Pharmacotherapies that specifically target ammonia for the prevention and treatment of hepatic encephalopathy in adults with cirrhosis.

BACKGROUND: Hepatic encephalopathy is a common complication of cirrhosis, with high related morbidity and mortality. Its presence is associated with a wide spectrum of change ranging from clinically obvious neuropsychiatric features, known as 'overt' hepatic encephalopathy, to abnormalities manifest only on psychometric or electrophysiological testing, 'minimal' hepatic encephalopathy. The exact pathogenesis of the syndrome is unknown but ammonia plays a key role. Drugs that specifically target ammonia include sodium benzoate, glycerol phenylbutyrate, ornithine phenylacetate, AST-120 (spherical carbon adsorbent), and polyethylene glycol. OBJECTIVES: To evaluate the beneficial and harmful effects of pharmacotherapies that specifically target ammonia versus placebo, no intervention, or other active interventions, for the prevention and treatment of hepatic encephalopathy in people with cirrhosis. SEARCH METHODS: We searched the Cochrane Hepato-Biliary Controlled Trials Register, CENTRAL, MEDLINE, Embase, and three other databases to March 2019. We also searched online trials registries such as ClinicalTrials.gov, European Medicines Agency, WHO International Clinical Trial Registry Platform, and the Food and Drug Administration for ongoing or unpublished trials. In addition, we searched conference proceedings, checked bibliographies, and corresponded with investigators. SELECTION CRITERIA: We included randomised clinical trials comparing sodium benzoate, glycerol phenylbutyrate, ornithine phenylacetate, AST-120, and polyethylene glycol versus placebo or non-absorbable disaccharides, irrespective of blinding, language, or publication status. We included participants with minimal or overt hepatic encephalopathy or participants who were at risk of developing hepatic encephalopathy. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data from the included reports. The primary outcomes were mortality, hepatic encephalopathy, and serious adverse events. We undertook meta-analyses and presented results using risk ratios (RR) or mean differences (MD), both with 95% confidence intervals (CIs), and I2 statistic values as a marker of heterogeneity. We assessed bias control using the Cochrane Hepato-Biliary domains and the certainty of the evidence using GRADE. MAIN RESULTS: We identified 11 randomised clinical trials that fulfilled our inclusion criteria. Two trials evaluated the prevention of hepatic encephalopathy while nine evaluated the treatment of hepatic encephalopathy. The trials assessed sodium benzoate (three trials), glycerol phenylbutyrate (one trial), ornithine phenylacetate (two trials), AST-120 (two trials), and polyethylene glycol (three trials). Overall, 499 participants received these pharmacotherapies while 444 participants received a placebo preparation or a non-absorbable disaccharide. We classified eight of the 11 trials as at 'high risk of bias' and downgraded the certainty of the evidence to very low for all outcomes.Eleven trials, involving 943 participants, reported mortality data, although there were no events in five trials. Our analyses found no beneficial or harmful effects of sodium benzoate versus non-absorbable disaccharides (RR 1.26, 95% CI 0.49 to 3.28; 101 participants; 2 trials; I2 = 0%), glycerol phenylbutyrate versus placebo (RR 0.65, 95% CI 0.11 to 3.81; 178 participants; 1 trial), ornithine phenylacetate versus placebo (RR 0.73, 95% CI 0.35 to 1.51; 269 participants; 2 trials; I2 = 0%), AST-120 versus lactulose (RR 1.05, 95% CI 0.59 to 1.85; 41 participants; 1 trial), or polyethylene glycol versus lactulose (RR 0.50, 95% CI 0.09 to 2.64; 190 participants; 3 trials; I2 = 0%).Seven trials involving 521 participants reported data on hepatic encephalopathy. Our analyses showed a beneficial effect of glycerol phenylbutyrate versus placebo (RR 0.57, 95% CI 0.36 to 0.90; 178 participants; 1 trial; number needed to treat for an additional beneficial outcome (NNTB) 6), and of polyethylene glycol versus lactulose (RR 0.19, 95% CI 0.08 to 0.44; 190 participants; 3 trials; NNTB 4). We did not observe beneficial effects in the remaining three trials with extractable data: sodium benzoate versus non-absorbable disaccharides (RR 1.22, 95% CI 0.51 to 2.93; 74 participants; 1 trial); ornithine phenylacetate versus placebo (RR 2.71, 95% CI 0.12 to 62.70; 38 participants; 1 trial); or AST-120 versus lactulose (RR 1.05, 95% CI 0.59 to 1.85; 41 participants; 1 trial).Ten trials, involving 790 participants, reported a total of 130 serious adverse events. Our analyses found no evidence of beneficial or harmful effects of sodium benzoate versus non-absorbable disaccharides (RR 1.08, 95% CI 0.44 to 2.68; 101 participants; 2 trials), glycerol phenylbutyrate versus placebo (RR 1.63, 95% CI 0.85 to 3.13; 178 participants; 1 trial), ornithine phenylacetate versus placebo (RR 0.92, 95% CI 0.62 to 1.36; 264 participants; 2 trials; I2 = 0%), or polyethylene glycol versus lactulose (RR 0.57, 95% CI 0.18 to 1.82; 190 participants; 3 trials; I2 = 0%). Likewise, eight trials, involving 782 participants, reported a total of 374 non-serious adverse events and again our analyses found no beneficial or harmful effects of the pharmacotherapies under review when compared to placebo or to lactulose/lactitol.Nine trials, involving 733 participants, reported data on blood ammonia. We observed significant reductions in blood ammonia in placebo-controlled trials evaluating sodium benzoate (MD -32.00, 95% CI -46.85 to -17.15; 16 participants; 1 trial), glycerol phenylbutyrate (MD -12.00, 95% CI -23.37 to -0.63; 178 participants; 1 trial), ornithine phenylacetate (MD -27.10, 95% CI -48.55 to -5.65; 231 participants; 1 trial), and AST-120 (MD -22.00, 95% CI -26.75 to -17.25; 98 participants; 1 trial). However, there were no significant differences in blood ammonia concentrations in comparison with lactulose/lactitol with sodium benzoate (MD 9.00, 95% CI -1.10 to 19.11; 85 participants; 2 trials; I2 = 0%), AST-120 (MD 5.20, 95% CI -2.75 to 13.15; 35 participants; 1 trial), and polyethylene glycol (MD -29.28, 95% CI -95.96 to 37.39; 90 participants; 2 trials; I2 = 88%). FUNDING: Five trials received support from pharmaceutical companies while four did not; two did not provide this information. AUTHORS' CONCLUSIONS: There is insufficient evidence to determine the effects of these pharmacotherapies on the prevention and treatment of hepatic encephalopathy in adults with cirrhosis. They have the potential to reduce blood ammonia concentrations when compared to placebo, but their overall effects on clinical outcomes of interest and the potential harms associated with their use remain uncertain. Further evidence is needed to evaluate the potential beneficial and harmful effects of these pharmacotherapies in this clinical setting.

Hypothesis: role for ammonia neutralization in the prevention and reversal of heart failure.

Ammonia plays a central role in the life and death of all living organisms and has been studied for over 100 yr. Ammonia is necessary for growth and development, but it is toxic in excess, and, as a result, differing methods of ammonia neutralization have evolved. After physiological and pathological stress to the heart, tissue ammonia levels rise. Local ammonia neutralization may be inadequate, and excess ammonia may exert its toxic effects. Phenylbutyrate (PBA), which is Federal Drug Administration approved for the treatment of elevated blood ammonia in urea cycle disorders, provides an accessory pathway for ammonia excretion. Recently, PBA has also been found to prevent specific cardiomyopathies. The central theme presents the hypothesis that stress to the myocardium from a variety of environmental sources causes injury, cell death, necrosis, and ammonia production. Ammonia, if not neutralized, exerts downstream toxic effects. Here, data are presented showing that neutralization with PBA alone and PBA combined with angiotensin-converting enzyme inhibition prevent and reverse pathophysiology associated with specific cardiomyopathies. NEW & NOTEWORTHY Ammonia produced after myocardial injury is hypothesized to be an upstream stress contributing to the pathophysiology of heart failure, effects that may be attenuated by a documented ammonia-reducing treatment. Reversal of heart failure can be achieved using an angiotensin-converting enzyme inhibitor combined with an ammonia-reducing treatment.

Efficient Anaerobic Digestion of Microalgae Biomass: Proteins as a Key Macromolecule.

Biogas generation is the least complex technology to transform microalgae biomass into bioenergy. Since hydrolysis has been pointed out as the rate limiting stage of anaerobic digestion, the main challenge for an efficient biogas production is the optimization of cell wall disruption/hydrolysis. Among all tested pretreatments, enzymatic treatments were demonstrated not only very effective in disruption/hydrolysis but they also revealed the impact of microalgae macromolecular composition in the anaerobic process. Although carbohydrates have been traditionally recognized as the polymers responsible for the low microalgae digestibility, protease addition resulted in the highest organic matter solubilization and the highest methane production. However, protein solubilization during the pretreatment can result in anaerobic digestion inhibition due to the release of large amounts of ammonium nitrogen. The possible solutions to overcome these negative effects include the reduction of protein biomass levels by culturing the microalgae in low nitrogen media and the use of ammonia tolerant anaerobic inocula. Overall, this review is intended to evidence the relevance of microalgae proteins in different stages of anaerobic digestion, namely hydrolysis and methanogenesis.

Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are vital to hepatocellular function and the liver response to injury. They share a phenotypic homology with astrocytes that are central in the pathogenesis of hepatic encephalopathy, a condition in which hyperammonemia plays a pathogenic role. This study tested the hypothesis that ammonia modulates human HSC activation in vitro and in vivo, and evaluated whether ammonia lowering, by using l-ornithine phenylacetate (OP), modifies HSC activation in vivo and reduces portal pressure in a bile duct ligation (BDL) model. METHODS: Primary human HSCs were isolated and cultured. Proliferation (BrdU), metabolic activity (MTS), morphology (transmission electron, light and immunofluorescence microscopy), HSC activation markers, ability to contract, changes in oxidative status (ROS) and endoplasmic reticulum (ER) were evaluated to identify effects of ammonia challenge (50 µM, 100 µM, 300 µM) over 24-72 h. Changes in plasma ammonia levels, markers of HSC activation, portal pressure and hepatic eNOS activity were quantified in hyperammonemic BDL animals, and after OP treatment. RESULTS: Pathophysiological ammonia concentrations caused significant and reversible changes in cell proliferation, metabolic activity and activation markers of hHSC in vitro. Ammonia also induced significant alterations in cellular morphology, characterised by cytoplasmic vacuolisation, ER enlargement, ROS production, hHSC contraction and changes in pro-inflammatory gene expression together with HSC-related activation markers such as α-SMA, myosin IIa, IIb, and PDGF-Rß. Treatment with OP significantly reduced plasma ammonia (BDL 199.1 µmol/L±43.65 vs. BDL+OP 149.27 µmol/L±51.1, p<0.05) and portal pressure (BDL 14±0.6 vs. BDL+OP 11±0.3 mmHg, p<0.01), which was associated with increased eNOS activity and abrogation of HSC activation markers. CONCLUSIONS: The results show for the first time that ammonia produces deleterious morphological and functional effects on HSCs in vitro. Targeting ammonia with the ammonia lowering drug OP reduces portal pressure and deactivates hHSC in vivo, highlighting the opportunity for evaluating ammonia lowering as a potential therapy in cirrhotic patients with portal hypertension.

Current state of knowledge of hepatic encephalopathy (part III): non-absorbable disaccharides.

Nonabsorbable disaccharides have been the mainstay of treatment for hepatic encephalopathy since introduced into clinical practice in 1966. Their beneficial effects reflect their ability to reduce the intestinal production/absorption of ammonia. A recent Cochrane review confirmed the efficacy and safety of nonabsorbable disaccharides for the treatment and prevention of hepatic encephalopathy in patients with cirrhosis. The findings were robust and support the use of nonabsorbable disaccharides as a first line treatment for hepatic encephalopathy, in this patient population, and for its prevention.

Current state of knowledge of hepatic encephalopathy (part I): newer treatment strategies for hyperammonemia in liver failure.

Alterations in interorgan metabolism of ammonia play an important role in the onset of hyperammonemia in liver failure. Glutamine synthetase (GS) in muscle is an important target for ammonia removal strategies in hyperammonemia. Ornithine Phenylacetate (OP) is hypothesized to remove ammonia by providing glutamate as a substrate for increased GS activity and hence glutamine production. The newly generated glutamine conjugates with phenylacetate forming phenylacetylglutamine which can be excreted in the urine, providing an excretion pathway for ammonia. We have also shown that OP targets glycine metabolism, providing an additional ammonia reducing effect.

Pathogenesis of hepatic encephalopathy in cirrhosis: the concept of synergism revisited.

The concept of synergistic mechanisms as the pathophysiologic basis of hepatic encephalopathy started with the pioneering work of Les Zieve in Minneapolis some 60 years ago where synergistic actions of the liver-derived toxins ammonia, methanethiol, and octanoic acid were described. More recently, synergistic actions of ammonia and manganese, a toxic metal that is normally eliminated via the hepatobiliary route and shown to accumulate in brain in liver failure, on the glutamatergic neurotransmitter system were described. The current upsurge of interest in brain inflammation (neuroinflammation) in relation to the CNS complications of liver failure has added a third dimension to the synergy debate. The combined actions of ammonia, manganese and pro-inflammatory cytokines in brain in liver failure result in oxidative/nitrosative stress resulting from activation of glutamate (NMDA) receptors and consequent nitration of key brain proteins. One such protein, glutamine synthetase, the sole enzyme responsible for brain ammonia removal is nitrated and inactivated in brain in liver failure. Consequently, brain ammonia levels increase disproportionately resulting in alterations of brain excitability, impaired brain energy metabolism, encephalopathy and brain swelling. Experimental therapeutic approaches for which proof-of-principle has been established include the NMDA receptor antagonist memantine, N-acetyl cysteine (recently shown to have antioxidant properties at both hepatic and cerebral levels) and probiotics.

Clinical science workshop: targeting the gut-liver-brain axis.

A clinical science workshop was held at the ISHEN meeting in London on Friday 11th September 2014 with the aim of thrashing out how we might translate what we know about the central role of the gut-liver-brain axis into targets which we can use in the treatment of hepatic encephalopathy (HE). This review summarises the integral role that inter-organ ammonia metabolism plays in the pathogenesis of HE with specific discussion of the roles that the small and large intestine, liver, brain, kidney and muscle assume in ammonia and glutamine metabolism. Most recently, the salivary and gut microbiome have been shown to underpin the pathophysiological changes which culminate in HE and patients with advanced cirrhosis present with enteric dysbiosis with small bowel bacterial overgrowth and translocation of bacteria and their products across a leaky gut epithelial barrier. Resident macrophages within the liver are able to sense bacterial degradation products initiating a pro-inflammatory response within the hepatic parenchyma and release of cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-8 into the systemic circulation. The endotoxemia and systemic inflammatory response that are generated predispose both to the development of infection as well as the manifestation of covert and overt HE. Co-morbidities such as diabetes and insulin resistance, which commonly accompany cirrhosis, may promote slow gut transit, promote bacterial overgrowth and increase glutaminase activity and may need to be acknowledged in HE risk stratification assessments and therapeutic regimens. Therapies are discussed which target ammonia production, utilisation or excretion at an individual organ level, or which reduce systemic inflammation and endotoxemia which are known to exacerbate the cerebral effects of ammonia in HE. The ideal therapeutic strategy would be to use an agent that can reduce hyperammonemia and reduce systemic inflammation or perhaps to adopt a combination of therapies that can address both.

Methanogenic community changes, and emissions of methane and other gases, during storage of acidified and untreated pig slurry.

AIMS: Acidification with concentrated H(2)SO(4) is a novel strategy to reduce NH(3) emissions from livestock slurry. It was recently found that also CH(4) emissions from acidified slurry are reduced. This study investigated the microbiological basis and temporal stability of these effects. METHODS AND RESULTS: Pig slurry from two farms, acidified by different techniques or untreated, was stored for 83 days in a pilot-scale facility. Methanogens were characterized before and after storage by T-RFLP and qPCR targeting mcrA. Emissions of NH(3) and CH(4) during storage were quantified. Acidified slurry pH was nearly constant at values of 5·5 and 6·5. Ammonia losses were reduced by 84 and 49%, respectively, while CH(4) emission with both acidification techniques was reduced by >90%. T-RFLP fingerprints showed little effect of acidification or storage time. A major T-RF of 105 bp could represent methanogens related to Thermoplasmata (Tp). No treatment effects on gene copy numbers were seen with universal methanogen primers, whereas effects were found with Tp-specific primers. CONCLUSION: Methane emissions were reduced >90% during storage. Thermoplasmata-related methanogens could be involved in CH(4) emissions from pig slurry. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of acidification on CH(4) emissions during storage of pig slurry was quantified for the first time. Acidification with sulphuric acid holds promise as a novel greenhouse gas mitigation strategy for confined livestock production.

Direct acting inhibitors of ammoniagenesis: a role in post-TIPS encephalopathy?

A limited number of medications are typically considered for the management of hepatic encephalopathy occurring as a complication of transjugular intrahepatic portosystemic shunt (TIPS) placement. Multiple alternative compounds aimed at disrupting ammoniagenesis are or will soon be available, though their use tends to be limited by a lack of large data sets and of clinical awareness. In this review, we provide a targeted overview of the mechanisms and availability of five anti-ammoniagenic compounds (sodium phenylbutyrate, glycerol phenylbutyrate, sodium benzoate, L-ornithine L-aspartate, and ornithine phenylacetate) identified as possibly useful alternative therapeutic agents for cirrhotic encephalopathy. Three of these medications have been FDA approved for use in congenital urea cycle disorders only, while two are under active investigation for use in cirrhotic patients. In spite of limitations posed by cost and comorbidities, familiarity with these options may prove beneficial in cases refractory to conventional management.

Ameliorative potential of alpha-ketoglutaric acid (AKG) on acute lung injuries induced by ammonia inhalation in rats.

INTRODUCTION: Toxicants such as ammonia, if inhaled, can damage respiratory tract leading to acute lung injury and pulmonary edema. Besides being a possible threat for the workers in chemical industry, easy availability and the toxic nature of ammonia may be used by terror groups for inflicting mass casualty among vulnerable population. In the present study, we have evaluated the therapeutic efficacy of alpha-ketoglutarate (AKG) to mitigate acute effects of ammonia on lung structure and antioxidant status in experimental animals. METHODS: Acute lung injury (ALI) models were developed by inhalation of aerosols of liquid ammonia in male Sprague Dawley rats. AKG (5%) respiratory fluid was inhaled by nebulization once daily for 5 days. Animals were euthanized and their blood samples were collected for hematology and serum biochemistry analysis. Total cell count, total protein (TP), lactate dehydrogenase (LDH), antioxidant enzyme activity (CAT, SOD, GSH), and malonaldialdehyde (MDA) formation were measured in bronchoalveolar lavage (BAL) fluid. RESULTS: Treatment with AKG showed significant lung protection by lowering the levels of total cell count, TP, LDH, superoxide dismutase (SOD), and MDA in BAL fluid. There was a marked increase in catalase (CAT) and glutathione (GSH) content of BAL fluid post-AKG inhalation. Histopathology of lung tissue correlated with cellular and biochemical findings indicate therapeutic efficacy of AKG against ammonia-induced lung injuries. CONCLUSIONS: The data suggest a possible therapeutic role of AKG inhalation against ammonia-induced structural and inflammatory changes in the lung.

[Hepatic encephalopathy and liver transplantation]. / Hepaticus encephalopathia és májátültetés.

About 6500-7000 people/year die in Hungary due to liver cirrhosis which is often complicated with hepatic encephalopathy (HE). While conventional interpretation is that hepatic encephalopathy is a consequence of high blood ammonia level, recent data indicate that the degree of encephalopathy is related to systemic inflammatory response during decompensation. In this review the authors overview and analyze the latest treatment modalities of hepatic encephalopathy based on most recent findings. They found that frequently used evidence based treatment which apply metronidazole, neomycine or disaccharides was only partially effective in clinical studies. Use of rifaximine only is supported by grade I evidence, however it is quite a costly drug. The authors could not identify a generally accepted guideline for the treatment of HE with a systematic literature review, although it has significant effect on survival after liver transplantation. Therefore, the authors urge to develop a consensus guideline for the treatment of HE.

Management of hepatic encephalopathy in patients with cirrhosis.

The term hepatic encephalopathy encompasses a spectrum of neuropsychiatric abnormalities seen in patients with liver dysfunction. Distinct syndromes are identified in acute liver failure and cirrhosis. Rapid deterioration in consciousness level and increased intracranial pressure that may result in brain herniation and death are a feature of acute liver failure whereas manifestations of hepatic encephalopathy in cirrhosis include psychomotor dysfunction, impaired memory, increased reaction time, sensory abnormalities, poor concentration and in severe forms, coma. In patients with acute-on-chronic liver failure the pathophysiology remains undefined. Ammonia has been considered central to its pathogenesis. In the brain, the astrocyte is the main site for ammonia detoxification, during the conversion of glutamate to glutamine. An increased ammonia level raises the amount of glutamine within astrocytes, causing an osmotic imbalance resulting in cell swelling and ultimately brain oedema. Recent studies suggest that inflammation and it modulators may play a synergistic role with ammonia in the pathogenesis of hepatic encephalopathy. Therapy of hepatic encephalopathy is directed primarily at reducing ammonia generation and increasing its detoxification. The currently accepted regimens to treat hepatic encephalopathy such as lactulose and protein restricted diets need further clinical trials and therefore placebo controlled clinical trials in hepatic encephalopathy are justified. In liver failure, ammonia metabolism involves multiple organs and therefore ammonia reduction will require simultaneous targeting of these organs. The present review describes the pathophysiological basis of hepatic encephalopathy and evaluates the available therapies.

Inhibitory effects of laminaran and alginate on production of putrefactive compounds from soy protein by intestinal microbiota in vitro and in rats.

Soybean is one of the major components of the Japanese diet. In traditional Japanese cuisine, soybean-based food items are often consumed with brown algae. In this study, we examined the effect of water-soluble and fermentable polysaccharides, laminaran and sodium alginate, from brown algae, on putrefactive compound production, by human faecal microbiota in broth containing 3% (w/v) soy protein. We also investigated the effect of 2% laminaran or alginate diet on caecal putrefactive compounds in rats maintained on diets containing 20% (w/w) soy protein. The caecal microbiota was also analysed using denaturing gradient gel electrophoresis and pyrosequencing with primers targeting the bacterial 16S rRNA gene. The polysaccharides, particularly laminaran, inhibited ammonia, phenol, and indole production by human faecal microbiota. Both the algal polysaccharides lowered the caecal indole content. Laminaran was found to increase the number of Coprobacter, whereas Helicobacter was found to decrease in the presence of both laminaran and sodium alginate.

Nitric oxide scavengers differentially inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria.

Differential inhibitors are important for measuring the relative contributions of microbial groups, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), to biogeochemical processes in environmental samples. In particular, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) represents a nitric oxide scavenger used for the specific inhibition of AOA, implicating nitric oxide as an intermediate of thaumarchaeotal ammonia oxidation. This study investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate and trolox were tested onNitrosopumilus maritimus, two unpublished AOA representatives (AOA-6f and AOA-G6) as well as the AOB representative Nitrosomonas europaea All four scavengers inhibited ammonia oxidation by AOA at lower concentrations than for AOB. In particular, differential inhibition of AOA and AOB by caffeic acid (100 µM) and methylene blue hydrate (3 µM) was comparable to carboxy-PTIO (100 µM) in pure and enrichment culture incubations. However, when added to aquarium sponge biofilm microcosms, both scavengers were unable to inhibit ammonia oxidation consistently, likely due to degradation of the inhibitors themselves. This study provides evidence that a variety of nitric oxide scavengers result in differential inhibition of ammonia oxidation in AOA and AOB, and provides support to the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway.

Augmentation of butyrate-induced differentiation of human hepatocyte by cyclin E over-expression.

In mammalian cells, cellular differentiation into specific cell types is usually preceded by growth arrest. On the other hand, the induced differentiation may also be preceded by an enhanced G1-S transition of the cell cycle prior to the growth arrest. This suggests that an early increase in proliferation is in some way a prerequisite for subsequent differentiation. We therefore attempted to assess whether we could produce human hepatocytes with further differentiated functions by promoting G1-S transition in a butyrate-treated human hepatocyte cell line. A cyclin E-over-expressing cell line was established by transfecting human cyclin E cDNA. Upon butyrate treatment, the cyclin E-over-expressing cells exhibited a significantly increased albumin-secreting and ammonia-detoxifying capacity when compared to the control cells. In particular, the ornithine transcarbamylase activity was increased in these cells. Collectively, these results implicate that the cyclin E over-expression may augment the hepatocyte-specific functions during the butyrate-induced differentiation process of human hepatocytes by enhancing G1-S cell cycle transition.

Adverse effect of urease on salt stress during seed germination in Arabidopsis thaliana.

Seed germination is a critical stage in the development of crops that grow in saline soils. We noticed that seeds of an Arabidopsis urease mutant have significantly increased salt stress tolerance. To understand why, we treated the wild type (WT) with a urease inhibitor and found that its salt stress tolerance was also improved. We hypothesized that urease acting on urea generates NH4⁺, which probably exacerbates salt stress. As expected, the urease inhibitor significantly decreased the NH4⁺ level in WT seeds. These findings suggest that blocking urease activity improves salt tolerance during seed germination by lowering the concentration of NH4⁺.

Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat.

Astrocyte hypertrophy and swelling occur in a variety of pathophysiological conditions, including diseases associated with hyperammonemia. Ammonia is rapidly incorporated into glutamine by glutamine synthetase localized in astrocytes. We tested the hypotheses that (1) 6 h of hyperammonemia (500-600 microM) is adequate for producing astrocyte enlargement, and (2) astrocyte enlargement is attenuated by inhibition of glutamine synthetase with methionine sulfoximine. Pentobarbital-anesthetized rats received an intravenous infusion of either sodium or ammonium acetate after intraperitoneal pretreatment with vehicle, methionine sulfoximine (0.8 mmol/kg) or buthionine sulfoximine (4 mmol/kg), an analogue that does not inhibit glutamine synthetase. Hyperammonemia produced enlarged cortical astrocytes characterized by (1) decreased electron density of cytoplasmic matrix in perikaryon, processes and perivascular endfeet, (2) increased circumference of nuclear membrane, (3) increased numbers of mitochondria and rough and smooth endoplasmic reticulum in perikarya and large processes, and (4) less compact bundles of intermediate filaments. Pretreatment with methionine sulfoximine, but not buthionine sulfoximine, attenuated the decrease in cytoplasmic density and the increase in nuclear circumference; most perivascular endfeet remained as dense as occurred with sodium acetate infusion. However, increased numbers of organelles in expanded perikarya and large processes occurred after methionine sulfoximine treatment with and without ammonium acetate infusion. In separate groups of rats, hyperammonemia produced an increase in cortical tissue water content which was inhibited by methionine sulfoximine, but not buthionine sulfoximine. We conclude that clinically-relevant levels of hyperammonemia can cause astrocyte enlargement within 6 h in vivo characterized by both watery cytoplasm and increased organelles indicative of a cellular metabolic stress and altered astrocyte function. The watery cytoplasm component of astrocyte enlargement depends on glutamine synthesis rather than on ammonium ions per se, and is possibly caused by the osmotic effect accumulated glutamine.

Characterization of N-methyl-D-aspartate receptor subunits involved in acute ammonia toxicity.

Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.