Effects of human intestinal flora on mutagenicity of and DNA adduct formation from food and environmental mutagens
Kazuhiro Hirayama1,5,6,
Pawel Baranczewski2,
Jan-Eric Åkerlund3,
Tore Midtvedt4,
Lennart Möller2 and
Joseph Rafter1
1 Departments of Medical Nutrition and
2 Biosciences, Karolinska Institutet, NOVUM, S-141 86 Huddinge,
3 Department of Surgery, Huddinge Hospital, S-141 86 Huddinge and
4 Department of Cell and Molecular Biology, Laboratory of Medical Microbial Ecology, Karolinska Institutet, S-171 77 Stockholm, Sweden
 |
Abstract
|
---|
Although the intestinal flora is believed to have a critical role in carcinogenesis, little is known about the role of the human intestinal flora on the effects of mutagens in vivo. The aim of the present study was to address a possible role of the human intestinal flora in carcinogenesis, by exploiting human-flora-associated (HFA) mice. The capacity of human faeces to activate or inactivate 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) and 2-nitrofluorene was determined using the Ames assay. Human faecal suspensions that were active in this regard were then selected and orally inoculated into germfree NMRI mice to generate HFA mice. HFA, germfree, conventionalized and conventional mice were administered IQ, 2-amino-9H-pyrido[2,3-b]indole (2-amino-
-carboline; AAC) and 2-nitrofluorene. The activity of human intestinal flora against mutagens could be transferred into the mice. In comparing germfree mice and mice harbouring an intestinal flora, the presence of a flora was essential for the activities of faeces against mutagens. After administration of IQ and 2-nitrofluorene, DNA adducts were observed in the mice with a flora, while adducts were extremely low or absent in germfree animals. DNA adducts after AAC treatment were higher in germfree mice in some tissues including colon than in mice with bacteria. Differences in DNA adduct formation were also observed between HFA mice and mice with mouse flora in many tissues. These results clearly indicate that the intestinal flora have an active role in DNA adduct formation and that the role is different for the different chemicals to which the animals are exposed. The results also demonstrate that the human intestinal flora have different effects from the mouse flora on DNA adduct formation as well as in vitro metabolic activities against mutagens. Studies using HFA mice could thus provide much-needed information on the role of the human intestinal flora on carcinogenesis in vivo.
Abbreviations: CV, conventional; GF, germfree; HFA, human-flora-associated; IQ, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline; AAC, 2-amino-9H-pyrido[2,3-b]indole (2-amino-
-carboline); NF, 2-nitrofluorene.
 |
Introduction
|
---|
It is now relatively well established that diet is an important aetiological factor in human carcinogenesis. For example, microbial mutation assays have shown that there are mutagenic/carcinogenic substances in cooked food; a series of heterocyclic amines has been isolated and identified as among the most potent of these mutagens (1,2). Mutagenic substances are also found in the environment, e.g. nitrated polycyclic hydrocarbons formed due to incomplete combustion of fossil fuels (35).
Epidemiological studies and risk estimates suggest that the intestinal flora increases the risk of cancer (6,7). A contribution of the intestinal bacteria to the metabolism of mutagenic compounds has also been reported (8,9). Comparison between germfree (GF) and conventional (CV) animals has demonstrated that the presence of the intestinal flora is associated with increased formation of colon tumours (10,11) and excretion of urinary mutagenic metabolites after carcinogen treatment (12,13). Intestinal flora may also contribute to carcinogenesis by cleaving conjugated mutagenic substances, which have been inactivated and excreted in bile, thus facilitating the enterohepatic recirculation of the mutagen (1416). On the other hand, it has also been reported that intestinal bacteria can bind mutagenic compounds in vitro and the binding correlates well with the reduction in mutagenicity observed after exposure to the bacterial strains (17,18).
Most of the work in this field has been performed using rodent models. There is surprisingly little information in the literature regarding the role of the human intestinal flora on the effects of dietary and environmental mutagens in vivo. This is largely due to the difficulties of studying the effects of the human intestinal flora, e.g. difficulties in controlling the environmental and dietary conditions of humans, and ethical issues associated with studies on the effects of carcinogenic or toxic substances with human volunteers. In terms of composition and metabolic activity, the intestinal flora of experimental animals is significantly different from that of humans (19), making extrapolation from results from animal studies to the human system questionable. In the present study, human-flora-associated (HFA) mice, whose intestinal flora are of human origin, were used as the model system. This in vivo model maintains the composition and metabolic characteristics of the human intestinal flora (1921) in conditions similar to those found in humans and, therefore, provides numerous advantages over the use of conventional (CV) experimental animals.
The aim of the present study was to address a possible role of the human intestinal flora in carcinogenesis, by exploiting HFA mice. The capacity of human faecal suspensions to activate or inactivate mutagens of dietary and environmental origin was determined and human faecal suspensions that were active in this regard were then inoculated into GF mice to generate HFA mice. The metabolic activities of faeces were compared among four groups of mice with different intestinal bacterial conditions, HFA, germfree (GF), conventionalized (ex-GF) and CV mice. These mice were then administered three mutagens, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (2-amino-
-carboline; AAC) and 2-nitrofluorene (NF), which are suspected to be cancer initiators, to determine the effects of flora of different origin in vivo. IQ, AAC and NF were chosen as models for human exposure to mutagens/carcinogens. IQ and AAC, both heterocyclic amines, are detected at significant levels in cooked foods and in cigarette smoke (2225). NF is a model compound for nitrated polycyclic hydrocarbons. DNA adduct formation was used as an in vivo biomarker of cancer risk in the present study. DNA adduct formation is regarded as a critical event in the development of cancer and the measurement of DNA adduct levels may be useful as a biomarker for early detection of cancer risk (2628).
 |
Materials and methods
|
---|
Sampling of human faeces
Human faeces were obtained from eight healthy adult male volunteers aged 2744 years. Stools were passed directly into a plastic bag, flushed with nitrogen, mixed and weighed. The samples were then placed in an anaerobic chamber for analysis of their metabolic activities against mutagens. Three samples were obtained from each volunteer, at
1 week intervals, resulting in 24 samples.
Animals
GF and CV NMRI mice were bred and maintained at The Laboratory of Medical Microbial Ecology, Karolinska Institutet. GF mice were raised using the methods described by Gustafsson (29) and kept in isolators. All animals were kept in cages with wood shavings and fed an autoclaved pelleted diet (R-36; Lactamin, Stockholm, Sweden) and water ad libitum. Five male and 10 female 812 week old mice were used in the preliminary experiment to generate HFA mice. Twelve female 814 week old mice were employed in the GF, ex-GF and CV groups for the investigation of DNA adduct formation and four female 25 week old and 8 male 1315 week old mice were used in the HFA group.
Preparation of different bacteriological conditions in mice
For the preliminary experiment to generate HFA mice, faeces were obtained from one of the volunteers (aged 27 years); a 10% suspension was prepared with prereduced 0.1 M potassium phosphate buffer (pH 7.2) in the anaerobic chamber. The suspension was divided into two aliquots. One was used immediately after preparation and the other was stored at 80°C for 1 week. A portion of the faeces was also stored at 80°C for 1 week and then processed anaerobically in the same way. Five GF mice were inoculated with 0.5 ml of one of these three kinds of suspension into the stomach by a metal catheter. Four weeks after inoculation, the metabolic activity of the caecal contents towards mutagens was analysed. This preliminary experiment showed that storage of the inoculum at 80°C had no marked effect on the metabolic activities of intestinal flora against mutagens in HFA mice, so portions of the faeces from another volunteer (aged 44 years) were prepared and stored at 80°C. A suspension was prepared from the frozen faeces, as described above, and GF mice were inoculated with 0.5 ml of the suspension orally to generate HFA mice for the investigation of DNA adduct formation. Ex-GF mice were produced by inoculation with the caecal contents from a CV mouse. Caecal contents were obtained from an adult female CV mouse and a 5% suspension was prepared under anaerobic conditions. GF mice were inoculated with 0.5 ml of the suspension into the stomach by a metal catheter. The HFA and ex-GF mice were kept in isolators for 4 weeks to stabilize their intestinal flora. GF and CV mice were kept for 4 weeks without any treatment. Before the first dose of the mutagens, faeces were collected from each group of mice and processed in the anaerobic chamber for the analysis of activities against the mutagens.
Preparation of samples for mutagenicity assay
Mutagens were dissolved in dimethylsulfoxide (Merck, Darmstadt, Germany) at 0.2 mg/ml for IQ and AAC (both from Toronto Research Chemicals, Toronto, Canada) and 1 mg/ml for NF (Aldrich-Chemie, Steinheim, Germany). In the anaerobic chamber, 10% (human) or 5% (mice) suspensions of the faecal/caecal samples were prepared with prereduced 0.1 M potassium phosphate buffer (pH 7.2) and mixed with mutagen solutions or dimethylsulfoxide (19:1). The mixtures were transferred into a GasPak System (BBL, Cockeysville, MD) and incubated for 18 h at 37°C. Mutagen solutions mixed with phosphate buffer without faecal/caecal samples were used as controls. Mixtures were centrifuged to clarify them after incubation and IQ, AAC and their derivatives were extracted from the supernatant with blue cotton (Schwanz/Mann Biotech, Cleveland, OH) (30). Extracts were dried under nitrogen flow and dissolved in phosphate buffer:dimethylsulfoxide (19:1). The supernatant from incubations with NF was filter-sterilized with an 0.45 µm filter (Whatman Inc., Clifton, NJ). All samples were stored at 20°C until used for the mutagenicity assay.
Mutagenicity assay
The mutagenicity of the samples on Salmonella typhimurium TA98 was tested using the preincubation method of the Ames assay (31). Rat liver S9 was prepared from rats induced with Aroclor 1254 (Monsanto, St Louis, MO). After activation by S9, IQ was much more mutagenic than it was without S9 activation, so samples with IQ were diluted 200 times for the assay with S9 activation. The numbers of revertant colonies after 48 h incubation at 37°C were counted and the results were compared with those from controls processed in the same way but incubated with phosphate buffer without faecal/caecal samples. The assays were performed at least in duplicate.
Treatment of the animals
Four weeks after inoculation of bacteria, each group of GF, HFA, ex-GF and CV mice was divided into four groups of three animals. Each group of HFA mice contained one female and two male mice. Mice were administered 40 mg/kg body weight of IQ or AAC or 20 mg/kg body weight of NF. Control groups were administered vehicle (corn oil) only. The mutagens or control vehicle were introduced into the stomach using a metal catheter once a day for 3 days. Animals were killed 24 h after the last dose. Whole blood was collected from the heart under diethyl ether anaesthesia and brain, lung, heart, liver, kidney, spleen, stomach, jejunum (3 cm below stomach) and colon were collected. Stomach, jejunum and colon were cut open and the contents were removed by rinsing with phosphate-buffered saline. Epithelial cells were scraped off from the intestine with a glass slide and epithelial cells and wall were stored separately. All samples were stored at 80°C until required for isolation of DNA.
DNA adduct analysis
The amounts of DNA adducts were analysed according to the procedures described by Möller and Zeisig (32) and Wohlin et al. (33). Samples were homogenized in buffer with 1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 1 M TrisHCl (pH 7.4). After incubation with RNase A and T1 (both from Sigma Chemical Co., St Louis, MO) and proteinase K (Merck), DNA was extracted with phenolchloroform. DNA was hydrolysed using micrococcal nuclease (Sigma) and spleen phosphodiesterase (Boehringer Mannheim, Mannheim, Germany) and DNA adducts were enriched by butanol extraction. The samples were post-labelled with [32P]ATP (Amersham, Little Chalfont, UK) using T4 polynucleotide kinase (Amersham) and analysed by HPLC with two 3.5 x 150 mm DeltaPak 5 µm C18-100A columns (Waters Chromatography, Milford, MA). The chromatograms obtained were compared with chromatograms of DNA from untreated control animals; peaks observed in treated animals but not in control animals were presumed to represent DNA adducts caused by the administered chemicals. Results were expressed as the number of nucleotides with DNA adducts per 108 normal nucleotides.
Statistical analysis
Student's t-test was used to compare the difference between two groups, and one-way analysis of variance (ANOVA) was used to evaluate the difference among three or more groups. The NewmanKeuls test was used to test the significance of the differences of mean values in ANOVA. Statistical significance was accepted at P < 0.05.
 |
Results
|
---|
Mutagenicity of mutagens after incubation with human faecal flora of healthy volunteers
The mutagenicity of IQ and NF after incubation with faeces of healthy volunteers is shown in Figure 1
. Human faeces incubated for 18 h without adding any mutagen did not show direct or S9-mediated mutagenicity in any of the samples.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1. Mutagenicity of IQ (above) and NF (below) after incubation with human faecal flora of healthy volunteers (AH). Values represent mean + SD (n = 3). Black bars, no metabolic activation (S9); striped bars, with metabolic activation (+S9). `Control', revertant colonies obtained in mutagen solutions incubated without faecal suspension.
|
|
IQ showed only weak mutagenicity in the Ames assay without S9 activation, but in the majority (20/24) of the faecal samples studied, it caused more direct mutagenicity (i.e. that without S9 activation) than controls after anaerobic incubation for 18 h. Although the differences were not statistically significant, due to the great day-to-day variation, volunteers A and B seemed to be more active in generation of direct-acting mutagens while volunteers G and H were less active. S9-mediated mutagenicity of IQ decreased after incubation with human faecal suspensions. All of the 24 samples showed decreased mutagenicity of IQ after activation by S9. Faeces from volunteers B, C, F and H were more effective in reducing mutagenicity than those from volunteers A and G.
NF was mutagenic both with and without S9 activation. In contrast to IQ, incubation with human faecal suspensions removed direct mutagenicity of NF almost completely in all volunteers. On the other hand, S9-mediated mutagenicity of NF, after incubation with faeces, sometimes increased (11/24 samples) and sometimes decreased (13/24).
Generation of HFA mice with a flora capable of activating mutagens, and the effect of freeze storage of the inoculum
Based on the results obtained from the experiments described above, an individual whose flora was highly capable of activating the mutagens was selected and HFA mice were generated. The caecal contents of the HFA mice inoculated with a fresh faecal suspension, a frozen suspension of fresh faeces or a suspension of frozen faeces increased direct mutagenicity of IQ and S9-mediated mutagenicity of NF after incubation for 18 h and decreased S9-mediated mutagenicity of IQ, direct mutagenicity of NF and S9-mediated mutagenicity of AAC (Figure 2
). Incubation with the caecal contents of HFA mice inoculated with a fresh faecal suspension always gave more revertant colonies than HFA mice inoculated with a frozen suspension of fresh faeces or a suspension of frozen faeces, except for direct-acting mutagenicity of NF which was almost completely eliminated by caecal contents of any of these three groups of HFA mice (Figure 2
).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2. Mutagenicity of IQ, NF and AAC after incubation with human faeces (black bars) and caecal contents of HFA mice inoculated with a suspension of fresh faeces (checked bars), a frozen suspension of fresh faeces (striped bars) or a suspension of frozen faeces (white bars). Values for HFA mice represent mean + SD (n = 5). AAC was not mutagenic without metabolic activation (S9) in the control. Dotted bars represent revertant colonies obtained in controls, i.e. mutagen solutions incubated without faecal suspension. Differences were significant (P < 0.05) between values with the same superscript.
|
|
The metabolic activities of the caecal contents of HFA mice produced by inoculation with a frozen suspension of fresh faeces or a suspension of frozen faeces against mutagens tested were comparable to those of human faeces, although the caecal contents of these two groups of HFA mice decreased S9-mediated mutagenicity of IQ more effectively (Figure 2
).
Alteration of mutagenicity of mutagens by faeces of mice from different groups
Alteration of mutagenicity by faeces obtained from GF, HFA, ex-GF and CV mice are shown in Figure 3
. The activities of faeces of HFA mice against mutagens were essentially the same as those of inoculated human faeces, except for a higher reduction of S9-mediated mutagenicity of IQ (data not shown). Faeces of mice harbouring human or mouse intestinal flora increased the direct mutagenicity of IQ. The highest increase in direct mutagenicity was observed in CV mice; the intestinal flora of ex-GF mice was less active. On the other hand, faeces of GF mice decreased the direct mutagenicity of IQ compared with the control, i.e. IQ incubated with phosphate buffer without faeces. The faeces from the three mouse groups harbouring bacteria decreased the S9-mediated mutagenicity of IQ, while faeces of GF mice did not affect this mutagenicity. HFA mice were the most active in reducing the S9-mediated mutagenicity of IQ.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3. Mutagenicity of IQ, NF and AAC after incubation with faeces from mice with different bacterial conditions, without (S9) and with (+S9) metabolic activation. Values represent the results of pooled faeces obtained from three mice. Black bars, GF mice; checked bars, HFA mice; striped bars, conventionalized (ex-GF) mice; white bars, CV mice. Dotted bars represent revertant colonies obtained in controls, i.e. mutagen solutions incubated without faecal suspension.
|
|
The direct mutagenicity of NF was almost completely eliminated by faeces obtained from mice with bacteria regardless of the origin of the flora. GF mice decreased the direct mutagenicity of NF to 44% of the control.
None of the faeces from the four mouse groups produced direct mutagens from AAC. Unlike the case of the other two mutagens, faeces of GF mice also had similar activity against S9-mediated mutagenicity of AAC to the other groups and all groups decreased this mutagenicity.
DNA adduct formation in different groups of mice
The pattern of detected adducts was essentially the same among different tissues and different groups of mice (GF, HFA, ex-GF and CV). DNA adduct formation after oral administration of IQ was very low in all four mouse groups (Figure 4
). Low levels of DNA adducts were detected in liver of the three mouse groups harbouring human or mouse intestinal flora, but DNA adduct formation in all other organs studied was undetectable or extremely low. In GF mice, adducts were not detected even in liver DNA.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4. DNA adduct formation in different groups of mice after oral administration of IQ. Values represent mean + SD (n = 3). Black bars, GF mice; checked bars, HFA mice; striped bars, conventionalized (ex-GF) mice; white bars, CV mice.
|
|
DNA adducts were detected, after administration of NF, in many of the organs of HFA, ex-GF and CV mice (Figure 5
). No adducts were observed in GF mice except in liver, in which only a trace amount was detected. In the three mouse groups with intestinal flora, higher levels of DNA adducts were detected in liver, colonic epithelium and kidney than in other organs. There were no or few DNA adducts in jejunum and stomach in all mouse groups. There were significantly more DNA adducts in the liver in ex-GF mice than in HFA mice, while there were significantly more adducts in colon, both epithelium and wall, in HFA mice than in ex-GF mice. There were significantly more DNA adducts in CV mice than in those detected in ex-GF mice in most of the organs studied.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5. DNA adduct formation in different groups of mice after oral administration of NF. Values represent mean + SD (n = 3). Black bars, GF mice; checked bars, HFA mice; striped bars, conventionalized (ex-GF) mice; white bars, CV mice. Differences were significant (P < 0.05) between GF and HFA (a), GF and ex-GF (b), GF and CV (c), HFA and ex-GF (d), HFA and CV (e) or ex-GF and CV (f) in the same tissue.
|
|
High levels of DNA adducts were detected in all of the four mouse groups after oral administration of AAC (Figure 6
). In contrast to IQ and NF, DNA adducts were also detected in GF mice in all organs studied, except for spleen. The highest adduct formation was observed in liver, followed by colonic epithelium. Jejunal epithelium, stomach epithelium, kidney and heart also showed relatively high levels of DNA adducts. DNA adduct formation in brain, lung and spleen was lower than in other organs. DNA adduct formation in liver, heart and stomach were comparable in GF and HFA mice, but colonization with human intestinal flora significantly reduced the formation of DNA adducts in colon, jejunum and kidney. Colonization with mouse flora in ex-GF mice did not affect the formation of adducts in liver, heart and stomach, and decreased DNA adduct formation significantly in colon, jejunum and kidney compared with GF mice. There was significantly more DNA adduct formation in the liver, heart and kidney of CV mice than of ex-GF mice. Liver DNA adducts in CV mice were the highest among the four mouse groups.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6. DNA adduct formation in different groups of mice after oral administration of AAC. Values represent mean + SD (n = 3). Black bars, GF mice; checked bars, HFA mice; striped bars, conventionalized (ex-GF) mice; white bars, CV mice. Differences were significant (P < 0.05) between GF and HFA (a), GF and ex-GF (b), GF and CV (c), HFA and ex-GF (d), HFA and CV (e) or ex-GF and CV (f) in the same tissue.
|
|
 |
Discussion
|
---|
As is shown in Figure 2
, the caecal contents of the HFA mice were active against the mutagens tested, as were human faeces. These results demonstrate that it is possible to transfer the activity of human intestinal flora against food and environmental mutagens into the mice by oral inoculation of a faecal suspension. The present study also showed that the activity of caecal contents of HFA mice inoculated with a suspension of frozen human faeces was similar to that of human faeces regarding the chemicals tested, although production of direct-acting mutagens from IQ and reduction of S9-mediated mutagenicity of IQ were more active in the HFA mice. This is of considerable importance practically because it makes it possible to generate HFA mice with the same metabolic activities repeatedly.
In comparing the activities of faeces against three mutagens, the results indicate that the presence of an intestinal flora is essential for the activities of faeces against the mutagens and that the effects of the faeces on mutagen metabolism appear to differ depending on the bacterial conditions and the mutagenic compound tested. However, the fact still remains that the production of direct-acting mutagens in the gut constitutes a carcinogenic risk for the host, not least in relation to colon cancer.
This study showed that the presence of an intestinal flora enhances DNA adduct formation by IQ and NF. This may be because of the metabolic capacity of intestinal bacteria to produce direct-acting mutagens or more mutagenic metabolites. It has been demonstrated that isolated bacteria, faeces or caecal contents from humans, mice and rats, but not caecal contents from GF rats, metabolize IQ to a direct-acting mutagen with the rate of metabolism being different among the different animal species (30,34,35). Nitroreduction of NF, for which the intestinal flora is responsible, is thought to be important for its toxicity (36). In addition, studies with GF rats demonstrated that the presence of the intestinal flora enhanced the formation of DNA adducts from this compound (15,37).
Many more DNA adducts were detected after treatment with AAC than after treatment with IQ or NF in most tissues studied, even though AAC showed much lower mutagenicity than did IQ in the Ames assay, after activation by the hepatic enzyme system, and had no direct-acting mutagenicity with or without incubation with intestinal bacteria. Unlike IQ and NF, AAC treatment resulted in high levels of DNA adducts in GF mice too, indicating that the presence of an intestinal flora is not essential for DNA adduct formation caused by AAC. In contrast to IQ and NF treatment, there were more DNA adducts after AAC treatment in some tissues in GF mice (including colon) than in mice with bacteria, while DNA adducts in liver and heart in GF mice were lower than those in CV mice. Regarding AAC administration, the intestinal flora appears to have a protective effect against DNA adduct formation in the colon. As DNA adduct formation in colonic mucosa is possibly caused by the absorption of carcinogens from the lumen (38), the physical binding of mutagens by bacteria could decrease their bioavailability and result in a protective effect on DNA adduct formation. Thus, the present study indicates that the intestinal flora has an active role in DNA adduct formation and the role varies according to the chemical to which the animals are exposed.
Differences in DNA adduct formation between GF mice and mice with an intestinal flora might be caused by differences in morphological and physiological characteristics of the intestine (39), slower transit time in GF animals (40), differences in host enzyme activities involved in mutagen metabolism (41) and properties of the intestinal contents such as pH (42). A lower epithelial cell turnover rate in GF mice might also increase DNA adduct formation. It has been reported that DNA damage in inactive genes is repaired more slowly than that in active genes (43). DNA adduct levels were higher in CV mice than in ex-GF mice when differences were found and DNA adduct formation in ex-GF mice was sometimes more similar to that of GF mice. The time we allowed for the intestinal flora to stabilize, 4 weeks in the present study, might not be long enough to normalize the condition of the GF mice, although it has been reported that the relative numbers of bacteria of different types stablize 28 weeks after intestinal flora has been introduced into GF mice (21, 44). The possibility that the presence of the intestinal flora in the early stages of life is important for normalization of the host cannot be excluded. Use of longer times to normalize the HFA mice before experiments or the use of offspring of HFA mice, which harbour a human intestinal flora from the beginning of their lives, should be considered.
The present study demonstrated that the human intestinal flora has different effects from mouse flora on DNA adduct formation in vivo as well as on metabolic activities against mutagens in vitro. Significant differences in DNA adduct formation were observed between HFA mice and ex-GF mice, which are identical except for the origin of the intestinal flora, in many tissues and the influences of the different origins of the intestinal flora were different among tissues. These results suggest that the role of the human intestinal flora in carcinogenesis could differ from that of experimental animals and indicate that studies using HFA mice might provide much needed information on the role of the human intestinal flora on carcinogenesis in vivo. Further studies on the composition of the flora activating or inactivating the mutagenicity of food and environmental mutagens and the identification of individual bacterial strains responsible for the enhancement or reduction of DNA adduct formation in vivo are needed. HFA mice can be used also to study the effects of dietary macrocomponents and administration of probiotics on the properties of human intestinal bacteria and carcinogenesis in vivo (45,46). The use of HFA mice could then contribute to prevention strategies for cancer involving improvement of the intestinal microbial balance by modulation of dietary habits or ingestion of probiotics.
 |
Notes
|
---|
5 Present address: Laboratory of Veterinary Public Health, Department of Veterinary Medical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 1138657, Japan 
6 To whom correspondence should be addressed Email: akazu{at}mail.ecc.u-tokyo.ac.jp 
 |
Acknowledgments
|
---|
This study was supported by grants from the Swedish Cancer Society and the Swedish Dairies Association. K.H. received a fellowship from the Swedish Cancer Society.
 |
References
|
---|
-
Wakabayashi,K., Nagao,M., Esumi,H. and Sugimura,T. (1992) Food-derived mutagens and carcinogens. Cancer Res., 52, 2092s2098s.[Abstract]
-
Sugimura,T. and Sato,S. (1983) Mutagens/carcinogens in foods. Cancer Res., 43, 2415s2421s.[Medline]
-
Hartong,A., Kraft,J., Schulze,J., Kiess,H. and Lewis,K.-H. (1984) The identification of nitrated polycyclic aromatic hydrocarbons in diesel particulate extracts and their potential formation as artifacts during particulate collection. Chromatographia, 19, 269273.[ISI]
-
Beije,B. and Möller,L. (1988) 2-Nitrofluorene and related compounds: prevalence and biological effects. Mutat. Res., 196, 177209.[ISI][Medline]
-
Kinouchi,T., Hideshi,T. and Ohnishi,Y. (1986) Detection of 1-nitropyrene in yakitori (grilled chicken). Mutat. Res., 171, 105113.[ISI][Medline]
-
Gorbach,S.L. and Goldin,B.R. (1990) The intestinal microflora and the colon cancer connection. Rev. Infect. Dis., 12, S252S261.[ISI][Medline]
-
Möller,L., Torndal,U.B., Eriksson,L.C. and Gustafsson,J.-Å. (1989) The air pollutant 2-nitrofluorene as initiator and promoter in a liver model for chemical carcinogenesis. Carcinogenesis, 10, 435440.[Abstract]
-
Osawa,T., Namiki,M., Suzuki,K. and Mitsuoka,T. (1983) Mutagen formation by intestinal bacteria. Mutat. Res., 122, 299304.[ISI][Medline]
-
Cerniglia,C.E., Howard,P.C., Fu,P.P. and Franklin,W. (1984) Metabolism of nitropolycyclic aromatic hydrocarbons by human intestinal microflora. Biochem. Biophys. Res. Commun., 123, 262270.[ISI][Medline]
-
Reddy,B.S., Narisawa,T., Wright,P., Vukusich,D., Weisburger,J.H. and Wynder,E.L. (1975) Colon carcinogenesis with azoxymethane and dimethylhydrazine in germ-free rats. Cancer Res., 35, 287290.[Abstract]
-
Reddy,B.S., Weisburger,J.H., Narisawa,T. and Wynder,E.L. (1974) Colon carcinogenesis in germ-free rats with 1,2-dimethylhydrazine and N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Res., 34, 23682372.[ISI][Medline]
-
Ball,L.M., Rafter,J.J., Gustafsson,J.-Å., Gustafsson,B.-E., Kohan,M.J. and Lewtas,J. (1991) Formation of mutagenic urinary metabolites from 1-nitropyrene in germ-free and conventional rats: role of the gut flora. Carcinogenesis, 12, 15.[Abstract]
-
George,S.E., Chadwick,R.W., Kohan,M.J., Allison,J.C., Williams,R.W. and Chang,J. (1994) Role of the intestinal microbiota in the activation of the promutagen 2,6-dinitrotoluene to mutagenic urine metabolites and comparison of GI enzyme activities in germ-free and conventionalized male Fischer 344 rats. Cancer Lett., 79, 181187.[ISI][Medline]
-
Larsen,G.L. (1988) Deconjugation of biliary metabolites by microflora, ß-glucuronidases, sulphatases, and cysteine conjugate ß-lyases and their subsequent enterohepatic circulation. In Rowland,I.R. (ed.), Role of the Gut Flora in Toxicity and Cancer. Academic Press, London, pp. 79107.
-
Scheepers,P.T.J., Velders,D.D., Steenwinkel,M.J.S.T., van Delft,J.H.M., Driessen,W., Stratemans,M.M.E., Baan,R.A., Koopman,J.P., Noordhoeck,J. and Bos,R.P. (1994) Role of the intestinal microflora in the formation of DNA and haemoglobin adducts in rats treated with 2-nitrofluorene and 2-aminofluorene by gavage. Carcinogenesis, 15, 14331441.[Abstract]
-
Möller,L. (1994) In vivo metabolism and genotoxic effects of nitrated polycyclic aromatic hydrocarbons. Environ. Health Perspect., 102 (Suppl. 4), 139146.
-
Orrhage,K., Sillerström,E., Gustafsson,J.-Å., Nord,C.E. and Rafter,J. (1994) Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria. Mutat. Res., 311, 239248.[ISI][Medline]
-
Morotomi,M. and Mutai,M. (1986) In vitro binding of potent mutagenic pyrolyzates to intestinal bacteria. J. Natl Cancer Inst., 77, 195201.[ISI][Medline]
-
Hirayama,K., Itoh,K., Takahashi,E. and Mitsuoka,T. (1995) Comparison of composition of faecal microbiota and metabolism of faecal bacteria among `human-flora-associated' mice inoculated with faeces from six different human donors. Microbial Ecol. Health Dis., 8, 199211.[ISI]
-
Mallett,A.K., Bearne,C.A., Rowland,I.R., Farthing,M.J.G., Cole,C.B. and Fuller,R. (1987) The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora. J. Appl. Bacteriol., 63, 3945.[ISI][Medline]
-
Hirayama,K., Kawamura,S. and Mitsuoka,T. (1991) Development and stability of human faecal flora in the intestine of ex-germ-free mice. Microbial Ecol. Health Dis., 4, 9599.
-
Felton,J.S., Knize,M.G., Wood,C., Wuebbles,B.J., Healy,S.K., Stuermer,D.H., Bjeldanes,L.F., Kimble,B.J. and Hatch,F.T. (1984) Isolation and characterization of new mutagens from fried ground beef. Carcinogenesis, 5, 95102.[Abstract]
-
Hayatsu,H., Matsui,Y., Ohara,Y., Oka,T. and Hayatsu,T. (1983) Characterization of mutagenic fractions in beef extract and in cooked ground beef. Use of blue-cotton for efficient extraction. Gann, 74, 472482.[ISI][Medline]
-
Matsumoto,T., Yoshida,D. and Tomita,H. (1981) Determination of mutagens, amino-
-carbolines in grilled foods and cigarette smoke condensate. Cancer Lett., 12, 105110.[ISI][Medline]
-
Layton,D.W., Bogen,K.T., Knize,M.G., Hatch,F.T., Johnson,V.M. and Felton,J.S. (1995) Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis, 16, 3952.[Abstract]
-
Umemoto,A., Kajikawa,A., Tanaka,M., Hamada,K., Seraj,M.J., Kubota,A., Nakayama,M., Kinouchi,T., Ohnishi,Y., Yamashita,K. and Monden,Y. (1994) Presence of mucosa-specific DNA adducts in human colon: possible implication for colorectal cancer. Carcinogenesis, 15, 901905.[Abstract]
-
Pfohl-Leszkowicz,A., Grosse,Y., Carrière,V., Cugnenc,P.-H., Berger,A., Carnot,F., Beaune,P. and de Waziers,I. (1995) High levels of DNA adducts in human colon are associated with colorectal cancer. Cancer Res., 55, 56115616.[Abstract]
-
Cui,X.-S., Bergman,J. and Möller,L. (1996) Preneoplastic lesions, DNA adduct formation and mutagenicity of 5-, 7- and 9-hydroxy-2-nitrofluorene, metabolites of the air pollutant 2-nitrofluorene. Mutat. Res., 369, 147155.[ISI][Medline]
-
Gustafsson,B.E. (1959) Light weight steel systems for rearing germfree animals. Ann. N.Y. Acad. Sci., 78, 166173.[ISI]
-
Carman,R.J., van Tassell,R.L., Kingston,D.G.I., Bashir,M. and Wilkins,T.D. (1988) Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans. Mutat. Res., 206, 335342.[ISI][Medline]
-
Maron,D.M. and Ames,B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113, 173215.[ISI][Medline]
-
Möller,L. and Zeisig,M. (1993) DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC. Carcinogenesis, 14, 5359.[Abstract]
-
Wohlin,P., Zeisig,M., Gustafsson,J.-Å. and Möller,L. (1996) 32P-HPLC analysis of DNA adducts formed in vitro and in vivo by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxalin, utilizing an improved adduct enrichment procedure. Chem. Res. Toxicol., 9, 10501056.[ISI][Medline]
-
Bashir,M., Kingston,D.G.I., Carman,R.J., van Tassell,R.L. and Wilkins,T.D. (1987) Anaerobic metabolism of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) by human fecal flora. Mutat. Res., 190, 187190.[ISI][Medline]
-
Rumney,C.J., Rowland,I.R. and O'Neill,I.K. (1993) Conversion of IQ to 7-OHIQ by gut microflora. Nutr. Cancer, 19, 6776.[ISI][Medline]
-
Möller,L., Corrie,M., Midtvedt,T., Rafter,J. and Gustafsson,J.-Å. (1988) The role of the intestinal microflora in the formation of mutagenic metabolites from the carcinogenic pollutant 2-nitrofluorene. Carcinogenesis, 9, 823830.[Abstract]
-
Möller,L., Zeisig,M., Midtvedt,T. and Gustafsson,J.-Å. (1994) Intestinal microflora enhances formation of DNA adducts following administration of 2-NF and 2-AF. Carcinogenesis, 15, 857861.[Abstract]
-
Kajikawa,A., Umemoto,A., Hamada,K., Tanaka,M., Kinouchi,T., Ohnishi,Y. and Monden,Y. (1995) Mucosa-preferential DNA adduct formation by 2-amino-3-methylimidazo[4,5-f]quinoline in the rat colonic wall. Cancer Res., 55, 27692773.[Abstract]
-
Henegham,J.B. (1988) Alimentary tract physiology: interactions between the host and its microbial flora. In Rowland,I.R. (ed.) Role of the Gut Flora in Toxicity and Cancer. Academic Press, London, pp. 3977.
-
Rickert,D.E., Long,R.M., Krakowka,S. and Dent,J.G. (1981) Metabolism and excretion of 2,4-[14C]dinitrotoluene in conventional and axenic Fischer 344 rats. Toxicol. Appl. Pharmacol., 59, 574579.[ISI][Medline]
-
Treptow-van Lishaut,S., Rechkemmer,G., Rowland,I., Dolara,P. and Pool-Zobel,B.L. (1999) The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats. Eur. J. Nutr., 38, 7683.[ISI][Medline]
-
Rowland,I.R. and Wise,A. (1985) The effect of diet on the mammalian gut flora and its metabolic activities. CRC Crit. Rev. Toxicol., 16, 31103.[ISI]
-
Leadon,S.A. and Lawrence,D.A. (1991) Preferential repair of DNA damage on the transcribed strand of the human metallothionein genes requires RNA polymerase II. Mutat. Res., 255, 6778.[ISI][Medline]
-
Hirayama,K., Mishima,M., Kawamura,S., Itoh,K., Takahashi,E. and Mitsuoka,T. (1994) Effects of dietary supplements on the composition of fecal flora of human-flora-associated (HFA) mice. Bifidobact. Microflora, 13, 17.
-
Rumney,C.J., Rowland,I.R., Coutts,T.M., Randerath,K., Reddy,R., Shah,A.B., Ellul,A. and O'Neill,I.K. (1993) Effects of risk-associated human dietary macrocomponents on processes related to carcinogenesis in human-flora-associated (HFA) rats. Carcinogenesis, 14, 7984.[Abstract]
-
Hambly,R.J., Rumney,C.J., Cunninghame,M., Fletcher,J.M.E., Rijken,P.J. and Rowland,I.R. (1997) Influence of diet containing high and low risk factors for colon cancer on early stages of carcinogenesis in human flora-associated (HFA) rats. Carcinogenesis, 18, 15351539.[Abstract]
Received February 23, 2000;
revised August 16, 2000;
accepted August 18, 2000.