The effect of exposure to chlorfenvinphos on lipid metabolism and apoptotic and necrotic cells death in the brain of rats

Experimental and Toxicologic Pathology 65 (2013) 531–539

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The effect of exposure to chlorfenvinphos on lipid metabolism and apoptotic and necrotic cells death in the brain of rats Alicja Roszczenko a,∗ , Joanna Rogalska a , Janina Moniuszko-Jakoniuk b , Malgorzata M. Brzóska a a b

Department of Toxicology, Medical University of Bialystok, Adama Mickiewicza 2C Street, 15-222 Bialystok, Poland School of Medical Science in Bialystok, Krakowska 9 Street, 15-875 Bialystok, Poland

a r t i c l e

i n f o

Article history: Received 1 September 2011 Accepted 11 March 2012 Keywords: Organophosphate pesticides Chlorfenvinphos Acetylcholinesterase Butyrylcholinesterase Brain Lipid metabolism Apoptosis Necrosis Rat

a b s t r a c t This study investigated the influence of chlorfenvinphos (0.3 mg/kg bw/24 h corresponding to 0.02 LD50 ; orally by gastric gavage for 14 and 28 days) on lipid metabolism, and apoptotic and necrotic cells death in the brain of rats as the possible mechanism of neurotoxic action of organophosphate (OP) pesticides at low exposure. Total cholesterol (TCh), triglycerides (TG), phospholipids (PL), and free fatty acids (FFA) were determined and apoptotic, necrotic, and living cells were quantified in the brain. Moreover, the serum and brain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were assayed as biomarkers of neurotoxicity. The treatment with chlorfenvinphos increased (duration dependently) the concentrations of TCh and TG and the ratio of TCh/PL, and decreased PL concentration. The prevalence of apoptotic and necrotic cells increased and that of the living brain cells depressed (by 10%) already after 14 days of the exposure. The brain activities of AChE and BChE decreased by 12% and 15%, and by 18% and 25% after 14 and 28 days, respectively, whereas the serum activities of these enzymes were inhibited (by 24% and 18%, respectively) only after the longer treatment. The changes in lipid metabolism and distribution of the living, apoptotic, and necrotic brain cells correlated with AChE and BChE activities in the serum and brain. The results show that chlorfenvinphos may disturb lipid metabolism and induce apoptosis and necrosis in the brain even at the exposure not affecting the serum activities of cholinesterases, and causing only moderate inhibition of their brain activities. Based on the findings it can be concluded that low repeated exposure to OP pesticides may influence the nervous system through disrupting the lipid profile of the nervous tissue and decreasing the number of the nervous cells. © 2012 Elsevier GmbH. All rights reserved.

1. Introduction Chlorfenvinphos (2-chloro-1-(2,4-dichlorophenyl) vinyl diethyl phosphate) belongs to organophosphate (OP) compounds being a group of pesticides the most commonly used nowadays. The worldwide production and using of OP pesticides for protection against many kinds of insects in agriculture, as insecticides in household, and for control of vector-borne diseases for public health programs results in their presence in the natural and occupational environment as well as in the dietary products. Thus, people are commonly exposed to these compounds, not infrequently in excessive amounts creating a serious hazard to the health (Gebara et al., 2011; Nougadˇcre et al., 2011; Singh et al., 2011; Wang et al., 2012). OP pesticides belong to the most toxic environmental pollutants and they cause numerous intoxications and fatalities all over the world (Griffith et al., 2011; Wang et al., 2012). Thus, growing

∗ Corresponding author. Tel.: +48 85 7485604; fax: +48 85 7485604. E-mail address: [email protected] (A. Roszczenko). 0940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2012.03.002

attention has been focused on the health effects of exposure to these compounds and explaining the mechanisms of their action. OP pesticides are characterized by high acute toxicity to mammals associated with an inhibition of activity of cholinesterases regulating the function of the nervous system such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (Lotti, 2001; Li et al., 2011). These compounds are known to damage to the different organs and systems at both acute and chronic exposure (Lasram et al., 2009; Singh et al., 2011; Dirican and Kalender, in press; Solati et al., in press); however, the main unfavorable effect of their action in mammals is the impact on the nervous system. Numerous experimental, epidemiological and clinical data show on the connection between exposure to OP pesticides and damage to the nervous system (Lotti, 2001; Sekar Babu et al., 2010; Kaur et al., 2007; Celik and Isik, 2009; Li et al., 2011; Solati et al., in press). The neurotoxic action of these compounds has been mainly explained by their ability to inhibit the activities of AChE and BChE. AChE is responsible for hydrolysis of acetylcholine playing a principal role in the creation and transmission of neural impulses in the central (CNS) and peripheral nervous system (Lotti, 2001). BChE is also able to catalyse the

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decomposition of acetylcholine; however, less efficiently (Darvesh et al., 2003). Moreover, this enzyme is not only of pharmacological, but also of toxicological importance because it scavenges cholinesterase inhibitors, including OP compounds, before they reach their synaptic targets (Darvesh et al., 2003; Doctor and Saxena, 2005). Thus, the inhibition of AChE and BChE results in the accumulation of acetylcholine in the nervous tissue with consequent over stimulation of specific cholinergic receptors and disruption of the function of the nervous system (Lotti, 2001; Darvesh et al., 2003). OP pesticides have also been reported to cause neuronal degeneration and contribute to the development of neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease (Kaur et al., 2007; Li et al., 2011). The acute impact of OP pesticides on the CNS is well-known (Lotti, 2001; Brocardo et al., 2005; Li et al., 2011); however, the influence of relatively low and moderate repeated intoxication with these compounds, corresponding to the exposure that may occur in human life, and its involvement in the development of diseases of the nervous system are not yet completely understood. An important causative role in the development of diseases of the nervous system has been played by the oxidative/antioxidative imbalance and disorders in lipid metabolism in the nervous tissue as well as a decrease in the number of the nervous cells (Refolo et al., 2000; Vance et al., 2005; Martins et al., 2009; Oliveira and Di Paolo, 2010; Ong et al., 2010; Ziv and Melamed, 2010). OP compounds have been reported to cause oxidative stress with consequent lipid peroxidation and to induce apoptosis and necrosis in the brain under acute and chronic exposure (Brocardo et al., 2005; Üner et al., 2006; Kaur et al., 2007; Dwivedi and Flora, 2011; El-Demerdash, 2011; Solati et al., in press). Moreover, it has been suggested that the OP pesticides-induced oxidative stress and lipid peroxidation may contribute to the neuronal degeneration and a relationship has been reported between these compounds-induced inhibition of AChE activity and lipid peroxidation in the brain (Brocardo et al., 2005; Üner et al., 2006; Kaur et al., 2007; Dwivedi and Flora, 2011; El-Demerdash, 2011). Taking into account that OP pesticides may affect some pathways playing a crucial role in the physiology and pathology of the CNS, including lipid metabolism and apoptosis being the “programmed cell death”, it seems possible that these compounds may influence the nervous system also through these mechanisms, but this question has not been adequately investigated up until now. It has been reported that exposure to OP pesticides modifies the concentrations of some lipid compounds, including mainly cholesterol, in the serum and different tissues, and a connection between these pesticides-induced BChE inhibition and changes in lipid metabolism has been hypothesized (Lucic´ et al., 2002; Lasram et al., 2009; Dwivedi and Flora, 2011; Solati et al., in press). However, the knowledge on the impact of OP compounds on the lipid profile of the nervous tissue is sparse. The only available data in this regard refers to the increased concentrations of products of lipid peroxidation in the brain (Brocardo et al., 2005; Üner et al., 2006; Dwivedi and Flora, 2011). The present study has been undertaken to investigate the hypothesis of involvement of disorders in lipid metabolism as well as apoptotic and necrotic cells death in the mechanisms of neurotoxic action of OP compounds at low repeated exposure. For this purpose, chosen indices of lipid metabolism were determined in the brain tissue and the distribution of living, apoptotic and necrotic cells among the total population of the brain cells was estimated in the rats exposed to chlorfenvinphos. Moreover, dependences between these parameters, and the serum and brain activities of AChE and BChE were estimated. Although both enzymes are sensitive biomarkers of neurotoxicity, data on the impact of OP pesticides on the activity of BChE, especially in the brain, and on the

relationship between these enzymes are still lacking. According to our knowledge, similar study has not been conducted up until now. 2. Materials and methods 2.1. Chemicals and reagents All the chemicals and reagents used were of the highestgrade purity or analytical purity. Chlorfenvinphos (purity 98.4%) was purchased from the Institute of Organic Industrial Chemistry in Annapol (Warsaw, Poland). Vetbutal was obtained from Biowet (Pulawy, Poland), sodium chloride (NaCl) from POCh (Gliwice, Poland), and physiological buffered saline (PBS) from Biomed-Lublin (Lublin, Poland). Acetylthicholine iodide, Sbutyrylthiocholine iodide and 5,5 -dithio-bis(2-nitrobenzoic acid) (DTNB) used for cholinesterases determination were purchased from POCh, Sigma–Aldrich (Schnelldorf, Germany), and BDH Chemical Ltd (Poole, England), respectively. The diagnostic kits for determination of total cholesterol (TCh) and triglycerides (TG) were obtained from BioMaxima (Lublin, Poland), whereas the kits for phospholipids (PL) and free fatty acids (FFA) assay from Wako Chemicals GmbH (Neuss, Germany). Bovine serum albumin was purchased from Sigma–Aldrich (St. Louis, MO, USA) and Reference Control Serum (No. 53010) from Medichem (Steinenbronn, Germany). The kit used for evaluation of apoptotic and necrotic cells (APOPTESTTM -FITC kit) was supplied by Dako Denmark A/S (Glostrup, Denmark). In all measurements ultra-pure water, received from two-way water purification MAXIMA system (ELGA, Bucks, UK), was used. 2.2. Animals The study was conducted on 40 adult albino male Wistar rats (Crl: WI (Han)) of initial body weight of 250–280 g purchased from the certified Laboratory Animal House (Brwinow, Poland). During the whole experiment the animals were kept at controlled conventional conditions (temperature 22 ± 2 ◦ C, relative humidity 50 ± 10%, 12-h light–dark cycle). They had ad libitum access to the nutritionally standard pellet rodent LSM diet (Labofeed, Kcynia, Poland) and drinking water. 2.3. Experimental protocol After a five-day acclimatization to the laboratory conditions, the rats were randomly divided into 4 groups of 10 animals each. Two of these groups were orally treated with an olive oil solution of chlorfenvinphos administered once a day by gastric gavage (using a gastric tube) in a dose of 0.3 mg/kg bw corresponding to 0.02 LD50 (LD50 – median lethal dose, the dose causing 50% lethality; the value of LD50 of chlorfenvinphos for a rat is 15 mg/kg bw) for 14 and 28 days. The oil solution of this pesticide was prepared immediately before dosage and was administered in a volume of 1 ml/100 g bw. The remaining two groups of rats received an equivalent amount of olive oil via a gastric tube for 14 and 28 days and served as respective control groups. During the experiment, the animals were weighed once a week and the amount of this pesticide was adjusted for the body weight to administer the dose of 0.3 mg/kg bw throughout the experiment. The used dose of chlorfenvinphos was more than twice lower than the lowest observed adverse effect level (LOAEL) of this pesticide for a rat established at 0.7 mg/kg bw/day (Toxicological profile for chlorfenvinphos, 1997). Since the study was aimed to investigate the impact of low repeated, but not long-term, exposure to chlorfenvinphos, the low level of dosage of this pesticide and relatively short time of its administration were used to ensure low to moderate extent of cholinesterases inhibition and avoid excessive accumulation of acetylcholine and

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development of poisoning. The exposure was designed to reach the inhibition of the serum activity of AChE not exceeding the value of 25% established as the cut-off level for biological surveillance of subjects occupationally exposed to OP pesticides (Lotti, 2001). Indeed, throughout the experiment, no mortality and signs of morbidity, including any symptoms of the nervous system disorders, were noted in the animals treated with chlorfenvinphos. At the termination, after overnight fasting and weighting, the rats were anesthetized with Vetbutal (pentobarbital sodium and pentobarbital 5:1, 30 mg/kg bw, ip). The whole blood was collected (without anticoagulant) by cardiac puncture and different organs and tissues, including the brain, were removed. The whole blood was centrifuged (MPW-350R centrifugator, Medical Instruments, Warsaw, Poland) after coagulation and serum was separated. The organs were immediately rinsed with ice-cold physiological saline solution (0.9% NaCl) and weighed with an automatic balance (OHAUS® , Nanikon, Switzerland; accuracy to 0.0001 g). The biological material not used immediately was stored frozen at −70 ◦ C until analysis. The serum samples and a half of the brain (containing cerebrum and cerebellum) were used in the present study. With the aim to quantitatively estimate the exposure to chlorfenvinphos, the serum and brain activities of AChE and BChE were determined, and the extent of this pesticide-induced inhibition of these enzymes (main indicator of exposure to OP compounds) as well as the ratio of their activities (AChE/BChE) were evaluated. TCh, TG, PL, and FFA were measured in the brain and the ratio of TCh/PL was calculated as the accepted index of membrane fluidity (Senault et al., 1990). Moreover, the occurrence of apoptotic, necrotic, aponecrotic and living cells in the total population of the brain cells was evaluated. The experimental protocol received approval of the Local Ethics Committee for Animal Experiments in Bialystok (Poland) for care and use of laboratory animals. 2.4. Analytical procedures 2.4.1. Preparation of brain homogenates With the aim to determine the activities of AChE and BChE, and the concentrations of chosen lipid compounds in the brain, 10% homogenates of the brain tissue were prepared. For this purpose, the brain tissue was homogenized in ice-cold 0.9% NaCl using a homogenizer (Schütt homgenplus ; Schütt Laboratechnik GmbH, Göttingen, Germany). Next, the homogenates were centrifuged (MPW-350R centrifugator, Medical Instruments) at 6000 rpm for 20 min at 4 ◦ C, and the aliquots were separated and used to measure AChE and BChE activities and the indices of lipid metabolism.

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as a coefficient of variation (CV), was <5% and 6%, respectively, whereas in the brain tissue it was <4% and 6%, respectively. The extent of the chlorfenvinphos-caused inhibition of AChE or BChE was calculated assuming activities of these enzymes in the respective control group as 100%. 2.4.3. Determination of chosen parameters of lipid metabolism The concentrations of TCh and TG in the aliquots of the brain homogenates were determined using diagnostic kits by BioMaxima based on the enzymatic methods described in the kit instructions. The analytical quality of TCh and TG measurements was checked by their simultaneous determination in the Reference Control Serum. The concentrations of TCh and TG determined by us were within the ranges of the certified values. The CV for these measurements was <6% and 5.5%, respectively. The concentrations of PL and FFA in the aliquots of the brain homogenates were determined colorimetrically using kits by Wako Chemicals GmbH based on the enzymatic methods described in the kit instructions. The CV was <5.3% and 6.5% for PL and FFA, respectively. The brain concentrations of TCh, TG, PL, and FFA were adjusted for protein concentration. The measurements of the lipid compounds were performed using the Hitachi U-3010 spectrophotometer. 2.4.4. Measurement of apoptotic, necrotic and living brain cells The occurrence of apoptotic, necrotic and living cells in the total population of the brain cells was quantified by flow cytometry method using APOPTESTTM -FITC kit and a Coulter Epics XL-MCL cytometer (Coulter Corporation, Miami, FL, USA). The brain cells for the assay were harvested by suspending in PBS according to the previously reported procedure (Rogalska et al., 2011). As it was described in the APOPTESTTM -FITC kit instruction, the harvested cells were suspended in an ice-cold binding buffer to obtain 105 –106 cells/ml (the cells were counted under a light microscope; Olympus, Tokyo, Japan). 1 ␮l of Annexin VFITC and 2.5 ␮l propidium iodide (PI) were added into 100 ␮l of the brain cell suspension, and the aliquot was placed in ice and incubated for 10 min in dark. Next, 250 ␮l of binding buffer were added to each sample and the stained cells were analyzed immediately (within 5 min) by a cytometer. The fluorescence of Annexin V was measured through FL-1 filter (530 nm) and that of PI by FL-2 filter (585 nm). Living cells (Annexin V− /PI− ), apoptotic cells (Annexin V+ /PI− ), necrotic cells (Annexin V− /PI+ ), and aponecrotic cells (Annexin V+ /PI+ ) were distinguished. 2.5. Statistical analysis

2.4.2. Determination of AChE and BChE activities The activities of AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8) in the serum and the aliquots of the brain homogenates were determined spectrophotometrically by the modified Ellman’s method using acetylthicholine iodide and S-butyrylthiocholine iodide as appropriate substrates, and DTNB as a chromogen (Ellman et al., 1961; Worek et al., 1999). The activities of AChE and BChE were expressed in the units of activity per liter in the serum (U/l) or per mg of protein in the brain tissue (U/mg protein). One unit of the AChE or BChE activity is the amount of the enzyme activity which catalyses the transformation of 1 ␮mol of the substrate per minute. In order to adjust the brain activities of these enzymes for protein concentration, total protein was assayed in the aliquots of the brain homogenates. Protein concentration was determined by the spectrophotometric Lowry method modified by Peterson (Peterson, 1977). Bovine serum albumin was used to prepare a standard curve. A Hitachi U-3010 spectrophotometer (Tokyo, Japan) was employed in the measurements of cholinesterases and protein. The precision of AChE and BChE determination in the serum, expressed

The results are presented as mean ± S.E. for ten rats in each group. A one-way analysis of variance (ANOVA, Kruskal–Wallis ranks test) was conducted to determine whether there were statistically significant differences among the experimental groups. Spearman rank correlation analysis was performed to investigate the relationship between the measured parameters. Differences and correlations were considered statistically significant at p < 0.05. All the calculations were performed using Statistica 9.0 package (StatSoft, Tulsa, OK, USA). 3. Results 3.1. AChE and BChE activities in the serum and brain The 14-days treatment with chlorfenvinphos in the dose of 0.02 LD50 had no influence on the activities of AChE and BChE in the serum; however, after 28 days of this pesticide administration the activities decreased (by 24% and 18%, respectively) compared to

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Fig. 1. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in the serum and brain of control rats and those exposed to chlorfenvinphos (0.02 LD50 ) for 14 and 28 days. Data is mean ± S.E. for 10 animals. Statistically significant differences (ANOVA, Kruskal–Wallis ranks test) are indicated by: a vs. respective control group and b vs. the group exposed to chlorfenvinphos for 14 days. *p < 0.05, † p < 0.01, ‡ p < 0.001. Numerical values above bars indicate percentage decrease (down-pointed arrow) compared to the respective control group.

the respective control group (Fig. 1). The activities of AChE and BChE in the serum after 28 days of the exposure to chlorfenvinphos were lower by 15% and 17%, respectively, than after 14 days of the treatment (Fig. 1). The brain activities of AChE and BChE were decreased (by 12% and 15%, respectively) already after 14 days of chlorfenvinphos administration and prolongation of the exposure up to 28 days enhanced the extent of these enzymes inhibition (Fig. 1). In the control animals there was no difference in the brain activities of AChE and BChE after 14 and 28 days, whereas in these treated with chlorfenvinphos for 28 days the activities were lower by 13% and 15%, respectively, than after 14 days of the exposure (Fig. 1). The ratio of AChE/BChE in the serum and brain of the control rats reached 0.617 ± 0.031 and 4.28 ± 0.48, respectively, after 14 days, and 0.625 ± 0.047 and 4.03 ± 0.19, respectively, after 28 days of the experiment. The exposure to chlorfenvinphos had no impact on the ratio of activities of these enzymes in the serum and brain (data not shown). The activities of AChE and BChE in the serum positively correlated with their brain activities (r = 0.431, p < 0.05 and r = 0.521, p < 0.01, respectively). A positive correlation was also noted

between the brain activities of the two enzymes (r = 0.343, p < 0.05); however, there was no such dependence in the serum. Moreover, positive correlations occurred between the brain AChE activity and BChE in the serum (r = 0.354, p < 0.05) as well as between AChE activity in the serum and the brain BChE activity (r = 0.463, p < 0.01). 3.2. Lipid metabolism in the brain The administration of chlorfenvinphos for 14 days increased the brain concentration of TG (by 73%), decreased that of PL (by 22%), and had no influence on the concentrations of TCh and FFA (Table 1). After 28 days of the treatment, the brain concentrations of TCh and TG were higher by 56% and 68%, respectively, the concentration of PL was lower by 21%, whereas the concentration of FFA still remained unchanged compared to the respective control group (Table 1). The ratio of TCh/PL increased by 12% and 2.1 times after 14 and 28 days of the exposure to chlorfenvinphos, respectively (Table 1). In the control rats there were no differences in the estimated indices of lipid metabolism after 14 and 28 days, whereas in the animals treated with chlorfenvinphos the brain concentrations of

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Table 1 Chosen parameters of lipid metabolism in the brain of control rats and those exposed to chlorfenvinphos (0.02 LD50 ) for 14 and 28 days. Parameter

Days of experiment 14 days

28 days

Control TCh (mg/g) TG (mg/g) PL (mg/g) FFA (mg/g) TCh/PL

13.07 1.29 20.69 1.55 0.60

± ± ± ± ±

Chlorfenvinphos 0.82 0.09 1.10 0.10 0.05

10.53 2.23 16.14 1.79 0.67

± ± ± ± ±

0.92 0.22a * 1.19a * 0.14 0.09a *

Control 12.15 2.14 19.73 1.82 0.60

Chlorfenvinphos

± ± ± ± ±

0.69 0.30 1.85 0.15 0.05

18.93 3.60 15.62 1.52 1.27

Data is mean ± S.E. for 10 animals. Statistically significant differences (ANOVA, Kruskal–Wallis ranks test) are indicated by: vs. respective control group and exposed to chlorfenvinphos for 14 days. TCh, total cholesterol; TG, triglycerides; PL, phospholipids; FFA, free fatty acids; TCh/PL, the ratio of TCh to PL. * p < 0.05. ‡ p < 0.001. a

TCh, TG, and the ratio of TCh/FL after 28 days were higher by 80%, 61%, and 89%, respectively, than after 14 days (Table 1). The brain concentration of TCh positively correlated with the concentration of TG and the ratio of TCh/PL. A positive correlation was also observed between the concentration of TG and TCh/PL ratio (Table 2). 3.3. Distribution of living, apoptotic, necrotic and aponecrotic cells in the brain The treatment with chlorfenvinphos for 14 and 28 days influenced the distribution of living (Annexin V− /PI− ), apoptotic (Annexin V+ /PI− ) and necrotic (Annexin V− /PI+ ) cells in the brain of rats, but had no impact on the number of aponecrotic cells (Annexin V+ /PI+ ; Fig. 2). The number of the living brain cells decreased (by 10%) already after 14 days of this pesticide administration and remained lower (by 5%), compared to the respective control group, after 28 days (Fig. 2). The percentage of both apoptotic and necrotic cells in the brain of the animals treated with chlorfenvinphos increased (Fig. 2). After 14 days of the experiment the percentage of apoptotic and necrotic cells was higher 2.9 times and by 84%, respectively, whereas after 28 days by 63% and 29%, respectively, than in the respective control group. The chlorfenvinphos-induced changes in the occurrence of the living and necrotic cells were independent of the duration of the treatment, whereas the percentage of apoptotic cells after 28 days was lower by 24% than after 14 days. The percentage of the living cells in the total population of the brain cells negatively correlated with that of the apoptotic and necrotic cells. Moreover, a high positive correlation occurred between the occurrence of apoptotic and necrotic cells in the brain (Table 2). 3.4. Dependences between AChE and BChE activities in the serum and brain, and indices of lipid metabolism as well as the number of living, apoptotic, and necrotic cells in the brain Numerous statistically significant correlations were noted between AChE and BChE activities in the serum and brain, and some of the estimated indices of lipid metabolism as well as the occurrence of the living, apoptotic and necrotic cells in the brain (Table 2). The serum and brain activity of AChE negatively correlated with the brain concentration of TCh and TCh/PL ratio. Moreover, a negative correlation occurred between the brain AChE activity and TG concentration, and a positive correlation was noted between the serum AChE activity and FFA concentration. The activity of BChE in the serum and brain negatively correlated with TG concentration and TCh/PL ratio in the brain. A negative dependence was also observed between BChE activity in the serum and the brain concentration of TCh, whereas the brain activity

b

± ± ± ± ±

1.61a ‡ 0.35a ‡ 1.64a * 0.12 0.30a ‡

b‡ b‡

b‡

vs. the group

of this enzyme positively correlated with FFA concentration. The activities of AChE and BChE in the serum and brain positively correlated with the prevalence of the living brain cells and negatively with the occurrence of apoptotic and necrotic cells. Statistically significant correlations were noted between some of the indices of lipid metabolism and the occurrence of the living, apoptotic and necrotic brain cells (Table 2). The brain concentrations of TCh and TG positively correlated with the prevalence of apoptotic and necrotic cells among the total population of the brain cells. A negative correlation occurred between the concentration of PL and the number of apoptotic cells. Moreover, the ratio of TCh/PL negatively correlated with the occurrence of the living brain cells, and positively with that of the apoptotic and necrotic cells. There were no dependences between the prevalence of the aponecrotic brain cells and all other estimated parameters (Table 2).

4. Discussion The present study has investigated the hypothesis that low repeated exposure to OP pesticides may cause disorders in lipid metabolism in the brain and induce apoptotic and necrotic death of the nervous cells contributing in this way to the CNS damage. Neurotoxicity of OP compounds has been mainly explained by accumulation of acetylcholine, due to the inhibition of AChE and BChE activities, that produces cholinergic effects (Lotti, 2001; Darvesh et al., 2003; Celik and Isik, 2009; Sekar Babu et al., 2010; Li et al., 2011). Other pathways, including changes of the lipid profile of the nervous tissue as well as induction of apoptosis and necrosis of the nervous cells seem also to be involved (Brocardo et al., 2005; Üner et al., 2006; Kaur et al., 2007; El-Demerdash, 2011; Li et al., 2011; Solati et al., in press); however, up to now the non-cholinergic (non-cholinesterases-related) mechanisms have not been appropriately evaluated. The present study is the first to investigate and confirm the possibility of involvement of these mechanisms in the development of the OP pesticides-induced damage to the CNS at low exposure. Moreover, the paper provides important data on the impact of these compounds on the brain activity of BChE and the relationship between this enzyme and AChE. The brain, being the lipid-rich tissue having relatively low antioxidative potential, is especially susceptible to the action of factors capable of inducing oxidative stress and disturbing lipid metabolism (Vance et al., 2005; Oliveira and Di Paolo, 2010; Ong et al., 2010). The nervous tissue due to high content of peroxidizable unsaturated lipids and high oxygen utilization is more vulnerable to peroxidative damage than other organs. The lipophilic properties of OP pesticides facilitate their interactions with cellular membranes. These compounds easily cross the blood–brain barrier and change

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Table 2 Dependences between investigated parameters in the serum and brain of rats. Variable

Indices of lipid metabolism in the brain TCh

AChE in serum −0.352* −0.421* AChE in brain −0.344* BChE in serum BChE in brain NS Indices of lipid metabolism in the brain TCh TG PL FFA TCh/PL Brain cells quantification Living Apoptotic Necrotic

Brain cells quantification

TG

PL

FFA

TCh/PL

Living

Apoptotic

Necrotic

Aponecrotic

NS −0.539† −0.468† −0.641‡

NS NS NS NS

0.390* NS NS 0.357*

−0.482† −0.641‡ −0.464† −0.516†

0.460† 0.486† 0.346* 0.379*

−0.350* −0.40* −0.483† −0.545†

−0.343* −0.563‡ −0.539† −0.659‡

NS NS NS NS

0.347*

NS NS

NS NS NS

0.521† 0.506† NS NS

NS NS NS NS −0.674‡

0.624† 0.525† −0.376* NS 0.481*

0.492# 0.583‡ NS NS 0.606‡

NS NS NS NS NS

−0.674‡

−0.725‡ 0.819‡

NS NS NS

Spearman rank correlation analysis was performed to investigate the relationship between variables. Data is presented as a correlation coefficient (r) and the level of statistical significance (p). NS, not statistically significant (p > 0.05). AChE, acetylcholinesterase; BChE, butyrylcholinesterase; TCh, total cholesterol; TG, triglycerides; PL, phospholipids; FFA, free fatty acids; TCh/PL, the ratio of TCh to PL. * p < 0.05. † p < 0.01. ‡ p < 0.001. # p = 0.05.

the fluidity of cellular and sub-cellular membranes in the CNS system leading to their improper function (Blasiak, 1993; Videira et al., 2001). The increased concentrations of TCh and TG with the elevated TCh/PL ratio, and decreased PL concentration observed in the brain of the rats exposed to chlorfenvinphos allow for the conclusion that this pesticide might decrease fluidity and disturb function of cellular membranes of the nervous cells. PL and cholesterol are the most abundant lipid components of cellular membranes in the nervous tissue and they play a central role in the physiology and pathology of the CNS (Blasiak, 1993; Refolo et al., 2000; Björkhem and Meaney, 2004; Dietschy and Turey, 2004; Pandya et al., 2004; Vance et al., 2005; Martins et al., 2009; Oliveira and Di Paolo, 2010; Ong et al., 2010). PL influence multiple aspects of the physiology of the nervous cells, including cytoskeleton regulation, membrane transport, and signal transduction (Oliveira and Di Paolo, 2010). Since these compounds form the stem structure of cellular membranes in the CNS, perturbations in their metabolism can lead to alterations in the dynamics of the cellular membranes and markedly influence viability of the nervous cells (Pandya et al., 2004). OP compounds can react with membrane PL resulting in a decrease in their concentration in neurons (Blasiak, 1993). The observation of decreased PL concentration and increased TCh/PL ratio in the brain tissue at the brain activities of AChE and BChE inhibited by only 12% and 15%, respectively, and unchanged activities of these enzymes in the serum shows that OP pesticides may affect the cellular membranes in the CNS at low repeated exposure. Cholesterol is essential for the proper function of synapses, synthesis of myelin, and transmembrane transport (Blasiak, 1993; Dietschy and Turey, 2004; Ong et al., 2010). Since cholesterol status in the brain is independent of its body turnover (Björkhem and Meaney, 2004), the chlorfenvinphos-induced increase in the concentration of TCh resulted from changes in the brain metabolism of this compound and might be caused by an inhibition of its excretion or induction of its synthesis in oligodendrocytes and astrocytes. Available literature data shows on the connection between cholesterol metabolism in the CNS and metabolic processes resulting in the synthesis of ␤-amyloid (Refolo et al., 2000; Vance et al., 2005; Ong et al., 2010). It has been suggested that increased content of cholesterol in the brain contributes to the development of neurodegenerative changes through enhancing synthesis and deposition of ␤-amyloid in the brain tissue (Refolo et al., 2000; Ong et al., 2010).

␤-Amyloid can disrupt lipid membranes by specific binding to PL and by forming pores permeable to various ions (Arispe et al., 1993; McLaurin and Chakrabartty, 1996). Since the lipid-mediated signalling is involved in the regulation of the brain function (Oliveira and Di Paolo, 2010; Ong et al., 2010), it can be concluded that the chlorfenvinphos-induced changes in the lipid profile of the brain may contribute to the CNS damage. The negative correlations noted between the activities of AChE and BChE in the serum and/or brain, and the brain concentrations of TCh and TG as well as the ratio of TCh/PL confirm the possibility of this pathway. Although the exposure to chlorfenvinphos had no statistically significant impact on the brain concentration of FFA, the positive correlations noted between the concentration of FFA and the activities of AChE in the serum and BChE in the brain suggest the dependence between these parameters. A very strong evidence of the toxic impact of the exposure to chlorfenvinphos on the brain is the decreased number of the living brain cells resulting from their enhanced apoptosis and necrosis. Necrosis is the toxic cell death, whereas apoptosis is a major biological mechanism for the removal of damaged cells. Necrosis is the most serious consequence of the injurious impact of any factor on a cell. Disturbed apoptosis in the CNS may contribute to neurodegenerative changes, such as Alzheimer’s and Parkinson’s disease. Moreover, intensified apoptosis may lead to structural changes in the brain causing disorders of memory and the learning process (Martins et al., 2009; Ziv and Melamed, 2010). A decrease in the number of the living brain cells, independently of a cause, results in disorders in the proper function of the CNS (Kaur et al., 2007; Ziv and Melamed, 2010; Li et al., 2011). The changes in the occurrence of the living, apoptotic and necrotic brain cells observed in the animals exposed to chlorfenvinphos may be explained, at least partly, by pro-oxidative properties of this compound. OP pesticides were reported to cause marked perturbations in the enzymatic and non-enzymatic antioxidant defense system, and induce oxidative stress and oxidative damages in the brain, including especially enhanced lipid peroxidation (Brocardo et al., 2005; Üner et al., 2006; Kaur et al., 2007; Lukaszewicz-Hussain, 2008; Dwivedi and Flora, 2011; ElDemerdash, 2011; Saafi et al., 2011). Chlorfenvinphos, at the exposure used in the present study, has been noted to influence the brain activity of antioxidant enzymes and diminish

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Fig. 2. Distribution of apoptotic, necrotic, aponecrotic and living cells in the brain of control rats and those exposed to chlorfenvinphos (0.02 LD50 ) for 14 and 28 days. Data is mean ± S.E. for 10 animals. Statistically significant differences (ANOVA, Kruskal–Wallis ranks test) are indicated by: a vs. respective control group and b vs. the group exposed to chlorfenvinphos for 14 days. *p < 0.05; ‡ p < 0.001. Numerical values above bars indicate percentage decrease (down-pointed arrow) or increase (up-pointed arrow) compared to the respective control group.

reduced glutathione concentration (Lukaszewicz-Hussain, 2008). Decreased level of reduced glutathione and the accompanying accumulation of reactive oxygen species (ROS) result in damage to the membranous components of the cells, including mitochondria that play a decisive role in the apoptotic cascade (Kaur et al., 2007). Kaur et al. (2007) have provided evidence that low-level chronic exposure to dichlorvos induces oxidative stress in the brain and causes neuronal apoptotic cell death. The study by these authors shows that the mechanisms of neurodegenerative impact of chronic exposure to OP pesticides involve pathological raise in the mitochondrial concentrations of calcium ions and increased production of ROS, which impair mitochondrial bioenergetics and physiological functions through release of cytochrome-c from mitochondria and its subsequent redistribution into cytosol and activation of caspase-3. Findings of other authors also indicate that oxidative stress is involved in the mechanisms of the OP pesticidesinduced CNS damage (Brocardo et al., 2005; Üner et al.,

2006; Lukaszewicz-Hussain, 2008; Dwivedi and Flora, 2011; ElDemerdash, 2011). The enhanced apoptosis and necrosis noted in the brain tissue of the rats exposed to chlorfenvinphos might also be a consequence of the increased concentration of TCh in this organ. Cholesterol accumulated in the brain has been reported to promote apoptosis and necrosis (Geng et al., 2003). The positive correlations noted in the present study between the prevalence of apoptotic and necrotic cells in the brain, and the concentrations of TCh and TG, and the TCh/PL ratio as well as between the occurrence of the living cells and the TCh/PL ratio confirm the relationship between the chlorfenvinphos-induced disruption of lipid metabolism and the decreased number of the living brain cells. The fact that increased apoptosis and necrosis were observed after 14 days of the exposure to chlorfenvinphos, whereas the concentration of TCh increased only after 28 days, shows that accumulation of TCh might be only one of the contributory factors, but not a sole cause of the decreased occurrence of the living brain cells. As it was above

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discussed, oxidative stress might be the important cause of the chlorfenvinphos-induced enhanced apoptosis and necrosis of the brain cells resulting in the diminished population of the living cells. The impact of OP compounds on the nervous system was a subject of numerous studies (Lotti, 2001; Sekar Babu et al., 2010; Kaur et al., 2007; Celik and Isik, 2009; Li et al., 2011; Solati et al., in press); however, still there is a lack of investigations focused on the effect of low oral exposure, being the main route of human intoxication with these compounds during the lifetime, on the non-cholinergic pathway. The measurements performed in the present study draw attention to the involvement in the OP pesticides-induced CNS damage of the non-related to cholinesterases inhibition pathways such as destroying the brain lipid profile and reduction of the living brain cells due to their enhanced apoptosis and necrosis. However, the fact that the activities of AChE and BChE in the brain and serum correlated with the indices of the brain lipid profile and prevalence of the living, apoptotic and necrotic brain cells seems to show on the connection between these enzymes inhibition and the extent of the non-cholinesterases-related CNS damage due to the exposure to OP pesticides. AChE and BChE are widely distributed within entire CNS and an inhibition of their activities decreases cellular metabolism, induces deformities of cellular membranes, and disturbs metabolic and nervous activity (Lotti, 2001; Darvesh et al., 2003). Although both enzymes are very useful biomarkers to estimate the damaging impact on the nervous system, AChE is still the mostly used indicator of exposure to OP pesticides in human (Lotti, 2001; Hernández et al., 2006; Singh et al., 2011) and experimental animals (Üner et al., 2006; Sekar Babu et al., 2010; Dwivedi and Flora, 2011; ElDemerdash, 2011). Some authors have determined BChE in the serum and various tissues under exposure to OP compounds (Celik and Isik, 2009; Karasova et al., 2009; Lajmanovich et al., 2009); however, only little data is available on the impact of these xenobiotics on the brain activity of this enzyme (Celik and Isik, 2009). The present study provides now and important data in this subject. The positive correlations noted between the brain activity of BChE and AChE in the serum and brain are an evidence for the relationship between these enzymes under exposure to PO pesticides. The worthy of notice is also the finding that chlorfenvinphos inhibits both enzymes to similar extent and thus their ratio in the serum and brain remains unaffected. Moreover, the positive correlations noted between BChE activity in the brain and serum, and between the brain BChE and AChE in the serum and brain as well as the numerous dependences between BChE and the indices of lipid metabolism and the distribution of the living, apoptotic and necrotic cells in the brain confirm usefulness of this enzyme to monitor low repeated exposure to OP pesticides and to assess their impact on the CNS. Unlike our finding, Celik and Isik reported that sublethal doses of dichlorvos and methyl parathion (5 and 10 mg/l for 28 days), that caused a marked inhibition of AChE activity, did not change the brain BChE activity, except for dichlorvos in the dose of 5 mg/l (Celik and Isik, 2009). The extent of inhibition of the serum activity of AChE after 28 days of the exposure to chlorfenvinphos was close to the 25% cut-off level for biological surveillance of subjects occupationally exposed to OP pesticides (Lotti, 2001). At this level of inhibition of the serum activity of this enzyme we have noted very clear disruption of the brain lipid profile and reduction in the population of the living brain cells. Moreover, it is important to underline that we have observed destroying of the lipid metabolism and changes in the occurrence of the living, apoptotic and necrotic brain cells at low inhibition of the brain activities of AChE and BChE, and their unaffected activities in the serum. Hernández et al. (2006) reported a lack of changes in the serum concentration of TCh and TG in the agriculture farmers chronically exposed to pesticides having the serum activity of AChE

inhibited by less and more than 25%; however, this observation does not exclude changes in the brain lipid profile. In the present study we have observed no symptoms of the CNS damage; however, taking into consideration the changes in the brain lipid profile, decreased number of the living brain cells and the extent of inhibition of the brain activities of AChE and BChE after 28 days of chlorfenvinphos administration, it seems very probable that longer exposure will more seriously destroy the function of the whole CNS. A lack of literature data closely related to the subject of the present study makes impossible wider discussion of the received results. In conclusion, the present study has revealed that low repeated intoxication with chlorfenvinphos inhibiting the brain activities of AChE and BChE by only a dozen or so percent (12–15%) disturbs lipid metabolism and decreases the number of the living cells through induction of apoptosis and necrosis in the brain of rats. A very important and practically useful finding of the study is providing evidence that chlorfenvinphos may influence the lipid profile and decrease the number of the living brain cells at the exposure not affecting the serum activities of cholinesterases and causing only moderate inhibition of their brain activities. Based on the findings it can be concluded that even low exposure to OP pesticides may contribute to the development of neurodegenerative diseases through disrupting the lipid profile of the nervous tissue and decreasing the number of the nervous cells. However, further research is needed to explain the non-cholinergic mechanisms of neurotoxic action of these compounds.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgment This study was financially supported by the Medical University of Bialystok (Poland).

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