Carotenoids: Considerations for Their Use in Functional Foods, Nutraceuticals, Nutricosmetics, Supplements, Botanicals, and Novel Foods in the Context of Sustainability, Circular Economy, and Climate Change.

Carotenoids are versatile isoprenoids that are important in food quality and health promotion. There is a need to establish recommended dietary intakes/nutritional reference values for carotenoids. Research on carotenoids in agro-food and health is being propelled by the two multidisciplinary international networks, the Ibero-American Network for the Study of Carotenoids as Functional Foods Ingredients (IBERCAROT; http://www.cyted.org) and the European Network to Advance Carotenoid Research and Applications in Agro-Food and Health (EUROCAROTEN; http://www.eurocaroten.eu). In this review, considerations for their safe and sustainable use in products mostly intended for health promotion are provided. Specifically, information about sources, intakes, and factors affecting bioavailability is summarized. Furthermore, their health-promoting actions and importance in public health in relation to the contribution of reducing the risk of diverse ailments are synthesized. Definitions and regulatory and safety information for carotenoid-containing products are provided. Lastly, recent trends in research in the context of sustainable healthy diets are summarized.

A Marine-Derived, Multi-mineral Supplement Influences Bacterial Fermentation and Short Chain Fatty Acid Profile In Vitro.

Aquamin is a calcium-rich multi-mineral supplement derived from the red marine algae, Lithothamnion species. Calcium supplementation has been shown to exert a prebiotic-like effect on the gut microbiota and has been associated with distinct changes in lactate and short chain fatty acid (SCFA) production. Irritable bowel syndrome (IBS) subtype is associated with changes in SCFA levels compared with healthy controls. Using an ex vivo simulation model, and a fecal inoculum from a patient diagnosed with IBS, we evaluated the effects of Aquamin (at 6 and 30 mg/mL) on SCFAs and lactate production, pH and gas production, and human microbiota composition. Our results demonstrate that Aquamin increased SCFA production (acetate and propionate by 8% and 24%, respectively, at 30 mg/mL dose), significantly decreased lactate production (30 mg/mL), and increased colonic fluid pH without inducing changes in colonic gas production or gastrointestinal (GI) microbiota composition. These results indicate that Aquamin may play a role in optimizing GI microbial function in an ex vivo setting.

Comparison of the nutritional composition of experimental fermented milk:wheat bulgur blends and commercially available kishk and tarhana products.

Dried, fermented blends of dairy products and cereals, such as kishk and tarhana, are foodstuffs traditionally consumed in many regions as they possess good nutritional qualities and extended storage stability. This study examined the nutritional composition of kishk or tarhana type products and compared with experimental blends of fermented milk and wheat bulgur containing 60-80% milk. The blends with higher milk contents had levels of protein (18.9%) and fat (5.8%) at the concentrations specified in fortified blended foods as outlined by the World Food Program. Higher milk contents were also associated with higher contents of calcium (323.2 mg/100 g), phosphorus (335.3 mg/100 g), vitamin A (486.7 µg/100 g) and α-tocopherol (174.5 µg/100 g). The nutritional content of the experimental fermented milk:wheat bulgur blends compared favourably with that of the commercial samples. These blends may be suitable as base products, to be fortified with micronutrients, for the development of fortified blended foods (FBFs) for humanitarian distribution.

Bioaccessibility and Bioavailability of a Marine-Derived Multimineral, Aquamin-Magnesium.

Introduction: Magnesium is an essential mineral involved in a range of key biochemical pathways. Several magnesium supplements are present on the market and their degree of bioavailability differs depending on the form of magnesium salt used. Aquamin-Mg is a natural source of magnesium, containing 72 additional trace minerals derived from the clean waters off the Irish coast. However, the in vitro bioaccessibility and bioavailability of Aquamin-Mg in comparison with other supplement sources of magnesium has yet to be tested. Method: Aquamin-Mg, magnesium chloride (MgCl2) and magnesium oxide (MgO) were subjected to gastrointestinal digestion according to the harmonized INFOGEST in vitro digestion method and in vitro bioavailability tested using the Caco-2 cell model. Magnesium concentration was measured by atomic absorption spectrophotometry (AAS). Results: Magnesium recovery from both Aquamin-Mg and MgCl2 was greater than for MgO. Magnesium from all three sources was transported across the epithelial monolayer with Aquamin-Mg displaying a comparable profile to the more bioavailable MgCl2. Conclusions: Our data support that magnesium derived from a marine-derived multimineral product is bioavailable to a significantly greater degree than MgO and displays a similar profile to the more bioavailable MgCl2 and may offer additional health benefits given its multimineral profile.

Concurrent iron and zinc deficiencies in lactating mothers and their children 6-23 months of age in two agro-ecological zones of rural Ethiopia.

PURPOSE: The aim of this study is to examine the co-occurrences of low serum ferritin and zinc and anaemia among mothers and their children in two agro-ecological zones of rural Ethiopia. METHODS: Data were collected from 162 lactating mothers and their breast fed children aged 6-23 months. The data were collected via a structured interview, anthropometric measurements, and blood tests for zinc, ferritin and anaemia. Correlation, Chi-square and multivariable analysis were used to determine the association between nutritional status of mothers and children, and agro-ecological zones. RESULTS: Low serum levels of iron and zinc, anaemia and iron deficiency anaemia were found in 44.4, 72.2, 52.5 and 29.6% of children and 19.8, 67.3, 21.8, 10.5% of mothers, respectively. There was a strong correlation between the micronutrient status of the mothers and the children for ferritin, zinc and anaemia (p < 0.005). Deficiency in both zinc and ferritin and one of the two was observed in 19.1, and 53.7% of the mothers and 32.7 and 46.3%, of their children, respectively. In the 24 h before the survey, 82.1% of mothers and 91.9% of their infants consumed foods that can decrease zinc bioavailability while only 2.5% of mothers and 3.7% of their infants consumed flesh foods. CONCLUSION: This study shows that micronutrient deficiencies were prevalent among lactating mothers and their children, with variation in prevalence across the agro-ecological zones. This finding calls for a need to design effective preventive public health nutrition programs to address both the mothers' and their children's needs.

Levels of potential bioactive compounds including carotenoids, vitamin C and phenolic compounds, and expression of their cognate biosynthetic genes vary significantly in different varieties of potato (Solanum tuberosum L.) grown under uniform cultural conditions.

BACKGROUND: In addition to their high carbohydrate content, potatoes are also an important dietary source of vitamin C and bioactive secondary metabolites, including phenolic compounds and carotenoids, which have been suggested to play a role in human health. The expression of genes encoding key enzymes involved in the synthesis of these compounds was assessed by reverse transcription-quantitative polymerase chain reaction and compared to the accumulation of the corresponding product in seven potato varieties showing contrasting levels of metabolite accumulation. RESULTS: Strong positive correlations were found between phenolic content in the flesh of tubers and transcript levels of phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. The expression of PAL and CHS was also related to that of AN1, a transcription factor involved in the synthesis of anthocyanins, suggesting that these genes are regulated in a coordinated manner. No clear relationship was found between transcript levels of phytoene synthase (PSY) or L-galactono-1,4-lactone dehydrogenase (GLDH) genes and total carotenoid or vitamin C accumulation, respectively. CONCLUSION: Data indicate that levels of total phenolic and flavonoid compounds in potato are controlled primarily by PAL and CHS gene expression. Transcript levels of PSY and GLDH did not control accumulation of carotenoids or vitamin C.

Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin (4-(ß-D-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde).

The cellular transport and bioactivity of the second major saffron apocarotenoid, picrocrocin, was examined in parallel to that of the major group, crocetin sugar esters, in aqueous extracts. The transport of pure picrocrocin was investigated in comparison to that of other saffron apocarotenoids, trans-crocetin (di-ß-D-gentiobiosyl) ester and crocetin using the Caco-2 cell model coupled with an in vitro digestion procedure. RP-HPLC-DAD was employed to quantify the bioaccessible and bioavailable amounts of individual apocarotenoids. Picrocrocin and crocetin sugar esters though highly bioaccessible (75% and 60%, respectively) were transported at minute quantities (0.2% and 0.5%, respectively; 10-fold lower than crocetin). Picrocrocin did not protect against oxidant-induced DNA damage in U937, human monocytic blood cells at the concentration investigated, however, it reduced the proliferation of human adenocarcinoma and hepatocarcinoma cells. Our findings may be useful for the requirements of food legislation regarding saffron preparations, in which both apocarotenoid groups coexist.

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

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-1ß 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.

A study of the ability of bioactive extracts from brewers' spent grain to enhance the antioxidant and immunomodulatory potential of food formulations following in vitro digestion.

Bioactivity of a snack-bar, chocolate-drink and yogurt fortified with brewers' spent grain (BSG) phenolic extracts (P2 or B2) or protein hydrolysates (barley protein hydrolysate (BPH), BPH < 3 kDa, BPH < 5 kDa, BPH > 5 kDa) was measured following gastrointestinal in vitro digestion. Concentrations of 0.5 and 0.1% (v/v) digestates were chosen for addition to Caco-2 and Jurkat T cells, respectively. Yogurt and B2 digestate protected against H2O2-induced DNA damage in Caco-2 cells (p < 0.05), by the comet assay. Snack-bar digestates possessed significant (p < 0.05) immunomodulatory effects, measured by ELISA in concanavalin-A stimulated Jurkat T cells. Addition of BPH enhanced (p < 0.05) the IFN-γ reducing capacity of the snack-bar while addition of BPH < 3 and < 5 kDa reduced IL-2 production to a greater extent than unfortified yogurt (p < 0.05). Selected BSG components can enhance the antioxidant and anti-inflammatory potential of foods.

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.

Phenolic-enriched fractions from brewers' spent grain possess cellular antioxidant and immunomodulatory effects in cell culture model systems.

BACKGROUND: Large quantities of brewers' spent grain (BSG), a co-product of the brewing industry, are produced annually. BSG contains hydroxycinnamic acids, and phenolic-rich extracts from BSG have previously demonstrated the ability to protect against oxidant-induced DNA damage. The present study investigated the anti-inflammatory potential of eight phenolic extracts from BSG: four pale (P1-P4) and four black (B1-B4) extracts. RESULTS: BSG extracts were more cytotoxic in Jurkat T than U937 cells, with lower IC50 values in Jurkat T cells, measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pale BSG extracts P2 and P3 showed the greatest anti-inflammatory potential, significantly (P < 0.05) reducing interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ) production. In addition, extracts P1-P3 and B2-B4 showed significant (P < 0.05) antioxidant effects, determined by the cellular antioxidant activity assays superoxide dismutase, catalase and glutathione content (GSH). CONCLUSION: Phenolic extracts from BSG, particularly the pale BSG extracts, have the ability to reduce a stimulated cytokine production and may also protect against cellular oxidative stress. Results of the present study highlight the potential of BSG phenolic extracts to act as functional food ingredients, providing an alternative use and improving the value of this brewing industry co-product.

Antioxidant and pro-apoptotic effects of marine-derived, multi-mineral aquamin supplemented with a pine bark extract, Enzogenol, and a green tea extract, Sunphenon.

A high dietary intake of polyphenols has been associated with a decreased risk of cardiovascular disease and cancer, attributed in part to their antioxidant activity and pro-apoptotic effects. Aquamin is a multi-mineral algal extract that enhances bone mineralization, relieves osteoarthritis, and aids digestion; however, Aquamin has not demonstrated antioxidant activity. In the present study, Aquamin was supplemented with 8% Enzogenol, a pine bark extract with a high phenolic content, and 2% Sunphenon, a green tea extract that also has a high phenolic content to produce a mixed product (A:E:S). The antioxidant activity of A:E:S was compared with that of its constituent compounds and also with catechin and epigallocatechin by measuring total phenol content, ferric-reducing antioxidant potential, and 2,2-diphenyl-2-picrylhydrazyl hydrate. The cytotoxic and apoptotic effects of the compounds were also measured in the U937, human monocytic blood cell line. A:E:S demonstrated an antioxidant activity that was equal to that of the compounds used in its preparation. Aquamin was not cytotoxic in the U937 cell line; however, A:E:S was cytotoxic and the primary mechanism of cell death was apoptosis. The biological effects of Aquamin were enhanced by supplementation with Enzogenol and Sunphenon to include antioxidant effects and the ability to induce apoptosis in U937 cells.

The hydroxycinnamic acid content of barley and brewers' spent grain (BSG) and the potential to incorporate phenolic extracts of BSG as antioxidants into fruit beverages.

The hydroxycinnamic acid (HA) content of starting barley for brewers' spent grains (BSG), whole BSG and phenolic extracts from BSG was measured using high performance liquid chromatography (HPLC) and correlated with antioxidant potential. The effect of BSG phenolic extracts on antioxidant activity of fruit beverages was also assessed (using the total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays). The concentration of HA present in barley extract and BSG was in the order of ferulic acid (FA), p-coumaric acid (p-CA) derivatives, FA derivatives, p-CA, caffeic acid (CA) and CA derivatives. Results suggested that brewing and roasting decreased the HA content. Antioxidant activity was significantly (P<0.05) correlated with caffeic acid (R(2)=0.8309) and total HA (R(2)=0.3942) concentrations. Addition of extracts to fruit beverages resulted in a significant (P<0.05) increase in antioxidant activity of cranberry juice, measured by the FRAP assay. In vitro digestion significantly (P<0.05) reduced TPC, DPPH and FRAP activity of the fruit beverages.

Changes in total and individual crocetin esters upon in vitro gastrointestinal digestion of saffron aqueous extracts.

Changes that may be expected in crocetin esters (crocins) upon digestion were examined in saffron aqueous extracts for the first time. Chemical characterization of total and individual crocins and other bioactive compounds was achieved by UV-vis spectrophotometry, RP-HPLC-DAD, and LC-ESI-MS. Antioxidant activity was evaluated using in vitro assays and the comet assay. The observed loss for both total and trans-crocins was higher in saffron (∼50%) than in gardenia extracts (∼30%), which were also examined for comparison. Loss was lower than that reported for hydrophobic carotenoids. cis-Isomers were less affected, leading to the hypothesis that trans/cis isomerization may occur in parallel to degradation reactions. Monitoring changes in the extracts at oral, gastric, or intestinal phases, separately, verified this view pointing out the critical effect of pH, temperature, and duration of process but not of digestive enzymes. No isomerization and less degradation (<20% loss) was evidenced when pure trans-crocetin (di-ß-D-gentiobiosyl) ester was subjected to gastric or intestinal conditions.

Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages.

AIMS: The potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. MAIN METHODS: Cytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25 µg/ml) and lipopolysaccaride (LPS; 1 µg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. KEY FINDINGS: α-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. SIGNIFICANCE: Our findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases.

Brewers' spent grain; bioactivity of phenolic component, its role in animal nutrition and potential for incorporation in functional foods: a review.

Brewers' spent grain (BSG) is a low-value co-product of the brewing industry produced in large quantities annually. This paper reviews the existing evidence regarding the phenolic component of BSG, focusing on composition, extraction and biofunctions such as antioxidant, anti-atherogenic, anti-inflammatory and anti-carcinogenic activities. Furthermore, the incorporation of BSG in foodstuffs will be discussed, including the use of BSG as an animal feed supplement and the potential of BSG to be incorporated into foods for human consumption. BSG contains hydroxycinnamic acids including ferulic acid, p-coumaric acid and caffeic acid; which have shown bioactivity in the pure form (antioxidant, anti-inflammatory, anti-atherogenic and anti-cancer). Phenolic extracts from BSG have also shown antioxidant potential, by protecting against oxidant-induced DNA damage, possibly by Fe chelation. Studies show that BSG has many benefits when used as a supplement in animal feed, such as increasing milk yield, milkfat content and in providing essential dietary amino acids. The incorporation of BSG in human foods such as cookies and ready-to-eat snacks has resulted in increased protein and fibre contents of the products, where the changes in organoleptic properties are controllable. It can be concluded that the phenolic component of BSG has potential bioactive effects, which are worth pursuing given that the inclusion of BSG into human foodstuffs is viable and beneficial.

Phenolic extracts of brewers' spent grain (BSG) as functional ingredients - assessment of their DNA protective effect against oxidant-induced DNA single strand breaks in U937 cells.

Brewers' spent grain (BSG), a by-product of the brewing industry, contains high amounts of phenolic acids, which have antioxidant effects. The present study examined the ability of BSG extracts to protect against the genotoxic effects of oxidants, hydrogen peroxide (H(2)O(2)), 3-morpholinosydnonimine hydrochloride (SIN-1), 4-nitroquinoline 1-oxide (4-NQO) and tert-butylhydroperoxide (t-BOOH) in U937 cells. Four pale (P1-P4) and four black (B1-B4) BSG extracts were investigated. U937 cells were pre-incubated with BSG extracts, exposed to the oxidants and the DNA damage was measured by the Comet assay. The black BSG extracts (B1-B4) significantly protected against H(2)O(2)-induced DNA damage. Extract B2, which had the highest phenol content, provided the greatest protection. Extracts P2, B2, B3 and B4 provided significant protection against SIN-1-induced DNA damage. None of the extracts protected against DNA damage induced by t-BOOH and 4-NQO. The DNA protective effects of the BSG phenolic extracts may be related to iron chelation.

Carotenoid content of commonly consumed herbs and assessment of their bioaccessibility using an in vitro digestion model.

Herbs are a rich source of bioactive phytochemicals such as carotenoids, which are known to exert various positive biological effects. However, there is very limited information in the literature regarding the content and bioavailability of carotenoids from commonly consumed herbs. Therefore, the objectives of the present study were first, to determine the carotenoid content of eight herbs namely basil (Ocimum basilicum), coriander (Coriandrum sativum), dill (Anethum graveolens), mint (Metha L.), parsley (Petroselinum crispum), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), and tarragon (Artemisia dracunculus L.); and second, to assess carotenoid bioaccessibility from these herbs using a simulated human in vitro digestion model. Carotenoid bioaccessibility is defined as the amount of carotenoids transferred to micelles after digestion when compared with the original amount present in the food. The content of individual carotenoids varied significantly among the herbs tested. Carotenoid bioaccessibility varied from 0 to 42.8%. Basil and coriander, and their respective micelles, contained the highest levels of beta-carotene, beta-cryptoxanthin, and lutein + zeaxanthin. Our findings show that herbs are rich sources of carotenoids and that these foods can significantly contribute to the intake of bioaccessible carotenoids.

Bioactivity of herb-enriched beef patties.

Interest exists in the manufacture of meat products with added functional ingredients to enhance consumer health. Because experimental evidence suggests that many herbs and spices, particularly those of the Lamiaceae family such as Salvia officinalis L. (sage) and Origanum vulgare L. (oregano), possess a wide range of biological and pharmacological activities, they represent promising functional ingredients for incorporation into meat and meat products. The present study aimed to determine the bioactivity of cooked beef patties that were enriched with or without sage or oregano extracts (1,200 microg/g). Cooked beef patties were subjected to an in vitro digestion procedure, and the resulting micelles isolated from the digested meats were added to human intestinal Caco-2 cells. The antioxidant potential (ferric reducing antioxidant power [FRAP] value) of enriched beef patties was significantly higher than the FRAP value of non-enriched beef patties, both before and after in vitro digestion. Cell viability significantly increased following treatment with certain concentrations of the micelle fractions from digested sage- or oregano-enriched beef patties. Pretreatment with micelles derived from sage- or oregano-enriched beef patties did not significantly protect against cell injury or DNA damage induced by H(2)O(2). However, micelles derived from digested sage-enriched beef patties (10% vol/vol) significantly increased cellular reduced glutathione (GSH) content. In addition, micelles derived from both sage- and oregano-enriched beef patties (10% vol/vol) significantly protected against H(2)O(2)-induced GSH depletion. Thus, it appears that sage and oregano exhibit some bioactivity within a meat system. Our findings suggest that herbal extracts have potential as possible functional ingredients in meat products.

Lack of genoprotective effect of phytosterols and conjugated linoleic acids on Caco-2 cells.

Much interest has focused on the cholesterol-lowering effects of phytosterols (plant sterols) but limited data suggests they may also possess anti-carcinogenic activity. Conjugated linoleic acids (CLA), sourced from meat and dairy products of ruminant animals, has also received considerable attention as a potential anti-cancer agent. Therefore, the aims of this project were to (i) examine the effects of phytosterols and CLA on the viability and growth of human intestinal Caco-2 cells and (ii) determine their potential genoprotective (comet assay), COX-2 modulatory (ELISA) and apoptotic (Hoechst staining) activities. Caco-2 cells were supplemented with the phytosterols campesterol, beta-sitosterol, or beta-sitostanol, or a CLA mixture, or individual CLA isomers (c10t12-CLA, t9t11-CLA) for 48 h. The three phytosterols, at the highest levels tested, were found to reduce both the viability and growth of Caco-2 cells while CLA exhibited isomer-specific effects. None of the phytosterols protected against DNA damage. At a concentration of 25 microM, both c10t12-CLA and t9t11-CLA enhanced (P<0.05) oxidant-induced, but not mutagen-induced, DNA damage. Neither the phytosterols nor CLA induced apoptosis or modulated COX-2 production. In conclusion, campesterol, beta-sitosterol, beta-sitostanol, c10t12-CLA, and t9t11-CLA were not toxic to Caco-2 cells, at the lower levels tested, and did not exhibit potential anti-carcinogenic activity.