In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

In silico and in vitro safety assessment of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

To address the growing world population and reduce the impact of environmental changes on the global food supply, ingredients are being produced using microorganisms to yield sustainable and innovative products. Food ingredients manufactured using modern biotechnology must be produced by non-toxigenic and nonpathogenic production organisms that do not harbor antimicrobial resistance (AMR). Several fungal species represent attractive targets as sources of alternative food products. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus strain CBS 143028. The whole genome sequence of this strain was annotated and subjected to sequence homology searches and in silico phenotype prediction tools to identify genetic elements encoding for protein toxins active via oral consumption, virulence factors associated with pathogenicity, and determinants of AMR. The in silico investigation revealed no genetic elements sharing significant sequence homology with putative virulence factors, protein toxins, or AMR determinants, including the absence of mucoricin, an essential toxin in the pathogenesis of mucormycosis. These in silico findings were corroborated in vitro based on the absence of clinically relevant mycotoxin or antibacterial secondary metabolites. Consequently, it is unlikely that R. pusillis strain CBS 143028 would pose a safety concern for use in food for human consumption.

Inoculation with enterococci does not affect colon inflammation in the multi-drug resistance 1a-deficient mouse model of IBD.

BACKGROUND: Intestinal bacteria are thought to play a role in the pathogenesis of human inflammatory bowel disease (IBD). We investigated whether oral inoculation with specific intestinal bacteria increased colon inflammation in the multi-drug resistance 1a-deficient (Mdr1a (-/-) ) mouse model of IBD. METHODS: Five-week-old Mdr1a (-/-) mice (FVB background) and FVB mice were randomly assigned to one of two treatment groups (Control or Inoculation, n = 12 per group). All mice were fed AIN-76A rodent diet, and mice in the Inoculation groups also received a single oral bacterial inoculation consisting of twelve cultured Enterococcus species combined with conventional intestinal flora obtained from the gastrointestinal tract of healthy mice (EF.CIF). Body weight, food intake, and disease activity index (DAI) were assessed throughout the study, and at 21 or 24 weeks of age, inflammation was assessed post-mortem by determining colon length and histological injury score (HIS), and plasma serum amyloid A (SAA). RESULTS: Mdr1a (-/-) mice consumed more food than FVB mice at 13 weeks of age (P < 0.05). There was also a significant effect of genotype on body weight, with Mdr1a (-/-) mice weighing less than FVB mice throughout the study (P < 0.05) regardless of treatment, but there was no effect of inoculation on body weight (P > 0.25). Colon HIS of Mdr1a (-/-) mice was significantly higher than that of FVB mice in the Control (9.3 ± 4.7 (mean ± SD) vs. 0.58 ± 0.51; P < 0.001) and Inoculation (6.7 ± 5.1 vs. 0.92 ± 0.39; P < 0.001) groups. There was no difference in colon HIS of Mdr1a (-/-) mice in the Control group compared with Mdr1a (-/-) mice in the Inoculation group (P = 0.25), nor was there any difference in within-group variation of colon HIS in these two Mdr1a (-/-) groups. DAI was higher in Mdr1a (-/-) mice than in FVB mice, but there was no effect of treatment in either strain, nor were there any differences in colon length or plasma SAA. CONCLUSIONS: Inoculation of Mdr1a (-/-) mice with the EF.CIF inoculum described here does not increase colon inflammation or reduce the observed variability of inflammation.

A combined omics approach to evaluate the effects of dietary curcumin on colon inflammation in the Mdr1a(-/-) mouse model of inflammatory bowel disease.

The aim of this study was to provide insight into how curcumin reduces colon inflammation in the Mdr1a(-/-) mouse model of human inflammatory bowel disease using a combined transcriptomics and proteomics approach. Mdr1a(-/-) and FVB control mice were randomly assigned to an AIN-76A (control) diet or AIN-76A+0.2% curcumin. At 21 or 24weeks of age, colonic histological injury score (HIS) was determined, colon mRNA transcript levels were assessed using microarrays and colon protein expression was measured using 2D gel electrophoresis and LCMS protein identification. Colonic HIS of Mdr1a(-/-) mice fed the AIN-76A diet was higher (P<.001) than FVB mice fed the same diet; the curcumin-supplemented diet reduced colonic HIS (P<.05) in Mdr1a(-/-) mice. Microarray and proteomics analyses combined with new data analysis tools, such as the Ingenuity Pathways Analysis regulator effects analysis, showed that curcumin's antiinflammatory activity in Mdr1a(-/-) mouse colon may be mediated by activation of α-catenin, which has not previously been reported. We also show evidence to support curcumin's action via multiple molecular pathways including reduced immune response, increased xenobiotic metabolism, resolution of inflammation through decreased neutrophil migration and increased barrier remodeling. Key transcription factors and other regulatory molecules (ERK, FN1, TNFSF12 and PI3K complex) activated in inflammation were down-regulated by dietary intervention with curcumin.

Modulation of colonic inflammation in Mdr1a(-/-) mice by green tea polyphenols and their effects on the colon transcriptome and proteome.

Animal models are an important tool to understand the complex pathogenesis of inflammatory bowel diseases (IBDs). This study tested the anti-inflammatory potential of a green tea extract rich in polyphenols (GrTP) in the colon of the multidrug resistance targeted mutation (Mdr1a(-/-)) mouse model of IBD. Insights into mechanisms responsible for this reduction in inflammation were gained using transcriptome and proteome analyses. Mice were randomly assigned to an AIN-76A (control) or GrTP-enriched diet. At 21 or 24 weeks of age, a colonic histological injury score was determined for each mouse, colon mRNA transcript levels were assessed using microarrays, and colon protein expression was measured using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry protein identification. Mean colonic histological injury score of GrTP-fed Mdr1a(-/-) mice was significantly lower compared to those fed the control diet. Microarray and proteomics analyses showed reduced abundance of transcripts and proteins associated with immune and inflammatory response and fibrinogenesis pathways, and increased abundance of those associated with xenobiotic metabolism pathways in response to GrTP, suggesting that its anti-inflammatory activity is mediated by multiple molecular pathways. Peroxisome proliferator-activated receptor-α and signal transducer and activator of transcription 1 appear to be two key molecules which regulate these effects. These results support the view that dietary intake of polyphenols derived from green tea can ameliorate intestinal inflammation in the colon of a mouse model of IBD, and are in agreement with studies suggesting that consumption of green tea may reduce IBD symptoms and therefore play a part in an overall IBD treatment regimen.

Bioactive food components, cancer cell growth limitation and reversal of glycolytic metabolism.

Cancer cells are resistant to apoptosis and show a shift in energy production from mitochondrial oxidative phosphorylation to cytosolic glycolysis. Apoptosis resistance and metabolic reprogramming are linked in many cancer cells and both processes center on mitochondria. Clearly, mutated cancer cells escape surveillance and turn into selfish cells. However, many of the mechanisms that operate cellular metabolic control still function in cancer cells. This review describes the metabolic importance of glucose and glutamine, glycolytic enzymes, oxygen, growth cofactors and mitochondria and focuses on the potential role of bioactive food components, including micronutrients. The role of B- and A-vitamin cofactors in (mitochondrial) metabolism is highlighted and the cancer protective potential of omega-3 fatty acids and several polyphenols is discussed in relation to metabolic reprogramming, including the mechanisms that may be involved. Furthermore, it is shown that cancer cell growth reduction by limiting the growth cofactor folic acid seems to be associated with reversal of metabolic reprogramming. Altogether, reversal of metabolic reprogramming may be an attractive strategy to increase susceptibility to apoptotic surveillance. Food bioactive components that affect various aspects of metabolism may be important tools to reverse glycolytic to oxidative metabolism and enhance sensitivity to apoptosis. The success of such a strategy may depend on several actors, acting in concert. Growth cofactors may be one of these, which call for careful (re)evaluation of their function in normal and in cancer metabolism.

Genome-wide analysis of dietary eicosapentaenoic acid- and oleic acid-induced modulation of colon inflammation in interleukin-10 gene-deficient mice.

BACKGROUND/AIMS: Dietary n-3 polyunsaturated fatty acids can reduce inflammation via a range of mechanisms. This study tested the effect of dietary eicosapentaenoic acid (EPA) on intestinal inflammation using interleukin-10 gene-deficient (Il10(-/-)) mice. METHODS: At 35 days of age, 12 weaned Il10(-/-) and 12 C57 mice were randomly assigned to one of two modified AIN-76A diets, supplemented with 3.7% purified ethyl esters of either EPA (n-3) or oleic acid (OA, control). To identify genes relevant to colon inflammation, transcription profiling (microarrays and qRT-PCR) and bioinformatic analyses were used. RESULTS: In this study, dietary EPA reversed the decrease in colon fatty acid beta-oxidation gene expression observed in OA-fed Il10(-/-) compared to C57 mice. Il10(-/-) mice fed the OA diet showed decreased expression of antioxidant enzyme genes, as well as those involved in detoxification of xenobiotics, compared to C57 mice on the same diet. In contrast, dietary EPA increased the expression of these genes in Il10(-/-) mice. CONCLUSIONS: These data indicate that dietary EPA-induced endogenous lipid oxidation which might have a potential anti-inflammatory effect on colon tissue. This is supported by the activation of the Ppara gene that regulates the expression of pro-inflammatory and immunomodulatory genes and proteins.

Survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG in the human gastrointestinal tract with daily consumption of a low-fat probiotic spread.

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (n = 13), 20 g of spread with a target dose of 1 x 10(9) CFU of L. reuteri DSM 17938 (n = 13), or 20 g of spread with a target dose of 5 x 10(9) CFU of L. rhamnosus GG (n = 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either L. reuteri DSM 17938 or L. rhamnosus GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.

The effects of dietary curcumin and rutin on colonic inflammation and gene expression in multidrug resistance gene-deficient (mdr1a-/-) mice, a model of inflammatory bowel diseases.

Damage of the intestinal epithelial barrier by xenobiotics or reactive oxygen species and a dysregulated immune response are both factors involved in the pathogenesis of inflammatory bowel diseases (IBD). Curcumin and rutin are polyphenolic compounds known to have antioxidant and anti-inflammatory activities, but their mechanism(s) of action are yet to be fully elucidated. Multidrug resistance gene-deficient (mdr1a-/- ) mice spontaneously develop intestinal inflammation, predominantly in the colon, with pathology similar to IBD, so this mouse model is relevant for studying diet-gene interactions and potential effects of foods on remission or development of IBD. The present study tested whether the addition of curcumin or rutin to the diet would alleviate colonic inflammation in mdr1a-/- mice. Using whole-genome microarrays, the effect of dietary curcumin on gene expression in colon tissue was also investigated. Twelve mice were randomly assigned to each of three diets (control (AIN-76A), control +0.2% curcumin or control +0.1% rutin) and monitored from the age of 7 to 24 weeks. Curcumin, but not rutin, significantly reduced histological signs of colonic inflammation in mdr1a-/- mice. Microarray and pathway analyses suggested that the effect of dietary curcumin on colon inflammation could be via an up-regulation of xenobiotic metabolism and a down-regulation of pro-inflammatory pathways, probably mediated by pregnane X receptor (Pxr) and peroxisome proliferator-activated receptor alpha (Ppara) activation of retinoid X receptor (Rxr). These results indicate the potential of global gene expression and pathway analyses to study and better understand the effect of foods in modulating colonic inflammation.

High folic acid increases cell turnover and lowers differentiation and iron content in human HT29 colon cancer cells.

Folate, a water-soluble B vitamin, is a cofactor in one-carbon metabolism and is essential for DNA synthesis, amino acid interconversion, methylation and, consequently, normal cell growth. In animals with existing pre-neoplastic and neoplastic lesions, folic acid supplementation increases the tumour burden. To identify processes that are affected by increased folic acid levels, we compared HT29 human colon cancer cells exposed to a chronic supplemental (100 ng/ml) level of folic acid to cells exposed to a normal (10 ng/ml) level of folic acid, in the presence of vitamin B12 and other micronutrients involved in the folate-methionine cycle. In addition to higher intracellular folate levels, HT29 cells at 100 ng folic acid/ml displayed faster growth and higher metabolic activity. cDNA microarray analysis indicated an effect on cell turnover and Fe metabolism. We fully confirmed these effects at the physiological level. At 100 ng/ml, cell assays showed higher proliferation and apoptosis, while gene expression analysis and a lower E-cadherin protein expression indicated decreased differentiation. These results are in agreement with the promoting effect of folic acid supplementation on established colorectal neoplasms. The lower expression of genes related to Fe metabolism at 100 ng folic acid/ml was confirmed by lower intracellular Fe levels in the cells exposed to folic acid at 100 ng/ml. This suggests an effect of folate on Fe metabolism.

Nutrigenomic approaches for benefit-risk analysis of foods and food components: defining markers of health.

To be able to perform a comprehensive and rigorous benefit-risk analysis of individual food components, and of foods, a number of fundamental questions need to be addressed first. These include whether it is feasible to detect all relevant biological effects of foods and individual food components, how such effects can confidently be categorised into benefits and risks in relation to health and, for that matter, how health can be quantified. This article examines the last of these issues, focusing upon concepts for the development of new biomarkers of health. Clearly, there is scope for refinement of classical biomarkers so that they may be used to detect even earlier signs of disease, but this approach defines health solely as the absence of detectable disease or disease risk. We suggest that the health of a biological system may better be reflected by its ability to withstand and manage relevant physiological challenges so that homeostasis is maintained. We discuss the potential for expanding the range of current challenge tests for use in conjunction with functional genomic technologies to develop new types of biomarkers of health.

Nutrigenomics applied to an animal model of Inflammatory Bowel Diseases: transcriptomic analysis of the effects of eicosapentaenoic acid- and arachidonic acid-enriched diets.

In vivo models of Inflammatory Bowel Diseases (IBD) elucidate important mechanisms of chronic inflammation. Complex intestinal responses to food components create a unique "fingerprint" discriminating health from disease. Five-week-old IL10(-/-) and C57BL/6J (C57; control) mice were inoculated orally with complex intestinal microflora (CIF) and/or pure cultures of Enterococcus faecalis and E. faecalis (EF) aiming for more consistent inflammation of the intestinal mucosa. Inoculation treatments were compared to non-inoculated IL10(-/-) and C57 mice, either kept in specific pathogen free (SPF) or conventional conditions (2x5 factorial design). At 12 weeks of age, mice were sacrificed for intestinal histological (HIS) and transcriptomic analysis using limma and Ingenuity Pathway Analysis Software. Colonic HIS was significantly affected (P<0.05) in inoculated IL10(-/-) mice and accounted for approximately 60% of total intestinal HIS. Inoculation showed a strong effect on colonic gene expression, with more than 2000 genes differentially expressed in EF.CIF-inoculated IL10(-/-) mice. Immune response gene expression was altered (P<0.05) in these mice. The second study investigated the effect of arachidonic (AA) and eicosapentaenoic acid (EPA) on colonic HIS and gene expression to test whether EPA, contrary to AA, diminished intestinal inflammation in EF.CIF IL10(-/-) mice (2 x 4 factorial design). AIN-76A (5% corn oil) and AIN-76A (fat-free) +1% corn oil supplemented with either 3.7% oleic acid (OA), AA or EPA were used. IL10(-/-) mice fed EPA- and AA-enriched diets had at least 40% lower colonic HIS (P<0.05) than those fed control diets (AIN-76A and OA diets). The expression of immune response and 'inflammatory disease' genes (down-regulated: TNFalpha, IL6, S100A8, FGF7, PTGS2; up-regulated: PPARalpha, MGLL, MYLK, PPSS23, ABCB4 with EPA and/or AA) was affected in IL10(-/-) mice fed EPA- and AA-enriched diets, compared to those fed AIN-76A diet.

Nutrient-gene interactions in benefit-risk analysis.

Individuals respond differently to nutrients and foods. This is reflected in different levels of benefits and risks at the same intake of a nutrient and, consequently, different 'windows of benefit' in terms of nutrient intake. This has led recently to the concept of 'personalised nutrition'. Genetic factors such as single nucleotide polymorphisms may be one source of this inter-individual variation in benefit-risk response to nutrients. In 2004 a European Union-funded network of excellence in the area of nutrigenomics (European Nutrigenomics Organisation; NuGO) organised a workshop on the role of nutrient-gene interactions in determining benefit-risk of nutrients and diet. The major issues discussed at the workshop are presented in the present paper and highlighted with examples from the presentations. The overall consensus was that although genetics provides a new vision where genetic information could in the future be used to provide knowledge on disease predisposition and nutritional requirements, such a goal is still far off and much more research is required before we can reliably include genetic factors in the risk-benefit assessment of nutrients and diets.

Biological role of glutathione in nitric oxide-induced toxicity in cell culture and animal models.

Glutathione (GSH) plays an important role in cellular defense response in many in vitro and in vivo models. Here we investigated its role in NO()-induced toxicity in cell culture and mouse models. Wild-type (TK6) and p53-null (NH32) human lymphoblastoid cells were treated with NO(.) at a steady-state concentration of 0.6 muM, similar to the level estimated to occur in inflamed tissues. In both cell types, GSH was depleted by this exposure in a dose- and time-dependent manner. Contrary to expectations, prior depletion of GSH by treatment with l-buthionine-SR-sulfoximine did not potentiate NO(.)-induced cell killing or DNA deamination in TK6 cells. In activated RAW264.7 murine macrophages producing NO(.), intracellular GSH content did not change, although gamma-glutamate-cysteine ligase was upregulated. NO(.) overproduction in RcsX lymphoma-bearing SJL mice resulted in significantly elevated GSH levels in various organs. Administration of the NO(.) synthase inhibitor N-methylarginine abolished the increase in GSH in these animals. Collectively, these data indicate a multifaceted and complex involvement of GSH in responses of cells and tissues to toxic levels of NO(.). NO(.) treatment effectively depleted GSH levels in human lymphoblastoid cells, but this alteration was not a critical initiating factor for NO(.)-mediated toxicity. Murine macrophages maintained GSH homeostasis when exposed to endogenously produced NO(.). In RcsX lymphoma-bearing mice, upregulation of de novo synthesis of GSH appeared to be a response to the toxic effects of NO(.).

Effects of high fat fish oil and high fat corn oil diets on initiation of AOM-induced colonic aberrant crypt foci in male F344 rats.

Modulating effects of high fat fish oil (HFFO) and high fat corn oil (HFCO) diets on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were studied in male F344 rats following 8 weeks of dietary treatment. The incidence of AOM-induced ACF was significantly lower in the proximal colon of rats fed the HFFO diets compared with rats fed the HFCO diets. No differential effects were found on enzyme activities that are involved in metabolic activation and detoxification of AOM. Activities of hepatic P450 IAI and P450 IIBI and hepatic and feacal levels of lipid peroxidation were increased by feeding the HFFO diet. Hepatic GST activity and plasma levels of PGE(2) were significantly lower in rats fed the HFFO diets compared with those fed the HFCO diets. These observations demonstrate that HFFO diets with high levels of n-3 PUFAs are also protective against preneoplastic lesions in the early stages of chemically induced colon carcinogenesis. It seems unlikely from our results that the inhibitory effect of a HFFO diet can be attributed to an altered metabolic activation and detoxification of AOM. Other mechanisms such as oxidative stress or reduction of PGE(2) levels may play an important role in the anticarcinogenic effects of n-3 PUFAs.

Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2.

Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of alpha-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase alpha (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.

The role of cyclooxygenase in n-6 and n-3 polyunsaturated fatty acid mediated effects on cell proliferation, PGE(2) synthesis and cytotoxicity in human colorectal carcinoma cell lines.

This study was conducted to investigate the role of the enzyme cyclooxygenase (COX) and its prostaglandin product PGE(2) in n-6 and n-3 polyunsaturated fatty acid (PUFA)-mediated effects on cellular proliferation of two human colorectal carcinoma cell lines. The long chain PUFAs eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (AA; 20:4n-6) both inhibited cell proliferation of Caco-2 cells compared with the long chain fatty acids alpha-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6). Neither incubation with PGE(2) nor reduction in PGE(2) synthesis by EPA compared with AA led to differential effects on cell proliferation in Caco-2 cells. This suggests that n-6 and n-3 PUFA-mediated cell proliferation in Caco-2 cells is not regulated via PGE(2) levels. AA and EPA had no effect on growth of HT-29 colon cancer cells with a low COX activity. However, stimulation of COX-2 activity by IL-1 beta resulted in a decrease in cell proliferation and an induction of cytotoxicity by AA as well as by EPA. Both inhibition of the COX pathway by indomethacin as well as inhibition of direct lipid peroxidation by antioxidants such as vitamin E and C diminished the anti-proliferative effects of AA as well as EPA. Also, malondialdehyde, a product of lipid peroxidation and COX-activity was decreased by addition of vitamin E and partially decreased by indomethacin. These data support the hypothesis that growth inhibitory and cytotoxic effects of PUFAs with methylene-interrupted double bonds such as AA and EPA are due to peroxidation products that are generated during lipid peroxidation and COX activity.

Effects of n-6 and n-3 polyunsaturated fatty acids on gap junctional intercellular communication during spontaneous differentiation of the human colon adenocarcinoma cell line Caco-2.

Gap junctional intercellular communication (GJIC), which modulates cell growth and differentiation, may play an important role in tumor growth. Cancer cells have dysfunctional GJIC, but it is not known whether GJIC is mechanistically involved in the carcinogenic and anti-carcinogenic effects of n-6 and n-3 polyunsaturated fatty acids (PUFAs) on colon tumor cells. Caco-2 cells were used as an in vitro model to study the effects of PUFAs on differentiated as well as undifferentiated human colon cells. The GJIC capacity of this cell line increased during spontaneous differentiation. However, no differential effects between n-6 and n-3 PUFAs on GJIC were observed. Short-term incubation with linoleic acid (18:2n-6), alpha-linolenic acid (18:3n-3), arachidonic acid (AA, 20:4n-6), and eicosapentaenoic acid (EPA, 20:5n-3) did not influence GJIC, while long-term incubation (> 10 days) with linoleic acid and alpha-linolenic acid inhibited GJIC of these colon cells. Long-chain metabolites such as AA and EPA were not formed after incubation with linoleic acid and alpha-linolenic acid, thus excluding the involvement of prostaglandins in the observed effects. Although the exact mechanism of GJIC inhibition is unclear, cytotoxicity probably mediated by lipid peroxidation products seems to be related, because incubation with more PUFAs (AA and EPA) completely abolished GJIC.

Dietary n-6 and n-3 polyunsaturated fatty acids and colorectal carcinogenesis: results from cultured colon cells, animal models and human studies.

During the past few decades, many studies have been conducted to evaluate the effects of n-6 and n-3 polyunsaturated fatty acids (PUFAs) on colorectal carcinogenesis. This report provides a brief overview of the recent studies that have been performed in cultured colon cells, animal models as well as of the population-based and short-term biomarker studies with humans. No differential effect between n-6 and n-3 PUFAs has been observed in vitro. Results from animal models indicate that n-6 PUFAs have a tumor enhancing effect, predominantly during the post-initiation phase. n-3 PUFAs may protect against colorectal carcinogenesis during both the initiation and post-initiation phase. Population-based human studies show little or no associations between n-6 or n-3 PUFA intake and colorectal cancer. Short-term biomarker studies in humans suggest though that fish oil (FO) supplementation with high amounts of n-3 PUFAs may protect against colorectal carcinogenesis and that n-6 PUFA supplementation may increase the risk.