hippocampus and 13
cerebellum, while 3NT and iNOS levels were elevated in the cortex. Whereas glutathione 14
peroxidase and catalase activity were decreased in the hippocampus, GR levels were 15
decreased in the cortex, and GPx and catalase levels were decreased in the cerebellum. The 16
GSH: GSSG ratio, an index of cellular redox status, was decreased in the hippocampus and 17
cerebellum. The results are in accordance with the observation that this nanoscale material 18
remains in this mammal model up to 30 days after its administration and the hypothesis that it 19
exerts pro-oxidant effects on the brain without crossing the BBB. These results have important 20
implications on the potential use of ceria ENM as therapeutic agents. 21
22
Key words: oxidative stress; ceria; brain; neurotoxicity; nanomaterial, nanoparticles; rat. 23
24
25
5
Introduction: 1
Engineered nanomaterials (ENM) can be manufactured in a variety of shapes and sizes and 2
physico-chemical, surface, as well as optical and magnetic properties. ENMs have numerous 3
applications in research, medicine, electronics and other industries. Physico-chemical properties 4
of nanomaterials differ from their bulk forms mainly because of the larger surface area to mass 5
ratio, which affects reactivity, strength and electrical properties of nanomaterials. Because of 6
their comparable size with biological molecules like proteins and DNA, ENMs can gain access 7
to usually difficult to reach biological compartments in cells (Fubini et al. 2010). Increased 8
surface activity can facilitate interactions with biological molecules, which may evoke greater 9
physiological responses, different from the same basic material with larger particle size, the bulk 10
form equivalent of ENMs (Donaldson et al. 2004; Landsiedel et al. 2009; Xia et al. 2009). One 11
effect exhibited by ENMs is the generation of free radicals or induction of oxidative stress, which 12
is also a primary mechanism of ENM toxicity (Xia et al. 2009). Oxidative stress effects are direct 13
consequences of imbalance in the rates of reactive oxygen and / or nitrogen species (ROS or 14
RNS) production verses scavenging of ROS and / or RNS and / or antioxidant levels 15
(Butterfield et al. 2007). 16
17
Ceria ENM (a.k.a. cerium oxide; CeO2), which is one of the most used ENM employed in 18
different industrial applications (Yokel et al. 2009; Hardas et al. 2010) has been shown to have 19
both anti-inflammatory properties as well as potent toxicity. However, there is no clear 20
understanding of what exactly controls ceria’s pro-or anti-oxidant effects. A recently published 21
report summarizes findings of in vitro and in vivo studies conducted with ceria ENM under basal 22
and induced oxidative stress conditions (Celardo et al. 2011). Ceria exhibited antioxidant 23
properties evidenced by scavenging free radicals, by reducing levels of peroxides, iNOS, TNF-24
α, NF-ĸβ, and interleukin, by promoting cell viability or protecting organelles from diesel exhaust 25
and cigarette smoke-induced oxidative stress, ROS generating chemical agents, or side effects 26
6
of radiation treatment. Ceria has been suggested for potential use in the treatment of diabetic 1
cardiomyopathy, cancer, stroke, retinal degradation and Alzheimer’s disease as well as to 2
prolong life span (Chen et al. 2006; Rzigalinski et al. 2006; Das et al. 2007; Xia et al. 2008; 3
D'Angelo et al. 2009; Hirst et al. 2009; Babu et al. 2010; Colon et al. 2010; Younce et al. 2010; 4
Estevez et al. 2011; Niu et al. 2011). Antioxidant properties of ceria may be related to its SOD- 5
and catalase-mimicking activity (Korsvik et al. 2007; Pirmohamed et al. 2010) attributed to 6
Ce3+/ Ce4+ redox coupling (Celardo et al. 2011). In contrast, there are reports of ceria induced 7
pro-oxidant effects under basal conditions. In different cell culture studies, ceria ENM mediated 8
ROS injury, induced lipid peroxidation, caused membrane damage, led to elevation of the 9
cytokine, IL-8, led to depletion of GSH, and led to reduced cell viability (Brunner et al. 2006; Lin 10
et al. 2006; Park et al. 2008; Auffan et al. 2009). 11
12
To utilize ceria for therapeutic and non-therapeutic applications, it is important to know the long 13
term effects of intended and un-intended ceria exposure on mammals. Most reports on effects 14
of ceria ENM were conducted using non-mammalian organisms or in cell culture, and none of 15
these addressed long-term effects or fate of ceria. In addition to our own previous studies 16
(Yokel et al. 2009; Hardas et al. 2010), a few ceria ENM studies were conducted in intact 17
animals (Chen et al. 2006; Niu et al. 2007; Hirst et al. 2009; Amin et al. 2011; Choi et al. 2011; 18
Hirst et al. 2011; Srinivas et al. 2011; Zhou et al. 2011). One study reports that deposition and 19
retention of ceria in various vital organs and increased WBC count were seen 30 days after 20
intraperitoneal and intravenous injection to mice, but otherwise ceria was tolerated by animals 21
(Hirst et al. 2011). Ceria reduced myocardial oxidative stress in transgenic mice for ischemic 22
cardiomyopathy, rat liver from monocrotaline-induced ROS injury by induction of GSH levels 23
and intravitreal injections of ceria inhibited retinal neovascular lesions (Niu et al. 2007; Amin et 24
al. 2011; Zhou et al. 2011). However, after pulmonary inhalation of ceria ENM, granulomatous 25
pathology and GSH depletion were seen in rat lungs (Cho et al. 2010; Srinivas et al. 2011). 26
7
Animal studies have also reported that ceria ENM can accumulate in various organs, including 1
the heart and lung, irrespective of the point of entry or distance (from injection point and organ 2
specifically examined) when supplied as intravenous or intra-peritoneal injections or as a food 3
additive (Chen et al. 2006; Niu et al. 2007; Hirst et al. 2009). This accumulation may lead to 4
systemic effects involving the inflammatory response (Celardo et al. 2011) or increased ROS 5
production under normal physiological conditions (Hirst et al. 2011). 6
7
To our knowledge there is no prior information available on the long-term effects (30 d or more 8
after administration) of ceria on brain and how these effects may contrast with an immediate 9
response after the initial ENM contact. Our previous study showed moderate pro-oxidant effects 10
on rat brain, 1 and 20 h after a single acute systemic instillation of 5 nm ceria ENM (Hardas et 11
al. 2010). The current study discusses residual effects of oxidative stress parameters in brain 30 12
days after one single acute ENM peripheral administration using 5 nm ceria ENM. To address 13
the objective, the levels and activities of the antioxidant enzymes catalase, manganese 14
superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), and glutathione reductase 15
(GR), were measured along with the ratio of reduced glutathione (GSH) to its oxidized form 16
(GSSG). To understand the extent of changes in cellular redox status, the levels of oxidative 17
stress endpoints, protein carbonyl (PC), 3-nitrotyrosine (3NT), and protein bound 4- hydroxyl-2-18
trans nonenal (HNE), were measured along with heat shock protein (Hsp70) levels. 19
20
Materials and Methods: 21
All the materials, methods including the well characterized 5 nm ceria ENM are same as that 22
used in our recently published study (Hardas et al. 2010). The rats used are of the same strain, 23
sex and approximately same weight as that used in the previous study with 5 nm ceria ENM 24
(Hardas et al. 2010). Therefore, only a brief overview is presented. 25
26
8
Nanomaterial: 1
Cerium chloride heptahydrate (Sigma-Aldrich # 228931, 99.9% metal basis), ammonium 2
hydroxide (Fisher #3256, ACS, 28-30%) and citric acid monohydrate (EMD Chemicals Inc # 3
CX1725-1, GR ACS) were used without further purification. A hydrothermal method was used to 4
synthesize ~5nm ceria aqueous suspension. Briefly, a 20 ml aqueous mixture of 0.01 mol 5
cerium chloride and 0.01 M citric acid was added to 20 ml of 3 M ammonium hydroxide. After 6
stirring for 24 h at 50C, the solution was transferred into a Teflon-lined stainless steel bomb 7
and heated at 80C for 24 h to complete the reaction. The final dispersion of ceria ENM was 8
infused intravenously to the rats over 1 h without any further treatment or purification. 9
10
Ceria characterization: 11
The details of ceria ENM characterization are published in our earlier study (Hardas et al. 2010). 12
In brief, the morphology and crystallinity of the ceria was evaluated using a 200-keV field 13
emission analytical transmission electron microscope (JEOL JEM-2010F, Tokyo, Japan) 14
equipped with an Oxford energy dispersive X-ray spectrometer. The particle size distributions 15
(PSDs) were determined using dynamic light scattering (DLS: Brookhaven Instruments 16
Corporation, 90Plus NanoParticle Size Distribution Analyzer, Holtsville, NY, USA). The surface 17
area of the dried ceria powder was determined using a BETASAP 2020 surface area analyzer 18
that determines particle surface area based on nitrogen adsorption (Micromeritics Instrument 19
Corporation, Norcross, GA, USA). The ceria content of the dispersion and the potential 20
presence of contaminating elements/metals were determined by digestion of ceria dispersion 21
samples and analysis by ICP-MS. Electron energy loss spectroscopy (EELS) was performed on 22
rat liver tissue using a JEOL 2010F STEM outfitted with a URP pole piece, GATAN 2000 GIF 23
(Pleasanton, CA, USA), GATAN DigiScan II, Fischione HAADF STEM detector (Export, PA, 24
USA), and EmiSpec EsVision software (Tempe, AZ, USA). 25
9
Animals: 1
In the current study 16 male Sprague Dawley rats, weighing 328 ± 21 g (mean ± SD), were 2
obtained from Harlan, Indianapolis, IN, and were housed individually prior to study and after 3
cannulae removal (a few days after the iv infusion) in the University of Kentucky Division of 4
Laboratory Animal Resources facility under a 12:12 h light:dark cycle at 70 ± 8°F and 30 to 70% 5
humidity. The rats had ad lib access to 2018 Harlan diet and RO water. All procedures involving 6
animals were approved by the University of Kentucky Institutional Animal Care and Use 7
Committee. The research was conducted in accordance with the Guiding Principles in the Use 8
of Animals in Toxicology (http://www.toxicology.org/ai/air/air6.asp).. 9
10
Ceria administration: 11
Rats were prepared with 2 cannulae that terminated in the vena cava to administer the ceria 12
dispersion or water (controls) in one and 1.8% sodium chloride in the second, to avoid 13
agglomeration induced by sodium chloride or 10% sucrose, which are commonly added to 14
prepare iso-osmotic solutions (Yokel et al. 2009); (Hardas et al. 2010). Seven rats received 0 15
and nine rats received 85 mg ceria/kg as a single acute dose of ENM. The rats were terminated 16
30 days after the infusion. Post-mortem samples including brain cortex, hippocampus, and 17
cerebellum were harvested rapidly and were frozen in liquid nitrogen and stored at -80 0C for 18
later oxidative stress measurements. Cerium concentrations in brain cortex, blood, and liver 19
were analyzed by ICP-MS; Agilent 7500cx, (Santa Clara, CA, USA) after microwave digestion 20
as described previously (Hardas et al., 2010). The detection limits of this method were 0.089 mg 21
Ce/kg. Mean spike recovery ranged from 97 to 105%. Relative percent difference between 22
replicates was ≤ 2% for 30-120 ng Ce/ml and 18% for 1.5 ng Ce/ml. 23
Light and electron microscopic assessment: 24
After animal termination brains were removed, sliced coronally in a brain matrix (Braintree 25
acrylic matrix BS-A 6000C). Sections containing hippocampus were fixed by immersion in 4% 26
10
buffered formalin. Cerebellum was similarly sliced and fixed. Samples were cut into 3 mm 1
cubes, dehydrated and embedded in Araldite 502. After polymerization, one micron thick 2
sections were cut and stained with toluidine blue for LM examination. Selected blocks were 3
sectioned at 80 nm, collected on 200 mesh copper grids and examined in a Philips CM 10 4
electron microscope at 60 kV. 5
6
Oxidative stress assessment, sample preparation: 7
All sample preparation and protein assays were carried out as described in our previous 8
publication (Hardas et al. 2010). Each sample was individually thawed and homogenized using 9
a 550 sonic dismembrator from Fischer Scientific for 10 to 20 s on ice. The buffer used for 10
homogenization contained 0.32 M sucrose, 0.125 M Tris, 0.6 mM MgCl2, and protease inhibitors 11
(4 µg/ul leupeptin, 4 µg/ul pepstatin A, 5 µg/ul aprotinin, and 0.2 mM phenylmethylsulfonyl 12
fluoride) at pH 8.0. Total protein concentration for each sample was measured using the 13
bicinchoninic acid assay and equal amounts of protein from control and treated samples were 14
used in each assay. 15
16
Oxidative stress markers: 17
The oxidative stress markers PC, 3NT, and HNE were assessed for each homogenized sample 18
using the slot blot technique. Specific antibodies were used to determine the levels of PC, 3NT, 19
HNE in controls and treated samples as previously described (Hardas et al. 2010). 20
21
GSH and GSSG levels: 22
The reduced (GSH) and oxidized glutathione (GSSG) levels were simultaneously measured in 23
each sample as previously described (Hissin and Hilf 1976). A small amount of brain tissue was 24
rapidly weighed, homogenized with metaphosphoric acid (25%) and sodium phosphate (0.1 M) - 25
ethylenediaminetetraacetic acid (0.005 M) buffer (pH 8) and then centrifuged. For GSH levels, 26
11
an aliquot of supernatant was further diluted with phosphate buffer and then incubated with o-1
phthaldehyde (OPT), before determination of fluorescence (λ excitation 350 nm and emission 2
420 nm). For GSSG levels equal volumes of supernatant were incubated with N-ethylmaleimide 3
(0.04 M) for 30 min and then diluted with sodium hydroxide (0.1 N), before assaying with OPT. 4
The GSH to GSSG ratio for each sample was calculated by comparing the fluorescence values 5
from each assay to their respective calibration curves. The final values are % control of mean ± 6
SEM of treated vs. control samples. 7
8
Western blot analysis: 9
The levels of the antioxidant enzymes GR, GPx, MnSOD, catalase, Hsp70 and iNOS were 10
measured using immunoblotting-Western blot techniques as described in our earlier 11
publications (Sultana et al. 2008; Hardas et al. 2010). In brief, 75 µg protein from each 12
homogenized sample was loaded and separated on SDS-PAGE alongside its respective 13
control. The separated proteins were transferred from poly-acrylamide gels to nitrocellulose 14
membranes, and then the band of specific protein identified using a specific antibody against 15
that protein. The band-intensity was quantified as previously described (Hardas et al., 2010) by 16
using the image analysis software, ImageQuant, purchased from GE Healthcare. 17
18
Enzyme activity assays: 19
The activities of GR, GPx, MnSOD and catalase enzymes were measured as described earlier 20
(Hardas et al. 2010), with commercially available kits from Cayman Chemical Company, Ann 21
Arbor, MI, USA as per the manufacturer’s instructions. Suitable protein samples from each 22
organ (i.e., 10-20 µg for brain homogenate) were mixed with assay buffer and with other specific 23
reagents for each assay on 96-well plates. Enzymatic reactions were initiated by addition of 24
reaction initiator reagents, NADPH for GR, cumene hydroperoxide for GPx, xanthine oxidase for 25
SOD, and hydrogen peroxide for the catalase assay. Progression of the each reaction was 26
12
studied separately by spectrophotometry at 340 nm (for GR and GPx), at 460 nm (for SOD) and 1
at 540 nm (for catalase). 2
Data and statistical analysis: 3
The slot blot, Western blot and enzyme assay results are presented as mean ± SEM. The 4
control mean was normalized to 100%. Grubb’s test was used to identify outliers in oxidative 5
stress parameter results. Student’s unpaired t-test was used to evaluate significant difference 6
between controls and ceria treated samples. Subsequently, two-way ANOVA was conducted to 7
determine the differential effect of ceria ENM treatment among the three brain regions studied. 8
Significance was accepted at p < 0.05. 9
10
Results: 11
Ceria composition 12
HR-TEM/HR-STEM showed the ceria ENM was polyhedral shaped (Figure 1). The XRD 13
patterns demonstrated the ceria was highly crystalline, with face centered cubic unit cells with 14
corresponding Miller indices of the most common faces of (111), (210) and (200). Evaluation of 15
a number of TEM images showed that the ceria had a number-average primary particle size of 16
~5 nm. The ceria ENM surface area was 121 m2/g, assuming the density of a ceria nanoparticle 17
7600 kg/m3 (the bulk density), the back-calculated average diameter should be 6.5nm that fits 18
our TEM observation quite well. Analysis by ICP-MS of the ceria dispersion used showed that 19
the ceria content of the as-synthesized dispersion was 4.35 ± 0.20% and the free cerium ion 20
content was 11.6 ± 0.3%. 21
Dispersion properties 22
The citrate ion was found to be a stabilizing electrolyte for ceria nanoparticles. At pH below 7.0 23
the ceria agglomerated; therefore, the ceria was maintained as an aqueous dispersion at pH 7.7 24
to 8. Zeta potential of the citrate-stabilized particles was -53 ± 7 mV at pH ~ 7.35 (Hardas et al. 25
2010). In general, dispersions with absolute values of zeta potential greater than 30 to 40 mV 26
13
are expected to be stable, and the stability of dispersion is better with higher zeta potential. The 1
cerium concentration in samples taken from the top and bottom of two ceria dispersion samples 2
in covered 15 ml centrifuge tubes that were un-disturbed for > 2 months were within 2.5% of 3
each other, demonstrating dispersion stability (data not shown). 4
5
Ceria concentration in brain and electron micrography 6
ICP-MS analysis showed that a very small amount of ceria was present in the brain compared 7
to the liver (Table 1). Electron micrographic studies suggested that ceria ENM was not present 8
in the brain, but located on the luminal side of the blood brain barrier (BBB) endothelium. The 9
hippocampus and cerebellum tissues did not show obvious ceria induced injury as no necrotic 10
neurons or elevated gliosis were observed and the BBB was visibly intact (data not shown). 11
12
EELS results 13
Electron energy loss spectroscopic (EELS) measurements on liver tissue were performed as a 14
representative organ. The 5 nm ceria agglomerates were located in the tissue 30 days after 15
infusion into rats. The ratio of Ce(3+) to Ce(4+) was evaluated using EELS measurements in 16
vivo 30 days post infusion. Further, this ratio was compared with the ratio of Ce3+/Ce4+ obtained 17
in freshly synthesized ceria. The high Ce3+/ Ce4+ ratio that was obtained in the as-synthesized, 18
fresh 5 nm ceria particles seems to have only been altered slightly in individual ceria measured 19
in liver after 30 days in vivo. However, this difference was not significant (data not shown). 20
21
Oxidative Stress Indices 22
The primary aim of these studies was to evaluate indices of oxidative stress in brain 30 days 23
following a single ceria ENM administration. 24
25
Ceria treatment affected catalase levels and activities 26
14
Previously ceria ENM was reported to have a H2O2-producing ability (Korsvik et al. 2007); 1
therefore, the effect of 5 nm ceria ENM on levels and activity of a primary H2O2-reducing 2
enzyme (catalase) was determined in the present study. Catalase activity was significantly 3
decreased in the hippocampus (~18%, *p<0.05, Figure 2b) and catalase levels were 4
significantly decreased in the cerebellum (~16%, *p<0.05, Figure 4a). To determine the 5
influence of ceria treatment on the SOD enzyme or contribution to H2O2 levels from SODs, if 6
any, the level of MnSOD and its activity were measured. There were no significant changes 7
observed in the levels or activity of MnSOD (data not shown). 8
9
Ceria treatment decreases GPx levels, activity and the GSH-GSSG ratio 10
GPx reduces H2O2 along with other peroxides using glutathione (GSH) as a source of reducing 11
equivalents. The activity of GPx was significantly decreased in the hippocampus (~69%, 12
**p<0.001, Figure 2b) and in cerebellum (~23 %, *p<0.05, Figure 4b). GPx levels showed a 13
decreasing trend in all brain regions, but was significantly decreased only in cerebellum (~27 %, 14
*p<0.05, Figure 4a). Within the three brain regions examined, hippocampal GPx activity was 15
significantly inhibited compared to that in cortex and cerebellum (**p < 0.01). GR levels were 16
significantly decreased (~24%, *p<0.05, Figure 3a) in the cortex. GR activity did not show any 17
significant change in any of the three brain regions studied. A marker of overall cellular redox 18
status was evaluated by comparing the GSH:GSSG ratio of ceria-treated samples. The GSH: 19
GSSG ratio was significantly decreased in the hippocampus (~13%, *p<0.05, Figure 2b) and 20
cerebellum (~15%, **p<0.01, Figure 4b) consistent with an increase in oxidative stress. 21
22
Ceria treatment induced protein oxidation 23
The levels of PC showed a significant increase in the hippocampus (~ 19%, *p<0.05, Figure 2c) 24
and cerebellum (~12 %, *p<0.05, Figure 4c) in treated vs. control samples. 3NT levels were 25
significantly increased in the cortex (~20%, *p<0.05, Figure 3c). There was no significant 26
15
change in protein-bound HNE levels in any of the three brain regions examined. Consistent with 1
increased 3NT levels in cortical region, iNOS levels were increased significantly (~27%, 2
*p<0.05, Figure 3d), and there was a positive correlation between 3NT and iNOS levels (r= 3
0.67, p < 0.05, Figure 3d). 4
5
Hsp-70 levels increased after ceria treatment 6
Hsp-70 is a member of the heat shock protein family and inducible by oxidative stress. Hsp-70 7
levels were significantly increased in the hippocampus (~49%, *p<0.05, Figure 2a), as well as 8
compared to that of cortical and cerebellar Hsp70 levels (*p<0.05). In the cerebellum Hsp70 9
levels were increased (~40% *p<0.05, Figure 4a). These results are consistent with diminution 10
of GSH levels indicated by the decreased GSH: GSSG ratio in these brain regions. 11
12
Discussion: 13
The present work was conducted to evaluate the oxidative stress effects of ceria ENM in brain 14
30 d after a single acute peripheral administration of 5 nm ceria ENM. ROS and RNS are 15
inevitable byproducts of all major metabolic cellular processes and conspicuous by their high 16
reactivity and high cytotoxicity. Apart from being a cause for many pathological conditions or 17
cellular disturbances, ROS/ RNS also play important roles in cell signaling pathways and 18
cellular homeostasis (Celardo et al. 2011; Butterfield et al., 2001). Therefore, balancing 19
excessive and insufficient ROS/RNS is critical, and endogenous enzymes like GPx, GR, 20
catalase and SOD as well as antioxidants like glutathione maintain this redox balance efficiently. 21
Any perturbation to this redox balance causes oxidative stress. One of the mechanisms by 22
which nanomaterials induce toxicity is by inducing ROS production, elevating oxidative stress, 23
which damages proteins, lipids or DNA (Butterfield and Stadtman, 1997; Nel et al. 2006; 24
Sharma Sharma 2007; Mocan et al. 2010). 25
16
Owing to its redox switching between two oxidation states, 3+ and 4+, ceria exhibits catalytic 1
activity, which has made it useful in industrial applications (Celardo et al. 2011). Ceria’s surface 2
redox capability can affect the immediate surroundings and has been strongly linked to particle 3
size (Gilliss et al. 2005). The characteristic oxidation and reduction in ceria ENM is linked to the 4
continued possibility to absorb and release oxygen by inducing oxygen vacancies close to the 5
particle surface. The Ce3+/ Ce4+ valance switch resembles redox behavior of some biological 6
antioxidant enzymes like SOD and catalase. The high Ce3+/ Ce4+ ratio is responsible for the 7
ceria ENM SOD-mimetic activity (Korsvik et al. 2007). 8
In our previous study, the same 5 nm ceria ENM at the same dose as used in the present study 9
showed moderate effects, on brain redox status, while catalase levels and activities were 10
increased in hippocampus after 1 and 20 h, respectively, and catalase activity was decreased in 11
cerebellum after 1 h. No change was seen in PC, 3NT and HNE levels (Hardas et al. 2010). 12
Although, ceria ENM was not found in the brain, but located on the luminal side of the BBB 13
endothelial cells and the BBB was intact, the current study demonstrated that 5 nm ceria ENM 14
produced significant pro-oxidant effects in the brain 30 days following administration and their 15
retention in peripheral organs (Figure 5). Based on the EELS analysis, it would appear that even 16
after a long-term exposure time of 30 days ceria continued to show a significant +3 valence on 17
the surface. Similar high +3 valence was observed on surface of ceria ENM after short-term 18
retention inside the rat, as seen in our previous study (Hardas et al. 2010), which was not 19
significantly different compared to freshly prepared ceria. This finding clearly defines an 20
enhanced oxygen storage capacity (Nesic et al.) of the ceria surfaces that does not diminish 21
greatly throughout the 30 day exposure period. Yet, ceria with enhanced OSC was shown in the 22
current study to have a significant pro-oxidative effect in the brain after the long-term exposure 23
following a single administration. 24
17
As depicted in Figure 5, a decline in the GSH: GSSG ratio (in hippocampus and cerebellum) in 1
the current study indicates elevated oxidative stress in the cellular environment. A similar 2
observation was reported in Park et. al. in which 30 nm ceria depleted GSH levels in human 3
lung epithelial cells in a dose-dependent manner (Park et al. 2008). Hydroxylated derivatives of 4
fullerenes also decreased the GSH: GSSG ratio and induced lipid peroxidation (Nakagawa et al. 5
2011). At the cellular level the GSH: GSSG ratio is dependent on GPx and GR enzymes, and in 6
the current study we observed decreased GPx activity (in hippocampus and cerebellum), 7
decreased GPx levels (in cerebellum) and decreased GR levels (cortex). Similarly, 15 nm silver 8
and 90 nm copper ENM down-regulated GPx gene expression (Wang et al. 2009), 25 nm silver 9
ENM (Rahman et al. 2009) and SWCNT (Wang et al. 2011) inhibited GPx activity, and silica 10
ENM reduced GR activity (Akhtar et al. 2010). Thus it may be possible that after 30 days 11
following administration, ENM have deleterious effects on enzymes needed for maintenance of 12
the reduced thiol status of the cells (Figure 5). 13
Figure 5 also shows that inhibition of catalase and GPx activities may lead to accumulation of 14
H2O2 and ultimately increased production of hydroxyl radicals (OH•). Activity of catalase can be 15
inhibited by hydroxyl radicals (OH•) and that of GPx by H2O2 and hydroperoxides (Pigeolet et al. 16
1990). Ceria ENM can produce H2O2 under abiotic conditions (Korsvik et al. 2007; Xia et al. 17
2008), and H2O2 has high membrane permeability (Halliwell 1992). Further H2O2 can undergo a 18
Fenton-type reaction to produce highly potent OH• radicals as noted above (Figure 5). A lack of 19
change in SOD activity and level in the current study may imply that endogenous SOD does not 20
account for ceria-induced elevated oxidative stress in brain. Therefore, ceria ENM treatment 21
may have caused induction in ROS that led to oxidative inhibition of antioxidant enzyme 22
activities, decreased the GSH: GSSG ratio and increased in PC and 3NT levels observed in the 23
present study. Similar consequences of oxidative stress were seen after exposure to various 24
18
other ENM, such as TiO2 (Hao et al. 2009; Liang et al. 2009; Xiong et al. 2011), SWCNT(Wang 1
et al. 2011), MWCNT(Guo et al. 2011), hematite (Radu et al. 2010), and ZnO (Xia et al. 2008). 2
3
The elevated 3NT levels, a marker for increased nitrosive stress, are consistent with elevated 4
levels of inducible nitric oxide synthase (iNOS). iNOS exists at extremely low levels under 5
normal physiological conditions, but it is inducible by endotoxin and inflammatory cytokines 6
among other stresses (Calabrese et al. 2000). We found a correlation between 3NT levels and 7
iNOS levels in the cortical region of rats examined 30 d after ceria ENM treatment. It may 8
indicate that increased 3NT levels are due to increased NO production in the cortex and we 9
speculate that iNOS levels concurrently may be induced following ceria treatment via increased 10
cytokine production. 11
12
Electron micrograph analysis showed an intact BBB and an absence of any significant amount 13
of ceria in the brain. However, cobalt-chromium ENMs have damaged DNAs without crossing 14
cellular membranes (Bhabra et al. 2009), and the chemotherapeutic drug doxorubicin led to 15
neurotoxic effects without ever crossing the BBB (Tangpong et al. 2006). Similarly, it is 16
conceivable that the observed oxidative stress response in brain regions could be due to the 17
accumulation of 5 nm ceria in peripheral organs and subsequent elevation of BBB-permeable 18
inflammatory cytokines. Studies to test this notion are in progress. 19
20
Induction of heat shock protein Hsp-70 levels as seen in the present study is in agreement with 21
other literature reports. Similar to effects of silver ENM (Ahamed et al. 2010) and fullerene C60 22
(Usenko et al. 2008) treatments, Hsp-70 levels and other oxidative stress markers were induced 23
with concomitant decrease in the GSH: GSSG ratio. In a transgenic mouse model for 24
cardiomyopathy, Hsp-70 levels were increased as an oxidative stress marker of ER stress. 25
Ceria ENM treatment rescued these cells from ER stress and as a result Hsp-70 expression 26
19
was down regulated (Niu et al. 2007). In the present study, 5 nm ceria ENM indirectly induced 1
ROS production in brain causing depletion of GSH, which initially induces antioxidant levels but 2
over 30 days eventually inhibits H2O2-scavenging catalase and GPx enzyme activities. This may 3
increase H2O2 levels and therefore OH• production via the Fenton reaction, which may cause 4
protein oxidation and induction of Hsp 70 levels (Figure 5). 5
6
The pro-oxidant effects observed in the brain 30 days after a single intravenous administration 7
of 5 nm ceria ENM are similar to those observed in age-related or Alzheimer disease-related 8
oxidative stress effects previously reported by our laboratory (Butterfield et al., 2001; Butterfield 9
and Stadtman, 1997). Although all three brain regions showed an effect of 5 nm ceria ENM 10
treatment, the extent of changes in oxidative stress indices was not same for all three brain 11
regions. Therefore, depending upon which brain region is affected, the function of that brain 12
region will be compromised. The EELS data suggests that mechanisms other than the valence 13
switching between Ce4+ and Ce3+ oxidation states and the possibility to absorb and release 14
oxygen by inducing oxygen vacancies must play a critically important role in pro-oxidant effects 15
of ceria ENM. At present it is difficult to speculate why 5 nm ceria ENM did not cross the BBB. 16
However, oxidative stress effects induced in brain may have been caused by some peripheral 17
inflammatory cytokines that cross the BBB or by ROS generated as a result of long-term 18
accumulation of 5 nm ceria ENM in peripheral organs. 19
20
Conclusions: 21
Although short term time exposure to ceria in vivo leads to a relatively small initial biochemical 22
response (Hardas et al. 2010), ceria administration may prove to be more harmful in the long 23
term. As reported here a single acute dose of 5 nm ceria ENM can adversely affect brain redox 24
status after 30 d. The important point to be noted is that, ceria may induce pro-oxidant effects 25
without crossing or disturbing BBB. The elevated oxidative stress in particular brain regions may 26
20
compromise brain functions and conceivably could even lead to neurodegeneration. Thus, 1
implications of our study are profound for the proposed use of ceria ENM in therapeutic/non-2
therapeutic applications, which may lead to human exposure. Therefore, caution is suggested 3
until and unless such effects as we demonstrated here can be mitigated. 4
5
Acknowledgements: This work was supported by United States Environmental Protection 6
Agency Science to Achieve Results [grant number RD-833772]. Although the research 7
described in this article has been funded wholly or in part by the United States Environmental 8
Protection Agency through STAR Grant RD-833772, it has not been subjected to the Agency’s 9
required peer and policy review and therefore does not necessarily reflect the views of the 10
Agency and no official endorsement should be inferred. 11
12
21
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15
25
Table legend: 1
Table 1: Cerium concentrations in blood, brain, and liver, expressed as concentration and as a 2
percentage of the ceria ENM dose. [Ce] in mg/kg wet weight of the blood or tissue (mean ± SD). 3
4
5
Table 1: Cerium concentrations in blood, brain, and liver 6
Cerium concentration [Ce] (mg/kg) wet weight and as a % of the ceria
ENM dosea
Blood (mg/kg)
Brain (mg/kg)
Liver (mg/kg)
[Ce]
0.11 ± 0.16
0.38 ± 0.52
505 ± 238
% dose
0.01 ± 0.02
0.008 ± 0.009
44 ± 27
7
a Based on reference volume of blood in the rat (7% of body weight) or weight of the brain or 8
liver times the ceria concentration. 9
10
11
12
26
Figure legends: 1
Figure 1: Ceria ENM imaged using HRTEM. The ceria were dispersed on a carbon film. 2
Visually they have a narrow size distribution ranging from 4 to 6 nm, the majority 5 nm. 3
4
Figure 2: In hippocampus 30 d after a single acute dose of 5 nm ceria ENM: a) histograms 5
showing GR, GPx, catalase, antioxidant enzymes and Hsp70 heat shock protein levels and 6
corresponding Western blot experiments showing protein levels in control [C] and treated [T] 7
samples. The intensity of each band was normalized with intensity of corresponding band of β-8
actin-loading control (not shown); b) GR, GPx catalase antioxidant activities, and GSH: GSSG 9
ratio measured in control and ceria treated samples; and c) oxidative stress markers PC, 3NT 10
and HNE levels. The values are calculated as % control for each measurement expressed as 11
mean ± SEM, control n = 7, treated n = 9, *p <0.05, **p < 0.01, compared to control. 12
13
Figure 3: In cortex 30 d after a single acute dose of 5 nm ceria ENM: a) histograms showing 14
GR, GPx, catalase, antioxidant enzymes and Hsp70 heat shock protein levels and 15
corresponding Western blot experiments showing protein levels in control [C] and treated [T] 16
samples. The intensity of each band was normalized with intensity of corresponding band of β-17
actin-loading control (not shown); b) GR, GPx, catalase, antioxidants activities and GSH: GSSG 18
ratio measured in control and ceria treated samples; c) oxidative stress markers PC, 3NT and 19
HNE levels, and d) iNOS levels and correlation between 3NT levels and iNOS levels in ceria 20
treated samples, n=9, r=0.67, p < 0.05. The values are calculated as % control for each 21
measurement expressed as mean ± SEM, control n = 7, treated n = 9, *p <0.05, **p < 0.01, 22
compared to control. 23
24
Figure 4: In cerebellum 30 d after a single acute dose of 5 nm ceria ENM: a) histograms 25
showing GR, GPx, catalase, antioxidant enzymes and Hsp70 heat shock protein levels and 26
27
corresponding Western blot experiments showing protein levels in control [C] and treated [T] 1
samples. The intensity of each band was normalized with intensity of corresponding band of β-2
actin-loading control (not shown); b) GR, GPx, catalase, antioxidants activities, and GSH: 3
GSSG ratio measured in control and ceria treated samples, and c) oxidative stress markers PC, 4
3NT and HNE levels. The values are calculated as % control for each measurement expressed 5
as mean ± SEM, control n = 7, treated n = 8, *p <0.05, **p < 0.01, compared to control. 6
7
Figure 5: In this proposed pathway ceria ENM induces pro-oxidant effects on rat brain without 8
crossing BBB. Ceria ENM indirectly induces ROS production leading to GSH depletion and 9
inhibition of catalase and GPx enzymes. This inhibition of H2O2 reducing enzyme activity can 10
induce H2O2 levels and therefore hydroxyl radicals (OH•) production mediated by Fenton 11
reaction. Increased OH• can further oxidize the proteins and may hamper their regular function. 12
Hydroxyl radicals may also inhibit H2O2 reducing catalase and GPx activity by way of oxidative 13
modification. These biochemical reactions could make cellular environment more oxidizing, 14
triggering a cellular stress response to induce Hsp-70 levels. Hsp-70 is a chaperone protein that 15
can shepherd oxidized proteins to the 20S proteosome for degradation for further cellular 16
clearance (shown as dotted arrow). If timely clearance of oxidized protein takes place then there 17
may not be any change in cellular PC levels. As there was no evidence of the presence of ceria 18
ENM inside the brain, it is further proposed that ceria ENM exert their pro-oxidant effect in the 19
brain secondary to its peripheral effects. 20
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