1-Methoxy-3-indolylmethyl DNA adducts in six tissues, and blood protein adducts, in mice under pak choi diet: time course and persistence

  • Melanie Wiesner-Reinhold
  • Gitte Barknowitz
  • Simone Florian
  • Inga Mewis
  • Fabian Schumacher
  • Monika Schreiner
  • Hansruedi GlattEmail author
Toxicokinetics and Metabolism


We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC–MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.


1-Methoxy-3-indolylmethyl glucosinolate Neoglucobrassicin DNA adducts Blood protein adducts Pak choi 



This study was financially supported by the German Federal Ministry of Education and Research (Grant 0315370D).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2019_2452_MOESM1_ESM.docx (195 kb)
Supplementary material 1 (DOCX 195 kb)


  1. Baasanjav-Gerber C, Engst W, Florian S et al (2010) Glucosinolates: DNA adduct formation in vivo and mutagenicity in vitro. In: Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (ed) Risk assessment of phytochemicals in food—novel approaches. Wiley-VCH, New York, pp 325–334Google Scholar
  2. Baasanjav-Gerber C, Hollnagel HM, Brauchmann J, Iori R, Glatt HR (2011a) Detection of genotoxicants in Brassicales using endogenous DNA as a surrogate target and adducts determined by 32P-postlabelling as an experimental end point. Mutagenesis 26(3):407–413. CrossRefGoogle Scholar
  3. Baasanjav-Gerber C, Monien BH, Mewis I et al (2011b) Identification of glucosinolate congeners able to form DNA adducts and to induce mutations upon activation by myrosinase. Mol Nutr Food Res 55(5):783–792. CrossRefGoogle Scholar
  4. Barknowitz G, Engst W, Schmidt S et al (2014) Identification and quantification of protein adducts formed by metabolites of 1-methoxy-3-indolylmethyl glucosinolate in vitro and in mouse models. Chem Res Toxicol 27(2):188–199. CrossRefGoogle Scholar
  5. Budnowski J, Hanske L, Schumacher F et al (2015) Glucosinolates are mainly absorbed intact in germfree and human microbiota-associated mice. J Agric Food Chem 63(38):8418–8428. CrossRefGoogle Scholar
  6. Burwell EL, Brickley BA, Finch CA (1953) Erythrocyte life span in small animals: comparison of two methods employing radioiron. Am J Physiol 172(3):718–724. CrossRefGoogle Scholar
  7. Clarke JD, Dashwood RH, Ho E (2008) Multi-targeted prevention of cancer by sulforaphane. Cancer Lett 269(2):291–304. CrossRefGoogle Scholar
  8. Dashwood RH, Xu M (2003) The disposition and metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline in the F344 rat at high versus low doses of indole-3-carbinol. Food Chem Toxicol 41(8):1185–1192. CrossRefGoogle Scholar
  9. Ehlers A, Florian S, Schumacher F et al (2015) The glucosinolate metabolite 1-methoxy-3-indolylmethyl alcohol induces a gene expression profile in mouse liver similar to the expression signature caused by known genotoxic hepatocarcinogens. Mol Nutr Food Res 59(4):685–697. CrossRefGoogle Scholar
  10. Fahey JW, Zhang YS, Talalay P (1997) Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci USA 94(19):10367–10372CrossRefGoogle Scholar
  11. Faust D, Nikolova T, Wätjen W, Kaina B, Dietrich C (2017) The Brassica-derived phytochemical indolo[3,2-b]carbazole protects against oxidative DNA damage by aryl hydrocarbon receptor activation. Arch Toxicol 91(2):967–982. CrossRefGoogle Scholar
  12. Förster N, Ulrichs C, Schreiner M, Müller CT, Mewis I (2015) Development of a reliable extraction and quantification method for glucosinolates in Moringa oleifera. Food Chem Toxicol 166:456–464. CrossRefGoogle Scholar
  13. Glatt HR (2000) Sulfotransferases in the bioactivation of xenobiotics. Chem-Biol Interact 129(1–2):141–170CrossRefGoogle Scholar
  14. Glatt HR, Baasanjav-Gerber C, Schumacher F et al (2011) 1-Methoxy-3-indolylmethyl glucosinolate: a potent genotoxicant in bacterial and mammalian cells—mechanisms of bioactivation. Chem-Biol Interact 192(1–2):81–86. CrossRefGoogle Scholar
  15. Gronke K, Hernandez PP, Zimmermann J et al (2019) Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature 566(7743):249–253. CrossRefGoogle Scholar
  16. Gupta RC (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc Natl Acad Sci USA 81(22):6943–6947. CrossRefGoogle Scholar
  17. Hanley AB, Parsley KR (1990) Identification of 1-methoxyindolyl-3-methyl isothiocyanate as an indole glucosinolate breakdown product. Phytochemistry 29(3):769–771CrossRefGoogle Scholar
  18. Hanschen FS, Lamy E, Schreiner M, Rohn S (2014) Reactivity and stability of glucosinolates and their breakdown products in foods. Angew Chem Int Ed Engl 53(43):11430–11450. CrossRefGoogle Scholar
  19. Herrmann K, Schumacher F, Engst W et al (2013) Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human liver samples. Carcinogenesis 34(5):1025–1030. CrossRefGoogle Scholar
  20. Hoelzl C, Glatt HR, Meinl W et al (2008) Consumption of Brussels sprouts protects peripheral human lymphocytes against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and oxidative DNA-damage: results of a controlled human intervention trial. Mol Nutr Food Res 52(3):330–341. CrossRefGoogle Scholar
  21. Jakubíková J, Sedlák J, Bod’o J, Bao Y (2006) Effect of isothiocyanates on nuclear accumulation of NF-κB, Nrf2, and thioredoxin in Caco-2 cells. J Agric Food Chem 54(5):1656–1662. CrossRefGoogle Scholar
  22. Jeffery EH, Araya M (2009) Physiological effects of broccoli consumption. Phytochem Rev 8(1):283–298CrossRefGoogle Scholar
  23. Kassie F, Knasmüller S (2000) Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC). Chem-Biol Interact 127(2):163–180. CrossRefGoogle Scholar
  24. Kassie F, Parzefall W, Musk S et al (1996) Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells. Chem-Biol Interact 102(1):1–16CrossRefGoogle Scholar
  25. Kensler TW, Egner PA, Agyeman AS et al (2013) Keap1-Nrf2 signaling: a target for cancer prevention by sulforaphane. Top Curr Chem 329:163–177. CrossRefGoogle Scholar
  26. Kumar A, Vineis P, Sacerdote C, Fiorini L, Sabbioni G (2010) Determination of new biomarkers to monitor the dietary consumption of isothiocyanates. Biomarkers 15(8):739–745. CrossRefGoogle Scholar
  27. Latté KP, Appel KE, Lampen A (2011) Health benefits and possible risks of broccoli: an overview. Food Chem Toxicol 49(12):3287–3309. CrossRefGoogle Scholar
  28. Lippmann D, Lehmann C, Florian S et al (2014) Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently. Food Funct 5(6):1073–1081. CrossRefGoogle Scholar
  29. Mewis I, Appel HM, Hom A, Raina R, Schultz JC (2005) Major signaling pathways modulate Arabidopsis glucosinolate accumulation and response to both phloem-feeding and chewing insects. Plant Physiol 138(2):1149–1162CrossRefGoogle Scholar
  30. Mewis I, Schreiner M, Nguyen CN et al (2012) UV-B irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors. Plant Cell Physiol 53(9):1546–1560CrossRefGoogle Scholar
  31. Musk SRR, Smith TK, Johnson IT (1995) On the cytotoxicity and genotoxicity of allyl and phenethyl isothiocyanates and their parent glucosinolates sinigrin and gluconasturtiin. Mutat Res 348(1):19–23CrossRefGoogle Scholar
  32. Neudecker T, Henschler D (1985) Allyl isothiocyanate is mutagenic in Salmonella typhimurium. Mutat Res 156(1–2):33–37. CrossRefGoogle Scholar
  33. Ratzka A, Vogel H, Kliebenstein DJ, Mitchell-Olds T, Kroymann J (2002) Disarming the mustard oil bomb. Proc Natl Acad Sci USA 99(17):11223–11228. CrossRefGoogle Scholar
  34. Ruvinsky A, Marshall Graves JA (2005) Mammalian genomics. CABI Publishing, WallingfordCrossRefGoogle Scholar
  35. Schumacher F, Engst W, Monien BH et al (2012) Detection of DNA adducts originating from 1-methoxy-3-indolylmethyl glucosinolate using isotope-dilution UPLC–ESI–MS/MS. Anal Chem 84(14):6256–6262. CrossRefGoogle Scholar
  36. Schumacher F, Herrmann K, Florian S, Engst W, Glatt HR (2013) Optimized enzymatic hydrolysis of DNA for LC–MS/MS analyses of adducts of 1-methoxy-3-indolylmethyl glucosinolate and methyleugenol. Anal Biochem 434(1):4–11. CrossRefGoogle Scholar
  37. Schumacher F, Florian S, Schnapper A et al (2014) A secondary metabolite of Brassicales, 1-methoxy-3-indolylmethyl glucosinolate, as well as its degradation product, 1-methoxy-3-indolylmethyl alcohol, forms DNA adducts in the mouse, but in varying tissues and cells. Arch Toxicol 88(3):823–836. Google Scholar
  38. Tretyakova N, Goggin M, Sangaraju D, Janis G (2012) Quantitation of DNA adducts by stable isotope dilution mass spectrometry. Chem Res Toxicol 25(10):2007–2035. CrossRefGoogle Scholar
  39. Vang O, Dragsted LO (1996) Naturally occurring antitumourigens. Nordic Council of Ministers, CopenhagenGoogle Scholar
  40. Verkerk R, Schreiner M, Krumbein A et al (2009) Glucosinolates in Brassica vegetables: the influence of the food supply chain on intake, bioavailability and human health. Mol Nutr Food Res 53:S219–S265. CrossRefGoogle Scholar
  41. Weigle WO (1957) Elimination of I131 labelled homologous and heterologous serum proteins from blood of various species. Proc Soc Exp Biol Med 94(2):306–309CrossRefGoogle Scholar
  42. Weissenberg S (2014) Analyse der biologischen Wirkung des Glucosinolatabbauprodukts 1-methoxy-3-indolylmethylalkohol (1-MIM-OH) in Abhängigkeit von der Expression der Sulfotransferase 1A1 in der Maus. Master thesis, Humboldt-Universität zu BerlinGoogle Scholar
  43. Wiesner M, Hanschen FS, Schreiner M, Glatt HR, Zrenner R (2013a) Induced production of 1-methoxy-indol-3-yl methyl glucosinolate by jasmonic acid and methyl jasmonate in sprouts and leaves of pak choi (Brassica rapa ssp. chinensis). Int J Mol Sci 14(7):14996–15016. CrossRefGoogle Scholar
  44. Wiesner M, Zrenner R, Krumbein A, Glatt HR, Schreiner M (2013b) Genotypic variation of the glucosinolate profile in pak choi (Brassica rapa ssp. chinensis). J Agric Food Chem 61(8):1943–1953. CrossRefGoogle Scholar
  45. Wiesner M, Schreiner M, Glatt HR (2014) High mutagenic activity of juice from pak choi (Brassica rapa ssp. chinensis) sprouts due to its content of 1-methoxy-3-indolylmethyl glucosinolate, and its enhancement by elicitation with methyl jasmonate. Food Chem Toxicol 67:10–16. CrossRefGoogle Scholar
  46. Winde I, Wittstock U (2011) Insect herbivore counter adaptations to the plant glucosinolate-myrosinase system. Phytochemistry 72(13):1566–1575. CrossRefGoogle Scholar
  47. Wu QJ, Xie L, Zheng W et al (2013a) Cruciferous vegetables consumption and the risk of female lung cancer: a prospective study and a meta-analysis. Ann Oncol 24(7):1918–1924CrossRefGoogle Scholar
  48. Wu QJ, Yang Y, Vogtmann E et al (2013b) Cruciferous vegetables intake and the risk of colorectal cancer: a meta-analysis of observational studies. Ann Oncol 24(4):1079–1087CrossRefGoogle Scholar
  49. Wu QJ, Yang Y, Wang J, Han LH, Xiang YB (2013c) Cruciferous vegetable consumption and gastric cancer risk: a meta-analysis of epidemiological studies. Cancer Sci 104(8):1067–1073CrossRefGoogle Scholar
  50. Xu MR, Orner GA, Bailey GS, Stoner GD, Horio DT, Dashwood RH (2001) Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methylimidazo[4,5-f]quinoline. Carcinogenesis 22(2):309–314. CrossRefGoogle Scholar
  51. Xu CJ, Huang MT, Shen GX et al (2006) Inhibition of 7,12-dimethylbenz[a]anthracene-induced skin tumorigenesis in C57bl/6 mice by sulforaphane is mediated by nuclear factor e2-related factor 2. Cancer Res 66(16):8293–8296. CrossRefGoogle Scholar
  52. Yamaguchi T (1980) Mutagenicity of isothiocyanates, isocyanates and thioureas on Salmonella typhimurium. Agric Biol Chem 44(12):3017–3018Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Leibniz Institute of Vegetable and Ornamental Crops e.V.GrossbeerenGermany
  2. 2.Department of Nutritional ToxicologyInstitute of Human Nutrition Potsdam-RehbrückeNuthetalGermany
  3. 3.Department Food SafetyFederal Institute of Risk AssessmentBerlinGermany
  4. 4.Faculty of Life Science, Urban Plant EcophysiologyHumboldt-Universität zu BerlinBerlinGermany
  5. 5.Department Nutritional Toxicology, Institute of Nutritional ScienceUniversity of PotsdamNuthetalGermany

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