Helicobacter pylori severely reduces expression of DNA repair proteins PMS2 and ERCC1 in gastritis and gastric cancer.

Gastric cancers are the third leading cause of cancer mortality in the world. Helicobacter pylori causes over 60 % of all stomach cancers. Colonization of the gastric mucosa by H. pylori results in increased DNA damage. Repair of DNA damage may also be reduced by H. pylori infection. Reduced DNA repair in combination with increased DNA damage can cause carcinogenic mutations. During progression to gastric cancer, gastric epithelium goes through stages of increasing pathology. Determining the levels of DNA repair enzymes during progression to gastric cancer could illuminate treatment approaches. Our aim is to determine the level of gastric expression of DNA repair proteins ERCC1 (a nucleotide excision repair enzyme) and PMS2 (a mismatch repair enzyme) in the presence of H. pylori infection at successive stages of gastric pathology and in gastric cancers. We analyzed gastric tissues of 300 individuals, including 30 without dyspepsia, 200 with dyspepsia and 70 with gastric cancers. The presence of H. pylori, gastric pathology and expression of DNA repair proteins ERCC1 and PMS2 were evaluated. Infection by H. pylori carrying the common cagA gene reduced median nuclear expression of ERCC1 and PMS2 to less than 20 % and 15 % of normal, respectively, in all pathologic stages preceding cancer. ERCC1 and PMS2 nuclear expression was 0-5 % of normal in gastric cancers. H. pylori can cause deficiency of ERCC1 and PMS2 protein expression. These deficiencies are associated with gastric pathology and cancer. This reduction in DNA repair likely causes carcinogenic mutations. Substantially reduced ERCC1 and PMS2 expression appears to be an early step in progression to H. pylori-induced gastric cancer.

Novel diet-related mouse model of colon cancer parallels human colon cancer.

AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer. METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin. RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile. CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics.

Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer.

BACKGROUND: Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations. PURPOSE: To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer. RESULTS: Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million. CONCLUSIONS: The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.

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.

Cancer and age related colonic crypt deficiencies in cytochrome c oxidase I.

AIM: To investigate whether deficiency of expression of cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer. METHODS: The pattern and level of expression of CcOI in non-neoplastic colonic crypts, and in dysplastic tissues, was assessed using standard immunohistochemical methods. Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason. Tissue samples were also obtained from surgical colonic resections. Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves. Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI. In most crypts the loss was apparent throughout the entire crypt, while in a small minority the loss was segmental. The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous. The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated. Data analyses were performed using SPSS statistical package 17.0. RESULTS: The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age, with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men. CcOI-DC increases after age 40 years, so that between the ages of 40 and 44 years the average frequency of CcOI-DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men. By 80-84 years of age, the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%. The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01. For women over age 60 years, deficiency of CcOI expression is greater in those women who have had a cancer in their colon. The frequency of CcOI-DC, measured in men, increased in tissues adjacent to colon cancer, being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 years and 14.13% ± 0.35% in resected histologically normal tissue of men with cancer in the same age range, P < 0.001. Similar significant differences were noted in older age ranges. The frequency of CcOI-DC crypts in the cecum and sigmoid colon of an individual are significantly correlated, with an R(2) = 0.414 for women and R(2) = 0.528 for men, P < 0.001. This suggests that the factors determining the level of CcOI deficiency act throughout the colon. Most defective crypts are in clusters of two or more, a likely consequence of crypt fission. In the non-neoplastic margins of cancers, crypts are frequently deficient for CcOI, and such crypts may appear in large clusters, some containing more than 100 deficient crypts. CcOI deficiency is also apparent in colon cancers and sometimes involves a large section of the tumor. Overall, CcOI deficient cells can be visualized in segments of crypts, in whole crypts that increase in frequency with age, in crypts undergoing fission, in clusters of crypts where the clusters increase in size with age, in increased frequency near tumors, in large clusters in the intimate margins of tumors, and in the tumors themselves. There is no clear dividing line between early stages that can be considered aspects of aging and later stages that can be considered aspects of the progression to cancer. This ambiguity may reflect a rather general situation leading to adult cancer where the early stages of cellular change appear to be relatively innocuous features of the aging process but over decades may evolve into malignancy. CONCLUSION: CcOI deficient crypts increase in frequency with age, and clusters of deficient crypts are associated with, and may give rise to, colon cancer.

Actinomycetes in relation to taste and odour in drinking water: myths, tenets and truths.

Actinomycetes are a complex group of bacteria present in a wide variety of environments, either as dormant spores or actively growing. Some actinomycetes produce two potent terpenoids (geosmin and 2-methylisoborneol (MIB)) and pyrazines, common causes of drinking water off flavours, and have been implicated in taste and odour episodes. However, isolation from a water source is not evidence that actinomycetes caused a taste and odour event. Dormant spores of actinomycetes may be isolated from aquatic environments in high concentrations, despite production in the terrestrial environment. Similarly, odourous compounds produced by actinomycetes may be produced terrestrially and washed into aquatic environments, with or without the actinomycetes that produced them. Actinomycetes may exist as actively growing mycelium in small, specialized habitats within an aquatic system, but their odourous compounds may influence a wider area. This paper attempts to elucidate the types and activities of actinomycetes that may be found in, or interact with, drinking water supplies.

Odours from pulp mill effluent treatment ponds: the origin of significant levels of geosmin and 2-methylisoborneol (MIB).

Pulp and paper mills are well known for their sharp, sulphurous stack emissions, but the secondary treatment units also can be significant contributors to local odour. This study investigated the source(s) of earthy/musty emissions from a mixed hardwood pulp mill in response to a high local odour. Samples from five sites in the mill over five months were analyzed for earthy/musty volatile organic compounds (VOCs), examined microscopically, and plated for bacteria and moulds. In all cases, activated sludge showed substantial geosmin levels and to a lesser extent 2-methylisoborneol (MIB) at 2000-9000 times their odour threshold concentrations (OTCs). These VOCs were lower or absent upstream and downstream, suggesting that they were produced within the bioreactor. Geosmin and MIB were highest in late summer and declined over winter, and correlated with different operating parameters. Geosmin was most closely coupled with temperature and MIB with nitrogen uptake. Cyanobacteria were present in all sludge samples, but actinomycetes were not found. Gram-negative bacteria and one fungal species isolated from the bioreactor and secondary outfall tested negative for geosmin or MIB. We conclude: (i) geosmin and MIB contribute significantly to airborne odours from this mill, but are diluted below OTC levels at the river; (ii) these VOCs are generated by biota in the activated sludge; and (iii) cyanobacteria are likely primary source(s). The growth of cyanobacteria in activated sludge represents a loss of energy to the heterotrophic population; thus earthy/musty odours may represent a diagnostic for less than optimal conditions.