Advertisement

Environmentally relevant exposures of male mice to carbendazim and thiram cause persistent genotoxicity in male mice

  • Bina Rai
  • Steven Don MercurioEmail author
Research Article

Abstract

Carbendazim and thiram are fungicides used in combination to prevent mold destruction of crops. Studies have demonstrated genotoxicity by these agents, but have not used concentrations below their water solubility limits in drinking water to test for persistence of genotoxicity due to chronic exposure. Ten 8-week old male Swiss-Webster mice were exposed to tap water, or nominal concentrations of 20 μM carbendazim, 20 μM thiram or 20 μM of both fungicides for 90 days (total of 40 mice). Five mice from tap water controls, carbendazim, thiram and combination-treated groups (20 mice total) had genotoxicity detected by comet assay of lymphocytes at the termination of the exposure period. The other 20 mice (4 treatment groups) were all switched to tap water and allowed a 45-day recovery period to check for persistence of DNA damage. The damage was compared with commercial control cells exposed to increasingly harsh treatment by etopside. Comet assay (mean % tail DNA + SE) of control mice (9.8 + 0.9) was similar to commercial control (CC0) cells (8.5 + 0.9). Carbendazim, thiram or the combination treatment caused similar mean % tail DNA with 33.0 + 2.9, 30.1 + 3.3 and 29.1 + 1.8, respectively, comparable with commercial cells slightly damaged by etopside (CC1 with 31.4 + 2.9) with no statistical change in water or food intake, body weight or liver or kidney weights. The key result was that a 45-day recovery period had no observable difference in the DNA damage as assessed by DNA % in comet tail with tap water controls and CCO control cells at 7.0 + 0.7 and 9.7 + 1.2 versus 27.5 + 1.9, 29.3 + 2.2 and 32.0 + 1.8, respectively, for carbendazim, thiram and combination treatments. It is of concern that the use of these agents in developing countries with little training or regulation results in water pollution that may cause significant persistent DNA damage in animal or human populations that may not be subject to repair.

Keywords

Fungicides Comet Genotoxicity Nepal Mice Carbendazim Thiram Persistence 

Notes

Acknowledgment

An undergraduate, Samantha J. Foltz, provided assistance with portions of the experiment. Dr. Marilyn Hart provided support in the function and maintenance of the fluorescence microscope.

Funding information

Funding was provided by a 2017–2018 Minnesota State University Faculty Research Grant.

References

  1. Agrawal RC, Shukla Y, Mehrotra NK (1997) Assessment of mutagenic potential of thiram. Food Chem Toxicol 35:523–525CrossRefGoogle Scholar
  2. Bentley KS, Kirkland D, Murphy M, Marshall R (2000) Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization. Mutat Res 464:41–51CrossRefGoogle Scholar
  3. Bhandari G, Atreya K, Yang X, Fan L, Geissen V (2018) Factors affecting pesticide safety behaviour: the perceptions of Nepalese farmers and retailers. Sci Total Environ 631-632:1560–1571.  https://doi.org/10.1016/j.scitotenv.2018.03.144 CrossRefGoogle Scholar
  4. Bjørge C, Brunborg G, Wiger R, Holme JA, Scholz T, Dybing E, Søderlund EJ (1996) A comparative study of chemically induced DNA damage in isolated human and rat testicular cells. Reprod Toxicol 10:509–519CrossRefGoogle Scholar
  5. Bowen DE, Whitwell JH, Lillford L, Henderson D, Kidd D, Mc Garry S, Pearce G, Beevers C, Kirkland DJ (2011) Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the comet assay and the flow-cytometric peripheral blood micronucleus test. Mutat Res 722:7–19.  https://doi.org/10.1016/j.mrgentox.2011.02.009 CrossRefGoogle Scholar
  6. Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151.  https://doi.org/10.1093/mutage/gem051 CrossRefGoogle Scholar
  7. Crebelli R, Zijno A, Conti L, Crochi B, Leopardi P, Marcon F, Renzi L, Carere A (1992) Further in vitro and in vivo mutagenicity assays with thiram and ziram fungicides: bacterial reversion assays and mouse micronucleus test. Teratog Carcinog Mutagen 12:97–112CrossRefGoogle Scholar
  8. da Silva J, de Freitas TRO, Marinho JR, Speit B, Erdtmann B (2000) An alkaline single-cell gel electrophoresis (comet) assay for environmental biomonitoring with native rodents. Genet Mol Biol 23:241–245CrossRefGoogle Scholar
  9. De Boer RJ, Perelson AS (2013) Quantifying T lymphocyte turnover. J Theor Biol 21;327:45–87.  https://doi.org/10.1016/j.jtbi.2012.12.025 CrossRefGoogle Scholar
  10. Ðikić D, Mojsović-Cuić A, Cupor I, Benković V, Horvat-Knezević A, Lisicić D, Orsolić N (2012) Carbendazim combined with imazalil or cypermethrin potentiate DNA damage in hepatocytes of mice. Hum Exp Toxicol 31:492–505.  https://doi.org/10.1177/0960327111417910 CrossRefGoogle Scholar
  11. Ermler S, Scholze M, Kortenkamp A (2013) Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro. Mutagenesis. 28:417–426.  https://doi.org/10.1093/mutage/get019 CrossRefGoogle Scholar
  12. European Food Safety Authority (EFSA) (2013) Peer review of the pesticide risk assessment of the active substance thiram. EFSA J 15:e04700.  https://doi.org/10.2903/j.efsa.2017.4700 CrossRefGoogle Scholar
  13. Evenson DP, Janca FC, Jost LK. (1987) Effects of the fungicide methyl-benzimidazol-2-yl carbamate (MBC) on mouse germ cells as determined by flow cytometry. J Toxicol Environ Health 1987;20(4):387–399CrossRefGoogle Scholar
  14. Franekić J, Bratulić N, Pavlica M, Papes D (1994) Genotoxicity of dithiocarbamates and their metabolites. Mutat Res 325:65–74CrossRefGoogle Scholar
  15. Gudmundsson B, Thormar HG, Sigurdsson A, Dankers W, Steinarsdottir M, Hermanowicz S, Sigurdsson S, Olafsson D, Halldorsdottir AM, Meyn S, Jonsson JJ (2018) Northern lights assay: a versatile method for comprehensive detection of DNA damage. Nucleic Acids Res 46:e118.  https://doi.org/10.1093/nar/gky645 CrossRefGoogle Scholar
  16. Gupta B, Rani M, Kumar R (2011) Degradation of thiram in water, soil and plants: a study by high-performance liquid chromatography. Biomed Chromatogr 26:69–75.  https://doi.org/10.1002/bmc.1627 CrossRefGoogle Scholar
  17. Hart RW, Hall KY, Daniel FB (1978) DNA repair and mutagenesis in mammalian cells. Photochem Photobiol 28:131–155CrossRefGoogle Scholar
  18. Heath RJ, Rubin JR, Holland DR, Zhang E, Snow ME, Rock CO (1999) Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J Biol Chem 274:11110–11114CrossRefGoogle Scholar
  19. Hemavathi E, Rahiman MA (1996) A comparative mutagenicity study of the three carbamate fungicides ziram, thiram and dithane M-45. J Environ Biol 17:171–180Google Scholar
  20. Huan Z, Luo J, Xu Z, Xie D (2016) Acute toxicity and genotoxicity of carbendazim, main impurities and metabolite to earthworms (Eisenia foetida). Bull Environ Contam Toxicol 96:62–69.  https://doi.org/10.1007/s00128-015-1653-y CrossRefGoogle Scholar
  21. Igarashi M, Setoguchi M, Takada S, Itoh S, Furuhama K (2007) Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice. Mutat Res 632:89–98.  https://doi.org/10.1016/j.mrgentox.2007.04.012 CrossRefGoogle Scholar
  22. International Programme on Chemical Safety (1993) Environmental health criteria 149 carbendazim. World Health Organization, GenevaGoogle Scholar
  23. Keml Swedish Chemicals Agency (2015) Substance evaluation conclusion as required by reach article 48 and evaluation report for thiram. SundbybergGoogle Scholar
  24. Kitagawa E, Takahashi J, Momose Y, Iwahashi H (2002) Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray. Environ Sci Technol 36:3908–3915.  https://doi.org/10.1021/es015705v CrossRefGoogle Scholar
  25. Kolenko VM et al (2001) Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes. Apoptosis 6:418–429CrossRefGoogle Scholar
  26. Langie SA, Koppen G, Desaulniers D, Al-Mulla F, Al-Temaimi R, Amedei A, Azqueta A, Bisson WH, Brown DG, Brunborg G, Charles AK, Chen T, Colacci A, Darroudi F, Forte S, Gonzalez L, Hamid RA, Knudsen LE, Leyns L, Lopez de Cerain Salsamendi A, Memeo L, Mondello C, Mothersill C, Olsen AK, Pavanello S, Raju J, Rojas E, Roy R, Ryan EP, Ostrosky-Wegman P, Salem HK, Scovassi AI, Singh N, Vaccari M, Van Schooten FJ, Valverde M, Woodrick J, Zhang L, van Larebeke N, Kirsch-Volders M, Collins AR (2015) Causes of genome instability: the effect of low dose chemical exposures in modern society. Carcinogenesis 36(Suppl 1):S61–S88.  https://doi.org/10.1093/carcin/bgv031 CrossRefGoogle Scholar
  27. Lebailly P, Vigreux C, Godard T, Sichel F, Bar E, LeTalaër JY, Henry-Amar M, Gauduchon P (1997) Assessment of DNA damage induced in vitro by etoposide and two fungicides (carbendazim and chlorothalonil) in human lymphocytes with the comet assay. Mutat Res 375(2):205–217CrossRefGoogle Scholar
  28. Liu J, Zhang P, Zhao Y, Zhang H (2019) Low dose carbendazim disrupts mouse spermatogenesis might be through estrogen receptor related histone and DNA methylation. Ecotoxicol Environ Saf 176:242–249.  https://doi.org/10.1016/j.ecoenv.2019.03.103 CrossRefGoogle Scholar
  29. MacBean C, Ed. (2010) Carbendazim (10605-21-7) (2008-2010). The e-pesticide manual, 15th edition, version 5.0.1., British crop protection council, Surrey, UKGoogle Scholar
  30. Mankato Water Treatment (2019) Water report. City of Mankato. https://www.mankatomn.gov/city-services-a-z/city-services-n-z/water/water-treatment/water-report.
  31. McCarroll NE, Protzel A, Ioannou Y, Frank Stack HF, Jackson MA, Waters MD, Dearfield KL (2002) A survey of EPA/OPP and open literature on selected pesticide chemicals. III Mutagenicity and carcinogenicity of benomyl and carbendazim. Mutat Res 512:1–35CrossRefGoogle Scholar
  32. Moffit JS, Bryant BH, Hall SJ, Boekelheide K (2007) Dose-dependent effects of sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis. Toxicol Pathol 35:719–727CrossRefGoogle Scholar
  33. Ngo LP, Parrish M, Ge J, Engelward (2016) Whole blood hemolysis with isotonic ammonium chloride solution. Internet. https://nextgen-protocols.org/wp-content/uploads/2016/08/Whole-blood-hemolysis-with-isotonic-ammonium-chloride-solution.pdf
  34. Novais SC, De Coen W, Amorim MJ (2012) Gene expression responses linked to reproduction effect concentrations (EC 10,20,50,90) of dimethoate, atrazine and carbendazim, in Enchytraeus albidus. PLoS One 7:e36068.  https://doi.org/10.1371/journal.pone.0036068 CrossRefGoogle Scholar
  35. Pande S, Singh F, Rao JN, Bakr MA, Chaurasia PCP, Joshi S, Johansen C, Singh SD, Kumar J, Rahman MM, Gowda CLL (2001) Integrated management of Botrytis gray mold of chickpea. Informational Bulletin No. 61. ICRISAT. 1-26Google Scholar
  36. Pande S, Stevenson P, Rao JN, Neupane RK, Chaudhary RN, Grzywacz D, Bourai VA, Kishore GK (2005) Reviving chickpea production in Nepal through integrated crop management, with emphasis on Botrytis gray mold. Plant Dis 89:1252–1262.  https://doi.org/10.1094/PD-89-1252 CrossRefGoogle Scholar
  37. Parazajder J (2011) Genotoxic effect of pesticide carbendazim in Swiss albino mice. Diploma thesis, University of Zagreb, CroatiaGoogle Scholar
  38. Prasad MH, Pushpavath K, Rita P, Reddy PP (1987) Effect of thiram on the germ cells of male mice. Food Chem Toxicol 25:709–711CrossRefGoogle Scholar
  39. Rajeswary S, Kumaran B, Ilangovan R, Yuvaraj S, Sridhar M, Venkataraman P, Srinivasan N, Aruldhas MM (2007) Modulation of antioxidant defense system by the environmental fungicide carbendazim in Leydig cells of rats. Reprod Toxicol 24:371–380CrossRefGoogle Scholar
  40. Rajput S, Kumari A, Arora S, Kaur R (2018) Multi-residue pesticides analysis in water samples using reverse phase high performance liquid chromatography (RP-HPLC). MethodsX. 5:744–751.  https://doi.org/10.1016/j.mex.2018.07.005 CrossRefGoogle Scholar
  41. Rama EM, Bortolan S, Vieira ML, Gerardin DC, Moreira EG (2014) Reproductive and possible hormonal effects of carbendazim. Regul Toxicol Pharmacol 69:476–486.  https://doi.org/10.1016/j.yrtph.2014.05.016 CrossRefGoogle Scholar
  42. Rath N, Rasputra KS, Liyanage R, Huff GR, Huff WE (2011) Dithiocarbamate toxicity – an appraisal. In: Stoytcheva M (ed) Pesticides in the modern world – effects of pesticides exposure. InTech, Rijeka, Crotia, pp 323–340.  https://doi.org/10.5772/1803
  43. Santovito A, Cervella P, Delpero M (2012) Chromosomal aberrations in cultured human lymphocytes treated with the fungicide, thiram. Drug Chem Toxicol 35:347–351.  https://doi.org/10.3109/01480545.2011.627862 CrossRefGoogle Scholar
  44. Serfilippi M, Pallman DR, Russell B (2003) Serum clinical chemistry and hematology reference values in outbred stocks of albino mice from three commonly used vendors and two inbred strains of albino mice. Contemp Top Lab Anim Sci 42(3):46–52Google Scholar
  45. Sharma DR, Thapa RB, Manandhar HK, Shrestha SM, Pradhan SB (2012) Use of pesticides in Nepal and impacts on human health and environment. J Agric Environ 13:67–72.  https://doi.org/10.3126/aej.v13i0.7590 CrossRefGoogle Scholar
  46. Silva AR, Cardoso DN, Cruz A, Lourenço J, Mendo S, Soares AM, Loureiro S (2015) Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna. Ecotoxicol Environ Saf 115:279–290.  https://doi.org/10.1016/j.ecoenv.2015.02.022 CrossRefGoogle Scholar
  47. Silva ARR, Cardoso DN, Cruz A, Mendo S, Soares AMVM, Loureiro S (2019) Long-term exposure of Daphnia magna to carbendazim: how it affects toxicity to another chemical or mixture. Environ Sci Pollut Res Int 26(Apr 12):16289–16302.  https://doi.org/10.1007/s11356-019-05040-1 CrossRefGoogle Scholar
  48. Stober W. (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol. Appendix3: appendix 3BGoogle Scholar
  49. Sykora P, Witt KL, Revanna P, Smith-Roe SL, Dismukes J, Lloyd DG, Engelward BP, Sobol RW (2018) Next generation high throughput DNA damage detection platform for genotoxic compound screening. Sci Rep 8:2771.  https://doi.org/10.1038/s41598-018-20995-w CrossRefGoogle Scholar
  50. Topè AM, Rogers PF (2009) Evaluation of protective effects of sulforaphane on DNA damage caused by exposure to low levels of pesticide mixture using comet assay. J Environ Sci Health B 44:657–662CrossRefGoogle Scholar
  51. Trevigen (2016a) CometAssay® 96 reagent kit for higher throughput single cell gel electrophoresis assay (96-well). Catalog # 4253–096-KGoogle Scholar
  52. Trevigen (2016b) CometAssay® control cells for single cell gel electrophoresis assay. Catalog # 4256–010-CCGoogle Scholar
  53. US Environmental Protection Agency (2004) Reregistration eligibility decision for thiram. EPA 738-R-04-012, Washington, DCGoogle Scholar
  54. Vanhauwaert A, Vanparys P, Kirsch-Volders M (2001) The in vivo gut micronucleus test detects clastogens and aneugens given by gavage. Mutagenesis. 16:39–50.  https://doi.org/10.1093/mutage/16.1.39 CrossRefGoogle Scholar
  55. Villani P, Andreoli C, Crebelli R, Pacchierotti F, Zijno A, Carere A (1998) Analysis of micronuclei and DNA single-strand breaks in mouse splenocytes and peripheral lymphocytes after oral administration of tetramethylthiuram disulfide (thiram). Food Chem Toxicol 36:155–164CrossRefGoogle Scholar
  56. Westera L, Drylewicz J, den Braber I, Mugwagwa T, van der Maas I, Kwast L, Volman T, van de Weg-Schrijver EH, Bartha I, Spierenburg G, Gaiser K, Ackermans MT, Asquith B, de Boer RJ, Tesselaar K, Borghans JA (2013) Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans. Blood 122:2205–2212.  https://doi.org/10.1182/blood-2013-03-488411 CrossRefGoogle Scholar
  57. Yalkowsky SH, He, Y. (2003) Handbook of aqueous solubility data: an extensive compilation of aqueous solubility data for organic compounds extracted from the AQUASOL dATAbASE. CRC press LLC, Boca Raton, FL., p. 299Google Scholar
  58. Yang C, Hamel C, Vujanovic GY (2011) Fungicide: modes of action and possible impact on nontarget microorganisms. ISRN Ecol:1–8.  https://doi.org/10.5402/2011/130289 CrossRefGoogle Scholar
  59. Yang WY, Cao Q, Callahan C, Galvis C, Sang QX, Alabugin IV. (2010) Intracellular DNA damage by lysine-acetylene conjugates. J nucleic acids. 2010. Pii: 931394. doi:  https://doi.org/10.4061/2010/931394 CrossRefGoogle Scholar
  60. Yu Y, Chu X, Pang G, Xiang Y, Fang H (2009) Effects of repeated applications of fungicide carbendazim on its persistence and microbial community in soil. J Environ Sci (China) 21:179–185CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Biological SciencesMinnesota State UniversityMankatoUSA

Personalised recommendations