Assessment of the ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells.

The ability of brown seaweed extracts, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus to protect against tert-butyl hydroperoxide (tert-BOOH) induced stress in Caco-2 cells was investigated. Oxidative stress was determined by measuring alteration in the enzymatic activity of catalase (CAT) and superoxide dismutases (SOD) and cellular levels of glutathione (GSH). L. hyperborea, P. canaliculata and F. serratus significantly protected against tert-BOOH induced SOD reduction but did not protect against the reduction in CAT activity or the increased cellular levels of GSH. The ability of F. serratus and F. vesiculosus to protect against H(2)O(2) and tert-BOOH induced DNA damage was also assessed. The DNA protective effects of the two seaweed extracts was compared to those of three metal chelators; deferoxamine mesylate (DFO), 1,10-phenanthroline (o-phen) and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM). F. serratus and F. vesiculosus significantly protected (P<0.05) against H(2)O(2) (50 µM) induced DNA damage but not tert-BOOH induced damage.

Evaluation of the antioxidant potential of grape seed and bearberry extracts in raw and cooked pork.

The effect of grape seed extract (GSE) and bearberry (BB), on lipid oxidation (TBARS, mg malondialdehyde (MDA)/kg muscle), colour (CIE 'a' redness value), pH, microbial status (log(10)CFU colony forming units/g pork) and sensorial properties of cooked pork patties was investigated. GSE (0-1000µg/g muscle) and BB (0-1000µg/g muscle) were added to raw pork (M. longissimus dorsi) patties which were stored in modified atmosphere packs (MAP) (75% O(2):25% CO(2)) for up to 12 days at 4°C. Cooked pork patties were stored in MAP (70% N(2):30% CO(2)) for up to 4 days at 4°C. Mesophilic plate counts and pork pH were unaffected by GSE and BB. GSE and BB addition decreased (P<0.05) lipid oxidation (TBARS) in raw pork patties on days 9 and 12 of storage, relative to controls. Antioxidant activity of GSE and BB was observed in cooked pork patties demonstrating the thermal stability of GSE and BB. The 'a' redness values of raw and cooked pork patties marginally increased with increasing GSE concentration. The sensory properties of cooked pork patties were unaffected by GSE and BB addition. Results obtained demonstrate the potential for using health promoting nutraceuticals in meat and meat products.

Involvement of calcium in 7beta -hydroxycholesterol and cholesterol-5beta,6beta -epoxide-induced apoptosis.

Oxidized low-density lipoprotein (oxLDL) is believed to play a central role in the development of atherosclerosis. The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7beta-Hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. The exact mechanism through which these oxysterols induce apoptosis remains to be fully elucidated. A perturbation of intracellular calcium homeostasis has been found to trigger apoptosis in many experimental systems. The aim of the present study was to determine the involvement of calcium signaling in 7beta-OH and beta-epoxide-induced apoptosis. To this end, the authors employed the calcium channel blockers verapamil and nifedipine and inhibitors of calpain activation, ALLM and ALLN. Verapamil protected against the decrease in viability induced by 7beta-OH whereas nifedipine had a protective effect in both 7beta-OH and beta-epoxide-treated cells, though these compounds did not restore viability to control levels. Verapamil, nifedipine, and ALLM prevented apoptosis induced by beta-epoxide. None of the compounds employed in the current study protected against 7beta-OH-induced apoptosis. Our results implicate calcium signaling in the apoptotic pathway induced by beta-epoxide and also highlight differences between apoptosis induced by 7beta-OH and beta-epoxide.

Phytosterols: lack of cytotoxicity but interference with beta-carotene uptake in Caco-2 cells in culture.

Ingestion of phytosterols has been shown to reduce plasma cholesterol in both animals and humans. The esterified forms of phytosterols are increasingly being incorporated into margarine and fat spreads, which are then marketed as functional foods. The aim was to assess the cytotoxicity and uptake of four phytosterols, beta-sitosterol, campesterol, stigmasterol and stigmastanol, in human intestinal cells in culture. Another aim was to determine if phytosterols would interfere with alpha-tocopherol or beta-carotene uptake by these cells. Human adenocarcinoma Caco-2 cells were supplemented for 24 h with increasing concentrations (0-12.5 microM) of each phytosterol. Cytotoxicity was assessed by neutral red uptake (NRU), lactate dehydrogenase release (LDH) and fluorescein diacetate/ethidium bromide (FDA/EtBr) assays. The phytosterols had no significant effects on Caco-2 cell viability assessed using LDH and FDA/EtBr assays. The highest concentrations of beta-sitosterol and campesterol tested (12.5 microM) resulted in decreased cell viability assessed using the NRU assay. All phytosterols were taken up by Caco-2 cells in culture. The results demonstrate a reduction in the uptake of beta-carotene when Caco-2 cells were supplemented with 20 microM beta-sitosterol. beta-Sitosterol did not interfere with alpha-tocopherol uptake by the cells. In conclusion, Caco-2 cells are a useful model system to study potential interactive effects of phytosterols with fat-soluble dietary components.

Comparison of the apoptotic processes induced by the oxysterols 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide.

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7beta-hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochrome c release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7beta-OH; however, in beta-epoxide-treated cells, the inhibitor of cytochrome c release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7beta-OH-treated cells but not in cells incubated with beta-epoxide. Trolox, a water-soluble synthetic analogue of alpha-tocopherol, prevented 7beta-OH-induced apoptosis but did not protect against cell death induced by beta-epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7beta-OH or beta-epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7beta-OH and beta-epoxide in U937 cells.

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Characteristics of 7 beta-hydroxycholesterol-induced cell death in a human monocytic blood cell line, U937, and a human hepatoma cell line, HepG2.

Oxysterols have been shown in a number of cell lines to induce apoptosis by a mechanism as yet unclear. The induction of apoptosis by certain agents has been associated with the generation of oxidative stress and the depletion of the endogenous antioxidant, glutathione, which may result in cytochrome c release and caspase activation. The aim of the present study was to determine whether 7 beta-hydroxycholesterol (7 beta-OH) alters glutathione levels or the activities of catalase, superoxide dismutase (SOD) or caspase-3 in association with cell death in either the U937 or the HepG2 cell lines. 7 beta-OH, which induced significant apoptosis at 12 h in the U937 cell line, was shown to cause a significant decrease in glutathione levels and an increase in the activity of SOD at this time point. An increase in caspase-3 activity was also observed in the U937 cell line following a 24-h incubation with 7 beta-OH. Glutathione concentration, SOD activity and caspase-3 activity were unchanged in the HepG2 cell line, which underwent necrosis following incubation with 7 beta-OH. The activity of the enzyme catalase remained unchanged in both cell lines. These results provide evidence that the generation of an oxidative stress may be a significant event occurring during 7 beta-OH-induced apoptosis.

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

The cytotoxicity of the oxysterols 25-hydroxycholesterol, 7beta-hydroxycholesterol, cholesterol-5alpha,6alphaepoxide, cholesterol-5beta,6beta-epoxide, 19-hydroxycholesterol and 7-ketocholesterol was examined in U937 cells, a human monocytic blood cell line. 7beta-Hydroxycholesterol, cholesterol-5beta,6beta-epoxide, and 7-ketocholesterol, at 30 micromol/L concentration, were found to be cytotoxic to this cell line and the mode of cell death was by apoptosis. 25-Hydroxycholesterol, cholesterol-5alpha,6beta-epoxide and 19-hydroxycholesterol (30 micromol/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. 7beta-Hydroxycholesterol was shown to increase the superoxide dismutase activity and decrease the glutathione concentration. However, cholesterol-5beta,6beta-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.

Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol.

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline single cell gel electrophoresis assay detected DNA strand breaks occurring during cell death, these breaks were observed only when the process was fairly well advanced and a major part of the cells had lost membrane permeability. Therefore the present report demonstrates that the single cell gel electrophoresis assay, used in isolation, cannot accurately be used to distinguish between the mode of cell death induced by 7betaOHC in U937 cells (apoptosis), or HepG2 cells (cell lysis).

Cytotoxicity, genotoxicity and oxidative reactions in cell-culture models: modulatory effects of phytochemicals.

Much research effort has focused on the identification of phytochemicals in fruit and vegetables which exert beneficial effects. Our research examines modulatory effects of phytochemicals on cytotoxicity, genotoxicity and oxidative reactions in cell systems. Two examples of our studies are discussed. First, the potential beneficial effects of flavonoids are demonstrated. Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free-radical-scavenging activities. The aim of the study was to determine if flavonoids could protect against H2O2-induced DNA damage, as measured by the comet assay, in Caco-2 and HepG2 cells. Both cell lines were supplemented with increasing concentrations of myricetin, quercetin and rutin for 24 h followed by exposure to H2O2 (50 microM) for 30 min. Exposure to H2O2 for 30 min at 37 degrees C resulted in significant DNA damage and pre-incubation with the flavonoids before H2O2 exposure significantly (P <0.05) protected Caco-2 and HepG2 cells against H2O2-induced DNA damage. Secondly, we illustrate the use of cellular models to study oxysterol-induced toxicity. Oxysterols are generated during the cooking and processing of foods and may be produced endogenously by the oxidation of membrane lipids. Recent findings suggest that oxysterols may modulate cytotoxicity by exerting effects on the induction of apoptosis. 7beta-Hydroxycholesterol (7beta-OHC) and 25-hydroxycholesterol, both of which are commonly found in foods, were investigated for their abilities to induce apoptosis in a human monocytic blood cell line, U937, and in the human hepatoma cell line, HepG2 cells. U937 and HepG2 cells were incubated for up to 48 h with 30 microM oxysterol. 7beta-OHC induced apoptosis in U937 cells as measured by non-random DNA fragmentation, condensed and fragmented nuclei, and the generation of hypodiploid cells. In contrast, oxysterols may induce cell death by a different mechanism in the hepatoma cells, possibly by necrosis.

Oxysterol-induced cell death in U937 and HepG2 cells at reduced and normal serum concentrations.

BACKGROUND AND AIMS: Cholesterol oxidation products (oxysterols) are commonly found in foods of animal origin and are also produced endogenously in the body. Oxysterols are cytotoxic to certain cell lines and in some cases have been shown to induce apoptosis. The aim of this study was to investigate the effects of 7beta-hydroxy-cholesterol (7beta-OHC) and 25-hydroxycholesterol (25-OHC) on cytotoxicity and induction of apoptosis in U937 and HepG2 cells, treated in media containing either 2.5% foetal calf serum (FCS) or 10% FCS to examine the effect of increasing the cholesterol level. METHODS: The cells were incubated for 24 and 48 h with 30 microM oxysterol. Viability was assessed by fluorescein diacetate/ethidium bromide staining and cell proliferation was determined by haemocytometer counting. Apoptosis was monitored by detection of DNA fragments (laddering) in 1.5% agarose gels. Cells with condensed or fragmented nuclei were identified by Hoechst 33342 staining. The percentage of cells with sub-G1 levels of DNA was measured by flow cytometry. RESULTS: Treatment of U937 cells with 7beta-OHC, in contrast to 25-OHC, resulted in a decrease in cell viability and proliferation at 24 and 48 h (P <.01). 25-OHC and 7beta-OHC were both equally cytotoxic to the HepG2 cell line. 7beta-OHC induced DNA laddering and an increase in the percentage of condensed or fragmented nuclei at both time points and at both serum concentrations in the U937 cell line. 25-OHC induced faint laddering in the U937 cells after 48 h in reduced serum media and resulted in a small increase in percentage condensed or fragmented nuclei which was independent of time of oxysterol exposure and serum concentration. The percentage of condensed or fragmented nuclei was low in the HepG2 cell line and no laddering was observed under any of the conditions studied. Flow cytometric analysis showed that only 7beta-OHC treated U937 cells had an increased level of hypodiploid cells. CONCLUSION: Both oxysterols appear to be equally cytotoxic to the HepG2 cell line. In U937 cells, 25-OHC is much less cytotoxic than 7beta-OHC. In addition, we have shown that 7beta-OHC induces apoptosis in U937 cells. 10% FCS displays a protective effect on cytotoxicity (as well as on 7beta-OHC induced apoptosis in U937 cells), although the data did not reach statistical significance.