Organosulfur compounds alone or in combination with vitamin C protect towards N-nitrosopiperidine- and N-nitrosodibutylamine-induced oxidative DNA damage in HepG2 cells.

The aim of this study was to investigate the protective effects of organosulfur compounds (OSCs) alone or in combination with vitamin C towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced oxidative DNA damage in the single cell gel electrophoresis (SCGE)/HepG2 assay. Diallyl sulfide (DAS) did not protect against NDBA-induced oxidized purines, but it reduced the oxidized purines induced by NPIP (1 microM, 29%). The formation of formamidopyridine-DNA glycosylase (Fpg) sensitive sites induced by NPIP or NDBA was prevented by dipropyl sulfide (DPS) at concentrations of 1-10 microM (55-24% and 66-15%, respectively). The maximum reduction of the formation of Fpg sensitive sites induced by NPIP was observed at the highest concentration of diallyl disulfide (DADS) (2.5 microM, 38%). However, the oxidative DNA damage induced by NDBA was strongly reduced by DADS at the lowest concentration tested (0.1 microM, 92%). The oxidative DNA damage induced by NPIP or NDBA was prevented by all the concentrations of dipropyl disulfide (DPDS) (0.1-2.5 microM, 59-80% and 51-64%, respectively). DADS and DPDS, in combination with vitamin C showed an overall protective effect towards the formation of Fpg sensitive sites induced by NPIP and NDBA. However, the contribution of OSCs to the protective effect found in combined experiments might not be relevant, because it could be caused by vitamin C alone. One feasible mechanism by which OSCs exert their protective effects towards N-nitrosamine-induced oxidative DNA damage could be by modulation of phase I and II enzyme activities. DADS and DPDS (0.1-2.5 microM) exerted greater inhibition on CYP2E1 and CYP2A6 activity than DAS and DPS (1-50 microM). However, DAS and DADS (1 microM) exerted greater inhibition on CYP1A1 activity than DPS and DPDS (1 microM). DAS/DPS (50 microM) and DADS (2.5 microM) exerted a moderate increase of UDP-glucuronyltransferase (UGT1A4) activity, whereas DPDS (2.5 microM) had the most pronounced effect.

A GWAS meta-analysis from 5 population-based cohorts implicates ion channel genes in the pathogenesis of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) shows genetic predisposition, however, large-scale, powered gene mapping studies are lacking. We sought to exploit existing genetic (genotype) and epidemiological (questionnaire) data from a series of population-based cohorts for IBS genome-wide association studies (GWAS) and their meta-analysis. METHODS: Based on questionnaire data compatible with Rome III Criteria, we identified a total of 1335 IBS cases and 9768 asymptomatic individuals from 5 independent European genotyped cohorts. Individual GWAS were carried out with sex-adjusted logistic regression under an additive model, followed by meta-analysis using the inverse variance method. Functional annotation of significant results was obtained via a computational pipeline exploiting ontology and interaction networks, and tissue-specific and gene set enrichment analyses. KEY RESULTS: Suggestive GWAS signals (P ≤ 5.0 × 10-6 ) were detected for 7 genomic regions, harboring 64 gene candidates to affect IBS risk via functional or expression changes. Functional annotation of this gene set convincingly (best FDR-corrected P = 3.1 × 10-10 ) highlighted regulation of ion channel activity as the most plausible pathway affecting IBS risk. CONCLUSION & INFERENCES: Our results confirm the feasibility of population-based studies for gene-discovery efforts in IBS, identify risk genes and loci to be prioritized in independent follow-ups, and pinpoint ion channels as important players and potential therapeutic targets warranting further investigation.

Effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells.

The aim of this study was to evaluate the effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced DNA damage (DNA strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2), using the Comet assay. Treatment with hydrogen peroxide (H(2)O(2)) increased the levels of DNA strand breaks and oxidized purine and pyrimidine bases, in a concentration and time dependent manner. Organosulfur compounds (OSCs) reduced DNA strand breaks induced by H(2)O(2). In addition, OSCs also decreased the levels of oxidized pyrimidines. However, none of the OSCs tested reduced the levels of oxidized purines. Isothiocyanates compounds (ITCs) and vitamin C showed protective effects towards H(2)O(2)-induced DNA strand breaks and oxidized purine and pyrimidine bases. The results indicate that removal of oxidized purine and pyrimidine bases by ITCs was more efficient than by OSCs and vitamin C. Our findings suggest that OSCs, ITCs and vitamin C could exert their protective effects towards H(2)O(2)-induced DNA strand breaks and oxidative DNA damage by the free radical-scavenging efficiency of these compounds.

Protective effects of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. N-Nitrosopyrrolidine (NPYR) and N-nitrosodimethylamine (NDMA) incubated with formamidopyrimidine-DNA glycosylase (Fpg), caused a significant increase in oxidative DNA damage in comparison to the solvent control, the lowest effective concentrations, being 5 and 27 mM, respectively. NPYR exerted greater genotoxic effects than NDMA. None of the organosulfur compounds (OSCs) concentrations tested in presence or absence of Fpg enzyme, caused DNA damage per se. OSCs (diallyl sulfide, DAS and dipropyl sulfide, DPS, 1-50 microM; diallyl disulfide, DADS and dipropyl disulfide, DPDS, 1-5 microM) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner when HepG2 cells were simultaneously treated with OSCs and N-nitrosamines. The effect of NPYR was attenuated by about 61-67%, respectively, with the highest concentration of DAS (50 microM) and DADS (5 microM). The protective effect of DADS (5 microM, 66%) was higher than DAS (50 microM, 53%) towards NDMA-induced oxidative DNA damage. A concentration of 50 microM DPS and 5 microM DPDS led to a 65-63% and 59-65% reduction in NPYR/NDMA-induced oxidative DNA damage, respectively. Our results indicate that OSCs protect human-derived cells against the oxidative DNA damaging effect of NPYR and NDMA, two carcinogenic compounds which occur in the environment.

Fecal diglycerides as selective endogenous mitogens for premalignant and malignant human colonic epithelial cells.

Diglycerides (DGs) have been found in fecal extracts at concentrations which induce mitogenesis of adenoma and some carcinoma cells but not normal cells in primary culture. DGs containing stearic, oleic, palmitic, and myristic acid side chains were found in fecal extracts from each of eight subjects. Synthetic 1,2-DGs, containing the fatty acids found in endogenous fecal DGs, induced mitogenesis in cultures of premalignant cells from each of 13 adenomas, covering all histological classes, and in cultures from two of four carcinomas. The potent adenoma mitogen, dimyristin, had no mitogenic activity on cultures of normal colonic epithelial cells from seven different subjects. These results suggest DGs may act as endogenous mitogens in the development of human colon cancer. The extent of adenoma mitogenesis was correlated with the chain length of the saturated R-groups: 16 greater than 14 greater than 12 greater than 10 greater than 8 much greater than 18. DGs with oleic acid residues, C18:1, were among the most active, while substitution of even one fatty acid residue with a stearic acid residue, C18:0, reduced or eliminated mitogenic activity. Dimyristin also induced enhanced levels of urokinase secretion from carcinoma cells, in parallel to the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. These results imply that DGs found in the colon induce a selective growth of benign colonic tumors and some carcinomas, and may enhance the invasive capacity of carcinomas, while leaving normal cells unaffected.

Calcium enhances the hemolytic action of bile salts.

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.

Metabolites in the developing rat liver--a proton nuclear magnetic resonance spectroscopic study.

We have used 1H-NMR spectroscopy in vitro to investigate metabolite changes in the rat liver in the first 21 days of life. The principle findings are firstly that betaine, a metabolite of choline, was relatively low (1-2 mumol/g) on days 1-7, then rose sharply to 5-6 mumol/g by day 19, whereas approximately reciprocal changes occurred in taurine levels. Secondly the lactate levels were remarkably low (0.1-0.8 mumol/g) on days 1-7. Changes in two other choline derivatives, phosphocholine (PC) and glycerophosphorylcholine (GPC) are also reported. The results are discussed in the context of the origin of these metabolites in the neonatal period, their levels in the adult (180 day-old) rat and the significance of the measured changes in metabolite levels during liver development.

The role of probiotic bacteria in cancer prevention.

Colorectal cancer is one of the most important causes of cancer morbidity and mortality in western countries [1]. A myriad of healthful effects have been attributed to the probiotic lactic acid bacteria; perhaps the most controversial remains that of anticancer activity. There is no direct experimental evidence for cancer suppression in humans as a result of consumption of lactic cultures in fermented or unfermented dairy products. However, there is a wealth of indirect evidence, based largely on laboratory studies, in the literature and this will be summarised in the present paper.

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.

Cellular toxicity of fecal water depends on diet.

To determine whether concentrations of potentially toxic lipids in the aqueous phase of human stool are responsive to changes in dietary fat, calcium, and fiber, 20 male volunteers were placed on a high-fat, low-calcium, low-fiber or a low-fat, high-calcium, high-fiber diet for 4 days. To assess toxicity of the fecal fractions, we examined the ability of fecal supernatants to lyse human erythrocytes. Bile acid concentrations in fecal water from the low-fat group were reduced significantly from 180 +/- 60 microM to 100 +/- 70 microM; in the high-fat group, increased from 190 +/- 60 microM to 250 +/- 100 microM. Erythrocyte lysis was 76% for the high-fat group, 37% for the low-fat group. There was a significant weak correlation between aqueous bile acid concentration and cell lysis. Results suggest that diet can influence concentrations of detergents in the aqueous phase of human stool and the potential toxicity of this fraction to cell membranes.

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.

The effect of scandium on the tissue distribution of Ga-67 in normal and tumor-bearing rodents.

In rats and mice the intravenous administration of scandium before or with Ga-67 produces an increase in Ga-67 excretion and bone deposition, coupled with pronounced decreases in the uptake of Ga-67 in soft tissues. These effects result from the blocking by scandium of Ga-67 plasma-protein binding sites, which forces Ga-67 into an unbound or loosely bound state. This increases Ga-67 excretion and bone deposition, which in turn acts to produce greatly reduced Ga-67 uptake in soft tissues. When tumor-bearing rats and mice are administered scandium, similar effects occur, but the uptake of Ga-67 by tumor tissue remains unchanged. This suggests that Ga-67 enters tumor and normal soft tissues by different routes. With tumor, an unbound or loosely bound form of gallium is primarily involved, whereas with normal soft tissues this route is apparently of minor importance.

Studies of the in vivo entry of Ga-67 into normal and malignant tissue.

Previous studies of the effect of scandium on the tissue distribution of Ga-67 suggest that Ga-67 makes its initial in vivo entry into normal and malignant tissues by different routes. (Scandium blocking of plasma protein Ga-67 binding increased Ga-67 excretion, decreased its uptake in normal tissues, but had little effect on rodent tumors.) In further studies we have used other methods to alter the plasma binding of Ga-67. Iron saturation of plasma produced effects on Ga-67 tissue distribution similar to those observed with scandium. On the other hand, increasing Ga-67 plasma binding through induction of anemia and administration of apotransferrin produced the reverse of the effects observed with scandium and iron. We conclude that the initial in vivo entry of Ga-67 into tumor tissue involves mainly an unbound or loosely bound form of Ga-67, whereas its uptake by normal soft tissues is strongly promoted by its binding to transferrin.

Studies of the vivo uptake of Ga-67 by an experimental abscess: concise communication.

The blocking of Ga -67 plasma protein-binding sites-by administration of scandium citrate, ferric citrate, and a colloidal hydrous ferric oxide preparation-reduced the uptake of Ga-67 in normal soft tissues and also that in the viable portion of an experimental abscess. On the other hand, enhancement of Ga-67 plasma protein binding by administration of rabbit apotransferrin increased Ga-67 uptake in both abscess and normal soft tissues. These results indicate that the pathways of Ga-67 from blood into inflammatory processes and normal soft tissues may be similar. However, when Ga-67 plasma protein binding was increased by inducing anemia, a markedly decreased Ga-67 uptake in the abscess resulted, whereas uptake in normal soft tissue was still elevated. It is possible that the discrepancy between the effects of apotransferrin and anemia on abscess-tissue uptake of Ga-67 resulted from a secondary effect produced by anemia, i.e., a decrease in the macrophage population in the abscess. Taken as a whole, the results obtained suggest that Ga-67 leaves the blood and enters inflammatory lesions by pathways that are probably quite different from those in a soft-tissue tumor, and that the routes for abscesses may be similar to those occurring in normal soft tissues.

A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples.

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtractive enrichment procedure for the cloning of differentially expressed genes. In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting material is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification step was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method, and also eliminated the risk of carry-over contamination between iterative steps of subtraction and PCR amplification. The modified protocol was evaluated in monitoring differences in gene expression in (i) a rat system consisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer.

Carcinogenesis in the colon: interaction between luminal factors and genetic factors.

At last, inroads are beginning to be made into the hitherto unknown and complex area of gene-environment interactions in the colon. Interestingly, many of the studies to date would suggest: that the Apc gene is a target for such interactions; that luminal factors can regulate the level of cellular proteins of central importance in the control of cell growth/arrest; and that some of the newly discovered members of the nuclear hormone receptor superfamily may be mediating gene-environment interactions in the colon. This is a very exciting area and will presumably be the subject of intense research in the near future. By characterizing the dietary/luminal factors that interact with the genes implicated in tumour development in the colon, we will reach another level of certainty regarding the dietary components responsible for tumour formation and their underlying mechanisms. It is gratifying to see at last the fields of epidemiology and molecular biology begin to overlap, and without doubt results from this new area of research will give a new and better status to the field of making dietary recommendations to decrease the risk of developing colorectal cancer.

Interactions between the environment and genes in the colon.

Colorectal cancer is now widely accepted to be the result of an accumulation of mutations in specific genes controlling cell division, apoptosis and DNA repair. There is also a wealth of evidence that dietary factors, including dietary fat and fibre, influence the development of colorectal cancer. However, until recently, there has been little understanding of how these dietary factors and genetic factors interact. It is generally believed that this interaction is mediated in part by events occurring in the lumen of the large bowel. By characterizing the dietary/luminal factors that interact with the genes implicated in tumour development in the colon, a new understanding of colorectal cancer is likely to emerge, hopefully leading to the formulation of dietary recommendations to decrease the risk of this cancer.

Lactobacilli, anticarcinogenic activities and human intestinal microflora.

Lactobacilli belong to the normal oropharyngeal and intestinal microflora in humans. These microorganisms contribute to the stabilization of the microflora and maintain the colonization resistance against pathogens. Lactobacilli have been used as dietary supplements in order to prevent gastrointestinal disturbances. Claims have been made that certain strains of lactobacilli possibly exert anticarcinogenic activities. The activity of bacterial enzymes, implicated in colon carcinogenesis may be elevated by a high meat, Western-type diet. Supplements of Lactobacillus acidophilus decreased these levels in both rats and humans. Colon cancer patients given L. acidophilus fermented milk showed a significant increase both in numbers of intestinal lactobacilli and dietary calcium intake, while decreasing trends in levels of both soluble faecal bile acids and faecal bacterial enzymes, two risk makers for colon cancer, were observed. In vitro studies have revealed that lactobacilli and other lactic acid bacteria have the ability to absorb cooked food mutagens. Recent studies in humans have shown that intake of L. acidophilus significantly reduced the mutagen excretion after consumption of fried meat. Several mechanisms by which lactobacilli might exert anticarcinogenic effects are discussed. Thus, certain strains of lactobacilli might lower the colon cancer risk in humans.

Probiotic mixture decreases DNA adduct formation in colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2,3-b]indole in a human-flora associated mouse model.

Consumption of probiotic bacteria such as bifidobacteria has been shown to reduce the risk of colon cancer in animal models. However, the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans. The aim of the study was to examine whether the probiotic mixture, which consisted of Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus, could decrease DNA adduct formation induced by 2-amino-9H-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) in the colonic epithelium of a human-flora-associated (HFA) mouse model. Ten HFA mice were divided into a control group (n=4) and a probiotic group (n=6). The control group was administered AAC for 3 days and sacrificed 24 h after the last dose. The probiotic group was administered the probiotic mixture for 2 weeks prior to the administration of AAC. Analysis of DNA adducts with the 32P-high-performance liquid chromatography method was performed on stomach, jejunum and colonic epithelium, representing direct exposure sites of AAC, and colon wall, liver and kidney, representing indirect exposure sites. The mean level of the DNA adducts in the colonic epithelium of the probiotic group was significantly lower than that of control group, while the mean levels at the other sites did not differ significantly between the groups. The results indicated that the probiotic mixture could decrease the DNA adduct formation in the colonic epithelium induced by AAC.

Association between asthenia and nutritional status, lean body mass, anemia, psychological status, and tumor mass in patients with advanced breast cancer.

Sixty-four consecutive patients with advanced breast cancer were included in a study designed to determine the prevalence of asthenia and its association with other clinical features. The Asthenia Score (AS, the average of four tests designed by our group to assess asthenia) was 59 +/- 9 for patients versus 88 +/- 7 for a group of 68 normal controls (p less than 0.001). Twenty-six patients (41%) scored below the tenth percentile of normal controls and were considered asthenics. AS was correlated with depression and the general severity index of the SCL-90 R test. No association was found between AS and nutritional status, lean body mass, tumor mass, anemia, or type of treatment. We conclude that asthenia is a frequent symptom in patients with advanced breast cancer, which, in our series, showed independent correlations only with psychological distress.