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® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-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°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 56days were reproducible between laboratories (68

Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of Rhesus glycoproteins in nitrogen metabolism and excretion.

Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (Rh) glycoproteins, a family of proteins that facilitate the movement of NH(3) across cell membranes. This study examines the effects of crowding and feeding on ammonia excretion in gulf toadfish (Opsanus beta) within the context of Rh glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). Four Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) were isolated from toadfish. Tissue distributions showed higher levels of mRNA expression in the gills and liver, moderate levels in the intestine and lower levels in the stomach. Crowding significantly lowered branchial Rh expression and ammonia excretion rates in fasted toadfish. A comparison of Rh expression in the digestive tract revealed relatively low levels of Rhcg1 and Rhcg2 in the stomach and high mRNA abundance of Rhbg, Rhcg1 and Rhcg2 in the intestine of fasted, crowded toadfish. We speculate that these trends may reduce secretion and enhance absorption, respectively, to minimize the amount of ammonia that is lost through gastrointestinal routes. By contrast, these patterns of expression were modified in response to an exogenous ammonia load via feeding. Post-prandial ammonia excretion rates were elevated twofold, paralleled by similar increases in branchial Rhcg1 mRNA, gastric Rhcg1 mRNA and mRNA of all intestinal Rh isoforms. These changes were interpreted as an attempt to increase post-prandial ammonia excretion rates into the environment owing to a gradient created by elevated circulating ammonia concentrations and acidification of the digestive tract. Overall, we provide evidence that toadfish modulate both the expression of Rh isoforms and urea synthesis pathways to tightly control and regulate nitrogen excretion.

Ammonia affects brain nitrogen metabolism but not hydration status in the Gulf toadfish (Opsanus beta).

Laboratory rodents made hyperammonemic by infusing ammonia into the blood show symptoms of brain cell swelling and increased intracranial pressure. These symptoms could be caused in part by an increase in brain glutamine formed when brain glutamine synthetase (GS) naturally detoxifies ammonia to glutamine. Previous studies on the Gulf toadfish (Opsanus beta) demonstrated that it is resistant to high ammonia exposure (HAE) (96 h LC(50)=10mM) despite an increase in brain glutamine. This study attempts to resolve whether the resistance of O. beta is mediated by special handling of brain water in the face of changing glutamine concentrations. Methionine sulfoximine (MSO), an inhibitor of GS, was used to pharmacologically manipulate glutamine concentrations, and magnetic resonance imaging (MRI) was used to assess the status of brain water. Ammonia or MSO treatment did not substantially affect blood acid-base parameters. Exposure to 3.5mM ammonium chloride in seawater for 16 or 40 h resulted in a parallel increase in brain ammonia (3-fold) and glutamine (2-fold) and a decrease in brain glutamate (1.3-fold). Pre-treatment with MSO prevented ammonia-induced changes in glutamine and glutamate concentrations. HAE also induced an increase in plasma osmolality (by 7%) which was probably due to a disturbance of osmoregulatory processes but which did not result in broader whole body dehydration as indicated by muscle water analysis. The increase in brain glutamine was not associated with any changes in brain water in toadfish exposed to 3.5 mM ammonia for up to 40 h or even at 10, 20 and 30 mM ammonia consecutively and for one hour in each concentration. The lack of brain water accumulation implies that ammonia toxicity in toadfish appears to be via pathways other than cerebral swelling. Furthermore, toadfish pre-treated with MSO did not survive a normally sub-lethal exposure to 3.5 mM ammonia for 40 h. The enhancement of ammonia toxicity by MSO suggests that GS function is critical to ammonia tolerance in this species.

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 1h 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'.

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.

Comparison of the effects of ammonia on brain mitochondrial function in rats and gulf toadfish.

We compared the effect of hyperammonemia on NADH levels in brain slices and on the rate of oxygen consumption from isolated nonsynaptic brain mitochondria in ammonia-sensitive Wistar rats with that in ammonia-tolerant gulf toadfish (Opsanus beta). The NADH content was significantly decreased (12% less than control after 45 min with 1 mM NH(4)Cl) in rat brain slices, but it was not affected in brain slices from toadfish (with both 1 and 6 mM NH(4)Cl). The rates of oxygen consumption of different sets of enzymes of the electron transport chain (ETC; complexes I, II, III, and IV; II, III, and IV; and IV alone) were unaltered by hyperammonemic conditions in isolated nonsynaptic mitochondria from either rats or toadfish. These results lead us to conclude that the differing effects of ammonia on NADH levels in rat and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these effects were seen in vitro, our studies enabled us to rule out the possibility that effects of ammonia on metabolism were via indirect systemic effects. These results are discussed in the context of current views on mechanisms of central nervous system damage in hyperammonemic states.