Oxidative Stress In Caco-2 Cells Research Articles

MITOCHONDRIAL DYSFUNCTIONS INDUCED BY OXIDATIVE STRESS IN CACO‐2CELLS

Despite the fact that oxidative stress (OS) has been implicated in IBD pathogenesis and mitochondria are the substantial source of ROS, little attention has been paid to these organelles. We hypothesize that OS can lead to mitochondrial dysfunctions recapitulating various IBD abnormalities. The present study will to investigate the impact of OS on mitochondrial antioxidant content, energy production, calcium homeostasis, cell death, transcription factors status and DNA integrity. We used FE/ASC to generate OS in Caco‐2 cells. Our results show that the administration of FE/ASC (0.2 mM/2 mM) (1) increased MDA levels; (2) reduced ATP production; (3) led to alterations in the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mTFB1, mTFB2) without any effects on POLRMT; (4) provoked dysregulation of mitochondrial calcium homeostasis; (5) elicited DNA lesions as illustrated by the raised levels of 8‐OHdG; (6) lowered, OGG1, one of the first lines of defence against 8‐oxodG mutagenicity; and (7) upregulated cytochrome C and AIF protein expressions, indicating exaggerated apoptosis. The presence of the powerful antioxidant BHT prevented the occurrence of OS and most of the mitochondrial abnormalities. In conclusion, our results suggest that OS causes mitochondrial dysfunctions and modulates mitochondrial transcription factors. We believe that the identification of OS‐mediated mitochondrion disorders adds new information regarding their implication in IBD pathogenesis.

Global Gene Expression Analysis Reveals Differences in Cellular Responses to Hydroxyl- and Superoxide Anion Radical–Induced Oxidative Stress in Caco-2 Cells

Reactive oxygen species-induced oxidative stress in the colon is involved in inflammatory bowel diseases and suggested to be associated with colorectal cancer risk. However, our insight in molecular responses to different oxygen radicals is still fragmentary. Therefore, we studied global gene expression by an extensive time series (0.08, 0.25, 0.5, 1, 2, 4, 8, 16, or 24 h) analyses in human colon cancer (caco-2) cells after exposure to H(2)O(2) or the superoxide anion donor menadione. Differences in gene expression were investigated by hybridization on two-color microarrays against nonexposed time-matched control cells. Next to gene expression, correlations with related phenotypic markers (8-oxodG levels and cell cycle arrest) were investigated. Gene expression analysis resulted in 1404 differentially expressed genes upon H(2)O(2) challenge and 979 genes after menadione treatment. Further analysis of gene expression data revealed how these oxidant responses can be discriminated. Time-dependent coregulated genes immediately showed a pulse-like response to H(2)O(2), while the menadione-induced expression is not restored over 24 h. Pathway analyses demonstrated that H(2)O(2) immediately influences pathways involved in the immune function, while menadione constantly regulated cell cycle-related pathways Altogether, this study offers a novel and detailed insight in the similarities and differences of the time-dependent oxidative stress responses induced by the oxidants H(2)O(2) and menadione and show that these can be discriminated regarding their modulation of particular colon carcinogenesis-related mechanisms.

Antioxidant effect of casein phosphopeptides compared with fruit beverages supplemented with skimmed milk against H2O2-induced oxidative stress in Caco-2 cells

Abstract Casein phosphopeptides (CPPs) have been proposed as potential dietary antioxidants on designing new functional products. A fruit beverage, with/without milk, was subjected to in vitro digestion. Caco-2 cultures were preincubated with soluble fractions or isolated CPPs, the latter obtained from skimmed milk. The mitochondrial activities (MTT test), intracellular GSH and GSH-reductase activity (GSH-Rd), cell cycle analysis and RNA distribution in cycle phases were studied after inducing oxidative stress status (5 mM H2O2). MTT conversion was better preserved by soluble fractions of fruit beverages, either with or without milk. GSH concentration was equally decreased, and GSH-Rd was increased in all preincubations. However, failed to inhibit a decreased G1 cell population. CPPs produced RNA accumulation in the G2 phase, suggesting a modulating effect on cell response. We can postulate an antioxidant effect derived from dietary CPPs on a biological system which can be synergistic with other antioxidant compounds derived from fruit beverage.

Antioxidant effect derived from bioaccessible fractions of fruit beverages against H 2O 2-induced oxidative stress in Caco-2 cells

This work evaluates the effect of bioaccessible fractions from fruit beverages against oxidative stress (OS) in Caco-2 cells. A fruit beverage (grape + orange + apricot) (with/without milk and/or iron/zinc) was subjected to in vitro gastrointestinal digestion, and bioaccessible fractions were incubated with Caco-2 cell cultures. Following preincubation, OS was induced with 5 mM H 2O 2. Intracellular reactive oxygen species (ROS), mitochondrial potential (Δ ψ m), mitochondrial metabolism (MTT test), intracellular reduced glutathione (GSH) and superoxide dismutase activity (SOD) were measured. The data evidenced viable cultures with increased mitochondrial metabolism and GSH-Rd activities, without alteration in SOD activity. Accordingly, more preserved mitochondrial integrity was also evidenced, allowing the action of antioxidant systems in preincubated cultures. Based on these data, we can conclude that a cytoprotective effect is derived from bioaccessible fractions of fruit beverages, though this effect failed to prevent intracellular ROS accumulation in Caco-2 cell cultures exposed to 5 mM H 2O 2.

Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Intestinal epithelial cells are confronted with many noxious stimuli which play an important role in the mucosal immune response. In the present study, Caco-2 cells treated with hydrogen peroxide were used as a model system for studying inflammatory responses and induction of cell death. Live lactobacilli and their concentrated spent culture supernatant (SCS) were applied for studying possible short- and long-term protective effects against hydrogen peroxide-mediated oxidative stress in Caco-2 cells. The secretion of pro-inflammatory cytokine interleukin-8 (IL-8) was investigated in non-filter grown Caco-2 cells, while transepithelial electrical resistance and cell death was studied in filter grown Caco-2 cells. Pre-incubation of Caco-2 cells with Lactobacillus plantarum 2142 did not decrease IL-8 levels induced by 1 mM hydrogen peroxide, nor did lactobacilli suppress IL-8 levels induced by hydrogen peroxide when Caco-2 cells were treated simultaneously with 1 mM hydrogen peroxide and L. plantarum 2142. Thus, lactobacilli did not exert a long-term protective effect against hydrogen peroxide in non-filter grown Caco-2 cells. However, the concentrated SCS of lactobacilli was able to reduce IL-8 levels by more than 6-fold, as determined 24 h after treatment. A short-term effect of lactobacilli was observed in filter grown Caco-2 cells, as they inhibited cell death induced by hydrogen peroxide in the concentration range 10-40 mM. Pre-incubation of epithelial cells with L. plantarum 2142 or simultaneous exposure to hydrogen peroxide and lactobacilli protected Caco-2 cells against cell death. In spite of the presence of lactobacilli, the permeability of membrane increased (transepithelial electrical resistance decreased), and exhibited a similar characteristic pattern as under treatment with hydrogen peroxide alone.

Oligophosphopeptides Derived from Egg Yolk Phosvitin Up-regulate γ-Glutamylcysteine Synthetase and Antioxidant Enzymes against Oxidative Stress in Caco-2 Cells

Previously, we have found phosphopeptides (PPPs) from hen egg yolk phosvitin possess a potent antioxidative activity against oxidative stress in human intestinal epithelial cells, Caco-2. However, their biological activity at the cellular level has not yet fully understood. The objective of this study is to evaluate the regulation of glutathione (GSH) biosynthesis-associated and antioxidant enzymes against oxidative stress in Caco-2 cells using an in vitro model. Treatment of 1 mM H2O2-induced Caco-2 cells with PPPs increased cellular GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit mRNA. Furthermore, intracellular glutathione reductase, glutathione S-transferase, and catalase activities were elevated by PPPs. In addition, PPPs with high content of phosphorus showed higher induction of these enzyme activities than PPPs without phosphorus. These data indicate that oligophosphopeptides from hen egg yolk phosvitin can up-regulate cellular GSH biosynthesis-associated enzymes activity and antioxidative activities, which play key roles against tissue oxidative stress in the human intestinal epithelial cells.

Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells

Experimental evidence suggests that most herbs and spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage. The objectives of the present study were: first, to determine the effects of plant extracts on the viability, membrane integrity, antioxidant status and DNA integrity of Caco-2 cells and second, to investigate the cytoprotective and genoprotective effects of these plant extracts against oxidative stress in Caco-2 cells. The plant extracts examined were rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), sage (Salvia officinalis L.) and echinacea (Echinacea purpurea L.). Cell membrane integrity was assessed by the lactate dehydrogenase release assay. Viability was determined by the neutral red uptake assay (NRUA) and the concentration of compound that resulted in 50 % cell death (IC50) was calculated. Antioxidant status of the cells was assessed by measuring GSH content, catalase activity and superoxide dismutase activity. To examine their cytoprotective and genoprotective effects, Caco-2 cells were pre-treated with each plant extract for 24 h followed by exposure to H2O2. DNA damage was assessed by the comet assay and cell injury was determined by the NRUA. Rosemary was the most toxic (IC50 123 microg/ml) and echinacea the least toxic (IC50 1421 microg/ml). Sage was the only plant extract to affect the antioxidant status of the cells by increasing GSH content. Sage, oregano and rosemary protected against H2O2-induced DNA damage (olive tail moment and percentage tail DNA), whereas protection against H2O2-induced cytotoxicity was afforded by sage only.

The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells

The increasing presence of toxic cyanobacteria in drinking and recreational water bodies, and their potential to impact on human and animal health is cause for concern. Recent work suggests that apoptosis plays a major role in the toxic effects induced by microcystin-LR (MCLR) in the gastrointestinal tract; however, the biochemical pathway remains elusive. Exposure of CaCo2, a human colon carcinoma cell line, and MCF-7, a cell line deficient in pro-caspase-3, cells to 50 μM MCLR induced similar reductions in cell viability as measured by MTT and LDH leakage. The role of MCLR induced oxidative stress in the initiation of apoptosis was investigated over a 2-hr period, and it was found that there was an increase in the release of H 2O 2 in the first 30 min of exposure for both cell lines. Both cell lines exhibited a dose-dependent increase in both micro- and millicalpain after 24 h exposure to MCLR suggesting a role for protease activation in MCLR-induced apoptosis in non-hepatic human derived cell lines.

Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells.

Oxidative stress, IL-1alpha, and IL-8 are known to contribute to mucosal inflammation of the gastrointestinal tract. We examined the IL-8 response after brief exposure to hydrogen peroxide induced oxidative stress in CaCo-2 cells (a human colon carcinoma cell line) and in human intestinal epithelial cells. In addition, we examined whether exposure to oxidative stress, followed by IL-1alpha, could modulate IL-8 production. A transient up-regulation of IL-8 mRNA expression was observed after hydrogen peroxide treatment. Hydrogen peroxide induced oxidative stress was also observed to promote IL-8 secretion. Exposure to hydrogen peroxide, followed by IL-1alpha, enhanced IL-8 production over that achieved with IL-1alpha alone. Thus, oxidative stress and IL-1alpha were observed to cooperatively enhance IL-8 production.