Mechanisms of ammonia tolerance in the gulf toadfish (Opsanus beta)

The gulf toadfish, Opsanus beta, has a greater tolerance than most other fish and mammals to ammonia. The aim of this thesis was to describe mechanisms that allow this ammonia tolerance using biochemical and molecular techniques. Guided by several hypotheses generated from studies in the medical field, namely, ammonia-induced mitochondrial dysfunction and alterations of the brain glutamate-glutamine cycle, we explored several hypotheses. We tested: (1) the effect of ammonia on mitochondrial energy metabolism and oxidative phosphorylation; (2) the effect of ammonia-induced increase in brain glutamine concentration on brain water content; (3) the effect of urea synthesis on the ability to prevent ammonia-induced increase in brain glutamine; and (4) the effect of ammonia treatment on the expression of the recently described putative ammonium channels, the Rh-like proteins. We observed that toadfish brain mitochondria, unlike rat brain mitochondria, did not experience a decrease in the concentration of NADH in the matrix when subjected to ammonia in the medium and therefore were able to maintain a stable energy metabolism. In contrast to mammals, toadfish did not experience an increase in brain water during sub-lethal or supra-lethal exposure to ammonia in the water. Also, over time toadfish were able to decrease the ammonia-induced increase in brain glutamine concentration which is believed to be toxic to brain cells and mitochondria. It was previously known that toadfish have the uncommon ability among other fish to synthesize urea de novo in the liver. This ability is also different from urea synthesis in mammals since the toadfish urea cycle prefers glutamine over ammonia as the nitrogen donor molecule. Toadfish were capable of decreasing the ammonia-induced increase in the concentration of urea throughout the whole body. Perhaps, its urea synthesis and excreting capabilities contributed to the observed decrease in brain glutamine. Finally, ammonia treatment had no effect on the relative expression of RhBG and RhCG mRNA when compared to control treated expression levels. We conclude that the tolerance of the toadfish to ammonia can be attributed to its special mitochondrial physiology and its particular manner of handling brain glutamine.</p

Piscine insights into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic encephalopathy

Although the number of fish species that have been studied for both hypoxia/anoxia tolerance and ammonia tolerance are few, there appears to be a correlation between the ability to survive these two insults. After establishing this correlation with examples from the literature, and after examining the role Peter Lutz played in catalyzing this convergent interest in two variables, this article explores potential mechanisms underpinning this correlation. We draw especially on the larger body of information for two human diseases with the same effected organ (brain), namely stroke and hepatic encephalopathy. While several dissimilarities exist between the responses of vertebrates to anoxia and hyperammonemia, one consistent observation in both conditions is an overactivation of NMDA receptors or glutamate neurotoxicity. We propose a glutamate excitotoxicity hypothesis to explain the correlation between ammonia and hypoxia resistance in fish. Furthermore, we suggest several experimental paths to test this hypothesis

Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1 h following IPC, and the other one that occurs 1–3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min ‘test’ ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N, N, N′, N′-tetramethyl- p-phenylenediamine ( P<0.01). The activities of complex I–III decreased in ischemic group by 16, 23 ( P<0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the ‘first window’

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda)

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys (R) exposed to nominal concentrations of cadmium chloride (from 53 to 608 mu g Cd L-1). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20 ± 1 degrees C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56 days were reproducible between laboratories (68 < EC50-56d < 124 mu g L-1) and were consistent with literature data. EC50-56d and EC10-56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC50-56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC50-56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin). (C) 2014 Elsevier Inc. All rights reserved