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Abstract: Increased atmospheric CO2 concentrations are causing greater dissolution of CO2 into seawater, and are ultimately responsible for today’s ongoing ocean acidification. We manipulated seawater acidity by addition of HCl and by increasing CO2 concentration and observed that two coastal harpacticoid copepods, Amphiascoides atopus and Schizopera knabeni were both more sensitive to increased acidity when generated by CO2. The present study indicates that copepods living in environments more prone to hypercapnia, such as mudflats where S. knabeni lives, may be less sensitive to future acidification. Ocean acidification is also expected to alter the toxicity of waterborne metals by influencing their speciation in seawater. CO2 enrichment did not affect the free-ion concentration of Cd but did increase the free-ion concentration of Cu. Antagonistic toxicities were observed between CO2 with Cd, Cu and Cu free-ion in A. atopus. This interaction could be due to a competition for H+ and metals for binding sites. [ABSTRACT FROM AUTHOR]

Toxicogenomics attempts to define how the regulation and expression of genes mediate the toxicological effects associated with exposure to a chemical. DNA microarrays are rapidly becoming one of the tools of choice for large-scale toxicogenomic studies. An approach in modern toxicogenomics has been to classify toxicity based on gene transcriptional patterns; comparing the transcriptional responses of a chemical with unknown toxicity to those for which the transcriptional profiles and toxicological endpoints have been well characterized. Recent evidence suggests that gene expression microarrays may be instrumental in defining mechanisms of action of toxicants. However, several assumptions are inherent to a toxicogenomic-based approach in toxicology, many of which remain to be validated. Gene expression profiling using DNA microarrays represents a snapshot of the gene transcriptional responses occurring at a particular time and within a particular tissue. Toxicity, on the other hand, represents a continuum of possible effects governed by both temporal and spatial factors that are inextricably contingent upon the exposure conditions. The perceived toxicological properties of any chemical are dependent on the route, dose, and duration of the exposure, and as such, gene expression patterns are also subject to these variables. Correct interpretation of DNA microarray data for the assessment of the toxicological properties of chemicals will require that temporal and spatial gene expression profiles be accounted for. These considerations are further compounded in ecotoxicological studies, during which altered gene expression patterns induced from exposure to an anthropogenic substance must be discernible over and above the complex effects that phenotypic, genotypic, and environmental variables have on gene expression. To this end, the greatest utility of DNA microarrays in the field of ecotoxicology may be in predicting the toxicological modes of action of anthropogenic substances on host physiology, particularly in non-model organisms. Predictable and accurate assessment of the impacts of a chemical substance in ecotoxicology will require that classical toxicological endpoints be used to validate any effects predicted based on gene expression profiling. Validated expression profiling may subsequently find utility in ecotoxicological-based computer simulation models, such as the Biotic Ligand Model (BLM), in which gene expression information may be integrated with geochemical, pharmacokinetic, and physiological data to accurately assess and predict toxicity of metals to aquatic organisms. [Copyright &y& Elsevier]

Potassium channel activity is important for modulating mammalian macrophage antimicrobial functions. The involvement of potassium channels in mediation of immune cell function in lower vertebrates, such as teleost, has not been explored. Since relatively little is known about the types of potassium channels present in fish macrophages, pharmacological blockers with broad ranges of activity were tested: 4-aminopyridine (4-AP), quinine, and tetraethylammonium chloride (TEA). The potassium channel blockers inhibited reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) production by goldfish macrophages activated with bacterial lipopolysaccharide (LPS) and/or macrophage activating factor (MAF)-containing supernatants. Quinine was the most potent inhibitor with an IC50 of 50 μM, while the other blockers, 4-AP and TEA, had IC50 of 1.2 and 0.6 mM, respectively. A reversible depolarization of the goldfish macrophage plasma membrane potential (Vm) was observed following treatments with potassium channel blockers, and was related to transcriptional changes in the inducible nitric oxide synthase gene (iNOS). Down-regulation of antimicrobial activities and depolarization of the goldfish macrophage plasma membrane were not a consequence of reduced cell number or viability, suggesting that potassium channels are required for generation of appropriate goldfish macrophage antimicrobial functions. [Copyright &y& Elsevier]

Juvenile rainbow trout (Oncorhynchus mykiss) were subjected to a 2-day radioactive pulse of 110mAg at 11.9 μg/l (as AgNO3), followed by a 19-day post-tracer exposure to non-radioactive Ag(I) (3.8 μg/l). The distribution of 110mAg in the gills, liver, intestine, kidney, brain and remaining carcass was investigated over a 19-day post-tracer period. Initially, the intestine contained the highest proportion of the 110mAg burden (34%), however, by day 8, less than 5% of the total radioactivity remained in this tissue. The majority of the 110mAg eliminated from the intestine appeared to distribute to the liver. Eventually, the 110mAg content in the liver accounted for as much as 65% of the total radioactivity in the fish. Apart from the liver and intestine, only the gills and carcass contained any appreciable amount (>5%) of the total body 110mAg content. Liver and gill samples were fractionated using differential centrifugation techniques to discern the subcellular distribution of 110mAg in these tissues. In the liver, the 110mAg levels in the cytosolic fraction increased from 35% to 72% of the total cellular burden between days 8 and 19, respectively. The radioactive pulse in the gills was predominantly found in a membrane compartment termed the nuclear fraction (∼60% of the total). Little change was observed over time (day 8 to day 19) to the subcellular distribution of Ag in the gills. Using size-exclusion chromatography, most (∼70%) of the 110mAg content in the liver cytosol eluted at a molecular weight characteristic of metallothionein. The cytosolic distribution of 110mAg in gills was quite diffuse, occurring primarily in the heavy molecular weight fractions. [Copyright &y& Elsevier]

Following the Deepwater Horizon oil spill, approximately 7 million liters of the dispersant Corexit 9500A were released to promote oil biodegradation by breaking up surface oil slick formation. This process is accomplished via amphipathic anionic surfactants within dispersants that facilitate the mixing of aqueous and lipid phases. However, the amphipathicity of Corexit may also cause it to interact with biological membranes like the gill, impairing gill function and ultimately disrupting physiological processes mediated by it, such as osmoregulation. The goal of this study was to investigate the osmoregulatory effects and toxicity of Corexit in Gulf killifish. Killifish at the embryonic, larval, juvenile, and adult life stages were exposed to Corexit in water of different salinities to assess the interactive effects of ontogeny and salinity on Corexit toxicity. Corexit was not toxic to embryos except when exposed in hyperosmotic water where it had negligible effects; however, its toxicity to killifish increased dramatically following hatch, showing its greatest deleterious effects in adults. Corexit tended to increase sodium and chloride burdens in killifish when exposed in hyperosmotic waters and reduced whole-body and plasma ion concentrations in fish exposed to hypoosmotic waters. However, Corexit exposure at hyperosmotic salinities resulted in an increased differential accumulation of sodium over chloride as killifish matured. These findings suggest that Corexit may impair gill structure or alter specific components of osmoregulatory function, thus impacting osmoregulation in hypersosmotic and hypoosmotic waters, potentially impairing survival during osmotic challenges. Furthermore, the magnitude of these impacts continues to increase concomitant with gill ontogeny. • Sensitivity to Corexit increases as Gulf killifish mature from embryos to adults. • Corexit was more toxic to Gulf killifish in hyperosmotic waters relative to hypoosmotic waters at all life stages. • The effect of Corexit on osmoregulatory physiology of killifish appears to change with maturation. [ABSTRACT FROM AUTHOR]

Abstract: We have studied intracellular pH (pHi) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH4Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na+-independent pHi recovery while the other cell type lacked a Na+-independent pHi recovery. Cells displaying Na+ independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na+-independent pHi recovery. Cells displaying Na+ independent recovery were identified as PNA+ (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA− (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na+ provides a direct functional marker for PNA+ and PNA− MR cells. Re-addition of Na+ to acidified cells resulted in a transient pHi recovery in both cell types. This event was abolished by amiloride (500µM) but it was insensitive to phenamil (50µM). The phorbol ester PMA (1µM) potentiated the Na+ induced pHi recovery suggesting that activation by PKC is required for continuous Na+/H+ exchanger activity in trout gill MR cells. This study is the first functional description of pHi recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pHi regulation in the gill epithelium. [Copyright &y& Elsevier]

The Fundulus genus of killifish includes species that inhabit marshes along the U.S. Atlantic coast and the Gulf of Mexico, but differ in their ability to adjust rapidly to fluctuations in salinity. Previous work suggests that euryhaline killifish stimulate polyamine biosynthesis and accumulate putrescine in the gills during acute hypoosmotic challenge. Despite evidence that polyamines have an osmoregulatory role in euryhaline killifish species, their function in marine species is unknown. Furthermore, the consequences of hypoosmotic-induced changes in polyamine synthesis on downstream pathways, such as ƴ-aminobutyric acid (Gaba) production, have yet to be explored. Here, we examined the effects of acute hypoosmotic exposure on polyamine, glutamate, and Gaba levels in the gills of a marine (F. majalis) and two euryhaline killifish species (F. heteroclitus and F. grandis). Fish acclimated to 32 ppt or 12 ppt water were transferred to fresh water, and concentrations of glutamate (Glu), Gaba, and the polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) were measured in the gills using high-performance liquid chromatography. F. heteroclitus and F. grandis exhibited an increase in gill Put concentration, but showed no change in Glu or Gaba levels following freshwater transfer. F. heteroclitus also accumulated Spd in the gills, whereas F. grandis showed transient increases in Spd and Spm levels. In contrast, gill Put, Spm, Glu, and Gaba levels decreased in F. majalis following freshwater transfer. Together, these findings suggest that increasing polyamine levels and maintaining Glu and Gaba levels in the gills may enable euryhaline teleosts to acclimate to shifts in environmental salinity. [Display omitted] • We measured gill polyamine and Gaba levels in killifish during freshwater exposure. • Euryhaline species (F. heteroclitus and F. grandis) had elevated levels of putrescine (Put) following freshwater transfer. • F. heteroclitus and F. grandis showed no change in glutamate (Glu) or Gaba levels. • Marine species (F. majalis) decreased Put, spermine, Glu, and Gaba levels. • Elevated gill polyamine synthesis may be indicative of osmotic plasticity in fishes. [ABSTRACT FROM AUTHOR]

Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis . Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na + concentration, and Cl − concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc , thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha- dl -difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock on active Cl − secretion. Spermidine partially reduced the decrease of short-circuit current ( I sc ) and membrane conductance ( G t ) produced by hypotonic exposure. These data suggest polyamines blunt the hypotonic inhibition of NaCl secretion and may lead to early apoptosis of seawater ionocytes and their replacement by FW-type ionocytes. [ABSTRACT FROM AUTHOR]

Abstract: The rate of acid-stimulated and phenamil-sensitive sodium (Na+) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA− MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na+ uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H+-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na+ transport in fish). In contrast, Na+ uptake in PNA− MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0mM external Na+. Acid-stimulated Na+ transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na+ transport in fish, were also responsible for inhibiting acid stimulated Na+ uptake in PNA− MR cells, but by themselves had no effect on basal Na+ transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na+ uptake in PNA− MR cells in a dose-dependent manner. We also demonstrate rapid (<1min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA− MR cells, but not in PVC. These data lend further support to the idea of a PNA− MR cell type as the primary site for Na+ uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na+ across the apical surface of the fish gill. [Copyright &y& Elsevier]