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Neuro-inflammatory response in rats chronically exposed to 137Cesium
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NeuroToxicology 29 (2008) 343–348
Short communication
Neuro-inflammatory response in rats chronically exposed to
137
Cesium
Philippe Lestaevel *, Line Grandcolas, Franc¸ois Paquet, Philippe Voisin, Jocelyne Aigueperse, Patrick Gourmelon Institut de Radioprotection et de Suˆrete´ Nucle´aire, Direction de la RadioProtection de l’Homme, Service de Radiobiologie et d’Epide´miologie, Laboratoire de Radiotoxicologie expe´rimentale, BP 17, 92262 Fontenay-aux-Roses, France Received 11 May 2007; accepted 4 January 2008 Available online 16 January 2008
Abstract After the Chernobyl nuclear accident, behavioural disorders and central nervous system diseases were frequently observed in populations living in the areas contaminated by 137Cs. Until now, these neurological disturbances were not elucidated, but the presence of a neuro-inflammatory response could be one explanation. Rats were exposed for 3 months to drinking water contaminated with 137Cs at a dose of 400 Bq kg 1, which is similar to that ingested by the population living in contaminated areas in the former USSR countries. Pro-inflammatory and anti-inflammatory cytokine genes were assessed by real-time PCR in the frontal cortex and the hippocampus. At this level of exposure, gene expression of TNF-a and IL-6 increased in the hippocampus and gene expression of IL-10 increased in the frontal cortex. Concentration of TNF-a, measured by ELISA assays, was also increased in the hippocampus. The central NO-ergic pathway was also studied: iNOS gene expression and cNOS activity were significantly increased in the hippocampus. In conclusion, this study showed for the first time that sub-chronic exposure with post-accidental doses of 137Cs leads to molecular modifications of pro- and anti-inflammatory cytokines and NO-ergic pathway in the brain. This neuro-inflammatory response could contribute to the electrophysiological and biochemical alterations observed after chronic exposure to 137Cs. # 2008 Elsevier Inc. All rights reserved. Keywords: Brain; Inflammation; Cytokines; Nitric oxide; Chernobyl
1. Introduction After the Chernobyl disaster, the population living in the contaminated areas was subjected to two types of radiation exposure, i.e., external radiation caused by the surface contamination of the environment and internal contamination actually caused by food and water consumption. At present, the most important contributor to both doses is 137Cs. The biological consequences on the health status for chronic exposure to 137Cs are not well identified. Several ex-USSR authors have described behavioural disorders and diseases of the central nervous system in populations living in radiocontaminated areas (Kamarli and Abdulina, 1996; Kryzhanovskaya, 1997; Titievsky et al., 1997; Bebeshko and Bobyliova, 2002). It has been suggested that 137Cs plays a role in these central effects, even if some of these neurological
* Corresponding author. Tel.: +33 1 58 35 82 84; fax: +33 1 58 35 84 67. E-mail address: [email protected] (P. Lestaevel). 0161-813X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.01.001
signs are similar to those seen with the post-traumatic stress syndrome. From an experimental viewpoint, it was conventionally admitted that 137Cs must not have effect on the central nervous system, except perhaps at very high doses. For example, the evaluation of neurobehavioural function failed to identify significant effect of chronic exposure to 137Cs (Houpert et al., 2007). However, one experimental study has shown significant modifications to the metabolism of some neurotransmitters in rats fed with oats contaminated with 137Cs at a very low dose (45 Bq kg 1) for 1 month (Bandazhevsky and Lelevich, 1995). Indeed, after sub-chronic ingestion during 3 months of 137Cs at 400 Bq kg 1, transient and subtle changes at electrophysiological level were also observed in the rat brain (Lestaevel et al., 2006). These experimental data raised the possibility that some neurological disturbances could occur in the brain after chronic exposure to 137Cs at low doses, but these changes have not so far been comprehensively described. The molecular targets and mechanism of 137Cs effects on the brain are completely unknown. In addition, chronic absorption of 137Cs could initiate
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a cascade of disturbances of brain homeostasis related to inflammatory reactions, which could explain some neurological disturbances observed after exposure to 137Cs. The present study was designed to measure the impact of chronic ingestion of 137Cs, at post-Chernobyl doses, on the brain inflammatory response. This study was performed in rats exposed during a 3month period to 137Cs in the drinking water at a dose similar to that ingested by the population living in contaminated areas just after the Chernobyl accident (400 Bq kg 1) (Handl et al., 2003). The experiments were carried out on the frontal cortex and the hippocampus, two structures strongly implicated in mental disorders observed after exposure to 137Cs. The inflammatory status of the brain was determined via a pattern of cytokines (TNF-a, IL-1b, IL-6, IL-8, TGF-b and IL-10) and via NO-ergic pathway. 2. Materials and methods 2.1. Animals Twenty Sprague–Dawley male rats (Charles River, France), 12 weeks old, weighing about 250 g each, were divided into two groups (control and exposed) of 10 rats each. The animals were housed in pairs, with a 12-h light/12-h dark cycle (light on: 08:00/20:00) and a temperature of 22 1 8C. Food and water were delivered ad libitum. Animal experiments were approved by the Animal Care Committee of the Institute of Radiation Protection and Nuclear Safety and conducted in accordance with the recommendations of the European Animal Care Commission (Act No. 87-848). 2.2. Exposure In the experimental group, the rats were exposed to 137Cs (CERCA, France) given in drinking water for 3 months, at a dose of 6500 Bq L 1 (about 400 Bq kg 1 day 1). This dose was similar to that ingested by the population living in the contaminated areas immediately after the accident (Handl et al., 2003). In the control group, rats drank uncontaminated water. A daily follow-up of animals (body weight, food and water intakes) was ensured until the end of the experiment.
2.3. Tissue collection After 3 months of exposure, animals were anesthetized by inhalation of 95% air/5% isoflurane (Fore`ne, Abobott, France) and euthanized by intracardiac puncture of blood. After decapitation, the brain was dissected on ice and the hippocampus and the frontal cortex were rapidly removed, frozen immediately in liquid nitrogen and stored at 80 8C until used. 2.4. mRNA extraction and RT-PCR experiment Total mRNA was isolated using the RNeasy Lipid Tissue kit (Qiagen, France) according to the manufacturer’s instructions. The cDNA was produced from 1 mg of total mRNA by reverse transcription with a BD Sprint PowerScript 96 Plate (BD Bioscience Clontech, Belgium). PCR amplification of cytokines and NOS used a Cyber PCR master mix. Primer sequences were designed with Primer Express software (Applied Biosystems). Sequences for the forward and reverse primers used in the present study are listed in Table 1. Real-time PCR was performed on an Abi Prism 7700 Sequence Detection System (Applied Biosystems, France) using 10 ng of template DNA for each reaction. PCR fluorescent signals were normalized to the fluorescent signal obtained by the housekeeping gene HPRT for each sample. 2.5. Cytokine assay IL-6 and TNF-a were measured in the supernatant of crushed tissue, in a phosphate buffer, with rat enzyme-linked immunosorbent assays (Cytoscreen rat IL-6 and TNF-a immunoassay, Biosource, France). The measurement of both IL-6 and TNF-a was performed step by step based on the protocol booklet of the ELISA kit. Results were expressed as pg mg 1 protein after a protein assay with Coomassie plus protein assay reagent (Pierce, France). 2.6. NOS activities Total NOS activity was measured by the conversion of L[14C] arginine to L-[14C] citrulline as described previously
Table 1 Primer sequence used for the real-time PCR analysis
IL-1b TNF-a IL-6 IL-8 TGF-b IL-10 cNOS nNOS iNOS HPRT
Forward sequence
Reverse sequence
CAA CAA AAA TGC CTC GTG C CAT CTT CTC AAA ATT CGA GTG ACA A ACA AGT CGG AGG CTT AAT TAC ACA T TTG GAG ACC CCT GCC TGG TCC AAA CGT CGA GGT GAC AGA GAA GCA TGG CCC AGA AAT GCT AAT GGT GTG GCC ATT GAC TGA CAA GGT GAC CAT CGT TGA CAG CTG GGC TGT ACA AAC CTT CTC GAG ATG TCA TGA AGG AGA
TGC TGA TGT ACC AGT TGG G TGG GAG TAG ACA AGG TAC AAC CC TTG CCA TTG CAC AAC TCT TTT C ACT TCT CCA CAA CCC TCT GC CAG GTG TTG AGC CCT TTC CA CGC ATC CTG AGG GTC TTC A GGT TCT ATC TCT TTG AGC AGT TCG T GTG ATG CTG CCC GAC ATG AT TGG AAG TGA AGC GTT TCG TCA GCG CTT TAA TGT AAT CCA GC
Abbreviations: IL-1b, interleukin 1b; TNF-a, tumor necrosis factor a; IL-6, interleukin 6; IL-8, interleukin 8; TGF-b, transforming growth factor b; IL-10, interleukin 10; cNOS, nitric oxide synthase constitutive; iNOS, nitric oxide synthase inducible; HPRT, housekeeping gene hypoxanthine-guanine phosphorybosyltransferase.
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(Iadecola et al., 1995). Briefly, brain samples were homogenized with 10 volumes of ice-cold homogenization buffer (250 mM Tris–HCl, 10 mM EDTA, 10 mM EGTA, pH 7.4) and centrifuged for 15 min at 10,000 g at 4 8C. Supernatants (10 ml) were then incubated for 60 min at 37 8C with 40 ml of reaction mixture containing 10 mM NADPH, 6 mM CaCl2, 50 mCi/ml L-[14C] arginine in a reaction buffer (50 mM Tris– HCl, 6 mM BH4, 2 mM flavin adenine dinucleotide, 2 mM flavin adenine mononucleotide, pH 7.4) and 40 ml of distilled water. The reaction was stopped by adding 400 ml of ice-cold stop buffer (50 mM HEPES and 5 mM EDTA, pH 5.5) and applied to DOWEX AG50WX8-400 columns to remove L-[14C] arginine. Columns were eluted and L-[14C] citrulline was measured by scintillation counting. For iNOS activity, which is Ca2+ independent, CaCl2 was substituted by distilled water. Activity for cNOS was calculated by subtracting the iNOS activity from total NOS activity. 2.7. Levels of NO metabolites (NOx ) Brain samples were homogenized in PBS (pH 7.4) and centrifuged at 10,000 g for 20 min at 4 8C. The supernatant fraction was used to determine the levels of NO metabolites (NOx ) by assaying nitrite (NO2 ) plus nitrate (NO3 ). Nitrate was reduced to nitrite with nitrate reductase. One hour after incubation at room temperature, the concentration of this final form was determined by a colorimetric assay based on the Griess reaction, as previously described (Ricart-Jane´ et al., 2002). The absorbance was read at 550 nm and data were expressed in mM. 2.8. Statistical analysis All data are expressed as the mean standard error of the mean (S.E.M.) for 6–10 animals. Statistical comparisons between control and exposed groups were performed using a Mann–Whitney test. Differences were considered significant when p < 0.05. 3. Results and discussion Brain inflammation is a common feature among many neuropathological processes and can be observed in Alzheimer’s disease, multiple sclerosis or chronic pain (Law et al., 2001; Chauvet et al., 2001). This neuro-inflammatory response can be characterized by expression of inflammatory molecules including cytokines and nitric oxide (Law et al., 2001). After a 137 Cs exposure during 3 months, our study showed that mRNA levels of TNF-a and IL-6 were not significantly modified in the frontal cortex (Fig. 1A). However, in the hippocampus, subchronic exposure to 137Cs for 3 months led to a significant increase in mRNA levels (+32%, p < 0.05) and concentrations (41.7 3.2 pg mg 1 protein for 137Cs group versus 32.4 2.1 pg mg 1 protein for control group, p < 0.05) of TNF-a compared to the controls (Fig. 1B). Similarly, 137Cs also led to a significant increase of +56% in IL-6 mRNA levels ( p < 0.05) (Fig. 1B). By ELISA assays, IL-6 concentrations
Fig. 1. Effect of 137Cs exposure on the gene expression of pro-inflammatory cytokines in the frontal cortex (A) and hippocampus (B). Measurements were carried out in control and exposed animals following a 3-month period of exposure. IL-1b, TNF-a, IL-6 and IL-8 were expressed as a ratio to the reference gene HPRT. Control values were normalized to 1. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
were not detectable in the hippocampus of exposed and control animals. These results are in accordance with previous studies that demonstrated an increase of TNF-a and IL-6 after very high dose of external ionizing irradiation (Hong et al., 1995; Marquette et al., 2003). TNF-a and IL-6 production may be a part of adaptive survival response by tissues to pathological situations. They could have both detrimental and beneficial effects. In vitro, TNF-a has been shown to be cytotoxic to oligodendrocytes (Selmaj and Raine, 1988), but also to protect neurons against glucose deprivation-induced injury and excitatory amino acid toxicity (Cheng et al., 1994), and to stimulate proliferation of microglial cells and astrocytes (Merrill, 1992). In vivo, TNF-a has been shown to increase vascular and blood–brain barrier permeability (Claudio et al., 1994). We could hypothesize that chronic exposure to 137Cs increased vascular permeability, via this inflammatory cytokine. Gene expression for other pro-inflammatory cytokines, IL-1b and IL-8, were not significantly altered by 137Cs in the frontal cortex and the hippocampus (Fig. 1A and B). These results demonstrate that the expression of IL-1b/IL-8 is differentially regulated in the brain compared to TNF-a/IL-6 after sub-chronic exposure to 137Cs. Concerning anti-inflammatory cytokines, the gene expression for TGF-b, which protects neurones by down-regulating the responses of glial cells to stimuli (Vitkovic et al., 2001), was not significantly modified by sub-chronic exposure to 137Cs in cerebral structures examined in the present study (Fig. 2A and B). The gene expression for IL-10, another anti-inflammatory
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Fig. 2. Effect of 137Cs exposure on the gene expression of anti-inflammatory cytokines in the frontal cortex (A) and hippocampus (B). Measurements were carried out in control and exposed animals following a 3-month period of exposure. IL-10 and TGF-b were expressed as a ratio to the reference gene HPRT. Control values were normalized to 1. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
cytokine, was significantly increased in the frontal cortex of exposed rats in comparison to the controls (+79%, p < 0.05), but not in the hippocampus (Fig. 2A and B). Most IL-10 functions are best described outside of the central nervous system, but recent findings extended the role of IL-10 in brain. For example, IL-10 plays a role in modulation of the microglial
function (Sawada et al., 1999). We could hypothesize that IL-10 has neuroprotective properties against 137Cs. Brain inflammation can be characterized by cytokine expression, but also by other molecules, such as nitric oxide (NO). The reactive nature of NO with oxygen free radicals suggests that NO participates in neuro-inflammatory response. In the frontal cortex, NO-ergic pathway, i.e. cNOS, nNOS, iNOS gene expression and activities were not significantly modified in comparison to the controls (Fig. 3A and C). In the hippocampus, the gene expression of iNOS, whose expression is stimulated by immunological molecules such as cytokines (Droge, 2002) was significantly increased (+62%, p < 0.05) (Fig. 3B). 137Cs increased also significantly the activity of cNOS in the hippocampus (+43%, p < 0.05) (Fig. 3D). This lack of correlation between the gene expression of iNOS and its activity in the hippocampus could be explained by the alteration of post-transcription events such as translation and transport of gene products. It could result in loss of functional molecules, minor post-transductional changes, and, finally, a lack of changes in iNOS enzyme activity. In order to evaluate the consequences of a chronic exposure to 137Cs on NO catabolism, NOx levels were measured in cortical and hippocampal tissues. No significant change in cortical (2.84 0.31 mM vs. 3.79 0.35 mM) and hippocampal (9.22 0.73 mM vs. 11.56 1.08 mM) NOx levels was observed in the exposed group compared to the control. These results exemplified perfectly that, in our experimental conditions, nitrites/nitrates in the cortex and the hippocampus do not closely reflect NOS changes in the brain. In other experimental conditions, such as after gamma-ray exposure (15 Gy), the same kind of result was observed, with an increase in gene expression of iNOS, without any significant effect on NOx (Lestaevel et al., 2003). However, the central NO-ergic pathway could constitute a good index of inflammatory response to 137Cs, at least in the hippocampus.
Fig. 3. Effect of 137Cs exposure on the gene expression (A and B) and enzymatic activities (C and D) of constitutive NOS (cNOS) and inducible NOS (iNOS) in the frontal cortex and hippocampus. Measurements were carried out in control and exposed animals following a 3-month period of exposure. Gene expressions were expressed as a ratio to the reference gene HPRT. For gene expression, control values were normalized to 1. Activities were expressed as a percent of the control. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
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The present investigation was mainly performed on cerebral cortex and hippocampus because there is a relation between the functions of these brain areas and mental disorders, such as lack of concentration, memory impairment, and high level of anxiety, observed in the population living in radio-contaminated areas after the Chernobyl accident (Havenaar et al., 2003; Kryzhanovskaya, 1997; Titievsky et al., 1997; Gamache et al., 2005). In others cerebral structures, such as the hypothalamus, no significant effect on inflammatory cytokines and NO pathway was observed (data not shown). In the brainstem, the only significant effect has been observed at level of IL-10 gene expression (+55%, p < 0.05, data not shown), as in the frontal cortex. These results strongly suggest that there is a dependantcerebral structure response after daily ingestion of 137Cs. These inflammatory changes could be related to a differential accumulation of 137Cs in these brain areas. But, after exposure over a 3-month period, the quantity of 137Cs in the cerebral structures was very similar (2.81 0.11 Bq g 1 in the frontal cortex; 2.94 0.15 Bq g 1 in the hippocampus; 4.26 0.12 Bq g 1 in the brainstem) (Lestaevel et al., 2006). Consequently, the differential effects on cytokines or NO-ergic pathway demonstrated here cannot be directly explained by local accumulations of 137Cs in the brain areas. Clearly, each brain region has its own specific blend of function, cell composition, connectivity and other relevant characteristics. This differential response to 137Cs exposure in these cerebral structures has been previously observed in other experimental situations. For example, a region-specific brain cytokine mRNA induction was demonstrated after instillation of ambient air ultrafine particles (Win-Shwe et al., 2005). The hippocampus appeared to be affected more than the cortex, a finding that is in line with other newer literature on the role of inflammation in neurodegeneration. A direct link between neuro-inflammation and hippocampal neurodegeneration during aging or Alzheimer disease has been recently demonstrated (Craft et al., 2006; Gavilan et al., 2007). Moreover, several cytokines and inflammatory mediators produced by activated glia in various areas of the hippocampal formation have the potential to initiate or exacerbate the progression of neuropathology of Alzheimer disease (Stepanichev et al., 2006). These recent findings suggest that the neuro-inflammation observed in the hippocampus after chronic ingestion of 137Cs could be a potential factor in a possible neurodegeneration. This point must be confirmed in the future. There is a relationship between the increase of pro- or antiinflammatory cytokines, and the kind of neurological symptoms shown by Chernobyl’s accident survivals or experimental chronic exposures. In prenatally irradiated children, who where born between April 1986 and February 1987 in regions of the Ukraine (Loganovskaja and Loganovsky, 1999) or after chronic exposure to 137Cs in rats (Lestaevel et al., 2006), a significant increase of slow frequency waves (<4 Hz) was observed. It was previously demonstrated that slow wave activity increased following administration of TNF-a (Opp, 2005). These results suggest that the 137Cs-induced inflammation response could contribute to electroencephalogram perturbation after exposure to 137Cs. Furthermore, one experimental study reports
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perturbations on the metabolism of serotonin, taurine, alanine, serine, glutamate and glycine, in rats after feeding with oats contaminated with 137Cs for 28 days (45 Bq kg 1) (Bandazhevsky and Lelevich, 1995). Among these disturbed metabolisms, some are regulated by cytokines or NO, such as serotonin or glutamate (Blatteis, 1990; Raber et al., 1998). Complementary studies should be carried out to better understand the role of pro- and anti-inflammatory response on neurotransmitters perturbations observed after chronic exposure to 137Cs. The increase of cytokines and NO in the brain after subchronic exposure to 137Cs underlies the existence of some inflammatory features but without histological evidence of such inflammation and any health effects (data not shown). Here, the rats were healthy throughout the experimental period: their food and water intakes and body weight gain were not affected. These results are in line with a previous study that also found no significant effect on general health parameters in rats in similar experimental conditions (Lestaevel et al., 2006). However, this apparent good state of health does not imply that the perturbation of central homeostasis is not in relation with a peripheral inflammation. The limited brain inflammatory response to 137Cs exposure observed in the present study could be a consequence of a systemic inflammatory response. There are a number of different routes by which a systemic inflammatory response may communicate with the central nervous system (Konsman and Dantzer, 2002). Among these routes, one involved the sensory afferents of the vagus nerve which communicate with neuronal populations within the brain following inflammation induced in the peritoneal cavity. This phenomenon has been already described after ionizing radiation, where the vagus nerve appears to be one of the major ascending pathways for a signal of systemic inflammation to the brain (Marquette et al., 2003). However, Dublineau et al. have previously demonstrated that after chronic exposure to 137Cs, inflammatory cytokines, i.e. IL-10, IL-8, IL-4 and TGF-b, on peripheral tissues, such as intestine, were not significantly modified compared to control (Dublineau et al., 2007). At plasma level, no renal and hepatic disturbances were observed (Tissandie et al., 2006). Moreover, in order to known the source of the increases cytokines in the brain, some animals have been perfused to eliminate blood cells. In these conditions, the same kinds of results have been obtained in ‘‘perfused rats’’ and in ‘‘not perfused rats’’ (data not shown). All these results suggested the specificity of the inflammatory changes to the brain. In conclusion, the present in vivo study demonstrated for the first time that a sub-chronic exposure to 137Cs at postChernobyl dose over a 3-month period leads to subtle molecular modifications on pro- and anti-inflammatory cytokines and on NO-ergic pathway. This inflammatory response seem to be cerebral structure dependant. The 137Cs-induced inflammation response may contribute to the sensitivity of the central nervous system to 137Cs. It could contribute to the perturbation of electroencephalogram and neurotransmitter synthesis and catalysis observed after chronic exposure to 137Cs (Lestaevel et al., 2006; Bandazhevsky and Lelevich, 1995). It will be crucial for people living in the radio-contaminated areas after
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the Chernobyl accident to determine experimentally whether 137 Cs exposure could induce similar phenomena after longer periods of exposure and at different exposure levels. Finally, this study remains a relatively novel undertaking and further experiments should be carried out to analyse genes involved in cytokine signalling as transcription factors or suppressors of cytokine signalling and to analyse cytokine-induced genes. Acknowledgements We thank C. Baudelin and T. Loiseau for their assistance during experimentation and animal exposure. This work is part of the Envirhom programme supported by the Institute for Radioprotection and Nuclear Safety. References Bandazhevsky YI, Lelevich VV. Clinical and experimental aspects of the effect of incorporated radionuclides upon the organism. Gomel, Belarus: Byelorussian Engineering Academy, Gomel State Medical Institute; 1995. Bebeshko VG, Bobyliova OA. Medical consequences of the Chernobyl nuclear power plant accident: experience of 15-year studies. Int Congr Series 2002;1234:267–79. Blatteis CM. Neuromodulative actions of cytokines. Yale J Biol Med 1990;63:133–46. Chauvet N, Palin K, Verrier D, Poole S, Dantzer R, Lestage J. Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide. Eur J Neurosci 2001;14:609–17. Cheng B, Christakos S, Mattson MP. Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron 1994;12:139–53. Claudio L, Martiney JA, Brosnan CF. Ultrastructural studies of the blood-retina barrier after exposure to interleukin-1 beta or tumor necrosis factor-alpha. Lab Invest 1994;70:850–61. Craft JM, Watterson DM, Van Eldik LJ. Human amyloid beta-induced neuroinflammation is an early event in neurodegeneration. Glia 2006;53:484–90. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47–95. Dublineau I, Grison S, Grandcolas L, Baudelin C, Paquet F, Voisin P, et al. Effects of chronic 137Cs ingestion on barrier properties of jejunal epithelium in rats. J Toxicol Environ Health A 2007;70:810–9. Gamache GL, Levinson DM, Reeves DL, Bidyuk PI, Brantley KK. Longitudinal neurocognitive assessments of Ukrainians exposed to ionizing radiation after the Chernobyl nuclear accident. Arch Clin Neuropsychol 2005;20:81–93. Gavilan MP, Revilla E, Pintado C, Castan˜o A, Vizuete ML, Moreno-Gonza´lez I, et al. Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons. J Neurochem 2007;103:984–96. Handl J, Beltz D, Botsh W, Harb S, Jakob D, Michel R, et al. Evaluation of radioactive exposure from 137Cs in contaminated areas of northern Ukraine. Health Phys 2003;84:502–17. Havenaar JM, De Wilde EJ, Van Den Bout J, Drottz-Sjoberg BM, Van Den Brink W. Perception of risk and subjective health among victims of the Chernobyl disaster. Soc Sci Med 2003;56:569–72.
Hong JH, Chiang CS, Campbell IL, Sun JR, Withers HR, McBridge WH. Induction of acute phase gene expression by brain irradiation. Int J Radiat Oncol Biol Phys 1995;33:619–26. Houpert P, Bizot JC, Bussy C, Dhieux B, Lestaevel P, Gourmelon P, et al. Comparison of the effects of enriched uranium and 137-cesium on the behaviour of rats after chronic exposure. Int J Radiat Biol 2007;83:99–104. Iadecola C, Xu X, Zhang F, el-Fakahany EE, Ross ME. Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia. J Cereb Blood Flow Metab 1995;15:52–9. Kamarli Z, Abdulina A. Health conditions among workers who participated in the cleanup of the Chernobyl accident. World Health Stat Q 1996;49:29–31. Konsman JP, Dantzer R. How the immune and nervous system interact during disease associated-anorexia. Nutrition 2002;17:664–8. Kryzhanovskaya L. Psychological dysfunction in the Chernobyl disaster victims. Eur Psychiatry 1997;12:161S. Law A, Gauthier S, Quirion R. Say NO to Alzheimer’s disease: the putative links between nitric oxide and dementia of the Alzheimer’s type. Brain Res Rev 2001;35:73–96. Lestaevel P, Clarenc¸on D, Gharib A, Peinnequin A, Cespuglio R, Gourmelon P, et al. Nitric oxide voltammetric measurements in the rat brain after gamma irradiation. Radiat Res 2003;160:631–6. Lestaevel P, Dhieux B, Tourlonias E, Houpert P, Paquet F, Voisin P, et al. Evaluation of the effect of chronic exposure to 137Cesium on sleep-wake cycle in rats. Toxicology 2006;226:118–25. Loganovskaja TK, Loganovsky KN. EEG, cognitive and psychopathological abnormalities in children irradiated in utero. Int J Psychophysiol 1999;34:213–24. Marquette C, Linard C, Galonnier M, Van Uye A, Mathieu J, Gourmelon P, et al. Il-1b, TNF-a and IL-6 induction in the rat brain after partial-body irradiation: role of vagal afferents. Int J Radiat Biol 2003;79:777–85. Merrill JE. Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological. Dev Neurosci 1992;14:1–10. Opp MR. Cytokines and sleep. Sleep Med Rev 2005;9:355–64. Raber J, Sorg O, Horn TF, Yu N, Koob GF, Campbell IL, et al. Inflammatory cytokines: putatives regulators of neuronal and neuro-endocrine function. Brain Res Rev 1998;26:320–6. Ricart-Jane´ D, Llobera M, Lopez-Tejero D. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Greiss method. Nitric Oxide 2002;6:178–85. Sawada M, Suzumura A, Hosoya H, Marunouchi T, Nagatsu T. Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J Neurochem 1999;72:1466–71. Selmaj KW, Raine CS. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol 1988;23:339–46. Stepanichev M, Zdobnova I, Zarubenko I, Lazareva N, Gulyaeva NV. Differential effects of tumor necrosis factor-alpha co-administered with amyloid beta-peptide (25-35) on memory function and hippocampal damage in rat. Behav Brain Res 2006;175:352–61. Tissandie E, Gue´guen Y, Lobaccaro JM, Aigueperse J, Gourmelon P, Paquet F, et al. Chronic contamination with 137Cesium affects Vitamin D3 metabolism in rats. Toxicology 2006;225:75–80. Titievsky SV, Tabachnikov SI, Kutko II. Complex approach to mental disorders after Chernobyl accident estimation. Biol Psychiatry 1997;42:23S. Vitkovic L, Maeda S, Sternberg E. Anti-inflammatory cytokines: expression and action in the brain. Neuroimmunomodulation 2001;9:295–312. Win-Shwe TT, Yamamoto S, Ahmed S, Kakeyama M, Kobayashi T, Fujimaki H. Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black. Toxicol Lett 2005;163: 153–60.
NeuroToxicology 29 (2008) 343–348
Short communication
Neuro-inflammatory response in rats chronically exposed to
137
Cesium
Philippe Lestaevel *, Line Grandcolas, Franc¸ois Paquet, Philippe Voisin, Jocelyne Aigueperse, Patrick Gourmelon Institut de Radioprotection et de Suˆrete´ Nucle´aire, Direction de la RadioProtection de l’Homme, Service de Radiobiologie et d’Epide´miologie, Laboratoire de Radiotoxicologie expe´rimentale, BP 17, 92262 Fontenay-aux-Roses, France Received 11 May 2007; accepted 4 January 2008 Available online 16 January 2008
Abstract After the Chernobyl nuclear accident, behavioural disorders and central nervous system diseases were frequently observed in populations living in the areas contaminated by 137Cs. Until now, these neurological disturbances were not elucidated, but the presence of a neuro-inflammatory response could be one explanation. Rats were exposed for 3 months to drinking water contaminated with 137Cs at a dose of 400 Bq kg 1, which is similar to that ingested by the population living in contaminated areas in the former USSR countries. Pro-inflammatory and anti-inflammatory cytokine genes were assessed by real-time PCR in the frontal cortex and the hippocampus. At this level of exposure, gene expression of TNF-a and IL-6 increased in the hippocampus and gene expression of IL-10 increased in the frontal cortex. Concentration of TNF-a, measured by ELISA assays, was also increased in the hippocampus. The central NO-ergic pathway was also studied: iNOS gene expression and cNOS activity were significantly increased in the hippocampus. In conclusion, this study showed for the first time that sub-chronic exposure with post-accidental doses of 137Cs leads to molecular modifications of pro- and anti-inflammatory cytokines and NO-ergic pathway in the brain. This neuro-inflammatory response could contribute to the electrophysiological and biochemical alterations observed after chronic exposure to 137Cs. # 2008 Elsevier Inc. All rights reserved. Keywords: Brain; Inflammation; Cytokines; Nitric oxide; Chernobyl
1. Introduction After the Chernobyl disaster, the population living in the contaminated areas was subjected to two types of radiation exposure, i.e., external radiation caused by the surface contamination of the environment and internal contamination actually caused by food and water consumption. At present, the most important contributor to both doses is 137Cs. The biological consequences on the health status for chronic exposure to 137Cs are not well identified. Several ex-USSR authors have described behavioural disorders and diseases of the central nervous system in populations living in radiocontaminated areas (Kamarli and Abdulina, 1996; Kryzhanovskaya, 1997; Titievsky et al., 1997; Bebeshko and Bobyliova, 2002). It has been suggested that 137Cs plays a role in these central effects, even if some of these neurological
* Corresponding author. Tel.: +33 1 58 35 82 84; fax: +33 1 58 35 84 67. E-mail address: [email protected] (P. Lestaevel). 0161-813X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.01.001
signs are similar to those seen with the post-traumatic stress syndrome. From an experimental viewpoint, it was conventionally admitted that 137Cs must not have effect on the central nervous system, except perhaps at very high doses. For example, the evaluation of neurobehavioural function failed to identify significant effect of chronic exposure to 137Cs (Houpert et al., 2007). However, one experimental study has shown significant modifications to the metabolism of some neurotransmitters in rats fed with oats contaminated with 137Cs at a very low dose (45 Bq kg 1) for 1 month (Bandazhevsky and Lelevich, 1995). Indeed, after sub-chronic ingestion during 3 months of 137Cs at 400 Bq kg 1, transient and subtle changes at electrophysiological level were also observed in the rat brain (Lestaevel et al., 2006). These experimental data raised the possibility that some neurological disturbances could occur in the brain after chronic exposure to 137Cs at low doses, but these changes have not so far been comprehensively described. The molecular targets and mechanism of 137Cs effects on the brain are completely unknown. In addition, chronic absorption of 137Cs could initiate
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a cascade of disturbances of brain homeostasis related to inflammatory reactions, which could explain some neurological disturbances observed after exposure to 137Cs. The present study was designed to measure the impact of chronic ingestion of 137Cs, at post-Chernobyl doses, on the brain inflammatory response. This study was performed in rats exposed during a 3month period to 137Cs in the drinking water at a dose similar to that ingested by the population living in contaminated areas just after the Chernobyl accident (400 Bq kg 1) (Handl et al., 2003). The experiments were carried out on the frontal cortex and the hippocampus, two structures strongly implicated in mental disorders observed after exposure to 137Cs. The inflammatory status of the brain was determined via a pattern of cytokines (TNF-a, IL-1b, IL-6, IL-8, TGF-b and IL-10) and via NO-ergic pathway. 2. Materials and methods 2.1. Animals Twenty Sprague–Dawley male rats (Charles River, France), 12 weeks old, weighing about 250 g each, were divided into two groups (control and exposed) of 10 rats each. The animals were housed in pairs, with a 12-h light/12-h dark cycle (light on: 08:00/20:00) and a temperature of 22 1 8C. Food and water were delivered ad libitum. Animal experiments were approved by the Animal Care Committee of the Institute of Radiation Protection and Nuclear Safety and conducted in accordance with the recommendations of the European Animal Care Commission (Act No. 87-848). 2.2. Exposure In the experimental group, the rats were exposed to 137Cs (CERCA, France) given in drinking water for 3 months, at a dose of 6500 Bq L 1 (about 400 Bq kg 1 day 1). This dose was similar to that ingested by the population living in the contaminated areas immediately after the accident (Handl et al., 2003). In the control group, rats drank uncontaminated water. A daily follow-up of animals (body weight, food and water intakes) was ensured until the end of the experiment.
2.3. Tissue collection After 3 months of exposure, animals were anesthetized by inhalation of 95% air/5% isoflurane (Fore`ne, Abobott, France) and euthanized by intracardiac puncture of blood. After decapitation, the brain was dissected on ice and the hippocampus and the frontal cortex were rapidly removed, frozen immediately in liquid nitrogen and stored at 80 8C until used. 2.4. mRNA extraction and RT-PCR experiment Total mRNA was isolated using the RNeasy Lipid Tissue kit (Qiagen, France) according to the manufacturer’s instructions. The cDNA was produced from 1 mg of total mRNA by reverse transcription with a BD Sprint PowerScript 96 Plate (BD Bioscience Clontech, Belgium). PCR amplification of cytokines and NOS used a Cyber PCR master mix. Primer sequences were designed with Primer Express software (Applied Biosystems). Sequences for the forward and reverse primers used in the present study are listed in Table 1. Real-time PCR was performed on an Abi Prism 7700 Sequence Detection System (Applied Biosystems, France) using 10 ng of template DNA for each reaction. PCR fluorescent signals were normalized to the fluorescent signal obtained by the housekeeping gene HPRT for each sample. 2.5. Cytokine assay IL-6 and TNF-a were measured in the supernatant of crushed tissue, in a phosphate buffer, with rat enzyme-linked immunosorbent assays (Cytoscreen rat IL-6 and TNF-a immunoassay, Biosource, France). The measurement of both IL-6 and TNF-a was performed step by step based on the protocol booklet of the ELISA kit. Results were expressed as pg mg 1 protein after a protein assay with Coomassie plus protein assay reagent (Pierce, France). 2.6. NOS activities Total NOS activity was measured by the conversion of L[14C] arginine to L-[14C] citrulline as described previously
Table 1 Primer sequence used for the real-time PCR analysis
IL-1b TNF-a IL-6 IL-8 TGF-b IL-10 cNOS nNOS iNOS HPRT
Forward sequence
Reverse sequence
CAA CAA AAA TGC CTC GTG C CAT CTT CTC AAA ATT CGA GTG ACA A ACA AGT CGG AGG CTT AAT TAC ACA T TTG GAG ACC CCT GCC TGG TCC AAA CGT CGA GGT GAC AGA GAA GCA TGG CCC AGA AAT GCT AAT GGT GTG GCC ATT GAC TGA CAA GGT GAC CAT CGT TGA CAG CTG GGC TGT ACA AAC CTT CTC GAG ATG TCA TGA AGG AGA
TGC TGA TGT ACC AGT TGG G TGG GAG TAG ACA AGG TAC AAC CC TTG CCA TTG CAC AAC TCT TTT C ACT TCT CCA CAA CCC TCT GC CAG GTG TTG AGC CCT TTC CA CGC ATC CTG AGG GTC TTC A GGT TCT ATC TCT TTG AGC AGT TCG T GTG ATG CTG CCC GAC ATG AT TGG AAG TGA AGC GTT TCG TCA GCG CTT TAA TGT AAT CCA GC
Abbreviations: IL-1b, interleukin 1b; TNF-a, tumor necrosis factor a; IL-6, interleukin 6; IL-8, interleukin 8; TGF-b, transforming growth factor b; IL-10, interleukin 10; cNOS, nitric oxide synthase constitutive; iNOS, nitric oxide synthase inducible; HPRT, housekeeping gene hypoxanthine-guanine phosphorybosyltransferase.
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(Iadecola et al., 1995). Briefly, brain samples were homogenized with 10 volumes of ice-cold homogenization buffer (250 mM Tris–HCl, 10 mM EDTA, 10 mM EGTA, pH 7.4) and centrifuged for 15 min at 10,000 g at 4 8C. Supernatants (10 ml) were then incubated for 60 min at 37 8C with 40 ml of reaction mixture containing 10 mM NADPH, 6 mM CaCl2, 50 mCi/ml L-[14C] arginine in a reaction buffer (50 mM Tris– HCl, 6 mM BH4, 2 mM flavin adenine dinucleotide, 2 mM flavin adenine mononucleotide, pH 7.4) and 40 ml of distilled water. The reaction was stopped by adding 400 ml of ice-cold stop buffer (50 mM HEPES and 5 mM EDTA, pH 5.5) and applied to DOWEX AG50WX8-400 columns to remove L-[14C] arginine. Columns were eluted and L-[14C] citrulline was measured by scintillation counting. For iNOS activity, which is Ca2+ independent, CaCl2 was substituted by distilled water. Activity for cNOS was calculated by subtracting the iNOS activity from total NOS activity. 2.7. Levels of NO metabolites (NOx ) Brain samples were homogenized in PBS (pH 7.4) and centrifuged at 10,000 g for 20 min at 4 8C. The supernatant fraction was used to determine the levels of NO metabolites (NOx ) by assaying nitrite (NO2 ) plus nitrate (NO3 ). Nitrate was reduced to nitrite with nitrate reductase. One hour after incubation at room temperature, the concentration of this final form was determined by a colorimetric assay based on the Griess reaction, as previously described (Ricart-Jane´ et al., 2002). The absorbance was read at 550 nm and data were expressed in mM. 2.8. Statistical analysis All data are expressed as the mean standard error of the mean (S.E.M.) for 6–10 animals. Statistical comparisons between control and exposed groups were performed using a Mann–Whitney test. Differences were considered significant when p < 0.05. 3. Results and discussion Brain inflammation is a common feature among many neuropathological processes and can be observed in Alzheimer’s disease, multiple sclerosis or chronic pain (Law et al., 2001; Chauvet et al., 2001). This neuro-inflammatory response can be characterized by expression of inflammatory molecules including cytokines and nitric oxide (Law et al., 2001). After a 137 Cs exposure during 3 months, our study showed that mRNA levels of TNF-a and IL-6 were not significantly modified in the frontal cortex (Fig. 1A). However, in the hippocampus, subchronic exposure to 137Cs for 3 months led to a significant increase in mRNA levels (+32%, p < 0.05) and concentrations (41.7 3.2 pg mg 1 protein for 137Cs group versus 32.4 2.1 pg mg 1 protein for control group, p < 0.05) of TNF-a compared to the controls (Fig. 1B). Similarly, 137Cs also led to a significant increase of +56% in IL-6 mRNA levels ( p < 0.05) (Fig. 1B). By ELISA assays, IL-6 concentrations
Fig. 1. Effect of 137Cs exposure on the gene expression of pro-inflammatory cytokines in the frontal cortex (A) and hippocampus (B). Measurements were carried out in control and exposed animals following a 3-month period of exposure. IL-1b, TNF-a, IL-6 and IL-8 were expressed as a ratio to the reference gene HPRT. Control values were normalized to 1. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
were not detectable in the hippocampus of exposed and control animals. These results are in accordance with previous studies that demonstrated an increase of TNF-a and IL-6 after very high dose of external ionizing irradiation (Hong et al., 1995; Marquette et al., 2003). TNF-a and IL-6 production may be a part of adaptive survival response by tissues to pathological situations. They could have both detrimental and beneficial effects. In vitro, TNF-a has been shown to be cytotoxic to oligodendrocytes (Selmaj and Raine, 1988), but also to protect neurons against glucose deprivation-induced injury and excitatory amino acid toxicity (Cheng et al., 1994), and to stimulate proliferation of microglial cells and astrocytes (Merrill, 1992). In vivo, TNF-a has been shown to increase vascular and blood–brain barrier permeability (Claudio et al., 1994). We could hypothesize that chronic exposure to 137Cs increased vascular permeability, via this inflammatory cytokine. Gene expression for other pro-inflammatory cytokines, IL-1b and IL-8, were not significantly altered by 137Cs in the frontal cortex and the hippocampus (Fig. 1A and B). These results demonstrate that the expression of IL-1b/IL-8 is differentially regulated in the brain compared to TNF-a/IL-6 after sub-chronic exposure to 137Cs. Concerning anti-inflammatory cytokines, the gene expression for TGF-b, which protects neurones by down-regulating the responses of glial cells to stimuli (Vitkovic et al., 2001), was not significantly modified by sub-chronic exposure to 137Cs in cerebral structures examined in the present study (Fig. 2A and B). The gene expression for IL-10, another anti-inflammatory
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Fig. 2. Effect of 137Cs exposure on the gene expression of anti-inflammatory cytokines in the frontal cortex (A) and hippocampus (B). Measurements were carried out in control and exposed animals following a 3-month period of exposure. IL-10 and TGF-b were expressed as a ratio to the reference gene HPRT. Control values were normalized to 1. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
cytokine, was significantly increased in the frontal cortex of exposed rats in comparison to the controls (+79%, p < 0.05), but not in the hippocampus (Fig. 2A and B). Most IL-10 functions are best described outside of the central nervous system, but recent findings extended the role of IL-10 in brain. For example, IL-10 plays a role in modulation of the microglial
function (Sawada et al., 1999). We could hypothesize that IL-10 has neuroprotective properties against 137Cs. Brain inflammation can be characterized by cytokine expression, but also by other molecules, such as nitric oxide (NO). The reactive nature of NO with oxygen free radicals suggests that NO participates in neuro-inflammatory response. In the frontal cortex, NO-ergic pathway, i.e. cNOS, nNOS, iNOS gene expression and activities were not significantly modified in comparison to the controls (Fig. 3A and C). In the hippocampus, the gene expression of iNOS, whose expression is stimulated by immunological molecules such as cytokines (Droge, 2002) was significantly increased (+62%, p < 0.05) (Fig. 3B). 137Cs increased also significantly the activity of cNOS in the hippocampus (+43%, p < 0.05) (Fig. 3D). This lack of correlation between the gene expression of iNOS and its activity in the hippocampus could be explained by the alteration of post-transcription events such as translation and transport of gene products. It could result in loss of functional molecules, minor post-transductional changes, and, finally, a lack of changes in iNOS enzyme activity. In order to evaluate the consequences of a chronic exposure to 137Cs on NO catabolism, NOx levels were measured in cortical and hippocampal tissues. No significant change in cortical (2.84 0.31 mM vs. 3.79 0.35 mM) and hippocampal (9.22 0.73 mM vs. 11.56 1.08 mM) NOx levels was observed in the exposed group compared to the control. These results exemplified perfectly that, in our experimental conditions, nitrites/nitrates in the cortex and the hippocampus do not closely reflect NOS changes in the brain. In other experimental conditions, such as after gamma-ray exposure (15 Gy), the same kind of result was observed, with an increase in gene expression of iNOS, without any significant effect on NOx (Lestaevel et al., 2003). However, the central NO-ergic pathway could constitute a good index of inflammatory response to 137Cs, at least in the hippocampus.
Fig. 3. Effect of 137Cs exposure on the gene expression (A and B) and enzymatic activities (C and D) of constitutive NOS (cNOS) and inducible NOS (iNOS) in the frontal cortex and hippocampus. Measurements were carried out in control and exposed animals following a 3-month period of exposure. Gene expressions were expressed as a ratio to the reference gene HPRT. For gene expression, control values were normalized to 1. Activities were expressed as a percent of the control. Data are expressed as mean S.E.M.; n = 6–10 for each group of rats; (&) control; (&) cesium; *significantly different from control ( p < 0.05).
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The present investigation was mainly performed on cerebral cortex and hippocampus because there is a relation between the functions of these brain areas and mental disorders, such as lack of concentration, memory impairment, and high level of anxiety, observed in the population living in radio-contaminated areas after the Chernobyl accident (Havenaar et al., 2003; Kryzhanovskaya, 1997; Titievsky et al., 1997; Gamache et al., 2005). In others cerebral structures, such as the hypothalamus, no significant effect on inflammatory cytokines and NO pathway was observed (data not shown). In the brainstem, the only significant effect has been observed at level of IL-10 gene expression (+55%, p < 0.05, data not shown), as in the frontal cortex. These results strongly suggest that there is a dependantcerebral structure response after daily ingestion of 137Cs. These inflammatory changes could be related to a differential accumulation of 137Cs in these brain areas. But, after exposure over a 3-month period, the quantity of 137Cs in the cerebral structures was very similar (2.81 0.11 Bq g 1 in the frontal cortex; 2.94 0.15 Bq g 1 in the hippocampus; 4.26 0.12 Bq g 1 in the brainstem) (Lestaevel et al., 2006). Consequently, the differential effects on cytokines or NO-ergic pathway demonstrated here cannot be directly explained by local accumulations of 137Cs in the brain areas. Clearly, each brain region has its own specific blend of function, cell composition, connectivity and other relevant characteristics. This differential response to 137Cs exposure in these cerebral structures has been previously observed in other experimental situations. For example, a region-specific brain cytokine mRNA induction was demonstrated after instillation of ambient air ultrafine particles (Win-Shwe et al., 2005). The hippocampus appeared to be affected more than the cortex, a finding that is in line with other newer literature on the role of inflammation in neurodegeneration. A direct link between neuro-inflammation and hippocampal neurodegeneration during aging or Alzheimer disease has been recently demonstrated (Craft et al., 2006; Gavilan et al., 2007). Moreover, several cytokines and inflammatory mediators produced by activated glia in various areas of the hippocampal formation have the potential to initiate or exacerbate the progression of neuropathology of Alzheimer disease (Stepanichev et al., 2006). These recent findings suggest that the neuro-inflammation observed in the hippocampus after chronic ingestion of 137Cs could be a potential factor in a possible neurodegeneration. This point must be confirmed in the future. There is a relationship between the increase of pro- or antiinflammatory cytokines, and the kind of neurological symptoms shown by Chernobyl’s accident survivals or experimental chronic exposures. In prenatally irradiated children, who where born between April 1986 and February 1987 in regions of the Ukraine (Loganovskaja and Loganovsky, 1999) or after chronic exposure to 137Cs in rats (Lestaevel et al., 2006), a significant increase of slow frequency waves (<4 Hz) was observed. It was previously demonstrated that slow wave activity increased following administration of TNF-a (Opp, 2005). These results suggest that the 137Cs-induced inflammation response could contribute to electroencephalogram perturbation after exposure to 137Cs. Furthermore, one experimental study reports
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perturbations on the metabolism of serotonin, taurine, alanine, serine, glutamate and glycine, in rats after feeding with oats contaminated with 137Cs for 28 days (45 Bq kg 1) (Bandazhevsky and Lelevich, 1995). Among these disturbed metabolisms, some are regulated by cytokines or NO, such as serotonin or glutamate (Blatteis, 1990; Raber et al., 1998). Complementary studies should be carried out to better understand the role of pro- and anti-inflammatory response on neurotransmitters perturbations observed after chronic exposure to 137Cs. The increase of cytokines and NO in the brain after subchronic exposure to 137Cs underlies the existence of some inflammatory features but without histological evidence of such inflammation and any health effects (data not shown). Here, the rats were healthy throughout the experimental period: their food and water intakes and body weight gain were not affected. These results are in line with a previous study that also found no significant effect on general health parameters in rats in similar experimental conditions (Lestaevel et al., 2006). However, this apparent good state of health does not imply that the perturbation of central homeostasis is not in relation with a peripheral inflammation. The limited brain inflammatory response to 137Cs exposure observed in the present study could be a consequence of a systemic inflammatory response. There are a number of different routes by which a systemic inflammatory response may communicate with the central nervous system (Konsman and Dantzer, 2002). Among these routes, one involved the sensory afferents of the vagus nerve which communicate with neuronal populations within the brain following inflammation induced in the peritoneal cavity. This phenomenon has been already described after ionizing radiation, where the vagus nerve appears to be one of the major ascending pathways for a signal of systemic inflammation to the brain (Marquette et al., 2003). However, Dublineau et al. have previously demonstrated that after chronic exposure to 137Cs, inflammatory cytokines, i.e. IL-10, IL-8, IL-4 and TGF-b, on peripheral tissues, such as intestine, were not significantly modified compared to control (Dublineau et al., 2007). At plasma level, no renal and hepatic disturbances were observed (Tissandie et al., 2006). Moreover, in order to known the source of the increases cytokines in the brain, some animals have been perfused to eliminate blood cells. In these conditions, the same kinds of results have been obtained in ‘‘perfused rats’’ and in ‘‘not perfused rats’’ (data not shown). All these results suggested the specificity of the inflammatory changes to the brain. In conclusion, the present in vivo study demonstrated for the first time that a sub-chronic exposure to 137Cs at postChernobyl dose over a 3-month period leads to subtle molecular modifications on pro- and anti-inflammatory cytokines and on NO-ergic pathway. This inflammatory response seem to be cerebral structure dependant. The 137Cs-induced inflammation response may contribute to the sensitivity of the central nervous system to 137Cs. It could contribute to the perturbation of electroencephalogram and neurotransmitter synthesis and catalysis observed after chronic exposure to 137Cs (Lestaevel et al., 2006; Bandazhevsky and Lelevich, 1995). It will be crucial for people living in the radio-contaminated areas after
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the Chernobyl accident to determine experimentally whether 137 Cs exposure could induce similar phenomena after longer periods of exposure and at different exposure levels. Finally, this study remains a relatively novel undertaking and further experiments should be carried out to analyse genes involved in cytokine signalling as transcription factors or suppressors of cytokine signalling and to analyse cytokine-induced genes. Acknowledgements We thank C. Baudelin and T. Loiseau for their assistance during experimentation and animal exposure. This work is part of the Envirhom programme supported by the Institute for Radioprotection and Nuclear Safety. References Bandazhevsky YI, Lelevich VV. Clinical and experimental aspects of the effect of incorporated radionuclides upon the organism. Gomel, Belarus: Byelorussian Engineering Academy, Gomel State Medical Institute; 1995. Bebeshko VG, Bobyliova OA. Medical consequences of the Chernobyl nuclear power plant accident: experience of 15-year studies. Int Congr Series 2002;1234:267–79. Blatteis CM. Neuromodulative actions of cytokines. Yale J Biol Med 1990;63:133–46. Chauvet N, Palin K, Verrier D, Poole S, Dantzer R, Lestage J. Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide. Eur J Neurosci 2001;14:609–17. Cheng B, Christakos S, Mattson MP. Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron 1994;12:139–53. Claudio L, Martiney JA, Brosnan CF. Ultrastructural studies of the blood-retina barrier after exposure to interleukin-1 beta or tumor necrosis factor-alpha. Lab Invest 1994;70:850–61. Craft JM, Watterson DM, Van Eldik LJ. Human amyloid beta-induced neuroinflammation is an early event in neurodegeneration. Glia 2006;53:484–90. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47–95. Dublineau I, Grison S, Grandcolas L, Baudelin C, Paquet F, Voisin P, et al. Effects of chronic 137Cs ingestion on barrier properties of jejunal epithelium in rats. J Toxicol Environ Health A 2007;70:810–9. Gamache GL, Levinson DM, Reeves DL, Bidyuk PI, Brantley KK. Longitudinal neurocognitive assessments of Ukrainians exposed to ionizing radiation after the Chernobyl nuclear accident. Arch Clin Neuropsychol 2005;20:81–93. Gavilan MP, Revilla E, Pintado C, Castan˜o A, Vizuete ML, Moreno-Gonza´lez I, et al. Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons. J Neurochem 2007;103:984–96. Handl J, Beltz D, Botsh W, Harb S, Jakob D, Michel R, et al. Evaluation of radioactive exposure from 137Cs in contaminated areas of northern Ukraine. Health Phys 2003;84:502–17. Havenaar JM, De Wilde EJ, Van Den Bout J, Drottz-Sjoberg BM, Van Den Brink W. Perception of risk and subjective health among victims of the Chernobyl disaster. Soc Sci Med 2003;56:569–72.
Hong JH, Chiang CS, Campbell IL, Sun JR, Withers HR, McBridge WH. Induction of acute phase gene expression by brain irradiation. Int J Radiat Oncol Biol Phys 1995;33:619–26. Houpert P, Bizot JC, Bussy C, Dhieux B, Lestaevel P, Gourmelon P, et al. Comparison of the effects of enriched uranium and 137-cesium on the behaviour of rats after chronic exposure. Int J Radiat Biol 2007;83:99–104. Iadecola C, Xu X, Zhang F, el-Fakahany EE, Ross ME. Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia. J Cereb Blood Flow Metab 1995;15:52–9. Kamarli Z, Abdulina A. Health conditions among workers who participated in the cleanup of the Chernobyl accident. World Health Stat Q 1996;49:29–31. Konsman JP, Dantzer R. How the immune and nervous system interact during disease associated-anorexia. Nutrition 2002;17:664–8. Kryzhanovskaya L. Psychological dysfunction in the Chernobyl disaster victims. Eur Psychiatry 1997;12:161S. Law A, Gauthier S, Quirion R. Say NO to Alzheimer’s disease: the putative links between nitric oxide and dementia of the Alzheimer’s type. Brain Res Rev 2001;35:73–96. Lestaevel P, Clarenc¸on D, Gharib A, Peinnequin A, Cespuglio R, Gourmelon P, et al. Nitric oxide voltammetric measurements in the rat brain after gamma irradiation. Radiat Res 2003;160:631–6. Lestaevel P, Dhieux B, Tourlonias E, Houpert P, Paquet F, Voisin P, et al. Evaluation of the effect of chronic exposure to 137Cesium on sleep-wake cycle in rats. Toxicology 2006;226:118–25. Loganovskaja TK, Loganovsky KN. EEG, cognitive and psychopathological abnormalities in children irradiated in utero. Int J Psychophysiol 1999;34:213–24. Marquette C, Linard C, Galonnier M, Van Uye A, Mathieu J, Gourmelon P, et al. Il-1b, TNF-a and IL-6 induction in the rat brain after partial-body irradiation: role of vagal afferents. Int J Radiat Biol 2003;79:777–85. Merrill JE. Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological. Dev Neurosci 1992;14:1–10. Opp MR. Cytokines and sleep. Sleep Med Rev 2005;9:355–64. Raber J, Sorg O, Horn TF, Yu N, Koob GF, Campbell IL, et al. Inflammatory cytokines: putatives regulators of neuronal and neuro-endocrine function. Brain Res Rev 1998;26:320–6. Ricart-Jane´ D, Llobera M, Lopez-Tejero D. Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Greiss method. Nitric Oxide 2002;6:178–85. Sawada M, Suzumura A, Hosoya H, Marunouchi T, Nagatsu T. Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J Neurochem 1999;72:1466–71. Selmaj KW, Raine CS. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol 1988;23:339–46. Stepanichev M, Zdobnova I, Zarubenko I, Lazareva N, Gulyaeva NV. Differential effects of tumor necrosis factor-alpha co-administered with amyloid beta-peptide (25-35) on memory function and hippocampal damage in rat. Behav Brain Res 2006;175:352–61. Tissandie E, Gue´guen Y, Lobaccaro JM, Aigueperse J, Gourmelon P, Paquet F, et al. Chronic contamination with 137Cesium affects Vitamin D3 metabolism in rats. Toxicology 2006;225:75–80. Titievsky SV, Tabachnikov SI, Kutko II. Complex approach to mental disorders after Chernobyl accident estimation. Biol Psychiatry 1997;42:23S. Vitkovic L, Maeda S, Sternberg E. Anti-inflammatory cytokines: expression and action in the brain. Neuroimmunomodulation 2001;9:295–312. Win-Shwe TT, Yamamoto S, Ahmed S, Kakeyama M, Kobayashi T, Fujimaki H. Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black. Toxicol Lett 2005;163: 153–60.