Rodney F. Bilton

Lycopene and β-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.

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.

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)

β-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 β‐carotene supplementation (10 μM) on peroxide‐initiated free radical‐mediated DNA damage in human HepG2 hepatoma cells. In supplemented cells, β‐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, β‐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.

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.

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, β-carotene and β-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 Cu2+-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.

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.

The degradation of β-sitosterol by pseudomonas sp. NCIB 10590 under aerobic conditions

Abstract The bacterial degradation of β-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 9α-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of β-sitosterol degradation by bacteria.

The degradation of lithocholic acid by Pseudomonas Spp NCIB 10590

Reports on the microbial degradation of lithocholic acid show a wide variety of transformations. A series of investigations with a selection of bacteria isolated from the rat intestine [ 1,2] has shown that many such bacteria have the ability to oxidise and then reduce the 3a-hydroxyl group. Characterised products include 3-oxo-Z

ESR spectra of dimeric copper(II) compounds of CuX2(H2O)2 and CuX2(pý) (X = 2-fluoro-6-chlorobenzoate; py = pyridine)

cholan-24-oic acid and 3/?-hydroxyS/3-cholan-24-oic acid. Nuclear dehydrogenation of lithocholic acid has also been noted with Corynebacterium simplex [3], where 3+xochola-1,4dien-24-oic acid was isolated. The microbial side-chain cleavage of lithocholic acid has been shown with Corynebacterium simplex [4] and Pseudomonas NCIB 10590 [5] yielding androsta-1,4-dien-3,17-dione in each case. A strain of Escherichia coZi isolated from a faecal sample of a colon cancer patient has the ability to degrade the side-chain of lithocholic acid yielding both Czz and C19 products [6]. Microbial degradation of the steroid nucleus has been observed when lithocholic acid was incubated with Cotynebacterium simplex [3]. In this case a nonsteroidal product, namely (4R)4-[4cz-(2-carboxyethyl)3acu-hexahydro-7-a/3-methyl-5-oxindan-l~-yl]valeric acid, was isolated. We outlined [7] a possible scheme for the microbial degradation of lithocholic acid. The isolation, identi-