Clinical Significance of Carbapenem-Tolerant Pseudomonas aeruginosa Isolated in the Respiratory Tract


CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4(+) T cells.

CD4(+) T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4(+) T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4(+) T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4(+)CD25(-) T cells into Rag2(-/-) mice did not cause colitis accompanied by increasing Foxp3(+) inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4(+)CD25(-) T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4(+) T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.

Biochemical and Biophysical Research Communications


The ARNT-STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαβ+CD8αα+cells.

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.


Reduced Diversity and Imbalance of Fecal Microbiota in Patients with Ulcerative Colitis

Clinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified. To determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults. Fresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data. T-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission. UC patients have imbalances in the intestinal environment-less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.


Notch2 regulates the development of marginal zone B cells through Fos.

B cells are classified into several subsets depending on their functions, marker expression pattern and localization. Marginal zone B (MZB) cells are a distinct lineage from follicular B cells, and regulate host defenses against blood-borne pathogens. Notch2/RBP-J signaling regulates the development of MZB cells by interacting with delta-like 1 ligand, although the target genes for Notch2 signaling remain unclear. We identified Fos as an upregulated gene in LPS-stimulated B cells that received Notch2 signaling. Fos is expressed in CD21(high)CD23(low) MZB cells at a higher level compared to CD21(Int)CD23(high) follicular B cells. Deleting the Notch2 gene in CD19(+) B cells decreased Fos expression in B cells. Overexpression of Fos in Notch2-deficient B cells or bone marrow cells partially restored MZB development. Fos promoter activity was upregulated by Notch2 signaling, indicating that Notch2 directly controls Fos transcription associated with MZB development. These data identify Fos as one of the target genes for Notch2 signaling that is crucial for MZB development.


Production of apoptosis-inducing substances from soybean protein by Clostridium butyricum: Characterization of their toxic effects on human colon carcinoma cells

Microbial metabolism of soybean constituents is known to produce novel active substances as a chemopreventive agent during the fermentation, and enterobacteria are expected to produce chemopreventive agents as a consequence of metabolizing soybean constituents in the intestinal tract. Then, the conditioned medium was prepared by culturing an enterobacterium Clostridium butyricum (C. butyricum) with soybean protein, and its direct effect on human colon carcinoma HCT116 cells was examined. The conditioned medium was shown to induce the cell death, and suggested to contain novel apoptosis-inducing substances. Thus, enterobacteria are predicted to produce anti-tumor substances from food-derived proteins within the intestinal tract.


Inhibitory effects of fermented brown rice on induction of acute colitis by dextran sulfate sodium in rats.

Although the pathogenic mechanisms of inflammatory bowel diseases are not fully understood, colonic microbiota may affect the induction of colonic inflammation, and some probiotics and prebiotics have been reported to suppress colitis. The inhibitory effects of brown rice fermented by Aspergillus oryzae (FBRA), a fiber-rich food, on the induction of acute colitis by dextran sulfate sodium (DSS) were examined. Feeding a 5% and 10% FBRA-containing diet significantly decreased the ulcer and erosion area in the rat colon stained with Alcian blue. In another experiment, 10% FBRA feeding decreased the ulcer index (percentage of the total length of ulcers in the full length of the colon) and colitis score, which were determined by macroscopic observation. It also decreased myeloperoxidase activity in the colonic mucosa. Viable cell numbers of Lactobacillus in the feces decreased after DSS administration and was reversely correlated with severity of colitis, while the cell number of Enterobacteriaceae increased after DSS treatment and was positively correlated with colitis severity. These results indicate that FBRA has a suppressive effect on the induction of colitis by DSS and suggest FBRA-mediated modification of colonic microbiota.


Modifying effects of fermented brown rice on fecal microbiota in rats

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.


Suppressive effect of Clostridium perfringens-produced heat-stable substance(s) on proliferation of human colon adenocarcinoma HT29 cells in culture

Clostridium perfringens has been regarded as one of the intestinal bacteria increasing colon cancer risk. In previous studies, we have shown that the oral administration of C. perfringens culture medium can inhibit the mutagen-induced formation of pre-neoplastic lesions in rat colon, thus proposing the existence of factor(s) preventing colon tumorigenesis in this culture medium. However, the properties of effective factor(s) and the mechanism of this inhibitory action still remain to be investigated. Then, the effect of C. perfringens culture medium on human colon adenocarcinoma HT29 cells was examined. The exposure of HT29 cells to C. perfringens culture medium was found to suppress the proliferation of these cells probably through the reduction of immediate early gene egr-1 expression. These observations suggest that C. perfringens culture medium has a cytostatic action on colon tumor cells, which may be responsible for the prevention of pre-neoplastic formation in rat colon.


Inhibitory effect of fluvastatin on ileal ulcer formation in rats induced by nonsteroidal antiinflammatory drug

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal damage as one of their side effects in humans and experimental animals. Lipid peroxidation plays an important role in NSAID-induced ulceration. The aim of this study was to investigate the inhibitory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on the ulceration in small intestines of rats. The effects of three HMG-CoA reductase inhibitors, fluvastatin, pravastatin and atorvastatin on ileal ulcer formation in 5-bromo-2-(4-fluorophenyl)-3-(4- methylsulfonylphenyl) thiophene (BFMeT)-treated rats were examined. Antioxidative activity of the inhibitors was measured by a redox-linked colorimetric method. Fluvastatin, which was reported to have antioxidative activity, repressed the ileal ulcer formation in rats treated with BFMeT an NSAIDs. However, the other HMG-CoA reductase inhibitors (pravastatin and atorvastatin) did not repress the ileal ulcer formation. Among these HMG-CoA reductase inhibitors, fluvastatin showed a significantly stronger reducing power than the others (pravastatin, atorvastatin). Fluvastatin having the antioxidaitive activity suppresses ulcer formation in rats induced by NSAIDs.


Simultaneous detection of Bacteroides fragilis group species by leuB-directed PCR

Bacteroides species, saccharolytic Gram-negative obligate anaerobes, are frequently isolated from human infections such as peritonitis, abscesses and bacteremia. Among the species in the genus Bacteroides, the species called "B. fragilis group" are particularly involved in human infections and are medically important because they account for a major part of anaerobic isolates from clinical specimens. The purpose of this study was to develop PCR primers that specifically and simultaneously amplify the beta -isopropylmalate dehydrogenase gene leuB in B. fragilis group species. We determined partial nucleotide sequences of leuB genes and compared them in seventeen strains of nine B. fragilis group species, and the regions that are conserved among Bacteroides strains but different from other species were used as a B. fragilis group-specific PCR primer set, BacLBF-BacLBR. Specificity tests of the primer set using 52 phenotypically characterized strains and 75 isolates from rat feces showed only one case each of false-positive and false-negative. The detection limit of the leuB-directed PCR using BacLBF and BacLBR was 3.9 x 10(3) colony-forming units. These results indicate that leuB amplification using BacLBF andBacLBR is a useful tool for rapid diagnosis of Bacteriodes infection and for rapid differential diagnosis of anaerobic infections.

The Journal of Medical Investigation : JMI


Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells


Inhibitory effects of Centella asiatica on azoxymethane-induced aberrant crypt focus formation and carcinogenesis in the intestines of F344 rats

Effects of the water extract of Centella asiatica Linn. on formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and intestinal tumorigenesis in male F344 rats were investigated. Treatment with the extract significantly decreased the number of larger ACF (with four or more crypts per focus) in the large intestine in the early stage, while the number of methylated DNA adducts was not decreased compared with that in the AOM-treated group. In the post-initiation stage, the extract significantly decreased the total number of ACF and the number of larger ACF, accompanied by a decrease in the 5-bromo-2'-deoxyuridine-labeling index and an increase in the induction of apoptotic cells in the colonic mucosa. The incidences of neoplasms, the numbers of adenocarcinomas in the small intestines and entire intestines, and sizes of neoplasms in the entire intestines in rats fed C. asiatica extract at a dose of 10 mg/kg were smaller than those in rats given AOM alone (p < 0.05). The extract at a dose of 100 mg/kg significantly reduced the multiplicity of neoplasms in the small intestine (p < 0.05). These results suggest that inhibition of the formation of AOM-induced ACF by C. asiatica extract is associated with modification of cell proliferation and induction of apoptosis in colonic crypts and that the extract has a chemopreventive effect on colon tumorigenesis.


Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.


Role of unbalanced growth of Gram-negative bacteria in ileal ulcer formation in rats treated with a nonsteroidal anti-inflammatory drug

Nonsteroidal anti-inflammatory drugs (NSAIDs) induced formation of intestinal ulcers as side effects, in which an unbalanced increase in the number of gram-negative bacteria in the small intestine plays an important role. To clarify how intestinal microflora are influenced by NSAIDs, we examined the effects of 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene (BFMeT), an NSAID, on intestinal motility and on the growth of Escherichia coli and Lactobacillus acidophilus. Transit index, a marker of peristalsis, was not different in BFMeT-treated and solvent-treated rats, indicating that BFMeT increased the number of gram-negative bacteria without suppression of peristalsis. The factors that affect the growth of intestinal bacteria were not found in intestinal contents of BFMeT-treated rats, because the growth of E. coli and that of L. acidophilus in the supernatants of small intestinal contents of BFMeT-treated rats and solvent-treated rats were not different. The mechanism of the increase in the number of gram-negative bacteria is still unclear, but heat-killed E. coli cells and their purified lipopolysaccharide (LPS) caused deterioration of BFMeT-induced ileal ulcers, while they could not cause the ulcers by themselves without the NSAID. Concentration of LPS and myeloperoxidase activity level were elevated correlatively in the intestinal mucosa of rats treated with LPS and BFMeT. These results suggest that an increase in the number of gram-negative bacteria and their LPS in the mucosa induces activation of neutrophils together with the help of NSAID action and causes ulcer formation.


Effects of high amylose maize starch and Clostridium butyricum on metabolism in colonic microbiota and formation of azoxymethane-induced aberrant crypt foci in the rat colon

High amylose maize starch (HAS) is not digested in the small intestine and most of it reaches the large intestine. In the large intestine, HAS is fermented by intestinal bacteria, resulting in production of short-chain fatty acids (SCFA), particularly butyrate. Clostridium butyricum can utilize HAS and produce butyrate and acetate. It has been proposed that butyrate inhibits carcinogenesis in the colon. In this study, we examined the inhibitory effects of HAS and C. butyricum strain MIYAIRI588 (CBM588) on azoxymethane-induced aberrant crypt foci (ACF) formation in rats. In the group of rats administered only CBM588 spores, the concentration of butyrate in the cecum increased, but there was no decrease in the number of ACF. In the group of rats fed an HAS diet, a decrease in the number of ACF was observed, and in the group of rats administered HAS and CBM588, the number of ACF decreased significantly. In these two groups, the concentrations of acetate and propionate in intestinal contents significantly increased, but the concentration of butyrate did not change. It was found that the beta-glucuronidase activity level of colonic contents decreased significantly in the two groups of rats fed HAS. This study showed that HAS and CBM588 changed the metabolism of colonic microbiota and decreased the level of beta-glucuronidase activity, phenomena that may play a role in the inhibition of ACF formation in the rat colon.


Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats

An 80% ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against 2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4-67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9 H-pyrido[2,3-b]indole, benzo[a]pyrene,N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract itself showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0 g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon.


Inhibitory effects of bitter melon (Momordica charantia Linn.) on bacterial mutagenesis and aberrant crypt focus formation in the rat colon

Antimutagenicity and chemopreventive activity of an 80%-ethanol extract of bitter melon (Momordica charantia Linn.) against the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) was investigated. The bitter melon extract was nonmutagenic and inhibited the mutagenicity of heterocyclic amines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and aflatoxin B1 in the Salmonella mutation assay. To examine the inhibitory effect of bitter melon on AOM-induced ACF formation, male F344 rats were fed various concentrations of the extract (0.1, 0.5, and 1.0 g/kg body weight) for five weeks during the initiation stage. One week after the administration of the plant extract, rats were subcutaneously given AOM at 15 mg/kg body weight once a week for two weeks. Three rats in each group were sacrificed 12 hr after the second AOM injection to analyze DNA adducts, O6-methylguanine (O6-meG) and N7-methylguanine in the liver and colon. The remaining rats were sacrificed 3 weeks after the second AOM injection to observe ACF. To examine the inhibitory effect of the extract on ACF formation in the postinitiation stage, rats were fed the extract at 0.1 and 1.0 g/kg body weight for 12 weeks starting two weeks after the second AOM injection. Treatment with bitter melon extract significantly inhibited ACF formation in the colon during the initiation stage and dose-dependently decreased the average of O6-meG DNA adduct in the colonic mucosa. During the postinitiation stage, bitter melon extract, at 1.0 g/kg body weight, significantly inhibited ACF formation in the colon, especially the formation of ACF with four or more crypts per focus. These findings suggest that bitter melon is a possible chemopreventive agent against colon carcinogenesis.


PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis

Bacteroides fragilis is a Gram-negative obligate anaerobe frequently isolated from clinical specimens and sometimes causes severe septicemia in compromised hosts. Increasing interest has been shown in the enterotoxigenicity and drug resistance of B. fragilis in the field of medical microbiology. We previously reported rapid detection of this anaerobe by nested PCR targeting a neuraminidase-encoding gene nanH. In the present study, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1, which is specific for nanH of B. fragilis, and we combined the hybridization assay using NH1 with the nanH-PCR to detect this anaerobe in a bacteremia model mice. In the specificity test, the oligonucleotide probe, NH1, hybridized only to amplification products from B. fragilis. PCR-dot blot hybridization based on nanH enabled detection of cells of B. fragilis in blood samples even when the number was as low as 2 x 10(3) colony-forming units/ml. These findings suggest that PCR-dot blot hybridization targeting nanH is a useful procedure for diagnosis of septicemia caused by B. fragilis when viable cells in blood cannot be detected by the traditional culture techniques.


Expression of Human Lactoferrin in Bacteroides uniformis and its Effect on Azoxymethane-induced Aberrant Crypt Focus Formation in the Rat Colon


Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.


Effects of β-Glucuronidase-Deficient and Lycopene-Producing Escherichia coli Strains on Formation of Azoxymethane-Induced Aberrant Crypt Foci in the Rat Colon

Biochemical and Biophysical Research Communications


Effects of roselle (Hibiscus sabdariffa Linn.), a Thai medicinal plant, on the mutagenicity of various known mutagens in Salmonella typhimurium and on formation of aberrant crypt foci induced by the colon carcinogens azoxymethane and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in F344 rats

The 80% ethanol extract of roselle (Hibiscus sabdariffa Linn.), a medicinal plant in Thailand, was examined for antimutagenic and chemopreventive activity in a colon carcinogenesis model. It reduced about 60-90% of the mutagenicity induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and other heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MelQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline(MelQx),3-amino-1,4-dimet hyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2),2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1),2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), at a concentration of 12.5 mg/plate in the Salmonella mutation assay. The extract showed no mutagenicity and no antibacterial activity below this dose. Mutagenicity of methylazoxymethanol (MAM) acetate, which, like PhIP, is a colon carcinogen,was also efficiently inhibited by the roselle extract. To investigate chemoprevention by roselle in a colon carcinogenesis model, we examined the inhibitory effects of the roselle extract in F344 rats in which aberrant crypt focus (ACF) formation was induced by azoxymethane (AOM) and PhIP. In the initiation stage, the number of AOM-induced ACF in the colon was significantly decreased by roselle (17-25%) compared with that in rats treated with AOM alone. The amount of O6-methylguanine in the colonic mucosa tended to be decreased in the roselle-treated rats. The number of PhIP-induced ACF was also significantly decreased by roselle treatment (22%) at a concentration of 1.0 g/kg body weight in the initiation stage. However, in the post-initiation stage of AOM-induced ACF formation, roselle increased the number of ACF, especially the number of foci which had more than three crypts/focus. These results indicate that roselle has antimutagenic activity against MAM acetate and heterocyclic amines and that it decreases the number of AOM- and PhIP-induced ACF in the initiation stage, although it rather increased the number of ACF in the post-initiation stage.


Protective effects of a culture supernatant of Lactobacillus acidophilus and antioxidants on ileal ulcer formation in rats treated with a nonsteroidal antiinflammatory drug


Culture supernatants of Lactobacillus acidophilus and Bifidobacterium adolescentis repress ileal ulcer formation in rats treated with a nonsteroidal antiinflammatory drug by suppressing unbalanced growth of aerobic bacteria and lipid peroxidation


Activation of 1-nitropyrene by nitroreductase increases the DNA adduct level and mutagenicity

1-Nitropyrene (1-NP) is a mutagenic nitro compound in the environment. We studied correlations between the mutagenicity of 1-NP for three strains of Salmonella typhimurium, the activity of bacterial nitroreductases and the amount of 1-NP-derived DNA adducts. Bacterial strains used in this study were S. typhimurium strains TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid. The mutagenicity of 1-NP was measured using the Ames assay, and the nitroreductase activities of these strains were assayed by quantification of 1-aminopyrene produced from 1-NP. The DNA adducts were measured by the 32P-postlabeling method. Among the three bacterial strains, strain YG1021 was the highest in mutagenicity of 1-NP, the nitroreductase activity and the DNA adduct level. However, S. typhimurium strain TA98NR had the lowest values of these three parameters. Nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other. These results indicate that bacterial nitroreductase plays an important role in forming the DNA adducts, and that the higher the adduct level the higher the level of mutagenicity.

The Journal of Medical Investigation : JMI


Effect of Intestinal Bacteria on Formation of Azoxymethane-Induced Aberrant Crypt Foci in the Rat Colon

The effect of intestinal bacteria on formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and DNA adducts in the rat colon was investigated. Male Sprague-Dawley rats were administered cultures of Lactobacillus acidophilus, Bifidobacterium adolescentis, Bacteroides fragilis, Escherichia coli and Clostridium perfringens for five weeks and given injections of AOM at 15 mg/kg body weight at the first and second weeks. The number of ACF five weeks after the start of the experiment was decreased in the rats treated with the cultures or culture supernatants of L. acidophilus and C. perfringens. The half-life of O6-methylguanine (O6-meG) in the L. acidophilus group was shorter than that in the GAM broth group. The half-life of 7-methylguanine did not differ among the groups. These results suggest that the metabolite(s) of L. acidophilus and C. perfringens inhibit(s) the ACF formation in rats treated with AOM and that the inhibitory effect of L. acidophilus is due to the enhanced removal of O6-meG from the colon mucosal DNA.


Intestinal anaerobic bacteria hydrolyse sorivudine, producing the high blood concentration of 5-(E)-(2-bromovinyl)uracil that increases the level and toxicity of 5-fluorouracil.

Sorivudine, 1-beta-D-arabinofuranosyl-5-(E)-(2-bromovinyl)uracil, is a potent antiviral agent against varicella-zoster virus and herpes simplex virus type 1. However, sorivudine should not be used in combination with anticancer drugs such as 5-fluorouracil (5-FU) because (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of sorivudine, inhibits the degradation of 5-FU, resulting in its accumulation in the blood and marked enhancement of the toxicity of 5-FU. Since phosphorolytic enzymes generate BVU from sorivudine, we investigated the distribution of the enzyme activity in rats. High activity was found in the cecal and large intestinal contents, while very low or no detectable activity in the liver, kidney, stomach, cecum, large intestine, and the stomach and small intestinal contents. These results suggest that intestinal microflora play an important role in BVU production. Therefore, we measured the phosphorylase activity in cell-free extracts from 23 aerobes, 16 anaerobes and a fungus. Bacteroides species B. vulgatus, B. thetaiotaomicron, B. fragilis, B. uniformis and B. eggerthii, dominant members of intestinal microflora, had high activity to convert sorivudine to BVU. To elucidate the contribution of intestinal microflora to BVU production in vivo, we administered sorivudine to rats treated with several antibiotics and measured the BVU concentration in the serum of rats. When sorivudine was given to rats treated with ampicillin or a mixture of bacitracin, neomycin and streptomycin, which decreased the numbers of viable aerobes and anaerobes, only a small amount of BVU was found in the serum. BVU concentration in the serum of rats treated with metronidazole to decrease the number of intestinal anaerobes was also very low. In contrast, BVU concentration in the serum of rats treated with kanamycin, which was used to decrease the number of aerobes selectively, was higher than that of non-treated rats. These results also suggest that BVU is produced by intestinal anaerobic bacteria especially Bacteroides species in vivo.


Preventive effect of fermented brown rice and rice bran on spontaneous type 1 diabetes in NOD female mice


日本臨床腸内微生物学会誌

日本臨床腸内微生物学会誌