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Basic & Clinical Pharmacology & Toxicology, 107, 893–898

Doi: 10.1111/j.1742-7843.2010.00593.x

Time Course of Serum S100B Protein and Neuron-Specific Enolase Levels of a Single Dose of Chlorpyrifos in Rats Ayhan Bozkurt1, Turker Yardan2, Engin Ciftcioglu3, Ahmet Baydin2, Aylin Hakligor4, Medine Bitigic4 and Sirri Bilge5 1

Department of Physiology, Ondokuz Mayıs University, School of Medicine, Samsun, Turkey, 2Department of Emergency Medicine, Ondokuz Mayıs University, School of Medicine, Samsun, Turkey, 3Department of Anatomy, Ondokuz Mayıs University, School of Medicine, Samsun, Turkey, 4Department of Biochemistry, Ankara Education and Research Hospital, Ankara, Turkey, and 5Department of Pharmacology, Ondokuz Mayıs University, School of Medicine, Samsun, Turkey (Received 5 November 2009; Accepted 11 March 2010)

Abstract: Organophosphate (OP) compounds are a large class of chemicals, many of which are used as pesticides. It is suggested that OPs specifically affect glia and neurons. Effects of acute exposure to chlorpyrifos (CPF), which is a common organophosphorus pesticide used worldwide, on neuron-specific enolase (NSE) and S100B levels in rat blood during 7 days were assessed. Rats were evaluated either before (0 hr) or 2, 12, 24, 48 and 168 hr (7 days) after injection of CPF (279 mg ⁄ kg, s.c.) or vehicle (peanut oil, 2 ml ⁄ kg, s.c.) for clinical signs of toxicity. Immediately after the evaluation of toxicity, blood samples were taken for biochemical assays. CPF administration produced decreases in body-weight and temperature, which were observed for first time at 12 hr after CPF administration and continued for 168 hr (p < 0.05–0.001). Serum S100B and NSE levels were acutely increased 2 hr after CPF administration and remained high at 12 hr (p < 0.01–0.001). NSE and S100B levels were not different in either CPF or vehicle groups at following time points. Serum butyrylcholinesterase (EC 3.1.1.8; BuChE) activity was dramatically reduced at 2 hr after CPF and remained low at each time points during 7 days (p < 0.01– 0.001). Our results suggest that the usefulness of serum levels of these glia- and neuron-specific marker proteins in assessing OP toxicity, specifically CPF-induced toxicity.

Organophosphate (OP) compounds are potent neurotoxic chemicals, many of which are used in the control of agricultural and household pests [1], and thus have a high potential for human exposure from various sources. Chlorpyrifos (CPF) is a widely used, broad spectrum, moderately toxic OP insecticide [2]. It has been well-established that one of the main effects of OPs, including CPF, is the inhibition of the enzyme acetylcholinesterase (AChE), the hydrolytic enzyme of acetylcholine (ACh) [2,3]. With extensive AChE inhibition, the neurotransmitter ACh accumulates in the synapses of the central and peripheral nervous systems, which in turn leads to overstimulation of post-synaptic cholinergic receptors and signs of cholinergic neurotoxicity such as autonomic dysfunction (e.g. excessive salivation, lacrimation, urination and defaecation) and involuntary movements (e.g. tremors and convulsions) [3,4]. In addition to the inhibition of AChE activity, glia and neurons may be alternative targets for OP compounds. In vitro studies indicate that CPF affects glia as well as neurons [5–10]. It is also suggested that glial cells provide neuroprotection against OP toxicity [10]. Additionally, early postnatal administration of CPF in rat pups disrupts the pattern of glial and neuronal cell development [11,12] and causes a generalized decrease in the total number of neurons and glia [13,14]. Author for correspondence: Ayhan Bozkurt, Ondokuz Mayis University, School of Medicine, Kurupelit, Samsun, Turkey (fax +90 362 4576041, email [email protected]).

Neurochemical and immunohistological studies have confirmed that some specific isoenzymes or isoproteins, e.g. S100B protein, and neuron-specific enolase (NSE) are specifically distributed in glial cell (S100B) and neuron (NSE) [15– 17]. Various clinical investigations have demonstrated the feasibility of using these marker proteins for evaluating the pathological changes in the nervous system [18–20]. However, no data are available on the serum levels of these marker proteins in OP poisoning in adult animals. Thus, the aim of our study was to assess the serum levels of glia- and neuron-specific marker proteins (S100B and NSE, respectively) in acute CPF poisoning. Materials and Methods Animals. Male Sprague–Dawley rats (3-month-old, body-weight 240–260 g) were obtained from Ondokuz Mayis University vivarium sources. Rats were housed 5–8 per cage in a quiet, temperature and humidity-controlled room (22 € 2C and 60 € 5%, respectively) in a 12-hr light:dark cycle, receiving food and water ad libitum. After a 1-week habituation to the housing conditions, the rats were accustomed to manipulation and handling (5 min. each day) in the test room for 5 days prior to experiments. Experiments were approved by the Institutional Animal Care and Use Committee of the Ondokuz Mayis University and adhered to the Guide for the Care and Use of Laboratory Animals (NIH publication 865–23, Bethesda, MD, USA). Experimental design. Chlorpyrifos (Riedel-de Hen, Germany) was prepared in peanut oil at a volume of 2 ml ⁄ kg. Rats were subcutaneously injected (between the shoulders along the midline of the back) with 279 mg ⁄ kg CPF (n = 48) or vehicle (n = 30) between 08:30

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a.m. and 09:30 a.m. This CPF dose was the maximum tolerated dose in adult rats as reported before [21]. Each group was divided into six subgroups (n = 5 for vehicle group and n = 8 for CPF group). The rats were evaluated either before (0 hr) or 2, 12, 24, 48 and 168 hr (7 days) after injection for clinical signs of toxicity. Immediately after the evaluation of toxicity, the animals were anaesthetized (100 mg ⁄ kg ketamine and 0.75 mg ⁄ kg chlorpromazine; i.p.), blood samples were taken by cardiac puncture for biochemical assays and the rats were killed. Toxicity evaluation. Clinical signs of toxicity including SLUD signs (acronym for salivation, lacrimation, urination and diarrhoea) and involuntary movements, were ranked by a ‘blind’ observer based on the method of Moser et al. [22]. Briefly, SLUD syndrome was scored as follows: 1 = normal; 2 = slight: one sign or very mild multiple SLUD signs; 3 = moderate: multiple signs; 4 = severe: multiple and extensive SLUD signs. For involuntary movements, 2 = normal quivering of vibrissae, head and limbs; 3 = mild fine tremors, seen typically in the forelimbs and head; 4 = whole-body tremors; 5 = myoclonic jerks; 6 = clonic convulsions. Other physiological measures such as body-weight and temperature, and locomotor activity were also measured.

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Locomotor activity. Spontaneous locomotor activity was measured in an activity cage (Ugo Basile, Varese, Italy) having dimensions of 39 · 28 · 26 cm. The values indicate pulses recorded by the apparatus as stainless steel bars tilt in response to animal movements. The activity of each rat was automatically recorded for 5 min. Biochemical assays. Blood samples processed to serum, centrifuged at 1467 g for 15 min. and stored at )20C until analysis. The S100B and NSE concentrations were measured with a commercial immunoluminometric assay (Liaison, DiaSorin S.p.A., Saluggia, Italy) in Liaison Analyzer (Liaison, DiaSorin S.p.A.). The detection limit of the assay is 0.02 lg ⁄ l for S100B and 0.04 lg ⁄ l for NSE. Butyrylcholinesterase (EC 3.1.1.8; BuChE) activity was measured by using Olympus OSR6114 kit (Olympus Italia, Segrate MI, Italy) as described earlier [23]. Thiocholine that forms during the reaction reduces yellow hexacyanoferrate to colourless hexacyanoferrate. The decrease in absorbance at 410 nm is directly proportional with the cholinesterase activity of the sample.

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Statistical analysis. All data are expressed as means € S.E.M. Statistical analyses were performed using the GraphPad InStat (v3.06) software (GraphPad Software, San Diego, CA, USA). Following the assurance of normal distribution of data, one-way analysis of variance (ANOVA) with the Tukey-Kramer post-hoc test was used in comparison to the baseline (0 hr) value for each group. The comparisons between treatments within a time point were evaluated with Student’s t-test. Differences were considered statistically significant if p < 0.05.

Results Toxicity evaluation. None of the rats exposed to the high dose of CPF (279 mg ⁄ kg, s.c.) either became moribund or died during the experiment. Measurements of behavioural signs of cholinergic toxicity indicated the absence of any effect of CPF on the variables recorded: SLUD signs and involuntary movement (data not shown). However, CPF administration produced decreases in body-weight and temperature, which were observed for first time at 12 hr after CPF administration and continued for 168 hr (p < 0.05–0.001), compared to CPF-0 and corresponding vehicle groups (figs 1A and B). Additionally, locomotor activity values (expressed as the number of move ⁄ 5 min.) were reduced (p < 0.05–0.01) at 24 and 48 hr after CPF administra-

Fig. 1. Time-dependent changes in body-weight (A), body temperature (B) and locomotor activity (C) of rats after a single high-dose chlorpyrifos (CPF, 279 mg ⁄ kg, s.c.) or vehicle (peanut oil, 2 ml ⁄ kg, s.c.) administration. Each bar is mean € S.E.M. n = 5 for vehicle group and n = 8 for CPF group. *p < 0.05, **p < 0.01, ***p < 0.001; compared to CPF group at 0 hr. ++p < 0.01, +++p < 0.001; compared to corresponding vehicle group.

tion, compared to CPF-0 and corresponding vehicle groups (fig. 1C). Locomotor activity returned to the basal level at 168 hr after CPF administration. Biochemical measures. Fig. 2A shows serum S100B levels in CPF or vehicle groups at different time points. S100B level was acutely increased 2 hr after CPF administration (p < 0.001) and remained

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NSE levels were also high at each time point but not statistically different from CPF-0 and corresponding vehicle groups. Fig. 2C shows serum BuChE activity in CPF or vehicle groups at different time points. BuChE activity was dramatically reduced at 2 hr after CPF and remained low at each time points during 7 days (p < 0.01–0.001), compared to CPF-0 and corresponding vehicle groups.

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Fig. 2. Time-dependent changes in serum S100B (A) and NSE levels (B) and butyrylcholinesterase activity (C) of rats after a single highdose chlorpyrifos (CPF, 279 mg ⁄ kg, s.c.) or vehicle (peanut oil, 2 ml ⁄ kg, s.c.) administration. Each bar is mean € S.E.M. n = 5 for vehicle group and n = 8 for CPF group. **p < 0.01, ***p < 0.001; compared to CPF group at 0 hr. ++p < 0.01, +++p < 0.001; compared to corresponding vehicle group.

high at 12 hr (p < 0.001), compared to CPF-0 and corresponding vehicle groups. At 24, 48 and 168 hr, S100B levels were not different in either CPF or vehicle groups. Fig. 2B shows serum NSE levels in CPF or vehicle groups at different time points. Similar to the S100B, NSE level was acutely increased 2 hr after CPF administration (p < 0.001) and remained high at 12 hr (p < 0.001), compared to CPF-0 and corresponding vehicle groups. After this time, serum

In the present study, we focused our attention on the patterns of serum levels of glial and neuronal markers (S100B and NSE, respectively) in CPF poisoning with a single dose (279 mg ⁄ kg, s.c.) in adult rats. This CPF dose was the maximum tolerated dose in adult rats and produces an extensive inhibition of brain AChE activity as reported before [21,24,25]. We showed that serum S100B and NSE levels elevate in the acute phase of CPF poisoning in adult rats given a single dose. The time to peak serum levels of S100B and NSE in our study was 2 hr after CPF exposure. We also evaluated time-course of clinical signs of toxicity by using behavioural signs (SLUD signs and involuntary movement) and some physiological measures (body-weight and temperature, and locomotor activity). CPF exposure resulted in decreases in body-weight and temperature, which were observed for the first time at 12 hr after CPF administration and continued for 7 days. Locomotor activity values were reduced at 24 and 48 hr after CPF administration and returned to the basal level at 7 days after CPF administration. No other signs of toxicity (i.e. SLUD signs and involuntary movement) were detected. Previous studies have also reported similar responses (i.e. relatively few signs of cholinergic toxicity) to acute exposure of CPF at the same or similar doses [24–29]. The primary mechanism of action and the most acutely life-threatening effect of the OP insecticides results from the inhibition of AChE with subsequent accumulation of ACh within the cholinergic synapses resulting in a wide range of neurotoxic effects [3,4,30]. Although there are some OPs, called direct inhibitors, which need not to undergo metabolic sulphoxidation to form oxon reactive form, i.e. dichlorvos, monocrotophos, heptenophos, most of the OPs inhibit AChE by their active oxon metabolites. These oxons also inhibit BuChE, which stoichiometrically detoxifies some of the oxon and prevents that fraction from inhibiting AChE [31–34]. Additionally, it is reported that BuChE is more sensitive to the inhibitory effect of CPF than is AChE [34–36]. Thus, BuChE represents a detoxification mechanism and a potential biomarker for OP insecticide exposure ⁄ response. We assessed the effect of CPF at the maximum tolerated dose (279 mg ⁄ kg, s.c.) on serum BuChE activity in adult rats in a time-dependent manner. In our study, serum BuChE activity decreased 2 hr after CPF exposure at this dose and remained low along the study period (7 days). During this period, we did not observe any difference in SLUD signs and involuntary movement. Furthermore, the decreases in

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body-weight and temperature were observed for first time at 12 hr after CPF administration and continued for 7 days. Although we were not able to assess the brain AChE activity, it is reported that CPF administration at this dose in adult rats produces an extensive inhibition (more than 70–80%) of brain AChE activity [21,24,25]. A statistically significant depression (starting from at least 50%) of brain AChE activity is usually associated with cholinergic signs and thus may be considered the actual critical, toxicologically relevant effect [37]. However, similar to our results, the correlation between percentage of brain cholinesterase activity depression and the appearance of cholinergic ‘classical signs’ (e.g. SLUD signs) has not always been good in animal models [37]. Indeed, in several studies in rats, a minimal expression of cholinergic toxicity, in the presence of extensive brain AChE inhibition, following CPF administration at the same or similar dose (250 mg ⁄ kg, s.c.) has been reported [24–29]. Several authors indicated that this effect could be related to direct modulation of muscarinic autoreceptor function leading to decreased ACh release and accumulation [25,38,39]. Organophosphorus pesticides are used in large quantities worldwide. CPF is a widely used, broad spectrum, moderately toxic OP insecticide [2]. Since its development in 1965, this compound has become used widely for a variety of agricultural and domestic applications. Following exposure to CPF, it is converted to an extremely toxic oxon metabolite in the liver. Many studies indicate that inhibition of AChE is the most sensitive macromolecular target of OP exposure, leading to systemic toxicity from cholinergic hyperstimulation [3,4]. Like other organophosphorus pesticides, CPF elicits toxicity by inhibiting AChE activity [2]. However, additional targets have been proposed for OPs, including DNA and RNA synthesis [40], the adenylate cyclase signalling cascade [41], and the expression of different transcription factors [42,43]. This suggests that OPs may also act through various mechanisms not necessarily related to AChE inhibition. It is now well-established that glial cells represent intimate partners to neurons throughout their lifespan. For example, during neurogenesis and early development, glial cells provide a scaffold for the proper migration of neurons and growth cones, a process mediated via the synthesis and secretion of a variety of growth factors and extracellular matrix components. Glial cells also provide guidance cues for neuronal proliferation and electrical differentiation of neurons. In the adult, glial cells maintain neuronal homeostasis, synaptic plasticity and repair [10]. Glia ordinarily protect neurons by releasing trophic factors, scavenging oxidative radicals, providing nutrients for repair, and guiding axonal outgrowth [44,45]. Although most studies on OP neurotoxicity have focused mainly on neurons, which are considered to be the primary target for neurotoxicity [24,40,46,47], recent in vitro and in vivo findings suggest that OPs may target glial cells as well. In aggregating brain cell cultures of foetal rat telencephalon, OPs affect glial enzymatic markers [7], alter glial fibrillary acidic protein (GFAP) immunoreactivity [9] and appear to be involved in neurotoxicity [10]. CPF inhibits replication ⁄ differentiation and elicits oxidative stress in C6 glioma

cells [5,8]. Guizzetti et al. [6] suggest that OP insecticides and their oxons affect astroglial cell proliferation and that the transition from the G0 ⁄ G1 to the S ⁄ G2 phase of the cell cycle may be particularly sensitive to the action of these compounds. Additionally, the results, indicating increased neurotoxicity in aggregate brain cell cultures of foetal rat telencephalon deprived of glial cells, suggest that glial cells provide neuroprotection against OP toxicity [10]. Glial cells have already been shown to protect neurons from the toxicity of lead acetate and methylmercury [48,49], and to reduce the neurotoxicity induced by trimethyltin [50]. These in vitro studies are supported by in vivo findings. Developmental exposure of rats to subtoxic doses of CPF has been shown to disrupt the pattern of glial development [11,12] and to alter the levels of GFAP, an astrocytic marker [11]. Early postnatal CPF exposure caused a generalized decrease in the total number of neurons and glia in the juvenile rat brain [13,14]. Slotkin and Seidler [12] reported that CPF elicited major transcriptional changes in genes involved in neural cell growth, development of glia, transcriptional factors involved in neural cell differentiation, and development of neurotransmitter synthesis. In the present study, we found that administration of a single high dose of CPF caused an acute increase in serum levels of glia- and neuronspecific marker proteins (S100B and NSE, respectively) in adult animals. Our results confirm and expand these studies which suggest the neurons and glia as alternative targets for OP compounds. S100B is a member of the S100 family of EF-hand Ca2+ binding protein, mainly expressed in astrocytes in the central nervous system, exerting intracellular and extracellular regulatory activities [15,51]. Once released into the brain extracellular space, S100B affects astrocytes, neurons and microglia in an autocrine and paracrine manner [52]. S100B release was shown to increase after brain damage, such as traumatic cortical injury, focal cerebral ischaemia, as well as in some psychiatric disorders [19]. However, although the enhanced S-100B immunoreactivity in the brain was related to neuronal damage, it is unclear whether this phenomenon is a pathogenic causative one or whether it reflects only the astroglial response to injury or whether it is involved in cellular defense mechanism against oxidative stress. S100B decreases cell death resulting from glucose deprivation in hippocampal neurons from rats [53], and protects neurons (from embryonic chick and neonatal rat) against glutamate- and staurosporin-induced damage in vitro [54] at 10–30 ng ⁄ ml and 1–10 ng ⁄ ml, respectively. By contrast, at high concentrations, S100B is found to be neurotoxic [55,56]. These results suggest that S-100B may act as a cellular survival factor at nanomolar concentrations and as an apoptosis-inducing agent at micromolar concentrations. Neuron-specific enolase is an enzyme localized in neuronal axons and cytoplasm and plays a crucial role in glycolysis [16,17]. NSE can indicate both neuronal cell loss by increased serum concentration [18]. However, the relationship between NSE and OP neurotoxicity is less studied. Matsuda et al. [57] found that the level of the NSE transcript

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in sciatic nerve significantly increases within 2 hr and remains high during 30 days after disulfoton, another OP, administration to rats and suggested that up-regulation of NSE mRNA may be a marker for the nervous system abnormality following OP exposure. In our study, serum NSE level was found to be acutely increased 2 and 12 hr after CPF administration. At other time points, serum NSE levels were also high but did not reach the statistically significant values. Since the half-life of NSE is approximately 24 hr [58], these high levels after 12 hr possibly reflect the initial increase in the acute phase. In conclusion, the present study suggests the usefulness of serum of S100B protein and NSE levels in assessing OP toxicity, specifically CPF-induced toxicity. However, our results, indicating that NSE and S100B increases only up to 12 hr after CPF administration, also suggest that NSE and S100B could not be valid after 12-hr CPF post-exposure. Based upon our study, further research, assessing the time- and dose-related effects of CPF on S100B and NSE levels and histopathology, is necessary to evaluate the role of S100B and NSE in OP poisoning. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement We express thanks to Selami Trel for his technical support. This work was supported by the Ondokuz Mayis University Scientific Research Foundation. References 1 Maroni M, Colosio C, Ferioli A, Fait A. Biological monitoring of pesticide exposure: a review. Organophosphorus pesticides. Toxicology 2000;143:9–37. 2 Barron MG, Woodburn KB. Ecotoxicology of chlorpyrifos. In: Ware GW (ed.). Rev Environ Contam Toxicol, vol. 144. Springer, New York, 1995;1-76. 3 Ecobichon DJ. Toxic effects of pesticides. In: Klassen CD (ed.). Casarett and Doull’s Toxicology, 5th edn. McGraw-Hill, New York, 1996;643–98. 4 Savolainen K. Understanding the toxic actions of organophosphates. In: Krieger R (ed.). Handbook of Pesticide Toxicology Agents, vol. 2. Academic Press, New York, 2001;2. 5 Garcia SJ, Seidler FJ, Crumpton TL, Slotkin TA. Does the developmental neurotoxicity of chlorpyrifos involve glial targets? Macromolecule synthesis, adenylyl cyclase signaling, nuclear transcription factors, and formation of reactive oxygen in C6 glioma cells Brain Res 2001;891:54–68. 6 Guizzetti M, Pathak S, Giordano G, Costa LG. Effect of organophosphorus insecticides and their metabolites on astroglial cell proliferation. Toxicology 2005;215:182–90. 7 Monnet-Tschudi F, Zurich MG, Schilter B, Costa LG, Honegger P. Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures. Toxicol Appl Pharmacol 2000;165:175–83. 8 Qiao D, Seidler FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos modeled in vitro: comparative effects of metabolites and other cholinesterase inhibitors on DNA synthesis in PC12 and C6 cells. Environ Health Perspect 2001;109:909–13.

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 2010 The Authors Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107, 893–898