Archives of Toxicology

, Volume 79, Issue 3, pp 169–176 | Cite as

Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage

  • Ü. ÜndeğerEmail author
  • N. Başaran


Because of the widespread use of pesticides for domestic and industrial applications the evaluation of their genotoxic effects is of major concern to public health. Although various experimental data have provided evidence that pesticides can possess genotoxic properties in animals and in in vitro test systems after acute and chronic exposure, the information on the genotoxic effects of some of pesticides is limited and inconsistent. In the present study, the genotoxic potential of commonly used pesticides (i.e., dimethoate and methyl parathion from the organophosphate class, propoxur and pirimicarb from carbamates, and cypermethrin and permethrin from pyrethroids) have been evaluated. The genotoxic effects of these substances were examined using the single cell gel electrophoresis (comet) assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 10, 50, 100 and 200 µg/ml concentrations of the test substances for 0.5 h at 37°C and DNA damage was compared with that obtained in lymphocytes from the same donor not treated with substances. Hydrogen peroxide, 100 µM, was used as a positive control. Within the concentration ranges studied, no significant cytotoxic effects were observed. Dimethoate and methyl parathion at 100 and 200 µg/ml; propoxur at 50, 100 and 200 µg/ml, and pirimicarb, cypermethrin and permethrin at 200 µg/ml significantly increased DNA damage in human lymphocytes.


Genotoxicity Comet assay Dimethoate Methyl parathion Propoxur Pirimicarb Cypermethrin Permethrin 



This study was supported by a grant from Research Foundation of Hacettepe University, Turkey, 01-02-301-005.


  1. Amer SM, Aboul-ela EI (1985) Cytogenetic effects of pesticides III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone. Mutat Res 155:135–142PubMedGoogle Scholar
  2. Amer SM, Ibrahim AA, el-Sherbeny KM (1993) Induction of chromosomal aberrations and sister chromatid exchange in vivo and in vitro by the insecticide cypermethrin. J Appl Toxicol 13:341–345PubMedGoogle Scholar
  3. Anderson D, Yu TW, Philips BJ, Schmezer P (1994) The effect of various antioxidants and other modifying agents on oxygen radical generated DNA damage in human lymphocytes in the comet assay. Mutat Res 307:261–271PubMedGoogle Scholar
  4. Anwar WA (1994) Assessment of cytogenetic changes in human populations at risk in Egypt. Mutati Res 313:183–191Google Scholar
  5. Au WW, Sierra-Torres CH, Cajas-Salazar N, Shipp BK, Legator MS (1999) Cytogenetic effects from exposure to mixed pesticides and the influence from genetic susceptibility. Environ Health Perspect 107:501–505Google Scholar
  6. Bhunya SP, Pati PC (1988) Genotoxic effects of a synthetic pyrethroid insecticide, cypermethrin, in mice in vivo. Toxicol Lett 41:223–230PubMedGoogle Scholar
  7. Bianchi L, Zannoli A, Pizzala R, Stivala RA, Chiesara E (1994) Genotoxicity assay of five pesticides and their mixtures in Saccharomyces cerevisia D7. Mutat Res 321:203–211PubMedGoogle Scholar
  8. Blasiak J, Jaloszynski P, Trzeciak A, Szyfter K (1999) In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutat Res 445:275–283PubMedGoogle Scholar
  9. Blevins RD, Lee M, Regan JD (1977) Mutagenicity screening of five methylcarbamate insecticides and their nitroso derivatives using mutants of Salmonella typhimurium LT2. Mutat Res 56:1–6PubMedGoogle Scholar
  10. Bolognesi C, Parrini M, Bonassi S, Ianello G, Salanitto A (1993) Cytogenetic analysis of a human population occupationally exposed to pesticides. Mutat Res 285:239–249PubMedGoogle Scholar
  11. Boyum A (1976) Isolation of lymphocytes, granulocytes, and macrophages. Scand J Immunol 5:9–15PubMedGoogle Scholar
  12. Carbonell E, Valbuena A, Xamena N, Creus A, Marcos R (1995) Temporary variations in chromosomal aberrations in a group of agricultural workers exposed to pesticides. Mutat Res 344:127–134PubMedGoogle Scholar
  13. Collins AR, Dusinska M, Franklin M, Somorovska M, Petrovska H, Fillion L, Panayiotidis Ö, Raslova K, Vaughan N (1997) Comet assay in human biomonitoring studies: reliability, validation and applications. Environ Mol Mutagen 30:139–146PubMedGoogle Scholar
  14. Crossen PE, Morgan WF, Horan JJ (1978) Cytogenetic studies of pesticide and herbicide sprayers. N Z Med J 88:192–195PubMedGoogle Scholar
  15. D’Arce LPG, Colus IMS (2000) Cytogenetic and molecular biomonitoring of agricultural workers exposed to pesticides in Brazil. Teratogen Carcinogen Mutagen 20:161–170CrossRefGoogle Scholar
  16. Das P, John G (1999) Induction of sister chromatid exchanges and chromosome aberrations in vivo in Etroplus suratensis (Bloch) following exposure to organophosphorus pesticides. Toxicol Lett 104:111–116CrossRefPubMedGoogle Scholar
  17. Dearfield KL, Stack HF, Quest JA, Whithing RJ, Waters MD (1993) A survey of EPA/OPP and open literature data on selected pesticide chemicals tested for mutagenicity: I. Introduction and first ten chemicals. Mutat Res 297:197–233PubMedGoogle Scholar
  18. Dulout FN, Pastori MC, Olivero OA, Gonzalez Cid M, Loria D, Matos E, Sobel N, de Bujan EC, Albiano N (1985) Sister chromatid exchanges and chromosomal aberrations in population exposed to pesticides. Mutat Res 143:237–244CrossRefPubMedGoogle Scholar
  19. Ecobichon DJ (2001) Toxic effects of pesticides. In: Klaassen CD (ed). Casarett and Doull’s toxicology. The basic science of poisons. McGraw-Hill, New York, pp 763–810Google Scholar
  20. Ellingham TJ, Christensen EA, Maddock MB (1986) In vitro induction of sister chromatid exchanges and chromosomal aberrations in peripheral lymphocytes of the oyster toadfish and American eel. Environ Mutagen 8:555–569PubMedGoogle Scholar
  21. Fahrig R (1974) Comparative mutagenicity studies with pesticides. IARC Sci Publ 10:161–181Google Scholar
  22. Fairbairn DW, Olive PL, O’Neill KL (1995) The COMET assay: a comprehensive review. Mutat Res 339:37–59PubMedGoogle Scholar
  23. Falck GCM, Hirvonen A, Scarpato R, Saarikoski ST, Migliore L, Norppa H (1999) Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers. Mutat Res 441:225–237PubMedGoogle Scholar
  24. Garaj-Vrhovac V, Zeljezic D (2000) Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay. Mutat Res 469:279–285PubMedGoogle Scholar
  25. Gillot-Delhalle J, Coluzzi A, Moutschen J, Moutschen-Dahmen M (1983) Mutagenicity of some organophosphorus compounds at the ade-6 locus of Scizosaccharomyces pombe. Mutat Res 117:139–148PubMedGoogle Scholar
  26. Giri S, Giri A, Sharma GD, Prasad SB (2003) Induction of sister chromatid exchanges by cypermethrin and carbosulfan in bone marrow cells of mice in vivo. Mutagenesis 18:53–58CrossRefPubMedGoogle Scholar
  27. Gonzales M, Loria D, Matos E (1990) Genotoxicity of the pesticide propoxur and its nitroso derivative, NO-propoxur, on human lymphocytes in vitro. Mutat Res 232:45–48PubMedGoogle Scholar
  28. Gonzales M, Soloneski S, Reigosa MA, Larramendy ML (2003) Effect of dithiocarbamate pesticide zineb and its commercial formulation, azzuro IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells. Mutat Res 534:145–154PubMedGoogle Scholar
  29. Grichner T, Badaev SA, Pospisil F, Velminsky J (1990) Effect of humic acids, para-amino benzoic acid on the N-nitrosation of carbamate insecticide propoxur and on the mutagenicity of nitroso propoxur. Mutat Res 229:37–41PubMedGoogle Scholar
  30. Grover IS, Malhi PK (1985) Genotoxic effects of some organophosphorus pesticides. I. Induction of micronuclei in bone marrow cells in rat. Mutat Res 155:131–134PubMedGoogle Scholar
  31. Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983) Salmonella mutagenesis test results for 250 chemicals. Environ Mutagen 5 [Suppl 1]:3–142Google Scholar
  32. Herrera A, Laborda E (1988) Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium. Mutagenesis 3:509–514PubMedGoogle Scholar
  33. Hoyos LS, Carvajal S, Solano L, Rodriguez J, Orozco L, Lopez Y, Au W (1996) Cytogenetic monitoring of farmers exposed to pesticides in Colombia. Environ Health Perspect 104:535–538PubMedGoogle Scholar
  34. Institoris L, Ündeğer U, Siroki O, Nehez M, Desi I (1999) Comparison of detection sensitivity of immuno- and genotoxicological effects of subacute cypermethrin and permethrin exposure in rats. Toxicology 137:47–55PubMedGoogle Scholar
  35. Kassie F, Laky B, Nobis E, Kundi M, Knasmüller S (2001) Genotoxic effects of methyl isothiocyanate. Mutat Res 490:1–9PubMedGoogle Scholar
  36. Kostka G, Palut D, Kopec-Szlezak J, Ludwicki JK (2000) Early hepatic chenges in rats induced by permethrin in comparison with DDT. Toxicology 142:135–143PubMedGoogle Scholar
  37. Kourakis A, Mouratidou M, Kokkinos G, Barbouti A, Kotsis A, Mourelatos D, Dozi-Vassiliades J (1992) Frequencies of chromosomal aberrations in pesticide sprayers working in plastic green houses. Mutat Res 279:145–148PubMedGoogle Scholar
  38. Ladhar SS, Grover IS, Radhawa SK (1990) Mutagenicity of systemic organophosphate pesticides metasystox and rogor. Ind J Exp Biol 28:390–391Google Scholar
  39. Lebailly P, Vigreux C, Godard T, Sichel F, Bar E, LeTalaer JY, Amar HM, Gauduchon P (1997) Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorthalonil) in human lymphocytes with the comet assay. Mutat Res 375:205–217PubMedGoogle Scholar
  40. Marrs TC, Dewhurst I. (1999) Toxicology of pesticides. In: Ballantyne B, Marrs TC, Syversen T (eds) General and applied toxicology. Macmillian Reference Ltd., London, pp 1993–2012.7Google Scholar
  41. Mathew G, Rahiman MA, Vijayalaxmi KK (1990) In vivo genotoxic effects in mice of Metacid 50, an organophosphorus insecticide. Mutagenesis 5:147–149PubMedGoogle Scholar
  42. Mohn G (1973) 5-Methyl tryptophan resisttance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides. Mutat Res 20:7–15PubMedGoogle Scholar
  43. Moretti M, Marcarelli M, Villarini M, Fatigoni C, Scassellati-Sforzolini G, Pasquini R (2002) In vitro testing for genotoxicity of the herbicide terbutryn:cytogenetic and primary DNA damage. Toxicol In Vitro 16:81–88PubMedGoogle Scholar
  44. Moriya M, Ohta T, Watanabe K, Miyazawa T, Kato K, Shirasu Y (1983) Further mutagenicity studies on pesticides in bacterial reversion assay system. Mutat Res 113:173–180PubMedGoogle Scholar
  45. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the ‘comet’ assay. Radiat Res 122:86–94PubMedGoogle Scholar
  46. Pluijmen M, Drevon C, Montesano RM, Malaveille C, Hautefeuille A, Bartsch H (1984) Lack of mutagenicity on synthetic pyrethroids in Salmonella typhimurium strains and in V79 Chinese hamster cells. Mutat Res 137:7–15PubMedGoogle Scholar
  47. Poli P, de Mello MA, Buschini A, de Castro VLSS, Restivo FM, Rossi C, Zucchi TMAD (2003) Evaluation of the genotoxicity induced by the fungicide fenarimol in mammalian and plant cells by use of the single-cell gel electrophoresis assay. Mutat Res 540:57–66PubMedGoogle Scholar
  48. Rupa DS, Reddy PP, Reddi OS (1989) Analysis of sister-chromatid exchanges, cell kinetics and mitotic index in lymphocytes of smoking pesticide sprayers. Mutat Res 223:253–258PubMedGoogle Scholar
  49. Scarpato R, Migliore L, Angotzi G, Fedi A, Miligi L, Loprieno N (1996) Cytogenetic monitoring of a group of Italian floriculturists: no evidence of DNA damage related to pesticide exposure. Mutat Res 367:73–82PubMedGoogle Scholar
  50. Scarpato R, Hirvonen A, Migliore L, Falck G, Norppa H (1997) Influence of GSTM1 and GSTT1 polymorphisms on the frequency of chromosome aberrations in lymphocytes of smokers and pesticide-exposed greenhouse workers. Mutat Res 389:227–235PubMedGoogle Scholar
  51. Siebert D, Lemperle E (1974) Induction of mitotic gene conversion in Saccharomyces cerevisia by N-nitroso pesticides. Mutat Res 22:121–126PubMedGoogle Scholar
  52. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191PubMedGoogle Scholar
  53. Sobti RC, Krishan A, Pfaffenberger CD (1982) Cytokinetic and cytogenetic effects of some agricultural chemicals on human lymphoid cells in vitro: organophosphates. Mutat Res 102:89–102PubMedGoogle Scholar
  54. Sordo M, Herrera LA, Ostrosky-Wegman P, Rojas E (2001) Cytotoxic and genotoxic effects of As, MMA, and DMA on leukocytes and stimulated human lymphocytesGoogle Scholar
  55. Speit G, Hartmann A (1999) The comet assay (single-cell gel test). A sensitive genotoxicity for the detection of DNA damage and repair. Methods Mol Biol 113:203–212PubMedGoogle Scholar
  56. Surrales J, Xamena N, Creus A, Catalan J, Norppa H, Marcos R (1995) Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures. Mutat Res 341:169–184PubMedGoogle Scholar
  57. Tisch M, Schmezer P, Faulde M, Groh A, Maier H (2002) Genotoxicity studies on permethrin, DEET and diazinon in primary human nasal mucosal cells. Eur Arch Otorhinolaryngol 259:150–153CrossRefPubMedGoogle Scholar
  58. Titenko-Holland N, Windham G, Kolachana P, Reinish F, Parvatham S, Osorio AM, Smith MT (1997) Genotoxicity of malathion in human lymphocytes assessed using the micronucleus assay in vitro and in vivo: a study of malathion-exposed workers. Mutat Res 388:85–95PubMedGoogle Scholar
  59. Ündeğer U, Başaran N (2002) Assessment of DNA damage in workers occupationally exposed to pesticide mixtures by the alkaline comet assay. Arch Toxicol 76:430–436Google Scholar
  60. Ündeğer U, Institoris L, Siroki O, Nehez M, Desi I (2000) Simultaneous geno- and immunotoxicological invesitgations for early detection of organophosphate toxicity in rats. Ecotoxicol Environ Saf 45:43–48Google Scholar
  61. Velazquez A, Xamena N, Creus A, Marcos R (1990) Mutagenic evaluation of the organophosphorus insecticides methyl parathion and triazophos in Drosophila melanogaster. J Toxicol Environ Health 31:313–325PubMedGoogle Scholar
  62. Villarini M, Moretti M, Pasquini R, Scassellati-Sforzolini G, Fatigoni C, Marcarelli M, Monarca S, Rodriguez AV (1998) In vitro genotoxic effects of the insecticide deltamethrin in human peripheral blood leukocytes: DNA damage (‘comet’ assay) in relation to the induction of sister-chromatid exchanges and micronuclei. Toxicology 130:129–139PubMedGoogle Scholar
  63. Villarini M, Scassellati-Sforzolini G, Moretti M, Pasquini R (2000) In vitro genotoxicity of terbutryn evaluated by the alkaline single-cell microgel-electrophoresis ‘comet’ assay. Cell Biol Toxicol 16:285–292CrossRefPubMedGoogle Scholar
  64. Wang TC, Lee TC, Lin MF, Lin SY (1987) Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells. Mutat Res 188:311–321PubMedGoogle Scholar
  65. WHO (1986) Environmental Health Criteria 64. Carbamate pesticides: a general introduction. World Health Organisation, GenevaGoogle Scholar
  66. WHO (1988) The WHO recommended classification of pesticides by hazard and guidelines to the classification 1988–1989. World Health Organisation, GenevaGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of Pharmaceutical Toxicology, Faculty of PharmacyHacettepe UniversityAnkaraTurkey

Personalised recommendations