Carcinogenicity of deoxycholate, a secondary bile acid.

High dietary fat causes increased bile acid secretion into the gastrointestinal tract and is associated with colon cancer. Since the bile acid deoxycholic acid (DOC) is suggested to be important in colon cancer etiology, this study investigated whether DOC, at a high physiologic level, could be a colon carcinogen. Addition of 0.2% DOC for 8-10 months to the diet of 18 wild-type mice induced colonic tumors in 17 mice, including 10 with cancers. Addition of the antioxidant chlorogenic acid at 0.007% to the DOC-supplemented diet significantly reduced tumor formation. These results indicate that a high fat diet in humans, associated with increased risk of colon cancer, may have its carcinogenic potential mediated through the action of bile acids, and that some dietary anti-oxidants may ameliorate this carcinogenicity.

Hydrophobic bile acid-induced micronuclei formation, mitotic perturbations, and decreases in spindle checkpoint proteins: relevance to genomic instability in colon carcinogenesis.

We show, for the first time, that hydrophobic bile acids cause aberrations of the mitotic machinery of colon cells that can give rise to aneuploidy, the chromosomal perturbations common in colon tumors. First, we show that DOC induces a statistically significant fourfold increase in the number of micronuclei in NCM-460 cells (a noncancerous colon cell line) and a threefold increase in the number of micronuclei in binucleated HT-29 colon cancer cells using the cytokinesis block micronucleus assay. Second, we observed mitotic aberrations after DOC treatment, including improper alignment of chromosomes at the metaphase plate, lagging chromosomes during anaphase, anaphase/telophase chromatin bridges, multipolar divisions, and formation of polynucleated cells. It was determined that there was a statistically significant threefold increase in the number of aberrant metaphases after short-term and long-term exposure of HT-29 and HCT-116 cells, respectively. Third, we showed with Western blots and immunohistochemistry that a likely basis for these mitosis-related perturbations included decreased expression of the spindle checkpoint proteins, Mad2, BubR1, and securin. Fourth, results of DOC treatment on nocodazole-challenged cells further indicated deficiencies in activation of the spindle assembly checkpoint. This study provides mechanisms by which hydrophobic bile acids can induce genomic instability in colon epithelial cells.

Expression of bile acid transporting proteins in Barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVES: Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC). METHODS: A total of 101 patients were included in this study. Immunohistochemistry (IHC) and reverse transcriptase (RT)-PCR were used to detect the expression of these transporters at the mRNA and protein levels. RESULTS: Our immunohistochemical studies showed that all three bile acid transporters are expressed in BE glands, but not in squamous epithelium. ASBT was found in the apical border in BE biopsies. The highest frequency of ASBT expression was in patients with nondysplastic BE (9 of 15, 60%), and a progressive loss of ASBT was observed through the stages of dysplasia. ASBT was not detected in EAC (0 of 15). IBABP staining was observed in the cytoplasm of BE epithelial surface cells. Expression of IBABP was found in 100% of nondysplastic BE (14 of 14), in 93% of low-grade dysplasia (LGD, 15 of 16), in 73% of high-grade dysplasia (HGD, 10 of 14), and in 33% of EAC (5 of 15). MRP3 was expressed in the basolateral membrane in 93% of nondysplastic BE (13 of 14), in 60% of LGD (10 of 16), and in 86% of HGD (11 of 13). Only weak MRP3 staining was detected in EAC biopsies (5 of 15, 33%). In addition, RT-PCR studies showed increased expression of mRNA coding for ASBT (6.1x), IBABP (9.1x), and MRP3 (2.4x) in BE (N=13) compared with normal squamous epithelium (N=15). Significantly increased mRNA levels of IBABP (10.1x) and MRP3 (2.5x) were also detected in EAC (N=21) compared with normal squamous epithelium. CONCLUSIONS: We found that bile acid transporters expression is increased in BE tissue at the mRNA and protein levels and that expression of bile acid transporter proteins decreased with progression to cancer.

Activation of the interleukin-6/STAT3 antiapoptotic pathway in esophageal cells by bile acids and low pH: relevance to barrett's esophagus.

OBJECTIVES: The molecular factors contributing to the development of Barrett's esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-x(L) and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway. MATERIALS AND METHODS: Immunohistochemistry was used to assess levels of phosphorylated STAT3 in esophageal tissue samples from BE patients with different grades of dysplasia. Seg-1 esophageal adenocarcinoma cells were evaluated for STAT3 activation and IL-6 and Bcl-x(L) expression by molecular biology techniques, including Western blot, reverse transcription-PCR, and ELISA after exposure to control media (pH 7.4), media supplemented with a 0.1 mmol/L bile acid cocktail with media at pH 4 or media at pH 4 with bile acid cocktail. RESULTS: Immunohistochemical analysis showed that activated, phosphorylated STAT3 is expressed in nuclei of dysplastic BE and cancer tissues. Treatment of Seg-1 cells with media containing bile acid cocktail and acidified to pH 4 resulted in increased activation of STAT3, IL-6 secretion, and increased expression of Bcl-x(L). Inhibition of the STAT3 pathway using STAT3 small interfering RNA or Janus-activated kinase inhibitor resulted in increased apoptosis. CONCLUSIONS: The IL-6/STAT3 antiapoptotic pathway is induced by short exposure to bile acid cocktail and low pH. This alteration, if persistent in vivo, may underlie the development of dysplastic BE and tumor progression.

Role of c-Myc in intestinal tumorigenesis of the ApcMin/+ mouse.

The c-MYC oncogene plays an important role in tumorigenesis and is commonly highly expressed in gastrointestinal cancers. In colon cells, c-MYC is regulated by the adenomatous polyposis coli (Apc) tumor suppressor gene. Multiple intestinal neoplasia (ApcMin/+ or Min) mice are heterozygous for a truncating Apc mutation and serve as a model of familial adenomatous polyposis (FAP) disease. To study the role of c-Myc in the mutant Apc-mediated colon tumorigenesis, we have developed a transgenic mouse with the conditional deletion of the floxed c-Myc alleles in the intestinal crypts of ApcMin/+ mice (ApcMin/+; c-Mycfl/fl). The floxed c-Myc deletion was initiated via a Cre recombinase controlled by the intestine-specific transcriptional regulatory elements of the liver fatty acid-binding protein gene (Fabpl4xat-132). Fabpl4xat-132-mediated Cre expression and recombination resulted in a two-fold decrease in c-MYC protein expression with no effect on intestinal tract morphology. Small intestinal tumorigenesis was significantly suppressed throughout the small intestinal tract of ApcMin/+; c-Mycfl/fl mice compared to c-Myc wild type littermates. In ApcMin/+; c-Mycfl/fl mice, the intestinal apoptosis was higher in the areas of the small intestine with the decreased c-Myc protein expression (P=0.0016, compared to their littermates with the wild type c-Myc). Thus, conditional inactivation of c-Myc, mediated by Fabpl4xat-132-driven Cre-recombinase, suppresses Apc-dependent intestinal tumorigenesis in adult ApcMin/+ mice, without apparent effect on normal intestinal mucosa.

Unique dietary-related mouse model of colitis.

BACKGROUND: A high-fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high-fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD. METHODS: Wild-type B6.129 mice were fed an AIN-93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses. RESULTS: Mice fed the DOC-supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin-proteasome pathway showed altered expression in the DOC-fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding. CONCLUSIONS: The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC-fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.

Reduced Pms2 expression in non-neoplastic flat mucosa from patients with colon cancer correlates with reduced apoptosis competence.

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

Crypt-restricted loss and decreased protein expression of cytochrome C oxidase subunit I as potential hypothesis-driven biomarkers of colon cancer risk.

There is an increasing demand for the development of intermediate biomarkers to assess colon cancer risk. We previously determined that a live cell bioassay, which assesses apoptosis resistance in the nonneoplastic colonic mucosa, detects approximately 50% of patients with colon cancer. A hypothesis-driven biomarker that reflects apoptosis resistance in routine formalin-fixed, paraffin-embedded tissue would be easier to use. Cytochrome c oxidase is a critical enzyme that controls mitochondrial respiration and is central to apoptosis. We did an immunohistochemical study of cytochrome c oxidase subunit I expression in 46 colonic mucosal samples from 16 patients who had undergone a colonic resection. These included five patients without evidence of colonic neoplasia (three normal and two diverticulitis), three patients with tubulovillous adenomas, and eight patients with colonic adenocarcinomas. Analysis of aberrancies in expression of cytochrome c oxidase subunit I showed that, compared with nonneoplasia, the patients with neoplasia had a higher mean incidence of crypts having decreased expression (1.7 versus 22.8, P = 0.03) and a higher mean incidence having crypt-restricted loss (0.6 versus 3.2, P = 0.06). The percentage with segmented loss was low and was similar in the two groups. Combining these results, the mean % normal (i.e., with none of the three types of abnormality) was 96.7 in nonneoplasia versus only 73.2 in patients with neoplasia (P = 0.02). It should be noted that a defect in cytochrome c oxidase subunit I immunostaining was not detected in all biopsy samples from each patient for whom some abnormality was found, indicating a "patchiness" in the cytochrome c oxidase subunit I field defect. As a result of this "patchiness," the increased variability in the incidence of crypt-restricted loss of cytochrome c oxidase subunit I expression was a statistically significant feature of the neoplasia group. Crypt-restricted loss of cytochrome c oxidase subunit I has not been previously reported in colonic mucosa and is presumably the result of a crypt-restricted stem cell mutation. Decreased cytochrome c oxidase subunit I expression also significantly correlated with apoptosis resistance, a factor known to contribute to carcinogenesis. The results suggest, however, that aberrant cytochrome c oxidase subunit I expression may be a better biomarker than loss of apoptosis competence for increased colon cancer risk.

Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

Increased expression and secretion of interleukin-6 in patients with Barrett's esophagus.

PURPOSE: Barrett's esophagus (BE) is a common premalignant lesion of the distal part of the esophagus that arises as a consequence of chronic duodenogastroesophageal reflux. Interleukin (IL)-6 is a pleiotropic cytokine that regulates immune defense mechanisms and hematopoiesis. In addition, IL-6 may also be involved in malignant transformation and tumor progression. IL-6 has been shown to inhibit apoptosis. The major aim of this study was to evaluate expression of IL-6 in BE at the protein and mRNA levels. In addition, we tested whether proteins that are associated with IL-6 signaling, phosphorylated signal transducer and activator of transcription 3 and two antiapoptotic proteins, Bcl-x(L) and Mcl-1, are also expressed in the same tissues. EXPERIMENTAL DESIGN: Biopsies of duodenum, BE, and squamous epithelium were evaluated by using a human cytokine protein array, ELISA, real-time PCR, and immunohistochemistry. RESULTS: Increased IL-6 levels were found to be secreted from BE tissue compared with duodenum or squamous epithelium from sites adjacent or 5 cm away from the BE lesion. IL-6 mRNA was also elevated in BE compared with duodenum or squamous epithelium in five of seven patients. Immunohistochemical studies confirmed IL-6 expression in intestinal glandular epithelium in BE tissue. Activated signal transducer and activator of transcription 3, Mcl-1, and Bcl-x(L) are present at higher levels in BE glands, with lower levels being found in duodenum or squamous epithelium CONCLUSIONS: These data, taken together, suggest that elevated IL-6 levels in BE may contribute to the development of apoptosis resistance, thereby placing this epithelium at higher risk of developing malignancy.

Molecular and cellular pathways associated with chromosome 1p deletions during colon carcinogenesis.

Chromosomal instability is a major pathway of sporadic colon carcinogenesis. Chromosome arm 1p appears to be one of the "hot spots" in the non-neoplastic mucosa that, when deleted, is associated with the initiation of carcinogenesis. Chromosome arm 1p contains genes associated with DNA repair, spindle checkpoint function, apoptosis, multiple microRNAs, the Wnt signaling pathway, tumor suppression, antioxidant activities, and defense against environmental toxins. Loss of 1p is dangerous since it would likely contribute to genomic instability leading to tumorigenesis. The 1p deletion-associated colon carcinogenesis pathways are reviewed at the molecular and cellular levels. Sporadic colon cancer is strongly linked to a high-fat/low-vegetable/low-micronutrient, Western-style diet. We also consider how selected dietary-related compounds (eg, excess hydrophobic bile acids, and low levels of folic acid, niacin, plant-derived antioxidants, and other modulatory compounds) might affect processes leading to chromosomal deletions, and to the molecular and cellular pathways specifically altered by chromosome 1p loss.

Maspin is a deoxycholate-inducible, anti-apoptotic stress-response protein differentially expressed during colon carcinogenesis.

Increased maspin expression in the colon is related to colon cancer risk and patient survival. Maspin is induced by the hydrophobic bile acid, deoxycholate (DOC), which is an endogenous carcinogen and inducer of oxidative stress and DNA damage in the colon. Persistent exposure of colon epithelial cells, in vitro, to high physiologic levels of DOC results in increased constitutive levels of maspin protein expression associated with the development of apoptosis resistance. When an apoptosis-resistant colon epithelial cell line (HCT-116RC) developed in the authors' laboratory was treated with a maspin-specific siRNA probe, there was a statistically significant increase in apoptosis compared to treatment with an siRNA control probe. These results indicate, for the first time, that maspin is an anti-apoptotic protein in the colon. Immunohistochemical evaluation of maspin expression in human colonic epithelial cells during sporadic colon carcinogenesis (131 human tissues evaluated) indicated a statistically significant increase in maspin protein expression beginning at the polyp stage of carcinogenesis. There was no statistically significant difference in maspin expression between hyperplastic/adenomatous polyps and colonic adenocarcinomas. The absence of "field defects" in the non-neoplastic colonic mucosa of patients with colonic neoplasia indicates that maspin may drive the growth of tumors, in part, through its anti-apoptotic function.

Deoxycholate, an endogenous cytotoxin/genotoxin, induces the autophagic stress-survival pathway: implications for colon carcinogenesis.

We report that deoxycholate (DOC), a hydrophobic bile acid associated with a high-fat diet, activates the autophagic pathway in non-cancer colon epithelial cells (NCM-460), and that this activation contributes to cell survival. The DOC-induced increase in autophagy was documented by an increase in autophagic vacuoles (detected using transmission electron microscopy, increased levels of LC3-I and LC3-II (western blotting), an increase in acidic vesicles (fluorescence spectroscopy of monodansycadaverine and lysotracker red probes), and increased expression of the autophagic protein, beclin-1 (immunohistochemistry/western blotting). The DOC-induced increase in beclin-1 expression was ROS-dependent. Rapamycin (activator of autophagy) pre-treatment of NCM-460 cells significantly (P < .05) decreased, and 3-MA (inhibitor of autophagy) significantly (P < .05) increased the cell loss caused by DOC treatment, alone. Rapamycin pre-treatment of the apoptosis-resistant colon cancer cell line, HCT-116RC (developed in our laboratory), resulted in a significant decrease in DOC-induced cell death. Bafilomycin A(1) and hydroxychloroquine (inhibitors of the autophagic process) increased the DOC-induced percentage of apoptotic cells in HCT-116RC cells. It was concluded that the activation of autophagy by DOC has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents.

Haploinsufficiency of the cdc2l gene contributes to skin cancer development in mice.

The Cdc2L gene encodes for the cyclin-dependent kinase 11 (CDK11) protein. Loss of one allele of Cdc2L and reduced CDK11 expression has been observed in several cancers, implicating its association with carcinogenesis. To directly investigate the role of CDK11 in carcinogenesis, we first generated cdc2l haploinsufficient mice by gene trap technology and then studied the susceptibility of these gene-trapped (cdc2l(GT)) mice to chemical-mediated skin carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage skin carcinogenesis model. Wild-type and cdc2l(GT) mice were subjected to a single topical application of initiation by DMBA and promotion twice a week for 19 weeks with TPA. At 19 weeks, 70% of the cdc2l(GT) mice and 60% of the cdc2l+/+ mice developed benign papillomas. However, there was an overall 3-fold increase in the average number of tumors per mouse observed in cdc2l(GT) mice as compared with cdc2l+/+ mice. There was also an increased frequency of larger papillomas in cdc2l(GT) mice. By using the polymerase chain reaction-restriction fragment length polymorphism assay, we found A to T transversion mutations at the 61st codon of H-ras gene in the papilloma tissue of both cdc2l(GT) mice and cdc2l+/+ mice. Ki-67 staining revealed increased proliferation in the papillomas of cdc2l(GT) (77.75%) as compared with cdc2l+/+ (30.84%) tumors. These studies are the first to show that loss of one allele of cdc2l gene, encoding CDK11, facilitates DMBA/TPA-induced skin carcinogenesis in vivo.

A novel dietary-related model of esophagitis and Barrett's esophagus, a premalignant lesion.

Barrett's esophagus (BE) is a premalignant lesion in which columnar epithelium (containing goblet cells) replaces esophageal squamous cells. Previous evidence suggested that hydrophobic bile acids and zinc deficiency each play a role in BE development. We fed wild-type C57BL/6 mice a zinc-deficient diet containing the hydrophobic bile acid, deoxycholic acid for various times up to 152 days. All mice fed this diet developed esophagitis by 69 days on the diet and 63% of the mice on this diet for 88 to 152 days also developed a BE-like lesion. Esophageal tissues showed thickened mucosa, increased proliferation, and increased expression of markers associated with oxidative and nitrosative stress. The newly formed BE-like lesions expressed Mucin-2, a marker of columnar differentiation. They also showed translocation of the p65 subunit of nuclear factor-kappaB and beta -catenin to the nucleus and typical histological changes associated with BE lesions. This mouse model of esophagitis and BE is expected to contribute to a deeper understanding of BE pathogenesis and to strategies for prevention of BE progression to cancer.

Deoxycholate-induced colitis is markedly attenuated in Nos2 knockout mice in association with modulation of gene expression profiles.

Nos2 knockout mice were compared to wild-type mice for susceptibility to colitis in response to a diet supplemented with deoxycholate, a bile acid increased in the colon of individuals on a high-fat diet. Wild-type mice fed a fat-related diet, supplemented with 0.2% DOC, develop colonic inflammation associated with increases in nitrosative stress, proliferation, oxidative DNA/RNA damage, and angiogenesis, as well as altered expression of numerous genes. However, Nos2 knockout mice fed a diet supplemented with deoxycholate were resistant to these alterations. In particular, 35 genes were identified whose expression was significantly altered at the mRNA level in deoxycholate-fed Nos2(+/+) mice but not in deoxycholate-fed Nos2(-/-) mice. Some of these alterations in NOS2-dependent gene expression correspond to those reported in human inflammatory bowel disease. Overall, our results indicate that NOS2 expression is necessary for the development of deoxycholate-induced colitis in mice, a unique dietary-related model of colitis.

Bile acids in combination with low pH induce oxidative stress and oxidative DNA damage: relevance to the pathogenesis of Barrett's oesophagus.

BACKGROUND: Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA. OBJECTIVE: To test the hypothesis that bile acids and gastric acid, two components of refluxate, can induce oxidative stress and oxidative DNA damage. METHODS: Oxidative stress was evaluated by staining Barrett's oesophagus tissues with different degrees of dysplasia with 8-hydroxy-deoxyguanosine (8-OH-dG) antibody. The levels of 8-OH-dG were also evaluated ex vivo in Barrett's oesophagus tissues incubated for 10 min with control medium and medium acidified to pH 4 and supplemented with 0.5 mM bile acid cocktail. Furthermore, three oesophageal cell lines (Seg-1 cells, Barrett's oesophagus cells and HET-1A cells) were exposed to control media, media containing 0.1 mM bile acid cocktail, media acidified to pH 4, and media at pH 4 supplemented with 0.1 mM bile acid cocktail, and evaluated for induction of reactive oxygen species (ROS). RESULTS: Immunohistochemical analysis showed that 8-OH-dG is formed mainly in the epithelial cells in dysplastic Barrett's oesophagus. Importantly, incubation of Barrett's oesophagus tissues with the combination of bile acid cocktail and acid leads to increased formation of 8-OH-dG. An increase in ROS in oesophageal cells was detected after exposure to pH 4 and bile acid cocktail. CONCLUSIONS: Oxidative stress and oxidative DNA damage can be induced in oesophageal tissues and cells by short exposures to bile acids and low pH. These alterations may underlie the development of Barrett's oesophagus and tumour progression.

Epidermal growth factor reduces hepatic sequelae in experimental necrotizing enterocolitis.

BACKGROUND AND AIM: Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We recently demonstrated that the gut/liver axis plays an important role in the pathophysiology of NEC through the release of inflammatory mediators into the intestinal lumen. We have also shown that supplementation of formula with epidermal growth factor (EGF) dramatically decreases ileal pathology associated with experimental NEC. In this study, we examined the effects of EGF on the liver portion of the gut/liver axis in the neonatal rat model of NEC. METHODS: Newborn rats were divided into three experimental groups, NEC, hand-fed with growth-factor free formula; NEC + EGF, hand-fed with formula supplemented with 500 ng/ml rat EGF; or DF, dam fed. All animals were exposed to asphyxia and cold stress twice daily for 4 days to develop NEC. RESULTS: EGF receptor expression was significantly (p

Bile acids induce ileal damage during experimental necrotizing enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats. METHODS: Premature rats were fed with cow's milk-based formula and subjected to asphyxia and cold stress to develop NEC. Jejunal and ileal luminal BAs, portal blood BAs, and messenger RNA and protein for the apical sodium-dependent bile acid transporter, the ileal bile acid binding protein, and the heteromeric organic solute transporter (Ostalpha/Ostbeta)were evaluated. RESULTS: Ileal luminal BAs levels were increased significantly during disease development and the removal of ileal BAs significantly decreased the incidence and severity of disease. Furthermore, when NEC was reduced via treatment with epidermal growth factor (EGF), BA levels were reduced significantly. Jejunal luminal BA levels were similar between animals with NEC and controls, but portal/ileal luminal BA ratios were decreased significantly in animals with NEC. The apical sodium-dependent bile acid transporter was up-regulated at the site of injury in animals with NEC and decreased after EGF treatment; however, the ileal bile acid binding protein was up-regulated only in the NEC and EGF group. Ostalpha/Ostbeta expression was low in all groups, and only slightly increased in the NEC group. CONCLUSIONS: These data strongly suggest that BAs play a role in the development of ileal damage in experimental NEC and that alterations in BA transport in the neonatal ileum may contribute to disease development.