Interactive Effects of Copper-Silver Mixtures at the Intestinal Epithelium of Rainbow Trout: An In Vitro Approach.

While metals are present in mixture in the environment, metal toxicity studies are usually conducted on an individual metal basis. There is a paucity of data in the existing literature regarding specific metal-metal interactions and their effect on metal toxicity and bioavailability. We studied interactions of a silver (Ag)-copper (Cu) mixture at the intestinal epithelium using an intestinal cell line derived from rainbow trout (Oncorhynchus mykiss), the RTgutGC. Exposures were conducted in media containing different chloride concentrations (low chloride, 1 mM; high chloride, 146 mM), thus resulting in different metal speciation. Cytotoxicity was evaluated based on two endpoints, cell metabolic activity and cell membrane integrity. The Ag-Cu mixture toxicity was assessed using two designs: independent action and concentration addition. Metal mixture bioavailability was studied by exposing cells to 500 nM of Ag or Cu as a single metal or a mixture (i.e., 500 nM of Cu plus 500 nM of Ag). We found an antagonistic effect in the low-chloride medium and an additive/synergistic effect in the high-chloride medium. We found that Cu dominates over Ag toxicity and bioavailability, indicating a competitive inhibition when both metals are present as free metal ions in the exposure media, which supports our hypothesis. Our study also suggests different mechanisms of uptake of free metal ions and metal complexes. The study adds valuable information to our understanding of the role of metal speciation on metal mixture toxicity and bioavailability. Environ Toxicol Chem 2024;43:105-114. © 2023 SETAC.

Alternatives to Fish Acute Whole Effluent Toxicity (WET) Testing: Predictability of RTgill-W1 Cells and Fathead Minnow Embryos with Actual Wastewater Samples.

Toxicity assays using fish cells and embryos continue to gain momentum as a more ethical and informative alternative to fish acute toxicity testing. The goal of our study was to test the accuracy of RTgill-W1 cells and the fathead minnow (Pimephales promelas) embryos to predict actual whole effluent toxicity (WET) in the fathead minnow larvae. The three models were compared concurrently using samples of various origins and treatment types. Additionally, the toxicity of reference toxicants (Cd, Cu, NH3-N, 3,4-dichloraniline, and benzalkonium chloride) spiked into a nontoxic wastewater was compared. The toxicity of reference toxicants was tested in isosmotic and hypoosmotic exposure media in RTgill-W1 cells. Of the 28 wastewater samples, 14 induced a toxic response in fish larvae. Embryos predicted 11 of the 14 wastewater samples toxic to the larvae, whereas RTgill-W1 cells predicted the toxicity of all 14 toxic samples to the larvae. In addition, embryos and RTgill-W1 cells predicted toxicity in two and six additional samples, respectively, that were nontoxic to larvae. Exposures in hypoosmotic medium significantly increased sensitivity of RTgill-W1 cells to all reference toxicants, excluding benzalkonium chloride, compared to exposures in isosmotic medium and showed toxicity levels similar to that in larvae. Thus, hypoosmotic exposure medium should be considered for aquatic toxicity testing applications. Overall, both gill cell and embryo models predicted toxicity in the majority of wastewater samples toxic to larvae and demonstrated their applicability for regulatory WET testing.

Characterization of RTgill-W1 cells epithelial development on transwell inserts: Evaluation of osmotic and toxic challenges.

RTgill-W1 cells cannot be directly exposed to freshwater (FW) or seawater (SW) due to osmotic stress. Adjustments of exposure solutions are needed, but these might reduce the bioavailability and toxicity of pollutants. To facilitate cell polarization and allow direct exposure of water samples, cells were cultured on transwell inserts. Monolayer formation was measured by trans-epithelial electrical resistance (TEER) and an apparent permeability (Papp) assay. At 14 days both TEER and Papp indicated the lowest permeability. Cell viability showed that cells can tolerate apical FW with complete medium (L-15/FBS) in the basolateral compartment but SW reduced cell viability. However, when reference toxicants, silver nitrate and sodium dodecyl benzene sulfonate, were added no toxicity was detected. Increased osmolality in the apical side and presence of proteins indicated diffusion from the basolateral to the apical side. Thus, reduced toxicity was likely caused by complexation with media salts and amino acids. A protein and amino acid free exposure medium (L-15/ex) was applied in the basolateral compartment. However, FW exposures with basolateral L-15/ex resulted in reduced cell viability. To reduce osmotic stress, mannitol was added to apical FW maintaining basolateral L-15/ex which improved cell viability and allowed detection of silver toxicity. Finally, RTgill-W1 cells did not show normal tight junction protein (ZO-1) immunocytochemical staining, which fits with the formation of a leaky epithelium. Overall, culturing of RTgill-W1 cells on transwell inserts allowed direct exposure to mannitol FW medium but showed a reduced sensitivity to toxicants. Thus, exposure on flat bottom wells is recommended for routine toxicity testing.

Role of the luminal composition on intestinal metal toxicity, bioavailability and bioreactivity: An in vitro approach based on the cell line RTgutGC.

The bioavailability of metal complexes is poorly understood. To evaluate bioavailability and toxicity of neutral and charged complexes as well as free metal ions, Visual Minteq, a chemical equilibrium model, was used to design media containing different metal species. Two non-essential (silver and cadmium) and two essential (copper and zinc) metals were selected. The rainbow trout (Oncorhynchus mykiss) gut cell line (RTgutGC) was used to investigate bioavailability, bioreactivity and toxicity of the different metal species. Toxicity was measured using a multiple endpoint cytotoxicity assay, bioavailability by measuring intracellular metal concentration, and bioreactivity by quantification of mRNA level of the metal responsive genes, metallothionein (MT), glutathione reductase (GR) and zinc transporter 1 (ZnT1). Speciation calculations showed that silver and cadmium preferentially bind chloride, copper phosphate and bicarbonate, and zinc remained primarily as a free ion. Cysteine avidly complexed with all metals reducing toxicity, bioavailability and bioreactivity. Silver and copper toxicity was not affected by inorganic metal speciation, whereas cadmium and zinc toxicity was decreased by chloride complexation. Moreover, reduction of calcium concentration in the medium increased toxicity and bioavailability of cadmium and zinc. Bioavailability of silver and zinc was reduced by low chloride while cadmium bioavailability was increased by low chloride and in presence of bicarbonate. Copper bioavailability was not affected by the medium composition. Cadmium and silver were more bioreactive, independently from the medium composition, in comparison to copper and zinc (i.e., higher induction of MT and GR). Cadmium was the only metal able to induce MT in presence of cysteine. ZnT1 was induced by cadmium in low-chloride, by zinc in low-chloride low-calcium and by cadmium and copper in the bicarbonate media. Overall, this study demonstrates that metal complexation alone is not sufficient to explain metal toxicity, and that anion exchange mechanisms play a role in metal uptake and bioreactivity.

Fish Embryo Acute Toxicity Testing and the RTgill-W1 Cell Line as In Vitro Models for Whole-Effluent Toxicity (WET) Testing: An In Vitro/In Vivo Comparison of Chemicals Relevant for WET Testing.

The fathead minnow (Pimephales promelas) fish embryo acute toxicity (FET) test was compared to the fish gill cells (RTgill-W1) in vitro assay and to the fish larvae acute toxicity test to evaluate their sensitivity for whole-effluent toxicity (WET) testing. The toxicity of 12 chemicals relevant for WET testing was compared as proof of principle. The concentrations lethal to 50% of a population (LC50) of embryos were compared to those in fish larvae and to the 50% effect concentration (EC50) in RTgill-W1 cells from previous literature. Along with traditional FET endpoints (coagulation, somite development, tail detachment, and heartbeat), cardiotoxicity was evaluated for WET applicability. Heart rate was measured at LC20 and LC50 values of six subselected chemicals (Cd, Cu, Ni, ammonia, 3,4-dichloraniline, and benzalkonium chloride). In addition, the toxicity of Cd and Ni was evaluated in RTgill-W1 cells exposed in a hypoosmotic medium to evaluate the effect that osmolarity may have on metal toxicity. A significant correlation was found between the FET and larvae LC50 values but not between the RTgill-W1 EC50 and FET LC50 values. Although sensitivity to Ni and Cd was found to increase with hypoosmotic conditions for FET and RTgill-W1 cells, a correlation was only found with removal of Ni from the analysis. Hypoosmotic conditions increased sensitivity with a significant correlation between RTgill-W1 cells and larvae. Cardiotoxicity was shown in three of the five subselected chemicals (Cd, Cu, and 3,4-dichloroaniline). Overall, both in vitro alternative models have shown good predictability of toxicity in fish in vivo for WET chemicals of interest. Environ Toxicol Chem 2022;41:2721-2731. © 2022 SETAC.

Effect of chloride concentration on the cytotoxicity, bioavailability, and bioreactivity of copper and silver in the rainbow trout gut cell line, RTgutGC.

Chloride (Cl-) influences the bioavailability and toxicity of metals in fish, but the mechanisms by which it influences these processes is poorly understood. Here, we investigated the effect of chloride on the cytotoxicity, bioavailability (i.e., accumulation) and bioreactivity (i.e., induction of mRNA levels of metal responsive genes) of copper (Cu) and silver (Ag) in the rainbow trout gut cell line (RTgutGC). Cells were exposed to metals in media with varying Cl- concentrations (0, 1, 5 and 146 mM). Metal speciation in exposure medium was analyzed using Visual MINTEQ software. Cytotoxicity of AgNO3 and CuSO4 was measured based on two endpoints: metabolic activity and membrane integrity. Cells were exposed to 500 nM of AgNO3 and CuSO4 for 24 h in respective media to determine metal bioavailability and bioreactivity. Ag speciation changes from free ionic (Ag+) to neutral (AgCl), to negatively charged chloride complexes (AgCl2-, AgCl3-) with increasing Cl- concentration in exposure media whereas Cu speciation remains in two forms (Cu2+ and CuHPO4) across all media. Chloride does not affect Ag bioavailability but decreases metal toxicity and bioreactivity. Cells exposed to Ag expressed significantly higher metallothionein mRNA levels in low Cl- media (0, 1, and 5 mM) than in high Cl- medium (146 mM). This suggests that chloride complexation reduces silver bioreactivity and toxicity. Conversely, Cu bioavailability and toxicity were higher in the high chloride medium (146 mM) than in the low Cl- (0, 1, and 5 mM) media, supporting the hypothesis that Cu uptake may occur via a chloride dependent mechanism. CLINICAL TRIALS REGISTRATION: This study did not require clinical trial registration.

In Vitro Digestion of Tire Particles in a Fish Model (Oncorhynchus mykiss): Solubilization Kinetics of Heavy Metals and Effects of Food Coingestion.

Tire and road wear particles (TRWP) have been shown to represent a large part of anthropogenic particles released into the environment. Nevertheless, the potential ecological risk of TRWP in the different environmental compartments and their potential toxic impacts on terrestrial and aquatic organisms remain largely underinvestigated. Several heavy metals compose TRWP, including Zn, which is used as a catalyst during the vulcanization process of rubber. This study investigated the solubilization potential of metals from cryogenically milled tire tread (CMTT) and TRWP in simulated gastric fluids (SFGASTRIC) and simulated intestinal fluids (SFINTESTINAL) designed to mimic rainbow trout (Oncorhynchus mykiss) gastrointestinal conditions. Our results indicate that the solubilization of heavy metals was greatly enhanced by gastrointestinal fluids compared to that by mineral water. After a 26 h in vitro digestion, 9.6 and 23.0% of total Zn content of CMTT and TRWP, respectively, were solubilized into the simulated gastrointestinal fluids. Coingestion of tire particles (performed with CMTT only) and surrogate prey items (Gammarus pulex) demonstrated that the animal organic matter reduced the amount of bioavailable Zn solubilized from CMTT. Contrastingly, in the coingestion scenario with vegetal organic matter (Lemna minor), high quantities of Zn were solubilized from L. minor and cumulated with Zn solubilized from CMTT.

Adsorption of progesterone onto microplastics and its desorption in simulated gastric and intestinal fluids.

The sorption of hydrophobic organic compounds (HOC) onto microplastics is relatively well reported in the literature, while their desorption remains poorly investigated, especially in biological fluids. The present study investigated the sorption and desorption of progesterone on polyethylene (PE), polypropylene (PP), and polystyrene (PS) microplastics. The sorption experiments showed that the equilibrium was reached in a few hours for all plastics. A sorption efficiency of 357.1 µg g-1 was found for PE and PS, and 322.6 µg g-1 for PP. Sorption experiments indicated that adsorption would certainly happen via surface sorption and a potentially pore-filling mechanism. The desorption was carried out in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), whose formulations were more complex than similar models reported so far. It has been found that the desorption was higher in SIF as compared to SGF, due to micelle formation in SIF promoting the pollutant solubilization. The sorption of pepsin onto microplastics has also been revealed, suggesting a competition between pollutants and pepsin for sorption sites and a potent reduction in pollutant solubilization. This study indicates that the ingestion of microplastics could be considered as an additional route of exposure to pollutants and therefore emphasizes pollutant bioavailability for aquatic organisms.

Applications of the RTgill-W1 Cell Line for Acute Whole-Effluent Toxicity Testing: In Vitro-In Vivo Correlation and Optimization of Exposure Conditions.

The cell line RTgill-W1 was evaluated as an in vitro alternative model for acute fish whole-effluent toxicity (WET) testing. We determined the 50% effective concentration (EC50) that reduces the viability of RTgill-W1 cells for selected toxicants commonly found in effluent samples and correlated those values with the respective 50% lethal concentration (LC50) of freshwater (fathead minnow, Pimephales promelas) and marine (sheepshead minnow, Cyprinodon variegatus) fish species obtained from the literature. Excluding low water-soluble organics and the volatile sodium hypochlorite, significant correlations were measured for metal, metalloids, ammonia, and higher water-soluble organics between in vitro EC50 values and in vivo LC50 values for both species. Typically, toxicity studies with RTgill-W1 cells are conducted by adding salts to the exposure medium, which may affect the bioavailability of toxicants. Osmotic tolerance of RTgill-W1 cells was found between 150 and 450 mOsm/kg, which were set as the hypoosmotic and hyperosmotic limits. A subset of the toxicants were then reexamined in hypoosmotic and hyperosmotic media. Copper toxicity decreased in hyperosmotic medium, and nickel toxicity increased in hypoosmotic and hyperosmotic media. Linear alkylbenzene sulfonate toxicity was not affected by the medium osmolality. Overall, RTgill-W1 cells have shown potential for applications in measuring metal, metalloids, ammonia, and water-soluble organic chemicals in acute WET tests, as well as complementing current toxicity identification and reduction evaluation strategies. In the present study, RTgill-W1 cells have been established as a valid animal alternative for WET testing, and we show that through manipulation of medium osmotic ranges, sensitivity to nickel was enhanced. Environ Toxicol Chem 2021;40:1050-1061. © 2020 SETAC.

Evaluation of the effect of silver and silver nanoparticles on the function of selenoproteins using an in-vitro model of the fish intestine: The cell line RTgutGC.

Emerging research in mammalian cells suggests that ionic (AgNO3) and nano silver (AgNP) can disrupt the metabolism of selenium which plays a vital role in oxidative stress control. However, the effect of silver (Ag) on selenoprotein function in fish is poorly understood. Here we evaluate the effects of AgNO3 and citrate coated AgNP (cit-AgNP) on selenoprotein function and oxidative stress using a fish cell line derived from the rainbow trout (Oncorhynchus mykiss) intestine (RTgutGC). Cell viability was evaluated using a cytotoxicity assay which measures simultaneously metabolic activity, membrane integrity and lysosome integrity. Cells exposed to equimolar amounts of AgNO3 and cit-AgNP accumulated the same amount of silver intracellularly, however AgNO3 was more toxic than cit-AgNP. Selenoenzymes glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) mRNA levels and enzyme activity were measured. While mRNA levels remained unaffected by AgNO3 or cit-AgNP, the enzyme activity of GPx was inhibited by AgNO3 (1 µM) and cit-AgNP (5 µM) and TrxR activity was inhibited by AgNO3 (0.4 µM) and cit-AgNP (1, 5 µM). Moreover, cells exposed to 1 µM of AgNO3 and cit-AgNP showed an increase in metallothionein b (MTb) mRNA levels at 24 h of exposure, confirming the uptake of silver, but returned to control levels at 72 h suggesting silver scavenging by MTb. Oxidative stress was not observed at any of the doses of AgNO3 or cit-AgNP tested. Overall, this study shows that AgNO3 or cit-AgNP can inhibit the activity of selenoenzymes but do not induce oxidative stress in RTgutGC cells.

Effects of Copper on the Neuromasts of Xenopus Laevis.

Fish and aquatic amphibians possess neuromasts on the surface of their body that constitute the lateral line, a sensory system used to detect water displacement. Copper is known to inactivate the neuromast organs of this system. Copper-induced neuromast loss in African clawed frogs, Xenopus laevis, was examined by exposing Nieuwkoop-Faber stage 54-55 larvae to copper concentrations of 0, 100, 200, 300, and 400 µg/L for 96 h, followed by an examination of neuromast counts, staining intensity, and behavioral responses. Neuromasts were counted using a novel imaging method across four different body regions: the whole body, partial body, head, and tail. Neuromast counts showed a decreasing, but nonsignificant, trend across increasing levels of copper exposure. Intensity of neuromast staining showed a stronger concentration-dependent decrease in all four body regions. The decrease in staining intensity, but not neuromast number, may indicate that although neuromasts are still functioning, they have a decreased number of viable hair cells. Potential loss of responsiveness related to neuromast damage was examined via sensitivity to puffs of air at varying distances. We detected little to no difference in response to the air puff stimulus between control tadpoles and tadpoles exposed to 400 µg/L of copper. Neuromasts of X. laevis may be more resistant to copper than those of North American tadpole species, possibly suggesting greater tolerance of the lateral line to environmental stressors in species that maintain this sensory system throughout their lifespan as compared with species that only have the lateral line during the larval period.

Role of metal speciation in the exposure medium on the toxicity, bioavailability and bio-reactivity of copper, silver, cadmium and zinc in the rainbow trout gut cell line (RTgutGC).

The role of metal speciation on metal bioavailability, bio-reactivity and toxicity at the fish intestine is poorly understood. To investigate these processes, we used an in vitro model of the rainbow trout (Oncorhynchus mykiss) intestine, the RTgutGC cell line. Cells were exposed to two essential metals (copper and zinc) and two non-essential metals (cadmium and silver) in a medium of well-defined composition, which allowed the determination of metal speciation in solution. Concentrations resulting in a 50% cell viability reduction (EC50) were measured using a viability assay based on two endpoints: metabolic activity and membrane integrity. Metal bioavailability and bio-reactivity was studied at non-toxic (300 nM all metals) and toxic (EC10; Ag-0.6, Cu-0.9, Cd-3, and Zn-9 µM) concentrations. Bioavailability (i.e. intracellular metal accumulation) was determined by ICP-MS, while bio-reactivity (i.e. induction of a metal specific transcriptional response) was determined by measuring the mRNA levels of a known biomarker of metal exposure (i.e. metallothionein) and of copper and zinc transporters (i.e. ATP7A and ZnT1). Dominant metal species in the exposure medium were Zn2+, CuHPO4, CdCl+, and AgCl2- respectively for Zn, Cu, Cd, and Ag. The EC50s showed the metal toxicity hierarchy: Ag > Cu > Cd > Zn. In RTgutGC cells, essential metal homeostasis was tightly regulated while non-essential metals accumulated more readily. Non-essential metals were also more bio-reactive inducing higher MT and ZnT1 mRNA levels. Taken together these findings indicate that metal toxicity in RTgutGC cannot solely be explained by extracellular metal speciation but requires the evaluation of metal bioavailability and bio-reactivity.

Bioenergetics-adverse outcome pathway: Linking organismal and suborganismal energetic endpoints to adverse outcomes.

Adverse outcome pathways (AOPs) link toxicity across levels of biological organization, and thereby facilitate the development of suborganismal responses predictive of whole-organism toxicity and provide the mechanistic information necessary for science-based extrapolation to population-level effects. Thus far AOPs have characterized various acute and chronic toxicity pathways; however, the potential for AOPs to explicitly characterize indirect, energy-mediated effects from toxicants has yet to be fully explored. Indeed, although exposure to contaminants can alter an organism's energy budget, energetic endpoints are rarely incorporated into ecological risk assessment because there is not an integrative framework for linking energetic effects to organismal endpoints relevant to risk assessment (e.g., survival, reproduction, growth). In the present analysis, we developed a generalized bioenergetics-AOP in an effort to make better use of energetic endpoints in risk assessment, specifically exposure scenarios that generate an energetic burden to organisms. To evaluate empirical support for a bioenergetics-AOP, we analyzed published data for links between energetic endpoints across levels of biological organization. We found correlations between 1) cellular energy allocation and whole-animal growth, and 2) metabolic rate and scope for growth. Moreover, we reviewed literature linking energy availability to nontraditional toxicological endpoints (e.g., locomotor performance), and found evidence that toxicants impair aerobic performance and activity. We conclude by highlighting current knowledge gaps that should be addressed to develop specific bioenergetics-AOPs. Environ Toxicol Chem 2019;38:27-45. © 2018 SETAC.

A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.

Ultrathin Alumina Membranes as Scaffold for Epithelial Cell Culture from the Intestine of Rainbow Trout.

Permeable membranes are indispensable for in vitro epithelial barrier models. However, currently available polymer-based membranes are low in porosity and relatively thick, resulting in a limited permeability and unrealistic culture conditions. In this study, we developed an ultrathin, nanoporous alumina membrane as novel cell culture interface for vertebrate cells, with focus on the rainbow trout (Onchorynchus mykiss) intestinal cell line RTgutGC. The new type of membrane is framed in a silicon chip for physical support and has a thickness of only 1 µm, with a porosity of 15% and homogeneous nanopores (Ø = 73 ± 21 nm). Permeability rates for small molecules, namely lucifer yellow, dextran 40, and bovine serum albumin, exceeded those of standard polyethylene terephthalate (PET) membranes by up to 27 fold. With the final goal to establish a representative model of the fish intestine for environmental toxicology, we engineered a simple culture setup, capable of testing the cellular response toward chemical exposure. Herein, cells were cultured in a monolayer on the alumina membranes and formed a polarized epithelium with apical expression of the tight junction protein ZO-1 within 14 days. Impedance spectroscopy, a noninvasive and real time electrical measurement, was used to determine cellular resistance during epithelial layer formation and chemical exposure to evaluate barrier functionality. Resistance values during epithelial development revealed different stages of epithelial maturity and were comparable with the in vivo situation. During chemical exposure, cellular resistance changed immediately when barrier tightness or cell viability was affected. Thus, our study demonstrates nanoporous alumina membranes as promising novel interface for alternative in vitro approaches, capable of allowing cell culture in a physiologically realistic manner and enabling high quality microscopy and sensitive measurement of cellular resistance.

Effect of media composition on bioavailability and toxicity of silver and silver nanoparticles in fish intestinal cells (RTgutGC).

To understand conditions affecting bioavailability and toxicity of citrate-coated silver nanoparticles (cit-AgNP) and dissolved silver at the luminal enterocyte interface, we exposed rainbow trout (Oncorhynchus mykiss) gut cells (RTgutGC) in media of contrasting composition: two amino acid-containing media, one of which was supplemented with proteins, as can be expected during digestion; and two protein and amino acid-free media contrasting low and high chloride content, as can be expected in the lumen of fish adapting to freshwater or seawater, respectively. Dose-response curves were generated measuring cell metabolic activity, membrane and lysosome integrity over a period of 72 hours. Then, nontoxic doses were applied and total silver accumulation, metallothionein and glutathione reductase mRNA levels were determined. The presence of proteins stabilized cit-AgNP keeping them in suspension. Conversely, in protein-free media, cit-AgNP agglomerated and settled, resulting in higher cellular accumulation of silver and toxicity. Chloride concentrations in exposure media modulated the toxicity of AgNO3 but not of cit-AgNP. Moreover, while amino acid-containing media are protective against AgNO3, likely due to the formation of thiolate complexes, they are only partially protective against cit-AgNP. Viability assays indicated that lysosomes are targets of cit-AgNP, supporting the hypothesis that cit-AgNP exert toxicity intracellularly. Metallothionein, a sensor of metal bioavailability, was induced by cit-AgNP in high chloride medium but not in low chloride medium, indicating that chloride might have a role in mobilizing silver from intercellular vesicles. Overall, this study shows that AgNP bioavailability and toxicity in the intestine is linked to its luminal content.

Daily rhythms in expression of genes of hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

In mammals, several genes involved in liver lipid and cholesterol homeostasis are rhythmically expressed with expression shown to be regulated by clock genes via Rev-erb 1α. In order to elucidate clock gene regulation of genes involved in lipid metabolism in Atlantic salmon (Salmo salar L.), the orphan nuclear receptor Rev-erb 1α was cloned and 24 h expression of clock genes, transcription factors and genes involved in cholesterol and lipid metabolism determined in liver of parr acclimated to a long-day photoperiod, which was previously shown to elicit rhythmic clock gene expression in the brain. Of the 31 genes analysed, significant daily expression was demonstrated in the clock gene Bmal1, transcription factor genes Srebp1, Lxr, Pparα and Pparγ, and several lipid metabolism genes Hmgcr, Ipi, ApoCII and El. The possible regulatory mechanisms and pathways, and the functional significance of these patterns of expression were discussed. Importantly and in contrast to mammals, Per1, Per2, Fas, Srebp2, Cyp71α and Rev-erb 1α did not display significant daily rhythmicity in salmon. The present study is the first report characterising 24 h profiles of gene expression in liver of Atlantic salmon. However, more importantly, the predominant role of lipids in the nutrition and metabolism of fish, and of feed efficiency in determining farming economics, means that daily rhythmicity in the regulation of lipid metabolism will be an area of considerable interest for future research in commercially important species.

A primary FIsh Gill Cell System (FIGCS) for environmental monitoring of river waters.

Studies were conducted to assess the feasibility of a primary FIsh Gill Cell culture system (FIGCS) for both laboratory and field based environmental monitoring of rivers known to be affected by metal contamination. FIGCS were exposed in the laboratory and in the field to water from the River Hayle, a metal-contaminated system in Cornwall, United Kingdom. Water chemistry, including transition metal concentrations, changes in transepithelial electrical resistance (TEER), cell viability and the expression of metal responsive genes, metallothionein A and B were measured. FIGCS tolerated river water in the laboratory showing no loss in TEER or cell viability following 24h exposure. The cells also tolerated transport to the field (∼1000 km and 30 h) and exposure to unfiltered and filtered river water. Metallothionein A and B, a measure of intracellular biologically active metals, expression was induced in the laboratory and field on exposure to water from sites with elevated metal concentrations compared to those sites where metal levels were below water metal Environmental Quality Standards. This demonstrates that FIGCS detects bioreactive metals in river waters on exposure in the laboratory or field and can be used for on-site environmental monitoring as well as investigations into bioavailability and toxicity of contaminant mixtures in natural waters.

Bioavailability of silver nanoparticles and ions: from a chemical and biochemical perspective.

Owing to their antimicrobial properties, silver nanoparticles (NPs) are the most commonly used engineered nanomaterial for use in a wide array of consumer and medical applications. Many discussions are currently ongoing as to whether or not exposure of silver NPs to the ecosystem (i.e. plants and animals) may be conceived as harmful or not. Metallic silver, if released into the environment, can undergo chemical and biochemical conversion which strongly influence its availability towards any biological system. During this process, in the presence of moisture, silver can be oxidized resulting in the release of silver ions. To date, it is still debatable as to whether any biological impact of nanosized silver is relative to either its size, or to its ionic constitution. The aim of this review therefore is to provide a comprehensive, interdisciplinary overview--for biologists, chemists, toxicologists as well as physicists--regarding the production of silver NPs, its (as well as in their ionic form) chemical and biochemical behaviours towards/within a multitude of relative and realistic biological environments and also how such interactions may be correlated across a plethora of different biological organisms.

Diablo/SMAC: a novel biomarker of pollutant exposure in European flounder (Platichthys flesus).

Diablo (or SMAC) is a protein released from mitochondria following apoptotic stimuli and inhibits the actions of Inhibitors of Apoptosis (IAP) proteins. IAPs regulate the activity of caspases and NFkB, the primary executioners of apoptosis and of inflammation, respectively. Thus, Diablo is important for the regulation of cellular responses to damage. In Northern Europe, statutory governmental marine monitoring programs measure various biomarkers in flounder to indicate biological effects of pollutant exposure. More recently transcriptomic techniques have been applied in flounder to gain a more comprehensive understanding of pollutant effects, and to discover novel biomarkers. In most of these studies utilising flounder, Diablo was amongst the most highly increased transcripts identified. The aim of this study was to further examine piscine Diablo, at the gene level and mRNA level, after exposure to prototypical pollutants, and in flounder caught from polluted environments. The results show that two genes encoding Diablo exist in fish species, and in flounder one of these genes is increased in liver after exposure to polyaromatic hydrocarbons and polychlorinated biphenyls, and also in livers from fish living on contaminated estuarine sediments. Therefore, Diablo measurement has potential as a biomarker of pollutant exposure, and could indicate damaging effects of chemical contaminants.