Oxygen free radical generating mechanisms in the colon: do the semiquinones of vitamin K play a role in the aetiology of colon cancer?

It is proposed that bile acids (deoxycholic acid), the K vitamins, iron(II) complexes and oxygen interact to induce an oncogenic effect in the colon by the generation of free radicals. In the relatively low oxidising/reducing conditions of the colonic lumen the K vitamins exist in the reduced form; however, if absorbed into the mucosa they have the capacity to be chemically oxidised and to enter into a redox cycle yielding oxygen radicals. The semiquinone radical of K(1) (phylloquinone) has been stabilised in bile acid mixed micelles and investigated by electron paramagnetic resonance spectroscopy and quantum chemical calculations. The estimated half-life of the radical was about 30 min which confirms a remarkably high stability in aqueous micellar solution. A model is presented in which the reduced K vitamins may initiate superoxide radical, O2(-*) generation leading to Fe(II) mediated Fenton reactions in the stem colon cells.

Dieting behavior among 11-15-year-old girls in Merseyside and the Northwest of England.

PURPOSE: To examine the general dieting behavior and characteristics of adolescent girls in the United Kingdom, and in particular, the Northwest of England. METHODS: A total of 569 girls, ages 11-15 years, from six schools in the Merseyside and Lancashire area, representing a cross-section of social status, completed a nonstandardized questionnaire concerning general dieting behavior. Data were analyzed using SPSS (p < .05 was considered significant). RESULTS: The incidence of dieting was 35.3%. The earliest reported age of starting to diet was 8 years. Significantly more girls from the independent schools (45.2%) had started to diet by the age of 10 years, compared to girls from the comprehensive schools (24%) (p = .03). Of those who had dieted, 30.3% had dieted up to two times during the previous 12 months, 17.4% had dieted up to four times, and 6% had dieted for most of the time. Most girls (33%) dieted for 2-4 weeks at a time, and 66% thought that dieting was good for their health. Only 52% said their parents did not approve of them dieting. Most girls (42.1%) dieted because they felt they were too fat. CONCLUSION: This study has shown that many young girls are engaging in potentially harmful dieting practices from a very early age, and are of the opinion that dieting is a healthy activity. This would suggest that many misconceptions are held with regard to nutritional advice and education, and that such information should be reviewed and changed accordingly.

Carotenoid composition and antioxidant potential in subfractions of human low-density lipoprotein.

Carotenoids and vitamin E are transported in human plasma complexed with lipoproteins. The bulk of them are associated with low-density lipoprotein (LDL), in which form they may act as antioxidants and thus delay the onset of atherosclerosis. We used a simple, rapid, ultracentrifugation technique to fractionate plasma lipoproteins in self-generating gradients of iodixanol (Optiprep), a non-ionic iodinated density gradient medium. The carotenoid content and composition of a number of LDL subfractions was determined by reversed-phase high-performance liquid chromatography. Lycopene, beta-carotene and beta-cryptoxanthin were mainly located in the larger, less-dense LDL particles whereas lutein and zeaxanthin were found preferentially in the smaller, more dense LDL particles. When the antioxidant content of these fractions was expressed per milligram of LDL protein, significantly lower concentrations of carotenoid and vitamin E were found to be associated with the smaller, protein-rich fractions of LDL. Strong positive correlations were found between total carotenoid and vitamin E plasma concentrations and the lag-time of Cu(2+)-mediated oxidation of LDL subfractions. The more dense LDL subfractions, which had lower levels of these antioxidants, were more readily oxidized, highlighting their possible role in atherosclerotic events.

Beta-carotene enhances hydrogen peroxide-induced DNA damage in human hepatocellular HepG2 cells.

In this study, the alkaline version of the comet assay has been used to determine the effect of beta-carotene supplementation (10 microM) on peroxide-initiated free radical-mediated DNA damage in human HepG2 hepatoma cells. In supplemented cells, beta-carotene failed to afford any protection against hydrogen peroxide-induced DNA strand breaks. Indeed, levels of strand breaks in supplemented cells were significantly higher than in cells exposed to hydrogen peroxide alone, especially after a long incubation period. In contrast, beta-carotene afforded significant levels of protection against DNA strand breaks when cells were treated with tert-butyl hydroperoxide. In this case, the level of protection increased as supplementation continued.

Lycopene and beta-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses.

Epidemiological studies have clearly demonstrated a link between dietary carotenoids and the reduced incidence of certain diseases, including some cancers. However recent intervention studies (e.g. ATBC, CARET and others) have shown that beta-carotene supplementation has little or no beneficial effect and may, in fact, increase the incidence of lung cancers in smokers. This presents a serious dilemma for the scientific community - are carotenoids at high concentrations actually harmful in certain circumstances? Currently, a significant number of intervention studies are on-going throughout the world involving carotenoids (of both natural and synthetic origin). Our approach has been to study the ability of supplementary carotenoids in protecting cells against oxidatively-induced DNA damage (as measured by the comet assay), and membrane integrity (as measured by ethidium bromide uptake). Both lycopene and beta-carotene only afforded protection against DNA damage (induced by xanthine/xanthine oxidase) at relatively low concentrations (1-3 microM). These levels are comparable with those seen in the plasma of individuals who consume a carotenoid-rich diet. However, at higher concentrations (4-10 microM), the ability to protect the cell against such oxidative damage was rapidly lost and, indeed, the presence of carotenoids may actually serve to increase the extent of DNA damage. Similar data were obtained when protection against membrane damage was studied. This would suggest that supplementation with individual carotenoids to significantly elevate blood and tissue levels is of little benefit and, may, in fact, be deleterious. This in vitro data presented maybe significant in the light of recent intervention trials.

Free radical stimulation of tyrosine kinase and phosphatase activity in human peripheral blood mononuclear cells.

Human lymphocytes were challenged with reactive oxygen species (ROS) generated by xanthine/xanthine oxidase leading to an increase in tyrosine phosphorylation, together with an increase in tyrosine phosphatase activity. In the presence of 50 microM vanadate and xanthine/xanthine oxidase, tyrosine phosphatase activity was inhibited and a marked increase in tyrosine phosphorylation was observed. The addition of catalase abolished the increase in tyrosine phosphorylation while the addition of superoxide dismutase had no effect. This suggests that vanadate together with hydrogen peroxide derived from xanthine/xanthine oxidase activity, interact to produce an agent that is an effective inhibitor of tyrosine phosphatase activity. When human lymphocytes were challenged with xanthine/xanthine oxidase in the presence of 50 microM CuCl2, an increase in both tyrosine phosphatase and kinase activity was observed. Cupric ions inhibited xanthine oxidase activity by 84%; neither superoxide or hydroxyl radicals could be detected, but traces of hydrogen peroxide were detected in the medium. We conclude that unbound metals can interact with ROS and readily influence signalling mechanisms in human lymphocytes.

Measurement of menadione-mediated DNA damage in human lymphocytes using the comet assay.

The model quinone compound menadione has been used to study the effects of oxidative stress in mammalian cells, and to investigate the mechanism of action of the quinone nucleus which is present in many anti-cancer drugs. We have used the alkaline single cell gel electrophoresis assay (comet assay) to investigate the effects of low doses of this compound on isolated human lymphocytes. We found that concentrations of menadione as low as 1 microM were sufficient to induce strand breaks in these cells. Pre-incubation with the NAD(P)H quinone oxidoreductase inhibitor dicoumarol, enhanced the production of menadione-induced strand breaks. In contrast, the metal ion chelator 1,10-phenanthroline inhibited formation of strand breaks, although prolonged incubation with 1,10-phenanthroline in combination with menadione resulted in an increase in a population of very severely damaged nuclei. A marked variation in the response of lymphocytes from different donors to menadione, and in different samples from the same donor was also observed.

Secondary bile acid induced DNA damage in HT29 cells: are free radicals involved?

Increased bile acid secretion, as a consequence of a high fat diet, results in the increased production of bile acids that may escape the enterohepatic circulation, and be subsequently metabolised by the colonic micro-flora to form the co-mutagenic and co-carcinogenic secondary bile acids. The potential of the secondary bile acids lithocholate (LOC) and deoxycholate (DOC), to induce DNA damage, in the colonocyte cell line HT29, at physiological concentrations both individually and in a 2:1 ratio was assessed. Results indicated significant levels of DNA damage induced by both bile acids, with LOC having the greater DNA damaging capacity. The potential role of vitamin A, and the antioxidant vitamin E, in reducing this damage was determined, over a range of vitamin concentrations. Both vitamins reduced the bile acid induced DNA damage. Vitamin A displayed a dose response relationship, whereas vitamin E reduced DNA damage close to negative control values at all concentrations above 50 microM. These results indicate a protective role for Vitamins A and E, against the DNA damaging capacity of LOC and DOC.

Quinone-induced DNA single strand breaks in a human colon carcinoma cell line.

It has been demonstrated that synthetic quinones, such as menadione, cause DNA damage in different cell systems, possibly being mediated by free radicals generated during redox cycling. It has been suggested that the damage caused could be related to tumor induction in different sites. To our knowledge it has not yet been demonstrated that the natural quinones, vitamin K1 and K2, exert the same activity. Using a colon carcinoma cell line, HT-29, we examined the extent of DNA damage induced by menadione, vitamin K1 and K2. Menadione caused significant DNA damage at low concentrations (25-200 microM) with a linear correlation of r = 0.95. In the presence of dicoumarol, a DT-diaphorase inhibitor, the damage was detected at concentrations five times lower indicating that free radicals generated during the redox cycling play a key role. Neither vitamin K1, incorporated in micelles, nor K2 caused detectable single strand breaks with respect to the controls either in the presence or in absence of dicoumarol. Our results demonstrate that, despite their redox cycling properties, the natural forms of vitamin K do not cause DNA damage in HT-29 cells as menadione does in the experimental conditions used.

Free radical generating mechanisms in the colon: their role in the induction and promotion of colorectal cancer?

A hypothesis is presented to account for the dietary induction and promotion of colorectal cancer. The principal agents are the secondary bile acids, lithocholic and deoxycholic acids, the vitamin K group and ferrous iron complexes. These metabolites may interact to subvert the normal free radical generating mechanisms involved in mucosal defence. Diets high in fat and red meat and low in fibre support a Bacteroides-dominated colonic microflora, which both synthesis and utilises vitamin K2 isoprenalogues or menaquinones as enzyme co-factors. Iron(II) complexes such as haemin from the breakdown of dietary haemoglobin and myoglobin also serve as growth factors for these bacteria and provide a rich source of haem-iron for intestinal uptake. Biliary secretion is stimulated by dietary fat and bile acids are essential for the intestinal uptake of vitamin K and possibly of iron complexes such as haemin. In the mature colonocyte, vitamin K and haemin may initiate redox cycling reactions which liberate superoxide (O2-.). Bile acids can activate the membrane bound phospholipase to liberate arachidonate and diacylglycerol. This leads in turn to the production of more O2-. which can enter the microcirculation and acts as a potent chemoattractant for the neutrophils that line the lamina propria. The released diacylglycerol can activate protein kinase C in the neutrophil membrane to switch on the respiratory burst oxidase system generating yet more O2-. and may stimulate the proliferation of transformed stem cells by a similar protein kinase C mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation of ursodeoxycholic acid by Pseudomonas sp NCIB 10590.

The metabolism of ursodeoxycholic acid by Pseudomonas sp NCIB 10590 has been studied in phosphate-buffered mineral salts. The organism completely metabolized ursodeoxycholic acid in 24 hr, and time-course experiments revealed that maximum product formation occurred at 14 hr. The major products detected and identified at 14 hr were 7 beta-hydroxychol-4-en-3-one-24-oic acid, 7 beta-hydroxy-3-oxo-pregna-1,4-diene-20-carboxylic acid, and 7 beta-hydroxyandrosta-1,4-diene-3,17-dione. Several minor intermediates were isolated and evidence is given for the following structures: 7 beta-hydroxy-5 beta-cholan-3-oxo-24-oic acid, 7 beta-hydroxyandrost-4-en-3,17-dione, 7 beta,17 beta-dihydroxyandrosta-1,4-diene-3-one, 3-hydroxy-1,3,5(10)-9,10-seco-androstatriene-3,17-dione-7 beta-ol, and 22 alpha-hydroxymethylpregna-1,4-diene-3-one-7 beta-ol.

Toxicity of paraquat to microorganisms.

The biochemical response of the microorganisms Lipomyces starkeyi (Lod & Rij), Escherichia coli K-12 W3110, Bacillus subtilis 168 (Marburg) and Pseudomonas sp. strain TTO1 to the presence of growth-inhibitory concentrations of paraquat was studied. Paraquat was added to each culture at a concentration previously determined to reduce the culture growth rate by up to 50%. The changes in activity of a number of enzymes previously shown to be associated with the defense of the mammalian system against the action of paraquat were studied. While the response of E. coli was in agreement with that found in other studies of this microorganism and supports a commonly accepted mechanism for paraquat toxicity, the results obtained with L. starkeyi, B. subtilis, and Pseudomonas sp. strain TTO1 suggest that other mechanisms exist for protection against the toxicity of paraquat.

The degradation of beta-sitosterol by Pseudomonas sp. NCIB 10590 under aerobic conditions.

The bacterial degradation of beta-sitosterol by Pseudomonas sp NCIB 10590 has been studied. Major biotransformation products included 24-ethylcholest-4-en-3-one, androsta-1,4-diene-3,17-dione, 3-oxochol-4-en-3-one-24-oic acid and 3-oxopregn-4-en-3-one-20-carboxylic acid. Minor products identified were 26-hydroxy-24-ethylcholest-4-en-3-one, androst-4-ene-3,17-dione, 3-oxo-24-ethylcholest-4-en-26-oic acid, 3-oxochola-1,4-dien-3-one-24-oic acid, 3-oxopregna-1,4-dien-3-one-20 carboxylic acid and 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of beta-sitosterol degradation by bacteria.

Metabolism of unsaturated bile acids and androstanes by human faecal bacteria.

The metabolism of unsaturated bile acids and androstanes by mixed human faecal cultures has been studied. The reactions observed were mainly reductive. Unsaturated 4-ene-3-oxo and 1,4-diene-3-oxo bile acids were reduced in Ring A. 5 beta-3-Oxo bile acids were reduced to 5 beta-3-hydroxy bile acids. 4-Ene, 1,4-diene and 4,6-diene-3,17-dioxo-androstanes were reduced in Ring A with concomitant reduction of oxo groups to hydroxyl groups. The Gram-negative facultative anaerobic faecal bacteria are implicated in the reductive process, whilst the genus Clostridium does not appear to be important. Inclusion of menadione, a synthetic form of vitamin K, retards the reductive process.

Mechanism of biodegradation of paraquat by Lipomyces starkeyi.

The biodegradation of ring-14C- and methyl-14C-labeled paraquat by the soil yeast Lipomyces starkeyi was studied in vitro. It was found that the degradation of paraquat (acting as a sole source of culture nitrogen) resulted in the accumulation in the extracellular medium of radiolabeled acetic acid. The culture also evolved radiolabeled CO2. The results suggest that the degradation of paraquat by L. starkeyi is associated with the integrity of the cell wall and that disruption or removal of the wall results in a complete loss of degradative capability. A mechanism for the degradation of paraquat by this organism is postulated.