ASSESSMENT OF THE GENOTOXIC AND MUTAGENIC EFFECT OF AL-TAIF POMEGRANATE (PUNICA GRANATUM L) PEEL EXTRACT ALONE AND COMBINED WITH MALATHION AND ATRAZINE PESTICIDES IN LIVER OF MALE ALBINO MICE
In our previous studies, we report the antioxidant, hepatoprotective and nephroprotective potential of of Al-Taif Pomegranate (Punica granatum L)
extracts against toxicity induced by Malathion (Mal) and Atrazine (Atra) pesticides in male albino mice. Hereby, we assess the genotoxic and mutagenic
potential of Al-Taif Pomegranate (P. granatum L) peel extract (PPE) alone and combined with Atra and Mal pesticides in the liver of male albino mice.
Our results report PPE genotoxicity and its failure to significantly decrease the genotoxic effect of the pesticides Mal and Atra. Genotoxic potential was
reported by using Comet assay, in which fifty isolated comets were randomly selected and used to measure tail length, % DNA of tail and tail moment
for each group in comparison with the negative control group. Moreover, PPE combined (Mal and Atra) groups show DNA point of mutation in P53
exon 5, that was detected by the highly sensitive and accurate assay single-strand conformation polymorphism (SSCP), represented by an extra third
band in comparison with the negative control group. This mutation was not detected by direct sequencing, means that it is a low-frequency mutation.
In conclusion, our results report Al-Taif PPE as a genotoxic extract and mutagenic in combination with Mal and Atra pesticides. Moreover, the present
results also confirm the sensitivity of SSCP technique in detection of point of mutation in comparison to direct sequencing.
Keywords: Malathion, Atrazine, Pomegranate peel extract, Mutagenic, Genotoxic.
1. Alam JB, Dikshit AK, Bandyopadhyay M. Efficacy of adsorbents for 2,4-D and atrazine removal from water environment. Glob Nest Int J 2000;2(2):139-48.
2. Shetty PK. Pesticide stewardship and food security in India. Socio-ecological implications of pesticide use in India. NIAS Report RP1. National Institute of Advanced Studies, Bangalore (India); 2004.
3. Huculeci R, Dinu D, Staicu AC, Munteanu MC, Costache M, Dinischiotu A. Malathion-induced alteration of the antioxidant defence system in kidney, gill, and intestine of Carassius auratus gibelio. Environ Toxicol 2009;24(6):523-30.
4. Yarsan E, Tanyuksel M, Celik S, Aydin A. Effects of aldicarb and malathion on lipid peroxidation. Bull Environ Contam Toxicol 1999;63(5):575-81.
5. Blasiak J, Jaloszynski P, Trzeciak A, Szyfter K. In vitro studies on the genotoxicity of the organophosphorus insecticide Malathion and its two analogues. Mutat Res 1999;445(2):275-83.
6. John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes. J Nutr Biochem 2001;12(9):500-504.
7. Fortunato JJ, Feier G, Vitali AM, Petronilho FC, Dal-Pizzol F, Quevedo J. Malathion-induced oxidative stress in rat brain regions. Neurochem Res 2006;31(5):671-8.
8. Filipov NM, Pinchuk LM, Boyd BL, Crittenden PL. Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice. Toxicol Sci 2005;86(2):324-32.
9. Cooper RL, Laws SC, Das PC, Narotsky MG, Goldman JM, Lee Tyrey E, et al. Atrazine and reproductive function: Mode and mechanism of action studies. Birth Defects Res B Dev Reprod Toxicol 2007;80(2):98-112.
10. Brusick DJ. An assessment of the genetic toxicity of atrazine: Relevance to human health and environmental effects. Mutat Res 1994;317(2):133-44.
11. Hayes T, Haston K, Tsui M, Hoang A, Haeffele C, Vonk A. Herbicides: Feminization of male frogs in the wild. Nature 2002;419(6910):895-6.
12. Bolle P, Mastrangelo S, Tucci P, Evandri MG. Clastogenicity of atrazine assessed with the Allium cepa test. Environ Mol Mutagen 2004;43(2):137-41.
13. Meisner LF, Belluck DA, Roloff BD. Cytogenetic effects of alachlor and/or atrazine in vivo and in vitro. Environ Mol Mutagen 1992;19(1):77â€‘82.
14. Mady NI, Allam AF, Salem AI. Evaluation of the addition of Nigella sativa oil-triclabendazole therapy in the treatment of human fascioliasis. J Egypt Pharmacol Exp Ther 2001;20:807-27.
15. Kaur G, Jabbar Z, Athar M, Alam MS. Punica granatum (pomegranate) flower extract possesses potent antioxidant activity and abrogates Fe-NTA induced hepatotoxicity in mice. Food Chem Toxicol 2006;44(7):984-93.
16. Das AK, Mandal SC, Banerjee SK, Sinha S, Das J, Saha BP, et al. Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. J Ethnopharmacol 1999;68(1-3):205-8.
17. Prashanth D, Asha MK, Amit A. Antibacterial activity of Punica granatum. Fitoterapia 2001;72:171-3.
18. Afaq F, Saleem M, Krueger CG, Reed JD, Mukhtar H. Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kappa B pathways and inhibits skin tumorigenesis in CD-1 mice. Int J Cancer 2005;113(3):423-33.
19. Toklu HZ, Dumlu MU, Sehirli O, Ercan F, Gedik N, GÃ¶kmen V, et al. Pomegranate peel extract prevents liver fibrosis in biliary-obstructed rats. J Pharm Pharmacol 2007;59(9):1287-95.
20. Toklu HZ, Sehirli O, Ozyurt H, Mayadagli AA, Eksioglu-Demiralp E, Cetinel S, et al. Punica granatum peel extract protects against ionizing radiation-induced enteritis and leukocyte apoptosis in rats. J Radiat Res 2009;50(4):345-53.
21. Uzun FG, Kalender S, Durak D, Demir F, Kalender Y. Malathion-induced testicular toxicity in male rats and the protective effect of vitamins C and E. Food Chem Toxicol 2009;47(8):1903-8.
22. Kniewald J, Jakominic M, Tomljenovic A, Simic B, Romac P, Vranesic D, et al. Disorders of male rat reproductive tract under the influence of atrazine. J Appl Toxicol 2000;20(1):61-8.
23. Bhaskar A, Kumar A. Antihyperglycemic, antioxidant and hypolipidemic effect of Punica granatum L flower extract in streptozotocin induced diabetic rats. Asian Pac J Trop Biomed 2012;2(3) 1764-9.
24. Morimura K, Yamamoto S, Murai T, Mori S, Chen TX, Wanibuchi H, et al. LOH and mutational analysis of p53 alleles in mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. Carcinogenesis 1999;20(4):715-8.
25. Sambrook J, Fritsch EF, Maniatis T. In: Sambrook J, Fritsch EF, Maniatis T, editors. Molecular Cloning: A Laboratory Manual. 2nd ed. NY: CSH Cold Spring Harbor Press; 1989.
26. Dai J, Wei H, Xiao Y. PCR-RFLP analysis on mitochondrial DNA D-Loop area of six inbred mice. J Med Coll PLA 1999;21:709-11.
27. Dai JG, Min JX, Xiao YB, Lei X, Shen WH, Wei H. The absence of mitochondrial DNA diversity among common laboratory inbred mouse strains. J Exp Biol 2005;208:4445-50.
28. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175(1):184-91.
29. Olive PL, BanÃ¡th JP. Induction and rejoining of radiation-induced DNA single-strand breaks: â€œTail momentâ€ as a function of position in the cell cycle. Mutat Res 1993;294(3):275-83.
30. Zahin M, Aqil F, Ahmad I. Broad spectrum antimutagenic activity of antioxidant active fraction of Punica granatum L. peel extracts. Mutat Res 2010;703(2):99-107.
31. FAO/WHO (Food and Agricultural Organization of the United Nations (FAO) and the World Health Organization (WHO). Joint FAO/WHO Meeting held in 2007. Pesticide Residues in Food, Toxicology Evaluation, Atrazine. Geneva, Switzerland: WHO; 2009. p. 37-138.
32. Ventura Campo de B, Angelis de Fransceschi de D, Marin-Morales MF. Mutagenic and genotoxic effects of the atrazine herbicide in Oreochromis niloticus (Perciformes Cichlidae) detected by the micronuclei test and the comet assay. Pest Biochem Physiol 2008;90(1):42-51.
33. Bosari S, Marchetti A, Buttitta F, Graziani D, Borsani G, Loda M, et al. Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive silver-stained SSCP analysis. Diagn Mol Pathol 1995;4(4):249-55.
34. Fan X, Furnari FB, Cavenee WK, Castresana JS. Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells. Int J Oncol 2001;18(5):1023-6.
35. Moore PD, Patlolla AK, Tchounwou PB. Cytogenetic evaluation of malathion-induced toxicity in Sprague-Dawley rats. Mutat Res 2011;725(1-2):78-82.
36. RÃ©us GZ, Valvassori SS, Nuernberg H, Comim CM, Stringari RB, Padilha PT, et al. DNA damage after acute and chronic treatment with Malathion in rats. J Agric Food Chem 2008;56(16):7560-5.
37. Moore PD, Yedjou CG, Tchounwou PB. Malathion-induced oxidative stress, cytotoxicity, and genotoxicity in human liver carcinoma (HepG2) cells. Environ Toxicol 2010;25(3):221-6.
38. Ali D, Nagpure NS, Kumar S, Kumar R, Kushwaha B. Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Chemosphere 2008;71(10):1823-31.
39. Mehta A, Verma RS, Srivastava N. Chlorpyrifos-induced DNA damage in rat liver and brain. Environ Mol Mutagen 2008;49(6):426-33.
40. Hamza RZ, Abd El-Azez AM, Hussien NA. Evaluation of the antioxidant potential for different extracts of Al-taif pomegranate (Punica granatum L) induced by Atrazine and Malathion pesticides in liver of male albino mice. Int J Pharm Pharm Sci 2015;7(1):89-94.
41. Nwani CD, Nagpure NS, Kumar R, Kushwaha B, Kumar P, Lakra WS. Mutagenic and genotoxic assessment of atrazine-based herbicide to freshwater fish Channa punctatus (Bloch) using micronucleus test and single cell gel electrophoresis. Environ Toxicol Pharmacol 2011;31(2):314-22.
42. Sharma S, Vig AP. Genotoxicity of atrazine, avenoxan, diuron and quizalofop-pethyl herbicides using the Allium cepa root chromosomal aberration assay. Terr Aquat Environ Toxicol 2012;6(2):90-5.
43. Foster S, Thomas M, Korth W. Laboratory-derived acute toxicity of selected pesticides to Ceriodaphnia dubia. Australas J Ecotoxicol 1998;4:53-9.
44. SÃ¡nchez-Lamar A, Fonseca G, Fuentes JL, Cozzi R, Cundari E, Fiore M, et al. Assessment of the genotoxic risk of Punica granatum L. (Punicaceae) whole fruit extracts. J Ethnopharmacol 2008;115(3):416â€‘22.
Asian J Pharm Clin Res, Vol 8, Issue 4, 2015, 302-307
Hussien et al.
45. Farkas D, Oleson LE, Zhao Y, Harmatz JS, Zinny MA, Court MH, et al. Pomegranate juice does not impair clearance of oral or intravenous midazolam, a probe for cytochrome P450-3A activity: Comparison with grapefruit juice. J Clin Pharmacol 2007;47(3):286-94.
46. Abdel Motaal A, Shakerb S. Anticancer and antioxidant activities of standardized whole fruit, pulp, and peel extracts of Egyptian pomegranate. Open Conf Proc J 2011;2:41-5.
47. Singh RP, Chidambara Murthy KN, Jayaprakasha GK. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agric Food Chem 2002;50(1):81-6.
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