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Biomedical subjects

J Rafter

Publications and source records attributed to J Rafter.

At least 19 recordsLinked to original sources
PubMed2003

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.

Animals↗
PubMed2002

Lactic acid bacteria and cancer: mechanistic perspective.

Colorectal cancer is one of the most important causes of cancer morbidity and mortality in Western countries. While a myriad of healthful effects have been attributed to the probiotic lactic acid bacteria (LAB), perhaps the most controversial remains that of anticancer activity. It should be pointed out that there is no direct experimental evidence for cancer suppression in man 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. The precise mechanisms by which LAB may inhibit colon cancer are presently unknown. However, such mechanisms might include: alteration of the metabolic activities of intestinal microflora; alteration of physico-chemical conditions in the colon; binding and degrading potential carcinogens; quantitative and/or qualitative alterations in the intestinal microflora incriminated in producing putative carcinogen(s) and promoters (e.g. bile acid-metabolising bacteria); production of antitumourigenic or antimutagenic compounds; enhancing the host's immune response; and effects on physiology of the host. These potential mechanisms are addressed in the present paper.

Colorectal Neoplasms↗
PubMed2001

Comparison of exhaust emissions from Swedish environmental classified diesel fuel (MK1) and European Program on Emissions, Fuels and Engine Technologies (EPEFE) reference fuel: a chemical and biological characterization, with viewpoints on cancer risk.

Diesel fuels, classified as environmentally friendly, have been available on the Swedish market since 1991. The Swedish diesel fuel classification is based upon the specification of selected fuel composition and physical properties to reduce potential environmental and health effects from direct human exposure to exhaust. The objective of the present investigation was to compare the most stringent, environmentally classified Swedish diesel fuel (MK1) to the reference diesel fuel used in the "European Program on Emissions, Fuels and Engine Technologies" (EPEFE) program. The study compares measurements of regulated emissions, unregulated emissions, and biological tests from a Volvo truck using these fuels. The regulated emissions from these two fuels (MK1 vs EPEFE) were CO (-2.2%), HC (12%), NOx (-11%), and particulates (-11%). The emissions of aldehydes, alkenes, and carbon dioxide were basically equivalent. The emissions of particle-associated polycyclic aromatic hydrocarbons (PAHs) and 1-nitropyrene were 88% and 98% lower than those of the EPEFE fuel, respectively. The emissions of semi-volatile PAHs and 1-nitropyrene were 77% and 80% lower than those from the EPEFE fuel, respectively. The reduction in mutagenicity of the particle extract varied from -75 to -90%, depending on the tester strain. The reduction of mutagenicity of the semi-volatile extract varied between -40 and -60%. Furthermore, the dioxin receptor binding activity was a factor of 8 times lower in the particle extracts and a factor of 4 times lower in the semi-volatile extract than that of the EPEFE fuel. In conclusion, the MK1 fuel was found to be more environmentally friendly than the EPEFE fuel.

Animals↗
PubMed2001

Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells.

BACKGROUND & AIMS: Evidence is accumulating that inhibitors of cyclooxygenase (COX)-2 activity are useful for preventing human colon cancer. Therefore, it is important to determine whether agents in the colonic luminal contents can influence the transcriptional regulation of COX-2 in colonic cells. METHODS: Transient transfections were performed, using a human COX-2 promoter-luciferase construct, in HCT 116 cells, and the effects of pure luminal compounds and components of fecal water, the fecal fraction in direct contact with the colonocytes, on luciferase activity studied. RESULTS: The luminal compounds deoxycholate, chenodeoxycholate, and butyrate all induced COX-2 promoter activity in HCT 116 cells. Lipid extracts of human fecal water also induced promoter activity in these cells, and the extent of induction varied between individuals. Induction of COX-2 promoter activity by the lipid extracts was positively correlated with induction of activator protein 1-dependent gene transcription. Results also indicated that protein kinase C and p38 mitogen-activated protein kinase mediated the effect of the luminal agents on COX-2 promoter activity. CONCLUSIONS: Components in the luminal contents can effect COX-2 transcription and may influence colonic tumor development. Available data suggest that the responsible components are under dietary influence.

Butyric Acid↗
PubMed2000

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites.

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Acrylic Resins↗
PubMed2000

Effects of human intestinal flora on mutagenicity of and DNA adduct formation from food and environmental mutagens.

Although the intestinal flora is believed to have a critical role in carcinogenesis, little is known about the role of the human intestinal flora on the effects of mutagens in vivo. The aim of the present study was to address a possible role of the human intestinal flora in carcinogenesis, by exploiting human-flora-associated (HFA) mice. The capacity of human faeces to activate or inactivate 2-amino-3-methyl-3H:-imidazo[4,5-f]quinoline (IQ) and 2-nitrofluorene was determined using the Ames assay. Human faecal suspensions that were active in this regard were then selected and orally inoculated into germfree NMRI mice to generate HFA mice. HFA, germfree, conventionalized and conventional mice were administered IQ, 2-amino-9H:-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) and 2-nitrofluorene. The activity of human intestinal flora against mutagens could be transferred into the mice. In comparing germfree mice and mice harbouring an intestinal flora, the presence of a flora was essential for the activities of faeces against mutagens. After administration of IQ and 2-nitrofluorene, DNA adducts were observed in the mice with a flora, while adducts were extremely low or absent in germfree animals. DNA adducts after AAC treatment were higher in germfree mice in some tissues including colon than in mice with bacteria. Differences in DNA adduct formation were also observed between HFA mice and mice with mouse flora in many tissues. These results clearly indicate that the intestinal flora have an active role in DNA adduct formation and that the role is different for the different chemicals to which the animals are exposed. The results also demonstrate that the human intestinal flora have different effects from the mouse flora on DNA adduct formation as well as in vitro metabolic activities against mutagens. Studies using HFA mice could thus provide much-needed information on the role of the human intestinal flora on carcinogenesis in vivo.

Adult↗
PubMed2000

Effect of colonic luminal components on induction of apoptosis in human colonic cell lines.

Apoptosis is central to cell number regulation in the colonic epithelium, and interest in its role in colon carcinogenesis has been growing rapidly. It thus becomes of interest to characterize luminal components, possibly of dietary origin, that may influence this process. We have investigated the sensitivity of two human colonic cell lines, the human adenocarcinoma cell line (HT-29) and the human fetal colonic mucosa cell line (FHC), to induction of apoptosis by sodium butyrate, bile acids, and human fecal water fractions. The apoptotic effect has been studied by 1) morphological changes in cells examined by fluorescence microscopy, 2) DNA fragmentation analysis by gel electrophoresis, 3) flow cytometry analysis of DNA strand breaks assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL), and 4) poly(ADP-ribose) polymerase cleavage by Western blot. Sodium butyrate and bile acids induced a time- and concentration-dependent apoptosis in both cell lines. Quantitation of this effect, by use of the TUNEL assay, indicated that deoxycholic acid was most effective in inducing this effect at lower concentrations and at shorter times. Apoptotic effects were also observed, in both cell lines, when the cells were exposed to intact human fecal waters (the fecal fraction in direct contact with the epithelium) and their lipid extracts, with the intact samples being more effective. Although all fecal waters examined induced apoptosis, quantitation of the effect by the TUNEL assay indicated that the ability to induce apoptosis differed markedly between samples. Induction of apoptosis by the fecal waters was not correlated to cytotoxicity but was negatively correlated to the pH of the samples. Interestingly, the cells derived from the fetal mucosa (FHC) were consistently less sensitive to apoptotic effects of the luminal components than the tumor-derived cells (HT-29). Thus human fecal water fractions induce apoptosis in colonic cells, and this effect is not due to lipid components alone.

Apoptosis↗
PubMed1999

Characterization of conjugated metabolites of benzo[a]pyrene in germ-free rat urine by liquid chromatography/electrospray tandem mass spectrometry.

The characterization of conjugated metabolites of benzo[a]pyrene (BP) in the urine of male germ-free rats given a single intraperitoneal dose of [(14)C]BP is described. Urinary metabolites, constituting 9% of the administered radioactivity, were extracted on a Sep-Pak C(18) cartridge and separated by lipophilic ion-exchange chromatography into neutral and acidic fractions (fractions I-V). Metabolites in the latter fractions, constituting more than 80% of the urinary radioactivity, were characterized by reversed-phase HPLC and capillary column liquid chromatography/electrospray mass spectrometry (LC/ESMS) and tandem mass spectrometry (MS/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity. Some coeluting compounds were semiquantified from the ion current chromatograms obtained in the capillary column LC/ESMS analyses. The major conjugated metabolites in fraction II, containing about 50% of the urinary radioactivity, consisted of three tetrahydrotrihydroxy-BP-S-N-acetylcysteines, the major isomer being 7,8,9,10-tetrahydro-8,9, 10-trihydroxy-BP-7-S-N-acetylcysteine, two dihydrotrihydroxy-BP-S-N-acetylcysteines, and a tetrahydrotetrahydroxy-BP-S-N-acetylcysteine. Fraction II also contained three apparently unconjugated compounds whose structures will be described elsewhere. Metabolites characterized in fractions III and IV, containing about 30% of the urinary radioactivity, included three BP-O,O'-disulfates, two monohydroxy-BP-O-sulfates, three dihydrodihydroxy-BP-O-sulfates, three BP-O,O'-diglucuronides, and a BP-O-sulfate-O'-glucuronide. Trace levels of a tetrahydrotrihydroxy-BP-S-glutathione conjugate were detected in fraction V.

Animals↗
PubMed1999

Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells.

We recently suggested that prolonged deregulated expression of AP-1 activity in colonic cells by bile acids may contribute to tumour promotion in the colon. In the present study, using two human colon carcinoma cell lines, HT-29 and HCT 116, transiently transfected with the AP-1-luciferase reporter construct, we showed that the bile acids, deoxycholate, chenodeoxycholate, ursodeoxycholate and lithocholate, induced AP-1-dependent gene transcription in a dose-dependent manner, whereas cholate was without effect. The greatest effect was observed with deoxycholate, and the ability of this bile acid to induce reporter gene activity was significantly correlated with its ability to induce cell proliferation (r = 0.91, P = 0.01). Cholesterol and the long chain fatty acids, myristate, palmitate and stearate, had no effect on AP-1-dependent gene transcription, whereas the short chain fatty acid, butyrate, exhibited a marked effect. Mindful of the fact that the concentrations of lumenal components that are actually in or entering the epithelial cells in the colon are presumably lower than lumenal values, we considered it of interest to determine the effect of dilution on the capacity of human faecal water to induce AP-1 activity and also cell proliferation. We demonstrated that diluted lipid extracts, from all of the faecal water samples examined, significantly induced AP-1-dependent gene transcription in the colonic cells, and that this effect differed markedly between the extracts. We confirmed that the faecal water lipid extracts, at the same dilution at which they increased AP-1 activity, significantly induced proliferation in the same cell line. These data suggest that lipid components of human faecal water, which is in direct contact with the colon epithelium and may be physiologically more active than the solid phase, can activate AP-1, a transcription factor whose activation has been associated with the promotion of neoplastic transformation.

Bile Acids and Salts↗
PubMed1999

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.

Animals↗
PubMed1998

Analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene found in rat urine by nano-electrospray mass spectrometry.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

Acetylcysteine↗
PubMed1998

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.

Acetylation↗
PubMed1996

Expression of the peroxisome proliferator-activated receptor (PPAR) in the mouse colonic mucosa.

The peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily, has been shown to be activated by various compounds such as fibrates, thiazolidinediones, prostaglandins, and fatty acids. Here we demonstrate expression of PPAR in mouse colonic and small intestinal mucosa by Western blot analysis and immunohistochemistry, indicating a higher expression level in the differentiated colonic epithelial cells facing the intestinal lumen. Quantification of PPAR mRNA by ribonuclease protection assay revealed relatively high expression of PPAR gamma and Nuc1 in the colon as compared to the small intestine. In contrast, PPAR alpha expression was higher in the small intestine as compared to the colon. These results demonstrate the presence of PPAR in the intestinal mucosa; however, the physiological roles of the various isoforms in the intestine remain to be established.

Animals↗
PubMed1996

Increased c-fos mRNA and binding to the AP-1 recognition sequence accompanies the proliferative response to deoxycholate of HT29 cells.

To further understand the molecular mechanisms of bile acid-mediated colon tumor promotion, we have examined the possible role of AP-1 activity in this process. The AP-1 complex has been reported to play an important role in control of cell growth. Our studies show that lithocholate, deoxycholate and ursodeoxycholate exhibited marked proliferative effects on a human adenocarcinoma cell line (HT29), while cholate was without effect. The proliferative effects appeared to be confined to narrow concentration windows which differed for the different bile acids. We demonstrate that deoxycholate caused an increase in expression of c-fos mRNA and increased binding to the AP-1 site, effects which were maximum at the concentration at which the bile acid induced the maximum proliferative effect on the cells. Cholate was without effect on AP-1 binding activity. In addition, we show that the AP-1 complex induced by treatment of the cells with the bile acid contained the c-fos protein. This could suggest that prolonged deregulated expression of AP-1 activity in colonic cells by certain bile acids may contribute to tumor promotion in the colon.

Base Sequence↗