Exposure to waterborne Cu inhibits cutaneous Na⁺ uptake in post-hatch larval rainbow trout (Oncorhynchus mykiss).

In freshwater rainbow trout (Oncorhynchus mykiss), two common responses to acute waterborne copper (Cu) exposure are reductions in ammonia excretion and Na(+) uptake at the gills, with the latter representing the likely lethal mechanism of action for Cu in adult fish. Larval fish, however, lack a functional gill following hatch and rely predominantly on cutaneous exchange, yet represent the most Cu-sensitive life stage. It is not known if Cu toxicity in larval fish occurs via the skin or gills. The present study utilized divided chambers to assess cutaneous and branchial Cu toxicity over larval development, using disruptions in ammonia excretion (Jamm) and Na(+) uptake (Jin(Na)) as toxicological endpoints. Early in development (early; 3 days post-hatch; dph), approximately 95% of Jamm and 78% of Jin(Na) occurred cutaneously, while in the late developmental stage (late; 25 dph), the gills were the dominant site of exchange (83 and 87% of Jamm and Jin(Na), respectively). Exposure to 50 μg/l Cu led to a 49% inhibition of Jamm in the late developmental stage only, while in the early and middle developmental (mid; 17 dph) stages, Cu had no effect on Jamm. Jin(Na), however, was significantly inhibited by Cu exposure at the early (53% reduction) and late (47% reduction) stages. Inhibition at the early stage of development was mediated by a reduction in cutaneous uptake, representing the first evidence of cutaneous metal toxicity in an intact aquatic organism. The inhibitions of both Jamm and Jin(Na) in the late developmental stage occurred via a reduction in branchial exchange only. The differential responses of the skin and gills to Cu exposure suggest that the mechanisms of Jamm and Jin(Na) and/or Cu toxicity differ between these tissues. Exposure to 20μg/l Cu revealed that Jamm is the more Cu-sensitive process. The results presented here have important implications in predicting metal toxicity in larval fish. The Biotic Ligand Model (BLM) is currently used to predict metal toxicity in aquatic organisms. However, for rainbow trout this is based on gill binding constants from juvenile fish. This may not be appropriate for post-hatch larval fish where the skin is the site of toxic action of Cu. Determining Cu binding constants and lethal accumulation concentrations for both skin and gills in larval fish may aid in developing a larval fish-specific BLM. Overall, the changing site of toxic action and physiology of developing larval fish present an interesting and exciting avenue for future research.

Zimmer AM ，Brauner CJ ，Wood CM 《-》

Acute exposure to waterborne copper inhibits both the excretion and uptake of ammonia in freshwater rainbow trout (Oncorhynchus mykiss).

In freshwater fish, exposure to sub-lethal concentrations of waterborne copper (Cu) results in inhibitions of ammonia excretion (Jamm) and Na(+) uptake (J(Na)in), yet the mechanisms by which these occur are not fully understood. In the present study, rainbow trout (Oncorhynchus mykiss) fry exposed to 50μg/l Cu for 24h displayed a sustained 40% decrease in Jamm and a transient 60% decrease in J(Na)in. Previously, these effects have been attributed to inhibitions of gill Na(+)/K(+)-ATPase and/or carbonic anhydrase (CA) activities by Cu. Trout fry did not display significant reductions in the branchial activities of these enzymes or H(+)-ATPase over 24h Cu exposure. Recently, Rhesus (Rh) glycoproteins, bi-directional NH3 gas channels, have been implicated in the mechanism of Cu toxicity. Juvenile trout were exposed to nominal 0, 50, and 200μg/l Cu for 3-6h under control conditions (ammonia-free water) followed by 6h exposure to high environmental ammonia (HEA; 1.5mmol/l NH4HCO3). HEA led to significant ammonia uptake in control fish (0μg/l Cu), and exposure to 50 and 200μg/l Cu resulted in significant reductions of ammonia uptake during HEA exposure. This is the first evidence that Cu inhibits both the excretion and uptake of ammonia, implicating bi-directional Rh glycoproteins as a target for Cu toxicity. We propose a model whereby Rh blockade by Cu causes the sustained inhibition of Jamm and transient inhibition of J(Na)in, with H(+)-ATPase potentially aiding in J(Na)in recovery. More work is needed to elucidate the role of Rh proteins in sub-lethal Cu toxicity.

Lim MY ，Zimmer AM ，Wood CM 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Interactive effects of waterborne metals in binary mixtures on short-term gill-metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss).

Metal binding to fish gills forms the basis of the biotic ligand model (BLM) approach, which has emerged as a useful tool for conducting site-specific water quality assessments for metals. The current BLMs are designed to assess the toxicity of individual metals, and cannot account for the interactive effects of metal mixtures to aquatic organisms including fish. The present study was designed mainly to examine the interactive effects of waterborne metals (Cd, Zn, Cu, Ag, and Ni) in specific binary combinations on short-term (3h) gill-metal binding and essential ion (Ca(2+) and Na(+)) uptake (a physiological index of toxicity) in fish, using juvenile freshwater rainbow trout (Oncorhynchus mykiss) as the model species. We hypothesized that binary mixtures of metals that share a common mode of uptake and toxicity (e.g., Cd and Zn - Ca(2+) antagonists, Cu and Ag - Na(+) antagonists) would reduce the gill binding of each other via competitive interactions and induce less than additive effects on ion transport. In addition, the mixture of metals that have different modes of uptake and toxicity (e.g., Cd and Cu, or Cd and Ni) would not exhibit any interactive effects either on gill-metal binding or ion transport. We found that both Zn and Cu reduced gill-Cd binding and vice versa, however, Ni did not influence gill-Cd binding in fish. Surprisingly, Ag was found to stimulate gill-Cu binding especially at high exposure concentrations, whereas, Cu had no effect on gill-Ag binding. The inhibitory effect of Cd and Zn in mixture on branchial Ca(2+) uptake was significantly greater than that of Cd or Zn alone. Similarly, the inhibitory effect of Cu and Ag in mixture on branchial Na(+) uptake was significantly greater than that of Cu or Ag alone. The inhibitory effects of Cd and Zn mixture on Ca(2+) uptake as well as Cu and Ag mixture on Na(+) uptake were found to follow the principles of simple additivity. In contrast, no significant additive effect on either Ca(2+) or Na(+) uptake was recorded in fish exposed to the mixture of Cd and Cu. Overall, we found that although the effects of metal mixture interactions on gill-metal binding did not always match with our original assumptions, the effects of metal mixtures on toxicity in fish were generally consistent with our predictions. The findings of the present study have important implications for improving the BLM approach to assess metal mixture toxicity in fish.

Niyogi S ，Nadella SR ，Wood CM 《-》

Different mechanisms of Na(+) uptake and ammonia excretion by the gill and yolk sac epithelium of early life stage rainbow trout.

In rainbow trout, the dominant site of Na+ uptake (JNa,in) and ammonia excretion (Jamm) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, JNa,in and Jamm occur via loose Na+/NH4+ exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, JNa,in by the yolk sac skin of PH trout occurs independently of Jamm Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l-1 NH4HCO3; 600 µmol l-1 Na+; pH 8), Jamm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in JNa,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in JNa,in [Na+/H+-exchanger-2 (NHE2) and H+-ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na+/K+-ATPase) in putative peanut lectin agglutinin-positive (PNA+) ionocytes in gill sections. Similar Na+/K+-ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of JNa,in and Jamm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different.

Zimmer AM ，Wilson JM ，Wright PA ，Hiroi J ，Wood CM ... -  《-》

Ammonia first? The transition from cutaneous to branchial ammonia excretion in developing rainbow trout is not altered by exposure to chronically high NaCl.

Larval rainbow trout (Oncorhynchus mykiss) were reared from hatch under control ([Na(+)]=0.60 mmol l(-1)) or high NaCl ([Na(+)]=60 mmol l(-1)) conditions to elucidate the driving force for the ontogeny of branchial Na(+)/NH4 (+) exchange, one of the earliest gill functions. We hypothesized that if Na(+) uptake is the driving force, then in high NaCl there would be a delay in the skin-to-gill shift in ammonia excretion (Jamm) and/or an elevation in whole-body total ammonia (Tamm). In both groups, however, the skin-to-gill shift for Jamm, determined using divided chambers, occurred at the same time (13 days post-hatch; dph) and whole-body Tamm was unchanged. Moreover, high NaCl larvae displayed elevated whole-body [Na(+)] relative to controls by 18 dph, suggesting that maintaining branchial Jamm occurs at the expense of Na(+) balance. Overall, these results support the 'ammonia hypothesis', which posits that ammonia excretion, probably as Na(+)/NH4 (+) exchange, is the primary function of the early fish gill.

Zimmer AM ，Wood CM 《-》