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》

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 《-》

Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia.

Sinha AK ，Liew HJ ，Nawata CM ，Blust R ，Wood CM ，De Boeck G ... -  《-》

Differential modulation of ammonia excretion, Rhesus glycoproteins and ion-regulation in common carp (Cyprinus carpio) following individual and combined exposure to waterborne copper and ammonia.

The main objective of this study was to understand the mode of interaction between waterborne copper (Cu) and high environmental ammonia (HEA) exposure on freshwater fish, and how they influence the toxicity of each other when present together. For this purpose, individual and combined effects of Cu and HEA were examined on selected physiological and ion-regulatory processes and changes at transcript level in the common carp (Cyprinus carpio). Juvenile carp were exposed to 2.6μM Cu (25% of the 96h LC50value) and to 0.65mM ammonia (25% of the 96h LC50value) singly and as a mixture for 12h, 24h, 48h, 84h and 180h. Responses such as ammonia (Jamm) and urea (Jurea) excretion rate, plasma ammonia and urea, plasma ions (Na(+), Cl(-) and K(+)), muscle water content (MWC) as well as branchial Na(+)/K(+)-ATPase (NKA) and H(+)-ATPase activity, and branchial mRNA expression of NKA, H(+)-ATPase, Na(+)/H(+) exchanger (NHE-3) and Rhesus (Rh) glycoproteins were investigated under experimental conditions. Results show that Jamm was inhibited during Cu exposure, while HEA exposed fish were able to increase excretion efficiently. In the combined exposure, Jamm remained at the control levels indicating that Cu and HEA abolished each other's effect. Expression of Rhcg (Rhcg-a and Rhcg-b) mRNA was upregulated during HEA, thereby facilitated ammonia efflux out of gills. On the contrary, Rhcg-a transcript level declined following Cu exposure which might account for Cu induced Jamm inhibition. Likewise, Rhcg-a was also down-regulated in Cu-HEA co-exposed fish whilst a temporary increment was noted for Rhch-b. Fish exposed to HEA displayed pronounced up-regulation in NKA expression and activity and stable plasma ion levels. In both the Cu exposure alone and combined Cu-HEA exposure, ion-osmo homeostasis was adversely affected, exemplified by the significant reduction in plasma [Na(+)] and [Cl(-)], and elevated plasma [K(+)], along with an elevation in MWC. These changes were accompanied by a decline in NKA activity. Gill H(+)-ATPase mRNA levels and activities were not affected by either Cu or HEA or both. Likewise, NHE-3 expression remained unaltered but tended to be numerically higher during HEA exposure. Overall, these data suggest that at equitoxic concentrations (25% of 96h LC50), the individual effect of Cu is more harmful while HEA induces quicker adaptive responses. Our findings also denote a competitive mode of interaction, exemplified by the inhibition of HEA -mediated adaptive responses in the presence of Cu.

Sinha AK ，Kapotwe M ，Dabi SB ，Montes CDS ，Shrivastava J ，Blust R ，Boeck G ... -  《-》

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 ... -  《-》