Phosphate Uptake and Its Relation to Arsenic Toxicity in Lactobacilli.

The use of probiotic lactobacilli has been proposed as a strategy to mitigate damage associated with exposure to toxic metals. Their protective effect against cationic metal ions, such as those of mercury or lead, is believed to stem from their chelating and accumulating potential. However, their retention of anionic toxic metalloids, such as inorganic arsenic, is generally low. Through the construction of mutants in phosphate transporter genes (pst) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains, coupled with arsenate [As(V)] uptake and toxicity assays, we determined that the incorporation of As(V), which structurally resembles phosphate, is likely facilitated by phosphate transporters. Surprisingly, inactivation in Lc. paracasei of PhoP, the transcriptional regulator of the two-component system PhoPR, a signal transducer involved in phosphate sensing, led to an increased resistance to arsenite [As(III)]. In comparison to the wild type, the phoP strain exhibited no differences in the ability to retain As(III), and there were no observed changes in the oxidation of As(III) to the less toxic As(V). These results reinforce the idea that specific transport, and not unspecific cell retention, plays a role in As(V) biosorption by lactobacilli, while they reveal an unexpected phenotype for the lack of the pleiotropic regulator PhoP.

Impact of Chronic Exposure to Arsenate through Drinking Water on the Intestinal Barrier.

Chronic exposure to inorganic arsenic (As) [As(III) + As(V)], which affects millions of people, increases the incidence of some kinds of cancer and other noncarcinogenic pathologies. Although the oral pathway is the main source of exposure, in vivo studies conducted to verify the intestinal toxicity of this metalloid are scarce and are mainly focused on evaluating the toxicity of As(III). The aim of this study was to evaluate the effect of chronic exposure (6 months) of BALB/c mice to As(V) (15-60 mg/L) via drinking water on the different components of the intestinal barrier and to determine the possible mechanisms involved. The results show that chronic exposure to As(V) generates a situation of oxidative stress (increased lipid peroxidation and reactive species) and inflammation (increased contents of several proinflammatory cytokines and neutrophil infiltrations) in the intestinal tissues. There is also evidence of an altered expression of constituent proteins of the intercellular junctions (Cldn1, Cldn3, and Ocln) and the mucus layer (Muc2) and changes in the composition of the gut microbiota and the metabolism of short-chain fatty acids. All of these toxic effects eventually may lead to the disruption of the intestinal barrier, which shows an increased paracellular permeability. Moreover, signs of endotoxemia are observed in the serum of As(V)-treated animals (increases in lipopolysaccharide-binding protein LBP and the proinflammatory cytokine IL-1ß). The data obtained suggest that chronic exposure to As(V) via drinking water affects the intestinal environment.

Dietary microplastics: Occurrence, exposure and health implications.

The increasing use of plastic materials generates an enormous amount of waste. In the aquatic environment, a significant part of this waste is present in the form of microplastics (MPs)- particles with a diameter of between 0.1 µm and 5 mm. The arrival of these small plastics in the food chain has been recently documented. MPs have been reported in fishery products, drinking water and sea salt among other foods. Their intestinal absorption is considered limited due to their size, however, they contain a mixture of chemicals intentionally added during their manufacture, which could cross the intestinal barrier. Currently there are not enough data to allow an accurate assessment of the risk associated with dietary exposure to MPs. The lack of robust methodologies is undoubtedly one of the main problems. There is limited information on occurrence in dietary sources (drinking water and food), human intake, toxicokinetics and long term toxicity of these contaminants. The present review describes the studies published so far and points to the need for improved knowledge in order to have a more accurate view of the problems posed by MPs.

Arsenic in Tissues and Prey Species of the Scalloped Hammerhead (Sphyrna lewini) from the SE Gulf of California.

The bioaccumulation of arsenic (As) in the muscle, liver, kidneys, and brain of the shark Sphyrna lewini was measured in 40 juvenile specimens from southeast Gulf of California. Additionally, the biomagnification factor was calculated through prey items from stomach contents of the analyzed specimens. The concentrations of As (mg kg-1, wet weight) were higher in the muscle (10.1 ± 0.3) and liver (9.4 ± 0.5) than in the brain (4.5 ± 0.3) and kidneys (4.2 ± 0.2), which may be attributed to the biological functions of each tissue. Positive correlations were found between the levels of As in muscle and liver with the biological parameters of S. lewini. Hammerhead sharks feed mainly of teleost fishes with low As values (Clupeidae fishes, 1.1 ± 0.5; Sciaenidae fishes, 1.0 ± 0.6; Scomber japonicus, 1.2 ± 0.6; and Etropus crossotus 2.1 ± 0.4) compared with the predator, indicating biomagnification. Inorganic arsenic (Asi) in muscle was estimated as 3% of the total As, although muscle consumption is unlikely to represent a risk (HQ < 1) in humans. Moreover, the probabilities of developing cancer were estimated as low (3.99 × 10-5 to 3.32 × 10-6). To avoid health risks related to As, a weekly ration must not exceed 69.3 and 484.8 g in children and adults, respectively.

Use of lactic acid bacteria and yeasts to reduce exposure to chemical food contaminants and toxicity.

Chemical contaminants that are present in food pose a health problem and their levels are controlled by national and international food safety organizations. Despite increasing regulation, foods that exceed legal limits reach the market. In Europe, the number of notifications of chemical contamination due to pesticide residues, mycotoxins and metals is particularly high. Moreover, in many parts of the world, drinking water contains high levels of chemical contaminants owing to geogenic or anthropogenic causes. Elimination of chemical contaminants from water and especially from food is quite complex. Drastic treatments are usually required, which can modify the food matrix or involve changes in the forms of cultivation and production of the food products. These modifications often make these treatments unfeasible. In recent years, efforts have been made to develop strategies based on the use of components of natural origin to reduce the quantity of contaminants in foods and drinking water, and to reduce the quantity that reaches the bloodstream after ingestion, and thus, their toxicity. This review provides a summary of the existing literature on strategies based on the use of lactic acid bacteria or yeasts belonging to the genus Saccharomyces that are employed in food industry or for dietary purposes.

In vivo evaluation of the effect of arsenite on the intestinal epithelium and associated microbiota in mice.

Chronic exposure to inorganic arsenic (As) [As(III) + As(V)], which affects millions of people, increases the incidence of some kinds of cancer and other non-carcinogenic pathologies. Although the oral pathway is the main form of exposure, in vivo studies have not been conducted to verify the intestinal toxicity of this metalloid. The aim of this study is to perform an in vivo evaluation of the intestinal toxicity of inorganic As, using female BALB/c mice exposed through drinking water to various concentrations of As(III) (20, 50, and 80 mg/L) for 2 months. An increase was observed in oxygen and/or nitrogen reactive species, and in gene and protein expression of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6) at concentrations equal to or greater than 50 mg/L. These changes were accompanied by a profound remodeling of the intestinal microbial profile in terms of diversity and global composition, which could be at the basis or exacerbate As(III) toxic effects. The histological study showed that there was moderate inflammation of the mucosa and submucosa, accompanied by hyperplasia of crypts at the highest administered dose. In addition, all the treatments with As(III) resulted in a decreased expression of Muc2, which encodes one of the main components of the intestinal layer of mucus. The effects described are compatible with the increased intestinal permeability observed at concentrations equal to or greater than 50 mg/L, indicative of loss of barrier function.

Effect of chronic exposure to inorganic arsenic on intestinal cells.

Chronic exposure to inorganic arsenic (As)-As(III) + As(V)-is associated with type 2 diabetes, vascular diseases and various types of cancer. Although the oral route is the main way of exposure to inorganic As, the adverse gastrointestinal effects produced by chronic exposure are not well documented. The aim of the present study is to evaluate the effect of chronic exposure to As(III) on the intestinal epithelium. For this purpose, NCM460 cells, non-transformed epithelial cells from the human colon, were exposed to As(III) (0.01-0.2 mg/L) for 6 months and monitored for acquisition of a tumor-like phenotype. Secretion of matrix metalloproteinases, histone modifications (H3 acetylation), hyperproliferation capacity, formation of floating spheres, anchorage-independent growth, release of cytokine interleukin-8 and expression of relevant genes in colon tumorigenesis were assessed. The results show a maintained proinflammatory response from the beginning, with an increase in interleukin-8 secretion (≤570%). Downregulation of CDX1 and CDX2 was also observed. After 14 weeks of exposure, cells presented marked increases in matrix metalloproteinase-2 secretion and histone modifications. As(III)-treated cells were hyperproliferative, grew in low-serum media and were able to form free-floating spheres. Overall, these data suggest that exposure of human colon epithelial cells to As(III) facilitates acquisition of transformed cell characteristics.

Dietary compounds as modulators of metals and metalloids toxicity.

A large part of the population is exposed to metals and metalloids through the diet. Most of the in vivo studies on its toxicokinetics and toxicity are conducted by means of exposure through drinking water or by intragastric or intraperitoneal administration of aqueous standards, and therefore they do not consider the effect of the food matrix on the exposure. Numerous studies show that some components of the diet can modulate the toxicity of these food contaminants, reducing their effect on a systemic level. Part of this protective role may be due to a reduction of intestinal absorption and subsequent tissue accumulation of the toxic element, although it may also be a consequence of their ability to counteract the toxicity directly by their antioxidant and/or anti-inflammatory activity, among other factors. The present review provides a compilation of existing information about the effect that certain components of the diet have on the toxicokinetics and toxicity of the metals and metalloids of greatest toxicological importance that are present in food (arsenic, cadmium, lead, and mercury), and of their most toxic chemical species.

Metal(loid) contamination in seafood products.

Seafood products are important sources of proteins, polyunsaturated lipids and phospholipids, and also of numerous micronutrients (vitamins and minerals). However, they may also present chemical contaminants that can constitute a health risk and that must be considered when evaluating the risk/benefit associated with consumption of this group of foods. Toxic metals and metalloids in seafood, such as mercury (Hg), cadmium (Cd), arsenic (As), and lead (Pb), are subjected to legislative control in order to provide the consumer with safe seafood. This review provides an exhaustive survey of the occurrence of these toxic metal(loid)s in seafood products, and of the risk resulting from their consumption. Consideration is given to aspects related to speciation, food processing, and bioavailability, which are key factors in evaluating the risk associated with the presence of these toxic trace elements in seafood products.

Characterization of the binding capacity of mercurial species in Lactobacillus strains.

BACKGROUND: Metal sequestration by bacteria has been proposed as a strategy to counteract metal contamination in foodstuffs. Lactobacilli can interact with metals, although studies with important foodborne metals such as inorganic [Hg(II)] or organic (CH3 Hg) mercury are lacking. Lactobacilli were evaluated for their potential to bind these contaminants and the nature of the interaction was assessed by the use of metal competitors, chemical and enzymatical treatments, and mutants affected in the cell wall structure. RESULTS: Lactobacillus strains efficiently bound Hg(II) and CH3 Hg. Mercury binding by Lactobacillus casei BL23 was independent of cell viability. In BL23, both forms of mercury were cell wall bound. Their interaction was not inhibited by cations and it was resistant to chelating agents and protein digestion. Lactobacillus casei mutants affected in genes involved in the modulation of the negative charge of the cell wall anionic polymer lipoteichoic acid showed increased mercury biosorption. In these mutants, mercury toxicity was enhanced compared to wild-type bacteria. These data suggest that lipoteichoic acid itself or the physicochemical characteristics that it confers to the cell wall play a major role in mercury complexation. CONCLUSION: This is the first example of the biosorption of Hg(II) and CH3 Hg in lactobacilli and it represents a first step towards their possible use as agents for diminishing mercury bioaccessibility from food at the gastrointestinal tract. © 2017 Society of Chemical Industry.

Impaired aquaporins expression in the gastrointestinal tract of rat after mercury exposure.

The main route of exposure to mercury in humans is through the diet. Consequently, the gastrointestinal mucosa is exposed to the mercurial forms, where they cause intestinal fluid accumulation, mucosal injuries and diarrhea. The relationship between inorganic mercury (HgCl2 ) and methylmercury (CH3 HgCl) exposure and water movement in the gastrointestinal tract is still unexplored. The leading role of aquaporins (AQPs) in the rapid bidirectional movement of fluid in the gastrointestinal tract of mammals is well established. The present study evaluates the effect of HgCl2 and CH3 HgCl exposure on AQP expression in different portions of the gastrointestinal tract of rats treated by gavage (5 mg kg(-1) of mercury species, single dose, 4 days). The results show that mercury species reduce mRNA and protein levels of AQPs in different parts of the gastrointestinal tract. In the stomach, treated rats show a significant reduction of expression of AQP3 (80-90% for mRNA and 50% for protein) and AQP4 (95-99% for mRNA and 20-40% for protein). In the small and large intestine, treated rats experience a significant reduction of AQP3 and AQP7 expression. Protein contents of both AQPs are reduced in similar proportions in jejunum (AQP3: 40-50%; AQP7: 45-50%) and colon (AQP3: 35-40%; AQP7: 45-60%), regardless of the treatment. Our results indicate that some AQPs are downregulated in the rat gastrointestinal tract by mercury exposure, suggesting a possible role of AQPs in the development of mercury gastrointestinal symptoms.

Proinflammatory effect of trivalent arsenical species in a co-culture of Caco-2 cells and peripheral blood mononuclear cells.

Chronic exposure to inorganic arsenic (As) is associated with type 2 diabetes, cardiovascular diseases and cancer. Ingested inorganic As is transformed within the gastrointestinal tract and can give rise to more toxic species such as monomethylarsonous acid [MMA(III)] and dimethylarsinous acid [DMA(III)]. Thus, the intestinal epithelium comes into contact with toxic arsenical species, and the effects of such exposure upon epithelial function are not clear. The present study has evaluated the effect of 1 µM arsenite [As(III)], 0.1 µM MMA(III) and 1 µM DMA(III) upon the release of cytokines [interleukin-6 (IL6), IL8, tumor necrosis factor alpha (TNFα)], using a compartmentalized co-culture model with differentiated Caco-2 cells in the apical compartment and peripheral blood mononuclear cells in the basolateral compartment. In addition, the combined effect of arsenical species and lipopolysaccharide (LPS), both added into the apical compartment, has been analyzed. The results indicate that exposure to the arsenical forms induces a proinflammatory response. An increase in cytokine secretion into the basolateral compartment was observed, particularly as regards TNFα (up to 1,600 %). The cytokine levels on the apical side also increased, though to a lesser extent. As/LPS co-exposure significantly affected the proinflammatory response as compared to treatment with As alone. Treatment with DMA(III) and As/LPS co-exposure increased the permeability of the intestinal monolayer. In addition, As/LPS treatments enhanced As(III) and MMA(III) transport through the intestinal monolayer.

In vitro characterization of the intestinal absorption of methylmercury using a Caco-2 cell model.

Methylmercury (CH3Hg) is one of the forms of mercury found in food, particularly in seafood. Exposure to CH3Hg is associated with neurotoxic effects during development. In addition, methylmercury has been classified by the International Agency for Research on Cancer as a possible human carcinogen. Although the diet is known to be the main source of exposure, few studies have characterized the mechanisms involved in the absorption of this contaminant. The present study examines the absorption process using the Caco-2 cell line as a model of the intestinal epithelium. The results indicate that transport across the intestinal cell monolayer in an absorptive direction occurs mainly through passive transcellular diffusion. This mechanism coexists with carrier-mediated transcellular transport, which has an active component. The participation of H(+)- and Na(+)-dependent transport was observed. Inhibition tests point to the possible participation of amino acid transporters (B(0,+) system, L system, and/or y(+)L system) and organic anion transporters (OATs). Our study suggests the participation in CH3Hg absorption of transporters that have already been identified as being responsible for the transport of this species in other systems, although further studies are needed to confirm their participation in intestinal absorption. It should be noted that CH3Hg experiences important cellular acumulation (48-78%). Considering the toxic nature of this contaminant, this fact could affect intestinal epithelium function.

Processes influencing the toxicity of microplastics ingested through the diet.

The present study assesses the effect of culinary treatment and gastrointestinal digestion upon the release of additives present in microplastics. Organic additives were determined by gas chromatography-mass spectrometry, and inorganic additives using inductively coupled plasma-mass spectrometry. The results revealed a large number of organic additives in the plastic samples, some being classified as possible carcinogens. Contents of Sb in PET (polyethylene terephthalate), Zn and Ba in LDPE (low-density polyethylene) and PVC (polyvinylchloride), and Ti and Pb in LDPE were also noteworthy. The culinary process promotes the release and solubilization of additives into the cooking liquid, with phthalates, benzophenone, N-butylbenzenesulfonamide (NBBS) and bisphenol A being of particular concern. The solubilization of phthalates and NBBS was also observed during gastrointestinal digestion. This study demonstrates that culinary treatment and gastrointestinal digestion promote release and solubilization of additives from plastics ingested with the diet. Such solubilization may facilitate their entry into the systemic circulation.

Protective effects of oral administration of lactic acid bacteria strains against methylmercury-induced intestinal toxicity in a murine model.

The utilization of lactic acid bacteria has been proposed to mitigate the burden of heavy metal exposure through processes probably involving chelation and reduced metal bioaccessibility. We evaluated the effects of daily intake of two strains of lactobacilli (Lactobacillus intestinalis LE1 or Lactobacillus johnsonii LE2) on intestinal toxicity during methylmercury (MeHg) exposure through drinking water (5 mg/L) for two months in mice. MeHg exposure resulted in inflammation and oxidative stress at the colon, as well as an increase in intestinal permeability accompanied by decreased fecal short-chain fatty acids (SCFA). The administration of the strains resulted in a differential protective effect that, based on their chelation capacity, supported the existence of additional mechanisms of action besides chelation. Both strains reduced IL-1ß levels and oxidative stress, while LE1 lowered TNF-α, diminished MeHg-induced mucus over-secretion triggered by the IL-4/IL-13/STAT6 pathway, reduced intestinal permeability, and ameliorated inflammation and oxidative stress, probably by acting on the Keap1/Nrf2/ARE pathway. Administration of LE1 partially restored SCFA contents, which could be partly responsible for the positive effects of this strain in alleviating MeHg toxicity. These results demonstrate that lactobacilli strains can be useful tools in reducing the intestinal toxicity of MeHg, the main mercurial form conveyed by food.

Lactic acid bacteria strains reduce in vitro mercury toxicity on the intestinal mucosa.

A bicameral model consisting of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1-derived macrophages has been used to test the ability of two strains of Lactobacillus to protect from damage caused by mercury. Exposure to 1 mg/ml mercury [Hg(II) or methyl-Hg] for seven days in this model resulted in an inflammatory and pro-oxidant response mainly driven by macrophages. This led to an impairment in the intestinal barrier, defective tight-junctions, increased permeability and mucus hypersecretion. In addition, the wound-healing capacity of the epithelial monolayer was also diminished. However, the presence of heat-killed Lactobacillus intestinalis or Lactobacillus johnsonii cells during Hg exposure reverted these effects, and most of the parameters recovered values similar to control cells. Both lactobacilli showed the capacity to bind Hg(II) and methyl-Hg under the cell culture conditions. This points to Hg sequestration as a likely mechanism that counteracted Hg toxicity. However, differences in the Hg binding capacity and in the effects between both strains suggest that other probiotic-mediated mechanisms may play a role in the alleviation of the damage elicited by Hg. These results show the potential of the bicameral intestinal epithelial model for screening of effective strains for their use in later in vivo studies.

Oral exposure to inorganic mercury or methylmercury elicits distinct pro-inflammatory and pro-oxidant intestinal responses in a mouse model system.

Humans are mainly exposed to mercury (Hg) through contaminated foodstuffs. However, the effects of Hg on the intestinal tract have received little attention. We performed a subchronic exposure to inorganic mercury or methylmercury in mice through drinking water (1, 5 or 10 mg/L for four months) to evaluate their intestinal impact. Histological, biochemical and gene expression analyses showed that both Hg species induced oxidative stress in small intestine and colon, while inflammation was mainly detected in the colon. Increased fecal albumin content indicated a compromised epithelial barrier. Mucus production was possibly also affected, as an increase in Muc2 expression was detected. However, differential effects were detected between both Hg species. Activation of p38 MAPK and increased crypt depth were detected in colon only with MeHg. Minor differences in microbiota composition were detected between unexposed and exposed mice. Although significant differences were detected between both Hg species at 10 mg/L, only the relative abundances of low abundance taxa were affected. Concentrations of microbial-derived short-chain fatty acids were decreased, suggesting an effect on microbial metabolism or increased demand by the intestinal epithelium. Results obtained confirm previous in vitro studies and highlights the intestinal mucosa as an initial target of Hg.

Challenges and strategies for preventing intestinal damage associated to mercury dietary exposure.

Food represents the major risk factor for exposure to mercury in most human populations. Therefore, passage through the gastrointestinal tract plays a fundamental role in its entry into the organism. Despite the intense research carried out on the toxicity of Hg, the effects at the intestinal level have received increased attention only recently. In this review we first provide a critical appraisal of the recent advances on the toxic effects of Hg at the intestinal epithelium. Next, dietary strategies aimed to diminish Hg bioavailability or modulate the epithelial and microbiota responses will be revised. Food components and additives, including probiotics, will be considered. Finally, limitations of current approaches to tackle this problem and future lines of research will be discussed.

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.

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.