Marta Calatayud

Mercury and selenium in fish and shellfish: occurrence, bioaccessibility and uptake by Caco-2 cells.

This study evaluates Hg and Se concentrations and bioaccessibility (element solubilised after simulated gastrointestinal digestion) in 16 raw seafood species consumed in Spain. The concentrations varied greatly (Hg, 3.8-1621 ng/g wet weight, ww; Se, 84-1817 ng/g ww). Only one sample of swordfish exceeded the Hg limit permitted in Spain (1mg/kg), and for this sample the Hg/Se molar ratio and Se Health Benefit Value food safety criteria also indicated the presence of a risk. Bioaccessibility of Hg (35-106%) and Se (17-125%) was very variable and the Hg/Se molar ratio in the bioaccessible fraction was less than one for all samples. Transport by Caco-2 cells, an intestinal epithelium model, was also evaluated from the swordfish bioaccessible fraction. Hg and Se transport from the food was less than 14%, and cell retention was much greater for Hg (49-69%) than Se (8-12%).

In Vitro Study of Transporters Involved in Intestinal Absorption of Inorganic Arsenic

Inorganic arsenic (iAs) [As(III)+As(V)] is a drinking water contaminant, and human exposure to these arsenic species has been linked with a wide range of health effects. The main path of exposure is the oral route, and the intestinal epithelium is the first physiological barrier that iAs must cross in order to be absorbed. However, there is a lack of information about intestinal iAs absorption. The aim of this study was to evaluate the participation of certain transporters [glucose transporters (GLUT and SGLT), organic anion transporting polypeptides (OATPs), aquaporins (AQPs), and phosphate transporters (NaPi and PiT)] in intestinal absorption of As(V) and As(III), using the Caco-2 cell line as a model of the intestinal epithelium. For this purpose, the effects of chemical inhibition and gene silencing of the transporters of interest on iAs uptake were evaluated, and also the differential expression of these transporters after treatment with iAs. The results show that chemical inhibition using rifamycin SV (OATP inhibitor), phloridzin (SGLT inhibitor), phloretin (GLUT and AQP inhibitor), and copper sulfate (AQP inhibitor) leads to a significant reduction in the apparent permeability and cellular retention of As(III). RT-qPCR indicates up-regulation of GLUT2, GLUT5, OATPB, AQP3, and AQP10 after exposure to As(III), while exposure to As(V) increases the expression of sodium-dependent phosphate transporters, especially NaPiIIb. Gene silencing of OATPB, AQP10, and GLUT5 for As(III) and NaPiIIb for As(V) significantly reduces uptake of the inorganic forms. These results indicate that these transporters may be involved in intestinal absorption of iAs.

Comparison of a static and a dynamic in vitro model to estimate the bioaccessibility of As, Cd, Pb and Hg from food reference materials Fucus sp. (IAEA-140/TM) and Lobster hepatopancreas (TORT-2)

Bioaccessibility, the fraction of an element solubilized during gastrointestinal digestion and available for absorption, is a factor that should be considered when evaluating the health risk of contaminants from food. Static and dynamic models that mimic human physiological conditions have been used to evaluate bioaccessibility. This preliminary study compares the bioaccessibility of arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) in two food certified reference materials (CRMs) (seaweed: Fucus sp., IAEA-140/TM; Lobster hepatopancreas: TORT-2), using two in vitro gastrointestinal digestion methods: a static method (SM) and a dynamic multicompartment method (TIM-1). There are significant differences (p<0.05) between the bioaccessible values of As, Cd, Pb and Hg obtained by SM and TIM-1 in the two CRMs. The specific form in which the elements studied are present in the CRM may help to explain the bioaccessibility values obtained.

Active films based on cocoa extract with antioxidant, antimicrobial and biological applications.

Novel films of ethylene-vinyl alcohol copolymer (EVOH) containing flavonoid-rich cocoa were developed. To understand their potential application as active packaging material, antioxidant and antimicrobial properties of the films were determined as well as the antioxidant activity of the release compounds in Caco-2 human epithelial colorectal adenocarcinoma cells. Exposure of the films to aqueous food simulant showed antioxidant capacity. The release of cocoa extract components was dependent on the antioxidant concentration incorporated in the film and on temperature. Cocoa extract and the fraction obtained after in vitro gastrointestinal digestion presented antioxidant activity against oxidative stress induced by hydrogen peroxide in Caco-2 cells. Films with 10%, 15%, and 20% cocoa extract produced bactericidal effect against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica. The application of films to an infant milk formula, previously inoculated with L. monocytogenes, inhibited the growth of bacteria 1.5 log units the first day and showed sustained release, inhibiting 0.52 and 0.76 log units, respectively, by the sixth day, while cocoa powder added directly did not produce any effect.

Characterization of the Intestinal Absorption of Arsenate, Monomethylarsonic Acid, and Dimethylarsinic Acid Using the Caco-2 Cell Line

Many toxicological studies have been conducted with arsenic species in target organ cell lines. However, although epithelial gastrointestinal cells constitute the first barrier to the absorption of contaminants, studies using intestinal cells are scarce. The present study examines absorption through the intestinal epithelium of the pentavalent arsenic species most commonly found in foods [arsenate, As(V); monomethylarsonic acid, MMA(V); and dimethylarsinic acid, DMA(V)], using the Caco-2 cell line as a model. Different concentrations (1.3-667.6 microM) and culture conditions (media, pH, addition of phosphates, and treatment with ethylenediaminetetraacetic acid) were evaluated to characterize such transport. The apparent permeabilities indicate that the methylated species show low absorption, whereas As(V) is a compound with moderate absorption. The kinetic study shows only a saturable component for MMA(V) transport in the range of concentrations assayed. The existence of paracellular transport was shown for all of the species, with greater significance in the case of the methylated forms. As(V) absorption was inhibited by 10 mM phosphate, and a phosphate transporter therefore could take part in intestinal absorption. Acidification of the medium (pH 5.5) resulted in a marked increase in As(V) and DMA(V) permeability (4-8 times, respectively) but not in MMA(V) permeability. This makes it necessary to consider the possible existence of absorption in the proximal intestine and even in the stomach, where the environment is acidic; alternatively, an H(+)-dependent transporter may be involved. The results obtained constitute the basis for future research on the mechanisms involved in the intestinal absorption of arsenic and its species, a decisive step in relation to their toxic action.

Differential toxicity and gene expression in Caco-2 cells exposed to arsenic species.

Inorganic arsenic [As(V)+As(III)] and its metabolites, especially the trivalent forms [monomethylarsonous acid, MMA(III), and dimethylarsinous acid, DMA(III)], are considered the forms of arsenic with the highest degree of toxicity, linked to certain types of cancer and other pathologies. The gastrointestinal mucosa is exposed to these forms of arsenic, but it is not known what toxic effect these species may have on it. The aim of the present work was to evaluate the toxicity and some mechanisms of action of inorganic arsenic and its metabolites [monomethylarsonic acid, MMA(V), dimethylarsinic acid, DMA(V), MMA(III) and DMA(III)] in intestinal epithelial cells, using the Caco-2 human cell line as a model. The results show that the pentavalent forms do not produce toxic effects on the intestinal monolayer, but the trivalent species have a different degree of toxicity. As(III) induces death mainly by necrosis, whereas only apoptotic cells are detected after exposure to MMA(III), and for DMA(III) the percentages of apoptosis and necrosis are similar. The three forms produce reactive oxygen species, accompanied by a reduction in intracellular GSH and lipid peroxidation, the latter being especially notable in the dimethylated form. They also alter the enzyme activity of glutathione peroxidase and catalase and induce expression of stress proteins and metallothioneins. The results indicate that the trivalent forms of arsenic can affect cell viability of intestinal cells by mechanisms related to the induction of oxidative stress. Further studies are needed to evaluate how the effects observed in this study affect the structure and functionality of the intestinal epithelium.

In vitro study of intestinal transport of inorganic and methylated arsenic species by Caco-2/HT29-MTX cocultures.

This study characterizes intestinal absorption of arsenic species using in vitro system Caco-2/HT29-MTX cocultures in various proportions (100/0 to 30/70). The species assayed were As(V), As(III), monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], and dimethylarsinous acid [DMA(III)]. The results show that the apparent permeability (P(app)) values of pentavalent species increase significantly in the Caco-2/HT29-MTX cocultures in comparison with the Caco-2 monoculture, probably because of enhancement of paracellular transport. For MMA(III) and DMA(III), P(app) decreases in the Caco-2/HT29-MTX cell model, and for As(III), there is no change in P(app) between the two culture models. Transport studies of arsenic solubilized from cooked foods (rice, garlic, and seaweed) after applying an in vitro gastrointestinal digestion showed that arsenic absorption also varies with the model used, increasing with the incorporation of HT29-MTX in the culture. These results show the importance of choosing a suitable in vitro model when evaluating intestinal arsenic absorption processes.

In vitro study of intestinal transport of arsenite, monomethylarsonous acid, and dimethylarsinous acid by Caco-2 cell line

Arsenic is a pollutant widely distributed in the environment. There are numerous studies on the toxicity of trivalent arsenic forms As(III), MMA(III), and DMA(III), but few data are available on the processes of digestion and absorption of these arsenic species and the mechanisms involved are unknown. The present study evaluated the processes involved in intestinal absorption of trivalent arsenic species, using the Caco-2 cell model as system. The apparent permeability values obtained for As(III) in apical-basolateral direction (4.6±0.3)×10(-6)cm/s, showing moderate intestinal absorption. Transport of MMA(III) [P(app)=(7.0±0.9)×10(-6)cm/s] and DMA(III) [P(app)=(10.6±1.4)×10(-6)cm/s] is greater than that of As(III). The cellular retention of As(III) (0.9-2.4%) was less than that observed for MMA(III) (30%) and DMA(III) (35%). A substantial paracellular component was observed in transport of As(III) and MMA(III), whereas DMA(III) does not use this pathway for its absorption. For all the trivalent species, transport depends on temperature, with an active transcellular component for MMA(III) and DMA(III). Variations in pH do not affect transport of these species. The presence of GSH and green tea extract significantly alters transport of As(III) across Caco-2 cells.

Trivalent arsenic species induce changes in expression and levels of proinflammatory cytokines in intestinal epithelial cells.

Chronic arsenic (As) toxicity in humans has been documented in many countries where exposure mostly occurs through drinking water. The As immunotoxic effects have been demonstrated in animal models as well as in humans. The studies of the immunotoxicity of As have centered on organs related to immune response or target organs, with few data being available at intestinal level. The present study has evaluated the changes in the expression and release of cytokines in Caco-2 cells, widely used as an intestinal epithelial model. Differentiated cells were exposed to 1 μM of As(III), 0.1 μM of monomethylarsonous acid [MMA(III)] and 1 μM of dimethylarsinous acid [DMA(III)] during 2, 4, 6 and 24 h. Additionally, the effect of As coexposure with lipopolysaccharide (LPS, 10 ng/mL) has been evaluated. The results show trivalent species to induce increases in the expression and release of the proinflammatory cytokines tumor necrosis factor alpha (TNFα), IL6, IL8 - the magnitude and time of response being different for each As species. The response of greatest magnitude corresponds to DMA(III), followed by As(III), while MMA(III) generates a limited response. Furthermore, the presence of LPS in the co-exposed cells could affect the expression and secretion of cytokines compared with individual exposure to arsenicals, especially for As(III)/LPS and DMA(III)/LPS.

Metabolism of inorganic arsenic in intestinal epithelial cell lines.

This study evaluates the metabolism of inorganic arsenic (iAs) [As(III) and As(V)] in human intestinal cells as a function of cell type, differentiation stage, type of support used for cell growth, and exposure time. Additionally, mRNA expression of arsenic (+3 oxidation state) methyltransferase (AS3MT) was evaluated. For this purpose, Caco-2 (absorptive type) and HT29-MTX (goblet type) cells were exposed at various stages of differentiation (5, 15, and 21 days post-seeding) with different concentrations of As(III) and As(V) (1 and 10 μM) and exposure times (24, 48, and 72 h), using multiwell plates or Transwells. The results show that both cell lines express AS3MT at all stages of differentiation and in all culture conditions. Caco-2 cells are capable of metabolizing iAs, As(III) metabolism being greater than that observed for As(V). Metabolism depends on the stage of differentiation, reaching 36% after 48 h of exposure of differentiated cells (15 days post-seeding), with the monomethylated species as the major metabolite. Analysis of the cell interior shows that the metabolites are present predominantly in trivalent form. The type of support is also an important factor, metabolism being greater in multiwell plates than in Transwells (36 ± 6% vs 11 ± 3%). Neither monomethylated arsenic species (MMA) nor dimethylated arsenic species (DMA) are detected in HT29-MTX cells after exposure to iAs, possibly because most of the iAs is retained in the mucus layer and does not internalize. These results show that the intestine is an organ that may take part in presystemic metabolism of iAs. Moreover, the transformation of iAs into more toxic species indicates the need to study the effects of this species on the intestinal epithelium.