Gastrointestinal dysmotility is associated with altered gut flora and septic mortality in patients with severe systemic inflammatory response syndrome: a preliminary study.

BACKGROUND: The gut is an important target organ for injury after severe insult, and resolution of feeding intolerance is crucial for critically ill patients. We investigated gut flora and motility to evaluate the impact of gastrointestinal dysmotility on septic complications in patients with severe systemic inflammatory response syndrome (SIRS). METHODS: Sixty-three ICU patients with severe SIRS were divided into two groups depending on their intestinal condition. Patients with feeding intolerance comprised patients who had feeding intolerance, defined as ≥ 300 mL reflux from nasal gastric feeding tube in 24 h, and patients without feeding intolerance comprised patients with no feeding intolerance. We compared fecal microflora, incidences of bacteremia, and mortality between these groups. KEY RESULTS: Analysis of feces showed that patients with feeding intolerance had significantly lower numbers of total obligate anaerobes including Bacteroidaceae and Bifidobacterium, higher numbers of Staphylococcus, lower concentrations of acetic acid and propionic acid, and higher concentrations of succinic acid and lactic acid than those in patients without feeding intolerance (P ≤ 0.05). Patients with feeding intolerance had higher incidences of bacteremia (86%vs 18%) and mortality (64%vs 20%) than did patients without feeding intolerance (P ≤ 0.05). CONCLUSIONS & INFERENCES: Gut flora and organic acids were significantly altered in patients with severe SIRS complicated by gastrointestinal dysmotility, which was associated with higher septic mortality in SIRS patients.

Protective effect of Lactobacillus casei strain Shirota against lethal infection with multi-drug resistant Salmonella enterica serovar Typhimurium DT104 in mice.

AIMS: The anti-infectious activity of lactobacilli against multi-drug resistant Salmonella enterica serovar Typhimurium DT104 (DT104) was examined in a murine model of an opportunistic antibiotic-induced infection. METHODS AND RESULTS: Explosive intestinal growth and subsequent lethal extra-intestinal translocation after oral infection with DT104 during fosfomycin (FOM) administration was significantly inhibited by continuous oral administration of Lactobacillus casei strain Shirota (LcS), which is naturally resistant to FOM, at a dose of 10(8) colony-forming units per mouse daily to mice. Comparison of the anti-Salmonella activity of several Lactobacillus type strains with natural resistance to FOM revealed that Lactobacillus brevis ATCC 14869(T) , Lactobacillus plantarum ATCC 14917(T) , Lactobacillus reuteri JCM 1112(T) , Lactobacillus rhamnosus ATCC 7469(T) and Lactobacillus salivarius ATCC 11741(T) conferred no activity even when they obtained the high population levels almost similar to those of the effective strains such as LcS, Lact. casei ATCC 334(T) and Lactobacillus zeae ATCC 15820(T) . The increase in concentration of organic acids and maintenance of the lower pH in the intestine because of Lactobacillus colonization were correlated with the anti-infectious activity. Moreover, heat-killed LcS was not protective against the infection, suggesting that the metabolic activity of lactobacilli is important for the anti-infectious activity. CONCLUSION: These results suggest that certain lactobacilli in combination with antibiotics may be useful for prophylaxis against opportunistic intestinal infections by multi-drug resistant pathogens, such as DT104. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotics such as FOM disrupt the metabolic activity of the intestinal microbiota that produce organic acids, and that only probiotic strains that are metabolically active in vivo should be selected to prevent intestinal infection when used clinically in combination with certain antibiotics.

Antitumor effects of the intravesical instillation of heat killed cells of the Lactobacillus casei strain Shirota on the murine orthotopic bladder tumor MBT-2.

PURPOSE: To characterize the potential of heat killed Lactobacillus casei, Shirota strain (LC9018), as an alternative to bacillus Calmette-Guerin (BCG) for treating patients with bladder cancer we investigated the antitumor effects of intravesical instillation of LC9018 in the MBT-2 orthotopic bladder tumor implantation model in C3H/He mice. MATERIALS AND METHODS: LC9018 or BCG, Tokyo 172 strain, was instilled once daily for 10 days starting on the day after orthotopic implantation of MBT-2. Tumor appearance and mean bladder weight on day 21 after tumor implantation were evaluated. Moreover, we investigated the augmentation of local cellular immunity in bladder mucosa by immunohistochemical staining and reverse transcription polymerase chain reaction. RESULTS: Intravesical LC9018 instillation significantly reduced the rate of tumor appearance in 8 of 38 subjects (p <0.001) and mean tumor growth plus or minus standard deviation with a bladder weight of 37 +/- 49 mg. (p <0.001) compared with tumor appearance in 41 of 58 subjects and mean bladder weight 146 +/- 183 mg. in controls. BCG had no significant antitumor activity in the orthotopic implantation model. Intravesical instillation of LC9018 augmented the local expression of antitumor cytokine messenger RNA (interferon-gamma and tumor necrosis factor-alpha) and induced the infiltration of neutrophils surrounded by macrophages that phagocytosed LC9018 cells at the bladder mucosa. CONCLUSIONS: These results suggest that LC9018 is potentially more potent and safer as a therapeutic agent than BCG for superficial bladder tumors. Furthermore, the antitumor effect of LC9018 is exerted via the augmentation of local cell mediated antitumor immunity.

Lactobacillus casei acquires the binding activity to fibronectin by the expression of the fibronectin binding domain of Streptococcus pyogenes on the cell surface.

Fibronectin binding domain was expressed on the cell surface of Lactobacillus casei strain Shirota which hardly adheres to fibronectin. DNA for the fibronectin binding domain of the sfbl gene, which encodes a fibronectin binding protein of Streptococcus pyogenes ATCC 21059, was amplified with polymerase chain reaction, cloned into a surface display vector pSAK332, and introduced into L. casei. The fibronectin binding domain was expressed as a fusion protein consisting of staphylokinase of Staphylococcus aureus and the anchor sequence of cell wall-associated 763 proteinase of Lactococcus lactis NCDO 763. The fibronectin binding ability of the resulting L. casei was confirmed with Western blot analysis, immunoelectron microscopic analysis, and adherence to fibroblast cells. These results indicate that L. casei has acquired a new phenotype to bind fibronectin upon the expression of the fibronectin binding domain on the cell surface. This L. casei also shows binding affinity to fibrinogen, indicating that fibronectin binding domain is involved in the binding to fibrinogen as well.

Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism.

Regulation of innate immunity may be an effective means of cancer control. Delaying cancer onset is regarded as an important mode of action in cancer prevention. We have been investigating the chemopreventive mechanisms of Lactobacillus casei Shirota (LcS), a probiotic strain. In this study, we evaluated the effect of LcS on tumor onset and the involvement of natural killer (NK) cells using a 3-methylcholanthrene-induced carcinogenesis model. C3H/HeN mice were divided into three groups, according to treatment: vehicle-treated, treated with vehicle only; control, 3-methylcholanthrene treated; LcS, 3-methylcholanthrene and LcS treated. 3-Methylcholanthrene was injected intradermally at 7 weeks of age. LcS was mixed into the diet (0.05%, w/w), which the mice were fed from the day of 3-methylcholanthrene injection onward. Tumor incidence was observed weekly. Profiles of splenic NK cells, in vitro cytotoxicity and the proportion, in the early stage of carcinogenesis were analyzed at 5 weeks after the injection. The tumor suppressive effect of LcS was also evaluated in a beige mouse model that is genetically deficient in NK cells. LcS delayed tumor onset and reduced tumor incidence in the results with C3H/HeN mice (P< 0.05). More specifically, tumor incidence in the control group was 33% at 6 weeks after the injection and 83% at 11 weeks as opposed to 0 and 42%, respectively, in the LcS group. NK cell cytotoxicity was significantly higher than in the control group, and the number of NK cells also increased in the LcS group of C3H/HeN mice. However, LcS failed to suppress tumorigenesis in the beige mouse. These findings suggest that enhancement of the cytotoxicity of NK cells by LcS delays tumor onset.

Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor beta chain and p53 double-knockout mice.

This study was conducted to confirm the hypothesis that intestinal microflora are required for the development of adenocarcinoma in the colon of the TCRbeta and p53 double-knockout (TCRbeta-/- p53-/-) mouse. Germ-free TCRbeta-/- p53-/- mice were produced. At 7 weeks of age, the animals were divided into two groups (n = 10/group), and one of these groups was conventionalized. Animals of both groups were subjected to histopathological examination for adenocarcinoma of the colon at 4 months of age. There was no development of adenocarcinoma of the colon among the germ-free mice, whereas in the conventionalized group, adenocarcinomas of the ileocecum and cecum were detected in 70% of animals. These results indicate the usefulness of the TCRbeta-/- p53-/- mouse as a colon cancer animal model that develops spontaneous adenocarcinoma of the colon early in life, and suggest that intestinal microflora play a major role in the development of adenocarcinoma of the colon in this animal model.

Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli.

Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L. crispatus, L. reuteri, and L. agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniques. These lactobacilli associated with the horse, except for L. reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats. A symbiotic relationship of these lactobacilli with the horse is suggested.

Comparison of the water-holding capacity of wheat bran products prepared by wet and dry smashing methods in vitro and effect on the gastrointestinal retention time in rats in vivo.

Microfibril wheat bran (MFW) prepared by wet smashing of wheat bran using a colloidal mill has the advantages of being more palatable than other wheat bran and easier to apply to various foods. In this study, we investigated water-holding capacity (WHC) and physiological effects of a novel food material, MFW, focusing on shortening of the retention time of the gastrointestinal contents compared to those of dry smashing of wheat bran (DWB) prepared by conventional method, and wheat bran (WB), which is the raw materials. The mean particle size of MFW was 35 microns, and WHC was 5.1 g/g. In contrast, those of DWB were 61 microns and 3.0 g/g, respectively. Those of WB were 420 microns and 5.0 g/g, respectively. The WHC of MFW was 1.7 times greater than that of DWB and comparable to that of WB. The dietary fiber content in MFW, DWB, and WB were 73.5, 66.9 and 70.2%, respectively. Six-week-old Fisher rats were divided into three groups, and fed for 20 days with AIN-76 chow supplemented with MFW, DWB, or WB to a dietary fiber content of 10%. On days 14-16 of the experimental period, the mean retention time (MRT) of gastrointestinal content and fecal weight were measured using solid phase and liquid phase markers. On day 20 of the experimental period, animals were killed, and the water content, pH, composition of short chain fatty acids (SCFAs) in the cecal content and total amounts of SCFAs in the cecum were investigated. MRT in the MFW group was 15.2 +/- 0.8 h in the solid phase, which was significantly shorter than that in the DWB group (18.0 +/- 0.9 h) (p < 0.05), and comparable to that in the WB (15.5 +/- 2.4 h). MRT in the liquid phase was almost the same as that in the solid phase: 14.7 +/- 1.0, 18.4 +/- 0.8, and 16.0 +/- 2.5 h in the MFW, DWB, and WB groups, respectively. The fecal weight, pH, the concentration of SCFA in the cecal content and total amounts of SCFAs in the cecum did not differ among the groups, but the cecal water content was in the order of MFW > WB > DWB, showing a significant difference between each group (p < 0.05). The above finding suggested that MFW is a novel food material with a greater WHC and the ability of shortening the retention time of the gastrointestinal contents compared to DWB.

The effect of an oral administration of Lactobacillus casei strain shirota on azoxymethane-induced colonic aberrant crypt foci and colon cancer in the rat.

The preventive effect of oral administration of viable Lactobacillus casei strain Shirota (LcS) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) and colon cancers in the rat was investigated. The study consisted of two experiments; in a short-term experiment (Exp-I), the inhibitory effect of 8- and 12-week treatments with LcS. Forty rats each received weekly a subcutaneous injection of AOM at a dose of 15 mg/kg of body weight for 5 weeks. Eight and twelve weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon and the mesenteric lymph nodes (MLN) were removed. The number of ACFs and the surface marker of lymphocytes derived from the MLN were investigated. The large ACF (those comprising four or more aberrant crypts per focus) had significantly decreased in the rats which had consumed the LcS diet. And oral administration of viable LcS significantly recovered CD8 positive lymphocytes to the levels in the control group. In a long-term experiment (Exp-II), 30 rats each received weekly a subcutaneous injection of AOM at a dose of 7. 4 mg/kg of body weight for 10 weeks. Twenty-five weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon were removed. The number and incidence of colon cancers were investigated. The number of rats with colon cancers and the number of colon cancers per rat, were significantly decreased in the rats which had consumed the LcS diet. LcS inhibited chemically-induced colon carcinogenesis in the rat. CD8 positive T lymphocytes may play a key role in the preventive effect against colon carcinogenesis.

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones.

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.

Inhibitory effect of microfibril wheat bran on azoxymethane-induced colon carcinogenesis in CF1 mice.

Microfibril wheat bran (MFW), a processed dietary fiber prepared by milling of coarse wheat bran (WB), is softer and has a more pleasant taste than WB. In this study, we examined the inhibitory effect of MFW on azoxymethane (AOM)-induced colon carcinogenesis in female CF1 mice and compared its effect with that of WB and cellulose (CL). The mice were fed a modified AIN 76 A diet supplemented with either MFW, WB, or CL at a final concentration of 20% (w/w). Six weekly s.c. injections of AOM (10 mg/kg body weight) were administered per mouse commencing 1 week after the start of the feeding period. Control mice were injected with saline only. Thirty-three weeks after the initial injection, the mice were sacrificed, examined for tumors, and the cecal contents were analyzed to determine the moisture content and the concentrations of short-chain fatty acids (SCFA). The average number of total tumors per mouse in the MFW (2.9 +/- 0.6, P = 0.017) and WB (5.3 +/- 1.3, P = 0.373) diet groups was lower than that of the CL diet group (7.5 +/- 1.3), though there was no significant difference in tumor incidence (94.7%, 90.0% and 94.7%, respectively) between the groups. More than 90% of the tumors in each group were adenocarcinomas. The incidence of adenoma and that of carcinoma in situ in the MFW diet group (5.3% and 0%, respectively) were also lower than those in the CL diet group (26.3 and 26.3%, respectively; P = 0.180 and P = 0.046, respectively). Analysis of the cecal contents revealed a significantly higher moisture content and significantly higher concentrations of SCFA, butyrate in particular, in the MFW and WB diet groups. The results of this study indicate that the source and texture of dietary fiber can influence tumor development in CF1 mice, and more specifically that MFW is a promising and useful dietary supplement with properties serving to protect against the development of colon cancer.

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies.

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.

Inhibitory effect of oral administration of Lactobacillus casei on 3-methylcholanthrene-induced carcinogenesis in mice.

The present study was designed to determine whether tumor induction by 3-methylcholanthrene (MC), a carcinogenic hydrocarbon, can be inhibited by oral administration of Lactobacillus casei strain Shirota (LC). C3H/HeN mice were divided into four groups and assigned to the following treatments: treated with MC and given control or LC-containing diet; treated with vehicle only and given control or LC-containing diet. MC (1 mg) was injected intradermally at 7 weeks of age and the tumor incidence was monitored; LC was mixed into a diet at a concentration of 0.05% (w/w) and the diet was fed from the day of MC injection throughout the study. Spleen cells were analyzed for the immune parameters at 12 and 16 weeks after the MC injection. Oral feeding of mice with LC reduced tumor incidence (P < 0.05). MC treatment lowered the in vitro response to concanavalin A (Con A) of spleen cells, the secretion of interleukin-2 in spleen cell culture after stimulation of the cells with Con A and the proportions of CD3+ CD4+ and CD8 + splenic cells. However, the analysis of the spleen cells obtained from the mice treated with MC and given the LC-containing diet revealed that these disrupted host immune parameters were maintained at the level of normal controls. These results suggest that oral feeding of mice with LC inhibits MC-induced tumorigenesis by modulating the disrupted host immune responses during MC carcinogenesis.

Effect of dietary deoxycholic acid and cholesterol on fecal steroid concentration and its impact on the colonic crypt cell proliferation in azoxymethane-treated rats.

To elucidate the role of fecal steroids in the malignant tumor formation of colonic epithelial cells, we examined the effects of dietary deoxycholic acid (DCA) and cholesterol (CHL) on fecal steroid concentrations and their impact on colonic crypt cell proliferation. Twenty 5-week-old male Fischer 344 rats were provided with either a control semisynthetic diet or the same diet supplemented with 0.15% DCA and 1% CHL (steroid diet) over a 5-week period. The effects of these two diets were compared among rats that were either injected with azoxymethane (AOM), a known gastrointestinal carcinogen, or saline. In a 2 x 2 factorial design, rats fed each of these diets were given two weekly subcutaneous injections of either AOM (15 mg/kg b.w.) or saline at 6 and 7 weeks of age. At 9 weeks of age, fecal samples were obtained for analysis of bile acids, CHL and its bacterial metabolites of intestinal microflora. At 10 weeks of age, animals were sacrificed and colonic proliferation was assessed as vincristine-accumulated mitotic figures per crypt. Rats fed the steroid diet had significantly elevated fecal bile acid (5x, P < 0.001) and neutral steroid (10x, P < 0.01) levels when compared to those fed the control diet. AOM treatment did not appear to influence these levels. However, rats injected with AOM had a significant increase (P < 0.001) in their rate of colonic cell proliferation as compared to saline-injected control animals on both diets. Furthermore, rats fed the steroid diet had a significantly higher (P < 0.001) cell proliferation rate than animals fed the control diet. The effects of AOM treatment and the steroid diet on cell proliferation were additive. Our results demonstrate that high concentrations of neutral and acid steroids in the colonic lumen can enhance carcinogen-induced elevated cell proliferation and thus may play a key role in the etiology of colon cancer.

Specific species of intestinal bacteria influence the induction of aberrant crypt foci by 1,2-dimethylhydrazine in rats.

To shed light on the association of intestinal microflora with the development of colon cancer, we studied the modifying effects of intestinal microflora on the occurrence of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF) in germfree (GF), gnotobiotic (GB) and conventionalized (Cvd) rats. In the first part of this study, 10 week old germfree Fischer-344 rats were randomly assigned to three groups and two groups of rats were orally inoculated with mixtures of pure culture of Escherichia coli, Enterococcus faecium, and several strains of Bacteroides and Clostridium species (GB), or feces from conventional rats (Cvd). Inoculated rats were given two weekly i.p. injections of DMH (20 mg/kg body wt) at 13 and 14 weeks of age. Rats were sacrificed 11 or 34 weeks after the last DMH injection for ACF scoring. The total number of ACF, ACF with four or more crypts/focus, and mean number of aberrant crypts per focus (crypt multiplicity) in GB rats sacrificed at week 34 were 168% (P < 0.001), 442% (P < 0.001) and 138% (P < 0.001) of those in GF rats, respectively. On the other hand, the same values in Cvd rats were 42% (P < 0.001), 147% (P = 0.246) and 159% (P < 0.001) of those in GF rats, respectively. Similar results were observed in rats that were sacrificed at week 11. In the second part of this study, the effect of colonization of Bifidobacterium breve on the ACF profiles was examined in GB rats. The number of ACF with four or more crypts/focus and crypt multiplicity in GB plus B. breve rats at week 11 were significantly lower than those of GB rats (P < 0.01, and P < 0.05, respectively), although the former was not statistically significant at week 34. These findings suggest that some intestinal bacteria might behave as promoters and some as anti-promoters in colon carcinogenesis.

Effects of a high-fat diet on azoxymethane-induced aberrant crypt foci and fecal biochemistry and microbial activity in rats.

To shed light on the association of dietary fat with the development of colon cancer, we studied the ability of azoxymethane (AOM) to induce aberrant crypt foci (ACF) and biochemical changes in rats fed high- or normal-fat diets. Six-week-old male Fischer 344 rats were placed on a high-fat [7% (wt/wt) soybean oil + 15% (wt/wt) beef tallow] or a normal-fat (7% soybean oil, AIN-93G) diet. Rats fed each of these diets were given two weekly subcutaneous injections of AOM (15 mg/kg body wt) or saline at seven and eight weeks of age. Fecal samples were obtained at 10 weeks of age, and animals were sacrificed for ACF scoring and analysis of cecal contents at 13 weeks of age. We observed greater numbers of ACF in the high- than in the low-fat group. Biochemically, rats fed the high-fat diet showed dramatically elevated fecal and cecal long-chain free fatty acid levels and intestinal alkaline phosphatase activity. These animals also showed increased cholesterol and decreased coprostanol levels. We did not detect significant differences in the fecal and cecal concentrations of total and soluble bile acids or total neutral sterols (cholesterol + coprostanol) between the two groups. Thus a high-fat diet does show certain striking effects on colon biochemistry in rats.

[Production of free bile acids by bacterial deconjugation from conjugated bile acids derived from peptone in a medium].

In the course of studies of in vitro bile acid transformation by lactic acid bacteria, we noticed that the medium used contained conjugated bile acids. HPLC analysis of the medium for each component indicated that these bile acids had originated from a peptone (Bacto Peptone, Difco). The concentration of these bile acids in a medium containing 2% Bacto Peptone was greater than 200 microM, higher than the concentration of bile acids usually added in in vitro bile acid transformation studies. Therefore, in such studies, it would be necessary to distinguish between the production of free secondary bile acids by bacterial deconjugation and that by 7 alpha-dehydroxylation. Since bile acids affect not only bacterial growth but also many metabolic activities, particular attention should be given to the use of Bacto Peptone in the medium.

Chemoprevention by pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, of N-methyl-N-nitrosourea-induced colon carcinogenesis in F344 rats.

A potential chemopreventive action of pravastatin (Pr), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on colon carcinogenesis was evaluated in F344 rats. All rats at 7 weeks of age received an intrarectal dose of 2 mg of N-methyl-N-nitrosourea 3 times weekly for 2 weeks in experiment I (2 groups of 16 rats each), and for 3 weeks in experiment II (4 groups of 30 rats each). They were given drinking water containing 0 ppm (control) or 200 ppm Pr during weeks 1 to 40 in experiment I, and containing 0 ppm (control), 25 ppm, 5 ppm and 1 ppm Pr during weeks 4 to 40 in experiment II. The body weight gains, and food and water intakes were similar in all the groups. The incidence of colon carcinomas at termination of the experiment at week 40 was not different in the 200 ppm Pr and control groups in experiment I (63% vs. 69%), while it was significantly lower in the 25 ppm and 5 ppm groups, but not in the 1 ppm Pr group, compared with the control group in experiment II (50%, 48%, and 77% vs. 80%). This inhibitory effect of Pr against colon carcinogenesis was not related to the cholesterol-lowering effect of this agent. We postulate that Pr inhibits the promotion stage of colon carcinogenesis, perhaps through modulation of cholesterol synthesis in situ in the colonic mucosa, thereby suppressing farnesyl isoprenylation of growth-regulating proteins such as p21 ras.