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Original Effects of Profenofos and Chlorpyrifos Pesticides on Oxidative Stress Biomarker, Biochemical Toxicity, DNA and RNA Content of Male Albino Mice

Haleema Al-Nahari*

Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Received 24 Dec. 2014 Received in revised form 11 Jan. 2015 Accepted 28 Jan. 2015 Objective: The present study aimed to evaluate the profenofos and Chlorpyrifos pesticides on oxidative stress biomarker, biochemical toxicity, DNA and RNA content of male Albino Keywords: The profenofos and Chlorpyrifos mice. Method: The animals were divided into three groups of pesticide, equal number, The control group (1) was orally and daily Laboratory-bred strain Swiss albino administered with an equivalent amount of the vehicle (distilled male mice, Oxidative stress biomarker, water) for two weeks, the second group was given drinking water Biochemical toxicity, with 1.5 mg/kg BW of profenofos during two weeks of oral and DNA and RNA content. daily administration and the third group were orally and daily

administered with Chlorpyrifos (1.5 mg /kg BW). At the ends of the experimental period (2 weeks), the mice were sacrificed under diethyl ether anesthesia at fasting state. Results: The present

results showed that the activities of Superoxide dismutase (SOD), Catalase and glutathione reductase (GR), enzymes in homogenate of snail’s tissues were decreased in the tissues of mice treated

profenofos and Chlorpyrifos while lipid peroxide (LP) activity increased. Also, the profenofos and Chlorpyrifos pesticide caused a gradual significant reduction of enzyme activities of ALT, AST

and ALP and increases in the glucose level and acid phosphatase in serum of male mice Also, the activity of glycolytic (PK, PFK

and GPI) and gluconeogenic enzyme activities (F-1, 6-D-Pase) were significantly increased in tissues of treated mice. The change in the intensity number and position of DNA bands resulting from

the profenofos and Chlorpyrifos pesticides can be attributed to the fact that herbicides can occur genotoxicity. Conclusion: The pollution the water environment by profenofos and pesticide

chlorpyrifos, would negatively impact on the metabolism of mice and genetic toxicity products at low concentration and treatment Corresponding author: Department of for a long time and demonstrated the risk addicting herbicides to Biological Sciences, Faculty of Science, King Abdulaziz University, human health. Saudi Arabia. E-mail address: [email protected] © 2015 British Biomedical Bulletin. All rights reserved

Al-Nahari ______ISSN-2347-5447 Introduction Pesticide application is an important agrochemical. Consequently, many of its procedure to enhance agricultural output. residues are frequently found in the However, the pesticide use that may environment. contaminate Drainage and Irrigation systems The objective of this study was to during the planting and control pests determine the impact pesticides, activities, and thus negatively affect vital chlorpyrifos and profenofos on oxidative components and is vital for the session stress biomarker biochemical toxicity, DNA polluted water1,2. and RNA content of male Albino mice. Some pesticides are a continuation of the environment, and can affect pollution of Materials and Methods aquatic species and the wild alike3-5. The pollutants, especially pesticide, Pesticides can disturb the aquatic organisms at the Profenofos (Selecron, 72% E.C.) are cellular, physiological and molecular levels6. an organophosphorous pesticide (O-(4- In spite of studies of the biological impacts Bromo-2-chlorophenyl) O-ethyl S-propyl of pesticides has increased over the last phosphorothioate; phosphorothioic acid). years, and the results for these products Relative molecular mass of the profenofos genotoxicity is often incomplete and is 373.65g/mol and the molecular formula sometimes conflicting7. (C11H15BrClO3PS) (Fig. 1). One of pesticides and profenofos is Chlorpyrifos is a chlorinated moderately toxic to the birds and mammals organophosphate (OP) pesticide, acaricide had low toxicity with insecticidal good8. and nematicide (0, 0-diethyl 0-(3, 5, 6- Also, it induced programmed cell trichloro-2-pyridinyl) -phosphorothioate). death and necrosis in cultured lymphocytes Relative molecular mass of the profenofos in human peripheral blood under the is 350.6 g/mol and a molecular formula circumstances in the laboratory using the (C9H11Cl3NO3PS). DNA diffusion examination9,10. Profenofos and Chlorpyrifos were Chlorpyrifos is an organophosphate obtained from the Plants Protection Center, insecticide. Clean chlorpyrifos composed of Ministry of Agriculture, Dokki, Giza, Egypt. white or colorless crystals. It has a skunk (See figure 1.) odor, like rotten eggs or garlic. Chlorpyrifos is used to control many Animals different types of pests, including termites, Swiss albino male mice by 10 weeks mosquitoes, and worms. Chlorpyrifos has old with an average weight of 28.5±2.5 g. been registered as a pesticide in 1965 and Mice were obtained from National Research the United States Environmental Protection Center, Cairo, Egypt and have been Agency (U.S. EPA) re-registered it in 2006. preserved in a good ventilated animal house. Using only in enclosed places of They were staying at a large polypropylene chlorpyrifos is legal in containers with the cage with free access to food and drinking bait treatment11. Chlorpyrifos (CPF), and water during the experiment and maintained organic phosphate applied widely in under standard conditions of temperature agriculture and aquaculture, pays oxidative (23°C to 25°C), a relative humidity of 65% stress due to the generation free radicals and to 86%. changes in antioxidant defense system12. Organophosphate pesticide represents one of the world’s most commonly used

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Animal treatment These parameters were determined The animals were divided into three spectrophotometrically by using reagent kits groups (n=6) of equal number, The control purchased from BioMerieux Company, group (1) was orally and daily administered France. RNA and DNA of liver tissue were with an equivalent amount of the vehicle determined according to Schneider25. (distilled water) for two weeks, the second DNAase and RNAase activities were group was given drinking water with 1.5 determined in liver tissues according to by mg/kg BW of profenofos during two weeks Bergmeyer26. of oral and daily administration and the third group were orally and daily administered Statistical analysis with Chlorpyrifos (1.5 mg /kg BW). The The Data obtained in this work were mice were sacrificed under diethyl ether analyzed using analysis of variance anesthesia at fasting state at the ends of the (ANOVA). The significance of difference experimental period (2 weeks). between the means was calculated using the Duncan Multiple Range Test27. Effect of profenofos and chlorpyrifos (pesticide) on oxidative stress biomarker, Results biochemical toxicity, DNA and RNA content of male albino mice The data present in Table (3) showed Serum samples were obtained by the that the effect of LC25 of Profenofos and centrifugation of blood of six rats of each Chlorpyrifos pesticides on some antioxidant group at 4000 rpm for 15 min at 4oC. Serum enzymes. The result indicated that thought, samples were divided into Eppendorf tubes. there was a significant reduction in Isolated sera from each group were stored at Superoxide dismutase (SOD), Catalase CAT –20oC until they were used for the analyses. and glutathione reductase (GR) while a For preparation of tissue homogenates of significant elevation in lipid peroxides LP in mouse liver tissue of six mice of each group, treated mice compared to the normal mice. one gram of liver tissues of mouse from The present results showed that SOD, CAT each group was homogenized in 5 ml and GR activity of mice treated with distilled water at pH 7.5. The homogenate Profenofos pesticide was reduced to - was centrifuged for 10 minutes at 3000 rpm 41.25%, -35.63% and -33.33% 29% and LP by used a glass homogenizer. Fresh was increased to 46.15%. The data in Table supernatant was used. 4 demonstrates that a clear inhibition in Estimation of antioxidant enzymes activities of enzymes (AST, ALT and ALP) such as Superoxide dismutase (SOD) was in serum of mice treated with profenofos measured according to Nishikimi et al. 13, and Chlorpyrifos pesticide as compared to Catalase (CAT)14, glutathione reductase the control mice. While a marked increase in (GR)15, lipid peroxides (LP)16. The levels of the glucose level and Acid phosphatase in serum alanine aminotransferase17 (ALT)16, serum of treated mice in comparison with aspartate aminotransferase (AST)17, the control group. Alkaline phosphatase (ALP)18, acid The data in tables (6, 7) showed that phosphatase (ACP)19,. Pyruvate kinase a significant increased level of glycolytic (PK)20, phosphofructokinase (PFK)21, (PK, PFK and GPI) and gluconeogenic Glucose phosphate isomerase (GPI)22, enzyme activities (F-1, 6-D-Pase) in tissue Fructose -1, 6-diphosphatase (F- 1, 6-ase)23 of mice treated with profenofos and and Serum glucose concentrations (GL)24. Chlorpyrifos as compared to the control. The increasing rates on the activities of PK,

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PFK, GPI and F-1-6, D-Pase enzymes were In good agreement with the present 58.2%, -81.81%, -27.11% and 28.6% results Maitlsteidt et al.,30 suggested in 2004 respectively for mice treated with that the nuclei and mitochondria as a key profenofos. business objectives toxic MG, perhaps by Regarding to nucleic acids system of increasing the generation of free radicals, the liver, the contents of DNA and RNA in lipid peroxidation and the formation of DNA tissues of mice were decreased by exposing adducts. to Profenofos and Chlorpyrifos to 50, Botros et al.,31 indicated that the 38.46% of DNA and 42.31%, 23.1% of complex mechanism of lipid peroxidation is RNA content, respectively, relative to those known that requires the participation of of normal healthy controls. The obtained highly reactive oxygen and other products of data are present in Table 3, showed that the reaction in a series of biochemical significant reductions were found in the reaction. The present results agree with DNAase and RNAase activity (nuclease Mittelstaedt et al.,30 indicated that the enzymes related to the nucleic acid system) nuclei and mitochondria act as major targets of tissue of mice treated with Profenofos and of MG toxic action, probably by increasing Chlorpyrifos related to healthy control. The the generation of free radicals, lipid inhibited activity in the DNAase was -39.91 peroxidation and DNA adduct formation. and -27.78% and in RNAase was 45.83%, - Thus, thioredoxin glutathione 19.79% for at normal control of the two reduced preserve high levels of reduced nucleases activity (DNAase and RNAase). glutathione either through direct reduced of Baraldi et al.,28 indicated that the oxidative glutathione disulfide by thioredoxin stress in diabetes coexists with an inhibition glutathione reduced or by reducing the in the antioxidant defense system and leads glutathione disulfide by thioredoxin. to the complications of diabetes. Therefore, thioredoxin system plays a significant role in redox balance and in the Discussion antioxidant defenses of S. mansoni (Algera et al.,32). Ganguly et al.,33 noted that the The present results found that, profenofos induced dramatic changes in significant decline in SOD, CAT, and GR mitotic index when compared with with significant increase in LP were nimbecidine. Kumar et al.,11 indicated that observed in the tissue treated with Chlorpyrifos induces oxidative stress in profenofos and Chlorpyrifos. The Chroococcus turgidus NTMS12. reduction in the activity of free radical A powerful inference to increased scavenging enzyme SOD may be due to its activity of antioxidant enzymes such as inactivation caused by excess ROS superoxide dismutase (SOD), catalase production. SOD neutralizes superoxide. (CAT) and the content of proline and Because it cannot cross the lipid membrane, reducing the level of unsaturated fatty acids and the production of hydrogen peroxide. with exposed to pesticides. These changes Hydrogen peroxide can penetrate biological are the defense mechanisms against the membranes. Catalase detoxifies hydrogen oxidative stress. Chlorpyrifos induces peroxide, which has the main role in tissue oxidative stress in the liver of Oreochromis damage. Therefore, the decrease in SOD niloticus29. may lead to the first line of defense against Concerning, ALT, AST and ALP enzymatic anion and hydrogen peroxide 29 enzyme activities, gradual significant Moustafa et al., . reduction were observed in serum of mice

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treated with Profenofos and Chlorpyrifos for DNAase) activity may be due to the two weeks. This results agree with Bakry et oxidative stress with a reduction in the al.34-36 and Bakry et al.,37. antioxidant defense system28. Lu and Yu,42 The present data showed that showed that Profenofos and Chlorpyrifos inhibition in AST and ALT ascribes to the induced cytotoxicity and DNA damage by hepatocellular damage resulting from oxidative stress in mice adrenal pesticide-toxic, where the transaminases pheochromocytoma (PC12) cells. Bakry et levels showed an intimate relationship to al.,43 indicated that highlights the potential cell necrosis and/or increased cell membrane ecological implications of ZnONP release in permeability which led to the discharge of aquatic environments and may serve to enzyme to the blood stream. The decrease in encourage regulatory agencies in Egypt to transaminase levels, providing additional more carefully monitor and regulate the support for the side effect of the Profenofos industrial use and disposal of ZnONPs. and Chlorpyrifos on the mitochondria of the hepatic cells30. In the present data showed References that acid phosphatase shows significant increasing in serum of treated mice. Levels 1. Kreuger, J. Peterson, M. Lundgren, E. of acid phosphatase have been observed Agricultural inputs of pesticide residues to higher in tissues by Abdel-Rahman et al38 stream, pond sediments in a small catchment Due to increased loss of enzyme inside cells in Southern Sweden, Bull. Environ. Contam. by diffusion through the cell membrane, Toxicol. 62 (1999) 55–62. 2. Mohamed, A.M. El-Emam, M.A. Osman, which show to serve as a stimulus to install G.Y. Abdel-Hamid, H. Ali, R.E. Effect of more of the enzyme. basudin, selecron and the phytoalkaloid With regard to sources of energy for colchicine (pesticides) on biological and mice, Profenofos and Chlorpyrifos molecular parameters of Biomphalaria significantly increased in the sugar level of alexandrina snails, Pest. Biochem. Physiol. the blood of treated mice. It can be 102 (2012) 68–78. attributed to the activity of pesticides that 3. Russo, J. Lagadic, L. Effects of impede consumption of oxygen of mice, environmental concentrations of atrazine on which induce anaerobic respiration. Under hemocyte density and phagocyte activity in hypoxic conditions, and animals take their the pond snail Lymnaea stagnalis energy from the anaerobic breakdown of (Gastropoda, Pulmonata), Environ. Pollut. 127 (2004) 303–311. glucose, which is available to the cells by 27,39 4. Bakry, F.A. Hasheesh W. A. Hamdi S.A.H. increasing glycogenolysis . (2011). Biological, biochemical, and The present data showed that molecular parameters of Helisoma duryi significantly increased of PK, PFK and GPI snails exposed to the pesticides Malathion (glycolytic enzymes) in liver tissue of and Deltamethrin. Pest. Bioch. Physiol. 101 treated mice with two pesticides compared (2011) 86–92. to control group accompanied by an increase 5. Bakry F.A., Abdelsalam H.A., Mahmoud in the gluconeogenesis; F, 1-6-diphosphtase. M.B and Hamdi S.A.H. 2012. Influence of The increase in gluconeogenic enzymes Atrazine and Roundup pesticides on might also be responsible for producing biochemical and molecular aspects of glucose during treatment40,41. Biomphalaria alexandrina snails. Pesticide Biochemistry and Physiology 104 (2012) 9– The present data indicated that 18. inhibited effects of Profenofos and 6. Fournier, M., Cyr, D., Blakley, B., Chlorpyrifos on nucleases (RNAase and Boermans, H., Brousseau, P., 2000.

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Phagocytosis as a biomarker of guinea pigs, Biochem. Med. 31 (1984) 217– immunotoxicity in wildlife species exposed 227. to environmental xenobiotics. Amer. Zool. 16. Buge, J.A. Aust, S.D. Microtonal lipid per 40, 412-420. oxidation, Methods Enzymol. 52 (1978) 302– 7. Siu, S.Y., Lam, P.K., Martin, M., Caldwell, 310. C.W., Richardson, B.J., 2008. The use of 17. Retiman S and Frankel S. 1957. Colorimetric selected genotoxicity assays in green-lipped method for the determination of serum mussel (Perna viridis): A validation study in glutamic oxaloacetic acid and glutamic Hong Kong coastal waters. Mar. Pollution pyruvic tranaminases. Am. J .Clin. Pathol. Bull. 57, 479-492. 28(1):56-65.[25] 8. Tomlin CDS (1997) the pesticide manual, 18. Belfield A and Goldberg DM. 1971. 11th Ed (ISBN 1 901396 11 8). UK: British Hydrolysis of adenosine-monophosphate by Crop Protection Council. acid phosphatase as measured by a 9. Alam M, Ghiasuddin, Sadat A, Muhammd continuous spectrophotometric assay. N, Khan AA and Siddiqui BS (2012) Enzyme. 4(2): 561-566.[26] Evaluation of Viburnum grandiflorum for its 19. Wattiaux R and De Duve C. 1956. Release in-vitro pharmacological screening. African of bounds hydrolase by mean of Triton X- Journal of Pharmacy and Pharmacology. 100. Biochem. J. 63: 606-608. 6(22): 1606–1610. 20. Trinder P. 1969. Determination of glucose in 10. Bakry F.A., El-Hommossany K., H. blood using glucose oxidase with an Mossalem H. (2012). Impact of Azardiracta alternative oxygen acceptor, Annals of indica Extract on Immunological and Clinical Biochemistry. 6: 24–27. Physiological Parameters of Biomphalaria 21. Bucher T and pflrider G. 1975. Pyruvate alexandrina Snails. European Review for kinase, In: Methods in Enzymology (SP Medical and Pharmacological Sciences. Colowick, SP Kaplan eds.) Academic Press 2012; 16(Suppl 3): 133-143, NY pp.435-440. 11. Kumar MS, Praveenkumar R, Jeon BH, 22. Zammit VA, Beis I, ENewshlome A. 1978. Thajuddin N.2014. Chlorpyrifos-induced Maximum activities and effects of fructose changes in the antioxidants and fatty acid biophosphate pyruvatekinase from muscle of compositions of Chroococcus turgidus vertebrates and invertebrates in relation to NTMS12. Lett. Appl. Microbiol. 2014 Nov; the control of glycolysis. Biochem. J. 174(3): 59 (5):535-541. 989-917. 12. Narra MR, 2014. Tissue-specific recovery of 23. King J. 1965. Glucose phosphate isomerase oxidative and antioxidant effects of In: practical Clinical Enzymology. London, chlorpyrifos in the freshwater crab, pp.1113-1117. Barytelphusaguerini. Arch Environ Contam 24. Sand O, .Petersen IM and Emmersen BK. Toxicol. 2014 Aug; 67(2):158-166. 1980. Changes in some carbohydrate 13. Nishikimi, M. Appaji, N. Yagi, K. The metabolizing enzymes and glycogen in liver, occurrence of superoxide anion in the glucose and lipid in serum during reaction of reduced phenazine methosulfate vitellogensis and after induction by estradiol- and molecular oxygen, Biochem. Biophys. 17-B in the flounder Platichtys flesusL. Res. Commun. 46 (1972) 849–854. Comp. Biochem. Phsiol. 65:327. 14. Monhanty, J.G. Jonathan, S. Jaffe, E. 25. Schneider W. C. Determination of acid in Edaward, S. Donald, G.R. A highly tissues by pentose analysis. In: “Method sensitive fluorescent microassay of H2O2 enzymology” (Edited by Gurler, B. H.Vural, release from activated human leukocytes N. Yilmaz, H. Oguz, A. N. Satici, The role using a dihydroxphenoxazine derivative, J. of oxidative stress in diabetic retinopathy. Immunol. Methods. 202 (1997) 133–137. Eye; 5: 730-735,2000 15. M. Erden, N.M. Bor, Changes in reduced 26. Bergmeyer, H.V. M. Horrder, R. Rej, glutathione, glutathione reductase and Approved recommendation on IFCC glutathione peroxidase after radiation in methods for the measurement of catalyst

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Table 1. The effect of prolonged exposure to Profenofos and Chlorpyrifos pesticides for 2 weeks on some antioxidant enzymes in tissues of male male mice

Chlorpyrifos pesticides Profenofos pesticide Control Parameters % Change Mean ± SD % Change Mean ± SD Mean ± SD 0.094±0.07 Superoxide dismutase -37.5% 0.10±0.8 -41.25% 0.16±0.04 2 (SOD) -29.69% 59.2±0.2 -35.63% 54.2±0.12 84.2+10.2a Catalase (CAT) 0.36+0.068 Glutathione reductase -42.59% 0.31+0.43 -33.33% 0.54+ 0.32a * (GR) 40% 9.1+ 0.5 46.15% 9.5+ 0.61 6.5+ 0.82 Lipid peroxides (LP)

* P<0.05 ** P < 0.01. Values are means ± SD of six replicates. Values are expressed μmol/min/mg protein while lipid peroxide is expressed in μg/g tissue

Table 2. Effect of profenofos and Chlorpyrifos pesticide on liver function enzymes in serum of male mice

Glucose (GL) Liver function enzymes (umole/mg protein/min.) mg/g tissue Acid ALP ALT AST phosphatase (ACP) % % % % %

Change Change Change Change Change 4.2 9.5 16.33 7.2 ±1.4 28.4 ±0.8 Control ±0.2 ±0.4 ±1.2 3.2 5.8±0. 13.3±0. 45.2±0.4 -26.38% 9.1 ±0.1 -23.81% 38.95% 18.55% 49.7% Profenofos ±0.23 12 2 4 3.8 6.4 12.6 42.5±0.3 -12.5% 8.1 ±0.6 -9.52% 32.63% 22.84% 49.6% Chlorpyrifos ±0.42 ±0.16 ±1.2 8

Data represent mean values of five replicates. Within columns for dose, time and (dose x time), mean values followed by different letters are statistically significantly different based on LSD at P = 0.05.

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Table 3. Effect of profenofos and Chlorpyrifos on some glycolytic and gluconeogenic enzymes in male mice liver

Glycolytic enzmes Gluconeogenic (umole/mg protein/min.) enzymes Fructose-1, 6 diphos-phatase GPI PFK PK (umole/mg protein/min.) % % % % change change change change 5.12 14.5±1.5 88.43 ±1.5 8.3 ±1.4 Control ±0.51 - 15.86% 16.8 ±1.4 112.4 ±1.5 -81.81% 12.6±1.6 -58.2% 8.1 ±1.7 Profenofos 27.11% 18.65 - 28.6% 103.6 ±3.2 -26.51% 10.5±1.5 -28.91% 6.6 ±1.3 Chlorpyrifos ±1.61 17.15%

Data represent mean values of five replicates. Within columns for dose, time and (dose x time), mean values followed by different letters are statistically significantly different based on LSD at P = 0.05.

Table 4. Effect of profenofos and Chlorpyrifos on on DNA and RNA contents in male mice

RNAase DNAase RNA % DNA mg/g Treatments IU/g IU/g mg/g 0.26 ± 0.52 ± Normal 48 ± 0.64 64.24 ± 1.2 0.08 0.034 control - 0.15 ± -45.83% 26 ± 0.64 38.6 ± 0.6 42.31 50 0.26 ± 0.14 Profenofos 39.91 0.003

- 0.20 ± -19.79% 38.5 ± 1.6 46.4 ± 1.8 23.1% -38.46% 0.32 ± 0.27 Chlorpyrifos 27.78 0.18

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Figure 1. Molecular structure of profenofos and chlorpyrifos

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