Lactobacillus strains reduce the toxic effects of a subchronic exposure to arsenite through drinking water.

The aim of the present study was to determine the efficacy of LAB strains in reducing the intestinal toxicity of arsenite [As(III)] and its tissue accumulation. For this purpose, Balb/c mice were randomly separated in four groups. One group received no treatment (control), one group received only As(III) (30 mg/L) via drinking water and the remaining two groups received As(III) via water and a daily dose of two LAB strains (Lactobacillus intestinalis LE1 and Lacticaseibacillus paracasei BL23) by gavage during 2 months. The results show that both strains reduce the pro-inflammatory and pro-oxidant response observed at the colonic level, partially restore the expression of the intercellular junction proteins (CLDN3 and OCLN) responsible for the maintenance of epithelial integrity, and increase the synthesis of the major mucin of the colonic mucus layer (MUC2), compared to animals treated with As(III) alone. Microbial metabolism of short-chain fatty acids also undergoes a recovery and the levels of fatty acids in the lumen reach values similar to those of untreated animals. All these positive effects imply the restoration of mucosal permeability, and a reduction of the marker of endotoxemia LPS binding protein (LBP). Treatment with the bacteria also has a direct impact on intestinal absorption, reducing the accumulation of As in the internal organs. The data suggest that the protective effect may be due to a reduced internalization of As(III) in intestinal tissues and to a possible antioxidant and anti-inflammatory activity of the bacteria through activation of pathways such as Nrf2 and IL-10. In vitro tests show that the protection may be the result of the combined action of structural and metabolic components of the LAB strains.

Intestinal homeostasis disruption in mice chronically exposed to arsenite-contaminated drinking water.

Chronic exposure to inorganic arsenic [As(III) and As(V)] affects about 200 million people, and is linked to a greater incidence of certain types of cancer. Drinking water is the main route of exposure, so, in endemic areas, the intestinal mucosa is constantly exposed to the metalloid. However, studies on the intestinal toxicity of inorganic As are scarce. The objective of this study was to evaluate the toxicity of a chronic exposure to As(III) on the intestinal mucosa and its associated microbiota. For this purpose, BALB/c mice were exposed during 6 months through drinking water to As(III) (15 and 30 mg/L). Treatment with As(III) increased reactive oxygen species (43-64%) and lipid peroxidation (8-51%). A pro-inflammatory response was also observed, evidenced by an increase in fecal lactoferrin (23-29%) and mucosal neutrophil infiltration. As(III) also induced an increase in the colonic levels of pro-inflammatory cytokines (24-201%) and the activation of some pro-inflammatory signaling pathways. Reductions in the number of goblet cells and mucus production were also observed. Moreover, As(III) exposure resulted in changes in gut microbial alpha diversity but no differences in beta diversity. This suggested that the abundance of some taxa was significantly affected by As(III), although the composition of the population did not show significant alterations. Analysis of differential taxa agreed with this, 21 ASVs were affected in abundance or variability, especially ASVs from the family Muribaculaceae. Intestinal microbiota metabolism was also affected, as reductions in fecal concentration of short-chain fatty acids were observed. The effects observed on different components of the intestinal barrier may be responsible of the increased permeability in As(III) treated mice, evidenced by an increase in fecal albumin (48-66%). Moreover, serum levels of Lipopolysaccharide binding proteins and TNF-α were increased in animals treated with 30 mg/L of As(III), suggesting a low-level systemic inflammation.

Psychometric properties of the Spanish version of the Frontal Assessment Battery (FAB-E) and normative values in a representative adult population sample.

BACKGROUND: The Frontal Assessment Battery is a short bedside test used to assess executive functions (EF). The aims of the present study were, first, to evaluate the psychometric proprieties of the Spanish version of the FAB (FAB-E) in a representative sample, and second, to establish cut-off points for impairment in executive function according to age and education level. METHODS: A sample of 798 healthy Spanish adult subjects aged 19 to 91 participated in this study. Neuropsychological assessment of participants was conducted using the FAB-E, Mini-Mental State Examination (MMSE) and Trail Making Test (TMT). We examined internal consistency, intraclass correlation, test-retest reliability, and concurrent and divergent validity. In addition, we established a cut-off point for detecting executive function impairment based on the 5th percentile by age group and education level. RESULTS: The analysis of the psychometric properties of the FAB-E showed good internal consistency (Cronbach's α = 0.60), intraclass correlation (0.72), test-retest reliability (0.70) and concurrent and divergent validity between the TMT (r = -0.523), MMSE (r = 0.426) and the FAB-E. The cut-off points for each age group were 16 points for the ≤ 29 group, 15 points for the 30-39 group, 14 points for the 40-49 and 50-59 groups, 12 points for the 60-69 group, and 10 points for the ≥ 70 age group. CONCLUSIONS: The psychometric analysis showed that the FAB-E has good validity and reliability. Thus, FAB-E may be a helpful tool to evaluate EF in a healthy Spanish population. In addition, this study provides information on reference data that will be very valuable for clinicians and researchers.

Lipoteichoic acid depletion in Lactobacillus impacts cell morphology and stress response but does not abolish mercury surface binding.

Lipoteichoic acid (LTA) is a key component of the cell wall of most Gram-positive bacteria and plays many structural and functional roles. In probiotic lactobacilli, the function of LTA in mediating bacteria/host cross-talk has been evidenced and it has been postulated that, owing to its anionic nature, LTA may play a role in toxic metal sequestration by these bacteria. However, studies on this last aspect employing strains unable to synthesise LTA are lacking. We have inactivated the LTA polymerase encoding gene ltaS in two different Lactobacillus plantarum strains. Analysis of LTA contents in wild-type and ltaS mutant strains corroborated the role of this gene as a major contributor to LTA synthesis in L. plantarum. The mutant strains displayed strain-dependent anomalous cell morphologies that resulted in elongated or irregular cells with aberrant septum formation. They also exhibited higher sensitivity to several stresses (osmotic and heat) and to antimicrobials that target the cell wall. The toxicity of inorganic [(Hg(II)] and organic mercury (methyl-Hg) was also increased upon ltaS mutation in a strain-dependent manner. However, the mutant strains showed 0 to 50% decrease in their capacity of Hg binding compared to their corresponding parental strains. This result suggests a partial contribution of LTA to Hg binding onto the cell surface that was dependent on the strain and the Hg form.

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.

In Vitro Reduction of Arsenic Bioavailability Using Dietary Strategies.

The main route of human exposure to inorganic arsenic (As) is through the consumption of food and water. Continued exposure to inorganic As [As(III) and As(V)] may cause a variety of diseases, including various types of cancer. The removal of As from these sources is complex, especially for food. One way to decrease As exposure could be by reducing intestinal absorption of it. The aim of this study is to seek dietary strategies (pure compounds, extracts, or supplements) that are capable of reducing the amount of As that is absorbed and reaches systemic circulation. Standard solutions of As(III) and As(V) and bioaccessible fractions of food samples with or without the dietary strategies to be tested were added to colon-derived human cells (NCM460 and HT-29MTX) to determine the apparent permeability (Papp) of As. Results show that transport across the intestinal monolayers is substantial, and the passage of As(III) (Papp = 4.2 × 10-5 cm/s) is greater than that of As(V) (Papp = 2.4 × 10-5 cm/s). Some of the treatments used (iron species, cysteine, grape extract) significantly reduce the transport of both inorganic As standards across the intestinal monolayer, thus decreasing absorption of them. In food samples, the effect of the dietary compounds on inorganic As bioavailability was also observed, especially in the cases of curcumin and cysteine. Compounds that proved effective in these in vitro assays could be the basis for intervention strategies aimed at reducing As toxicity in chronically exposed populations or regular consumers of food products with high As contents.

The use of lactic acid bacteria to reduce mercury bioaccessibility.

Mercury in food is present in either inorganic [Hg(II)] or methylmercury (CH3Hg) form. Intestinal absorption of mercury is influenced by interactions with other food components. The use of dietary components to reduce mercury bioavailability has been previously proposed. The aim of this work is to explore the use of lactic acid bacteria to reduce the amount of mercury solubilized after gastrointestinal digestion and available for absorption (bioaccessibility). Ten strains were tested by addition to aqueous solutions containing Hg(II) or CH3Hg, or to food samples, and submission of the mixtures to gastrointestinal digestion. All of the strains assayed reduce the soluble fraction from standards of mercury species under gastrointestinal digestion conditions (72-98%). However their effectiveness is lower in food, and reductions in bioaccessibility are only observed with mushrooms (⩽68%). It is hypothesized that bioaccessible mercury in seafood forms part of complexes that do not interact with lactic acid bacteria.

Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.

A collaborative trial was conducted to determine the performance characteristics of an analytical method for the quantification of inorganic arsenic (iAs) in food. The method is based on (i) solubilisation of the protein matrix with concentrated hydrochloric acid to denature proteins and allow the release of all arsenic species into solution, and (ii) subsequent extraction of the inorganic arsenic present in the acid medium using chloroform followed by back-extraction to acidic medium. The final detection and quantification is done by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS). The seven test items used in this exercise were reference materials covering a broad range of matrices: mussels, cabbage, seaweed (hijiki), fish protein, rice, wheat, mushrooms, with concentrations ranging from 0.074 to 7.55mgkg(-1). The relative standard deviation for repeatability (RSDr) ranged from 4.1 to 10.3%, while the relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 22.8%.

Toxic trace elements at gastrointestinal level.

Many trace elements are considered essential [iron (Fe), zinc (Zn), copper (Cu)], whereas others may be harmful [lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As)], depending on their concentration and chemical form. In most cases, the diet is the main pathway by which they enter our organism. The presence of toxic trace elements in food has been known for a long time, and many of the food matrices that carry them have been identified. This has led to the appearance of legislation and recommendations concerning consumption. Given that the main route of exposure is oral, passage through the gastrointestinal tract plays a fundamental role in their entry into the organism, where they exert their toxic effect. Although the digestive system can be considered to be of crucial importance in their toxicity, in most cases we do not know the events that occur during the passage of these elements through the gastrointestinal tract and of ascertaining whether they may have some kind of toxic effect on it. The aim of this review is to summarize available information on this subject, concentrating on the toxic trace elements that are of greatest interest for organizations concerned with food safety and health: Pb, Cd, Hg and As.

Gastrointestinal stromal tumor of the stomach and hepatocellular carcinoma: An unusual association.

INTRODUCTION: Gastrointestinal stromal tumors (GIST) are uncommon, potentially malignant tumors, that arise in the wall of the gastrointestinal tract. Up to 50% can develop metastasis, mainly in the liver, but the occurrence of synchronous primary liver tumors is a rare event in these patients. PRESENTATION OF CASE: The authors report a case of the association of gastric GIST and hepatocellular carcinoma (HCC) in a non-cirrhotic liver in a 76 year-old patient. DISCUSSION: The appearance of an hepatic lesion in a GIST patient does not necessarily imply its secondary nature. CONCLUSION: In diagnosed GIST patients, all efforts should be pursued to characterize synchronous hepatic lesions, in order to plan a correct and tailored treatment of the patients.

Evaluation of traction stirrup distraction technique to increase the joint space of the shoulder joint in the dog: A cadaveric study.

The objective of this study was to evaluate technical feasibility and efficacy of a joint distraction technique by traction stirrup to facilitate shoulder arthroscopy and assess potential soft tissue damage. Twenty shoulders were evaluated radiographically before distraction. Distraction was applied with loads from 40 N up to 200 N, in 40 N increments, and the joint space was recorded at each step by radiographic images. The effects of joint flexion and intra-articular air injection at maximum load were evaluated. Radiographic evaluation was performed after distraction to evaluate ensuing joint laxity. Joint distraction by traction stirrup technique produces a significant increase in the joint space; an increase in joint laxity could not be inferred by standard and stress radiographs. However, further clinical studies are required to evaluate potential neurovascular complications. A wider joint space may be useful to facilitate arthroscopy, reducing the likelihood for iatrogenic damage to intra-articular structures.

Participation of divalent cation transporter DMT1 in the uptake of inorganic mercury.

Mercury (Hg) is found in food in various chemical forms, which differ in terms of accumulation, transport, and toxicity. Although methylmercury (CH3Hg) is the predominant mercury species in the diet, contributed mostly by seafood products, there is also a contribution of inorganic mercury [Hg(II)] from vegetables, cereals, and seafood products. The main pathway for exposure to mercury is oral, and therefore the gastrointestinal mucosa is the first barrier that the contaminant meets when it enters the systemic circulation. However, the transport mechanisms responsible for the process of mercury absorption are not known. The aim of this study is to evaluate the possible participation of divalent metal transporter 1 (DMT1) in Hg(II) intestinal uptake. For this purpose, we have used various complementary approaches. We have studied mercury acquisition in a Saccharomyces cerevisiae strain expressing murine DMT1. Moreover, we have evaluated the effect of a reduction of DMT1 expression in Caco-2 cells, by means of small interfering RNA and of treatment with hepcidin, on mercury uptake and transport. The results show that expression of the transporter DMT1 in yeast produces an increase in Hg(II) accumulation. Furthermore, a decrease in the levels of DMT1 mRNA in Caco-2 cells in various stages of differentiation leads to a reduction in cellular accumulation and apical-basolateral transport of Hg(II). These data point clearly to the mediation of the divalent cation transporter DMT1 in the entry of Hg(II) into the intestinal epithelium.

Characterization of the intestinal absorption of inorganic mercury in Caco-2 cells.

The main form of mercury exposure in the general population is through food. Intestinal absorption is therefore a key step in the penetration of mercury into the systemic circulation, and should be considered when evaluating exposure risk. Many studies have investigated the transport of mercury species in different cell lines, though the mechanisms underlying their intestinal absorption are not clear. This study evaluates the accumulation and transport of Hg(II), one of the mercury species ingested in food, using Caco-2 cells as intestinal epithelium model with the purpose of clarifying the mechanisms involved in its absorption. Hg(II) shows moderate absorption, and its transport fundamentally takes place via a carrier-mediated transcellular mechanism. The experiments indicate the participation of an energy-dependent transport mechanism. In addition, H(+)- and Na(+)-dependent transport is also observed. These data, together with those obtained from inhibition studies using specific substrates or inhibitors of different transporter families, suggest the participation of divalent cation and amino acid transporters, and even some organic anion transporters, in Hg(II) intestinal transport. An important cellular accumulation of up to 51% is observed - a situation which in view of the toxic nature of this species could affect intestinal mucosal function. This study contributes new information on the mechanisms of transport of Hg(II) at intestinal level, and which may be responsible for penetration of this mercurial form into the systemic circulation.

Determination of total cadmium, lead, arsenic, mercury and inorganic arsenic in mushrooms: outcome of IMEP-116 and IMEP-39.

The Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre (JRC), a Directorate General of the European Commission, operates the International Measurement Evaluation Program (IMEP). IMEP organises inter-laboratory comparisons in support of European Union policies. This paper presents the results of two proficiency tests (PTs): IMEP-116 and IMEP-39, organised for the determination of total Cd, Pb, As, Hg and inorganic As (iAs) in mushrooms. Participation in IMEP-116 was restricted to National Reference Laboratories (NRLs) officially appointed by national authorities in European Union member states. IMEP-39 was open to all other laboratories wishing to participate. Thirty-seven participants from 25 countries reported results in IMEP-116, and 62 laboratories from 36 countries reported for the IMEP-39 study. Both PTs were organised in support to Regulation (EC) No. 1881/2006, which sets the maximum levels for certain contaminants in food. The test item used in both PTs was a blend of mushrooms of the variety shiitake (Lentinula edodes). Five laboratories, with demonstrated measurement capability in the field, provided results to establish the assigned values (Xref). The standard uncertainties associated to the assigned values (uref) were calculated by combining the uncertainty of the characterisation (uchar) with a contribution for homogeneity (ubb) and for stability (ust), whilst uchar was calculated following ISO 13528. Laboratory results were rated with z- and zeta (ζ)-scores in accordance with ISO 13528. The standard deviation for proficiency assessment, σp, ranged from 10% to 20% depending on the analyte. The percentage of satisfactory z-scores ranged from 81% (iAs) to 97% (total Cd) in IMEP-116 and from 64% (iAs) to 84% (total Hg) in IMEP-39.

In vitro evaluation of inorganic mercury and methylmercury effects on the intestinal epithelium permeability.

The mercurial forms [inorganic divalent mercury, Hg(II) and methylmercury, CH3Hg] produce neurological and immune effects as well as hematological and renal alterations. The main route of exposure is through the diet. Consequently, the gastrointestinal mucosa is exposed to these mercurial forms, though the potential toxic effects upon the mucosa are not clear. The present study evaluates the toxicity of Hg(II) and CH3Hg (0.1-2 mg/L) in an intestinal epithelium model using the differentiated and undifferentiated human Caco-2 cell line.The experiments made show the mercurial forms generate reactive oxygen and/or nitrogen species and a significant decrease in glutathione contents. This redox imbalance could be the cause of the lipid peroxidation observed after short exposure times. Such conditions of stress lead to a modulation of stress proteins, intercellular junction proteins and tumor necrosis factor-alpha expression and to a redistribution of F-actin and ZO1 protein in the intestinal monolayer. The abovementioned effects may be the cause of the increase in permeability in the differentiated cells observed at concentrations similar to those found in food products (0.5-1 mg/L). The increase in permeability could produce an impairment of the barrier function of the intestinal epithelium.

Evaluation of a joint distractor to facilitate arthroscopy of the hip joint in dogs.

OBJECTIVE: To evaluate the technical feasibility and efficacy of a hip joint distraction technique, any potential ligamentous damage linked to the procedure, and the effect of joint venting on the maximum distraction achieved. METHODS: Twenty hip joints from 11 canine cadavers were evaluated radiographically by standard and stressed projections. Joint distraction was applied with loads from 40 up to 200 N, in 40 N increments, and fluoroscopic images were obtained at each load. At 200 N, a needle was inserted into the joint to achieve a venting effect, and the space was measured again. Standard and stressed radiographs were performed to evaluate potential laxity changes. RESULTS: Distraction caused a significant increase in joint space at each load of distraction, although there were some variations. Joint venting produced a significant increase in joint space. A statistically significant difference in joint laxity evaluated radiographically before and after the procedure was recorded. CLINICAL SIGNIFICANCE: Use of the distraction apparatus resulted in an increase in joint space. This could be useful for clinical situations where a larger joint space is required such as for arthroscopic procedures. However, loads in excess of 200 N may induce significant increases in joint laxity.

Evaluation of a joint distractor to facilitate arthroscopy of the tibio-tarsal joint in dogs.

OBJECTIVE: To evaluate the technical feasibility, efficacy and potential soft tissue damage of a joint distraction technique to facilitate arthroscopy of the tibio-tarsal joint and to test the effect of joint venting on the maximum distraction achieved. METHODS: Twenty hocks were preliminarily evaluated radiographically by standard and stressed projections. Joint distraction was applied with loads from 40 N up to 200 N, in 40 N increments, and radiographic images were obtained at each load. The effect of joint venting by an intra-articular needle was evaluated at the maximum load. Standard and stressed radiographs were repeated to evaluate potential laxity changes. RESULTS: Joint distraction caused a significant increase in the joint space at each load of distraction except for the 40 to 80 N load increase. Joint venting produced a significant increase in the joint space. No increase of joint laxity could be inferred from the postdistraction radiographic evaluation. CLINICAL SIGNIFICANCE: Hock distraction can be useful to facilitate arthroscopic procedures, increasing the joint space available for intra-articular manoeuvres. Further clinical studies are required to evaluate potential neurovascular complications.

Glutathione-enriched baker's yeast: production, bioaccessibility and intestinal transport assays.

AIMS: A glutathione (GSH) yeast-based biomass (Saccharomyces cerevisiae) was used to investigate GSH stability, solubilization during gastrointestinal digestion and GSH intestinal transport. METHODS AND RESULTS: A postgrowing procedure was applied to improve intracellular GSH yeast content. The presence of adenine (ADE) in the biotransformation solution (CYS-GLY-GLU mixture) and alternatively, a glucose shot after 4-h incubation, allowed to obtain cells containing about GSH 1.6-1.7% dcw (dry cell weight) (control 0.5%). Yeast samples were subjected to in vitro gastrointestinal digestion and absorption assays employing Caco-2 and HT29-MTX cell lines in different proportions (100/0, 70/30 and 50/50). Trials were also performed to verify intestinal cell viability. CONCLUSIONS: At least 87% of ingested GSH is available in reduced form for intestinal absorption. In vitro GSH transport assays indicated that GSH is poorly absorbed (<20%). Nevertheless, studies in response to oxidative stress induced by H2 O2 demonstrated a protective role of the GSH-enriched biomass towards intestinal cell viability. SIGNIFICANCE AND IMPACT OF STUDY: An enriched GSH yeast-based biomass has been obtained using a postgrowing procedure. Although GSH present in enriched yeasts is poorly absorbed by intestinal cells, this biomass showed an intestinal local protective effect, improving cells viability when a simulated oxidative stress was applied.

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.