Environmentally relevant exposures of male mice to carbendazim and thiram cause persistent genotoxicity in male mice
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.
KeywordsFungicides Comet Genotoxicity Nepal Mice Carbendazim Thiram Persistence
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 was provided by a 2017–2018 Minnesota State University Faculty Research Grant.
- 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
- 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
- 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
- International Programme on Chemical Safety (1993) Environmental health criteria 149 carbendazim. World Health Organization, GenevaGoogle Scholar
- Keml Swedish Chemicals Agency (2015) Substance evaluation conclusion as required by reach article 48 and evaluation report for thiram. SundbybergGoogle Scholar
- 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
- 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
- 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.
- 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
- 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
- Parazajder J (2011) Genotoxic effect of pesticide carbendazim in Swiss albino mice. Diploma thesis, University of Zagreb, CroatiaGoogle Scholar
- 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
- 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
- Stober W. (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol. Appendix3: appendix 3BGoogle Scholar
- Trevigen (2016a) CometAssay® 96 reagent kit for higher throughput single cell gel electrophoresis assay (96-well). Catalog # 4253–096-KGoogle Scholar
- Trevigen (2016b) CometAssay® control cells for single cell gel electrophoresis assay. Catalog # 4256–010-CCGoogle Scholar
- US Environmental Protection Agency (2004) Reregistration eligibility decision for thiram. EPA 738-R-04-012, Washington, DCGoogle Scholar
- 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
- 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