Cent. Eur. J. Med. • 4(4) • 2009 • 506-511 DOI: 10.2478/s11536-009-0072-z

Central European Journal of Medicine

Changes in antioxidative parameters in the kidney of rats subchronically intoxicated with chlorfenvinphos – an organophosphate insecticide

Research Article Anna Łukaszewicz-Hussain*

Department of Toxicology, Medical University of Bialystok, 15-222 Białystok, Poland

Received 7 November 2008; Accepted 14 April 2009

Abstract: Chlorfenvinphos is an organophosphate insecticide, posing a risk to those who are professionally involved in its production and use in agriculture, as well as to the general population. Organophosphates (OPs) are the class of insecticides, whose primary target is acetylcholinesterase (AChE) that hydrolyzes acetylcholine, a major neurotransmitter at the central and peripheral neuronal synapses. Moreover, many authors postulate that these compounds, both in acute and chronic intoxication, change the activities of antioxidative enzymes, thus leading to the enhancement of lipid peroxidation in many tissues. In the current study, animals received once a day, intragastrically with a stomach tube, 0.1ml/100g of olive oil (control groups) and oil solution of chlorfenvinphos at a dose of 0.02LD50 (0.3 mg/kg b. w.) – the experimental groups. The animals were sacrificed on day 14 or on day 28 of exposure. In the kidneys of rats, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) as well as reduced glutathione level (GSH) were determined. Chlorfenvinphos administration resulted in increased activities of antioxidative enzymes in the kidney of rats. Renal activities of SOD, GPx and GR were more pronounced on day 28 of chlorfenvinphos exposure than on day 14. The kidney reduced glutathione level (GSH) did not change in comparison to the control level. The current experimental find- ings indicate that subchronic administration of chlorfenvinphos leads to an adaptive response in the kidney of rats and this response is mostly due to reduced glutathione level and glutathione metabolism. Keywords: Subchronic intoxication • Chlorfenvinphos • Kidney • Oxidative stress

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction brain [6-12]. However, changes in lipid peroxidation index in different organs are due to for example, exposure duration. In subacute chlorfenvinphos intoxication, we Chlorfenvinphos (2-chloro-1-(2,4-dichlorophenyl) demonstrated enhancement of lipid peroxidation index vinyl diethyl phosphate, CVP) is an organophosphate in the liver along with exposure duration. In contrast, the insecticide, posing a risk to those who are professionally level of malonyldialdehyde in the kidney decreased on involved in its production and use in agriculture, as day 28 of CVP exposure as compared to day 14 [11]. well as to the general population that may be exposed Thus, there is a tissue-specific response to oxidative through the consumption of polluted food products or stress and this response may be due to different tissue water [1-3]. antioxidant levels [13]. Organophosphates (OPs) constitute a class of After acute human OP poisoning, higher levels of OP insecticides, whose primary target is acetylcholinesterase were found in the kidneys than in the blood [6,14]. In rats, (AChE) that hydrolyzes acetylcholine, a major OPs were shown to cause acute renal tubular injury and neurotransmitter at the central and peripheral neuronal renal dysfunction, although the changes do not correlate synapses [3-5]. Moreover, many authors postulate that with cholinesterase inhibition degree [6,15]. Similar these compounds, both in acute and chronic intoxication, observations were made by Łukaszewicz-Hussain change the activities of antioxidative enzymes and cause [11], who in an earlier work observed enhancement enhancement of lipid peroxidation in the kidney, liver and

* E-mail: [email protected] 506 A. Łukaszewicz-Hussain

of lipid peroxidation in the kidney before reduction in glutathione peroxidase (GPx) and glutathione reductase serum cholinesterase activity, i.e. an index assessed in (GR) as well as reduced glutathione level (GSH) were professional OP exposure. determined. Previously, we also found a very high level of The Local Ethical Committee approved the study. lipid peroxidation index – determined as the level of thiobarbituric acid reactive substances (TBARS) in 2.3. Biochemical estimation the kidney of subchronically low-level chlorfenvinphos SOD activity was measured in 10% kidney homogenates exposed rats. The TBARS level on day 28 of prepared in 0.25M sacharose, centrifuged at 8500xg, chlorfenvinphos administration was lower as compared 40C for 10 min; next, the assay was performed in the to the values observed in the early phase (on day 14) of supernatant using BIOXYTECH SOD-525TM Assay kit intoxication [11]. Thus, results of the above-cited paper produced by OXIS International, Inc., Portland, USA. indicate that an adaptive process takes place in this Renal CAT activity was also measured in 10% organ and suggest elevated activity/level of antioxidative homogenates prepared in phosphate buffer, centrifuged parameters. On the other hand, reactive oxygen species at 9000xg, 40C for 15 min. The activity was determined are known to mediate many toxin-induced renal injuries in the supernatant as described by Aebi [18]. [6,16,17]. Renal GPx activity was measured in a supernatant For the reasons mentioned above, the objective using BIOXYTECH GPx-340TM Assay kit produced of the present study was to determine antioxidative- by OXIS International, Inc., Portland, USA. For this enzymatic and nonenzymatic parameters, and verify determination, the supernatant was prepared by the hypothesis that the decrease in TBARS level homogenization of kidney tissues in 8 volumes of cold with exposure duration is due to the enhancement of buffer (50mM Tris-HCl, pH 7.5, containing 5mM EDTA antioxidative barrier. and 1mM 2-mercaptoethanol), and then centrifuged at 8500xg for 10 min., 40C. Renal GR activity was determined with the use of 2. Materials and Methods BIOXYTECH GR-340TM Assay kit produced by OXIS International, Inc., Portland, USA, in a supernatant of 2.1. Animals tissue homogenate obtained by mincing the kidney Male Wistar rats (from a certified Laboratory Animal in cold buffer (50mM potassium phosphate pH 7.5, House, Brwinow, Poland), with a 250-280g body weight, containing 1mM EDTA) and centrifuging the homogenate were housed in metal cages with free access to drinking at 8500xg for 10 min., 40C. water and standard pellet diet. For determination of GSH level, the kidneys were minced in ice-cold metaphoshate acid solution and 2.2. Treatment and tissue collection centrifuged at 3000xg for 10 min. The assay was The animals received once a day, intragastrically performed in a clear supernatant of the homogenate TM with a stomach tube (ones daily), 0.1ml/100g of with use of BIOXYTECH GSH-400 Assay kit produced olive oil (control groups – I and III) and oil solution of by OXIS International, Inc., Portland, USA. chlorfenvinphos i.e. 2-chloro-1-(2,4-dichlorophenyl) The enzymatic activities were expressed as units of the activity per gram of protein and for this reason, vinyl diethyl phosphate (CVP) at a dose of 0.02LD50 (0.3 mg/kg b. w.) – the experimental groups II and IV. The renal protein concentration was measured according to the method of Lowry et al. [19], using bovine serum as LD50 for chlorfenvinphos was 15 mg/kg b. w. [2]. Chlorfenvinphos – the purity value – min. 98.2% a standard. (m/m) based on determinations made on samples Reduced glutathione level was expressed in mmol of using gas chromatography (GLC) and thin layer GSH per gram of kidney tissue. chromatography (TLC), was obtained from the Institute of Organic Industrial Chemistry, Warsaw, Poland. The 2.4. Statistical analysis identity of the product was established by infrared Data for all groups of animals were compared using one spectroscopy. way analysis of variance (ANOVA) followed by the Tukey- The animals were sacrificed on day 14 (groups I and Kramer multiple comparison tests. Pearson correlation II) or on day 28 (groups III and IV) of exposure. Kidneys between the study parameters was calculated. were removed and washed with ice-cold 0.9% NaCl The probability of p < 0.05 was considered solution containing 0.16-mg/ml heparin. Renal activities significant. of superoxide dismutase (SOD), catalase (CAT),

507 Changes in antioxidative parameters in the kidney of rats subchronically intoxicated with chlorfenvinphos – an organophosphate insecticide

Table 1. Activities of antioxidative enzymes and reduced glutathione level in kidneys of rats in subchronic intoxication with chlorfenvinphos.

Days of exposure to CVP 14 days 28 days parameter 0 0.02 LD50 0 0.02 LD50 Group I Group II Group III Group IV SOD activity 39.71±2.29 57.38±4.47a 41.75±3.15 63.09±3.08ab (U/mg protein) n=8 n=7 n=7 n=8 CAT activity 52.81±7.02 92.24±7.21a 48.30±6.79 81.31±12.63a (U/mg protein) n=6 n=6 n=6 n=7 GPx activity 42.09±5.37 60.85±6.50a 43.55±2.54 77.55±8.31ab (U/g protein) n=8 n=8 n=8 n=8 GR activity 12.25±1.16 14.77±0.92a 12.05±0.59 18.84±2.24ab (U/g protein) n=7 n=7 n=6 n=7 GSH concentration 3.93±0.81 3.93±0.51 3.65±0.38 4.22±0.40 (mmol/g tissue) n=6 n=6 n=7 n=8

Data represent mean ± SD of 6 to 8 individual values a – statistically significant compared to control; b – statistically significant compared to value observed on day 14 then on day 14. However, the activity of CAT – hydrogen 3. Results peroxide removal enzyme, remained at the same elevated level throughout the experiment. No statistically Activities of the examined antioxidative enzymes significant differences were found in the levels of reduced increased, statistically significant in comparison to glutathione in the rat kidney between chlorfenvinphos the control groups, in the kidney of rats on day 14 of exposed rats and control groups. chlorfenvinphos administration as well as on day The source or the mechanism for increased levels 28. Renal SOD activity on day 28 of exposure to the of reactive oxygen species in CVP-treated rats is insecticide was also statistically significant higher as not explored in this work. However, the increased compared to day 14. However, CAT activity remained at activities of antioxidative enzymes in the kidney of the the same elevated level throughout the exposure. insecticide exposed rats as well as enhancement of lipid In this study, the activities of GPx as well as GR, i.e. peroxidation index reported in our earlier study seem glutathione related enzymes, increased in comparison to indicate generation of superoxide radicals, hydrogen to the control groups throughout the experiment, and peroxide and other reactive oxygen species [11]. Thus, were higher on day 28 than on day 14 of chlorfenvinphos it can be assumed that oxidative stress occurs in the administration. kidneys of chlorfenvinphos exposed rats. Other authors At the same time, kidney GSH level did not change in vivo observed similar effects, i.e. induction of oxidative in comparison to the control level. stress in the kidney by other organophosphates, as well in vitro [6,7,20,21]. The oxidative stress in the kidney is most likely caused by oxygen metabolites of insecticide 4. Discussion as was reported from intoxication with bidrin – another organophosphate compound [6]. In the current study, the rats received chlorfenvinphos, Poovala et al. [6] reported that bidrin (BD) induces an organophosphate insecticide, at a dose of 0.3 mg/ production of hydrogen peroxide and lipid peroxidation in a kidney cell line (LLC-PK – analogue of proximal kg b. w. /day (0.02LD50). The experimental model and 1 chlorfenvinphos dose in this study was the same as tubular cells) and that antioxidant suppressors of lipid used in our earlier investigations [11,12]. LOAEL for peroxidation and free radicals generation protect against chlorfenvinphos was established at 0.7 mg/kg/day, oxidative cell injury. Both the increased production based on adverse neurological effects in rats [2]. of reactive oxygen species and attenuation of the Chlorfenvinphos administration resulted in increased antioxidant barrier of the organism are likely to induce activities of antioxidative enzymes in the kidney of rats. oxidative stress. In other words, oxidative stress can be The renal activities of SOD, GPx and GR were more defined as an imbalance between the production of free pronounced on day 28 of chlorfenvinphos exposure radicals and the body antioxidant defence system – both enzymatic and nonenzymatic. Oxidative stress leads to

508 A. Łukaszewicz-Hussain

oxidation of lipids, proteins and others important cellular study the effect on the activities of renal antioxidative biomolecules [22]. enzymes was observed at a dose smaller than LOAEL It can be assumed, based on the current study findings level for rats [2]. and other reports, that increased activities of antioxidative The changes observed in GSH level with enzymes are due to generation of reactive oxygen chlorfenvinphos treatment were generally tissue-specific species in the course of chlorfenvinphos administration as it was demonstrated by Lukaszewicz-Hussain in an and that oxidative stress may be responsible for some earlier [12] as well as in the current study. of the morphological and pathophysiological changes As reported previously, subchronic administration of observed by others in the kidneys of human and rat chlorfenvinphos leads to changes in brain antioxidative exposed to organophosphates [14,20,23,24]. enzymes - their levels were found to increase throughout Previously, we observed increased level of TBARS the experiment but did not change with time of exposure. in the kidneys of CVP exposed rats as compared to At the same time, brain reduced glutathione level the control group [11]. Accumulation of lipid peroxide is decreased [12]. In turn, the concentration of TBARS considered to be a major contributor to the loss of cell remained at an elevated level up to day 28 of CVP function under oxidative stress conditions [21]. However, exposure [11]. This finding is in agreement with Kaur renal concentration of TBARS decreased statistically et al. [32], who demonstrated that low-level long-term significant with chlorfenvinphos exposure duration [11]. organophosphate exposure led to decrease in GSH As shown in the current study, decreased levels in the mitochondria isolated from dichlorvos concentration of lipid oxidation index is accompanied by treated rat brain. Therefore, the decreased GSH level increased activities of antioxidative enzymes, especially finally results in enhancement of lipid peroxidation those responsible for hydrogen peroxide removal, i.e. [11,12,32]. catalase and glutathione peroxidase. These enzymes The experimental findings of the current study break down hydrogen peroxide. GPx seems to play a suggest that consequences of oxidative stress in tissues more important role in peroxide removal, as it converts depend mostly on tissue GSH level. The unchanged not only hydrogen peroxide but also lipid peroxides to renal level of reduced glutathione leads to decrease water and alcohol. Therefore, GPx “monitors” the rate in TBARS concentration in a later period of low-level of lipid peroxidation and participates in cell membrane chlorfenvinphos administration. Similar observations stabilization [25,26]. were made by other authors, who, in a study on fish, In the current study, renal GPx activity increased with observed a tissue-specific adaptive response to chlorfenvinphos exposure duration, in contrast to CAT neutralize oxidative stress following diazinon exposure activity. This suggests that the change in GPx activity is ; this response was due to different antioxidant levels in associated with the changes in TBARS level observed in tissues [13]. Also, Pena-Llopis et al. [33] demonstrated our earlier study [11]. On the other hand, increased renal that tolerance to the OP dichlorvos could be explained as GPx activity was accompanied by increased activity of individual capacity of maintaining glutathione level. GSH glutathione reductase. Enhancement of this process as the dominant nonprotein thiol in mammalian cells is was demonstrated along with exposure duration. Thus, essential in maintaining the intracellular redox balance activities of GPx and GR go in the same way. and also plays an important function in detoxification of Glutathione peroxidase removes peroxides using xenobiotics or their metabolites [28]. reduced glutathione. In this reaction, GPx converts GSH It can be assumed that GSH level may have to oxidized form (GSSG). In return, to recycle GSSG to controlled the extent of radical-induced lipid peroxidation GSH, cells utilize glutathione reductase [27-30]. Thus, in the kidney to a level that is probably physiologically changes in the activity of GPx are connected with GR tolerable for this organ. activity and with changes in tissue GSH level. In the Thus, based on experimental findings of this study, current study, renal GSH remained at the same level it can be suggested that subchronic administration of as in the control group. Therefore, the simultaneous chlorfenvinphos leads to an adaptive response in the rat rise in GPx and GR activities allowed them to maintain kidney and that this response is mostly due to reduced unchanged concentration of renal GSH and led to glutathione level and its metabolism. decrease in level of kidney thiobarbituric acid reactive substances, as shown in our earlier study [11]. This finding has an important toxicological implication since oxidative stress leads to kidney damage, as it was reported by others [6,21,24,31]. Moreover, in the present

509 Changes in antioxidative parameters in the kidney of rats subchronically intoxicated with chlorfenvinphos – an organophosphate insecticide

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