An Organophosphate Insecticide
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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.