How important are glutathione and thiol reductases to oyster hemocyte function?

Bivalves are animals with worldwide distribution. Although they play key roles in economic activities, human feeding and environmental studies, there is a considerable lack of knowledge about the relationship between their immune system and antioxidant defenses. Here, we performed an in vitro experiment where Crassostrea gigas hemocytes were exposed to the electrophilic compound 1-chloro-2,4-dinitrobenzene (CDNB, 0.1-50 µM) for one hour. CDNB treatment clearly disturbed thiol homeostasis, causing a concentration-dependent decrease in the glutathione (GSH) content and a decrease in the activity of two thiol reductases, glutathione reductase (GR - 2.5 and 50 µM CDNB) and thioredoxin reductase (TrxR - only 50 µM CDNB). The MTT reduction assay showed that none of the CDNB concentrations tested significantly altered cell viability. However, there was a decrease in the hemocyte's ability to uptake the neutral red dye, which indicates lysosomal impairment (≥12.5 µM CDNB). Cellular immunocompetence was further investigated and, despite the lower GSH content, GR activity and impairment in lysosome integrity, hemocyte functions (adhesion capacity, phagocytosis of latex beads and laminarin-induced ROS production) were preserved in the 2.5 and 12.5 µM CDNB treatments. These results suggest a minor importance of thiol pools and GR activity in C. gigas hemocyte's immunocompetence, in an in vitro acute exposure model. The 50 µM CDNB treatment, however, significantly compromised all the measured hemocyte functions. This response was associated with TrxR inhibition, increased lysosome impairment, decreased GSH content, and lower GR activity. Therefore, it seems that TrxR may be particularly important for the hemocyte function, or, alternatively, it is only affected when a deeply aggravated scenario in thiol homeostasis is set up. Such findings point out the need for further studies towards a better understanding of antioxidant and immune defenses interactions in bivalve cellular systems.

Comparison of cytotoxicity of α-Al2O3 and η-Al2O3 nanoparticles toward neuronal and bronchial cells.

The role of the crystalline structure on the toxicity of two phases of Al2O3 NPs, alpha (α-Al2O3 NPs) and eta (η-Al2O3 NPs), was investigated in this study. Different techniques were employed for the characterization of the Al2O3 NPs and multiple toxicological endpoints were used to assess the toxicity toward mouse neuroblastoma (N2A) and human bronchial epithelial (BEAS-2B) cells. Based on the results of the multiple toxicological endpoints, revealed differences in the toxic potential results for α-Al2O3 NPs and η-Al2O3 NPs, with the latter showing a more pronounced effect. These effects could be due to the high uptake of the η-Al2O3 NPs in the cytoplasmic vesicles, as evidenced by TEM and ICP-MS. Hence, the results demonstrate the potential toxicity of both α-Al2O3 NPs and η-Al2O3 NPs, although the N2A and BEAS-2B cells showed greater susceptibility toward η-Al2O3 NPs. Thus, our study demonstrates the important role of the crystalline structure in relation to the nanotoxicity of Al2O3 NPs.

Tattoo inks: Characterization and in vivo and in vitro toxicological evaluation.

Tattoo inks represent a growing market in the world economy, but this growth is associated with an increase in reports of adverse effects caused by the use of this product. In this study, four commercial tattoo inks (blue, green, red and black) were studied to characterize the composition and particle size and identify possible in vivo and in vitro toxicological effects on Daphnia magna and HaCaT cells, respectively. Compositional analysis confirmed the functional groups in the vehicles and organic pigments. The presence of nanoparticles was confirmed by image analysis. The toxicological evaluation indicated distinct results for blue and green inks for the parameters tested, despite the presence of similar levels of metals. The red ink, followed by the green, presented the highest toxicity, which may be related to pigments containing azo compounds and not to the metal fraction. Black ink was found to be the safest toxicologically. This paper provides an overview of the composition of tattoo inks and their toxicological effects in epidermal cells and in the environment.

Toxicological Evaluation and Quantification of Ingested Metal-Core Nanoplastic by Daphnia magna Through Fluorescence and Inductively Coupled Plasma-Mass Spectrometric Methods.

There are few studies on nanoplastic that propose quantification of the amount ingested combined with evaluation of the toxic effects on aquatic organisms. We propose 2 methods to quantify the amount of polystyrene nanoplastic (PSNP) ingested by Daphnia magna: fluorescence intensity, where a fluorescent monomer (F) is added to the PSNP and quantified through fluorescence light microscopy, and total aluminum quantification, where PSNP is synthesized with Al2 O3 metal-core nanoparticles and used for quantification of the nanoplastic ingested by the organism Daphnia magna using inductively coupled plasma-mass spectrometry. In addition, the PSNP was functionalized with palmitic acid to simulate the environmental conditions leading to biological and chemical transformations. Acute and chronic toxicity tests were performed with fluorescent PSNP (PSNP/F) and palmitic acid-functionalized PSNP/F (PSNP/F-PA). The ingestion quantified was higher by factors of 2.8 and 3.0 for PSNP/F-PA and 1.9 and 1.7 for PSNP/F applying the fluorescence intensity and total Al quantifying methods, respectively, when compared to PSNP. These results are consistent with the data obtained in the toxicity tests, which showed an approximately 3 times increase in the adverse effect of PSNP/F-PA on the mobility and reproduction of the organisms. Thus, the strong inhibition of D. magna reproduction caused by PSNP/F-PA in the chronic toxicity tests could be associated with a greater amount of this nanoplastic being ingested by the organisms. Environ Toxicol Chem 2019;38:2101-2110. © 2019 SETAC.

Gills as a glutathione-dependent metabolic barrier in Pacific oysters Crassostrea gigas: Absorption, metabolism and excretion of a model electrophile.

The mercapturic acid pathway (MAP) is a major phase II detoxification route, comprising the conjugation of electrophilic substances to glutathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) enzymes. In mammals, GSH-conjugates are exported from cells, and the GSH-constituent amino acids (Glu/Gly) are subsequently removed by ectopeptidases. The resulting Cys-conjugates are reabsorbed and, finally, a mercapturic acid is generated through N-acetylation. This pathway, though very well characterized in mammals, is poorly studied in non-mammalian biological models, such as bivalve mollusks, which are key organisms in aquatic ecosystems, aquaculture activities and environmental studies. In the present work, the compound 1-chloro-2,4-dinitrobenzene (CDNB) was used as a model electrophile to study the MAP in Pacific oysters Crassostrea gigas. Animals were exposed to 10µM CDNB and MAP metabolites were followed over 24h in the seawater and in oyster tissues (gills, digestive gland and hemolymph). A rapid decay was detected for CDNB in the seawater (half-life 1.7h), and MAP metabolites peaked in oyster tissues as soon as 15min for the GSH-conjugate, 1h for the Cys-conjugate, and 4h for the final metabolite (mercapturic acid). Biokinetic modeling of the MAP supports the fast CDNB uptake and metabolism, and indicated that while gills are a key organ for absorption, initial biotransformation, and likely metabolite excretion, hemolymph is a possible milieu for metabolite transport along different tissues. CDNB-induced GSH depletion (4h) was followed by increased GST activity (24h) in the gills, but not in the digestive gland. Furthermore, the transcript levels of glutamate-cysteine ligase, coding for the rate limiting enzyme in GSH synthesis, and two phase II biotransformation genes (GSTpi and GSTo), presented a fast (4h) and robust (∼6-70 fold) increase in the gills. Waterborne exposure to electrophilic compounds affected gills, but not digestive gland, while intramuscular exposure was able to modulate biochemical parameters in both tissues. This study is the first evidence of a fully functional and interorgan MAP pathway in bivalves. Hemolymph was shown to be responsible for the metabolic interplay among tissues, and gills, acting as a powerful GSH-dependent metabolic barrier against waterborne electrophilic substances, possibly also participating in metabolite excretion into the sea water. Altogether, experimental and modeled data fully agree with the existence of a classical mechanism for phase II xenobiotic metabolism and excretion in bivalves.

The biological importance of glutathione peroxidase and peroxiredoxin backup systems in bivalves during peroxide exposure.

Organic peroxide elimination in eukaryotes essentially depends on glutathione peroxidase (GPx) and peroxiredoxin (Prx) enzymes, which are supported by their respective electron donors, glutathione (GSH) and thioredoxin (Trx). This system depends on the ancillary enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) to maintain GSH and Trx in their reduced state. This study discusses the biological importance of GR and TrxR in supporting GPx and Prx during cumene hydroperoxide (CHP) exposure in brown mussel Perna perna. ZnCl2 or 1-chloro-2,4-dinitrobenze (CDNB) was used to decrease GR and TrxR activities in gills, as already reported with mammals and bivalves. ZnCl2 exposure lowered GR activity (28%), impaired the in vivo CHP decomposition and decreased the survival rates under CHP exposure. CDNB decreased GR (54%) and TrxR (73%) activities and induced glutathione depletion (99%), promoting diminished peroxide elimination and survival rates at a greater extent than ZnCl2. CDNB also increased the susceptibility of hemocytes to CHP toxicity. Despite being toxic and causing mortality at longer exposures, short (2 h) exposure to CHP promoted an up regulation of GSH (50 and 100 µM CHP) and protein-thiol (100 µM CHP) levels, which was blocked by ZnCl2 or CDNB pre-exposure. Results highlight the biological importance of GSH, GR and TrxR in supporting GPx and Prx activities, contributing to organic peroxides elimination and mussel survival under oxidative challenges. To our knowledge, this is the first work that demonstrates, albeit indirectly, the biological importance of GPx/GR/GSH and Prx/TrxR/Trx systems on in vivo organic peroxide elimination in bivalves.

Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress.

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 µm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24 h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48 h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48 h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24 h) to digestive gland (48 h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas.

Antioxidant deficit in gills of Pacific oyster (Crassostrea gigas) exposed to chlorodinitrobenzene increases menadione toxicity.

Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 µM for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH-CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH-CDNB conjugates.