Effects on cell proliferation, activator protein-1 and genotoxicity by fecal water from patients with colorectal adenomas.

BACKGROUND: The free water phase of feces (fecal water) may mediate the effects of diet on colon carcinogenesis. We examined the effects of fecal water from adenoma patients and controls on three parameters in colonocytes believed to be relevant to tumorigenesis, i.e. genotoxicity in intact cells and on isolated DNA, proliferative activity and activator protein-1 (AP-1) activity. METHODS: Genotoxicity in intact colonic cells was assayed using the single-cell gel electrophoresis assay ('comet' assay) and on isolated DNA using double-stranded DNA from the X-174 RF plasmid. Cell proliferation was assessed using the commercially available 'alamar blue' proliferation kit and AP-1 activity using cells transiently transfected with an AP-1-luciferase reporter construct. RESULTS: The data showed that lipid extracts of fecal water samples from the adenoma patients had a significantly higher capacity to induce cell proliferation than those from controls, and that this effect could be explained to a large extent by the concentrations of deoxycholic and chenodeoxycholic acids in the fecal water using regression models. No difference between patients and controls was observed for induction of AP-1 activity or induction of DNA strand breaks in intact cells. However, induction of DNA strand breaks in isolated DNA was significantly higher for the fecal waters from patients than for those from controls, which could be explained in part in a regression model by concentrations of lithocholic acid in fecal water and fecapentaene-12 in feces. CONCLUSIONS: Our results support the hypothesis that the biochemistry of fecal waters from adenoma patients and controls differs.

Effects on Cell Proliferation, Activator Protein-1 and Genotoxicity by Fecal Water from Patients with Colorectal Adenomas.

BACKGROUND: The free water phase of feces (fecal water) may mediate the effects of diet on colon carcinogenesis. We examined the effects of fecal water from adenoma patients and controls on three parameters in colonocytes believed to be relevant to tumorigenesis, i.e. genotoxicity in intact cells and on isolated DNA, proliferative activity and activator protein-1 (AP-1) activity. METHODS: Genotoxicity in intact colonic cells was assayed using the single-cell gel electrophoresis assay (`comet' assay) and on isolated DNA using double-stranded DNA from the X-174 RF plasmid. Cell proliferation was assessed using the commercially available `alamar blue' proliferation kit and AP-1 activity using cells transiently transfected with an AP-1-luciferase reporter construct. RESULTS: The data showed that lipid extracts of fecal water samples from the adenoma patients had a significantly higher capacity to induce cell proliferation than those from controls, and that this effect could be explained to a large extent by the concentrations of deoxycholic and chenodeoxycholic acids in the fecal water using regression models. No difference between patients and controls was observed for induction of AP-1 activity or induction of DNA strand breaks in intact cells. However, induction of DNA strand breaks in isolated DNA was significantly higher for the fecal waters from patients than for those from controls, which could be explained in part in a regression model by concentrations of lithocholic acid in fecal water and fecapentaene-12 in feces. CONCLUSIONS: Our results support the hypothesis that the biochemistry of fecal waters from adenoma patients and controls differs.

Deoxycholic acid causes DNA damage in colonic cells with subsequent induction of caspases, COX-2 promoter activity and the transcription factors NF-kB and AP-1.

Evidence is accumulating that bile acids induce apoptosis in colonic cells. Therefore, it becomes important to study the underlying molecular mechanisms and the role of this phenomenon in tumor promotion. Minutes after exposure of HCT 116 and HT-29 cells to deoxycholate (DCA), DNA damage, measured using the COMET assay, was evident. Caspase-3 was rapidly activated in HCT 116 cells exposed to DCA, whereas in HT-29 cells, caspase-3 activation was delayed. Using transient transfections with reporter constructs, we showed that the transcription factors activator protein-1 (AP-1) and NF-kB were increased in HCT 116 cells, in a dose-dependent fashion, by DCA COX-2 promoter activity was also induced by DCA and using mutant COX-2 promoter plasmids, we showed that the ability of DCA to induce promoter activity was partly dependent upon a functional NF-kB and C/EBP site, and completely dependent on a functional c-AMP response element site. DNA damage thus appears to be the initiating event in DCA-induced apoptosis. In conclusion, the bile acid, DCA, has a major impact on apoptotic mechanisms in colonic cells and this may be contributing to its effect as a tumor promoter.

Effects of lactic acid bacteria on the uptake and distribution of the food mutagen Trp-P-2 in mice.

BACKGROUND: Lactic acid bacteria have been reported to have antimutagenic and anticarcinogenic properties in vivo and in vitro. Lactobacillus acidophilus and Bifidobacterium longum have earlier been shown to bind the food mutagen Trp-P-2 in vitro. METHODS: The influence of oral supplementation with L. acidophilus NCFB 1748 and B. longum BB 536 on the uptake and distribution of 14C-labelled Trp-P-2 in several mouse tissues was quantified by liquid scintillation measurements and examined by tape section autoradiography (gives an unbiased qualitative registration of differences in overall tissue distribution) in the present investigation. Furthermore, the effect of 13-naphthoflavone (BNF), a cytochrome P4501A (CYP1A)-inducing agent, on the distribution of 14C-labelled Trp-P-2 was examined. RESULTS: After oral (6 mg/kg; 5 microCi) or iv (1.2 mg/kg; 1 microCi) administration of 14C-labelled Trp-P-2, high levels of radioactivity were observed in the bile, urine and contents of the gastrointestinal tract. Lower levels were present in the liver, lung, kidney, intestines, brown fat, submaxillary salivary gland and thymus. In mice supplemented with lactic acid bacteria there was a significantly decreased level (29%-73%) of radioactivity in the lung, thymus, liver, kidney, submaxillary salivary gland and small intestine as compared with controls. In mice pretreated with BNF, a low but distinct localization of radioactivity in the lung was observed, whereas no similar localization occurred in controls. CONCLUSIONS: The results suggest that (i) there is a decreased bioavailability of the Trp-P-2 in the majority of the tissues examined in bacteria supplemented mice and (ii) there is a low but distinct CYP1A-dependent activation of Trp-P-2 in the lung of BNF-treated mice.

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein.

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.

Bile acid concentrations, cytotoxicity, and pH of fecal water from patients with colorectal adenomas.

In the multistage model of human colorectal tumorigenesis, both genetic and environmental factors play an important role. The identity of the environmental factors involved, however, still remains to be elucidated. As fecal bile acids are proposed as candidates, we compared the concentration of bile acids in fecal water from patients at different risk of developing colorectal cancer. In addition, pH of fecal water as well as its cytotoxicity to HT-29 colonic cells was determined. The high-risk group consisted of individuals diagnosed with one or more (tubulo)villous colorectal adenomas larger than 1 cm in diameter and containing moderate or severe dysplasia (N = 20). Subjects with colorectal adenomas smaller than 1 cm and showing only minor dysplasia were assigned to the medium risk group (N = 19). The control group consisted of persons with normal findings by colonoscopy (N = 25). The results show no significant differences in fecal water bile acid concentrations between the three groups. However, 46% of the observed cytotoxicity is explained in a regression model that includes pH and the concentrations of deoxycholic acid, cholic acid, and ursodeoxycholic acid. The pH of fecal water is found to be significantly lower in the high risk group as compared to the controls, suggesting that a relatively high fecal pH has a protective effect on the development of colorectal adenomas. Although hyperproliferation as a result of cytotoxicity has been suggested to contribute to tumor formation in the colon, the pH-dependent cytotoxicity of bile acids in fecal water was not found to be associated with adenoma formation in the present study.

Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water--potential risk factors for colon cancer.

Several epidemiologic studies have suggested that dairy product intake is associated with a decreased incidence of colon cancer. To determine whether the cytotoxicity and genotoxicity of the aqueous portion of human stool (two potential risk markers for the disease) were affected by a change in dairy product intake, 18 healthy male and female volunteers were randomly divided into two groups. In a crossover design, the volunteers shifted from their normal dairy product-rich diet to a dairy product-free diet. Nutritional analysis of the food consumed during the study period showed a significant decrease in energy intake from 9000 to 7866 kJ/d because of a decreased intake of protein and fat. Carbohydrate and fiber intakes remained unchanged during the intervention. Calcium intake decreased significantly from 1488 to 372 mg/d, with similar significant decreases in phosphate and vitamin D intakes. Cytotoxicity of fecal water, analyzed by the HT-29 cytotoxicity assay, indicated a significant decrease in cell survival from 34% to 20% when dairy products were excluded from the participants' diets. Single-cell gel electrophoresis (COMET assay), used to analyze genotoxicity of fecal waters, indicated no differences brought about by the dietary intervention. In conclusion, our findings indicate that a shift from a dairy product-rich to a dairy product-free diet resulted in a significant effect on an accepted risk marker for colon cancer and may suggest that the mechanism by which dairy products are protective is at the level of tumor promotion rather than initiation.

Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay.

Human faecal waters from 35 healthy non-smoking volunteers (23 from England and 12 from Sweden) consuming their habitual diet were screened for genotoxicity by the single-cell gel electrophoresis (comet) assay using a human colon adenocarcinoma cell line (CACO-2) as the target. Hydrogen peroxide induced DNA damage was categorized as low, intermediate or high for tail moments greater than 5, 17 and 32, respectively: 11 samples were highly genotoxic, four were intermediate, one was low and 19 showed no activity. Endonuclease III treatment significantly increased DNA damage for all except the non-genotoxic faecal waters, suggesting that faecal water genotoxicity may be due, at least in part, to oxidative damage. Faecal water cytotoxicity has previously been attributed to the bile and fatty acid content. In the comet assay no DNA damage was induced by deoxycholate or lithocholate at normal physiological concentrations, suggesting that the genotoxicity of faecal water was due to other substances. Both bile acids induced DNA damage above 300 microM, levels often found in patients with colonic polyps and there was a significant increase in genotoxicity after endonuclease III treatment indicative of oxidative DNA damage.

Effect of lipid extracts of fecal water from colorectal tumor patients on DNA synthesis in Swiss 3T3 cells.

BACKGROUND: Alterations in the proliferative activity of the colonic epithelium play a central role in the development of human colon cancer. It has been suggested that crypt cell hyperproliferation may partly be due to cell regeneration after exposure to cytotoxic surfactants in fecal water. METHODS: The present study determined whether lipid extracts of fecal water from colorectal tumor patients had the capacity to influence DNA synthesis in growing and quiescent Swiss 3T3 cells, at concentrations at which no cytotoxicity was evident. RESULTS: Four of five such extracts significantly increased DNA synthesis in growing cells but were without effect on quiescent cells. Comparison of the effects of the lipid extracts on the cells with that of bile acids, compounds that have received much attention as potential regulators of proliferation, indicated that the effect of the lipid extracts could not solely be attributed to bile acids. CONCLUSIONS: Lipid components of fecal water may also influence cell proliferation through mechanisms other than cytotoxicity.

Fatty acid activation of the peroxisome proliferator activated receptor, a member of the nuclear receptor gene superfamily.

The peroxisome proliferator activated receptor is a member of the steroid receptor gene superfamily, sharing amino acid sequence homology with other receptors and also showing similarities at the level of gene structure. This receptor is activated both by xenobiotic compounds that induce peroxisome proliferation and by fatty acids at physiological concentrations. Upon activation the receptor mediates transcription of responsive genes through binding to peroxisome proliferator response elements. These genes include those encoding peroxisomal enzymes and members of the cytochrome P450 family of drug metabolizing enzymes. It is therefore possible that the peroxisome proliferator activated receptor may play a crucial role in regulating lipid homeostasis.

A systematic analytical chemistry/cell assay approach to isolate activators of orphan nuclear receptors from biological extracts: characterization of peroxisome proliferator-activated receptor activators in plasma.

Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAR/glucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction by gas chromatography alone and in combination with mass spectrometry (GC-MS), indicated the presence of free fatty acids. Individual active components in this mixture were isolated by a final fractionation using high performance liquid chromatography (HPLC). GC-MS analyses of HPLC fractions able to activate the chimeric receptor identified palmitic acid, oleic acid, linoleic acid, and arachidonic acid as endogenous activators of rPPAR. No other activators were identified. This approach is able to specifically extract and identify endogenous activators of PPAR from a complex biological extract and as such may be valuable in the identification of activators of other orphan receptors in the steroid hormone receptor superfamily.

A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily--isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain.

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

Faecal bile acid excretion in children with inflammatory bowel disease.

Faecal bile acid excretion and intestinal transit time were studied in 18 children with inflammatory bowel disease in clinical remission and with normal stools: 16 with ulcerative colitis, two with Crohn's colitis, mean age 14 years (range 10-17 years). Five healthy children, mean age 12.4 years (range 10-17 years), were studied as control subjects. Most patients were taking sulphasalazine, but none were taking steroids. Transit time was determined by carmine and did not differ between groups. Faeces were collected for 72 hours, and faecal water was prepared by centrifugation of faeces at 15,000 x g for two hours. Bile acids in total faeces and faecal water were studied using capillary gas-liquid chromatography-mass spectrometry. Faecal excretion of total bile acids, unconjugated bile acids, and glycine and taurine conjugates were significantly increased in patients as was faecal water excretion of total bile acids, particularly the taurine conjugates and cholic and chenodeoxycholic acids. Total concentrations of bile acids in faeces and faecal water were two to five times higher in patients. The children with inflammatory bowel disease in clinical remission had high excretion and concentration rates of bile acids, especially taurine conjugates, in both total faeces and faecal water, a finding of considerable interest in the pathogenesis of malignancy in these diseases.

Soluble fecal acidic lipids and colorectal epithelial cell proliferation in normal subjects and in patients with colon cancer.

Soluble fecal acidic lipid concentrations and colonic epithelial cell proliferation rates, two biologic markers for colon cancer, were compared in a group of colon cancer patients and a group of healthy controls. No significant difference was found in the concentration of bile acids (155 (136) microM, n = 33, versus 103 (89) microM, n = 19) in the aqueous phase of feces from patients and controls or in proliferation rates between the two groups, the volume density of tritiated thymidine-labeled epithelial cells being 0.153 (0.050), n = 8, for the patients and 0.164 (0.072), n = 11, for the controls. When the dietary intake of three food components known to influence both of the above factors (that is, fat, fiber, and calcium) was ascertained for the year preceding the study, the only significant difference observed was the higher calcium intake in the female patients than in the controls. The authors conclude that it may be premature to rely too heavily on either of the above markers to predict risk for developing this disease.

Formation of mutagenic urinary metabolites from 1-nitropyrene in germ-free and conventional rats: role of the gut flora.

1-Nitropyrene (NP), an environmental pollutant, a potent mutagen and an animal carcinogen, undergoes reduction, acetylation, ring-hydroxylation and conjugation in the rat in vivo to form mutagenic metabolites which are excreted in the urine. In order to investigate the role of the gut flora in the generation of these metabolites, germ-free rats of the AGUS strain, and conventional AGUS rats matched for sex and age, were injected i.p. with NP labelled with 14C. The germ-free rats excreted significantly less of the dose in urine than did the conventional rats. When urines were examined for mutagenicity with the Ames plate incorporation assay, the highest mutagenic activity was seen in the presence of S9 in 8-24 h urine from conventional rats. The conventional urines exceeded the germ-free urines by 10-fold in their content of 6-hydroxy-1-acetamidopyrene (NAAP-6-OH), previously identified as the predominant contributor to the mutagenicity of the urines of rats dosed with NP and excreted mainly as its beta-glucuronide conjugate. Conventional Charles River CD rats treated orally with D-glucaro-1,4-lactone, an inhibitor of beta-glucuronidase activity, excreted somewhat less NP-derived 14C in their urines over 48 h than did matched untreated rats, and their 8-24 h urines contained less than half as much of the mutagenic NAAP-6-OH as was found in the urines of the control rats. These results indicate that the gut flora are necessarily involved in the formation of NAAP-6-OH, and that both nitroreduction and the hydrolysis of glucuronides released for enterohepatic recirculation are essential in generating mutagenic metabolites from NP.

Shift from a mixed to a lactovegetarian diet: influence on acidic lipids in fecal water--a potential risk factor for colon cancer.

Although there have recently been reports in the literature indicating that vegetarian-type diets are protective against the development of human colon cancer, this is still far from clear. It was also recently indicated that the concentration of acidic lipids in the aqueous phase of stool constitutes a risk factor for the development of colon cancer. Thus, we examined the effect of a change from a mixed to a lactovegetarian diet on this fecal variable. The dietary change caused a decrease in the total concentration of soluble fecal fatty acids (4310 +/- 3020 to 1080 +/- 1040 mumol/L, p less than 0.05) and deoxycholic acid (125 +/- 42 to 73 +/- 35 mumol/L, p less than 0.05). However, there was no change in either the total bile acid concentration in (164 +/- 54 to 107 +/- 41 mumol/L) or the cellular toxicity of (0.94 +/- 0.55 to 1.60 +/- 0.63 mumol/L, relative survival) the aqueous phase of stool. Thus, the consumption of a lactovegetarian diet may reduce certain risk factors of potential significance in colon carcinogenesis.