Schumacher et al.: 1-Methoxy-3-indolylmethyl DNA adducts in mice

A secondary metabolite of Brassicales, 1-methoxy-3-indolylmethyl glucosinolate, as well as its degradation product, 1-methoxy-3-indolylmethyl alcohol, form DNA adducts in the mouse, but in varying tissues and cells

Supplementary Material

Fabian Schumacher1,§, Simone Florian1,§, Anke Schnapper1,2, Bernhard H. Monien1, Inga Mewis3, Monika Schreiner3, Albrecht Seidel4, Wolfram Engst1, and Hansruedi Glatt1,*

1 German Institute of Human Nutrition, Potsdam-Rehbrücke, 14558 Nuthetal, Germany

2 Institute for Functional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany

3 Leibniz-Institute of Vegetable and Ornamental Crops Grossbeeren/Erfurt e.V., 14979 Grossbeeren, Germany

4 Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer-Foundation, 22927 Grosshansdorf, Germany

§ These authors contributed equally to the study.

* To whom correspondence should be addressed. Phone: (+49) 33200 882321. Fax: (+49) 33200-882426. E-mail:

Supplementary Fig. S1. Time dependence of the levels of the N6-(1-MIM)-dAdo in DNA of caecum (left), colon (middle) and liver (right) of mice after a single oral treatment with 1-MIM glucosinolate (upper row) or 1-MIM alcohol (lower row) at a dose of 600 µmol/kg body mass. Data are means ± SE of 5 mice. If the adduct levels were below LOD, they were set as LOD for the presentation and the further calculations. This procedure had to be used after 1-MIM glucosinolate treatment for 1 colonic and 4 hepatic samples in the 3-h group, 4 hepatic samples in the 8-h group, and 1 caecal and 1 colonic sample in the 48-h group, and for all samples of the bars labelled “n.d.”. The bars (then in grey) indicate the LOD in this case.

We postulated that the elimination of adducts might follow first order kinetics. Therefore, we used the logarithms of the adduct levels for a one-way ANOVA analysis with a post test for linear trend in the period between the peak level (at 8 h) and the last time point (48 h). Similar time courses were observed for the N2-(1-MIM)-dGuo adducts (Main document, Fig. 2).

Supplementary Fig. S2. Dose dependence of the levels of the N6-(1-MIM)-dAdo adduct in DNA of caecum (left), colon (middle) and liver (right) of mice after a single oral treatment with 1-MIM glucosinolate (upper row) or 1-MIM alcohol (lower row). The animals were sacrificed 8 h after the treatment. Data are means ± SE of 5 mice. If the adduct levels were below LOD, they were set as LOD for the presentation and the further calculations. This procedure had to be used for 3 caecal samples in the 60-µmol 1-MIM glucosinolate/kg group, 4 hepatic samples in the 600-µmol 1-MIM glucosinolate/kg group, 1 caecal and 3 hepatic samples in the 20-µmol 1-MIM alcohol/kg group, 2 colonic samples in the 60-µmol 1-MIM alcohol/kg group, and for all samples of the bars labelled “n.d.”. The bars (then marked in grey) describe the LOD in this case.

Numbers indicated above the bars represent adduct levels (per 108 dN) divided by the dose used (in µmol per kg body mass). Similar results were observed for N2-(1-MIM)-dGuo adducts (Main document, Fig. 3), with their levels being nearly 3.3-fold higher than those of the N6-(1-MIM)-dAdo adducts.

Supplementary Table 1

Tissue distribution of dAdo adducts in DNA of mice treated with an equimolar dose of 1-MIM glucosinolate or 1-MIM alcohol a

Tissue / N 6-(1-MIM)-dAdo per 108 dN
1-MIM glucosinolate / 1-MIM alcohol
Stomach / <LOD / 1250 ± 240
Jejunum / 46 ± 16 b / 54 ± 18 b
Caecum / 320 ± 60 / 1640 ± 310
Colon / 110 ± 20 / 230 ± 40
Liver / 19 ± 9 d / 1510 ± 230
Lung / <LOD / <LOD
Kidney / <LOD / 22 ± 7 c

a The animals received a single oral treatment with the test compounds at a dose of 600 µmol per kg body mass. They were sacrificed 8 h after the treatment. Values are means ± SE from 5 mice. Similar results were observed for the N2-(1-MIM)-dGuo adduct (main document, Table 1), with their levels being higher by a factor of nearly 1.2 (stomach) and 3.3 (remaining tissues) than those of the N 6-(1-MIM)-dAdo adducts.

b,c,d Adduct values for 1, 2 and 4 mice, respectively, out of the 5 mice were below the LOD (10 adducts per 108 dN). Levels in these animals were set as LOD for the calculation of the mean and SE.

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