Chlorpyrifos, 0,0' -dicthyl-0-3,5,6-trichloro-2-pyridyl phosphorothionate, exposure in rats caused signiftcant inhibi- tion of acctylcholincsterac activity in different ...
Indian Jo urnal of Experimental Biology Vol. 39, February 2001, pp. 174- 177
Chlorpyrifos induced alterations in levels of thiobarbituric acid reactive substances and glutathione in rat brain Radhey Shyam Verma & Nalini Srivastava· School of Studies in Biochemistry, Jiwaji University, Gwalior 474 0 II, Indi a
Received 19 January 2000; revised 13 October 2000 Chlorpyrifos, 0,0' -dicthyl-0-3,5,6-trichloro-2-pyridyl phosphorothionate, exposure in rats caused signiftca nt inhibition of acctylcholincsterac activity in different regi o ns of brain and generated oxidative stress as evidenced by increase in the level of thiobarbituric acid reacti ve subs tances and decrease in th e ratio of reduced to oxid ized glutathione in all the three regions of brain. Malondialdchydc leve l was increased sign ificantly in all reg io ns of brain and the in c rease was dose dependent. Mid brain showed hi ghest leve l of lipid pcroxidation.
Chlropyrifos (CPF), a broad spectrum organophosphate insecticide, has been widely used throughout the world including India . Unlike other organophosphate (OP) compounds, which have short retention 1 periods, CPF remain s in the soil for quite long time • OP Insecticides have been shown to exert direct effects on neurotransmitter receptors 2, membra ne signal transduction pathways 3 and on the release of neurotransmitters4. CPF is metabolized in liver by microsomal xenobiotic metabolizing enzymes. These enzymes cata lyse oxidative desulfuration of CPF to chlorpyrifos oxon which eli cit neurotoxicity by inhibiting acetylcholinesterase. Both CPF and oxon are rapidly hydrolysed to 3,5,6-trichloro-2-pyrid inol by hepatic mixed function oxidases 5 . The compounds metabolized through mixed function oxidase system induce oxidative stress 6 which may contribute significantly in the overall toxicity. The redox state of the tissues is reported to be indicative of oxidative stress . The redox status is reported to be altered during ageing7, excessive exercise 8 and by exposure to variety of chemicals 9· 10 . The present study has been undertaken to determine the toxic effects of intramuscularly administered CPF on different regions of rat brain . The potential of CPF to generate oxidative stress was assessed by measuring lipid peroxidation and ratio of reduced to oxidised gl utathione (GSH/GSSG) in different parts of brain of exposed an imals as GSH plays a pivotal role in the maintenance of intracellular redox status and antioxidant enzyme functions .
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Adu lt male albino rats of Wistar strain (weighing about 125 ± 5g) were obtained from the anim al hou se of Defense Research Development Establishment, Gwalior and maintained in a light and temperature contro ll ed (25EC) animal room of ou department on standard pellet diet (Hindustan Le vers, India) and water ad libitum. The animals were acclimatized for one week prior to start of the experiment. The animals were divided into 4 groups of 6 animals each. Three groups were given CPF [Lupin Agrochemical (India) Ltd, Bharuch, Gujrat] dissolved in dimethylsu lfoxide (DMSO) intramuscularly in thigh muscles for 3 days at a dose of 50, I 00 and 200 rng/kg body weight and sacrificed on fourth day. The fourth, control group was g iven only DMSO. A nimal were sacrificed by cervical dislocation and brain was excised out. Different reg~ons of the brain were separated and washed with 0 .9% NaCl, homogenized in Potter - Elvehjem homogeniser and used for different estimations. Lipid peroxidation 11 , acetylcholi nesterase 12 , GSH 14 and GSSG 13 and protein were estimated. Results were expressed as mean ± SE of different set of observations taken on different days. A ll the statistical analyses were performed using one-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparison test applied across treatment groups for each brain area. Significance was based on a P< 0.05 . The results are presented in Tables I and 2. Administration of CPF in rats produced a significant loss in weight of the animals. It was observed that the weight loss was dose dependent. The acute 15 oral LD 50 for rats is reported 165 mg/kg body weight therefore the doses of CPF in the present experiment
NOTES
were selected as 50, 100 and 200 mg/kg body weight. These doses cover the exposure levels ranging from less than LD 50 to more th an LD 50. The decrease in acetyl cholinesterase activity was also significant even at lowest dose in all the three regions of the brain. Fore brain showed maximum decrease among the three regions tested. Decrease in acetylcholinesterase acti vity in different tissues has been reported earli er. Kaur et al. 16 reported decrease in cholinesterase activity in erythrocytes and serum of goat kids. The neurotoxic behaviour of CPF and many other OP compounds (whether insecticides, warfare agents or simple metabolic inhibitors) is well reported 17 • The present study corroborates the reported results. Chemicals which are metabolized by mixed function oxidation or redox cycled, express their toxicity partially or totally via oxidative stress. Thi s insecticide is biotransformed to its oxygen analog, i.e. CPF oxon, before it functions as potent cholinesterase inhibitor 18• Phosphorothionate compounds undergo oxidative desulfuration by cyt P-450 dependent monooxygenase system to exhibit anticholinesterase activity 19 • Present results showed that CPF generated oxidative stress in all parts of brain as evidenced by increase in the level of thiobarbituric acid reactive substances (TBARS) in exposed ani mals. This indicates the activation of lipid
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peroxidation system which is associated with fall of 0 2 tension and increase tn NADPH level. Further, tilcreased MDA level may lead to peroxidative damages, deteriorating the structural and functional integrity of the neuronal membrane. In addition to the primary defense against oxidative stress by antioxidant enzymes, secondary defense against it is also offered by small molecules which react with radicals to produce another radi cal compound, the 'scavengers'. When these scavengers produce a less harmful radical species, they are called 'antioxidant'. Reduced glutathione is a powerful and known antioxidant in the cells and its metabolism is Table 2-Effect o f ch lorpyrifos on the leve ls of GSH and GSSG and GSH/GSSG rat io of different parts of rat bra in [Values are mean± SE of 6 different set of obse rvati ons]
Fore Bra in
Table !-E ffect of chlorpyrifos on lipid peroxidation and acetylcho linesterase activity of different parts of ra t brain [Values are mean± SE of 6 different set of obse rvat ions ! Brain Regi on
Fore Brain
Group
Acetylcholinesterase (nmole/min/ mg protein)
GSSG (!lglg ti ssue)
GSH/ GSSG
A
Control
11 7.22 ±1.03
424.92 ± 13.40
0.276
B
50 mg/kg
90.39 ±1.42
542 .97 ±37.20
0. 166
C
IOOmg/kg
75 .28 ±!.55
562.46 ± 13.60
0.134
D
200 mg/kg
74.34 ±2 .27
658 .06 ±3 1.30
0.113
Mid Brain
A
Contro l
13 1.45 ± 6.08
440.0 1 ±38.31
0.293
B
50 mg/kg
106.43 ± 4.3 1
570.80 ±22.00
0.186
I 00 rng/kg
97.68 ± 2.55
657.3 1 21.80
0.149
D
200 rng/kg
92.67 ± 3.76
672.23 ±25.60
0.1 38
A
Control
47.29 ±0.92
66 1.00 ±26.30
0.072
Control
54.7 1±0.76
45.69±1. 17
B D
50 mg/k g 100 mg/kg 200 mg/kg
69.83±4.37 88.49±2.74 10:153±3.04
21.6 1±0.45 12.23±0.4 1 8.4 1±0. 14
A
Control
37.54±0.52
20.05±0.34
B
46 .87±1.80 73 .09± 1.44 84.22±2.79
11 .40±0.16 8.44±0.09 7.30±0.14
B
50 mg/kg
38.40 ±0.79
741.93 ±28 .1 0
0.052
D
50 mg/k g IOOmg/kg 200 mg/kg
A
Control
74.64± 1.65
50.90± 1.99
C
100 mg/kg
30.9 1 ±3.57
801.96 ±35.8 1
0.039
B
50 mg/kg 100 mg/kg 200 mg/kg
90. 17±2 .1 4 I 08.72±5 .11 128.56±2.83
22.55±0.15 20.22±0.34 16.00±0.22
D
200 mg/kg
29.63 ±0.77
853.09 ± 15.50
0.035
13 rain
c Hind Brain
GSH (!lglg ti ssue)
A
c Mid
Lipid perox ida ti on (nmole malo ndialdehyde fo rm ed/g o f ti ssue)
Group
Brain Region
c D
A= control, B =50 mg/kg, C = 100 mg/k g, D = 200 mg/kg P < 0.05 in each case whe n compared with correspo ndin g co ntrol
Hind Brain
A= Control, B =50 mg/k g, C = I 00 mg/kg, D = 200 mg/kg P < 0.05 in each case when co mpared wi th correspo ndin g control
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INDIAN J EXP BlOL, JA NUARY 200 1
very sensitive to normal/oxidised state of the cells 20 . It plays an important role in protecting cells against radical and oxyradical damage. Relationship between extent of lipid perox idation and glutathione status of the li ver has a significant inverse rel ation 21. The results of present study showed that level of GSH was decreased and GSSG was increased after CPF exposure in all pa1ts of brain (Table 2). Vodela and Dalvi22 reported a very slight increase in hepatic GSH level in rats but decrease in its level in chiken liver. They observed a significant increase in glutathione-S-transferase activity in rats after CPF administration and concluded that it is detoxified through GSH mediation. Decline in GSH may result in increasing the lipid perox idation causing stimulation of radical form ation rendering brain tissues to peroxidative damage and make the system inefficient. The protective effects of antioxidant enzymes and GSH had been reported on iron medi ated lipid perox idati on in gastrointestinal tracts of rat23 . Bagchi et al 9 have also rep01t ed depletion of GSH and increase in lipid peroxid ation in liver and brain of mice after 12-0tetradecanosylphorbal-13-acetate (TPA) treatment. The mechanism by which GSH is depleted and in tum prevented the formation of malondialdehyde (MDA) is not clear. Rel atively high ratios of GSH/GSSG are maintained intracellularly through the action of glutathione reductase in an NADPH dependent reaction . Decreased GSH may result in the impairment of mechanisms of metabolic detoxication. Decrease in this ratio is indicative of oxidative stress as reported in aging, ethanol intoxication, lindane exposure and excessive exercise etc.7.8·24 ·25 . This fall may be due to decrease in the activity of glutathione reductase or depletion in the supply of NADPH. Further investigations on the mechanism of oxidative stress generation and the role of GSH/GSSG as antioxidant are warranted. This work is supported by a grant from Department of Science and Technology, New Delhi. Help and cooperation of Dr R.Vij ay raghvan and Dr P.V .L. Rao, Scientists, Defense Research Development Establishment, Gwalior in fluorimetric estimations is thankfully acknowledged. We thank Dr Vijayraghavan for ex pert statistical advice.
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NOTES
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