João Canário

Mercury and selenium interaction in vivo: Effects on thioredoxin reductase and glutathione peroxidase

Mercury compounds exert toxic effects via interaction with many vital enzymes involved in antioxidant regulation, such as selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). Selenium supplementation can reactivate the mercury-inhibited TrxR and recover the cell viability in vitro. To gain an insight on how selenium supplementation affects mercury toxicity in vertebrates, we investigated the effects of selenium on the mercury accumulation and TrxR and GPx activities in a fish model. Juvenile zebra-seabreams were exposed either to methylmercury (MeHg) or inorganic mercury (Hg(2+)) in the presence or absence of sodium selenite (Se) for 28 days followed by 14 days of depuration. Mercury accumulation was found to be 10-fold higher under MeHg exposure than under Hg(2+) exposure. Selenium supplementation caused a half decrease of the accumulation of MeHg but did not influence Hg(2+) accumulation. Exposure to both mercurials led to a decrease of the activity of TrxR (<50% of control) in all organs. Se supplementation coincident with Hg(2+) exposure protected the thioredoxin system in fish liver. However, supplementation of Se during the depuration phase had no effects. The activity of GPx was only affected in the brain of fishes upon the exposure to MeHg and coexposure to MeHg and Se. Selenium supplementation has a limited capacity to prevent mercury effects in brain and kidney. These results demonstrate that Se supplementation plays a protective role in a tissue-specific manner and also highlight the importance of TrxR as a main target for mercurials in vivo.

Inhibition of the thioredoxin system in the brain and liver of zebra-seabreams exposed to waterborne methylmercury.

Mercury compounds were recently found to interact in vitro with the thioredoxin system, inhibiting both Thioredoxin (Trx) and Thioredoxin reductase (TrxR). In order to evaluate if Trx and TrxR are affected in vivo by methylmercury (MeHg), we exposed juvenile zebra-seabreams to different concentrations of this toxicant in water for 28days followed by a 14-day depuration period. Methylmercury accumulated to a larger extent in the kidney and liver of fishes, but decreased significantly during the depuration. During the exposure, MeHg percentage in the liver reached levels above 90% of total mercury (HgT) decreasing to 60% of HgT by the end of the depuration period. In the kidney, MeHg accounted for 50-70% of HgT. In the brain and muscle, mercury accumulated throughout the exposure with all mercury being MeHg. The total mercury kept increasing in these organs during the depuration period. However, in the brain, this increase in HgT was accompanied by a decrease in the MeHg percentage (~10%). In the liver, both Trx and TrxR activities were significantly reduced (TrxR--40%; Trx--70%) by the end of the exposure, but recovered to control levels (100%) during the depuration. In the brain, both enzymes where inhibited during the depuration period (TrxR--75%; Trx--70%) when some production of inorganic mercury was detected. Activity of glutathione reductase showed increased levels when TrxR activity was low, suggesting complementarity between both systems. These results indicate that in vivo the thioredoxin system is a toxicological target for MeHg with TrxR being particularly affected.

Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining (1)H NMR metabolomics and conventional biochemical assays.

Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg toxicity in gills of wild fish, providing new insights into the toxicological pathways underlying the oxidative stress.

Mercury in contaminated sediments and pore waters enriched in sulphate (Tagus Estuary, Portugal)

Three sediment cores, collected nearby the effluent of a chlor-alkali industry, were sliced in 0.5-cm layers and centrifuged for pore water extraction. Mercury, Fe and Mn were determined in the solids as total concentration, hydroxylamine extractable fraction and HCl extractable fraction. Sulphur was determined in the HCl extraction. Total and reactive mercury, chlorinity, S(2-), SO(4)(2-), total Fe, and total Mn were measured in pore waters. The solids contained 3.0-60 nmol g(-1) of total Hg and pore waters 70-5800 pM of total Hg and 1.8-76 pM of reactive mercury. Pore waters presented 2.3-94 times more sulphate than the overlying estuarine waters due to the input from the industry. In layers where hydroxylamine extractable Fe exhibited a broad maximum (precipitation of Fe-oxides) sulphate was reduced to S(2-). The competition between the high content of SO(4)(2-) and Fe(III) as electron acceptors, in chemical reactions occurring in the upper sediments, may explain the co-existence of S(2-) and Fe-oxides in the same layers. Mercury was detected in the hydroxylamine extracts (20-29 nmolg(-1)) in the layers where Fe-oxides were formed, and reactive dissolved Hg showed minimum concentrations. The excess of sulphate in pore waters favoured the abundant Fe-oxides in the upper solid sediments, which appear to work as a barrier limiting the escape of mercury to the water column.

Mercury in sediments and vegetation in a moderately contaminated salt marsh (Tagus Estuary, Portugal).

Depth variations of total mercury (Hg) and methylmercury (MeHg) concentrations were studied in cores from non-colonized sediments, sediments colonized by Halimione portulacoides, Sarcocorniafruticosa and Spartina maritima and belowground biomass, in a moderately contaminated salt marsh (Tagus Estuary, Portugal). Concentrations in belowground biomass exceeded up to 3 (Hg) and 15 (MeHg) times the levels in sediments, and up to 198 (Hg) and 308 (MeHg) times those found in aboveground parts. Methylmercury in colonized sediments reached 3% of the total Hg, 50 times above the maximum values found in non-colonized sediments. The absence of correlations between total Hg concentrations in sediments and the corresponding MeHg levels suggested that methylation was only dependent on the environmental and microbiological factors. The analysis of belowground biomass at high-depth resolution (2 cm) provided evidence that Hg and MeHg were actively absorbed from sediments, with higher enrichment factors at layers where higher microbial activity was probably occurring. The results obtained in this study indicated that the biotransformation of Hg to the toxic MeHg could increase the toxicity of plant-colonized sediments.

The relevance of defining trace metal baselines in coastal waters at a regional scale: the case of the Portuguese coast (SW Europe).

The Water Framework Directives aims a reduction in concentration of hazardous substances in the marine environment. Consequently, there is a need to distinguish between anthropogenically influenced metal concentrations from natural background levels. To better achieve this goal in the Portuguese coast, dissolved and particulate trace metal (TM) concentrations along the Portuguese coast were determined in 46 sites distance 1-3 km from the shoreline. Dissolved values ranged within the following intervals: 0.01-0.89 nM for Cd, 0.01-3.37 nM for Co, 0.90-45.4 nM for Cu, 3.30-140 pM for Hg, 1.88-15.1 nM for Ni, 0.01-0.15 nM for Pb and 1.40-62.0 nM for Zn. Whereas Cd, Co, Cu, Ni and Zn were enhanced in the southern coast, while Pb values were higher in the central part of the western coast. Mercury concentrations showed punctual increases all along the coast. Values of trace metals in suspended particulate matter varied in a broad range: 36-2902 μmol g(-1) for Al, 0.10-15.1 nmol g(-1) for Cd, 1.50-165 nmol g(-1) for Co, 50.0-990 nmol g(-1) for Cu, 2.80-76.4 nmol g(-1) for Hg, 22-1471 nmol g(-1) for Ni, 10.0-347 nmol g(-1) for Pb and 416-10,981 nmol g(-1) for Zn. Higher values for Al, Ni and Co were found in the central part of the western coast. However, Cd, Cu, Pb and Zn increased their levels from the north coast towards the central and south areas. The variability of both dissolved and particulate metals appears to be mainly associated with oceanographic conditions and continental inputs at North and central areas of the coast, and in the south coast to geological features rather than to anthropogenic pressures. On the basis of these results, regional baseline concentrations are proposed for the three typologies in Portuguese coastal waters defined under the Water Framework Directive.

Pathways and speciation of mercury in the environmental compartments of Deception Island, Antarctica.

This work reports the first integrated mercury study in an Antarctic ecosystem. Sample collection took place in Deception Island, an active volcano in the South Shetland Islands, in several environmental compartments (water, snow, sediments and vegetation) and different locations, during December 2011. The results suggest that volcanic activity is the most important Hg source. Mercury levels in water and sediments sampled at two fumaroles were up to 10,000 times higher than in the other sampling sites. Dissolved methylmercury (MeHg) is below the detection limit in those samples, probably due to the very high temperature found in fumaroles (above 80 °C). On the other hand MeHg accounted for, on average, 23% of total dissolved Hg in the saline waters of Foster bay, which suggests exceptional conditions for Hg methylation. Combined with the high residence time of the water in Foster bay, the results point to the existence of a MeHg pool available for aquatic living organisms.

Mercury and methylmercury bioaccumulation by polychaete worms is governed by both feeding ecology and mercury bioavailability in coastal mudflats

Polychaete worms are abundant in many mudflats but their importance to coastal food web Hg biomagnification is not known. We sampled sediments and polychaete worms from mudflats in the Bay of Fundy to investigate the bioaccumulation of mercury (Hg) and methylmercury (MeHg) in the coastal invertebrate food web. Hg concentrations in the sediments were low (<20 μg kg(-1)). Labile Hg (methanol/KOH sediment extraction) in surface sediments (0-1 cm) was positively correlated with Hg bioaccumulation by surface sediment-ingesting polychaetes but, surprisingly, there was a negative correlation between δ(15)N (i.e. trophic level) and THg bioaccumulation factors in polychaete worms. Worms feeding on deeper sediments contained the greatest MeHg concentrations (69.6 μg kg(-1)). Polychaetes are an important vector for Hg biomagnification to the coastal avian food web. This research demonstrates that feeding depth and method of feeding are more important than trophic position or sediment Hg concentrations for predicting Hg bioaccumulation.

Assessing trace element contamination in Fildes Peninsula (King George Island) and Ardley Island, Antarctic

King George Island, situated in the South Shetland Islands archipelago, is one of the most visited sites in Antarctica. This has contributed to a high density of scientific stations and shelters in the region, especially in Fildes Peninsula. In order to evaluate the natural and anthropogenic sources of trace elements (As, Cd, Cu, Zn, Pb and Hg) soil and moss samples were collected from different sites in January 2013. In general, the results revealed homogeneous concentrations (μgg(-)(1)) for each element in the majority of collected samples (As: 3.8±1.4; Cd: 0.4±0.9; Cu: 34±4; Zn: 115±13; Pb: 20±5; Hg; 0.011±0.009). However, some samples in specific areas of Fildes Bay showed the existence of local anthropogenic activities that have contributed to the enrichment of contaminants in soils and moss samples that correlated to one another (e.g. Pb: 1101μgg(-)(1)). Human presence is linked to examples of contamination and environmental perturbation, making essential the implementation of this type of study in order to understand and protect unique places in Antarctica.

Fish eyes and brain as primary targets for mercury accumulation — A new insight on environmental risk assessment

Fish eyes and brain are highly susceptible to environmental Hg exposure but this issue is still scarcely investigated, mainly regarding methylmercury (MeHg) accumulation. Yet, Hg levels in fish lens have not been previously examined under field conditions. Total Hg (tHg), MeHg and inorganic Hg (iHg) levels were assessed in the brain, eye wall and lens of the golden grey mullet (Liza aurata) from an Hg contaminated area, both in winter and summer, together with water and sediment levels. Sampling was performed at Aveiro lagoon (Portugal) where a confined area (LAR) is severely contaminated by Hg. Fish brain, eye wall and lens accumulated higher levels of tHg, MeHg and iHg at LAR than the reference site, reflecting faithfully environmental spatial differences. The brain and eye wall responded also to the winter-summer changes found in water and sediment, accumulating higher levels of MeHg (and tHg) in winter. Contrarily, lens was unable to reflect seasonal changes, probably due to its composition and structural stability over time. The three neurosensory structures accumulated preferentially MeHg than iHg (MeHg was higher than 77% of tHg). Lens exhibited a higher retention capacity of MeHg (mean around 1 μg g(-1) at LAR), accumulating higher levels than the other two tissues. Interestingly, MeHg and iHg levels were significantly correlated for the brain and eye wall but poorly associated within the two analysed eye components. The high levels of MeHg found in the brain, eye wall and lens could compromise their functions and this needs further research.