Administration Of Ammonium Acetate Research Articles

Severe Acute Liver Dysfunction Induces Delayed Hepatocyte Swelling and Cytoplasmic Vacuolization, and Delayed Cortical Neuronal Cell Death.

Liver dysfunction is the main cause of hepatic encephalopathy. However, histopathological changes in the brain associated with hepatic encephalopathy remain unclear. Therefore, we investigated pathological changes in the liver and brain using an acute hepatic encephalopathy mouse model. After administering ammonium acetate, a transient increase in the blood ammonia level was observed, which returned to normal levels after 24 h. Consciousness and motor levels also returned to normal. It was revealed that hepatocyte swelling, and cytoplasmic vacuolization progressed over time in the liver tissue. Blood biochemistry also suggested hepatocyte dysfunction. In the brain, histopathological changes, such as perivascular astrocyte swelling, were observed 3 h after ammonium acetate administration. Abnormalities in neuronal organelles, especially mitochondria and rough endoplasmic reticulum, were also observed. Additionally, neuronal cell death was observed 24 h post-ammonia treatment when blood ammonia levels had returned to normal. Activation of reactive microglia and increased expression of inducible nitric oxide synthase (iNOS) were also observed seven days after a transient increase in blood ammonia. These results suggest that delayed neuronal atrophy could be iNOS-mediated cell death due to activation of reactive microglia. The findings also suggest that severe acute hepatic encephalopathy causes continued delayed brain cytotoxicity even after consciousness recovery.

Severe Acute Hepatic Dysfunction Induced by Ammonium Acetate Treatment Results in Choroid Plexus Swelling and Ventricle Enlargement in the Brain.

Hepatic encephalopathy is a major cause of liver failure. However, the pathophysiological role of ventricle enlargement in brain edema remains unclear. Here, we used an acute hepatic encephalopathy mouse model to examine the sequential pathological changes in the brain associated with this condition. We collected tissue samples from experimental animals treated with ammonium acetate at 3 and 24 h post-injection. Despite the normalization of the animal’s ammonia levels, samples taken at 24 h after injection exhibited distinct enlargement of lateral ventricles. The choroid plexus samples obtained at 3 h post-ammonium acetate treatment indicated enlargement; however, this swelling was reduced at the later timepoint. The aquaporin-1 proteins that regulate the choroid plexus were localized both in the apical membrane and the cytoplasm of the epithelia in the control; however, they translocated to the apical membranes of the epithelia in response to ammonia treatment. Therefore, severe acute hepatic encephalopathy induced by ammonium acetate administration caused enlargement of the ventricles, through swelling of the choroid plexus and aquaporin-1 transport and aggregation within the apical membranes.

Cerebellar neurodegeneration in a new rat model of episodic hepatic encephalopathy

Hepatic encephalopathy has traditionally been considered a reversible disorder. However, recent studies suggested that repeated episodes of hepatic encephalopathy cause persistent impairment leading to neuronal loss. The aims of our study were the development of a new animal model that reproduces the course of episodic hepatic encephalopathy and the identification of neurodegeneration evidences. Rats with portacaval anastomosis underwent simulated episodes of hepatic encephalopathy, triggered by the regular administration of ammonium acetate, and/or lipopolysaccharide. The neurological status was assessed and neuronal loss stereologically quantified in motor areas. During the simulated episodes, ammonia induced reversible motor impairment in portacaval anastomosis rats. In cerebellum, stereology showed a reduction in Purkinje cell population in portacaval anastomosis and PCA+NH3 groups and morphological changes. An increase in astrocyte size in PCA+NH3 group and activated microglia in groups treated with ammonium acetate and/or lipopolysaccharide was observed. A modulation of neurodegeneration-related genes and the presence of apoptosis in Bergmann glia were observed. This new animal model reproduces the clinical course of episodic hepatic encephalopathy when ammonia is the precipitant factor and demonstrates the existence of neuronal loss in cerebellum. The persistence of over-activated microglia and reactive astrocytes could participate in the apoptosis of Bergmann glia and therefore Purkinje cell degeneration.

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60mg/kg, followed 20min later by an i.p. injection of vehicle or 550mg/kg of ammonium acetate. Afterwards, animals were observed for 45min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.

Antioxidant enzymes, hydrogen peroxide metabolism, and respiration in rat heart during experimental hyperammonemia

The effects of toxic ammonia doses on H2O2 metabolism, energy metabolism, and antioxidant enzyme activities in rat heart were studied. Ammonium acetate administration to animals proved to increase total superoxide dismutase (SOD), catalase, and glutathione peroxidase activities in the heart cytoplasmic fraction as well as Mn-SOD, catalase, and glutathione reductase in heart mitochondria. Conversely, ammonia inhibited the same activities in the brain, liver, and erythrocytes. Hyperammonemia had no effect on the levels of ATP, ADP and total adenine nucleotides in the heart but decreased them in the brain. Ammonia impaired oxidative phosphorylation and increased the rate of H202 production in heart and brain mitochondria. The ammonia concentration inhibiting antioxidant enzymes in the liver and brain can be insufficient for such effect in the heart.

Membrane alterations and fluidity changes in cerebral cortex during acute ammonia intoxication

Acute ammonia intoxication is known to cause alterations in activities of several membrane bound enzymes like Na + K + ATPase, acetylcholine esterase and glutamate uptake in brain. The alteration in these membrane associated activities could be a consequence of altered membrane architecture. To probe this, the effect of pathophysiological concentrations of ammonia on lipid composition and fluidity of membranes isolated from cerebral cortex of rats, were investigated in the present study. Administration of acute doses of ammonium acetate caused depletion of membrane sphingomyelin and cholesterol levels thereby reducing cholesterol: phospholipid (C: PL) ratio. Levels of phosphatidylserine increased while those of phosphatidylcholine and phosphatidylethanolamine remain unaltered. Membrane fluidity estimations using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) indicated no changes in core and surface membrane fluidity following ammonium acetate administration. Acute ammonia toxicity induced no alteration in bulk fluidity but a decrease in annular fluidity of membranes, as determined using pyrene fluorescence. Elevated levels of malondialdehyde and declined level of total thiols in cerebral cortex membranes of rats under acute ammonia intoxication indicated the existence of oxidative stress.

Tolerance of neonatal rat brain to acute hyperammonemia

The aim of the present work was to study the effects of hyperammonemia on brain energy metabolism in neonatal rats. Rats were rendered hyperammonemic by ammonium acetate administration. This decreased brain ATP concentrations but enhanced brain ammonia and lactate levels in both adult and neonatal rats. In adult rats, the decrease in brain ATP concentrations was accompanied by a plunge in the respiratory control rate (RCR) of brain mitochondria. However, the ammonia-induced effect on RCR was not observed in neonatal rats, suggesting that the fall in ATP levels observed in neonatal rats would not be due to an impairment of mitochondrial respiratory efficiency. However, in neonatal rats the increase in blood and brain ammonia concentrations did not change brain glutamate concentrations but decreased glutamine contents. These results may be of relevance for the understanding of the resistance of neonatal rats observed in this work to acute ammonia toxicity

Hyperammonemia and chronic hepatic encephalopathy: an in vivo PMRS study of the rat brain.

The brain energy metabolism of rats affected by chronic hepatic encephalopathy due to portacaval shunting was monitored by in vivo 31P-nuclear magnetic resonance spectroscopy before and after ammonium acetate administration. With respect to healthy unoperated and to sham operated controls, portacaval shunting decreased the levels of the nuclear magnetic resonance (NMR) visible brain phosphocreatine and nucleoside phosphates, and the intracellular [free Mg(2+)]. Ammonium acetate induced a further decrease of the levels of the NMR detectable phosphocreatine and nucleoside triphosphates and of the [free Mg(2+)], while the PMR spectra of the brain of non-shunted rats did not show any significant change even after treatment with ammonium acetate.

Plasma and brain levels of oxindole in experimental chronic hepatic encephalopathy: effects of systemic ammonium acetate and L-tryptophan.

It has previously been shown that the neurodepressant L-tryptophan metabolite oxindole is increased in the blood and brain of rats with fulminant hepatic failure and in the blood of cirrhotic patients affected by chronic hepatic encephalopathy. In the present investigation, we found that oxindole levels were significantly increased in the blood and brain of portacaval-shunted rats, an animal model of chronic hepatic encephalopathy, compared with sham-operated controls. A further increase in plasma and brain oxindole content was found after oral administration of L-tryptophan (300 mg/kg) to both portacaval-shunted or sham-operated animals, while intraperitoneal injection of the amino acid did not modify oxindole content either in brain or blood. Ammonium acetate administration (4.0 mmol/kg, intraperitoneal) reversibly deteriorated the neurological status of portacaval-shunted animals, but did not modify, in a directly related manner, plasma and brain oxindole content. The present findings are in line with the possibility that oxindole may be an additional L-tryptophan-related candidate in the pathogenesis of chronic hepatic encephalopathy.

In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure

Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.

In vivo Investigation of Glutamate–Glutamine Metabolism in Hyperammonemic Monkey Brain Using 13C-Magnetic Resonance Spectroscopy

To investigate the metabolism of glutamate and glutamine in living monkey brain, a system of in vivo ¹³C magnetic resonance spectroscopy (MRS) using ¹H-decoupled ¹³C spectroscopy combined with monitoring temperature changes in the brain by MR phase mapping was developed. Serial ¹³C-NMR spectra of the amino acids glutamate and glutamine were acquired non-invasively over 4 h from anesthetized monkey brain after the intravenous administration of [1-¹³C]glucose (0.5–1.0 g/kg). In the acute hyperammonemic state induced by the administration of ammonium acetate (77 mg/kg bolus), it was observed that ¹³C incorporation into glutamine-4 was clearly accelerated, without changes of ¹³C incorporation into glutamate-4. During hyperammonemia, it was shown directly by [2-¹³C]glucose administration that the anaplerotic pathway for the TCA cycle was also augmented, contributing to the formation of glutamine in the astroglia.

L-ornithine-L-aspartate in experimental portal-systemic encephalopathy: therapeutic efficacy and mechanism of action.

Strategies aimed at the lowering of blood ammonia remain the treatment of choice in portal-systemic encephalopathy (PSE). L-ornithine-L-aspartate (OA) has recently been shown to be effective in the prevention of ammonia-precipitated coma in humans with PSE. These findings prompted the study of mechanisms of the protective effect of OA in portacaval-shunted rats in which reversible coma was precipitated by ammonium acetate administration (3.85 mmol/kg i.p.). OA infusions (300 mg/kg/h, i.v) offered complete protection in 12/12 animals compared to 0/12 saline-infused controls. This protective effect was accompanied by significant reductions of blood ammonia, concomitant increases of urea production and significant increases in blood and cerebrospinal fluid (CSF) glutamate and glutamine. Increased CSF concentrations of leucine and alanine also accompanied the protective effect of OA. These findings demonstrate the therapeutic efficacy of OA in the prevention of ammonia-precipitated coma in portacaval-shunted rats and suggest that this protective effect is both peripherally-mediated (increased urea and glutamine synthesis) and centrally-mediated (increased glutamine synthesis).

Effects of acute hyperammonemia in vivo on oxidative metabolism in nonsynaptic rat brain mitochondria.

The effects of hyperammonemia induced in vivo by injecting rats with ammonium acetate on oxidative phosphorylation, malate-aspartate shuttle, some related enzyme activities and metabolite levels in brain mitochondria were studied ex vivo. Rats were found to be either ammonia-sensitive (showing convulsions) or ammonia-resistant (without convulsions) after intraperitoneal injection of ammonium acetate (7 mmol/kg). Ammonium acetate administration to ammonia-sensitive rats led to inhibition of State 3 rates of brain mitochondria utilizing pyruvate, glutamate, isocitrate, and succinate as substrates and to decreased respiratory control index. In brain mitochondria isolated from ammonia-resistant animals, the ammonia-induced effect on such State 3 rates was not observed. In brain mitochondria from hyperammonemic rats without convulsions, a small increase in the activity of malate dehydrogenase was observed; glutamate dehydrogenase, succinate dehydrogenase, and aspartate aminotransferase were not affected. In brain mitochondria from rats with ammonia-induced convulsions, the activities of malate dehydrogenase and succinate dehydrogenase were reduced significantly. Ammonium acetate injection to rats was associated with a 5-fold increase in the brain mitochondrial ammonium ion content and a decrease (ca. 50%) in brain mitochondrial glutamate and aspartate; brain mitochondrial malate and 2-oxoglutarate levels remained unchanged. The rate of the malate-aspartate shuttle in brain mitochondria of hyperammonemic rats was decreased by 20% as compared to corresponding rate in control rats. We conclude that acute administration of ammonium acetate induces serious disturbances in the electron-transport chain, interferences of the malate-aspartate shuttle, alterations of the levels of shuttle intermediates and inhibition of the activities of malate and succinate dehydrogenases in brain mitochondria.

Ammonium acetate challenge in experimental chronic hepatic encephalopathy induces a transient increase of brain 5-HT release in vivo

Ammonia has been shown to cause release of neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) from synaptosomal preparations in vitro. In the present study, frontal neocortical extracellular levels of 5-HT and its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were determined in vivo by the use of microdialysis in portacaval shunted (PCS) rats, an experimental model of chronic hepatic encephalopathy (HE), prior to and after an acute coma-inducing administration of ammonium acetate (NH 4Ac; 5.2 mmol/kg, i.p.). PCS rats displayed elevated ( P<0.01) 5-HIAA but unaltered 5-HT extracellular levels compared with controls, supporting the contention of an increased neocortical 5-HT metabolism but unaltered neuronal 5-HT output in chronic HE. However, a transient elevation of extracellular 5-HT levels was observed when PCS-NH 4 Ac rats were in coma. Increased brain ammonia may thus augment neuronal 5-HT release in chronic HE, which in turn could be a causative factor for precipitation of more severe stages of HE.

Relation of taurine transport and brain edema in rats with simple hyperammonemia or liver failure.

Taurine (Tau), an amino acid that abounds in brain, has been implicated in inhibitory neuromodulation and osmoregulation, the latter function being manifested by Tau release along with osmotically obligated water in response to brain tissue edema. A previous study (Hilgier and Olson: J. Neurochem. 62:197-204, 1994) had shown that simple hyperammonemia (HA) induced in rats by daily administration of ammonium acetate resulted in a decrease of both tissue specific gravity indicative of edema and Tau content, in basal ganglia (BG) but not in cerebral cortex (CC). By contrast, rats with hepatic encephalopathy (HE) following administration of a hepatotoxin, thioacetamide, were characterized by CC edema and an increased Tau content in both BG and CC. In the present study, we tested the following parameters that may potentially have affected Tau distribution in the two models: a) spontaneous, and stimulated (hypoosmolarity-induced) release of loaded [3H] Tau in vitro from CC and BG slices; b) blood Tau content; and c) uptake of [14C] Tau in vivo from blood to brain corrected for [3H] water passage-the so-called brain uptake index (BUI). The two edema-affected structures: BG in the HA model and CC in the HE model, showed increased spontaneous Tau release. Edema-associated spontaneous release of Tau may favor inhibitory neurotransmission contributing to the pathomechanism of HA or HE. Stimulated release, reflecting the ability of the tissue to reduce water content, was decreased in the BG from HA rats, in agreement with the postulated role of Tau in osmoregulation. Stimulated release was unchanged in CC of HE rats. Neither spontaneous nor stimulated release of Tau were affected in CC of HA rats or in BG of HE rats. HE, but not HA, was associated with elevated blood content and increased BUI for TAU, which in combination, contributed to the increase of Tau content in CC. The latter phenomenon adds to the list of metabolic changes distinguishing simple HA from toxic liver damage, reemphasizing the crucial role of factors other than ammonia in the pathomechanism of HE.

Selective Alterations of Extracellular Brain Amino Acids in Relation to Function in Experimental Portal‐Systemic Encephalopathy: Results of an In Vivo Microdialysis Study

Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.

The benzodiazepine antagonist CGS 8216 prevents hyperammonemia-induced somatostatin receptor reduction in the brain.

Previous results from our group showed that hyperammonemia decreases the number of somatostatin (SS) receptors and that benzodiazepine receptors might regulate the number of SS receptors in rat brain. These findings together with the supersensitivity of benzodiazepine receptors in the hyperammonemic rat brain suggest that benzodiazepine receptors might mediate the effect of hyperammonemia on SS receptors. To assess this hypothesis we tested whether 2-phenylpyrazolo[3,4-c]-quinolin-3(5H)-one (CGS 8216), a benzodiazepine antagonist, prevented the effect of ammonium acetate on rat brain SS receptors. Administration of ammonium acetate (5 mmol/kg, i.p.) for 7 days did not affect the levels of somatostatin-like immunoreactivity but decreased the number of SS receptors in synaptosomes from the frontoparietal cortex and hippocampus without affecting their apparent affinity. This decrease could be blocked by the concomitant administration of CGS 8216 (10 mg/kg, i.p.). The benzodiazepine antagonist alone had no observable effect on the somatostatinergic system. These results suggested that the effect of hyperammonemia on SS receptors could be mediated, at least in part, through the benzodiazepine receptors.

Regional alterations of dopamine and its metabolites in rat brain following portacaval anastomosis

Hyperammonemia and changes in brain monoamine metabolism have been proposed to contribute to the pathogenesis of the neuropsychiatric symptoms characteristic of human portal-systemic encephalopathy (PSE) resulting from chronic liver disease. Portacaval anastomosis (PCA) in the rat leads to sustained hyperammonemia and mild encephalopathy. In order to evaluate the role of dopamine (DA) metabolism in PSE, levels of DA and its metabolites were measured by HPLC with electrochemical detection in brain regions of rats with PCA at various stages of encephalopathy precipitated by ammonium acetate administration. Following ammonium acetate administration, rats with PCA rapidly develop severe neurological signs of encephalopathy progressing through loss of righting reflex to coma; sham-operated control animals administered ammonium acetate showed no such neurological deterioration. Concentrations of the DA metabolites DOPAC and HVA as well as [DA metabolites]/[DA] ratios, an indirect measure of DA turnover in brain, were increased in caudate-putamen, in cingulate and pyriform entorhinal cortices as well as in raphe nucleus and locus coeruleus. Increased DA metabolites, however, did not worsen at coma stages of PSE. Increased DA turnover thus appears to relate to early neuropsychiatric and extrapyramidal symptoms of PSE.

Mechanism for lowering blood ammonia levels by lactitol.

Lactitol has been reported to decrease the blood ammonia concentration in various experimental hyperammonemia models such as portacaval shunted rats. The mechanism responsible for this lowering of blood ammonia levels was investigated in rats. When lactitol was given orally twice a day for 7 days at doses of 3 and 10 g/kg/day and at half that daily dose on day 8, it significantly decreased the ammonium concentration of the portal blood by 27.3-43.2%, cecal ammonia contents by 49.2-57.6% and the pH of the cecal contents from 6.52 to 5.92-5.54, 4 hr after the final administration. Lactitol also inhibited increases in the portal ammonia concentration induced by the intracecal administration of ammonium acetate (300 mg/kg) 4 hr after the final administration. When lactitol was given orally at bolus doses of 1 and 3 g/kg simultaneously with a charcoal meal, lactitol significantly facilitated small intestinal transit by 12-13%. At a bolus dose of 3 g/kg, given 1 hr before the administration of a charcoal meal into the proximal colon, lactitol significantly facilitated colonic transit by 29.5%. These effects of lactitol were similar to those of lactulose. These findings suggest that lactitol decreases blood ammonia concentration by inhibiting both the production and the absorption of ammonia through reducing intestinal pH and shortening the residence time of intestinal contents in the intestinal tract.

Attenuation of ammonia toxicity in mice by PK 11195 and pregnenolone sulfate

Ammonia and benzodiazepines are thought to be involved in the pathogenesis of hepatic encephalopathy. The present study was undertaken to evaluate the effect of various benzodiazepine-receptor ligands and neurosteroids on ammonia toxicity in mice. Administration of ammonium acetate (8–15 mmole/kg; i.p.) to Swiss Webster mice resulted in a dose-dependent increase in mortality. Pretreatment with the central benzodiazepine receptor agonist clonazepam or the antagonist Ro15-1788 (7 mg/kg each; i.p.) had no significant effect on the lethal response to 10 mmole/kg ammonium acetate. However, pretreatment with the putative antagonist of the peripheral-type benzodiazepine receptor, PK 11195 (10 mg/kg; i.p.), reduced mortality from 50 to 10%. Ro5-4864 (10 mg/kg; i.p.), an agonist of the peripheral-type benzodiazepine receptors, had no effect on ammonia toxicity. The neurosteroid, pregnenolone sulfate (20 mg/kg; i.p.) reduced mortality from 50 to 25%. The non-competitive N- methyl- d-aspartate (NMDA) receptor antagonist, MK-801 (2 mg/kg; i.p.), had no effect on the lethal response to ammonium acetate. The results from the present study suggest a role for peripheral-type benzodiazepine receptors and specific neurosteroids in the alleviation of ammonia toxicity in mice.