Yuen K. Ip

Ammonia toxicity, tolerance, and excretion

Ammonia is an unusual toxicant in that it is produced by, as well as being poisonous to, animals. In aqueous solution ammonia has two species, NH3 and NH4+, total ammonia is the sum of [NH3] + [NH4+] and the pK of this ammonia/ammonium ion reaction is around 9.5. The NH3/NH4+ equilibrium both internally in animals and in ambient water depends on temperature, pressure, ionic strength, and pH; pH is most often of greatest significance to animals. Elevated ammonia levels in the environment are toxic. Temperature has only minor effects on ammonia toxicity expressed as total ammonia in water, and ionic strength of the water can influence ammonia toxicity, but pH has a very marked effect on toxicity. Acid waters ameliorate, whereas alkaline waters exacerbate ammonia toxicity. The threshold concentration of total ammonia ([NH3] + [NH4+]) resulting in unacceptable biological effects in freshwater, promulgated by the EPA (1998), is 3.48 mg N/liter at pH 6.5 and 0.25 mg N/liter at pH 9.0. There is only a relatively small saltwater data set, and a paucity of data on ammonia toxicity in marine environments, particularly chronic toxicity. The national criteria promulgated in the EPA (1989) saltwater document is a criterion continuous concentration (chronic value) of 0.99 mg N/liter total ammonia and a criterion maximum concentration (half the mean acute value) of 6.58 mg N/liter total ammonia, somewhat less than the equivalent freshwater pH 8.0 values of 1.27 and 8.4 mg N/liter total ammonia, respectively. This is consistent with marine species being somewhat more sensitive to ammonia than freshwater species. Toxicity studies are usually carried out on unfed, resting fish in order to facilitate comparison of results. Based on recent studies, however, environmental stresses, including swimming, can have dramatic effects on ammonia toxicity. It is also clear that feeding results in elevated postprandial body ammonia levels. Thus, feeding will probably also exacerbate ammonia toxicity. Fish may be more susceptible to elevated ammonia levels during and following feeding or when swimming. Thus, present ammonia criteria may fail to protect migrating fish and may be inappropriate for fish fed on a regular basis. Most teleost fish are ammonotelic, producing and excreting ammonia by diffusion of NH3 across the gills. They are very susceptible to elevated tissue ammonia levels under adverse conditions. Some fish avoid ammonia toxicity by utilizing several physiologic mechanisms. Suppression of proteolysis and/or amino acid catabolism may be a general mechanism adopted by some fishes during aerial exposure or ammonia loading. Others, like the mudskipper, can undergo partial amino acid catabolism and use amino acids as an energy source, leading to the accumulation of alanine, while active on land. Some fish convert excess ammonia to less toxic compounds including glutamine and other amino acids for storage. A few species have active ornithine—urea cycles and convert ammonia to urea for both storage and excretion. Under conditions of elevated ambient ammonia, the mudskipper P. schlosseri can continue to excrete ammonia by active transport of ammonium ions. There are indications that some fish may be able to manipulate the pH of the body surface to facilitate NH3 volatilization during aerial exposure, or that of the external medium to lower the toxicity of ammonia during ammonia loading. Future investigation of these aspects of “environmental ammonia detoxification” may produce new information on how fish avoid ammonia intoxication.

Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land

SUMMARY This study aimed to elucidate the strategies adopted by the African slender lungfish, Protopterus dolloi, to ameliorate the toxicity of ammonia during short (6 days) or long (40 days) periods of aestivation in a layer of dried mucus in open air in the laboratory. Despite decreases in rates of ammonia and urea excretion, the ammonia content in the muscle, liver, brain and gut of P. dolloi remained unchanged after 6 days of aestivation compared with the control fasted for 6 days. For specimens aestivated for 40 days, the ammonia contents in the muscle, liver and gut were significantly lower than those of the control fasted for 40 days, which suggests a decrease in the rate of ammonia production. In addition, there were significant increases in contents of alanine, aspartate and glutamate in the muscle, which suggests decreases in their catabolism. During the first 6 days and the last 34 days of aestivation, the rate of ammonia production was reduced to 26% and 28%, respectively, of the control rate (6.83 μmol day–1 g–1 on day 0). During the first 6 days and the next 34 days of aestivation, the averaged urea synthesis rate was 2.39-fold and 3.8-fold, respectively, greater than the value of 0.25 μmol day–1 g–1 for the day 0 control kept in water. No induction of activities of the ornithine-urea cycle (OUC) enzymes was observed in specimens aestivated for 6 days, because the suppression of ammonia production led to a light demand on the OUC capacity. For specimens aestivated for 40 days, the activities of carbamoyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase + lyase were significantly greater than those of the control fasted for 40 days. This is in agreement with the observation that the rate of urea synthesis in the last 34 days was greater than that in the first 6 days of aestivation. P. dolloi aestivated in a thin layer of dried mucus in open air with high O2 tension throughout the 40 days of aestivation, which could be the reason why it was able to sustain a high rate of urea synthesis despite this being an energy-intensive process. Our results indicate that a reduction in ammonia production and decreases in hepatic arginine and cranial tryptophan contents are important facets of aestivation in P. dolloi.

Urea synthesis in the African lungfish Protopterus dolloi - hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 ·days of aerial exposure

SUMMARY Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1) endogenous ammonia production was drastically reduced and (2) endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.

Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddarti (mudskipper).

This study aimed to elucidate whether exposure to a sublethal concentration (8mmoll(-1)) of NH(4)Cl (pH 7.0) for 12 or 48h would induce oxidative stress in gills and brain of the mudskipper Boleophthalmus boddarti which has high tolerance of environmental and brain ammonia. The gills of B. boddarti experienced a transient oxidative stress after 12h of ammonia exposure as evidenced by an increase in lipid hydroperoxide content, decreases in contents of reduced glutathione (GSH) and total GSH equivalent, and in activities of total glutathione peroxidase, glutathione reductase and catalase. There were also transient increases in protein abundance of p53 and p38 in gills of fish exposed to ammonia for 12h, although the protein abundance of phosphorylated p53 remained unchanged and there was a decrease in the protein abundance of phosphorylated p38, at hour 12. Since the majority of these oxidative parameters returned to control levels at hour 48, the ability of the gills of B. boddarti to recover from ammonia-induced oxidative stress might contribute to its high environmental ammonia tolerance. Ammonia also induced oxidative stress in the brain of B. boddarti at hours 12 and 48 as evidenced by the accumulation of carbonyl proteins, elevation in oxidized glutathione (GSSG) content and GSSG/GSH, decreases in activities of glutathione reductase and catalase, and an increase in the activity of superoxide dismutase. The capacity to increase glutathione synthesis and GSH content could alleviate severe ammonia-induced oxidative and nitrosative stress in the brain. Furthermore, the ability to decrease the protein abundance of p38 and phosphorylated p53 might prevent cell swelling, contributing in part to the high ammonia tolerance in the brain of B. boddarti. Overall, our results indicate that there could be multiple routes through which ammonia induced oxidative stress in and outside the brain.

The Loach Misgurnus anguillicaudatus Reduces Amino Acid Catabolism and Accumulates Alanine and Glutamine during Aerial Exposure

The loach Misgurnus anguillicaudatus inhabits rice fields in Southern China. It encounters drought during summer and ammonia loading during agricultural fertilization. In the laboratory, aerial exposure led to decreases in its ammonia and urea excretion. Ammonia accumulated to very high levels in the muscle and liver. Urea synthesis through the ornithine‐urea cycle was not involved in ammonia detoxification in M. anguillicaudatus. However, M. anguillicaudatus was capable of partial amino acid catabolism leading to the accumulation of alanine in the first 24 h of aerial exposure. This was apparently coupled to a possible decrease in protein/amino acid catabolism. These are not detoxification mechanisms but mechanisms that avoid internal fouling by ammonia. Misgurnus anguillicaudatus was also capable of detoxifying internally produced ammonia in part to glutamine, which appears to be an important adaptation after 24 h of aerial exposure. However, unlike the case of the marble goby (Oxyeleotris marmoratus), there was no alteration to the kinetic properties of the hepatic glutamine synthetase. During dry seasons, M. anguillicaudatus moves actively on land until it encounters soft mud in which it can bury itself through several strong wriggling actions of the body. Hence, it is possible that M. anguillicaudatus uses partial amino acid catabolism to fuel its short period of activities on land and switches to the formation of glutamine to detoxify internally produced ammonia when it remains relatively inactive in the mud.

The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays

SUMMARY The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7‰) indefinitely or in brackish water (20‰) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l-1 of urea in the plasma, with the rest of the osmolality made up mainly by Na+ and Cl-. In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20‰ water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg-1, in which urea contributed 83 mmol l-1. Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20‰ water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5‰, 10‰ and 15‰ water. Although the local T. lymma in full-strength sea water (30‰) had a much greater plasma urea concentration (380 mmol l-1), its urea excretion rate (4.7 μmol day-1 g-1) was comparable with that of H. signifier in 20‰ water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.

The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during 144 h of aerial exposure

SUMMARY The swamp eel Monopterus albus inhabits muddy ponds, swamps, canals and rice fields, where it can burrow within the moist earth during the dry summer season, thus surviving for long periods without water. This study aimed to elucidate the strategies adopted by M. albus to defend against endogenous ammonia toxicity when kept out of water for 144 h (6 days). Like any other fish, M. albus has difficulties in excreting ammonia during aerial exposure. In fact, the rates of ammonia and urea excretions decreased significantly in specimens throughout the 144 h of aerial exposure. At 144 h, the ammonia and urea excretion rates decreased to 20% and 25%, respectively, of the corresponding control values. Consequently, ammonia accumulated to high levels in the tissues and plasma of the experimental specimens. Apparently, M. albus has developed relatively higher ammonia tolerance at the cellular and subcellular levels compared with many other teleost fish. Since the urea concentration in the tissues of specimens exposed to air remained low, urea synthesis was apparently not adopted as a strategy to detoxify endogenous ammonia during 144 h of aerial exposure. Instead, ammonia produced through amino acid catabolism was detoxified to glutamine, leading to the accumulation of glutamine in the body during the first 72 h of aerial exposure. Complimenting the increased glutamine formation was a significant increase in glutamine synthetase activity in the liver of specimens exposed to air for 144 h. Formation of glutamine is energetically expensive. It is probably because M. albus remained relatively inactive on land that the reduction in energy demand for locomotory activity facilitated its exploitation of glutamine formation to detoxify endogenous ammonia. There was a slight decrease in the glutamine level in the body of the experimental animals between 72 h and 144 h of aerial exposure, which indicates that glutamine might not be the end product of nitrogen metabolism. In addition, these results suggest that suppression of endogenous ammonia production, possibly through reductions in proteolysis and amino acid catabolism, acts as the major strategy to avoid ammonia intoxication in specimens exposed to air for ≥72 h. It is concluded that glutamine formation and reduction in ammonia production together served as effective strategies to avoid the excessive accumulation of ammonia in the body of M. albus during 144 h of aerial exposure. However, these strategies might not be adequate to sustain the survival of M. albus in the mud for longer periods during drought because ammonia and glutamine concentrations had already built up to high levels in the body of specimens exposed to air for 144 h.

Strategies for Surviving High Concentrations of Environmental Ammonia in the Swamp Eel Monopterus albus

The swamp eel Monopterus albus lives in muddy ponds, swamps, canals, and rice fields in the tropics. It encounters high concentrations of environmental ammonia (HEA) during dry seasons or during agricultural fertilization in rice fields. This study aimed at determining the tolerance of M. albus to environmental ammonia and at elucidating the strategies that it adopts to defend against ammonia toxicity in HEA. In the laboratory, M. albus exhibited very high environmental ammonia tolerance; the 48‐, 72‐, and 96‐h median lethal concentrations of total ammonia at pH 7.0 and 28°C were 209.9, 198.7, and 193.2 mM, respectively. It was apparently incapable of actively excreting ammonia against a concentration gradient. In addition, it did not detoxify ammonia to urea, the excretion of which would lead to a loss of nitrogen and carbon, during ammonia loading. The high tolerance of M. albus to HEA was attributable partially to its exceptionally high tolerance to ammonia at the cellular and subcellular levels. During the 144 h of exposure to 75 mM NH4Cl at pH 7.0, the ammonia contents in the muscle, liver, brain, and gut of M. albus reached 11.49, 15.18, 6.48, and 7.51 μmol g−1, respectively. Such a capability allowed the accumulation of high concentrations of ammonia in the plasma (3.54 μmol mL−1) of M. albus exposed to HEA, which would reduce the net influx of exogenous ammonia. Subsequent to the buildup of internal ammonia levels, M. albus detoxified ammonia produced endogenously to glutamine. The glutamine contents in the muscle and liver reached 10.84 and 17.06 μmol g−1, respectively, after 144 h of exposure to HEA, which happened to be the highest known for fish. Unlike urea, the storage of glutamine in the muscle during ammonia loading allowed its usage for anabolic purposes when the adverse environmental condition subsides. Glutamine synthetase activity increased significantly in the liver and gut (2.8‐ and 1.5‐fold, respectively) of specimens exposed to HEA for 144 h. These results suggest that the liver was the main site of ammonia detoxification and the gut was more than a digestive/absorptive organ in M. albus. Monopterus albus did not undergo a reduction in amino acid catabolism during the first 24 h of ammonia exposure. However, assuming a total inhibition of excretion of endogenous ammonia, there was a deficit of −312 μmol N between the reduction in nitrogenous excretion (3,360 μmol N) and the retention of nitrogen (3,048 μmol N) after 72 h of aerial exposure. The deficit became much greater after 144 h, reaching a value of −3,243 μmol N. These results suggest that endogenous ammonia production in M. albus was suppressed in order to prevent the newly established internal steady state concentration of ammonia from rising to an intolerable level after an extended period of exposure to HEA.

Chronic and acute ammonia toxicity in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti: brain ammonia and glutamine contents, and effects of methionine sulfoximine and MK801.

SUMMARY The objective of this study was to elucidate if chronic and acute ammonia intoxication in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti, were associated with high levels of ammonia and/or glutamine in their brains, and if acute ammonia intoxication could be prevented by the administration of methionine sulfoximine [MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of n-methyl d-aspartate type glutamate (NMDA) receptors]. For P. schlosseri and B. boddaerti exposed to sublethal concentrations (100 and 8 mmol l-1 NH4Cl, respectively, at pH 7.0) of environmental ammonia for 4 days, brain ammonia contents increased drastically during the first 24 h, and they reached 18 and 14.5 μmol g-1, respectively, at hour 96. Simultaneously, there were increases in brain glutamine contents, but brain glutamate contents were unchanged. Because glutamine accumulated to exceptionally high levels in brains of P. schlosseri (29.8 μmol g-1) and B. boddaerti (12.1μ mol g-1) without causing death, it can be concluded that these two mudskippers could ameliorate those problems associated with glutamine synthesis and accumulation as observed in patients suffering from hyperammonemia. P. schlosseri and B. boddaerti could tolerate high doses of ammonium acetate (CH3COONH4) injected into their peritoneal cavities, with 24 h LC50 of 15.6 and 12.3 μmol g-1 fish, respectively. After the injection with a sublethal dose of CH3COONH4 (8 μmol g-1 fish), there were significant increases in ammonia (5.11 and 8.36 μmol g-1, respectively) and glutamine (4.22 and 3.54 μmol g-1, respectively) levels in their brains at hour 0.5, but these levels returned to normal at hour 24. By contrast, for P. schlosseri and B. boddaerti that succumbed within 15-50 min to a dose of CH3COONH4 (15 and 12 μmol g-1 fish, respectively) close to the LC50 values, the ammonia contents in the brains reached much higher levels (12.8 and 14.9 μmol g-1, respectively), while the glutamine level remained relatively low (3.93 and 2.67 μmol g-1, respectively). Thus, glutamine synthesis and accumulation in the brain was not the major cause of death in these two mudskippers confronted with acute ammonia toxicity. Indeed, MSO, at a dosage (100 μg g-1 fish) protective for rats, did not protect B. boddaerti against acute ammonia toxicity, although it was an inhibitor of GS activities from the brains of both mudskippers. In the case of P. schlosseri, MSO only prolonged the time to death but did not reduce the mortality rate (100%). In addition, MK801 (2 μg g-1 fish) had no protective effect on P. schlosseri and B. boddaerti injected with a lethal dose of CH3COONH4, indicating that activation of NMDA receptors was not the major cause of death during acute ammonia intoxication. Thus, it can be concluded that there are major differences in mechanisms of chronic and acute ammonia toxicity between brains of these two mudskippers and mammalian brains.

The African Lungfish (Protopterus dolloi): Ionoregulation and Osmoregulation in a Fish out of Water

Although urea production and metabolism in lungfish have been thoroughly studied, we have little knowledge of how internal osmotic and electrolyte balance are controlled during estivation or in water. We tested the hypothesis that, compared with the body surface of teleosts, the slender African lungfish (Protopterus dolloi) body surface was relatively impermeable to water, Na+, and Cl− due to its greatly reduced gills. Accordingly, we measured the tritiated water (3H‐H2O) flux in P. dolloi in water and during air exposure. In water, 3H‐H2O efflux was comparable with the lowest measurements reported in freshwater teleosts, with a rate constant (K) of 17.6% body water h−1. Unidirectional ion fluxes, measured using 22Na+ and 36Cl−, indicated that Na+ and Cl− influx was more than 90% lower than values reported in most freshwater teleosts. During air exposure, a cocoon formed within 1 wk that completely covered the dorsolateral body surface. However, there were no disturbances to blood osmotic or ion (Na+, Cl−) balance, despite seven‐ to eightfold increases in plasma urea after 20 wk. Up to 13‐fold increases in muscle urea (on a dry‐weight basis) were the likely explanation for the 56% increase in muscle water content observed after 20 wk of air exposure. The possibility that muscle acted as a “water reservoir” during air exposure was supported by the 20% decline in body mass observed during subsequent reimmersion in water. This decline in body mass was equivalent to 28 mL water in a 100‐g animal and was very close to the calculated net water gain (approximately 32 mL) observed during the 20‐wk period of air exposure. Tritiated water and unidirectional ion fluxes on air‐exposed lungfish revealed that the majority of water and ion exchange was via the ventral body surface at rates that were initially similar to aquatic rates. The 3H‐H2O flux declined over time but increased upon reimmersion. We conclude that the slender lungfish body surface, including the gills, has relatively low permeability to water and ions but that the ventral surface is an important site of osmoregulation and ionoregulation. We further propose that an amphibian‐like combination of ventral skin water and ion permeability, plus internal urea accumulation during air exposure, allows P. dolloi to extract water from its surroundings and to store water in the muscle when the water supply becomes limited.