V. Devesa

Accumulation of heavy metals and As in wetland birds in the area around Doñana National Park affected by the Aznalcollar toxic spill

The impact of the spill from the mine in Aznalcollar (Seville, Spain) on waterfowl in the Doñana National Park is assessed. The concentrations of Cu, Pb, Cd, Zn nd As in the liver and eggs of 16 species of waterfowl found dead in the Park between April and November 1998 were determined. The highest levels were found for Zn, followed by Cu, Pb, Cd and As. The main parameters related to the accumulation of these elements in the waterfowl studied were species and trophic level. The other variables studied--distance from the spill, days of exposure, sex, size, and age--are important, although this depends on the element studied. Zn and Cu from the spill have entered the food chain of the aquatic birds studied, but Cd, Pb and As have not. There is currently no evidence to suggest that the trace element concentrations measured have reached toxic levels.

Trace elements in blood collected from birds feeding in the area around Donana National Park affected by the toxic spill from the Aznalcollar mine

A long-term monitoring plan was established to study if bird populations around Doñana National Park were affected by the toxic spill from the Aznalcóllar mine. The concentrations of Zn, Pb, As, Cu, Sb, Co, Tl and Cd in the blood of 11 bird species feeding in the area were determined. The parameters which most affect the accumulation of trace elements in the birds studied are, firstly, species and secondly, trophic position, sex, days of exposure and weight. In some individuals, Zn and Cu occurred at higher levels than the reference values for contaminated areas. Concentrations of Pb and Cd in a considerable number of individuals were higher than those found in birds from uncontaminated areas. The present data, together with the lack of data on blood metal concentration prior to the spill, do not offer any conclusive evidence of the influence of the spill on avian blood metal concentrations.

Total and inorganic arsenic in the fauna of the Guadalquivir estuary: environmental and human health implications.

To evaluate the impact on fauna of the release of toxic waste from the tailings dam operated by the Boliden Apirsa S.L company at Aznalcóllar, Seville (Spain) a study was carried out of total and inorganic arsenic contents in 164 samples from six different estuary species, including molluscs, crustaceans and fish, collected at six sampling stations distributed along the estuary and mouth of the River Guadalquivir. The contents found, expressed in micrograms per gram wet weight, were as follows. Total arsenic: Crassostrea angulata--giant cupped oyster (2.44 +/- 0.45); Scrobicularia plana--peppery furrow (2.50 +/- 0.73); Palaemon longirostris--delta prawn (1.33 +/- 0.54); Uca tangeri--AfroEuropean fiddler crab (1.76 +/- 0.08); Melicertus kerathurus--shrimp (3.60 +/- 1.92); and Liza ramada--mullet (0.65 +/- 0.38). Inorganic arsenic: C. angulata (0.09 +/- 0.02); S. plana (0.38 +/- 0.23); P. longirostris (0.04 +/- 0.01); U. tangeri (0.22 +/- 0.03); M. kerathurus (0.03 +/- 0.01); and L. ramada (0.03 +/- 0.03). The levels of total As are comparable to those obtained by other authors. With respect to inorganic arsenic, only S. plana and U. tangeri present high levels of inorganic arsenic. This may be due to the fact that these organisms live in estuary sediments, reservoirs of inorganic arsenic, and ingest particles of sediments during feeding. Because of the lack of information for this area concerning previous levels of total and inorganic arsenic in the species analysed, it was not possible to establish the impact on the fauna of the River Guadalquivir estuary of the toxic spill resulting from the failure of the mine tailings dam at Aznalcóllar. With respect to the implications to human health as a result of consumption of species from the Guadalquivir estuary, only with the species Scrobicularia plana, as a high consumption of this mollusc might, in some cases, exceed the maximum tolerable intake for inorganic arsenic indicated by the FAO/WHO. Consumption of the liver of L. ramada does not appear to present problems to human health.

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%).

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.

Arsenic and fluoride induce neural progenitor cell apoptosis

The aim of the present study is to determine the effect of inorganic arsenic (As) and its metabolites on the viability of the neural progenitor cell (NPC) line C17.2, in order to evaluate cellular mechanisms involved in As developmental neurotoxicity. Moreover, we analyzed the effects of the coexposure to As and fluoride (F), a situation to which some populations are commonly exposed. Our results show that NPCs are not susceptible to pentavalent As species [arsenate, monomethylarsonic acid, and dimethylarsinic acid] and F alone. However, the trivalent metabolites of arsenate [arsenite, monomethylarsonous acid, and dimethylarsinous acid] are toxic at concentrations below 1 mg/l, and this susceptibility increases when there is coexposure with F (≥ 5 mg/l). Arsenite triggers apoptosis after 24 h of exposure, whereas monomethylarsonous acid produces necrosis at very short times (2 h). Arsenite leads to an increase in intracellular Ca levels and generation of reactive oxygen species, which may cause a decrease in mitochondrial transmembrane potential, release of cytochrome c, and consequent activation of caspases. A slight activation of calpain also takes place, which might favor activation of the mitochondrial pathway or might activate other pathways. The treatment with some antioxidants such as quercetin and α-tocopherol shows only a partial reduction of the cytotoxicity.

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.

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.

Intestinal transport of methylmercury and inorganic mercury in various models of Caco-2 and HT29-MTX cells.

Food is the main pathway of exposure to mercury for most of the population. In food, mercury is generally present as inorganic mercury [Hg(II)] or methylmercury [MeHg]. Both chemical forms have some degree of toxicity, especially MeHg, which is considered a powerful neurotoxicant during development and is classified as a possible human carcinogen. Since the main exposure pathway is oral, gastrointestinal absorption is a decisive step in the process by which mercury reaches the systemic circulation. However, there are few studies that characterize this absorption process. The present work evaluates transport and cellular retention of Hg(II) and MeHg, using various models of the intestinal epithelium (Caco-2 monocultures and Caco-2/HT29-MTX co-cultures in various proportions). Additionally, a study was made of the influence of the mucus secreted by HT29-MTX cells and of substances normally present in the gastrointestinal tract (l-cysteine, bile salts and food components) on mercury transport and accumulation. The results show that incorporation of HT29-MTX reduces the permeability coefficient of Hg(II) and MeHg. This decrease coincides with an increase in cellular accumulation, since mercury is retained in the layer of mucus secreted by HT29-MTX cells [Hg(II): 40%; MeHg: 70%]. The presence of l-cysteine, bile salts and food matrix components increases the percentage of both species that is not absorbed. It is noteworthy that in all the conditions assayed the intracellular accumulation of mercury was very high (37-77%). This study shows the importance of the cell model and assay conditions for an in vitro evaluation of intestinal transport of mercury species.