Estuarine fish assemblages present a species-specific difference in the multixenobiotics resistance activity.

We investigated the activity of the multixenobiotic resistance (MXR) phenotype, a biological defense system in aquatic organisms, in the fish assemblages of two tropical estuaries with different degrees of environmental impacts, the Paraiba River and Mamanguape River Estuaries. The aim of this work was to compare the activity of the MXR phenotype of different fishes to test the hypothesis that each species has an inherent activity level and to use this activity as a bioindicator of aquatic contamination. We assessed the MXR activity of the gills, using rhodamine B (RB) accumulation assay. The results demonstrated a species-specific difference in the MXR activity of fishes caught in the same estuarine zone. Also, the pelagic species Eucinostomus melanopterus, Eucinostomus argenteus, and Lutjanus jocu had higher RB accumulation, while the demersal species Sphoeroides testudineus and Sphoeroides greeleyi had the lowest RB accumulation, suggesting that the ecological characteristic of fish in the water column exerts an influence on MXR activity. Besides, we demonstrated the potential of using the gill MXR activity of the key estuarine species, the Brazilian silversides Atherinella brasiliensis, as a tool for biomonitoring estuaries.

Tolerance of native and invasive bivalves under herbicide and metal contamination: an ex vivo approach.

The literature indicates that exotic species have a greater tolerance to environmental stressors compared with native species. In recent decades, the introduction of contaminants into the environment has increased as a result of industrialization. The objective of this study was to verify the resistance of bivalve mollusks from freshwater native (Anodontites trapesialis) and exotic (Limnoperna fortunei) species to chemical contamination using an ex vivo/in vitro approach. Gill and muscle tissues were exposed to two different types of environmental stressors, copper (metal), and Roundup Transorb® (herbicide). The tissues were submitted to a cytotoxicity test in which the lysosomal integrity was assessed, from the adaptation of a method to isolated cells, and multixenobiotic resistance (MXR) test which evaluated cellular defense. In the exotic species, only copper at 9000 µg/L and Roundup Transorb® at 5000 µg/L were cytotoxic. In the native species, copper cytotoxicity at 900 and 9000 µg/L and Roundup Transorb® at 50 and 5000 µg/L were observed. Results were the same in both tissues. The MXR, responsible for the extrusion of contaminants (cell defense), was inhibited in both species when exposed to the contaminants, this cell defense system seems to be more inhibited in the native species, when exposed to both pollutants, indicating greater sensitivity. Therefore, cytotoxicity may be related to the lack of capacity of cellular defense. In relation to lysosomal integrity, the native species was more sensitive to cytotoxic pollutants, where a greater number of experimental conditions of metals and herbicide showed cytotoxicity, as well as more experimental situations inhibited its ability to defend itself.

Differential activities of the multixenobiotic resistance mechanism in freshwater fishes inhabiting environments of Patagonia Argentina.

Environmental impairment resulted from urbanizations can produce damage on freshwater species including strong physiological effects at individual or population level. The multixenobiotic resistance (MXR) is a defence mechanism which has been demonstrated in several aquatic organisms. The key mediators of MXR activity are ATP-binding cassette (ABC) proteins like P-glycoprotein (P-gp). This system protects aquatic organisms against the accumulation of xenobiotics by extruding them from cells in an energy-dependent manner. MXR has been pointed out as relevant in the ecotoxicological context and has been proposed as a biomarker for pollution assessment. Since fish species are common target in freshwater biomonitoring programs, the purpose of the study was to evaluate the MXR mechanism in native Hatcheria macraei (Patagonian catfish) and exotics Salmo trutta (brown trout), Oncorhynchus mykiss (rainbow trout) and Oncorhynchus tshawytscha (Chinook salmon) freshwater fishes widespread in Argentine Patagonia. We characterized the MXR mechanism using a combination of functional assays and Western blot analysis. Our results in different tissues such as liver, gills, muscle and epidermis indicate that the fishes studied have different species-specific levels of MXR activity, being gills and liver the tissues with greater detoxifying activity. Induction of MXR transport activity was also identified in liver tissue from rainbow trout from urban stream suggesting their suitability in the biomonitoring of aquatic environments subjected to urban contaminants.

Capacity of tissue water regulation is impaired in an osmoconformer living in impacted estuaries?

Estuarine osmoconformes rely on their ability to perform tissue and cell water regulation to cope with daily osmotic challenges that occur in the estuary. In addition, these animals currently must deal with pollutants present in the estuarine environment, which can disturb their capacity of water regulation. We collected the mangrove oyster Crassostrea rhizophorae in two tropical estuaries in the Northeast region of Brazil with different degrees of human interference: the Paraíba Estuary (impacted) and the Mamanguape Estuary (preserved). Tissue water content was analyzed after exposure to salinities 12, 24 and 36 for 24 h. Gill cell volume regulation was analyzed in vitro upon hypo- and hyper-osmotic conditions. We also analyzed gill MXR (multi-xenobiotic resistance) mechanism, as reference of environmental pollution. Gill and muscle of oysters from two sites of Paraíba Estuary, and from one site of Mamanguape Estuary were not able to maintain tissue water content upon hypo- and hyper-osmotic conditions. Gill cells of oyster from the same sites exhibited swelling followed by regulatory volume decrease upon hypo-osmotic condition. Gill MXR activity was increased in oysters from these sites. The best tissue and cell water regulation, and the lowest MXR activity, was found in oyster from downstream of Mamanguape Estuary, our reference site and the one most preserved. Tissue and cell water regulation proved to be a sensitive parameter to environmental pollution and could be considered as biomarker of aquatic contamination.

Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype.

In order to assess the impact of nanoplastics on marine species, polystyrene nanoparticles (PS NPs) have been largely used as model particles. Here we studied the effects of 50 nm amino-modified PS-NH2 on Mediterranean sea urchin Paracentrotus lividus immune system cells (coelomocytes) in the presence of celomic fluid (CF) and at different NP concentrations (1, 5, 10, and 25 µg mL-1) and experimental conditions (absence or presence of EDTA). PS-NH2 acquired a protein corona once incubated with CF, dominated by the toposome precursor protein (TPP). In short-term cultures, a significant concentration- and time-dependent decrease in lysosomal membrane stability and apoptotic-like nuclear alterations were observed in phagocytes upon exposure to PS-NH2 (10 and 25 µg mL-1) in CF but they resulted abolished in the presence of EDTA confirming the role of TPP in triggering PS-NH2-coelomocytes interaction and toxicity. PS-NH2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH2 to affect pump's efflux activity. Overall results encourage additional studies on positively charged nanoplastics, since the observed effects on sea urchin coelomocytes as well as the TPP corona formation might represent a first step for addressing their impact on sensitive marine species.

Influence of Water Temperature on the MXR Activity and P-glycoprotein Expression in the Freshwater Snail, Physa acuta (Draparnaud, 1805).

Cristina N. Horak and Yanina A. Assef (2017) P-glycoprotein (P-gp) mediated multixenobiotic resistance (MXR) is a mechanism analogous to multidrug resistance, which has been extensively characterized in mammalian tumours. The expression and function of the MXR mechanism has been demonstrated in numerous aquatic organisms and has been proposed as a biomarker for pollution assessment. A close relationship between thermal stress and MXR response has been reported in some aquatic organisms. Seasonal studies in freshwater organisms are scarce and conducted mainly in zebra mussel (Dreissena polymorpha), whose presence has not been reported in South America. The general purpose of the present study was to evaluate seasonal variation of a biomarker, the MXR mechanism, in the worldwide distributed freshwater snail P. acuta. We analyzed the in situ influence of temperature on the biomarker response over an 18-month field study. MXR defence system was evaluated by a combination of functional assays (RB accumulation) and molecular approaches to analyse P-gp expression. The results demonstrated a linear correlation between MXR response, at activity and expression level, and water temperature at sample site, in P. acuta snails. The characterization of the MXR system in worldwide distributed species, including the study of their seasonal fluctuations, could contribute to the increasing interest to incorporate this biomarker to provide an integrated assessment of mussel health status. This work supports the possible use of P. acuta snails with this purpose and also highlights that the occurrence of variations in MXR response related to water temperature has to be taken into account in the interpretation of in situ monitoring studies.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells.

The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes.

Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers.

In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

Genotoxic and biochemical effects of atrazine and Roundup(®), alone and in combination, on the Asian clam Corbicula fluminea.

The present study aimed to evaluate biochemical and genotoxic effects of the herbicides atrazine (ATZ) and Roundup(®) (RD) separately, as well as their mixture, on the freshwater clam Corbicula fluminea after 96 h exposure. Animals were exposed to 2 and 10 ppb of ATZ (ATZ2 and ATZ10), 2 and 10 ppm of RD (RD2 and RD10) and the following mixtures: 2 ppb ATZ+2ppm RD (AR2) and 10 ppb ATZ+10 ppm RD (AR10). Activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the multixenobiotic resistance mechanism (MXR), reduced glutathione concentrations (GSH) and lipid peroxidation (LPO) were measured in gills and digestive gland. DNA damage was determined in clams hemocytes through the comet assay. The gills were more susceptible to the action of the herbicides and the results showed that ATZ2 and ATZ10 caused a significant reduction in EROD and the mixture leads to a significant decrease in EROD and MXR. No significant change in the biotransformation parameters was observed in the digestive gland. Regarding the primary antioxidant defenses, SOD activity increased in the gills of clams exposed to ATZ10 and RD10 and in the digestive gland of animals exposed to RD2 and RD10, CAT activity was significantly reduced only in digestive gland of clams exposed RD10 while GPX increased in the gills after exposure to ATZ2 and RD10. The exposure to RD10 caused a significant increase in LPO in both gills and digestive gland. While the exposure to ATZ and RD separately did not increase DNA damage, the exposure to AR2 and AR10 caused a significant increase in the occurrence of DNA damage. In conclusion, this study showed that both herbicides applied alone caused effects on C. fluminea; ATZ interfered mostly in biotransformation while RD interfered mainly in antioxidant defenses leading to lipid peroxidation. The herbicides mixture showed antagonistic effects on the gills EROD and on lipid peroxidation in gills and digestive gland and synergistic effects on the gills MXR and on DNA damage in the hemocytes.

Cell damage induced by copper: an explant model to study anemone cells.

Sea anemones are benthic organisms, of low mobility and can be directly affected by water pollution. This work studied the defense mechanisms and DNA damage caused by copper toxicity in cells from the anemone Bunodosoma cangicum. For this, exposure of anemones cells were held, kept in primary culture through explant of podal disk to copper (7.8 and 15.6 µg/L), and the control group, for 6 and 24h. Cytotoxicity was seen through the viability and cell number, MXR phenotype through the accumulation of rhodamine-B, ROS generation by H2DCF-DA and DNA damage by comet assay. The results obtained show that there is a drop in viability and number of cells, especially after exposure of 24h in 15.6 µg/L. There is an induction of the MXR activity only at 7.8 µg/L for 24h. As for ROS, there is an increase in the generation of reactive species in greatest concentration of copper for 6h, and in both for 24h, which leads to oxidative stress, which culminates with a DNA damage. What was evidenced by the increase of the tail size, % DNA presented and moment of tail. Therefore, the copper represents an adversity to the anemones cells, being cytotoxic and genotoxic.

Variability of filtration and food assimilation rates, respiratory activity and multixenobiotic resistance (MXR) mechanism in the mussel Perna perna under lead influence

The economic importance that myticulture is conquering in Santa Catarina State (South of Brazil) explains the crescent search for new coastal sites for farming. Physiological and biochemical studies of the mussel Perna perna are important to the establishment of methodologies for program assessment and environmental monitoring, allowing to infer about site quality and possible influences of xenobiotic agents on coastal areas. In order to evaluate effects caused by lead poisoning (1.21 mumol.L-1), the mussels were maintained at constant temperature (25ºC) and fed with Chaetoceros gracilis for 15 days. The control group was acclimatized in sea water 30‰. At the end of this period time, physiological measurements were carried out along with statistic analysis for filtration rates, lead assimilation and overall respiratory activity. The mechanism of multixenobiotic resistance (MXR) was particularly evaluated in standardized gill fragments using rhodamine B accumulation and its quantification under fluorescence optical microscopy. Regarding the control group, results had shown that the mussels maintenance in a lead-poisoned environment caused higher filtration rates (1.04 and 2.3 and L.h-1.g-1; p 0.05) and lower assimilation rates (71.96% and 54.1%, respectively). Also it was confirmed a lesser rhodamine B accumulation in the assays under influence of lead, suggesting that this metal induces the MXR mechanism expression in mussel P. perna. These results indicate that such physiological and biochemical alterations in the mussels can modify the energy fluxes of its metabolism, resulting in possible problems on the coastal systems used as cultivating sites.

A importância sócio-econômica que a mitilicultura conquistou no Estado de Santa Catarina explica a crescente procura por novos sítios costeiros para o seu uso. Estudos fisiológicos e bioquímicos do mexilhão Perna perna se apresentam como metodologias viáveis para programas de avaliação e monitoramento ambiental, permitindo inferir sobre a qualidade dos sítios e possíveis influências de agentes xenobióticos nas áreas costeiras. Com o objetivo de determinar a existência de variabilidade nesses parâmetros evocada pelo chumbo (1.21 mimol.L-1), mexilhões Perna perna foram mantidos à temperatura constante e alimentados com Chaetoceros gracilis por 15 dias. O grupo controle foi aclimatado em água do mar a 30‰. No final desse período foram realizados experimentos estáticos fisiológicos de filtração, respiração e assimilação, como também foi quantificada a atividade do mecanismo MXR em fragmentos de brânquias, pelo acúmulo de rodamina B, por microscopia óptica de fluorescência. Em relação ao grupo controle, a manutenção dos organismos em solução de chumbo causou maiores taxas de filtração (1,04 e 2,3 e L.h-1.g-1; p 0,05) e menores taxas de assimilação (71,96 e 54,1%, respectivamente). Também foi confirmado menor acúmulo de rodamina nos organismos sob influência do chumbo, sugerindo que este metal induz a expressão do mecanismo MXR no mexilhão Perna perna. Os resultados indicam que tais alterações fisiológicas e bioquímicas do mexilhão podem alterar os fluxos de energia no metabolismo, resultando em possíveis problemas nos sistemas costeiros utilizados como sítios de cultivos.

Variability of filtration and food assimilation rates, respiratory activity and multixenobiotic resistance (MXR) mechanism in the mussel Perna perna under lead influence

The economic importance that myticulture is conquering in Santa Catarina State (South of Brazil) explains the crescent search for new coastal sites for farming. Physiological and biochemical studies of the mussel Perna perna are important to the establishment of methodologies for program assessment and environmental monitoring, allowing to infer about site quality and possible influences of xenobiotic agents on coastal areas. In order to evaluate effects caused by lead poisoning (1.21 mumol.L-1), the mussels were maintained at constant temperature (25ºC) and fed with Chaetoceros gracilis for 15 days. The control group was acclimatized in sea water 30‰. At the end of this period time, physiological measurements were carried out along with statistic analysis for filtration rates, lead assimilation and overall respiratory activity. The mechanism of multixenobiotic resistance (MXR) was particularly evaluated in standardized gill fragments using rhodamine B accumulation and its quantification under fluorescence optical microscopy. Regarding the control group, results had shown that the mussels maintenance in a lead-poisoned environment caused higher filtration rates (1.04 and 2.3 and L.h-1.g-1; p 0.05) and lower assimilation rates (71.96% and 54.1%, respectively). Also it was confirmed a lesser rhodamine B accumulation in the assays under influence of lead, suggesting that this metal induces the MXR mechanism expression in mussel P. perna. These results indicate that such physiological and biochemical alterations in the mussels can modify the energy fluxes of its metabolism, resulting in possible problems on the coastal systems used as cultivating sites.

A importância sócio-econômica que a mitilicultura conquistou no Estado de Santa Catarina explica a crescente procura por novos sítios costeiros para o seu uso. Estudos fisiológicos e bioquímicos do mexilhão Perna perna se apresentam como metodologias viáveis para programas de avaliação e monitoramento ambiental, permitindo inferir sobre a qualidade dos sítios e possíveis influências de agentes xenobióticos nas áreas costeiras. Com o objetivo de determinar a existência de variabilidade nesses parâmetros evocada pelo chumbo (1.21 mimol.L-1), mexilhões Perna perna foram mantidos à temperatura constante e alimentados com Chaetoceros gracilis por 15 dias. O grupo controle foi aclimatado em água do mar a 30‰. No final desse período foram realizados experimentos estáticos fisiológicos de filtração, respiração e assimilação, como também foi quantificada a atividade do mecanismo MXR em fragmentos de brânquias, pelo acúmulo de rodamina B, por microscopia óptica de fluorescência. Em relação ao grupo controle, a manutenção dos organismos em solução de chumbo causou maiores taxas de filtração (1,04 e 2,3 e L.h-1.g-1; p 0,05) e menores taxas de assimilação (71,96 e 54,1%, respectivamente). Também foi confirmado menor acúmulo de rodamina nos organismos sob influência do chumbo, sugerindo que este metal induz a expressão do mecanismo MXR no mexilhão Perna perna. Os resultados indicam que tais alterações fisiológicas e bioquímicas do mexilhão podem alterar os fluxos de energia no metabolismo, resultando em possíveis problemas nos sistemas costeiros utilizados como sítios de cultivos.