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Carcinogenesis Advance Access originally published online on July 28, 2007
Carcinogenesis 2007 28(11):2419-2425; doi:10.1093/carcin/bgm170
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

ß-Glucuronidase in human intestinal microbiota is necessary for the colonic genotoxicity of the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in rats

Christèle Humblot1,3, Michaël Murkovic2, Lionel Rigottier-Gois1,4, Martine Bensaada1, Anthony Bouclet1, Claude Andrieux1, Jamila Anba1 and Sylvie Rabot1,*

1 UR910 Ecology and Physiology of the Digestive Tract, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas Cedex, France
2 Department of Food Chemistry and Technology, Graz University of Technology, Petersgasse 12/2, A-8010 Graz, Austria
3 Present address: UR106 Nutrition, Food, Societies, Institut de Recherche pour le Developpement, BP 64501, 911 avenue Agropolis, F-34394 Montpellier Cedex 05, France
4 Present address: UR888 Dairy Research and Applied Genetics, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas Cedex, France

* To whom correspondence should be addressed. Tel: +33 1 3465 2465; Fax: +33 1 3465 2462;Email: sylvie.rabot{at}jouy.inra.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a genotoxic/carcinogenic compound formed in meat and fish during cooking. Following absorption in the upper part of the gastrointestinal tract, IQ is mainly metabolized in the liver by xenobiotic-metabolizing enzymes. Among them, UDP-glucuronosyl transferases lead to harmless glucuronidated derivatives that are partly excreted via the bile into the digestive lumen, where they come into contact with the resident microbiota. The purpose of this study is to investigate if microbial ß-glucuronidase could contribute to IQ genotoxicity by releasing reactive intermediates from IQ glucuronides. We constructed a ß-glucuronidase-deficient isogenic mutant from a wild-type Escherichia coli strain carrying the gene uidA encoding this enzyme and compared the genotoxicity of IQ in gnotobiotic rats monoassociated with the wild-type or the mutant strain. The Comet assay performed on colonocytes and hepatocytes showed that the presence of ß-glucuronidase in the digestive lumen dramatically increased (3-fold) the genotoxicity of IQ in the colon. This deleterious effect was paralleled by slight modifications of the pharmacokinetics of IQ. The urinary and faecal excretion of the parent compound and its conjugated derivatives reached a maximum 24–48 h after gavage in rats harbouring the ß-glucuronidase-deficient strain. In rats associated with the wild-type strain, the kinetics of urinary excretion showed a biphasic curve with a second, smaller peak after 144 h. This is the first in vivo demonstration that bacterial ß-glucuronidase plays a pivotal role in the genotoxicity of a common food-borne carcinogen.

Abbreviations: ANOVA, analysis of variance; HA, heterocyclic aromatic amine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; IQ-N-glucuronide, N2-(ß-1-glucosiduronyl)-2-amino-3-methylimidazo[4,5-f]quinoline; IQ-sulphamate, N-(3-methylimidazo[4,5-f]quinolin-2-yl)sulphamic acid; IQ-5-O-glucuronide, 2-amino-5-(ß-1-glucosiduronyloxy)-3-methylimidazo[4,5-f]quinoline; IQ-5-O-sulphate, 2-amino-3-methylimidazo[4,5-f]quinolin-5-yl sulphate; p.a., peak area; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Several epidemiological studies indicate that the incidence of gastrointestinal cancer is lower in vegetarians compared with meat eaters (1). One factor that might explain this observation is heterocyclic aromatic amines (HAs). These genotoxic/carcinogenic compounds are formed during cooking of meats and evidence is accumulating that they are involved in the aetiology of human colon cancer (2). For instance, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), one of the most widespread HAs in the human diet, is a potent carcinogen in rodents and non-human primates, with liver and colon being the main target organs for tumour formation (3,4).

A few studies have highlighted the crucial impact of intestinal microbiota on the genotoxicity of HAs. Following IQ administration, DNA adducts have been observed in mice harbouring their native or a human-originating microbiota while adducts were extremely low or absent in germ-free animals (5). Similarly, the extent of IQ-induced DNA damage in colonocytes and hepatocytes, measured with the Comet assay, is 2- to 3-fold higher in human microbiota-associated rats, and 4- to 5-fold higher in conventional rats, than in germ-free counterparts (6). Hence, the presence of the intestinal microbiota seems essential for the expression of the genotoxic effects of IQ.

In contrast, studies investigating the interactions between intestinal microbiota and HAs have mainly emphasized detoxification processes. Several authors have reported that HAs can be passively detoxified by direct binding to the cell walls of bacteria (7). However, most of the studies were performed in vitro and the few in vivo experiments have focused on food-borne lactic acid bacteria but not on the colonic-resident microbiota (7). For example, the frequency of HA-induced colonic aberrant crypt foci in rats was reduced by 96% by feeding them fermented milk containing the species Bifidobacterium animalis (8). Similarly, Zsivkovits et al. (9) reported a reduction of HA-induced DNA damage in the colon of rats after gavage with Bifidobacterium longum. The ring hydroxylation of HAs has been suggested as an alternative detoxification mechanism. Indeed, 7-OH-IQ, a specific derivative of IQ produced by intestinal microbiota (10,11), is a direct-acting mutagen (12). However, in bioassays involving administration of 7-OH-IQ by long-term intrarectal infusion in adult rats or by intra-peritoneal injection followed by long-term dietary administration in newborn mice, no carcinogenic effects were observed (13).

Such data cannot account for the aggravating effect of intestinal microbiota on the genotoxicity of HAs. One possible explanation is based on the activity of ß-glucuronidase (E.C. 3.2.1.3 [EC] 1). Indeed, feeding rats the fructose polymer inulin lowers the ß-glucuronidase activity in intestinal microbiota and, in parallel, reduces IQ-induced DNA damage in colon and liver cells (14). Several other studies have highlighted the correlation between dietary protection against precancerous lesions induced by chemical carcinogens and lowering of bacterial ß-glucuronidase activity. For example, Rowland et al. (15) demonstrated that the combined administration of B.longum and inulin to rats decreased the incidence of aberrant crypt foci induced by azoxymethane by 59%; concurrently, consumption of these dietary compounds was associated with a significant decrease in the ß-glucuronidase activity of the rats’ caecal contents. Generally, high-risk diets for colorectal cancer have been shown consistently to increase ß-glucuronidase activity in intestinal microbiota relative to low-risk diets (16).

Despite this correlation, direct experimental proofs of the involvement of bacterial ß-glucuronidase in the intestinal detoxification of chemical carcinogens are still lacking. In view of this, we have undertaken to demonstrate the role of this enzyme in the genotoxicity of IQ. For this purpose, we have compared the extent of DNA damage in the colon and liver of gnotobiotic rats harbouring a ‘ß-glucuronidase+’ bacterium with those occurring in counterparts inoculated with an isogenic ß-glucuronidase-null mutant. Escherichia coli is an abundant member of the human intestinal microbiota and unlike other intestinal bacteria, genes encoding ß-glucuronidase in this species have been identified and characterized (17). Different E.coli strains described as defective in the uidA gene encoding ß-glucuronidase are reported in the literature. However, none of them are strictly isogenic: some carry a large 50 kb deletion from addA to manA including the uidA locus, whereas others have undefined mutations (18,19). Consequently, we decided to create an isogenic uidA– mutant from a wild-type uidA+ strain to ascertain that differences in the genotoxicity of IQ would be due strictly to the presence of this gene. In addition, we have analysed the excretion profile of IQ to correlate the intensity of its genotoxicity with its metabolic fate and pharmacokinetics.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Generation of a ß-glucuronidase-deficient isogenic mutant
The wild-type E.coli strain TG1 that carries an active ß-glucuronidase (20) was purchased from Biolabs (Ozyme, St Quentin-en-Yvelines, France). As the sequence of the gene locus encoding the ß-glucuronidase (uidA) was not available for TG1, we used polymerase chain reaction (PCR) to determine the similarity of its uidA sequence with those of other E.coli strains. Primers BW3 and BW4 (Table I) were designed according to the published E.coli uidA gene accession number (GB/S69414) and used to amplify the complete gene (17). First establishing that the PCR amplification yielded a DNA product of the predicted size, we next proceeded with the deletion of the uidA gene using the one-step inactivation procedure described by Datsenko et al. (19) for chromosomal genes of E.coli K-12. This method requires the strain BW25141 carrying the plasmid pKD46 and the strain DH5{alpha} carrying the plasmid pKD4, provided by Mary Berlyn (New Haven, CT). TG1 electro-competent cells were transformed with the plasmid pKD46 (bla) encoding the arabinose-inducible {lambda} Red recombinase and were selected for ampicillin resistance at 30°C. PCR amplification of the Flp recombinase target-flanked kanamycin resistance gene on pKD4 (kan) was performed with the primers BWH1 and BWH2 (Table I). The purified PCR fragment was digested with DpnI and electroporated into TG1/pKD46; transformants were selected for kanamycin resistance at 37°C. A kanamycin-resistant/ampicillin-sensitive clone was selected and replacement of the uidA gene by the kanamycin cassette was verified by PCR with primer BW4 flanking the uidA gene and primer k2 inside the kanamycin cassette (19). This strain was named TG1 {Delta} uidA::KmR.


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  		Table I. Oligonucleotide primers used for PCR amplification

 
Colonization of germ-free F344 rats
Germ-free 12- to 14-week old male F344 rats, weighing ~280 g at the start of the experiment, were obtained from our Germ-Free Rodent Breeding Facilities. Rats were randomly separated into two groups and housed in distinct Plexiglas isolators (Ingénia, Vitry-sur-Seine, France). Animals of one group were inoculated intragastrically with 1 ml of an overnight culture of the wild-type strain TG1 (Luria Bertani broth, 37°C, 150 r.p.m.); they were subsequently designated as ß-glucuronidase+ rats. Animals belonging to the other group were inoculated with 1 ml of a culture of the ß-glucuronidase-deficient strain TG1 {Delta} uidA::KmR grown under the same conditions; they were named ‘ß-glucuronidase–’ rats. Throughout the study, animals were given free access to autoclaved tap water and to a pelleted semi-synthetic diet (SAFE, Augy, France) sterilized by {gamma} irradiation at 45 kGy (IBA Mediris, Fleurus, Belgium) (21). The animal room was maintained at a temperature of 21 ± 1°C and a relative humidity of 50 ± 5% with a 12-h light–dark cycle. The experiment began after a 3-week acclimatization period.

Experimental design of in vivo studies
All procedures were carried out in accordance with the European guidelines for the care and use of laboratory animals. For the genotoxicity assays, eight ß-glucuronidase+ and eight ß-glucuronidase– rats were used. Four rats of each experimental status were orally dosed with 90 mg/kg IQ (Research Chemicals, Toronto, Canada) whereas the remaining four animals received vehicle only (corn oil). Four hours later, all rats were killed by CO2 asphyxiation and the liver and colon were collected for the Comet assay. Liver and colon cells were isolated as described by Bradley et al. (22) and Brendler et al. (23), respectively. Caecal contents were collected for spreading onto Luria Bertani agar plates to enumerate living bacterial cells and for assaying ß-glucuronidase activity. Before enzyme analysis, bacterial cells were separated from the other components of the caecal content using a Nycodenz® cushion (24). Briefly, caecal contents were diluted in PBS (K2HPO4 0.1 M, NaCl 0.15 M, pH 7.5) and the suspensions were introduced into Nycodenz® (d = 1.3)-containing vials. After ultracentrifugation in a swinging rotor (10 000g, 40 min, 4°C), bacterial cells were removed from the interface, washed with PBS and frozen without liquid at –80°C until the time of analysis. For the follow-up of IQ metabolic fate, six ß-glucuronidase+ and six ß-glucuronidase– rats were individually housed in metabolism cages within the isolators. One day prior to IQ treatment, excreta collection receptacles were cleaned. Urine and faeces were subsequently collected at 0, 24, 48, 72, 96, 120, 144 and 168 h after the gavage with IQ (90 mg/kg). To avoid bacterial degradation of parent IQ and metabolites excreted in urine, ampicillin (100 µl of a 25 mg/ml solution) was added to the urine collection receptacles at each time point. Urine and faecal samples were frozen at –20°C pending analyses. At the end of experiment, animals were killed by CO2 asphyxiation and caecal contents were collected and treated as described above for bacterial cell enumeration and for ß-glucuronidase assay.

Assay of ß-glucuronidase activity and enumeration of active cells
We measured the ß-glucuronidase activity and the proportion of active cells in the cultures of strains TG1 and TG1 {Delta} uidA::KmR used for the rat inoculation (Luria Bertani broth, overnight, 37°C, 150 r.p.m.) and in the caecal bacteria isolated from the gnotobiotic animals. Cells were broken open with glass beads and ß-glucuronidase activity was determined spectrophotometrically ({lambda} = 400 nm) by measuring the rate of release of p-nitrophenol from the p-nitrophenylglucuronide (25). Protein concentration was determined according to the method of Lowry et al. (26). The proportion of active cells was determined by fluorescent in situ hybridization. After washing twice with PBS (130 mM NaCl, 3 mM NaH2PO4, 7 mM Na2HPO4, pH 7.2), pellets from 1 ml aliquots of the overnight cultures were suspended in 600 µl PBS; caecal bacteria were gently thawed and similarly suspended in 600 µl PBS. Three hundred microlitres of each suspension was added with 5-dodecanoylaminofluorescein di-ß-D-glucuronide (C12FDGlcU, Molecular Probes, Eugene, OR) as a substrate (final concentration 100 µM) and incubated overnight at 37°C in the dark; the remaining suspension was used as a negative control. After incubation, the bacterial suspensions were washed with PBS to remove excess substrate and subsequently analysed by flow cytometry as described by Rigottier-Gois et al. (27). Data acquisition was performed with a FACSCalibur flow cytometer (Becton Dickinson France, Le Pont-de-Claix, France) equipped with an air-cooled argon ion laser providing 15 mW at 488 nm. All parameters, i.e. the forward-angle light scatter (in the 488 nm band pass filter), the side-angle light scatter (in the 488 nm band pass) and the green fluorescence intensity conferred by the labelling with C12FDGlcU substrate (FL1, in the 530 nm band pass filter), were collected as logarithmic signals.

Determination of IQ genotoxicity by the Comet assay
Trevigen® slides were used and PBS, alkali (electrolysis) buffer, lysis solution, electrophoresis solution and SYBR® Green stain (Roche Diagnostics, Mannheim, Germany) were prepared following the manufacturer’s recommendations (Interchim, Montluçon, France). Microgel electrophoresis was subsequently performed according to Singh et al. (28). Briefly, 1 x 104 cells suspended in 90 µl of 0.5% low melting point agarose were transferred to the slides. After allowing the agarose to solidify by placing the slides on a cooled metal plate for 2 min, the slides were submersed into lysis solution for 24 h. Subsequent to alkali treatment (pH 13.00, 20 min) and electrophoresis (300 mA, 25 mV, 20 min; Biometra Standard Power Pack P25), the slides were removed from the electrophoresis chamber, washed and stained with SYBR® Green. Analysis of DNA damage was made by measuring the comet tail lengths of the indicator cells with a fluorescence microscope (Olympus BX40, 125-fold magnification) connected to a monitor, using a specific macro from the NIH-public domain image analysis program (29). Three slides were prepared from each organ, and 50 cells per slide were analysed (total 150 cells per organ) (30).

Determination of IQ and its metabolites in the faeces and urine
Before chromatographic analysis, urine samples were clarified by centrifugation (8000g, 10 min) and faeces were freeze-dried, weighed and suspended in the High performance liquid chromatography (HPLC) buffer (0.02 M K2HPO4, pH 4.5). Urine and faeces were extracted with Blue rayon trisulfonated (Sigma–Aldrich, Saint-Quentin-Fallavier, France) using the protocol of Bashir et al. (10), except that the residue was re-suspended in 0.02 M K2HPO4, pH 4.5. Using these conditions, the average yield of extraction was 36%. Conjugated metabolites were revealed by submitting half of each extract to hydrolysis with 4800 U/ml of ß-glucuronidase from Helix pomatia containing 50–205 U aryl sulfatase (Sigma–Aldrich) (31). The enzymatic reaction was performed overnight at 37°C and stopped by the addition of acetonitrile. Crude and deconjugated extracts were analysed by diode-array detector–HPLC using the method described by Rafter et al. (32) with slight modifications. Briefly, extracts were injected to HPLC with an autosampler 2690 (Waters, Milford, MA) onto a reversed-phase column packed with LiChrospher® 100 RP-18e (5 µm, 250 mm) (Merck, Paris, France), equipped with a pre-column LiChrospher® 100 RP-18e (5 µm, Merck). The separation was performed at a flow rate of 1.0 ml/min with a 0–30% acetonitrile linear gradient in 0.02 M K2HPO4, pH 4.5 for 20 min. IQ was monitored at 252 nm and eluted at 10.6 min; quantification was determined by using a standard curve from 0 to 500 µM. Production of IQ-conjugated metabolites was estimated by measuring their peak areas since standards were not available. Samples exhibiting the greatest peaks of IQ conjugates were further analysed by mass spectrometry–HPLC for structural assignment as described by Messner et al. (33). Briefly, the HPLC analyses were performed on a Hewlett Packard HP 1100 MSD (Waldborn, Germany) using a reversed-phase material (Semi Micro ODS-80 TS column, 5 µm, 250 x 2 mm I.D.) from Tosoh Bioscience GmbH (Stuttgart, Germany) as an analytical column. The separation was performed at a flow rate of 0.3 ml/min by gradient elution with methanol/acetonitrile/water/acetic acid (8/14/76/2, vol/vol/vol/vol) at pH 5.0 (adjusted with ammonium hydroxide 25%) as solvent A and acetonitrile as solvent B. The gradient programme was as follows: 0% B, 0–12 min; 0–30% B, 12–20 min; 30% B, 20–35 min and the injection volume 10 µl. The mass selective detector was equipped with an atmospheric pressure ionization electrospray using a fragmentation voltage of 80 V for negative ionization of IQ-conjugated metabolites. Drying nitrogen was heated to 350°C and the drying gas flow was 10 l/min. The data were acquired in the selected ion mode [m/z 373 for N2-(ß-1-glucosiduronyl)-2-amino-3-methylimidazo[4,5-f]quinoline (IQ-N-glucuronide), 277 for N-(3-methylimidazo[4,5-f]quinolin-2-yl)sulphamic acid (IQ-sulphamate), 389 for 2-amino-5-(ß-1-glucosiduronyloxy)-3-methylimidazo[4,5-f]quinoline (IQ-5-O-glucuronide) and 293 for 2-amino-3-methylimidazo[4,5-f]quinolin-5-yl sulphate (IQ-5-O-sulphate)].

Statistical methods
Results are reported as means ± standard errors. The effect of the bacterial status on the bacterial concentration in rats’ caeca, and on the ß-glucuronidase activity and proportion of active cells in batch cultures and in rats’ caeca, was analysed using a Student’s t-test. Differences between ß-glucuronidase+ and ß-glucuronidase– rats with regard to IQ genotoxicity were assessed using a one-way analysis of variance (ANOVA); when ANOVA indicated significant differences, groups were compared in pairs with the Student–Newman–Keuls multiple comparison test. The urinary and faecal excretion of native and conjugated forms of IQ was analysed using ANOVA for repeated measurements. Terms analysed as treatments in the ANOVA model were bacterial status and bacterial status x excretion kinetics. The former term represents the effect of bacterial status on the total excretion of each form of IQ and the latter term represents the effect of bacterial status on the excretion kinetics. They were analysed in the individual stratum and in the individual x kinetics stratum, respectively. The level of significance was set at P < 0.05. Calculations were performed using the Statview® software (version 5.0, SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The caecal bacterial concentration was high and similar in both groups of rats, i.e. (2.6 ± 0.6) x 108 and (6.2 ± 2.0) x 108 cfu/g for ß-glucuronidase+ and ß-glucuronidase– animals (n = 10), respectively.

ß-Glucuronidase activity and proportion of active cells in bacterial cultures and caecal contents
In batch cultures, the ß-glucuronidase activity was 85.1 ± 5.8 nmol/min/mg protein for the wild-type strain, TG1, and 0 for the ß-glucuronidase-defective mutant, TG1 {Delta} uidA::KmR (n = 3). The activity of bacterial cells isolated from the caecal contents was 51.7 ± 10.5 nmol/min/mg protein in ß-glucuronidase+ rats versus 0 in ß-glucuronidase– rats (n = 10). Typical flow cytometry histograms from the measurement of the proportion of active cells are presented in Figure 1. When the ß-glucuronidase-defective strain TG1 {Delta} uidA::KmR was incubated with the fluorogenic substrate C12FDGlcU, autofluorescent level ranged from 0 to 102. These data were used to define the threshold. When cells of the wild-type TG1 strain were incubated with the substrate, we observed a shift of at least 1 log unit, which accounted for a specific metabolic labelling with the fluorogenic substrate, allowing us to conclude that these cells had a ß-glucuronidase activity. In pure cultures (n = 3), the proportion of fluorescent cells was 69.6 ± 3.4% with the wild-type TG1 strain and 3.4 ± 0.2% with the ß-glucuronidase-defective isogenic mutant. In caecal contents (n = 3), the proportion of fluorescent cells was 70.4 ± 0.8% with ß-glucuronidase+ rats and 1.1 ± 0.2% with ß-glucuronidase– rats. Overall, the proportion of active cells was significantly higher with the wild-type ß-glucuronidase+ TG1 strain than with the ß-glucuronidase-defective mutant (P = 0.027 for pure cultures and P = 0.0001 for rats’ caecal contents).


Figure 1
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  		Fig. 1. Example of flow cytometry histograms of bacterial populations isolated from the caecum of rats monoassociated with the wild-type ß-glucuronidase+ TG1 strain or with its ß-glucuronidase-deficient isogenic mutant. When the ß-glucuronidase– cell suspensions were incubated with the fluorogenic substrate C12FDGlcU, autofluorescence levels ranged from 0 to 102, and the region M1 was defined. When C12FDGlcU was added to the suspensions of ß-glucuronidase+ bacterial cells, a shift of fluorescence to higher intensities was observed and the area in the M1 region could be integrated. This area represents the number of cells that were specifically metabolically labelled with the fluorogenic substrate. For additional details see Materials and methods.

 
Effect of ß-glucuronidase on the genotoxicity of IQ in liver and colon
Results of the Comet assay in colonocytes and hepatocytes are shown in Figure 2. In colon cells (Figure 2A), the average tail length of the comets ranged from 17 to 21 µm in untreated rats, regardless of whether the animals harboured the ß-glucuronidase– or ß-glucuronidase+ strain. IQ treatment did not cause any significant DNA damage in ß-glucuronidase– rats. In contrast, IQ treatment dramatically increased the tail length of the comets in ß-glucuronidase+ rats, reaching 60 µm in length (P < 0.05). No significant increase in comet tail length was observed in hepatocytes from IQ-treated rats (Figure 2B) compared with the background level observed in untreated animals (comet tail length ~10 µm), regardless of the bacterial status.


Figure 2
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  		Fig. 2. Induction of DNA damage in colonocytes (A) and hepatocytes (B) of rats monoassociated with the wild-type ß-glucuronidase+ TG1 strain or with its ß-glucuronidase-deficient isogenic mutant. Animals were killed 4 h after IQ or vehicle treatment and cells isolated from liver and colon were submitted to the Comet assay. Bars indicate means + SEMs of comet tail lengths measured with four animals per treatment group (three slides per organ, 50 cells per slides). *significant increase compared with each other group (P < 0.05). For additional details see Materials and methods.

 
Effect of ß-glucuronidase on the excretion of IQ and metabolites in faeces and urine
Several small peaks unrelated to IQ metabolism were detected in urine prior to IQ treatment. Following gavage with IQ, diode-array detector–HPLC analysis revealed the presence of seven new peaks in all animals, regardless of the bacterial status. One of these peaks was identified as native IQ, based on the identity of its retention time (10.6 min) and ultraviolet spectrum with those of the authentic standard. The four compounds that eluted at 7.0, 7.4, 8.9 and 10.1 min were affected by the hydrolysis treatment with ß-glucuronidase/aryl sulphatase; we focused on these compounds since they were very likely to be IQ conjugates. As shown in Figure 3, mass spectrometry–HPLC analysis enabled us to identify the compounds eluting at 7.0, 7.4, 8.9 and 10.1 min as IQ-5-O-glucuronide (m/z 389), IQ-5-O-sulphate (m/z 293), IQ-sulphamate (m/z 277) and IQ-N-glucuronide (m/z 373), respectively. On the chromatograms of faecal extracts, no background peak could be observed prior to IQ treatment. Following gavage with IQ, diode-array detector–HPLC analysis revealed the presence of three new peaks in all animals. One of these was IQ and another, which eluted at 7.0 min, was sensitive to the ß-glucuronidase/aryl sulphatase treatment. We could infer from mass spectrometry–HPLC analysis that this compound was IQ-5-O-glucuronide (m/z 389).


Figure 3
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  		Fig. 3. Structure of IQ and metabolic routes leading to the four IQ conjugates found in the urine of rats [adapted from Snyderwine et al. (34) and Kestell et al. (35)].

 
The total excretion of native IQ was similar in ß-glucuronidase+ and ß-glucuronidase– rats: 678 ± 152 nmol (1.8% of the dosage) and 314 ± 94 nmol (1.0% of the dosage) in urine (P = 0.07) and 440 ± 79 nmol (1.2% of the dosage) and 609 ± 61 nmol (1.6% of the dosage) in faeces (P = 0.12). The excretion kinetics are represented in Figure 4. In the faeces, they were similar in both groups of rats (P = 0.32), with most of the excretion occurring within 48 h. In the urine, IQ excretion followed a biphasic curve, with a maximum at 24 h and a second, smaller peak at 144 h. This pattern tended to be more pronounced in ß-glucuronidase+ rats (P = 0.04).


Figure 4
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  		Fig. 4. Excretion kinetics of IQ in urine (A) and faeces (B) of rats monoassociated with the wild-type ß-glucuronidase+ TG1 strain or with its ß-glucuronidase-deficient isogenic mutant. Animals were individually housed in metabolism cages and gavaged once with IQ (90 mg/kg). Urine and faeces were collected prior to IQ treatment (time 0) and at intervals thereafter. Data squares and bars are means and SEM of six rats. The box indicates the P values of the comparisons between ß-glucuronidase+ and ß-glucuronidase– rats (total excretion and pattern of kinetics excretion). For additional details see Materials and methods and Results.

 
ß-Glucuronidase+ and ß-glucuronidase– rats excreted the same quantities of IQ glucuronoconjugates: 0.43 ± 0.23 and 0.13 ± 0.05 peak area (p.a.) units for IQ-N-glucuronide (P = 0.22), which was found only in urine; 4.3 ± 1.5 and 1.9 ± 0.7 p.a. units for IQ-5-O-glucuronide present in urine (P = 0.16), and much greater quantities, namely (5 ± 1) x 105 and (3 ± 1) x 105 p.a. units, for IQ-5-O-glucuronide (P = 0.13) excreted in faeces. In the urine of ß-glucuronidase– rats, excretion of N- and O-glucuronoconjugates was completed within 48 h (Figure 5A and B). On the other hand, in the ß-glucuronidase+ group, a biphasic curve was observed with a second peak at 144 h; however, as this phenomenon occurred only in two out of the six rats, the patterns of the excretion kinetics were not significantly different between ß-glucuronidase+ and ß-glucuronidase– rats: P = 0.13 and 0.08 for IQ-N-glucuronide and IQ-5-O-glucuronide, respectively. In the faeces, the major amount of IQ-5-O-glucuronide was excreted within 72 h, similarly for both groups of rats (Figure 5C, P = 0.13).


Figure 5
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  		Fig. 5. Excretion kinetics of IQ glucuronoconjugates recovered in urine (A: IQ-N-glucuronide, B: IQ-5-O-glucuronide) and faeces (C: IQ-5-O-glucuronide) of rats monoassociated with the wild-type ß-glucuronidase+ TG1 strain or with its ß-glucuronidase-deficient isogenic mutant. Same legend as Figure 4.

 
As with native IQ and glucuronoconjugates, the bacterial status had no significant effect on the total excretion of sulphoconjugates: 96.3 ± 26.9 and 40.6 ± 10.6 p.a. units for IQ-sulphamate (P = 0.08) and 33.5 ± 6.3 and 17.4 ± 5.0 p.a. units for IQ-5-O-sulphate (P = 0.07) in ß-glucuronidase+ and ß-glucuronidase– rats, respectively. Both sulphoconjugates followed a biphasic excretion kinetics, with most of the excretion occurring ~24 h and a second, smaller peak at 144 h (Figure 6). This biphasic pattern was significantly more marked in ß-glucuronidase+ rats: P = 0.01 and 0.005 for N- and O-sulphoconjugates, respectively.


Figure 6
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  		Fig. 6. Excretion kinetics of IQ sulphoconjugates (A: IQ-sulphamate, B: IQ-5-O-sulphate) recovered in urine of rats monoassociated with the wild-type ß-glucuronidase+ TG1 strain or with its ß-glucuronidase-deficient isogenic mutant. Same legend as Figure 4.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
A high colonization density was achieved in the caecum of gnotobiotic rats, regardless of their association with the wild-type TG1 strain or the TG1 {Delta} uidA::KmR mutant. This density was consistent with the population level typically reported for this species in the human digestive ecosystem (36), though the strain TG1 does not originate from this environment. As expected, disruption of the uidA gene abolished the expression of ß-glucuronidase in pure cultures. In the caecal lumen, we found that the wild-type strain expressed the ß-glucuronidase activity whereas the mutant was consistently inactive. Thus, by colonizing germ-free rats with E.coli strains that either carry the ß-glucuronidase activity or lack it, we have been able to generate a simplified ecosystem model that allows for the specific investigation of the role of this enzyme in the metabolic fate and bioactivity of a potent carcinogen.

Assessment of the effect of ß-glucuronidase on the genotoxicity of IQ in liver and colon revealed that, in the absence of IQ, the average comet tail length was basal, corroborating data reported from previous experiments with germ-free, conventional or human microbiota-associated rats (6,14,30). Following dosing with IQ, only those rats that harboured the ß-glucuronidase+ strain exhibited an increased tail length of up to 60 µm in colonocytes. The degree of damage was of the same order as the one observed in F344 conventional and human microbiota-associated rats receiving the same dose of IQ, i.e. 90 mg/kg (6,14,30). These results clearly indicate that bacterial ß-glucuronidase plays a pivotal role in the ability of IQ to induce DNA damage in colonocytes. Moreover, they are consistent with observations made by others suggesting that IQ cannot induce DNA damage in the colonocytes of germ-free rodents (5,6) and help to elucidate the chemoprotective effect of dietary compounds capable of lowering ß-glucuronidase activity in the colon (14).

The ability of ß-glucuronidase to enhance IQ genotoxicity was paralleled by slight modifications of the pharmacokinetics of IQ. About 3% of ingested IQ was recovered in its native state in rats’ excreta, regardless of the bacterial status, suggesting that IQ was extensively metabolized and/or stored in body tissues. These findings are consistent with those of Embola et al. (37,38), who found that the urinary excretion of IQ in rats did not exceed 3% of a single dose (40 mg/kg body wt) administered by gavage. The four IQ conjugates that we identified in rats’ excreta, namely IQ-N-glucuronide, IQ-5-O-glucuronide, IQ-sulphamate and IQ-5-O-sulphate, have been regularly reported in the literature (38–40). In the present experiment, all were found in urine and one of them, IQ-5-O-glucuronide, was also found in faeces. Furthermore, whether the rats were associated with the wild-type E.coli strain or with the ß-glucuronidase-deficient mutant, faeces were, by far, the main route of excretion for this metabolite. This finding contrasts with results reported by Luks et al. (40) who did not detect IQ conjugates in rats’ faeces, using experimental conditions close to ours (same rat strain, oral administration of a dose of 40 mg/kg body wt of IQ); as the conjugates were present in bile, they concluded that they were split by hydrolytic enzymes in the intestinal microbiota. Therefore, while it makes sense that ß-glucuronidase– rats excreted IQ-5-O-glucuronide in faeces, recovering similar amounts of this compound in the faeces of ß-glucuronidase+ companions was unexpected. An insufficient level of ß-glucuronidase able to hydrolyse IQ-5-O-glucuronide extensively may be responsible for this apparent inconsistency. Indeed, the bacterial concentration achieved in the intestinal lumen of the gnotobiotic rats used in the present study was 100-fold lower than that occurring in conventional rats. Furthermore, only the ß-glucuronidase isoform encoded by the E.coli strain TG1 was present, whereas a high diversity of ß-glucuronidase isoforms, some of which may have a greater affinity for IQ-5-O-glucuronide, are likely to be present in a multicomponent microbiota (41). Nevertheless, the absence of marked differences in the total excretion and day by day excretion kinetics of IQ and IQ glucuronoconjugates of ß-glucuronidase+ versus ß-glucuronidase– rats does not preclude more subtle changes in the dynamics of IQ metabolism, likely to influence its genotoxicity. Indeed, one can reasonably hypothesize that, in ß-glucuronidase+ rats, the flux of IQ-5-O-glucuronide excreted into the intestinal lumen via the bile was partially hydrolysed to reactive intermediates capable of causing genetic damage to the colonocytes and re-entered the enterohepatic circulation to undergo further cycles of conjugation and excretion. Depending on its rate, we may have been unable to reveal this turnover since we collected faeces and urine at set 24-h intervals. Measurement of the flux of IQ and metabolites in the enterohepatic circulation would help to confirm this hypothesis. A microbial hydrolysis of IQ-5-O-glucuronide could also explain why bacterial status affected the excretion kinetics of IQ sulphoconjugates. Turesky et al. (39) have shown that sulphoconjugation is a major route for IQ metabolism in conventional rats. Therefore, IQ and intermediates released from IQ-5-O-glucuronide in the intestinal lumen of ß-glucuronidase+ rats could be further conjugated by sulphotransferases. On the opposite, IQ-5-O-glucuronide excreted in the intestinal lumen of ß-glucuronidase– rats is eliminated from the body, with no recycling likely to lead to other conjugated forms.

Whether the rats were associated with the wild-type TG1 strain or with the ß-glucuronidase-defective mutant, IQ did not induce any DNA damage in the liver. A previous experiment by Hirayama et al. (5) reported an absence of DNA adducts in the liver of germ-free mice treated with IQ and Kassie et al. (6), using the Comet assay, found a low-level genotoxic effect of IQ in germ-free F344 rats, compared with animals harbouring their native or a human intestinal microbiota. In this regard, the gnotobiotic used in the present study is functionally similar to the germ-free paradigm, suggesting that other bacterial functions are required to trigger the genotoxic effect of IQ in the liver. In particular, a down-regulation of hepatic transferases by intestinal microbiota could decrease IQ detoxification, thus supporting the higher level of IQ genotoxicity in the liver of conventional rodents, in contrast to germ-free animals. The gnotobiotic rats used in the present study were colonized by a single bacterial strain, at a 100-fold lower level than the average bacterial concentration achieved in the large intestine of rats harbouring a multicomponent microbiota (36); therefore, they could have behaved as germ-free animals with regard to a putative regulation of xenobiotic metabolism in the liver. Hooper et al. (42) have shown that the intestinal microbiota regulates the transcription of genes encoding glutathione-S-transferase and multidrug resistance protein-1a in the ileal mucosa of mice. Although it is easier to conceive an influence of the intestinal microbiota on neighbouring enterocytes than on distant hepatocytes, the chemical connections between the intestinal lumen and the liver, through enterohepatic circulation, make this influence possible. Overall, the contrasting results, observed in the colon and liver of the ß-glucuronidase+ rats, highlight the complexity of the role of intestinal microbiota; this one could act directly by generating harmful metabolites and indirectly by regulating the expression of mammalian signals involved in the detoxification of IQ.

One might speculate whether the central role of ß-glucuronidase in the colonic genotoxicity of IQ may apply to other HAs, since this family includes molecules with very diverse chemical structures. Ligation of the biliary duct in rat does not alter the genotoxic effect of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (43), suggesting that the involvement of bacterial ß-glucuronidase in the metabolic fate of PhIP has no influence on its bioactivity. Hirayama et al. (5) investigated the effect of human intestinal microbiota on DNA adducts induced by 2-amino-9H-pyrido[2,3-b]indole and found a higher level of damage in germ-free mice than in mice with microbiota. Therefore, the impact of ß-glucuronidase would vary depending on the animals’ exposure to different chemicals. This could arise from different susceptibilities of HA glucuronoconjugates to ß-glucuronidase hydrolysis. For example, Styczynski et al. (44) showed that PhIP-glucuronide originating from conjugation by human enzymes was a substrate for bacterial ß-glucuronidase, whereas PhIP-glucuronide from rabbit did not undergo ß-glucuronidase-catalysed hydrolysis. The authors relate this difference to the different sites of glucuronidation targeted by the two mammalian species.

In summary, the present study provides evidence that bacterial ß-glucuronidase can play a pivotal role in the genotoxicity of a food-borne carcinogen commonly occurring in a Western-type diet. These findings provide a rationale for dietary manipulations aimed at reducing the risk of colorectal cancer. In particular, they shed light on a mechanism whereby oligosaccharides, which are regularly reported to lower ß-glucuronidase (45), can protect against chemically induced DNA damage and precancerous lesions in the colon of laboratory rodents (14,15,46).


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
European Community under the Research and Technological Development programs Quality of Life and Management of Living Resources ‘Heterocyclic Amines in Cooked Foods—Role in Human Health’ ‘(QLK1-CT99-01197) and Microbe Diagnostics (QLK1-2000-108)’; PhD grant from the French Ministry in charge of Education and Research to C.H.


   Acknowledgments
 
The authors would like to thank Mary Berlyn for the strains BW25141 (pkD46) and DH5{alpha} (pkD4), Catherine Philippe for helpful advice with diode-array detector–HPLC analyses, Rosa Durao for breeding the germ-free rats and Gérard Corthier, Joël Doré, Maria-José Flores-Sanabria, Philippe Langella and Marja-Liisa Väisänen for critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received March 10, 2007; revised July 19, 2007; accepted July 20, 2007.


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