Yvonne C. O'Callaghan

Comparative study of the cytotoxicity and apoptosis-inducing potential of commonly occurring oxysterols

The cytotoxicity of the oxysterols 25-hydroxycholesterol, 7β-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 19-hydroxycholesterol and 7-ketocholesterol was examined in U937 cells, a human monocytic blood cell line. 7β-Hydroxycholesterol, cholesterol-5β,6β-epoxide, and 7-ketocholesterol, at 30 μmol/L concentration, were found to be cytotoxic to this cell line and the mode of cell death was by apoptosis. 25-Hydroxycholesterol, cholesterol-5α,6α-epoxide and 19-hydroxycholesterol (30 μmol/L) did not induce apoptosis in this cell line. Since it has been suggested that the generation of an oxidative stress may occur in the early stages of the apoptotic process, the glutathione concentration and the activity of superoxide dismutase were also measured in the oxysterol-treated cells. 7β-Hydroxycholesterol was shown to increase the superoxide dismutase activity and decrease the glutathione concentration. However, cholesterol-5β,6β-epoxide and 7-ketocholesterol, which were also shown to induce apoptosis, did not affect the glutathione concentration or the superoxide dismutase activity in the U937 cells. Therefore, oxysterol-induced apoptosis may not be dependent on the generation of an oxidative stress.

The role of the mitochondria in apoptosis induced by 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols are oxygenated derivatives of cholesterol that may be formed endogenously or absorbed from the diet. Significant amounts of oxysterols have frequently been identified in foods of animal origin, in particular highly processed foods. To date, oxysterols have been shown to possess diverse biological activities; however, recent attention has focused on their potential role in the development of atherosclerosis. Oxysterols have been reported to induce apoptosis in cells of the arterial wall, a primary process in the development of atheroma. The aim of the present study was to identify the role of the mitochondria in the apoptotic pathways induced by the oxysterols 7beta-hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) in U937 cells. To this end, we investigated the effects of these oxysterols on mitochondrial membrane potential, caspase-8 activity, the mitochondrial permeability transition pore and cytochrome c release. 7beta-OH-induced apoptosis was associated with a loss in mitochondrial membrane potential after 2 h, accompanied by cytochrome c release from the mitochondria into the cytosol after 16 h. Pre-treatment with a range of inhibitors of the mitochondrial permeability transition pore protected against 7beta-OH-induced cell death. In contrast, beta-epoxide induced a slight increase in caspase-8 activity but had no effect on mitochondrial membrane potential or cytochrome c release. The present results confirm that 7beta-OH-induced apoptosis occurs via the mitochondrial pathway and highlights differences in the apoptotic pathways induced by 7beta-OH and beta-epoxide in U937 cells.

Cytotoxic and Apoptotic Effects of the Oxidized Derivatives of Stigmasterol in the U937 Human Monocytic Cell Line

Dietary exposure to phytosterols has increased in recent years due to the incorporation of these compounds into cholesterol-lowering products. Previous studies have investigated the cytotoxic effects of the oxidized derivatives of β-sitosterol and determined that phytosterol oxidation products (POP) have a similar but less potent toxicity compared to their cholesterol equivalents. In the present study, the cytotoxicity of the oxidized derivatives of stigmasterol were investigated in the U937 cell line. The stigmasta-5,22-diene-3β,7β-diol (7β-OH), 5,6-epoxystigmasta-22,23-diol (epoxydiol), 5,6,22,23-diepoxystigmastane (diepoxide), and (22R,23R)-stigmast-5-ene-3β,22,23-triol (22R,23R-triol) derivatives were identified as the most cytotoxic, and the mode of cell death was identified as apoptosis in cells incubated with 7β-OH, epoxydiol, and diepoxide stigmasterol. The antioxidants α-tocopherol, γ-tocopherol, and β-carotene did not protect against apoptosis induced by 7β-OH and diepoxide stigmasterol; however, α-tocopherol was found to protect against epoxydiol-induced apoptosis. The cellular antioxidant, glutathione, was depleted and the apoptotic protein, Bcl-2, was down-regulated by the stigmasterol oxides identified as apoptotic.

Synthesis and Characterization of Stigmasterol Oxidation Products

The synthesis and structural characterization of a series of oxides of stigmasterol is described providing a valuable series of reference standards for these oxides, analogous to the cholesterol oxidation products (COPs) which have been shown to have detrimental biological effects. Biological evaluation of the oxides of phytosterols is significant in the context of increased dietary use of phytosterols in the drive to reduce cholesterol absorption.

Antioxidant, immunomodulatory and antiproliferative effects of gelatin hydrolysate from unicorn leatherjacket skin

BACKGROUND The in vitro cellular bioactivities including, antioxidant, immunomodulatory and antiproliferative effects of a gelatin hydrolysate (GH) prepared from unicorn leatherjacket skin, using partially purified glycyl endopeptidase, were investigated in order to optimize the use of fish skin waste products as functional food ingredients. RESULTS GH under the tested concentrations (750-1500 µg mL(-1) ) protected against H2 O2 -induced DNA damage in U937 cells. GH also protected against the H2 O2 -induced reduction in cellular antioxidant enzyme activities, superoxide dismutase and catalase, in HepG2 cells. GH demonstrated immunomodulatory potential by reducing pro-inflammatory cytokine (interleukin-6 (IL-6) and IL-1β) production and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Cell proliferation in human colon cancer (Caco-2) cells was significantly reduced in a dose-dependent manner following incubation with GH. CONCLUSION These results indicate that GH has several bioactivities which support its potential as a promising functional food ingredient with various health benefits.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells.

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and β-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, β-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7β-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3β,5α,6β-triol and 2CMP:1DHB-5β,6β-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Anti-Inflammatory Effects of Wild Irish Mushroom Extracts in RAW264.7 Mouse Macrophage Cells

Mushrooms and mushroom extracts have traditionally been used as therapies for a wide variety of ailments, including allergy, arthritis, and other inflammatory disorders. However, more evidence is required on the mechanism by which mushrooms exert these effects. In the present study, the anti-inflammatory properties of ethanol and hot water extracts prepared from 27 fungal samples collected between October and November 2011 at various forest locations in the southwest of Ireland were investigated using the lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7 cells) model of inflammation. LPS-stimulated cells were incubated in the presence of mushroom extracts at nontoxic concentrations for 24 h and the production of interleukin-6 (IL-6) was quantified by ELISA. Seven ethanolic and one hot water extract that decreased IL-6 production were selected for further study. The extracts were then incubated with LPS-stimulated cells for 24 h and the production of IL-6, tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) was measured. Ethanolic extracts prepared from Russula mairei, Lactarius blennius, Craterellus tubaeformis, Russula fellea, and Craterellus cornucopioides demonstrated selective anti-inflammatory activity by decreasing the production of NO and IL-6 but not TNF-α in LPS-stimulated RAW264.7 cells. These findings support existing evidence of the anti-inflammatory potential of mushroom extracts.

Immunomodulatory potential of a brewers’ spent grain protein hydrolysate incorporated into low-fat milk following in vitro gastrointestinal digestion

Abstract Brewers’ spent grain (BSG) protein rich fraction was previously hydrolysed using Alcalase (U) and three additional fractions were prepared by membrane fractionation; a 5-kDa retentate (U > 5), a 5-kDa permeate (U < 5) and a 3-kDa permeate (U < 3). In the present study, these fractions were added to milk, subjected to simulated gastrointestinal digestion (SGID) and their anti-inflammatory potential was investigated. The digestates caused a significant reduction (p < 0.05) in interleukin-6 (IL-6) production in Concanavalin-A (ConA)-stimulated Jurkat T cells. The samples did not significantly alter the production of IL-6 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. IL-2 and interferon-γ (IFN-γ) production in stimulated Jurkat T cells and IL-1β and tumor necrosis factor-α (TNF-α) production in stimulated RAW 264.7 cells were not affected in the presence of the digestates. Results show that a SGID milk product supplemented with BSG hydrolysate and its associated ultrafiltered fractions can confer anti-inflammatory effects in Jurkat T cells.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50μg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50μg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50μg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.

Bioaccessibility of polyphenol and carotenoid from wild Irish berries subjected to an in vitro digestion procedure

Berries are a relatively rich dietary source of fibre, vitamins, minerals and phytochemicals, which may have a wide range of human health benefits such as anti-cancer, anti-inflammatory and antioxidant activities. Little information is available on the phytochemical content of edible wild Irish berries. In addition, the effect of digestion on the bioaccessibility of antioxidants and carotenoids has previously been examined in a wide range of foods; however, there is little available information relating to berries. Therefore, we measured the content and bioassessibility of phenols and carotenoids in hawthorn (Crataegus monogyna), whitebeam (Sorbus aria), rowan (Sorbus aucuparia), rosehip (Rosa super excelsa) and sloe (Prunus spinosa). Berries were picked locally. For each berry, a 5 g sample was weighed, homogenised and subjected to in-vitro digestion, which simulated human gastric and intestinal digestion. Ultracentrifugation was used to separate micelle fractions from the digested material. Total phenol content (TPC) of the undigested and micelle samples was measured using the Folin–Ciocalteu method. Both the undigested and micelle samples were saponified and carotenoids were extracted using hexane. HPLC was used to quantify the carotenoid content of the fruits. Bioaccessibility is defined as the amount of phenols or carotenoids transferred to micelles after digestion when compared with the original amount present in the food.