Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

Investigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracids

The present study investigated the genotoxic potential of the marine biotoxins okadaic acid (OA) and azaspiracids (AZAs). Harmful algae blooms (HABs) are an increasing global problem with implications for the ecosystem, economy and human health. Most data available on human intoxication are based on acute toxicity. To date, limited data has been published on possible long term effects, carcinogenicity and genotoxicity. To investigate genotoxicity in the present study, DNA fragmentation was detected using the COMET assay. In contrast to most other available studies, two further endpoints were included. The Trypan Blue Exclusion assay was used to provide information on possible cytotoxicity and assess the right concentration range. Flow cytometer analysis was included to detect the possible involvement of apoptotic processes. In house background data for all endpoints were established using positive controls. Three different cell lines, Jurkat T cells, CaCo-2 cells and HepG-2 cells, representing the main target organs, were exposed to OA and AZA1-3 at different concentrations and exposure times. Data obtained from the COMET assay showed an increase in DNA fragmentation for all phycotoxins, indicating a modest genotoxic effect. However, the data obtained from the Trypan Blue Exclusion assay showed a clear reduction in cell viability and cell number, indicating the involvement of cytotoxic and/or apoptotic processes. This is supported by data obtained by flow cytometer analysis. All phycotoxins investigated showed signs of early/late apoptosis. Therefore, the combined observations made in the present study indicate that OA and AZA1-3 are not genotoxic per se. Apoptotic processes appear to make a major contribution to the observed DNA fragmentation. The information obtained in this study stresses the importance of inclusion of additional endpoints and appropriate positive controls in genotoxicity studies. Furthermore, these data can assist in future considerations on risk assessment, especially regarding repeated exposure and exposure at sub-clinical doses.

The use of different blood sample preparations in the development of biomarkers for the environmental monitoring of domestic farm animals

The application of biological effect monitoring for the detection of environmental chemical exposure in domestic animals is still in its infancy. This study investigated blood sample preparations in vitro for their use in biological effect monitoring. When peripheral blood mononuclear cells (PBMCs), isolated following the collection of multiple blood samples from sheep in the field, were cryopreserved and subsequently cultured for 24 hours a reduction in cell viability (<80%) was attributed to delays in the processing following collection. Alternative blood sample preparations using rat and sheep blood demonstrated that 3 to 5 hour incubations can be undertaken without significant alterations in the viability of the lymphocytes; however, a substantial reduction in viability was observed after 24 hours in frozen blood. Detectable levels of early and late apoptosis as well as increased levels of ROS were detectable in frozen sheep blood samples. The addition of ascorbic acid partly reversed this effect and reduced the loss in cell viability. The response of the rat and sheep blood sample preparations to genotoxic compounds ex vivo showed that EMS caused comparable dose-dependent genotoxic effects in all sample preparations (fresh and frozen) as detected by the Comet assay. In contrast, the effects of CdCl2 were dependent on the duration of exposure as well as the sample preparation. The analysis of leukocyte subsets in frozen sheep blood showed no alterations in the percentages of T and B lymphocytes but led to a major decrease in the percentage of granulocytes compared to those in the fresh samples. The percentages of IFN-γ and IL-4 but not IL-6 positive cells were comparable between fresh and frozen sheep blood after 4 hour stimulation with phorbol 12-myrisate 13-acetate and ionomycin (PMA+I). These results show that frozen blood gives comparable responses to fresh blood samples in the toxicological and immune assays used.