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Glucosinolates Are Mainly Absorbed Intact in Germfree and Human Microbiota-Associated Mice Julia Budnowski,†,⊥ Laura Hanske,†,⊥ Fabian Schumacher,‡,∥ Hansruedi Glatt,‡ Stefanie Platz,§ Sascha Rohn,§ and Michael Blaut*,† †

Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Department of Nutritional Toxicology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany § Institute of Food Chemistry, Hamburg School of Food Science, University of Hamburg, Hamburg, Germany ‡

ABSTRACT: Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-L-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small. KEYWORDS: glucosinolates, intestinal microbiota, bioactivation, isothiocyanates, DNA adducts

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GLS as a source for glucose.10,11 In these studies, microbiota hydrolyzed sinigrin and glucotropaeolin into their corresponding isothiocyanates, namely, allyl isothiocyanate and benzyl isothiocyanate, respectively, and partly further into their corresponding nitriles, both in gnotobiotic rats and in vitro. Observations regarding enzyme activation by ascorbic acid and pH dependence suggested that the enzyme activity of Bif idobacterium is attributable to an enzyme similar to myrosinase of plant origin.10 The aim of the present study was to investigate to which extent human gut bacteria contribute to the bioactivation of selected GLS in vivo. For that purpose, the conversion of GRA and NGBS was compared between mice associated with complex human intestinal microbiota (human microbiota-associated, HMA) and germ free mice. Chemical structures of GRA, NGBS, and their metabolites following myrosinase-catalyzed hydrolysis and further reaction products investigated in the present study are depicted in Figure 1.

INTRODUCTION Brassica vegetables are rich in glucosinolates (GLS).1 Some of them including glucoraphanin [4-(methylsulfinyl)butyl GLS, GRA], highly abundant in, e.g., broccoli,2 have been proposed to exert chemoprotective properties. In contrast, other GLS such as neoglucobrassicin [N-methoxyindole-3-ylmethyl GLS, NGBS] for instance, from Pak Choi, are mutagenic to cultured cells after metabolic activation3 and form DNA adducts in these cells as well as in various tissues of mice after oral administration.4 Effects of GLS are mainly attributed to their hydrolysis products, which are formed by an endogenous plant enzyme called myrosinase (thioglucosidase, EC 3.2.1.147). Bioactive metabolites of GLS are isothiocyanates and indoles, which have been investigated intensively during the last decades.5 Although myrosinase is largely inactivated during the thermal treatment of vegetables, isothiocyanate metabolites were nevertheless excreted,6 and cardioprotective effects of GLS as well as the induction of phase II enzymes by GLS were observed following the intake of cooked Brassica vegetables.7 Mammalian thioglucosidase activity has so far not convincingly been demonstrated. Therefore, gut bacteria were assumed to play a role in the activation of GLS in the intestinal tract after the ingestion of cooked brassica vegetables. This was already proposed by Greer and Deeney in 1959.8 In agreement with this notion, the excretion of GLS metabolites was strongly decreased by decimation of the intestinal microbiota by mechanical cleansing or antibiotic treatment.9 All of these findings support an activation of GLS in the gut, presumably by intestinal bacteria. Various intestinal bacteria including Bifidobacterium and Bacteroides have been shown to degrade © 2015 American Chemical Society

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MATERIALS AND METHODS

Chemicals. GRA was purified from ripe seeds of Brassica oleracea as described by Haack et al.12 and provided by Dr. Renato Iori (Agricultural Research Council, Industrial Crop Research Centre, Italy). D,L-Sulforaphane [4-(methylsulfinyl)butyl isothiocyanate, SFN] and erucin [4-(methylthio)butyl isothiocyanate, ERU] were purchased from Enzo Life Sciences GmbH (Lörrach, Germany). Glucoerucin [4Received: Revised: Accepted: Published: 8418

June 16, 2015 September 7, 2015 September 13, 2015 September 13, 2015 DOI: 10.1021/acs.jafc.5b02948 J. Agric. Food Chem. 2015, 63, 8418−8428

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of (A) glucoraphanin, (B) neoglucobrassicin, and their metabolites following myrosinase-catalyzed hydrolysis and further reaction products (mercapturic acids, DNA adducts) investigated in the present study. SULT, sulfotransferase. (methylthio)butyl glucosinolate, GER] was from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Germany). NGBS was isolated from Pak Choi with a purity of >99% according to Baasanjav-Gerber et al.3 NMethoxyindole-3-carbinol (NI3C) and N-methoxyindole-3-acetonitrile were synthesized and provided by Dr. Albrecht Seidel (Biochemical Institute for Environmental Carcinogens, Professor Dr. Gernot Grimmer-Foundation, Germany) as described previously.13 Myrosinase, N-(tert-butoxycarbonyl)-L-cysteine methyl ester (N-tBocCys-ME), cysteinylglycine, N-acetyl-L-cysteine, L-glutathione, and trifluoroacetic acid were purchased from Sigma-Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ethyl acetate, L-cysteine, acetonitrile, and MeOH (both HPLC-grade) were obtained from Roth (Karlsruhe, Germany). Ethanol (HPLC-grade) was obtained from VWR (Darmstadt, Germany). All aqueous solutions were prepared with ultrapure water purified with Ultra Clear UV UF (Barsbüttel, Germany). N-tBoc-CysME stock solution (1.068 M in MeOH) was prepared fresh daily with 100% MeOH and was further diluted to a 427 mM working solution with 60% MeOH (v/v). Mercapturic acid conjugates of SFN and ERU were prepared as reference substances for the detection and quantification as described by Kassahun14 and Vermeulen.15 Briefly, isothiocyanates were dissolved in ethanol and gradually added to the corresponding reagent solution (N-acetyl-L-cysteine, L-cysteine, cysteinylglycine, or Lglutathione). The mixture was allowed to react during 24 h of stirring at room temperature. After removal of the solvents using a vacuum centrifuge (Jouan SpeedVac RC 1020, Saint Herblain, France), the structure and purity of all analytical standards were investigated by HPLC-flow-inject electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Animals and Treatments. To investigate the role of human gut bacteria in the bioactivation of GRA and NGBS in vivo, animal experiments were carried out using HMA mice in comparison to germ free mice. Animal experiments were conducted in accordance with the

institutional and national guidelines for the care and use of laboratory animals. All experimental procedures were approved by the Animal Welfare Committee of the local authority (Landesamt fü r Verbraucherschutz, Landwirtschaft and Flurneuordnung, Frankfurt/ Oder, Germany) under approval number 23-2347-4-2010. The mice were housed in isolators under controlled temperature and lighting conditions (23 °C, 12 h light/dark cycle) and had free access to irradiated food (50 kGy) and autoclaved water. Eight week old germ free mice were associated with fecal microbiota from a healthy human female donor. A fresh fecal sample was transferred to an anaerobic hood, diluted 1:10 with reduced phosphate buffer (PBS, in g/L: NaCl 8.5, KH2PO4 0.3, Na2HPO4 0.6, cysteine × HCl × H2O 0.25, Bacto peptone 0.1, and resazurine 0.001, pH 7) and the resulting slurry subsequently diluted 1:5 with glycerol and stored at −80 °C until use. For association of the germ free mice, 200 μL of the suspension was applied to each animal by gavage. The ability of the donor’s microbiota to activate GLS was not affected by this procedure as cecal and colonic contents of the recipient mice displayed similar activities (data not shown). For practical reasons, animal experiments were carried out consecutively. Inoculation with frozen material ensured the same composition and quality of inoculation material for all experiments, as it is known that the microbial composition of a human donor changes over time.16 We previously demonstrated that fecal material for inoculation can be stored after freezing and be transferred to germ free rodents by inoculation of the frozen fecal material. Counts of bacterial strains in cecal, colonic, and fecal material were comparable to those of gnotobiotic animals that received the bacteria live.17 The stability of the intestinal microbiota composition in mice was monitored throughout the study by PCR-coupled denaturing gradient gel electrophoresis as described previously.18 The microbiological status of germ free mice was confirmed as described previously.19 Throughout the study, the fecal microbiota of the HMA mice had up to 51% similarity to that of the human fecal donor sample. Similarity of 8419

DOI: 10.1021/acs.jafc.5b02948 J. Agric. Food Chem. 2015, 63, 8418−8428

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Journal of Agricultural and Food Chemistry fecal microbiota within a human individual usually varies between 65 and 88% within a certain time frame.16 Similarities did not differ significantly between time points. These data correspond to those reported in a previous long-term study with rats.20 Glucoraphanin Treatment. Male, 8-week-old germ free C3H/ HeOuJ mice (Charles River Laboratories International, Sulzfeld, Germany, n = 30) were associated with human intestinal microbiota by intragastric application of a human fecal suspension (diluted 1:50 in reduced phosphate buffer, pH 7, containing in g/L of 80% glycerol: NaCl 8.5, KH2PO4 0.3, Na2HPO4 0.6, cysteine × HCl × H2O 0.25, Bacto peptone 0.1, resazurine 0.001) or water as a control. We investigated fecal samples from several human donors for their glucosinolate-degrading activity in relation to their habitual Brassica intake. However, we did not observe any correlation between the intake of Brassica vegetables and the rate of glucosinolate hydrolysis in their fecal slurries. The animals were fed the standard diet (Altromin, Lage, Germany) for 2 weeks after association to allow bacteria to colonize the animals’ gut. Thereafter, they were switched to a GLS-free semisynthetic diet (Altromin C1000f, Lage, Germany). After 2 weeks on this diet, at 12 weeks of age, 10 mice per group received once an oral dose of glucoraphanin (172 mg/kg body weight) or glucoraphanin in combination with myrosinase (17 U/kg body weight) as a positive control or water as a negative control. The latter served to assess the absorbance by proteins in the sample. For application, glucoraphanin and myrosinase were dissolved in autoclaved ultrapure water and sterilized by membrane filtration (0.22 μm pore diameter). The GRA dose of 7.2 μmol corresponds to approximately 35 or 60% of the total daily GLS dose administered in a recent study to mice in the form of GLS-enriched broccoli or pak choi.21 The activity of myrosinase administered together with GRA was sufficient to cleave the GRA completely within 22 min in vitro (data not shown). Urine and feces were collected using metabolic cages (Tecniplast, Hohenpeißenberg, Germany) over 48 h after application. The collected samples were pooled for every 12 h. Urine samples were centrifuged for 5 min at 20.000g and 5 °C. The resulting supernatants and the feces were frozen at −80 °C until further analysis. Finally, mice were anesthetized, and blood was taken by cardiac puncture. The blood was centrifuged at 2.000g for 10 min at 4 °C, and the resulting plasma was stored at −80 °C. The gastrointestinal tract was removed and divided into three segments: cecum, small intestine, and colon. The tissue and contents from each segment were collected and immediately frozen at −80 °C until further analysis. Neoglucobrassicin Treatment. The NGBS experiment was conducted in the same way as the GRA experiment but with the following modifications: Since NGBS was not commercially available and isolation from plant material was very time-consuming, the number of animals was reduced to n = 5 per group. NGBS (297 mg/kg body weight) or water as a negative control was orally administered to HMA and germ free animals. The latter served to assess the absorbance of proteins in the sample. To identify a dose that causes detectable adduct levels in colonic and cecal tissue of mice in response to the oral administration of neoglucobrassicin, we performed preliminary experiments. We tested doses of 60, 200, and 600 μmol/kg body weight, and the latter dose was considered to be most suitable. The germ free animals received NGBS in combination with myrosinase (29 U/kg body weight) as a positive control. For HMA mice, the positive control was omitted. The activity of myrosinase administered together with NGBS was sufficient to cleave the NGBS completely within 22 min in vitro (data not shown). The mice were killed 8 h after NGBS application, and urine and feces were pooled during this period. Cecal and colonic tissues were shock-frozen in liquid nitrogen and stored at −80 °C until DNA adduct analysis. Analysis of Glucosinolates and Their Metabolites in Biological Samples. Glucoraphanin. The analysis of GRA and its potential metabolites GER, SFN, and ERU was performed as described previously.22 Briefly, each sample was analyzed twice: SFN and ERU were determined directly after derivatization with N-tBoc-Cys-ME. GRA and GER were determined indirectly after enzymatic hydrolysis to SFN and ERU, respectively. The GRA and GER contents of a

sample were calculated from the difference of the concentrations of the SFN-N-tBoc-Cys-ME derivative or the ERU-N-tBoc-Cys-ME derivative before and after myrosinase treatment. The latter represented the sum of the initial GLS and isothiocyanate content, and the former represented the initial sulforaphane or erucin content. Neoglucobrassicin. For analysis of intact NGBS and its metabolites NI3C and N-methoxyindole-3-acetonitrile, urine (30 μL) and plasma (30 μL) were diluted 1:2 (v/v) with phosphate buffer (1 M, pH 6.7) and briefly vortexed. The diluted samples were extracted three times with 300 μL of 1-butanol. After adding 1-butanol, samples were vortexed for 5 min at room temperature and centrifuged at 3.000g and 4 °C for 10 min. Feces and intestinal contents (each 30 mg) were diluted 1:10 (w/v) with phosphate buffer (0.1 M, pH 6.7), vortexed for 5 min at room temperature, and then centrifuged for 10 min at 24.652g and 4 °C. The resulting supernatants were extracted three times with 900 μL of 1-butanol, vortexed three times, and centrifuged at 3.000g at 4 °C for 10 min. The pellets were extracted three times with 300 μL of 20% MeOH in water (v/v), vortexed for 5 min at room temperature, and subsequently centrifuged at 20.000g at 4 °C for 10 min. Every supernatant obtained was evaporated until dryness using a vacuum centrifuge. The residue was suspended in 30 μL of 60% acetonitrile in water (v/v), vortexed for 5 min at room temperature, centrifuged at 20.000g and 4 °C for 5 min, and the supernatant subjected to HPLC analysis. Mercapturic Acid Conjugates in Urine. For the analysis of mercapturic acids, urine samples were centrifuged at 20.000g and 4 °C for 5 min. Supernatants were diluted with water (1:5, v/v), centrifuged again at 20.000g and 4 °C for 5 min, and analyzed immediately by HPLC/DAD. The analysis of potential mercapturic acid conjugates of NGBS was not performed due to the unavailability of reference substances. DNA-Adduct Analysis. DNA adducts are formed by reaction of nucleophilic sites of the DNA with exogenous or endogenous electrophilic metabolites. Such a highly reactive electrophile, precisely a benzylic carbocation, can be formed after NGBS hydrolysis either from the resulting short-lived isothiocyanate or from an also unstable sulfoconjugate, whose formation from NI3C is catalyzed by sulfotransferase.13 Adducts in DNA isolated from cecum and colon tissues were determined using isotope-dilution ultraperformance liquid chromatography−electrospray ionization−tandem mass spectrometry (UPLC-ESI-MS/MS) as described previously.23,24 Briefly, DNA samples (25 μg) were spiked with [15N5]N2-(1-methoxy-3-indolylmethyl)-2′-deoxyguanosine ([15N5]N2-(1-MIM)-dG) and [15N5]N6(1-methoxy-3-indolylmethyl)-2′-deoxyadenosine ([15N5]N6-(1-MIM)dA) as internal adduct standards, hydrolyzed to 2′-deoxynucleosides, and quantified by isotope-dilution multiple reaction monitoring (MRM). The use of a standard DNA isolation protocol25 resulted in considerable contamination with RNA despite RNase treatment. Therefore, dG was quantified in the DNA hydrolysate by UPLC-ESIMS/MS using [15N5]dG as an internal standard, and the actual DNA content of the samples was subsequently determined knowing that dG constitutes 21% of all nucleosides in the murine genome.26 HPLC and UPLC-ESI-MS/MS Apparatus and Conditions. Metabolites were separated and quantified with a Summit HPLC system (Dionex, Idstein, Germany) consisting of a pump (P680A LPG), an autosampler (ASI-100T), a column oven (TCC-100), and a diodearray detector (UVD 340U PDA). The HPLC was equipped with an analytical C18 LiChrospher reversed-phase column (5 μm, 4.6 × 250 mm, Merck, Darmstadt, Germany) and a C18 LiChrospher guard column (5 μm). The autosampler temperature was set at 5 °C, the flow rate was 1.0 mL/min, and the column temperature was 30 °C. Instrument control, data acquisition, and peak area analysis were done using the Chromeleon 6.40 Chromatography data system (Dionex, Idstein, Germany). GLS and their metabolites were identified by their retention times, and their UV-spectra in comparison to reference substances and calibration curves were used for their quantification. For the analysis of isothiocyanate-N-(tert-butoxycarbonyl)-L-cysteine methyl ester (ITC-N-tBoc-Cys-ME) derivatives, NGBS and its metabolites, the mobile phase consisted of potassium dihydrogen phosphate (50 mM) acidified with phosphoric acid to pH 2.3 (solvent 8420


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Figure 2. Renal excretion of glucoraphanin (GRA) and its potential metabolites glucoerucin (GER), sulforaphane (SFN), erucin (ERU), sulforaphane-N-acetyl-L-cysteine (SFN-NAC), and erucin-N-acetyl-L-cysteine (ERU-NAC) within 48 h after GRA application (172 mg/kg body weight) depicted as % recovery of applied GRA in germ free mice without (a) and with (b) myrosinase application and human microbiota-associated (HMA) mice without (c) and with (d) myrosinase application. Each point represents an experimental animal (n = 9 to 10 per group). Depicted are the mean and standard deviation. * translates into the significant difference (p < 0.05) between germ free and HMA mice. A), acetonitrile (solvent B), and ultrapure water (solvent C). ITC-NtBoc-Cys-ME derivatives were analyzed as described previously.22 For the measurement of NGBS and its metabolites, the gradient started with 100% A, changed to 3% B within 4.5 min, from 3% to 25% B within 20.5 min, and from 25% to 60% B within 20 min. The gradient was subsequently switched from 60% B to 100% C and held for 5 min. Then, the column was rinsed with 100% C for 5 min. Finally, the mobile phase returned to the initial solvent composition for 5 min for re-equilibration of the column between single runs. For the analysis of mercapturic acid conjugates, which were detected at 254 nm, the mobile phase consisted of 0.05% (v/v) trifluoroacetic acid in water (A), 0.1% (v/v) trifluoroacetic acid in acetonitrile (B) and ultrapure water (C). A linear gradient from 0% B to 20% B within 10 min, which was held for 5 min, changed to 35% B within 7.5 min and to 100% B within 5 min, which was held for 5 min. Finally, the mobile phase returned to the initial composition for 5 min for re-equilibration of the column between single runs. The analysis of the DNA adducts formed from NGBS metabolites was conducted using an Acquity UPLC system equipped with an Acquity UPLC BEH Phenyl column (1.7 μm, 2.1 × 100 mm) coupled to a Xevo TQ triple quadrupole mass spectrometer (all from Waters, Eschborn, Germany) operating in the positive ion mode (ESI+). Conditions for LC separation and MS/MS detection of the analytes have been published previously.23 Statistical Analysis. The excretion of GRA, NGBS, and their metabolites via urine, feces, and gut contents of mice was calculated for each animal as percentage recovery of the ingested GLS dose based on molar amount of GLS applied. Statistical analysis was performed using the software SPSS 20.0 (IBM, NY, USA). Values were tested for normal distribution using the Kolmogorov−Smirnov test.27 Differences were analyzed for normally distributed data on their statistical significance using the unpaired t test and for dependent measurements using the paired t test. Differences of non-normally distributed data were checked for significance using the Mann−Whitney-U-test for

unpaired data and the Wilcoxon-test for paired data. Differences in excretion between HMA and germ free mice were considered significant at p < 0.05, and a trend was defined as p < 0.1. Data are presented as mean values.

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RESULTS Glucoraphanin Is Absorbed Intact and Mainly Excreted via Urine. Plasma and Urine. Forty-eight hours after application of GRA with or without myrosinase, no GRAderived metabolites could be detected in plasma from germ free or HMA mice. Instead, approximately 30% of the applied GRA was excreted in urine in its intact form, mainly within 24 h. The application of GRA (172 mg/kg body weight, corresponding to 362 μmol/kg body weight) to germ free and HMA mice resulted within 12 h in the total renal excretion of GRA and its metabolites in amounts that corresponded to 24 (±17) and 29 (±19)% of administered dose of GRA, respectively, when no myrosinase had additionally been applied and 22 (±17) and 21 (±12)%, respectively, when myrosinase had been applied. When the observation time was extended to 48 h, these values were only slightly increased; they were 29 (±18) and 37 (±23)% in germ free and HMA mice, respectively, without myrosinase, and 43 (±17) and 30 (±12)%, respectively, with myrosinase (Figures 2a−d). Thus, GRA was absorbed intact regardless of the colonization status of the mice and whether myrosinase had been applied. In contrast to the procedure used in our study, Lai et al. administered GRA or SFN directly to the cecal lumen of anesthetized rats using a surgical procedure. In this way, these substances bypassed the small intestine where absorption of GRA preferentially takes place as may be deduced from the appearance of intact GRA in the animals’ urine within 8421


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Figure 3. Fecal excretion of glucoraphanin (GRA) and its potential metabolites glucoerucin (GER), sulforaphane (SFN), and erucin (ERU) within 48 h after its application (172 mg/kg body weight) depicted as % recovery of applied GRA in germ free mice without (a) and with (b) myrosinase application and human microbiota-associated (HMA) mice without (c) and with (d) myrosinase application. Each point represents an experimental animal (n = 9 to 10 per group). Depicted are the mean and standard deviation.

12 h after application.28 A small amount of GER was detected in all groups (the mean varying from 0.8 to 1.6% recovery within 48 h after GRA application, based on the dose of GRA applied to the animals), which suggests that GRA was reduced to GER by the animals’ metabolism. The isothiocyanates, SFN and ERU, were excreted in the urine of germ free and HMA mice. However, renal excretion was low for SFN and almost negligible for ERU. The amount of SFN recovered within 48 h after GRA application in germ free and HMA mice corresponded to 0.23 (±0.23) and 0.24 (±0.13)% of the applied GRA dose, when no myrosinase had been applied, and to 0.62 (±0.53) and 0.44 (±0.29)%, respectively, when myrosinase had been applied. Thus, GLS underwent hydrolysis to isothiocyanates in the animals irrespective of the presence or absence of myrosinase. ERU may have been formed either by the reduction of SFN or by hydrolysis of GER, the reduction product of GRA. SFN and ERU excreted in urine within the first 12 h after application of GRA were mainly present as N-acetyl-L-cysteine (NAC) conjugates, also referred to as mercapturic acids. Glutathione or cysteine conjugates were not detected. The amount of NAC-conjugates excreted by the experimental groups differed: Within 48 h after the application of GRA, germ free mice and HMA mice excreted SFN-NAC in amounts corresponding to 0.57 (±0.62) and 1.1 (±1.4)% of the initial administered GRA dose, respectively, as well as ERU-NAC in amounts corresponding to 0.20 (±0.18) and 0.76 (±0.39)% of the initial administered GRA dose. Thus, germ free animals excreted significantly less ERU-NAC than HMA mice (p < 0.05). As expected, the application of myrosinase resulted in increased amounts of SFN-NAC and ERU-NAC excreted by germ free mice and HMA mice: 7.9 (±6.7)% of the GRA dose was recovered within 48 h as SFN-NAC and 1.8 (±1.3)% as

ERU-NAC in germ free mice as well as 2.5 (±2.6)% as SFNNAC and 1.4 (±0.6)% as ERU-NAC in HMA mice (p < 0.05 for SFN-NAC). Gut Contents and Feces. Neither intact GRA nor its metabolites were detectable in small intestinal, cecal, and colonic contents of HMA mice 48 h after GRA application. However, they were present at low concentrations in small intestinal, cecal, and colonic contents of germ free animals corresponding to 0.05 (±0.03)% with myrosinase and 0.64 (±0.41)% without myrosinase of the applied GRA dose (p < 0.05). Thus, in germ free mice, myrosinase reduced the GRA content in the small intestine, cecum, and colon. In HMA mice, the absence of GRA or isothiocyanates from gut contents was unrelated to the application of myrosinase. Thus, the presence of myrosinase, whether administered orally or as a consequence of bacterial colonization, led to a reduced recovery of GRA in the gut contents. From a total of 10 animals per experimental group, SFN was only detected in the intestinal contents of two germ free animals without and one germ free animal with myrosinase administration (0.004, 0.006 and 0.024%, respectively, of the initial dose of GRA applied to the animals were recovered as SFN). Fecal excretion of GRA and its metabolites over 48 h was determined for both mouse groups (Figures 3a−d). The germ free mice excreted more intact GRA in their feces than HMA mice, namely, 4.4 (±4.2)% versus 0.12 (±0.19)% of the initial dose of GRA, p < 0.001, without myrosinase supplementation, as well as 2.3 (±3.1)% versus 0.36 (±0.63)% with myrosinase supplementation. As observed for the gut contents, myrosinase administered orally or provided by intestinal bacteria, resulted in an overall reduced recovery of GRA in the feces. GER was detected at very low levels in all groups (the mean varying from 8422


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Figure 4. Renal excretion of neoglucobrassicin (NGBS) and its potential metabolites N-methoxyindole-3-carbinol (NI3C) and N-methoxyindole-3acetonitrile (Nitrile) within 8 h after its application (297 mg/kg body weight) depicted as % recovery of applied NGBS in germ free mice without (a) and with (b) myrosinase application and in human microbiota-associated (HMA) mice (c). Each point represents an experimental animal (n = 5 per group). Depicted are the mean and standard deviation.

NGBS dose, respectively, and 38 (±9)% in the urine of HMA mice (Figures 4a−c). NI3C, a stable degradation product of NGBS, which under neutral pH conditions is formed from the respective isothiocyanate, was not detected in urine samples. NMethoxyindole-3-acetonitrile, which is formed under acidic conditions from the isothiocyanate of NGBS, was excreted in very small amounts in all experimental groups. Germfree animals with and without myrosinase application excreted the nitrile at concentrations that correspond to 0.18 (±0.16) and 0.30 (±0.37)% of the administered NGBS dose, respectively, and HMA animals to 0.46 (±0.18)%. Thus, renal excretion of N-methoxyindole-3-acetonitrile was higher in HMA mice than in germ free animals that received myrosinase (p < 0.05). Gut Contents and Feces. The recovery of NGBS in gut contents of HMA mice was very low and corresponded to only 0.01 (±0.005)% of the administered NGBS dose. The combined gut contents of germ free mice contained NGBS mainly in its intact form, no matter whether myrosinase had been applied (2.5 ± 5.5% of applied dose) or not (4.2 ± 4.5, p < 0.05, Figures 5a−c). Germfree mice contained only small amounts of NI3C in their intestines, corresponding to 0.12 (±0.05) and 0.03 (±0.04)% of the administered NGBS dose, with and without myrosinase application, respectively. However, HMA mice contained even less NI3C in their intestines, corresponding to 0.0002 (±0.0002)% of the applied NGBS dose (p < 0.05). N-Methoxyindole-3-acetonitrile contents in the cecum and colon of germ free animals were low and corresponded to 0.01 (±0.02)% of the administered NGBS dose with myrosinase application and 0.02 (±0.02)% without, while it was not detected in HMA mice. Neither NGBS nor its metabolites were detected in feces from HMA mice within 8 h after its application. Independent from the application of myrosinase, fecal excretion of NGBS by

0.007 to 0.09% recovery within 48 h after GRA application, based on the dose of GRA applied to the animals). Fecal excretion of SFN and ERU by HMA mice corresponded for both isothiocyanates together to 0.07 (±0.12) and 0.025 (±0.035)% of the initial GRA dose, with or without oral myrosinase application, respectively. In germ free mice that received myrosinase, the proportion of orally applied GRA recovered as SFN and ERU was 0.12 (±0.10)%. SFN was also detected in germ free animals, which did not receive myrosinase, but only in very low amounts, corresponding to 0.014 (±0.019)% of the initial GRA dose applied to the animals. Overall, the recovery of GRA and its metabolites in the feces 48 h after its oral administration was low, independent of bacterial colonization of the animals: in germ free and HMA mice, 4.5 (±4.4) and 0.15 (±0.22)% of the applied GRA dose, when no myrosinase had been applied, and 2.5 (±3.1) and 0.43 (±0.75)%, respectively, when myrosinase had been applied. Neoglucobrassicin Is Also Absorbed Intact and Mainly Excreted via Urine. Urine and Plasma. Eight hours after its oral application, NGBS was detected in the plasma of mice from all groups and hence, similar to GRA, was also absorbed intact. The recovery of NGBS in plasma was higher in germ free mice that did not receive myrosinase than in germ free animals that received myrosinase, namely, 0.022 (±0.015) versus 0.005 (±0.004)% of the administered NGBS dose, p < 0.05. The recovery of NGBS in the plasma of HMA mice was 0.007%. However, the detection of NGBS was overall low, and metabolites derived from NGBS, i.e., NI3C and Nmethoxyindole-3-acetonitrile, were not observed. Independent from bacterial colonization and the application of myrosinase, NGBS was excreted in urine intact. The recovery in urine of germ free animals with and without myrosinase application was 28 (±18) and 32 (±23)% of the administered 8423


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Figure 5. Recovery of neoglucobrassicin (NGBS) and its metabolites N-methoxyindole-3-carbinol (NI3C) and N-methoxyindole-3-acetonitrile (Nitrile) in gut contents of germ free mice without (a) and with (b) myrosinase application and of human microbiota-associated (HMA) mice (c) 8 h after its application (297 mg/kg body weight). Each point represents an experimental animal (n = 5 per group). Depicted are the mean and standard deviation.

Figure 6. Fecal excretion of neoglucobrassicin (NGBS) and its potential metabolites N-methoxyindole-3-carbinol (NI3C) and N-methoxyindole-3acetonitrile (Nitrile) within 8 h after its application (297 mg/kg body weight) depicted as % recovery of applied NGBS in germ free mice without (a) and with (b) myrosinase application. Data from human microbiota-associated (HMA) mice are not depicted as neither NGBS nor its metabolites were detected in their feces. Each point represents an experimental animal (n = 5 per group). Depicted are the mean and standard deviation.

DNA Adducts. The genotoxic activity of NGBS depending on the colonization status and/or the myrosinase treatment was studied by LC-MS/MS analysis of adducts in DNA isolated from murine cecum and colon 8 h after administration of NGBS. DNA adducts were detected in the cecum and colon of all NGBS-treated HMA and germ free mice: HMA, 78 (±49) and 54 (±45) DNA adducts (sum of dA- and dG-adducts) per 108 nucleotides, respectively. They were also detected in the cecum and colon of germ free mice: 441 (±625) and 333 (±547) DNA adducts per 108 nucleotides, respectively, with

germ free mice in its intact form was low and corresponded to 2.7 (±5.9)% of the administered NGBS dose with myrosinase treatment and 1.7 (±2.4)% without (Figure 6a−b). NI3C was excreted in very small quantities in the myrosinase-treated germ free animals, corresponding to 0.006 (±0.008)% of the applied NGBS dose. Germfree mice also contained very small fecal amounts of N-methoxyindole-3-acetonitrile corresponding to 0.06 (±0.12)% of the NGBS dose applied to mice with myrosinase application and 0.04 (±0.04)% without. 8424


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Journal of Agricultural and Food Chemistry myrosinase, and 55 (±51) and 33 (±26) DNA adducts per 108 nucleotides, respectively, without myrosinase. Control animals, germ free or HMA, that received the vehicle only showed no DNA adducts originating from NGBS. Thus, without oral application of myrosinase, germ free and HMA mice did not differ from each other with respect to the amount of DNA adducts formed. It may be concluded that the oral application of myrosinase to germ free mice was responsible for the increased numbers of cecal and colonic DNA adducts (p < 0.05 and p < 0.1, respectively). However, as the amount of DNA adducts formed in the cecum and colon of HMA mice was not significantly different from that of germ free mice not treated with myrosinase (Figure 7), a role of the human-derived bacteria colonizing the gut of HMA mice in NGBS activation is negligible.

the present study urinary excretion of indolic NGBS 8 h after its application was similar to that of aliphatic GRA after 12 h. This indicates a rather fast absorption, distribution, and excretion for both GLS. This is in agreement with data obtained in rats, in which 95% of the total excretion of GRA was observed in urine within 8 h after its application.30 Detection of intact GLS in plasma and urine in the present study possibly indicates that physiological effects previously reported for GLS may be at least in part exerted by GLS itself rather than by its degradation products. This has not been studied to any great extent, and the results are in part contradictory. For example, GRA induces chemoprotective phase II enzymes34 and has shown cytotoxic and genotoxic properties in vitro.35 Isothiocyanates Are Excreted in Urine Mainly as Mercapturic Acid Conjugates. After the application of GRA, SFN and ERU appeared in the urine of germ free and HMA mice. ERU is formed either by the reduction of SFN or hydrolysis of GER. The latter is formed by the reduction of GRA.36 Urinary SFN and ERU were mainly excreted as their NAC-conjugates within 12 h after GRA application. A rapid renal excretion of mercapturic acid conjugates was also reported for humans; it occurred within the first 8 h after the ingestion of broccoli sprouts.37 However, whereas in humans 40% of the applied GRA dose was detected as SFN-NAC,6 the excretion of NAC-conjugates in the present mouse study was low, unless myrosinase was given to the mice. These differences might be due to differences between animal species in the catalytic activity and specificity of xenobiotic metabolizing enzymes or to the fact that in the present study GRA was applied as a pure substance, whereas in human studies broccoli or other GLScontaining vegetables were given, in which GRA is embedded in a plant matrix.38 Studies of the metabolism of isothiocyanate in various species have shown differences in the metabolite profile. The cyclic mercaptopyruvic acid (4-hydroxy-4-carboxy3-benzylthiazolidin-2-thione) occurred in the urine from guinea-pigs and rabbits after the oral or intravenous administration of benzyl isothiocyanate or its cysteine metabolite.39 In human individuals, the isothiocyanates are mainly metabolized via the mercapturic acid pathway.40 However, to the best of our knowledge, mercaptopyruvic acid has not been observed in human intervention studies up to now, and we also did not detect it in our study. Interindividual Differences in Metabolism of the Glucosinolates. In the present study, considerable differences in the excreted amounts of GRA, NGBS, and their metabolites were observed between individual animals. High interindividual variation in excretion was also observed in human studies after the intake of GLS via Brassica vegetables.41,42 The interindividual differences in the excretion of GLS metabolites observed in the present study may be due to different times of feed intake, as the amount of feed in the stomach varies when animals are fed ad libitum, which in turn would affect transit time and thereby the metabolism of GLS.43 Impact of Intestinal Microbiota on the Bioactivation of Glucosinolates Is Low. This is the first study that examines the impact of human intestinal bacteria on the bioavailability of GLS in vivo using HMA mice and germ free animals as a control. As a positive control, myrosinase was applied orally to the mice to achieve enzymatic cleavage of GLS in the animals’ digestive tract. This is the first time that intact GLS are reported to be present in feces and gut contents after their oral application, even though in low amounts. Germ free

Figure 7. DNA adduct levels (sum of N6-(1-MIM)-dA and N2-(1MIM)-dG) in cecum and colon of germ free mice with and without myrosinase application and of human microbiota-associated (HMA) mice 8 h after application of neoglucobrassicin (297 mg/kg body weight). Each point represents an experimental animal (n = 5 per group). Depicted are the mean and standard deviation. *p < 0.05.

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DISCUSSION Glucosinolates Are Absorbed Intact and Mainly Excreted via Urine. Mice excreted approximately one-quarter to one-third of orally applied GLS, GRA, and NGBS, intact in their urine. Thus, the amount of hydrolysis products recovered was lower than expected. This finding may be of importance as potential health effects of GLS have mainly been attributed to the hydrolysis products of GLS. Intact GLS were detected not only in the urine of mice following their application but also in plasma. For example, intact NGBS appeared in the plasma of mice 8 h after NGBS had been administered confirming the absorption of intact GLS into the circulation. Until now, only a few studies have addressed the absorption of intact GLS: intact GRA was observed in plasma of dogs and rats.29 Rats were also reported to excrete intact GRA, but the proportion of GRA recovered in urine was only 5% of the administered dose.30 Following the consumption of broccoli, GRA and sinigrin were detected in human urine and plasma, respectively.31,3235,36 Studies on everted intestinal sacs of rats provided evidence for a passive transport of intact GLS from the mucosal to the serosal side of the epithelial cells in the small intestine and colon.33 The transport rate of GLS depends on their chemical structure: transport of the aromatic GLS glucotropaeolin was slightly faster than that of the aliphatic GLS sinigrin. This has been attributed to the lipophilic side chain of sinigrin.33 However, in 8425


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community had not lost this activity (data not shown). Communication between host and microbiota plays an important role in this ecosystem.47 Therefore, the bacterial metabolism of GLS may have been affected in vivo by the specific host environment. The composition of the intestinal microbiota and the catalytic activity of selected enzymes in the gut of HMA mice are largely comparable to those of the human donor. Therefore, comparing HMA mice with germ free mice is a suitable experimental approach to investigate the complex human gut microbiota in vivo.48 However, it should be kept in mind that not all human bacteria are able to establish in the mouse gut49 and that the digestive tract of humans differs from that of mice. Owing to these limitations, the results obtained in this mouse study cannot simply be extrapolated to humans without additional experiments. Nevertheless, our experiments clearly show that the intestinal microbiota plays a minor role in the bioactivation of GLS in HMA mice.

mice had higher amounts of intact GLS in their feces and gut contents than HMA mice. This difference may result from an increased hydrolysis or a higher absorption of GLS in HMA animals and supports the hypothesis that human intestinal bacteria convert GLS. However, these differences may also be the result of a shortened intestinal transit time reported for colonized animals in comparison to that of their germ free counterparts.44 The intestinal microbiota modulates the intestinal nervous system and thus stimulates the motility of the intestine45 such that GLS would stay longer in the intestinal tract of germ free animals. Only very small amounts of metabolites derived from orally applied GRA or NGBS were detected in plasma, urine, or feces. Unexpectedly, HMA and germ free mice did not differ substantially in the spectra of GLS metabolites formed and hence in their ability to hydrolyze GLS into their respective metabolites. Basal GLS activation in germ free animals was only slightly increased by oral myrosinase application or by bacterial colonization with HMA. This is in agreement with a gnotobiotic rat study, in which intestinal microbiota in contrast to plant myrosinase hardly contributed to the hydrolysis of GLS, which was adminstered as freeze-dried Brussels sprouts (mainly sinigrin).46 On the basis of these results and the outcome of our study, the impact of human intestinal bacteria on activation of GLS appears to be rather weak in vivo, at least in HMA rodents. Other factors that could possibly account for GLS hydrolysis in germ free mice were investigated and excluded: an in vitro incubation of GLS with synthetic gastric and intestinal juices and various mouse tissues did not lead to their hydrolysis (data not shown). The detection of intact GRA and NGBS in intestinal contents and feces of mice also argues in favor of a considerable stability of GLS during gastrointestinal passage. Moreover, GRA and NGBS preparations used in the animal experiments remained stable for at least 24 h of incubation at 37 °C (data not shown). A spontaneous cleavage of GLS in germ free mice is therefore unlikely. There was also no indication that the germ free mice investigated in the present study were contaminated with bacteria. Their germ free status was confirmed by Gram staining and checking fecal samples from these animals for growth (data not shown). Thus, the presented results indicate a thioglucosidase activity of the host, which is in contradiction to the literature to date. However, it cannot be excluded that the HMA animal model is not well suited for studying the bacterial hydrolysis of GLS in vivo. This conclusion may be drawn from the fact that the number of DNA adducts detected in the intestinal tissue did not differ significantly between HMA and germ free mice after the application of NGBS and overall were very low. In contrast, conventional C3H and FVB/N mice treated in the same way as the HMA and germ free C3H mice in the present study showed 5- to 8-fold higher numbers of DNA adducts in the cecum compared to the HMA mice of the present study (conventional C3H mice, 392 (±43) DNA adducts per 108 nucleotides (n = 5); conventional FVB/N mice, 616 (±283) DNA adducts per 108 nucleotides (n = 10)). This indicates that bacterial hydrolysis of NGBS was higher in conventionally colonized mice than in HMA mice.4 This difference between conventional and HMA mice indicates that the human microbiota transferred to germ free mice may have lost its ability to hydrolyze GLS following colonization. However, the fact that the fecal samples from both the human donor and the recipient mice efficiently hydrolyzed GRA and NGBS in vitro indicates that the microbial

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AUTHOR INFORMATION

Corresponding Author

*Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. Phone +49-33200-882470. Fax: +49-33200882407. E-mail: [email protected]. Present Address ∥

F.S.: Department of Nutritional Toxicology, University of Potsdam, Nuthetal, Germany. Author Contributions ⊥

J.B. and L.H. contributed equally to this work.

Funding

This study was funded by the German Federal Ministry of Education and Research (01EA1310D) and the Gottfried Wilhelm Leibniz Society (SAW 2009). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ines Gruener and Ute Lehmann for good animal care and Sabine Schmidt for excellent technical assistance. We acknowledge Dr. Renato Iori (Agricultural Research Council, Industrial Crop Research Centre, Italy) for kindly providing GRA and Dr. Albrecht Seidel (Biochemical Institute for Environmental Carcinogens, Professor Dr. Gernot GrimmerFoundation, Germany) for kindly providing NI3C and Nmethoxyindole-3-acetonitrile.

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ABBREVIATIONS USED GLS, glucosinolate(s); HMA, human microbiota-associated; GRA, glucoraphanin; SFN, sulforaphane; GER, glucoerucin; ERU, erucin; NGBS, neoglucobrassicin; NI3C, N-methoxyindole-3-carbinol; ITC-N-tBoc-Cys-ME, isothiocyanate-N-(tertbutoxycarbonyl)-L-cysteine methyl ester

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REFERENCES

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