Ethyl pyruvate modulates delayed paralysis following thoracic aortic ischemia reperfusion in mice.

OBJECTIVE: Delayed paralysis is an unpredictable problem for patients undergoing complex repair of the thoracic/thoracoabdominal aorta. These experiments were designed to determine whether ethyl pyruvate (EP), a potent anti-inflammatory and antioxidant agent, might ameliorate delayed paralysis following thoracic aortic ischemia reperfusion (TAR). METHODS: C57BL6 mice were subjected to 5 minutes of thoracic aortic ischemia followed by reperfusion for up to 48 hours. Mice received either 300 mg/kg EP or lactated ringers (LR) at 30 minutes before ischemia and 3 hours after reperfusion. Neurologic function was assessed using an established rodent scale. Spinal cord tissue was analyzed for markers of inflammation (keratinocyte chemoattractant [KC], interleukin-6 [IL-6]), microglial activation (ionized calcium-binding adapter molecule-1 [Iba-1]), and apoptosis (Bcl-2, Bax, and terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] staining) at 24 and 48 hours after TAR. Nissl body stained motor neurons were counted in the anterior horns sections from L1-L5 segments. RESULTS: Ninety-three percent of the LR mice developed dense delayed paralysis between 40 and 48 hours after TAR, whereas only 39% of EP mice developed delayed paralysis (P < .01). Bcl-2 expression was higher (P < .05) and Iba-1 expression was lower (P < .05) in the EP group only at 24 hours reperfusion. At 48 hours, the number of motor neurons was higher (P < .01) and the number and TUNEL-positive cells was lower (P < .001) in the EP-treated mice. EP decreased the expression of KC (P < .01) and IL-6 (P < .001) at 48 hours after TAR. CONCLUSIONS: The protection provided by EP against delayed paralysis correlated with preservation of motor neurons, higher expression of antiapoptotic molecules, decreased microglial cell activation, and decreased spinal cord inflammation. EP may be a treatment for humans at risk for delayed paralysis.

Successful use of extracorporeal membrane oxygenation in an adult patient with toxic shock-induced heart failure.

Cardiomyopathy secondary to toxic shock syndrome (TSS) is an uncommon but potentially life-threatening problem. We report the case of a 51-year-old male who presented with profound cardiogenic shock and multiorgan failure that could not be managed by conventional therapy with intravenous fluids, vasopressors and inotropes. Venoarterial extracorporeal membrane oxygenation (VA ECMO) was instituted as a bridge to recovery. After administration of antibiotics and intravenous immunoglobulin, the patient's condition improved and he was successfully weaned off ECMO after 6 days. The patient recovered from multiorgan failure, and left ventricular ejection fraction improved from <10% pre-ECMO to 65% 8 months after discharge. This case supports the view that VA ECMO can be used successfully to support vital organ perfusion in patients with profound but reversible cardiomyopathy attributed to TSS.

HMGB1 as a drug target in staphylococcal pneumonia.

High mobility group box (HMGB)1 is a small DNA-binding protein. In the nucleus, HMGB1 plays a role in gene expression and DNA replication. When it is released or secreted into the extracellular milieu, HMGB1 functions as a pro-inflammatory cytokine-like mediator. Recently reported data support the view that treatment with a neutralizing anti-HMGB1 antibody ameliorated pulmonary damage in a murine model of pneumonia caused by a pathogenic strain of Staphylococcus aureus. These findings suggest that HMGB1 may be an important drug target as scientists, clinical investigators and pharmaceutical companies seek to develop better agents for the treatment of staphylococcal pneumonia. Unfortunately, however, encouraging results from murine models of human disease often fail to translate into positive findings in clinical trials. Thus, before moving from pre-clinical into clinical studies, it may be prudent to validate and extend the recent experimental findings by carrying out additional studies, using a large animal model of pneumonia.

Analyzing the value of monitoring duodenal mucosal perfusion using photoplethysmography.

Photoplethysmography (PPG) is a technique that permits noninvasive measurement of changes in the volume of tissues. A novel device uses PPG to assess changes in duodenal mucosal perfusion. When tested in septic piglets, data obtained using this device correlate with the blood lactate concentration and duodenal serosal microvascular blood flow as measured with a laser Doppler flowmeter. This new PPG-based approach for continuously monitoring gut mucosal perfusion warrants further development, leading to prospective clinical trials in patients.

Identification of a novel pathway of transforming growth factor-ß1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

Extracellular ß-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-ß1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-ß1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-ß1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-ß1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-ß1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-ß1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-ß1.

A comparison of critical care research funding and the financial burden of critical illness in the United States.

OBJECTIVES: To estimate federal dollars spent on critical care research, the cost of providing critical care, and to determine whether the percentage of federal research dollars spent on critical care research is commensurate with the financial burden of critical care. DESIGN AND DATA SOURCES: The National Institutes of Health Computer Retrieval of Information on Scientific Projects database was queried to identify funded grants whose title or abstract contained a key word potentially related to critical care. Each grant identified was analyzed by two reviewers (three if the analysis was discordant) to subjectively determine whether it was definitely, possibly, or definitely not related to critical care. Hospital and total costs of critical care were estimated from the Premier Database, state discharge data, and Medicare data. To estimate healthcare expenditures associated with caring for critically ill patients, total costs were calculated as the combination of hospitalization costs that included critical illness as well as additional costs in the year after hospital discharge. MEASUREMENTS AND MAIN RESULTS: Of 19,257 grants funded by the National Institutes of Health, 332 (1.7%) were definitely related to critical care and a maximum of 1212 (6.3%) grants were possibly related to critical care. Between 17.4% and 39.0% of total hospital costs were spent on critical care, and a total of between $121 and $263 billion was estimated to be spent on patients who required intensive care. This represents 5.2% to 11.2%, respectively, of total U.S. healthcare spending. CONCLUSIONS: The proportion of research dollars spent on critical care is lower than the percentage of healthcare expenditures related to critical illness.

Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis.

Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics.

The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.

High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH(2)-terminal kinase and higher nuclear factor-kappaB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process.

Effects of ethyl pyruvate and other α-keto carboxylic acid derivatives in a rat model of multivisceral ischemia and reperfusion.

BACKGROUND: Ethyl pyruvate (EP) has been shown to ameliorate hepatic, renal, and intestinal mucosal injury and down-regulate expression of several pro-inflammatory mediators in a wide variety of preclinical models of critical illnesses, such as sepsis, burn injury, acute pancreatitis, stroke, and hemorrhagic shock. The molecular mechanisms responsible for the therapeutic effects of EP remain poorly understood, but might be related to the compound's structure as the ester of an α-keto carboxylic acid. Herein, we tested the hypothesis that EP and other α-keto carboxylic acid derivatives can modulate organ injury after lower torso ischemia/reperfusion (I/R). METHODS: Rats were subjected to 50 min of supraceliac aortic occlusion. Over a 20-min period, starting 2 min before the release of the aortic clamp, the animals received 2 µL/g of Ringer's lactate solution (RL, n = 5) or an equivalent volume of a solution containing EP (n = 5), benzoyl formate (BF, n = 5), parahydroxyphenyl pyruvate (PHPP, n = 5) or sodium pyruvate (NaPyr, n = 5). The total dose of each compound was 0.86 mMol/kg. After 1h of reperfusion, we measured ileal mucosal permeability to fluorescein-labeled dextran (mw 4000 Da), liver malondialdehyde (MDA) content, and plasma levels of alanine aminotransferase (ALT) and TNF. Rats in the control group (CT, n = 4) were subjected to laparotomy and surgical isolation of the supraceliac aorta, but not visceral I/R. RESULTS: Ileal mucosal permeability, plasma levels of ALT and TNF, and hepatic MDA content increased significantly in the RL group relative to the CT group. Both EP and BF significantly ameliorated the development of systemic arterial hypotension, mucosal hyperpermeability, and significantly decreased plasma levels of TNF. MDA content was significantly decreased by EP, PHPP, BF, and NaPyr. CONCLUSIONS: In general, EP is more efficacious in this model than is NaPyr. Although more remains to be learned about the pharmacologic differences between EP and pyruvate, one important factor may the greater lipophilicity of the former compound. This insight may permit the development of even more effective cytoprotective and anti-inflammatory agents based on the pyruvoyl moiety.

Postischemic treatment with ethyl pyruvate prevents adenosine triphosphate depletion, ameliorates inflammation, and decreases thrombosis in a murine model of hind-limb ischemia and reperfusion.

INTRODUCTION: Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury. METHODS: C57BL6 mice underwent 90 minutes of unilateral ischemia followed by 24 hours of reperfusion using two treatment protocols. For the preischemic treatment (pre-I) protocol, mice (n=6) were given 300 mg/kg EP before ischemia, followed by 150 mg/kg of EP just before reperfusion and at 6 hours and 12 hours after reperfusion. In a postischemic treatment (post-I) protocol, mice (n=7) were treated with 300 mg/kg EP at the end of the ischemic period, then 15 minutes later, and 2 hours after reperfusion and 150 mg/kg of EP at 4 hours, 6 hours, 10 hours, 16 hours, and 22 hours after reperfusion. Controls mice for both protocols were treated with lactated Ringers alone at time intervals identical to EP. Skeletal muscle levels of adenosine triphosphate (ATP), interleukin-1ß, keratinocyte chemoattractant protein, and thrombin antithrombin-3 complex were measured. Skeletal muscle architectural integrity was assessed microscopically. RESULTS: ATP levels were higher in mice treated with EP compared with controls under the both treatment protocols (p=0.02). Interleukin-1ß, keratinocyte chemoattractant protein, thrombin antithrombin-3 complex (p<0.05), and the percentage of injured fibers (p<0.0001) were significantly decreased in treated versus control mice under the both protocols. CONCLUSION: Muscle fiber injury and markers of tissue thrombosis and inflammation were reduced, and ATP was preserved with EP in pre-I and post-I protocols. Further investigation of the efficacy of EP to modulate IR injury in a larger animal model of IR injury is warranted.

Ethyl pyruvate reduces the development of zymosan-induced generalized inflammation in mice.

OBJECTIVE: Ethyl pyruvate (EP) is a simple aliphatic ester, which has been shown to have anti-inflammatory effects in previous numerous cell culture and animal studies. In the present study, we investigated the effects of EP (75 mg/kg i.p.) on the development of shock caused by zymosan. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: Male CD mice. INTERVENTIONS: Mice received either intraperitoneally zymosan (500 mg/kg, administered i.p. as a suspension in saline) or vehicle (0.25 mL/mouse saline). EP (75 mg/kg i.p. was administered 1 and 6 hrs after zymosan administration. Organ failure and systemic inflammation in mice was assessed 18 hrs after administration of zymosan and/or EP. MEASUREMENTS AND MAIN RESULTS: Treatment of mice with EP attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. EP also attenuated the lung, liver, and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase activity in the lung and intestine caused by zymosan. Immunohistochemical analysis for inducible nitric oxide synthase, nitrotyrosine, poly (ADP-ribose), tumor necrosis factor-alpha, and interleukin-1beta revealed positive staining in pancreatic and intestinal tissue obtained from zymosan-injected mice. The degree of staining for nitrotyrosine, inducible nitric oxide synthase, poly (ADP-ribose), tumor necrosis factor-alpha, and interleukin-1beta were markedly reduced in tissue sections obtained from zymosan-injected mice, which had received EP. In addition, administration of zymosan caused a severe illness in the mice characterized by a systemic toxicity, significant loss of body weight, and a 60% of mortality at the end of observation period (7 days). Treatment with EP significantly reduced the development of systemic toxicity, the loss in body weight, and the mortality (20%) caused by zymosan. CONCLUSIONS: This study provides evidence that EP attenuates the degree of zymosan-induced shock in mice.

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines.

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and alphaES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (alphaES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (alphaES). Each of these isoforms were absent in tissue extracts prepared from RAGE(-/-) mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (alphaES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the alphaES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope.

Bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia.

Lipopolysaccharide (LPS) is an important factor in sepsis. LPS given by intraperitoneal injection induces intestinal hyperpermeability and bacterial translocation in animals and stimulates hepatic Kupffer cells to release TNF-alpha into the bile. This study aims to test the hypothesis that in response to LPS stimulation, hepatic Kupffer cells and extrahepatic macrophages release a large amount of the inflammatory cytokine high-mobility group box 1 (HMGB1) into the bile and that bile containing HMGB1 contributes to gut barrier dysfunction in experimental endotoxemia. To test this, rat common bile ducts were catheterized and bile flow rate was monitored before and during the LPS administration. Eight hours after LPS challenge, anti-HMGB1 neutralizing antibody or nonimmune (sham) IgG was injected into the duodenal lumen of endotoxemic rats; normal mice were also gavaged with normal or endotoxemic rat bile (bile collected from LPS-treated rats). We found that after LPS challenge, the bile flow rate in rats was significantly decreased at the 4- to 12-h time points, TNF-alpha concentration in the bile was markedly elevated at the 3- to 4-h time points, and bile HMGB1 levels were significantly increased at the 8- to 12-h time points. Duodenal injection with anti-HMGB1 antibody reversed LPS-induced gut barrier dysfunction in rats. In addition, feeding endotoxemic rat bile to normal mice significantly increased both mucosal permeability and bacterial translocation. The increase in permeability and bacterial translocation was reversible following removal of HMGB1 from the endotoxemic rat bile. These findings document that bile HMGB1 mediates gut barrier dysfunction in experimental endotoxemia.

Targeting mitochondria.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely linked to degenerative diseases such as Alzheimer's disease, Parkinson's, neuronal death including ischemic and hemorrhagic stroke, acute and chronic degenerative cardiac myocyte death, and cancer. As a byproduct of oxidative phosphorylation, a steady stream of reactive species emerge from our cellular energy plants, the mitochondria. ROS and RNS potentially cause damage to all cellular components. Structure alteration, biomolecule fragmentation, and oxidation of side chains are trade-offs of cellular energy production. ROS and RNS escape results in the activation of cytosolic stress pathways, DNA damage, and the upregulation of JNK, p38, and p53. Incomplete scavenging of ROS and RNS particularly affects the mitochondrial lipid cardiolipin (CL), triggers the release of mitochondrial cytochrome c, and activates the intrinsic death pathway. Due to the active redox environment and the excess of NADH and ATP at the inner mitochondrial membrane, a broad range of agents including electron acceptors, electron donors, and hydride acceptors can be used to influence the biochemical pathways. The key to therapeutic value is to enrich selective redox modulators at the target sites. Our approach is based on conjugating nitroxides to segments of natural products with relatively high affinity for mitochondrial membranes. For example, a modified gramicidin S segment was successfully used for this purpose and proven to be effective in preventing superoxide production in cells and CL oxidation in mitochondria and in protecting cells against a range of pro-apoptotic triggers such as actinomycin D, radiation, and staurosporine. More importantly, these mitochondria-targeted nitroxide/gramicidin conjugates were able to protect against apoptosis in vivo by preventing CL oxidation induced by intestinal hemorrhagic shock. Optimization of nitroxide carriers could lead to a new generation of effective antiapoptotic agents acting at an early mitochondrial stage. Alternative chemistry-based approaches to targeting mitochondria include the use of proteins and peptides, as well as the attachment of payloads to lipophilic cationic compounds, sulfonylureas, anthracyclines, and other agents with proven or hypothetical affinities for mitochondria. Manganese superoxide dismutase (MnSOD), SS tetrapeptides with 2',6'-dimethyltyrosine (Dmt) residues, rhodamine, triphenylphosphonium salts, nonopioid analgesics, adriamycin, and diverse electron-rich aromatics and stilbenes were used to influence mitochondrial biochemistry and the biology of aging. Some general structural principles for effective therapeutic agents are now emerging. Among these are the presence of basic or positively charged functional groups, hydrophobic substructures, and, most promising for future selective strategies, classes of compounds that are actively shuttled into mitochondria, bind to mitochondria-specific proteins, or show preferential affinity to mitochondria-specific lipids.

Ethyl pyruvate ameliorates liver injury secondary to severe acute pancreatitis.

BACKGROUND: Ethyl pyruvate (EP) is capable of significantly decreasing serum alanine aminotransferase and reducing hepatic necrosis in a murine model of severe acute pancreatitis (SAP); however, the working mechanism is still unclear. This study aims to elucidate the underlying mechanism of EP solution ameliorating SAP-induced liver injury and provide a new therapeutic agent to treat liver injury. MATERIALS AND METHODS: Acute necrotizing pancreatitis was induced in C57Bl/6 male mice by feeding the animals a choline-deficient diet supplemented with 0.5% ethionine for 24 h; then the animals were challenged with 7 hourly 50 mug/kg cerulein i.p. injections and a single i.p. injection of Escherichia coli lipopolysaccharide (4 mg/kg). Two hours after the injection of lipopolysaccharide, 40 mg/kg EP, the same volume of Ringers lactate solution (RLS), or saline solution were i.p. injected to animals of EP, RLS, and control groups every 6 h for a total 48-h period. RESULTS: When mice were treated with EP, hepatic mRNA expression of tumor necrosis factor-alpha, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 was significantly lower than that in pancreatitis mice treated with RLS. Compared to RLS treatment, treatment with EP significantly decreased the number of inflammatory cell infiltration and markedly inhibited hepatic nuclear factor-kappa B DNA binding; EP therapy dramatically inhibited high motility group B1 release from inflamed hepatic tissue and significantly decreased the concentration of hepatic tissue malondialdehyde, an oxidative stress parameter. EP treatment also significantly improved body circulating blood volume. CONCLUSION: EP is a potent anti-inflammatory and anti-oxidative agent to ameliorate hepatic local inflammatory response and resultantly decreases liver injury secondary to SAP.

Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity.

INTRODUCTION: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP. METHODS: ALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours. RESULTS: Seventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-kappaB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration. CONCLUSIONS: Prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.