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J Rafter

Publications and source records attributed to J Rafter.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role of protein kinase C in growth stimulation of primary mouse colonic epithelial cells.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (160 nM) and the secondary bile acid, deoxycholic acid (50 microM) stimulated DNA synthesis in quiescent primary epithelial cells from the normal mouse colon as measured by autoradiographic analysis of [3H]thymidine incorporation. The purpose of this present study was to investigate the involvement of protein kinase C in the proliferative response of the normal colonic cells. The protein kinase C inhibitor, bisindolyl-maleimide GF 109203X, efficiently blocked the proliferative response of the cells to the phorbol ester and caused a dose-dependent decrease in the response to deoxycholic acid. While the phorbol ester-induced proliferation was unaffected by another inhibitor, H-7, the response of the cells to deoxycholic acid was blocked. Pretreatment of the cells with the phorbol ester (160 nM) for 24 h blocked the proliferative response to deoxycholic acid. Measurement of the intracellular distribution of protein kinase C activity showed a time-dependent and significant translocation of the enzyme activity from the soluble to the particulate cell fractions after exposure to 12-O-tetradecanoylphorbol-13-acetate. While exposure to the bile acid indicated a similar time-dependent translocation of the enzyme activity, the effect was not significant. The phorbol ester induced a time-dependent accumulation of c-fos mRNA and protein was measured by solution hybridization and immunocytochemistry, respectively. No effect of deoxycholic acid on c-fos expression could be observed in the present study. The data support a role for protein kinase C in the growth stimulating effect of physiological concentrations of deoxycholic acid on normal colonic epithelial cells. However, differences in the mechanisms underlying phorbol ester- and bile acid-induced proliferation are indicated.

Animals↗
PubMed1994

Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria.

Lactic acid bacteria have been reported to have antimutagenic/anticarcinogenic properties in vitro and in vivo. One possible mechanism for this effect involves a physical binding of the mutagenic compounds to the bacteria. The purpose of the present investigation was to study the binding capacity of eight human intestinal or lactic acid bacterial strains for mutagenic heterocyclic amines formed during cooking of protein-rich food. Binding of the mutagens Trp-P-2, PhIP, IQ and MeIQx by the bacterial strains was analyzed by HPLC. There were only minor differences in the binding capacities of the tested strains but the mutagenic compounds were bound with markedly different efficiencies. Trp-P-2 was almost completely bound and the binding tended not to be of a reversible nature. The binding of PhIP, which reached about 50%, was important as PhIP is a major mutagen in the western diet. IQ and MeIQx were slightly less well bound. pH appeared to be of importance for the binding efficacy. Binding correlated well with the reduction in mutagenicity observed after exposure of the heterocyclic amines to the bacterial strains. The results indicate that cooked food mutagenic compounds, commonly found in the western meat-rich diet, can be bound to bacteria from the normal intestinal microflora in vitro.

Amines↗
PubMed1994

Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats.

[14C]Benzo[a]pyrene (BP) was administered to male germfree rats. Urinary metabolites, constituting 9% of the administered radioactivity, were fractionated by lipophilic ion exchange chromatography. More than 80% of the urinary metabolites were conjugated, while neutral metabolites constituted 13-18%. The latter group was characterized by reversed-phase HPLC, ultraviolet spectrometry (UV) and gas chromatography/mass spectrometry (GC/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity upon HPLC fractionation. Some coeluted peaks were further quantitated from the total ion current chromatograms obtained in the GC/MS analysis. Two 7,8,9,10-tetrols which might be produced from the ultimate carcinogen r-7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP (anti-7,8-diol-9,10-epoxide) were detected at trace levels and indicated to be the r-7-t-8,9-c-10-(7,10/8,9-) and r-7-t-8,9,10-(7/8,9,10-) isomers. A trans-11,12-dihydrodiol was characterized as a major metabolite, while a trans-7,8-dihydrodiol was present at trace levels. Three additional quantitatively important metabolites were identified as isomeric trihydroxy-BPs. Two metabolites coeluting with BP quinones on HPLC were detected with relatively high abundance and tentatively identified as carboxylic methyl ester derivatives of BP quinones. Three quinones were detected with 1,6-, 3,6- and 6,12-substitutions. The 6,12- and 11,12-dihydroxy-BPs were also found at trace levels. A group of quinone-like metabolites were tentatively identified as trioxo-BPs. No monohydroxy-BP was detected in the neutral fraction of the urine extract. The time course of excretion was also studied and found to differ between individual metabolites.

Animals↗
PubMed1993

Increases in 13-hydroxyoctadecadienoic acid dehydrogenase activity during differentiation of cultured cells.

Recently, oxidation products of linoleic acid such as 13-hydroxyoctadecadienoic acid (HODE) have been implicated in the regulation of cellular physiology including the proliferative response to growth factor treatment. In addition, an NAD(+)-dependent 13-HODE dehydrogenase was recently described. To evaluate the contribution of this enzyme to cellular processes we have examined the behavior of the enzyme under different conditions. In the present report, changes in the activity of 13-hydroxyoctadecadienoic acid dehydrogenase during in vitro differentiation of two different cell lines were examined. The cell line HT-29 undergoes induced differentiation via manipulation of the medium while the Caco-2 line undergoes spontaneous differentiation upon attainment of confluence. In both cell lines, longer culture times were accompanied by increases in 13-HODE dehydrogenase activity. The increase in enzyme activity continued even after cell proliferation had ceased. Cellular differentiation was verified by the observation of increases in sucrase and alkaline phosphatase activities. In addition, the activity of 13-HODE dehydrogenase was measured in growing, early confluent and late confluent cultures of undifferentiating Swiss mouse 3T3 fibroblasts. In the fibroblast line, no significant changes in 13-HODE dehydrogenase activity were observed during the course of the experiment. The specific activity of 13-HODE dehydrogenase was also significantly different between the three cell lines, consistent with the extent of differentiation. Highest levels of activity were found in Caco-2 cells (200-400 pmol/min/mg) and barely detectable levels in the fibroblasts (0.6-2 pmol/min/mg). The correlation between 13-HODE dehydrogenase and cell differentiation suggests the enzyme may have a role to play in the partitioning of cells between proliferation and differentiation pathways.

3T3 Cells↗
PubMed1992

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.

Bacterial Physiological Phenomena↗
PubMed1991

Proliferative potential and expression of cell type specific functions in primary mouse colonic epithelial cells.

Primary cultures of mouse colonic epithelial cells have been obtained that are typically epithelial by morphology and moreover express keratins and endogenous beta-galactosidase; this latter activity was also demonstrated in the epithelial lining of the mouse colonic mucosa. The proliferative response of the primary colonic epithelial cells to epidermal growth factor, insulin, and the bile acid, deoxycholic acid, has been studied. Using primary cultures maintained at suboptimal growth conditions, which yielded 96 to 100% quiescent cells, epidermal growth factor, insulin, and the bile acid, deoxycholic acid, at concentrations at which it normally occurs in the aqueous phase of human feces, stimulated proliferation as measured by autoradiography. Exposure of the cells to combinations of these factors resulted in additive increases in growth. In conclusion, cells from the normal mouse colon can now be cultured while retaining at least two normal marker functions and moreover respond to some known mitogens and the potential tumor promoter deoxycholic acid. The cells can also be subcultivated while maintaining their epithelial morphology and marker functions for at least 3 passages.

Animals↗
PubMed1990

Progesterone metabolism in the microsomal fraction of the testis, head kidney, and trunk kidney from the rainbow trout.

In the present study on male rainbow trout, well-defined microsomal fractions from gonad, trunk kidney, and head kidney were used to study enzymes active on progesterone. The metabolites produced were identified by mass spectrometric analysis. In the testis the main metabolite was 17 alpha-hydroxyprogesterone which is an intermediate in the steroid biosynthetic pathway. 17 alpha-Hydroxyprogesterone was also identified in incubations from head and trunk kidney. The 17 alpha-hydroxylase activity was higher in the head kidney than in the trunk kidney which probably reflects the presence of steroid producing interrenal cells in this part of the kidney. The conversion of progesterone to 17 alpha-hydroxylated products in the testis and head kidney was NADPH dependent and inhibited by carbon monoxide, indicating the participation of cytochrome P450 monooxygenase in this reaction. NADH supported the reaction to some extent (27% of the NADPH-dependent activity) in the testis but not in the head kidney. In addition to 17 alpha-hydroxyprogesterone the head and trunk kidney microsomes gave rise to 16 alpha- and 6 beta-hydroxyprogesterone. These activities were low or absent in testis microsomes. Progesterone 5 alpha-reductase activity was only detected in trunk kidney microsomes.

17-alpha-Hydroxyprogesterone↗
PubMed1989

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.

Adenoma↗
PubMed1989

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.

Aged↗
PubMed1989

Tissue localization of the carcinogenic glutamic acid pyrolysis product Glu-P-1 in control and beta-naphthoflavone-treated mice and rats.

Autoradiograms obtained after i.v. injection of the 14C-labelled carcinogenic glutamic acid pyrolysis product Glu-P-1 to mice and rats showed a pronounced uptake of radioactivity in the liver, kidney, thyroid and nasal mucosa. High concentrations of radioactivity were present in the bile and intestinal contents at short post-injection times. In the male rat, the Zymbal's gland and the preputial gland were identified as sites of high and specific binding at all post-injection times examined. The liver and nasal mucosa were identified as sites of retention of non-extractable radioactivity. In the pigmented mouse, Glu-P-1 and/or its metabolites were accumulated in melanin. Glu-P-1 is known to be activated by cytochrome P-448. Pretreatment with beta-naphthoflavone (a cytochrome P-448 inducer) did not change the tissue localization of radioactivity in either species except for the liver where the overall labelling was decreased. Neither did pretreatment of mice with the glutathione-depleting agent phorone change the distribution pattern significantly. However, combined pretreatments of mice with either phorone or beta-naphthoflavone and the cytochrome P-448 inhibitor 9-hydroxyellipticine resulted in an increased overall retention of radioactivity in the body.

Animals↗
PubMed1988

The role of the intestinal microflora in the formation of mutagenic metabolites from the carcinogenic air pollutant 2-nitrofluorene.

After a single oral dose of 2-nitro[9-14C]fluorene (NF) to germfree and conventional rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and feces. The mutagenicity excreted from germfree animals exceeded the mutagenicity excreted from conventional animals. Absence of the microflora resulted in lack of nitroreduction and excreted metabolites from germfree rats were mono- or dihydroxylated nitrofluorenes, as assessed by liquid chromatography/mass spectrometry (LC/MS). These hydroxy-NFs were associated with the high direct-acting mutagenicity that was excreted in germfree animals. Only traces of NF were found in urine from germfree rats and no NF was detected in urine from conventional animals. Thus, in contrast to a number of reports on other mutagenic compounds, these results would tend to indicate that the intestinal microflora causes a reduced excretion of mutagenic metabolites of NF.

Air Pollutants↗
PubMed1988

Effects of triethyl lead chloride on oestradiol metabolism in the female rat liver microsomal fraction.

Triethyl lead chloride (TEL) was added directly to female rat liver microsomal fractions at final concentrations of 0.0, 0.005, 0.05 and 0.5 mM, respectively. The effect of oestradiol on liver microsomal metabolism was studied in vitro by incubating the microsomes with radioactive oestradiol-17 beta. The oestradiol metabolites were separated, identified and quantified. The highest treatment concentrations resulted in decreased activities of enzymes that metabolize oestradiol-17 beta.

Animals↗
PubMed1987

Studies on the chromatographic fractionation of metabolites of benzo[a]pyrene in faeces and urine from germfree and conventional rats.

Rats, germfree and conventional, were dosed with 14C-labelled benzo[a]pyrene. Faeces and urine were collected. Metabolites in faeces were effectively extracted with a new method using a combination of solvents and solid sorbents. Metabolites in urine were extracted with octadecylsilane-bonded silica. The metabolites were fractionated into groups by chromatography on a cation exchanger (SP-LH-20 or SP-Sephadex C-25) and an anion exchanger (TEAP-LH-20). Some of the groups were further purified by column chromatography and analysed by HPLC and TLC. The analyses show a complex pattern of metabolism. A large part of the metabolites (9-24% depending on animal type and route of excretion) had amphoteric properties, e.g. like glutathione and cysteine conjugates. The abundance of conjugates sensitive to beta-glucuronidase and sulphatase was low. The relative amount of acidic conjugates in faeces was much higher in the germfree than in the conventional rats indicating the influence of the intestinal flora on the metabolism. The results support the view that the mercapturic acid pathway is a quantitatively important metabolic route for benzo[a]pyrene in rats. The methods of extraction and group fractionation were designed to be generally applicable to the analysis of lipophilic xenobiotics and their metabolites.

Animals↗
PubMed1987

Metabolism of the carcinogenic air pollutant 2-nitrofluorene in the isolated perfused rat lung and liver.

The metabolism of 2-nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, was studied in the isolated perfused rat lung and liver. NF has been identified in urban air and diesel exhaust and occurs in the gas, as well as in the particulate phase. Therefore, it is conceivable that the lung represents one point of entry of this compound into the body. The lung metabolizes NF to hydroxylated NFs, mainly 9-hydroxy-NF, independently of the route of administration (intravascular or intratracheal). After intratracheal administration, NF is rapidly excreted into the perfusate, indicating that other organs might be exposed to unmetabolized NF. The liver excretes NF metabolites as biliary glucuronides. Untreated bile is not mutagenic. However, after beta-glucuronidase treatment of bile, direct-acting mutagens were detected. The mutagenic metabolites in beta-glucuronidase-treated bile were the same as identified in the perfusate of the isolated lung. Since beta-glucuronidase is an enzyme found in the human intestinal microflora, inhalation of NF could result in the liberation of genotoxic metabolites in the colon.

Air Pollutants↗
PubMed1987

Metabolism of the carcinogenic air pollutant 2-nitrofluorene in the rat.

After a single oral dose of 2-nitro [9-14C]fluorene to rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and faeces, urine being the major excretory route. The major part of the urinary mutagenicity was associated with the unconjugated fraction of metabolites which consisted of a range of hydroxylated acetylaminofluorenes in addition to a number of hydroxylated nitrofluorenes. The formation of hydroxylated acetylaminofluorenes supports the contention that 2-nitrofluorene, in vivo, enters the metabolic pathway of the well-known carcinogen 2-acetylaminofluorene. The single most mutagenic metabolite of 2-nitrofluorene was a hydroxylated nitrofluorene; the formation of this metabolite was markedly increased when the rats had been treated with beta-naphthoflavone. Finally, an alternative pathway is discussed for the formation of 1- and 3-hydroxyacetylaminofluorenes.

2-Acetylaminofluorene↗