Alleviating Impact of Taurine on Renal Lipid Peroxidation and Oxidative Stress in Lambda-Cyhalothrin Exposed Rat

Sujata Maiti Choudhury, MSc, MPhil, PhD

Associate Professor, Department of Human Physiology with Community Health, Vidyasagar University, Midnapore, West Bengal 721102, India; E-mail: sujata.vu2009@gmail.com

INTRODUCTION

Now-a-days the use of pesticides in agriculture has been increasing continuously. The harmful effects of many pesticides, such as organophosphates, organochlorine and carbamates, have led to use of pyrethroids as alternatives. Pyrethroids analogs of naturally occurring pyrethrins is widely used in agriculture in many countries because pyrethroids are highly effective, low toxic to non-target organisms and have easy biodegradability.¹ Lambda-cyhalothrin (LCT) is a potent, synthetic, type II pyrethroid pesticide and is worldwide used to control a different variety of insect pests in agricultural and domestic fields and public health sectors.^2,3,4 It is reported that lambda-cyhalothrin is moderately toxic^5,6 for mammals and highly toxic for fish, bees and aquatic invertebrates at low concentrations.^7,8 Through the use of agricultural foodstuff, pesticide residues have the ability to affect directly on human health.⁹ People those are living in proximity to farms and exposed heavily to the home application of pesticides or eat foods rich in pesticide residues, are highly vulnerable to pesticides intoxication in addition to the workers of pesticides manufacturers, agriculture workers and their families.¹⁰ It is well documented that the accidental poisonings and death of humans occurred by the use of pesticides especially in developing countries.¹¹

Many pesticides generate their toxicity through the induction of oxidative stress.¹² The free radicals generate oxidative damage to all biomolecules and initiate a chain reaction that leads to damage in physiological systems and accumulating free radicals over a period of time cause degenerative diseases.¹³ Several research studies reported the induction of oxidative stress by synthetic pyrethroids such as fenvalerate and cypermethrin.^14,15,16

Taurine possesses antioxidant and membrane-stabilizing properties. Several studies reported that taurine exhibited protective activity against renal toxicity by its antioxidative role.¹⁷ On the other study, it was reported that taurine supplementation became resistant to kidney damage and also proteinuria caused by either streptozotocin-induced type 1 diabetes or aminonucleoside-induced glomerulopathy.¹⁸ In a related study, chronic taurine treatment prevented aging-related up regulation of transforming growth factor beta (TGF-β1), collagen types I and IV and fibronectin messenger ribonucleic acid (mRNAs), proteins involved in the development of renal fibrosis in aging rat.¹⁹ Renal function, especially the oxidative status of the renal system may be altered due to the exposure of pyrethroids. The present study was conducted to evaluate the toxic effect of orally administered lambda-cyhalothrin on renal lipid peroxidation and antioxidant status in male Wistar rat and to find out the ameliorative potential of taurine in this toxic condition. 

MATERIALS AND METHODS

Chemicals and Reagents

Lambda-cyhalothrin 5% emulsifiable concentrate (EC) was procured from RPC Agro Industries, Kolkata. Taurine, 1, 2dichloro-4-nitrobenzene (CDNB) were purchased from Sigma-Aldrich. Thiobarbituric acid, 5, 5’ Dithiobis-2-nitrobenzoic acid (DTNB), ethylene di tetraacetic acid (EDTA), and hydrogen peroxide were all purchased from Sigma Chemical, USA. All other chemicals used were of the finest analytical grade.

Animal Care and Treatment

In this study, 36 mature male albino rats (Wistar) weighing 130±15 g were acclimatized for 1 week before the start of the treatments at a suitable temperature of 25 °C±2 °C with 12 hours light-dark cycle. Animals were provided with accessible dry food pellets and water sufficiently. Rats were randomly divided into six groups, and each group contains six animals. Institutional Animal Ethical Committee approved the experimental protocol. The experimental six groups were designed as:

Group-I: DW-Control (Distilled Water, 2 ml/kg body weight)
Group-II: TAU-Control (TAU, 50 mg/kg body wt.)
Group-III: LCT-Low (LCT, 10.83 mg/kg body wt.)
Group-IV: TAU+LCT-Low (TAU, 50 mg/kg body wt.+LCT, 10.83 mg/kg body wt.)
Group –V: LCT-High (LCT, 15.17 mg/kg body wt.)
Group-VI: TAU+LCT-High (TAU, 50 mg/kg body wt.+LCT, 15.17 mg/kg body wt.)

Two respective doses 10.83(1/7^th LD50 dose) and 15.17(1/5^th LD50 dose) mg/kg body wt. of LCT were applied.²⁰ After one hour of the treatment of taurine (50 mg/kg body wt.), lambda-cyhalothrin was administered at two dose levels (10.83 mg/kg body wt, and 15.17 mg/kg body wt.) for consecutive 14 days. Animal’s weight was taken daily and the dose was adjusted accordingly to weight.

Sample Collection

The total body weight of each animal was recorded at the end of the experimental period. All rats were sacrificed by rapid decapitation after 24 hours of the last dose. Then weights of the kidney tissues were recorded and stored properly for the determination of oxidative stress biomarkers.

Estimation of Renal Index

Renal index was measured by using the following formula

                Kidneys weight (g)
Renal index = —————————— x 100
Body weight (g)

Estimation of Serum and Tissue Protein

Different dilutions of BSA solutions are prepared by mixing stock BSA solution (1 mg/ ml) and water. From these different dilutions, protein reagents (98:1:1) consisting of sodium carbonate (Na₂CO₃) in 0.1 N sodium hydroxide (NaOH), sodium potassium tartrate in distilled water, copper sulphate (Cu₂SO₄) in distilled water were added to different test tubes and 10 µl of serum or tissue homogenate and 500 µl of normal saline (0.9 g%) were also added. The solutions were mixed well. Then 500 µl of Folin-Ciocalteau solution was added to each tube and incubated at 37 °C for 30 min. The standards were prepared similarly. The optical density was measured at 660 nm.²¹ The absorbance was plotted against protein concentration to get a standard calibration curve.

Estimation of Oxidative Stress Parameters

Malondialdehyde (MDA): MDA of kidney tissue homogenate was assayed by using the method of Ohkawa et al.²² One ml of homogenate (20 mg/ml phosphate buffer) was mixed with 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of acetate buffer (20% pH 3.5), and 1.5 ml of aqueous solution of thiobarbituric acid (0.8%). Red pigment was produced after heating of that mixture at 95 °C for 60 min. Then it was extracted with 5 ml of n-butanol-pyridine mixture (15: 1) and centrifuged at 5000 rpm for 10 min at room temperature. The absorbance of the supernatant was measured at 535 nm.

Reduced glutathione (GSH): The reduced glutathione in kidney tissue homogenate was measured according to the method of Griffith.²³ The assay mixture contained 200 μl of kidney tissue homogenate and 100 μl of sulfosalicylic acid (4 gm %). The mixture was centrifuged for 10 min at 3000 rpm. Then 1.8 ml of DTNB (4 mg %) was added with the supernatant and was shaken well. Reading was taken at 412-420 nm.

Oxidized glutathione (GSSG): Oxidized glutathione of kidney tissue homogenate was measured by using the method of Griffith.²⁴ At first, 100 μl of kidney tissue homogenate was mixed with 2 μl of 2-vinyl pyridine and was incubated for 1h at 37 °C. Then 250 μl of sulfosalicylic acid (4 gm %) was added with it and was kept in room temperature for 30 min. It is centrifuged at 2000 rpm for 10 min. Then 200 μl of the supernatant was added with 2 ml of DTNB (4 mg %) and the reading was taken at 412 nm within 1min.

Catalase (CAT): CAT content was measured according to the method of Aebi.²⁵ The reaction mixture consisted of 0.5 ml of H₂O₂, 2.5 ml of double distilled water and 40 μl of kidney tissue homogenate prepared in 0.05 M trisHCl and was taken in a cuvette. After mixing, six readings were noted at 240 nm in 30 sec interval.

Glutathione peroxidase (GPx): Glutathione peroxidase was assayed according to Rotruck et al.²⁶ At first, homogenates (0.2 ml) were mixed with 0.1 ml of 2.5 mM hydrogen peroxide (H₂O₂), 0.2 ml of 0.4 M sodium phosphate buffer, 0.1 ml of 10 mM sodium azide and 0.2 ml of 4 mM reduced glutathione and was incubated for 5 min at 37 °C. After that 0.4 ml of 10% trichloroacetic acid (TCA) was added to that mixture to stop the reaction and centrifuged at 3200 rpm for 20 min. Then 3 ml of disodium hydrogen phosphate (Na₂HPO₄) and 1 ml of 5, 5′-dithiobisnitrobenzoic acid (DTNB) were added to 0.5 ml of supernatant.

Statistical Analysis

All data were analyzed by One-Way analysis of variance (ANOVA) followed by two-tail t-test using the Origin 6.0 Scientific Data Analysis. The results were expressed as the Mean±Standard Error of Mean (SEM). The difference between group means was considered significant when p<0.05.

RESULTS 

Effects on Renal Index

Results are expressed as Mean±SEM (N=6). The analysis is done by ANOVA followed by multiple comparison two-tail t-tests. Superscript a: Group-I versus all other groups; Superscript b: Group-III versus Group-IV; Superscript c: Group-V versus Group-VI. Asterisks represent the different level of significance (** indicates p<0.01, *** indicates p<0.001).

The renal index of LCT exposed rats was decreased significantly (p<0.001) in a dose-dependent manner compared to a control group (Table 1). Taurine increased the renal index of LCT induced rats significantly.

Effects on Kidney and Serum Protein Content

In the present study, lambda-cyhalothrin caused a reduction in total kidney protein level compared to control rats in a dose-dependent manner. At the same time, LCT increased the serum protein level in LCT-exposed rat. Taurine restored back the respective protein levels towards normal in both of the cases (Figures 1 and 2).

Figure 1. The Effect of Taurine on Kidney Tissue Protein in Lambda-cyhalothrin Exposed Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups; Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI. Asterisks Represent the Different Level of Significance (* indicates p<0.05, **indicates p<0.01)

Figure 2. The Effect of Taurine on Serum Protein in Lambda-cyhalothrin Induced Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups;
Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI. Asterisks Represent the Different Level of Significance (* indicates p<0.05, **indicates p<0.01)

Effects on Enzymatic Parameters for Lipid Peroxidation

The effect of taurine on kidney malondialdehyde (MDA) level of lambda-cyhalothrin exposed male albino rat is shown in Figure 3. In LCT treated group, MDA content increased significantly (p<0.001) compared to the control group in a dose-dependent manner where treatment of taurine decreased the LCT toxicity and restored the normal level of the MDA to a great extent.

Figure 3. The Effect of Taurine on Kidney Malon-di-Aldehyde (MDA) Level in Lambda-cyhalothrin Exposed Male Albno Rat.

Results are Expressed as Mean±SEM (N=6). SuperScript a, Group-I versus all Other Groups; Superscript b Group-III versus Group-IV;
Superscript c Group-V versus Group-VI. Asterisks Represent the Different Level of Significance (**indicates p<0.01,*** indicates p<0.001)

Kidney GSH level was decreased in LCT low and high dose treated animal groups significantly (p<0.001) but pre-treatment of taurine causes significant (p<0.001) elevation in GSH level in LCT intoxicated animals (Figure 4).

Figure 4. The Effect of Taurine on Kidney GSH Level in Lambda-cyhalothrin Exposed Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA
Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups;
Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI.
Asterisks Represent the Different Level of Significance (* indicates p<0.05, *** indicates p<0.001))

Figure 5 shown that the kidney GSSG level in LCT low and high dose treated rats were increased significantly (p<0.05 and p<0.01) compared to control rats. GSSG level was decreased by taurine pre-treatment in LCT exposed rats.

Figure 5. The Effect of Taurine on Kidney GSSH Level in Lambda-cyhalothrin Exposed Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups;
Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI. Asterisks
Represent the Different Level of Significance (*indicates p<0.05,**indicates p<0.01,***indicates p<0.001)

Effects on Antioxidant Enzymes

As presented in Figure 6, the activities of CAT in the LCT treated low and high dose groups were significantly (p<0.001) decreased compared to the control group. However, the activity of CAT was significantly increased by taurine pre-treatment in low (p<0.01) and (p<0.05) high dose group animals.

Figure 6. The Effect of Taurine on Kidney Catalase (CAT) in Lambda-cyhalothrin Exposed Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA
Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups; Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI.
Asterisks Represent the Different Level of Significance (**indicates p<0.01, *** indicates p<0.001)

Activities of glutathione peroxidase (GPx) in kidney of LCT treated low and high dose animals were significantly (p<0.05) and (p<0.001) decreased than the control group rats. Taurine treatment significantly increased GPx levels, in low dose (p<0.05) and high dose (p<0.01) animals (Figure 7).

Figure 7. The Effect of Taurine on Kidney Glutathione Peroxidase (GPx) in Lambda-cyhalothrin Exposed Male Albino Rat.

Results are Expressed as Mean±SEM (N=6). Analysis is Done by ANOVA Followed by Multiple Comparison Two-Tail t-tests. Superscript a, Group-I versus all Other Groups; Superscript b Group-III versus Group-IV; Superscript c Group-V versus Group-VI. Asterisks Represent the Different Level of Significance (* indicates p<0.05,**indicates p<0.01)

DISCUSSION

The present study was designed to evaluate the toxic effects of lambda-cyhalothrin on male albino rat kidney and its attenuation by taurine. It has been reported that in toxicological studies, body and organs weights are considered as important criteria for evaluating organ toxicity. The body weight change is considered as a sign of toxicity of any chemical substance.²⁷ In this study, we have evaluated the renal index as the basic marker for the renal toxicity. The renal index of lambda-cyhalothrin intoxicated rats was significantly lower than control rats but attenuation of renal index by taurine was seen and this may be due to its antioxidant activity. Ingestion of chlorpyrifos, diazinon and their mixture resulted in the reduction in relative kidney weight in male rats which is in agreement with our findings.²⁸ In addition; one of the most common reasons for renal tissue damage is oxidative stress which was observed in lambda-cyhalothrin exposed rats.

Proteins, the essential organic macromolecules for cellular structure and function, are expected to react first with pesticide after the entry of pesticide into body cells. Pesticides have the ability to alter very quickly the buffering system of the intracellular environment. Pesticides impair protein metabolism leading perhaps to a disarray of functional and structural status of the cell.²⁹ In the present study, lambda-cyhalothrin had a significant lowering effect on total serum protein levels in dose-dependent manner compared to control rats.

Toxicants can cause a defect in protein synthesis and that may lead to a decrease in tissue protein content. The exposure of toxins to living organisms may alter the hormonal balance that can results a direct or indirect decrease in tissue protein content.^30,31 The effect of different pesticides such as endosulfan,³² organchlorins,³³ chlorpyrifos,³⁴ phosphorothionate,³⁵ imidacloprid,³⁶ and cypermethrin³⁷ poisoning on protein metabolic profiles of rats has been studied by different researchers. In current investigation exposure of lambda-cyhalothrin to albino rats resulted in a gradual decrease in the protein content of kidney tissue.

Oxygen free radical induced lipid peroxidation, which causes damage to cell membranes and consequently develops tissue injury.³⁸ In this study, lambda-cyhalothrin elevated renal malondialdehyde (MDA) level and reduced renal glutathione (GSH) contents as well as inhibited glutathione-s-transferase activity of the kidney tissue. The decrease in tissue GSH due to the enhancement in lipid peroxidation is considered as an antioxidant defence role of GSH. GSH, glutathione-s-transferase are used in the cell as antioxidant defence mechanism. Reduced glutathione serves as an antioxidant against free radicals and organic peroxide.³⁹

LCT induced toxic manifestations may also be associated with the induction of oxidative stress through the formation of free radicals and alteration in antioxidant systems. It was observed that LCT significantly increased the level of MDA in the kidneys of rats, whereas the activity of antioxidant enzymes (CAT) was decreased.⁴⁰ Treatment with taurine caused a significant reduction in the toxic effects of this pesticide. The administration of LCT in different periods of postnatal ontogenesis was also reported to enhance oxidative stress by a significant increase in MDA level and suppressed activity of antioxidant enzymes (CAT) in brain tissue.⁴¹ In our study, we found that administration of LCT to rats resulted in a marked dose-dependent increase in the lipid peroxidation as indicated by the increase in the level of malondialdehyde (MDA) and that may be due to LCT induced increase in ROS level. GSH, one of the most important biological molecules, play a key role in the detoxification of the reactive toxic metabolites. Decline in GSH levels in the kidney after LCT treatment may be an indication of oxidative stress, whereas GSH is utilized for the detoxification of reactive toxic substances. An increased level of GSSG also reflects the oxidative stress of ovary. Normal cellular functioning depends on a balance between ROS production and antioxidant defence mechanisms present in the cell.

Antioxidant enzymes cause a primary defence that prevents oxidative damage of biological macromolecules. According to the results, the activities of CAT, a glutathione peroxidase in the kidney of LCT treated rats were significantly decreased. These results suggested that LCT has the capability to induce free radicals and oxidative damage as evidenced by alterations in various antioxidant enzymes.⁴² Reduction of antioxidant enzymes levels may be due to the direct effect on the enzymes against LCT-induced ROS generation. Taurine administration reversed all these abnormalities of above mentioned renal parameters to a good extent. It diminished lipid peroxidation either by scavenging or quenching oxygen-derived free radicals, hydrogen peroxide or hypochlorous acid directly, or by binding free metal ion species like Fe²⁺ or Cu²⁺ by its sulfonic acid group. It was also suggested that by decreasing carbonyl group production, enhanced oxidative damage⁴³ was reduced by taurine.¹⁷ 

CONCLUSION

The present findings demonstrated that taurine was able to reverse the pathological parameters of renal damage induced by lambda-cyhalothrin. Pre-treatment of taurine maintained the antioxidant status of kidney due to its free radical scavenging action. Taurine undoubtedly restored the renal function by blocking lambda-cyhalothrin induced renal oxidative stress. So, taurine may be considered useful against lambda-cyhalothrin induced toxicity in renal system. 

ACKNOWLEDGEMENTS

The authors would like to thank the authority of Vidyasagar University, Midnapore, India for providing all the facilities to execute this study.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

1. Fetoui H, Garoui el M, Makni-Ayadi F, Zeghal N. Oxidative stress induced by lambda-cyhalothrin in rat erythrocytes and brain: Attenuation by vitamin C. Environ Toxicol Pharmacol. 2008; 26(2): 225-231. doi: 10.1016/j.etap.2008.04.002

2. Amweg EL, Weston DP, Ureda NM. Use and toxicity of pyrethroid pesticides in the central valley, California, USA. Environ Toxicol Chem. 2005; 24(4): 966-972. doi: 10.1897/04-146R1.1

3. Oros DR, Werner I. Pyrethroid insecticides: An analysis of use patterns, distributions, potential toxicity and fate in the sacramento-san joaquin delta and central valley. White paper for the interagency ecological program. SFEI contribution 415. San Francisco Estuary Institute, Oakland, CA. 2005.

4. International programme on chemical safety (IPCS). Environmental Health Criteria-99, Cyhalothrin, Geneva. WHO. 1990. 1-99.

5. Anadon A, Martinez M, Martinez MA, Diaz MJ, Martinez-Larranaga MR. Toxicokinetics of lambda-cyhalothrin in rats. Toxicol Lett. 2006; 165(1): 47-56. doi: 10.1016/j.toxlet.2006.01.014

6. Ratnasooriya WD, Ratnayake SS, Jayatunga YN. Effects of Icon, a pyrethroid insecticide on early pregnancy of rats. Hum Exp Toxicol. 2003; 22(10): 523-533. doi: 10.1191/0960327103ht381oa

7. Barata C, Baird DJ, Nogueira AJ, Soares AM, Riva MC. Toxicity of binary mixtures of metals and pyrethroid insecticides to daphnia magna straus. Implications for multi-substance risks assessment. Aquat Toxicol. 2006; 78(1): 1-14. doi: 10.1016/j.aquatox.2006.01.013

8. Schroer AF, Belgers JD, Brock TC, Matser AM, Maund SJ, Van den Brink PJ. Comparison of laboratory single species and field population-level effects of the pyrethroid insecticide lambda-cyhalothrin on freshwater invertebrates. Arch Environ Contam Toxicol. 2004; 46(3): 324-335. doi: 10.1007/s00244-003-2315-3

9. Sanborn M, Cole D, Kerr K, Vakil C, Sanin LH, Basil K. Systematic review of pesticides human health effects. The ontario college of family physicians. Ontario, CA, 2004.

10. AL-Gehani SA. Effect of sub chronic exposure to malathion on haematological parameters in the quail (Coturnixcoturnix). Global Adv Res J Environ Sci Toxicol. 2013; 2: 77-81.

11. Bertolote JM, Fleischmann A, Eddleston M, Gunnell D. Deaths from pesticide poisoning: A global response. Br J Psychiatry. 2006; 189: 201-203. doi: 10.1192/bjp.bp.105.020834

12. Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, Rezaie A. Pesticides and oxidative stress: A review. Med Sci Monit. 2004; 10: 141-148.

13. Ames BN, Shigenaga MK, Hagen TM.Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci. 1993; 90: 7915-7922. doi: 10.1073/pnas.90.17.7915

14. Giray B, Gurbay A, Hincal F. Cypermethrin-induced oxidative stress in rat brain and liver is prevented by vitamin E or allopurinol. Toxicol Lett. 2001; 118: 139-146. doi: 10.1016/S0378-4274(00)00277-0

15. El-Demerdash FM, Yousef MI, Kedwany FS, Baghdadi HH. Role of alpha tochopherol and beta-carotene in ameliorating the fenvalerate induced changes in oxidative stress, hematobiochemical parameters and semen quality of male rats. J Environ Sci Heal B. 2004; 39: 443-459. doi: 10.1081/PFC-120035929

16. Prasanthi K, Muralidhara, Rajini PS. Fenvalerate-induced oxidative damage in rat tissues and its attenuation by dietary sesame oil. Food Chem Toxicol. 2005; 43: 299-306. doi: 10.1016/j.fct.2004.10.005

17. Schaffer S, Azuma J, Takahashi K, Mozaffari M. Why is taurinecytoprotective? Adv Exp Med Biol. 2003; 526: 307-321. doi: 10.1007/978-1-4615-0077-3_39

18. Trachtman H, Lu P, Sturman JA. Immunohistochemical localization of taurine in rat renal tissue: Studies in experimental disease states. J Histochem Cytochem. 1993; 41: 1209-1216. doi: 10.1177/41.8.8331284

19. Cruz CI, Ruiz-Torres P, del Moral RG, Rodriguez-Puyol M, Rodriguez-Puyol D. Age-related progressive renal fibrosis in rats and its prevention with ACE inhibitors and taurine. Am J Physiol Renal Physiol. 2000; 278: F122-F129. doi: 10.1152/ajprenal.2000.278.1.F122

20. Sharma CD, Saxena NP, Sharma R. Assessment of clastogenicity of lambda-cyhalothrin, a synthetic pyrethroid in cultured lymphocytes of albino rats. World Appl Sci J. 2010; 8: 1093-1099.

21. Lowry OH, Roseborough NJ, Farr AL, Randall RL. Protein measurement with Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.

22. Ohkawa H, Onishi N, Yagi K. Assay for lipid per-oxidation in animal tissue by thiobarbituric acid reaction. Anal Biochem. 1979; 95: 351-358. doi: 10.1016/0003-2697(79)90738-3

23. Griffith OW. Glutathione turnover in human erythrocytes. J Biol Chem. 1981; 256: 4900-4904.

24. Griffith MP. Determination of glutathione and glutathione disulphide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980; 106: 207-212. doi: 10.1016/0003-2697(80)90139-6

25. Aebi H. Catalase. In: Bergmeyer HU (Ed). Method of Enzymetic Analysis. NewYork, US: Academic Press. 1974. 674-684.

26. Rotruck JT, Pope AL, Ganther HC, Hafeman DG, Hoekstro WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science. 1973; 179: 588-590. doi: 10.1126/science.179.4073.588

27. Crissman JW, Goodman DJ, Hildebrandt PK, et al. Best practice guideline: Toxicological histopathology. Toxicol Pathol. 2004; 32: 126-131. doi: 10.1080/01926230490268756

28. Mansour SAK, Abbassy MAL, Shaldam HA. Hepato-renal toxicity induced by chlorpyrifos, diazinon and their mixture to male rats with special concern to the effect of zinc supplementation. J Toxicol Pharmacol. 2017; 1: 15.

29. Shukla OP, Omkar AK. Kulshrestha. In: Pesticides, Man and Biosphere, 1^st Edn. New Delhi, India: APH Publishing Corporation. 1998.

30. Singh R, Pathak DN. Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, Catalase and glucose-6-phosphate dehydrogenase activities in Fecl3 induced epileptogenic foci in the rat brain. Epilepsia. 1990; 31: 15-26. doi: 10.1111/j.1528-1157.1990.tb05354.x

31. Murthy AS, Priyamvada DA. The effects of endosulfon and its isomers on tissue protein glycogen and lipids in the fish Channapunctatus. Pestic Biochem Physiol. 1982; 17: 280-286. doi: 10.1016/0048-3575 (82)90138-9

32. Choudhary N, Joshi SC. Reproductive toxicity of endosulfan in male albino rats. Bull Environ Contam toxicol. 2003; 70: 285-289. doi: 10.1007/s00128-002-0189-0

33. Wade MG, Parent S, Finnson KW, et al. Thyroid toxicity due to subchronic exposure to a complex mixture of 16 organochlorines, lead, and cadmium. Toxicol Sci. 2002; 67: 207-218. doi: 10.1093/toxsci/67.2.207

34. Prasad SR. Neurochemical and Histological Studies During the Development of Behavioural Tolerance to Organophosphate Compound Chlorpyrifos Toxicity in Albino Rats [dissertation]. Tirupati, India: University of Sri Venkateswara; 2007.

35. Rahman MF, Siddiqui MKJ. Hematological and clinical chemistry changes induced by subchronic dosing of a novel phosphorothionate (RPR-V) in Wistar male and female rats. Drug Chem Toxicol. 2006; 29: 95-110. doi: 10.1080/01480540500408697

36. Kishandar N. Neurotoxicity Effects of Neonicotinoid Insecticide Imidacloprid in Albino Rat. Insedicidebideclopid in Albin, Rat [dissertation]. Tirupati, India: University of Sri Venkateswara; 2007; 7: 83-90.

37. Sukanya N. Neurotoxic Effect of Cypermethrin in Wistar Strain Rats: A Biochemical, Behavioral and Histological Study [dissertation]. Tirupati, India: University of Sri Venkateswara; 2007.

38. Sener G, Sehirli O, Cetinel S, et al. Amelioration of sepsis-induced hepatic and ileal injury in rats by the leukotriene receptor blocker montelukast. Prostaglandins Leukot Essent Fatty Acids. 2005; 73: 453-460. doi: 10.1016/j.plefa.2005.07.008

39. El-Deib KM, Ahmed MM, Ahmed NZ. Biochemical evaluation of the protective impact of silymarin against cyclophosphamide induced hepatotoxicity in rats. Egypt J Biochem Mol Biol. 2011; 29: 291-310. doi: 10.4314/ejbmb.v29i2.72440

40. Fetoui H, Makni M, Garoui el M, Zeghal N. Toxic effects of lambda-cyhalothrin, a synthetic pyrethroid pesticide, on the rat kidney: Involvement of oxidative stress and protective role of ascorbicacid. Exp Toxicol Pathol. 2010; 62: 593-599. doi: 10.1016/j.etp.2009.08.004

41. Ansari RW, Shukla RK, Yadav RS, et al. Cholinergic dysfunctions and enhanced oxidative stress in the neurobehavioral toxicity of lambda-cyhalothrin in developing rats. Neurotox Res. 2012; 22: 292-309. doi: 10.1007/s12640-012-9313-z

42. Salama AK, Osman KA, Saber NA, Soliman SA. Oxidative stress induced by different pesticides in the land snails, Helix aspersa. Pak J Biol Sci. 2005; 8: 92-96. doi: 10.3923/pjbs.2005.92.96

43. Franconi F, Di Leo MA, Bennardini F, Ghirlanda G. Is taurine beneficial in reducing risk factors for diabetes mellitus. Neurochem Res. 2004; 29: 143-150. doi: 10.1023/B:NERE.0000010443.05899.2f