Intestinal transport of Cylindrospermopsin using the Caco-2 cell line

Cylindrospermopsin (CYN) is a cyanotoxin produced by various cyanobacterial species. It is a water soluble zwitterion, stable at extreme temperatures and pH. Despite the main route of exposure to CYN is through drinking water and food, there is a lack of data concerning its intestinal absorption and the mechanisms implicated. The aim of this study was to characterize the mechanisms involved in the intestinal absorption of CYN, using Caco-2 human cell line as a model of the intestinal epithelium. The results obtained in the present work increases the limited knowledge regarding CYN transport across the intestinal epithelium and identifies the paracellular route as an important pathway in CYN absorption. A minor carrier-mediated transcellular transport has been evidenced. This transport is not affected by low temperatures, suggesting that an active mechanism is not involved. Moreover, the transport through the intestinal monolayer is H+ and GSH dependent and Na + independent. The transport characteristics elucidated in this study prepare the ground for future studies directed at identifying transporters involved in the intestinal absorption of this toxin.Ministerio de Economía y Competitividad AGL2015-64558R (MINECO/FEDER, UE) y AGL2012-3346

Polyphosphate in Lactobacillus and Its Link to Stress Tolerance and Probiotic Properties

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of Lactobacillus and their corresponding mutants devoid of poly-P synthesis capacity (defective in the poly-P kinase, ppk, gene). Results showed that resistance to salt (NaCl) and acidic (pH 4) stresses upon ppk mutation was affected in Lactobacillus casei, while no effect was observed in two different Lactobacillus plantarum strains. Inorganic [Hg(II)] and organic (CH3Hg) mercury toxicity was generally increased upon ppk mutation, but no influence was seen on the capacity to retain both mercurial forms by the bacteria. Notwithstanding, the culture supernatants of ppk-defective L. plantarum strains possessed a diminished capacity to induce HSP27 expression, a marker for cell protection, in cultured Caco-2 cells compared to wild-type strains. In summary, our results illustrate that the role of poly-P in stress tolerance can vary between strains and they reinforce the idea of probiotic-derived poly-P as a molecule that modulates host-signaling pathways. They also question the relevance of this polymer to the capacity to retain mercury of probiotics

Desarrollo y aplicacion de metodologias analiticas para la determinacion de especies arsenicales en productos de la pesca

Tesis doctoral presentad para lograr el título de Doctor por la Universidad de Valencia en 1997Desde hace tiempo se conoce que los pescados son la fuente mas significativa de arsénico en la dieta, de forma que la cantidad total de arsénico ingerida por el hombre depende de la cantidad de alimentos de origen marino que consume. El arsénico puede encontrarse en diferentes formas químicas que difieren mucho en su grado de toxicidad. Hasta el momento actual las metodologías existentes para la especiación de arsénico en productos de la pesca son escasas y pocos los datos sobre contenidos de las mismas. En la presente tesis doctoral se han desarrollado metodologías analiticas para la determinación de tres especies arsenicales, arsenobetaina (ab), acido monometilarsonico (mma) y acido dimetilarsinico en productos de la pesca. Para la arsenobetaina se han desarrollado y optimizado dos metodologías diferentes basadas en cromatografía liquida de alta resolucion y deteccion por espectrofotometria de emision atomica de plasma generado por induccion o en cromatografia liquida de alta resolucion, oxidacion en horno microondas y deteccion por espectrofotometria de absorcion atomica con generacion de hidruros. Para mma y dma se ha desarrollado y optimizado una metodologia basada en cromatografia liquida de alta resolucion y posterior deteccion por espectrofotometria de absorcion atomica con generacion de hidruros. Las caracteristicas analiticas de todas las metodologias indican la adecuación de las mismas para su aplicación a matrices reales. Por ello, se han cuantificado estas tres especies así como el arsénico total, en un amplio muestrario de productos de la pesca tanto frescos como procesados. Del análisis de los resultados obtenidos puede concluirse que la arsenobetaina, especie atoxica, no es la especie mayoritaria en los productos procesados y que dentro de ellos, los bivalvos son los que presentan los menores porcentajes de esta especie respecto al arsénico total.Peer reviewe

Arsenic speciation in manufactured seafood products

The literature on the speciation of arsenic (As) in seafoods was critically reviewed. Most research has been directed toward fresh seafood products with few papers dealing with As speciation in manufactured seafoods. Predictions concerning As species made on the basis of fresh seafood products cannot be extrapolated to manufactured seafoods. Therefore, due to the numerous species of As, the scarcity of data concerning their presence in foods, the transformations each species may undergo during industrial processing and cooking, and the lack of legislation on permitted As levels in seafood products, As species in manufactured seafood products need to be determined and quantified.Funds to carry out this work were provided by the Comision Interministerial de Ciencia y Tecnologia (CICyT), Project ALI96-0511. D.V. received a Research Personnel Training Grant from the Ministerio de Educaci6n y Ciencia

Optimization of the solubilization, extraction and determination of inorganic arsenic [As(III)+As(V)] in seafood products by acid digestion, solvent extraction and hydride generation atomic absorption spectrometry

A method for the selective quantitative determination of inorganic arsenic [As(III) + As(v)] in seafood was developed. In order to do so, various procedures for the solubilization and extraction of inorganic arsenic quoted in the literature were tested. None provided satisfactory recoveries for As(III) and As(v) in real samples. Consequently, a methodology was developed which included solubilization with HCl and subsequent extraction with chloroform. The arsenic was solubilized in 9 mol 1(-1) hydrochloric acid. After reduction by hydrobromic acid and hydrazine sulfate, the inorganic arsenic was extracted into chloroform, back-extracted into 1 mol 1(-1) HCl, dry-ashed, and quantified by hydride generation-atomic absorption spectrometry (HG-AAS). The analytical features of the method are as follows: detection limit, 3.07 ng g(-1) As (fresh mass); precision (RSD), 4.0%; recovery, As(III) 99%, As(v) 96%. In the optimized conditions, other arsenic species-dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC) and tetramethylarsonium-ion (TMA(+))-were not co-extracted. However, different percentages of minor species were extracted with chloroform: monomethylarsonic acid (MMA) 100%, and trimethylarsine oxide (TMAO) 3-10%. Real samples and reference materials of seafood (DORM-1, DORM-2, TORT-2, CRM-278 and SRM-1566a) were analyzed. The analysis of DORM-1 provided an inorganic arsenic value of 124 +/- 4 ng g(-1) As, dry mass (dm), which is very close to the value obtained by other authors using high performance liquid chromatography -inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and ionic chromatography-hydride generation-atomic absorption spectrometry (IC-HG -AAS)

Effect of subchronic exposure to inorganic arsenic on the structure and function of the intestinal epithelium

Inorganic arsenic (As), the most toxic form of As found in water and food, is considered a human carcinogen. Numerous studies show its systemic toxicity, describing pathologies associated with chronic exposure. The main pathway of exposure to inorganic As is oral, but many of the events that occur during its passage through the gastrointestinal tract are unknown. This study evaluates the effect of subchronic exposure to inorganic As [As(III): 0.025–0.1 mg/L; As(V): 0.25–1 mg/L, up to 21 days] on the intestinal epithelium, using Caco-2 cells as in vitro model. Inorganic As produces a pro-inflammatory response throughout the exposure time, with an increase in IL-8 release (up to 488%). It also causes changes in the program of cell proliferation and differentiation, which leads to impairment of the cell repair process. In addition, subchronic exposure affects the epithelial structure, causing loss of microvilli, fundamental structures in the processes of intestinal absorption and digestion. Moreover, the exposure affects the epithelial barrier function, evidenced by an increase of Lucifer Yellow transport (103–199%). Therefore, it can be concluded that subchronic exposure to inorganic As can alter intestinal homeostasis, affecting the mucosal layer, which performs the most important functions of the intestinal wall.This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2015-68920-R), for which the authors are deeply indebted. Gabriela de Matuoka e Chiocchetti received a fellowship from the Brazilian Government (CAPES-proceso BEX1086/14-6).Peer reviewe

In vitro evaluation of dietary compounds to reduce mercury bioavailability

Mercury in foods, in inorganic form [Hg(II)] or as methylmercury (CH3Hg), can have adverse effects. Its elimination from foods is not technologically viable. To reduce human exposure, possible alternatives might be based on reducing its intestinal absorption. This study evaluates the ability of 23 dietary components to reduce the amount of mercury that is absorbed and reaches the bloodstream (bioavailability). We determined their effect on uptake of mercury in Caco-2 cells, a model of intestinal epithelium, exposed to Hg(II) and CH3Hg standards and to swordfish bioaccessible fractions. Cysteine, homocysteine, glutathione, quercetin, albumin and tannic reduce bioavailability of both mercury species. Fe(II), lipoic acid, pectin, epigallocatechin and thiamine are also effective for Hg(II). Some of these strategies also reduce Hg bioavailability in swordfish (glutathione, cysteine, homocysteine). Moreover, extracts and supplements rich in these compounds are also effective. This knowledge may help to define dietary strategies to reduce in vivo mercury bioavailability.This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2015-68920), for which the authors are deeply indebted. Carlos Jadán Piedra received a Personnel Training Grant from SENESCYT (Ecuadorian Ministry of Higher Education, Science, Technology and Innovation) to carry out this study.Peer reviewe

Evaluation of the safety of seaweed consumption: heavy metals and arsenic

Trabajo presentado en la 11th International Conference on Applied Phycology, celebrad en Galway (Irlanda) del 21 al 27 al de junio de 200

Monomethylarsonic and dimethylarsinic acid contents in seafood products

The aim of the present study is to obtain information about the levels of monomethylarsonic acid (MMA) and dimethylarsmic acid (DMA) in popular seafood products and to study the possible effect of the manufacturing process on the levels of arsenical species. The methodology employed couples high-performance liquid chromatography with hydride generation atomic absorption spectrometric detection. Among all of the samples analyzed (n = 29), only 3 mollusks presented MMA levels above the limit of detection for the method, 0.6-3.7 ng g-1, fresh mass (fm), expressed as arsenic compound, and according to the dilution employed. The DMA levels ranged between <2 and 475 ng g-1 (fm), with the highest values occurring in the fish group. In 10 of the samples analyzed the total arsenic (fm) represented by unknown species [other than arsenobetaine (AB), MMA, and DMA] was around 1 μg g-1 or considerably higher (1.7-4.8 μg g-1) mostly in frozen and preserved fish. In the fish group the highest level of DMA expressed as a percentage of total arsenic was found in the preserved fish. More research is needed to identify all arsenic compounds in seafood and to confirm whether AB is degraded during the manufacturing processes, producing DMA and other arsenical compounds

Migration of Arsenobetaine from Canned Seafood to Brine

A study was carried out to determine arsenobetaine (AB) in brines from canned seafood products by coupling high-performance liquid chromatography (HPLC), microwave-assisted oxidation (MO), and hydride generation-atomic absorption spectrometry (HG-AAS). Conditions were established for cleanup of the brines prior to HPLC determination of AB. The analytical features of the method for AB determination in brines were as follows: the detection limit was 2.0 ng g-1 As (fresh mass), the mean relative standard deviation 3%, and the mean recovery percentage 99 ± 4%. The proposed procedure was used to analyze AB in brines from canned seafood purchased at local retail outlets. The ranges of total arsenic and AB expressed as As (both in μg g-1 of fresh mass) were as follows: total arsenic, 0.06-3.64; AB, 0.05-3.87. The percentage of total arsenic represented by AB in brines varied from 48 to 110%. The migration of AB from muscle tissue to the brine does not rule out the possible presence in brines of other toxic species. Migrations of arsenic to other kinds of brine, oil, or sauce should also be studied