Metallothionein determination can be applied to learn about aquatic metal pollution and oxidative stress detoxification mechanisms through Problem-based Learning.

A simple, repeatable and inexpensive laboratory practice applied to teach and discuss aquatic metal pollution and oxidative stress detoxification mechanisms through biomarker analyses, as well as important ecotoxicology concepts, is presented herein. It has been implemented in a university in Brazil to both undergraduate and Master's and PhD students, indicating usefulness to all these levels. Students learned to detect metallothionein and reduced glutathione concentrations in biological samples and investigate several variables of interest in biomonitoring assessments. In addition, statistical correlations were used to indicate the potential dual role played by MT in aquatic organisms, allowing for biological inferences regarding both aquatic metal pollution and oxidative stress detoxification mechanisms and maturing of ecotoxicological and biomonitoring concepts discussed and presented both theoretically and integrated to the laboratory findings.

Ecological Risks of Metal and Metalloid Contamination in the Rio Doce Estuary.

The rupture of a mining dam in southeastern Brazil in 2015 was the country's greatest environmental tragedy. In order to evaluate the ecological risks of the mine tailings on the Rio Doce estuary, this study assessed trace metal contamination and sediment quality indices up to 2.9 y after the dam rupture. Surface sediments were collected from 17 stations on the Rio Doce estuary and Cd, Pb, Cr, Zn, Cu, and As concentrations were determined by inductively coupled plasma atomic emission spectroscopy (ICP-OES). Four ecological risk indices (modified contamination degree [mCd], pollution load index [PLI], risk index [RI], and sediment quality guideline quotient [SQG-Q]) suggest a high risk by metal contamination with possible adverse biological effects, with moderate seasonal variability. Based on a precautionary approach, our results support fisheries closures and the need for public health monitoring in the affected areas, and support other studies that suggest chronic metal contamination of the Rio Doce estuary. Integr Environ Assess Manag 2020;16:655-660. © 2020 SETAC.

A ruptura de uma barragem de mineração no sudeste do Brasil em 2015 causou a maior tragédia ambiental do país. Para avaliar os riscos ecológicos dos rejeitos de minério no estuário do Rio Doce, este estudo avaliou os índices de contaminação por metais traços e qualidade dos sedimentos até 2,9 anos após a ruptura da barragem. Os sedimentos de superfície foram coletados em 17 estações no estuário do Rio Doce e as concentrações de Cd, Pb, Cr, Zn, Cu e As foram determinadas por ICP-OES. Quatro índices de risco ecológico (mCd, PLI, RI e SQG-Q) sugerem um alto risco de contaminação por metais com possíveis efeitos biológicos adversos, com variabilidade sazonal moderada. Com base em uma abordagem preventiva, nossos resultados apoiam o fechamento da pesca e a necessidade de monitoramento da saúde pública nas áreas afetadas, além de apoiar outros estudos que sugerem contaminação crônica por metais no estuário do Rio Doce. Integr Environ Assess Manag 2020;16:655-660.

Role of Sonic hedgehog signaling in cell cycle, oxidative stress, and autophagy of temozolomide resistant glioblastoma.

The first-line chemotherapy treatment for Glioblastoma (GBM) - the most aggressive and frequent brain tumor - is temozolomide (TMZ). The Sonic hedgehog (SHH) pathway is involved with GBM tumorigenesis and TMZ chemoresistance. The role of SHH pathway inhibition in the potentiation of TMZ's effects using T98G, U251, and GBM11 cell lines is investigated herein. The combination of GANT-61 and TMZ over 72 hr suggested a synergistic effect. All TMZ-resistant cell lines displayed a significant decrease in cell viability, increased DNA fragmentation and loss of membrane integrity. For T98G cells, G2 /M arrest was observed, while U251 cells presented a significant increase in reactive oxygen species production and catalase activity. All the cell lines presented acidic vesicles formation correlated to Beclin-1 overexpression. The combined treatment also enhanced GLI1 expression, indicating the presence of select resistant cells. The selective inhibition of the SHH pathway potentiated the cytotoxic effect of TMZ, thus becoming a promising in vitro strategy for GBM treatment.

Proteomic characterization of medicinal plants used in the treatment of diabetes.

Several plants have been studied for their medicinal properties, especially concerning the management of chronic diseases, such as diabetes, aiming at a more accessible form of treatment. In this context, the aim of this study was to characterize plant proteins used in folk medicine as hypoglycemic agents for the treatment of diabetes, namely "abajerú" (Chrysobalanus icaco) and "cow's paw" (Bauhinia forficata and Bauhinia variegata). The species were differentiated by proteome characterization. Proteins were in-solution digested using trypsin by the filter-assisted sample preparation (FASP) method. Peptides were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for protein characterization. In total, 131 proteins were identified. The main biological functions of these proteins were cellular respiration, transport, metabolism and photosynthesis. Insulin-like proteins were not detected, but proteins involved in controlling glucose levels were. The results are of value in the proteomic characterization of phytotherapeutic plants, and may serve as baseline for the assessed species in Brazil, where a lack of knowledge in this regard is observed.

Biliary and hepatic metallothionein, metals and trace elements in environmentally exposed neotropical cichlids Geophagus brasiliensis.

One of the metal detoxifying mechanisms that occurs in fish is metallothionein (MT) induction and metal binding. Hepatic MT induction has been well described, but biliary MT metal detoxification has only recently been described in fish. In this scenario, metal-metal interactions have been increasingly evaluated to further understand the behavior of these contaminants regarding homeostasis and biological functions, as well as their toxic effects. Studies, however, have been mainly conducted concerning the elemental pair Se-Hg, and scarce reports are available concerning other metal pairs. Therefore, this study aimed to evaluate biliary and hepatic MT metal detoxification mechanisms in a territorial neotropical cichlid, Geophagus brasiliensis. Fish were sampled from the anthropogenically impacted estuarine Rodrigo de Freitas Lagoon, located in Southern Rio de Janeiro, and trace elements and MT were determined by inductively coupled plasma mass spectrometry (ICP-MS) and UV-Vis spectrophotometry, respectively, in fish liver and bile. MT in bile were significantly lower than in liver. Significant differences between bile and liver were observed for many trace elements, and, although most were higher in liver, Cd and Ni were significantly higher in bile, indicating efficient excretion from the body via the biliary route. A significant correlation was observed between MT and Fe in bile, and between MT in liver and Cu and Zn in bile. Molar ratio calculations demonstrated protective elements effects against Al, As, Cd, Hg, Pb and V in both bile and liver, as well as some novel interrelationships, indicating the importance of these investigations regarding the elucidation of element detoxifying mechanisms. Furthermore, investigation of other elemental associations may aid in decision-making processes regarding environmental contamination scenarios linked to public health.

Investigating heavy metal bioaccumulation by macrofauna species from different feeding guilds from sandy beaches in Rio de Janeiro, Brazil.

The relationship between metal accumulation and feeding behavior of macrofauna species is a key concept to understand the bioavailability of different metals in the marine environment. We examined and compared the concentrations of eight heavy metals (Cr, Zn, Pb, Ni, Cu, Cd, Co and V) in different feeding guilds of macrofauna species, from a data set including 68 sandy beaches along the Rio de Janeiro coast. For this purpose, macrofauna species were classified in five feeding guild categories: carnivorous, herbivorous, detritivorous, suspensivorous and filter feeders. The coast of Rio de Janeiro was divided into seven regions according to environmental characteristics and historical human activities. For each region, generalized linear models were adjusted to test for differences between feeding guild abundances. Redundancy Analysis was performed to explore the relationship among the feeding guilds composition and the environmental variables. We found high variability in abundance and composition among feeding guilds, linked with environmental heterogeneity. In general, carnivorous species showed a higher heavy metal concentrations compared to other trophic guilds evaluated. However, bioaccumulation across the feeding guild was not the rule and patterns varied across regions. Our hypothesis is that variations are probably related to the different magnitudes of metal contamination along the coast as also in to the trophic structure found in each beach. This data highlighted the crucial role of the relationship between variability of environmental drivers and bioaccumulation in macrofauna species in sandy beaches ecosystem.

Variation in mercury concentration in juvenile Magellanic penguins during their migration path along the Southwest Atlantic Ocean.

The vulnerability of seabirds related to their migratory dynamics is frequently linked to environmental problems along the migration path. In this context, Magellanic penguins (Sphenicus magellanicus) seem to be vulnerable to an extensive range of environmental disturbances during their northward migration along the Atlantic waters of South America, which include by catch, marine debris ingestion, overfishing and environmental contamination. In this study, we investigate mercury accumulation in muscle and hepatic tissues of juveniles penguins collected along the Brazilian coast during three migratory seasonal years (2006, 2008 and 2012) and three areas along a latitudinal gradient. We found significant differences in Hg levels across the years, with higher hepatic Hg levels found in tissues of penguins sampled in 2008. The higher Hg levels in samples of penguins from 2008 might be attributed to variations in body condition or Hg uptake, associated with the trophic imbalance linked to an extreme El Niño event during that year. Significant differences in Hg accumulation across the latitudinal areas were also observed. The penguins sampled at the farthest area from the breeding ground presented the higher levels of Hg and also the poorest body condition. Body condition and other traits may influence the levels of chemical pollutants and decrease the migratory success rate in the juvenile age phase, compromising population dynamics.

The importance of an efficient extraction protocol for the use of fish muscle cholinesterases as biomarkers.

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL-1 NaCl or 1molL-1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed.

A moderate metal-binding hydrazone meets the criteria for a bioinorganic approach towards Parkinson's disease: Therapeutic potential, blood-brain barrier crossing evaluation and preliminary toxicological studies.

Alzheimer's and Parkinson's diseases share similar amyloidogenic mechanisms, in which metal ions might play an important role. In this last neuropathy, misfolding and aggregation of α-synuclein (α-Syn) are crucial pathological events. A moderate metal-binding compound, namely, 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone (INHHQ), which was previously reported as a potential 'Metal-Protein Attenuating Compound' for Alzheimer's treatment, is well-tolerated by healthy Wistar rats and does not alter their major organ weights, as well as the tissues' reduced glutathione and biometal levels, at a concentration of 200mgkg-1. INHHQ definitively crosses the blood-brain barrier and can be detected in the brain of rats so late as 24h after intraperitoneal administration. After 48h, brain clearance is complete. INHHQ is able to disrupt, in vitro, anomalous copper-α-Syn interactions, through a mechanism probably involving metal ions sequestering. This compound is non-toxic to H4 (human neuroglioma) cells and partially inhibits intracellular α-Syn oligomerization. INHHQ, thus, shows definite potential as a therapeutic agent against Parkinson's as well.

Heavy metal contamination in sandy beach macrofauna communities from the Rio de Janeiro coast, Southeastern Brazil.

We evaluated concentrations of eight heavy metals Cr, Zn, Pb, Ni, Cu, Cd, Co and V, in tissues of representative macrofauna species from 68 sandy beaches from the coast of Rio de Janeiro state. The links between contamination levels and community descriptors such as diversity, evenness, density and biomass, were also investigated. Metal concentrations from macrofaunal tissues were compared to maximum permissible limits for human ingestion stipulated by the Brazilian regulatory agency (ANVISA). Generalized linear models (GLM's) were used to investigate the variability in macrofauna density, richness, eveness and biomass in the seven different regions. A non-metric multidimensional scaling analysis (n-MDS) was used to investigate the spatial pattern of heavy metal concentrations along the seven regions of Rio de Janeiro coast. Variation partitioning was applied to evaluate the variance in the community assemblage explained by the environmental variables and the heavy metal concentrations. Our data suggested high spatial variation in the concentration of heavy metals in macrofauna species from the beaches of Rio de Janeiro. This result highlighted a diffuse source of contamination along the coast. Most of the metals concentrations were under the limits established by ANVISA. The variability in community descriptors was related to morphodynamic variables, but not with metal contamination values, indicating the lack of direct relationships at the community level. Concentration levels of eight heavy metals in macrofauna species from 68 sandy beaches on Rio de Janeiro coast (Brazil) were spatially correlated with anthropogenic activities such as industrialization and urbanization.

Mercury, selenium and stable isotopes in four small cetaceans from the Southeastern Brazilian coast: Influence of feeding strategy.

Dolphins are good bioindicators of the contamination status of marine ecosystems, since their dietary and habitat plasticity in both coastal and offshore ecotypes provide information on the trace elements levels originated from natural and anthropogenic sources. In this context, this study aimed to investigate provides mercury (Hg), selenium (Se) levels, trophic ecology and feeding environments of four small cetaceans (Tursiops truncatus, Steno bredanensis, Sotalia guianensis and Pontoporia blainvillei) inhabiting the central-northern coast of Rio de Janeiro State, southeastern Brazil. For the latter, δ15N and δ13C stable isotopes were used as indicators in this regard. Stable isotope values indicate that the four studied species have distinctive foraging habitats, coastal and least coastal, and occupy different trophic positions. The significant relationship found between muscle Hg and δ15N suggests that individual foraging preference remains relatively constant for the studied dolphin species over extended periods. The individual prey size and species are probably responsible for the differences found in Hg and Se concentrations in muscle tissue among all dolphin species. The vulnerable small coastal cetacean, P. blainvillei, which feeds on small teleost fish and squid, presented the lowest muscular Hg concentrations (less than 3.5 µg g-1 dry wt.). Meanwhile, S. bredanensis is more likely to uptake large amounts of trace elements among the four dolphin species, due to its feeding habits mainly being large offshore fish that accumulate high amounts of trace elements in organs and tissues. Differences found between Hg concentrations in fetus-mother pairs were much higher in S. guianensis than in P. blainvillei, suggesting that maternal contribution of Hg via placenta was more significant for the former.

Mercury species, selenium, metallothioneins and glutathione in two dolphins from the southeastern Brazilian coast: Mercury detoxification and physiological differences in diving capacity.

In the present study, the concentration of trace elements, total mercury (Hg) and selenium (Se) and mercury forms (MeHg, Hginorg and HgSe) in the vulnerable coastal dolphins Pontoporia blainvillei and Sotalia guianensis were appraised and compared, using metallothioneins (MT) and glutathione (GSH) as biomarkers for trace element exposure. The trace element concentrations varied between muscle and liver tissues, with liver of all dolphin specimens showing higher Hg and Se concentrations than those found in muscle. Hg, MeHg and Hginorg molar concentrations showed a clear increase with Se molar concentrations in the liver of both dolphins, and Se concentrations were higher than those of Hg on a molar basis. Se plays a relevant role in the detoxification of MeHg in the hepatic tissue of both dolphins, forming Hg-Se amorphous crystals in liver. In contrast, MT were involved in the detoxification process of Hginorg in liver. GSH levels in P. blainvillei and S. guianensis muscle tissue suggest that these dolphins have different diving capacities. Muscle Hg concentrations were associated to this tripeptide, which protects dolphin cells against Hg stress.

Differential metallothionein, reduced glutathione and metal levels in Perna perna mussels in two environmentally impacted tropical bays in southeastern Brazil.

Mussel farming is an important economic activity in Brazil, and these organisms are consumed by the majority of the population in most coastal zones in the country. However, despite the increasing pollution of aquatic ecosystems in Brazil, little is known about the biochemical activity in mussels in response to metal exposure. In this context, the aim of the present study was to investigate metal and metalloid exposure effects in Perna perna mussels, by determining metal levels, the induction of metallothionein (MT) synthesis, and oxidative stress, in the form of reduced glutathione (GSH) in 3 contaminated areas from the Guanabara Bay in comparison to a reference site, Ilha Grande Bay, both in summer and winter. Metal and metalloid concentrations were also compared to Brazilian and international guidelines, to verify potential health risks to human consumers. Mussels from all sampling sites were shown to be improper for human consumption due to metal contamination, including Ilha Grande Bay, which has previously been considered a reference site. Several statistically significant correlations and seasonal differences were observed between MT, GSH and metals and metalloids in both analyzed tissues. A Discriminant Canonical Analysis indicated that the digestive gland is a better bioindicator for environmental contamination by metals and metalloids in this species and offers further proof that MT variations observed are due to metal exposure and not oxidative stress, since GSH influence for both muscle tissue and the digestive glands was non-significant in this analysis. These results show that P. perna mussels are an adequate sentinel species for metal contamination with significant effects on oxidative stress and metal exposure biomarkers. To the best of our knowledge, this is the first study to report metals, metalloids, MT and GSH levels in the muscle tissue of this species.

Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast.

Magellanic penguins have been reported as good biomonitors for several types of pollutants, including trace-elements. In this context, selenium (Se), total mercury, methylmercury, inorganic mercury (Hg(inorg)), cadmium (Cd) and lead (Pb), as well as metallothionein (MT) levels, were evaluated in the feathers, liver and kidney of juvenile Magellanic penguins found stranded along the coast of Southern Brazil. The highest concentrations of all trace-elements and methylmercury were found in internal organs. Concentrations of Cd and Se in feathers were extremely low in comparison with their concentrations in soft tissues. The results showed that both Se and MT are involved in the detoxification of trace-elements (Cd, Pb and Hg(inorg)) since statistically significant relationships were found in liver. Conversely, hepatic Se was shown to be the only detoxifying agent for methylmercury.

Bile and liver metallothionein behavior in copper-exposed fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.