Mass Spectrometry-Based Metabolite Profiling in the Mouse Liver following Exposure to Ultraviolet B Radiation

Although many studies have been performed on the effects of ultraviolet (UV) radiation on the skin, only a limited number of reports have investigated these effects on non-skin tissue. This study aimed to describe the metabolite changes in the liver of hairless mice following chronic exposure to UVB radiation. We did not observe significant macroscopic changes or alterations in hepatic cholesterol and triglyceride levels in the liver of UVB-irradiated mice, compared with those for normal mice. In this study, we detected hepatic metabolite changes by UVB exposure and identified several amino acids, fatty acids, nucleosides, carbohydrates, phospholipids, lysophospholipids, and taurine-conjugated cholic acids as candidate biomarkers in response to UVB radiation in the mouse liver by using various mass spectrometry (MS)-based metabolite profiling including ultra-performance liquid chromatography-quadrupole time-of-flight (TOF)-MS, gas chromatography-TOF-MS and nanomate LTQ-MS. Glutamine exhibited the most dramatic change with a 5-fold increase in quantity. The results from altering several types of metabolites suggest that chronic UVB irradiation may impact significantly on major hepatic metabolism processes, despite the fact that the liver is not directly exposed to UVB radiation. MS-based metabolomic approach for determining regulatory hepatic metabolites following UV irradiation will provide a better understanding of the relationship between internal organs and UV light.     

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.     

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.     

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.     

Overexpression of miR-155 in the Liver of Transgenic Mice Alters the Expression Profiling of Hepatic Genes Associated with Lipid Metabolism

Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.     

Short term regulation of hepatocyte glutathione content by hepatic sinusoidal cells in co-culture

An inadequate balance between oxidant species and antioxidant mechanisms may constitute the primary mechanisms of a number of pathologies. The liver plays a central role in this balance: parenchymal hepatic cells contain and export especially high levels of the antioxidant glutathione and activated Kupffer cells release inflammation mediators and reactive oxygen species. There is growing evidence of a paracrine regulation of hepatic function by means of a fluent intercellular communication which must still be fully elucidated, especially in basal conditions. In vivo models provide often too complex results but, in vitro, tissue interactions are left aside; therefore it is important to find new experimental models to address cell communication studies. Here we propose the complementary use of three models to study liver glutathione system regulation in basal conditions: pure parenchymal cells primary cultures, addition of sinusoidal cell conditioned media to parenchymal cells and co-culture of sinusoidal cells using porous membranes. We have also developed a high specifity immunofluorescent method for the complete characterization of sinusoidal cell populations by flow cytometry and confocal microscopy. Our results show that Kupffer cells possess higher levels of reactive oxygen species than sinusoidal endothelial cells even in basal conditions. We also report that the glutathione content of hepatic parenchymal cells in basal conditions is regulated by a sinusoidal-parenchymal cells cross-talk and suggest the existence of a paracrine circuit in the management of liver oxidative stress.     

Nitric oxide modulates glutathione synthesis during endotoxemia

Nitric oxide is known to modulate intracellular glutathione levels, but the relationship between nitric oxide synthesis and glutathione metabolism during endotoxemia is unknown. The present study was designed to examine the effects of increased nitric oxide formation on hepatic glutathione synthesis and antioxidant defense in endotoxemic mice. Our results demonstrate that hepatic glutathione synthesis is decreased for 24 h following injection of lipopolysaccharide (LPS). Administration of the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTZ), failed to normalize hepatic glutathione concentration, and suggests that decreased γ-glutamylcysteine ligase activity is primarily responsible for the decrease in hepatic glutathione levels during endotoxemia. Inhibition of nitric oxide synthesis prevented the endotoxin-induced changes in hepatic and plasma glutathione status and up-regulated liver glutathione and cysteine synthesis pathways at the level of gene expression. Furthermore, whereas the activity of glutathione peroxidase and glutathione S-transferase decreased during endotoxemia, both of these changes were prevented by inhibition of nitric oxide synthesis. In conclusion, increased nitric oxide synthesis during endotoxemia causes marked changes in glutathione flux and defenses against oxidative stress in the liver.     

Nitric oxide modulates glutathione synthesis during endotoxemia

Nitric oxide is known to modulate intracellular glutathione levels, but the relationship between nitric oxide synthesis and glutathione metabolism during endotoxemia is unknown. The present study was designed to examine the effects of increased nitric oxide formation on hepatic glutathione synthesis and antioxidant defense in endotoxemic mice. Our results demonstrate that hepatic glutathione synthesis is decreased for 24 h following injection of lipopolysaccharide (LPS). Administration of the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTZ), failed to normalize hepatic glutathione concentration, and suggests that decreased γ-glutamylcysteine ligase activity is primarily responsible for the decrease in hepatic glutathione levels during endotoxemia. Inhibition of nitric oxide synthesis prevented the endotoxin-induced changes in hepatic and plasma glutathione status and up-regulated liver glutathione and cysteine synthesis pathways at the level of gene expression. Furthermore, whereas the activity of glutathione peroxidase and glutathione S-transferase decreased during endotoxemia, both of these changes were prevented by inhibition of nitric oxide synthesis. In conclusion, increased nitric oxide synthesis during endotoxemia causes marked changes in glutathione flux and defenses against oxidative stress in the liver.     

Protective effects of cassia seed ethanol extract against carbon tetrachloride-induced liver injury in mice

Oxidative stress has been recognized as a critical pathogenetic mechanism for the initiation and the progression of hepatic injury in a variety of liver disorders. Antioxidants, including many natural compounds or extracts, have been used to cope with liver disorders. The present study was designed to investigate the hepatoprotective effects of cassia seed ethanol extract (CSE) in carbon tetrachloride (CCl(4))-induced liver injury in mice. The animals were pre-treated with different doses of CSE (0.5, 1.0, 2.0 g/kg body weight) or distilled water for 5 days, then were injected intraperitoneally with CCl(4) (0.1% in corn oil, v/v, 20 ml/kg body weight), and sacrificed at 16 hours after CCl(4) exposure. The serum aminotransferase activities, histopathological changes, hepatic and mitochondrial antioxidant indexes, and cytochrome P450 2E1 (CYP2E1) activities were examined. Consistent with previous studies, acute CCl(4) administration caused great lesion to the liver, shown by the elevation of the serum aminotransferase activities, mitochondria membrane permeability transition (MPT), and the ballooning degeneration of hepatocytes. However, these adverse effects were all significantly inhibited by CSE pretreatment. CCl(4)-induced decrease of the CYP2E1 activity was dose-dependently inhibited by CSE pretreatment. Furthermore, CSE dramatically decreased the hepatic and mitochondrial malondialdehyde (MDA) levels, increased the hepatic and mitochondrial glutathione (GSH) levels, and restored the activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione S-transferase (GST). These results suggested that CSE could protect mice against CCl(4)-induced liver injury via enhancement of the antioxidant capacity.     

Characterization of a new endogenous vitamin A metabolite

Here, we describe the discovery of a new major endogenous vitamin A metabolite with particularly high hepatic concentrations. This metabolite was isolated from mouse livers and was characterized as 9-cis-4-oxo-13,14-dihydro-retinoic acid (RA) based on mass spectral, ultraviolet, and nuclear magnetic resonance analyses. It was also detected in one human liver. To gain further insight into endogenous retinoid metabolism, mice were fed over a period of 14 days ad libitum with diets enriched with different amounts of retinyl palmitate [15,000, 45,000 or 150,000 international units (IU)/kg diet]. Higher retinyl palmitate amounts in the diet resulted surprisingly in a dose-dependent decrease in all-trans-RA levels in serum, kidney, and brain, whereas levels of 9-cis-4-oxo-13,14-dihydro-RA, retinol, and retinyl esters were dose-dependently elevated in serum, kidney, and liver. 13-cis-RA levels could be detected in serum, liver, and kidney, but were unaffected by the dietary vitamin A status. 9-cis-RA levels were below the detection limit of 0.2 ng/ml serum or 0.4 ng/g tissue. This study indicates that the oxidation at C4 of the cyclohexenyl ring, isomerization of the C9/C10 double bond, and reduction of the C13/C14 double bond are major endogenous metabolic pathways of vitamin A.     

Serum IGF-1 levels correlate negatively to liver damage in diabetic rats

Abstract

Diabetes and insulin resistance frequently cause liver damage. Diabetes also causes reduction in liver and blood IGF-1 levels. We investigated the relation between liver damage and IGF-1 levels in diabetic rats. Fourteen Wistar albino rats were divided into control and diabetic groups. Diabetes was induced by streptozotocin. Rats were sacrificed for biochemical and histologic examinations 2 weeks after streptozotocin injection. Serum and liver IGF-1 levels were decreased, liver malondialdehyde (MDA) levels were increased, glutathione peroxidase (GPx) enzymes activities were decreased and serum alanine aminotransferase (ALT) levels were increased in diabetic group. Microscopic examination of liver revealed that normal tissue organization was disrupted in streptozotocin-induced diabetic rats. There was a strongly positive correlation between blood glucose levels and liver injury, and blood and liver IGF-1 levels. There was a strongly negative correlation between blood IGF-1 levels and hepatic injury. Our results suggest that reduction of blood IGF-1 levels correlates with hepatic injury and circulating IGF-1 levels may have predictive value for determining hepatic damage that results from diabetes. In addition, circulating IGF-1 levels are correlated with glutathione levels and the oxidative stress status of diabetic rat liver.     

Bromobenzene and p-bromophenol toxicity and covalent binding invivo

A hepatotoxic dose of bromobenzene (3 mmoles/kg) decreases hepatic glutathione concentration in rats by approximately 80% within 5 hr following ip injection. A major bromobenzene metabolite, p-bromophenol at a similar dose did not significantly alter hepatic glutathione levels compared to controls. Twenty four hr after administration, serum glutamate pyruvate transaminase (SGPT) levels were significantly increased by bromobenzene but not by p-bromophenol. After ¹⁴C-bromobenzene administration, a significant amount of covalently bound radiolabel was detected in liver, kidney and small intestine. A small amount of covalently bound radiolabel was also detected in the lung. After a similar dose of ¹⁴C-bromophenol, covalently bound radiolabel was found in liver (62% of the amount detected with ¹⁴C-bromobenzene) and smaller amounts were detected in kidney, small intestine and lung. These data are consistent with the view that the hepatotoxity and glutathione depleting ability of bromobenzene are mediated mainly by bromobenzene-3, 4-oxide rather than by chemically reactive metabolites of p-bromophenol derived from bromobenzene. Covalently bound radiolabel from ¹⁴C-bromobenzene, however, may be derived from both bromobenzene-3, 4-oxide and the nontoxic reactive metabolites of p-bromophenol.     

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.     

Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver

Infection is associated with low serum thyroid hormones and thyrotropin levels. Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression. In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration. Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha(1), TR-alpha(2), and TR-beta levels were reduced by 55, 87, and 41%, respectively. We previously reported that LPS rapidly decreases RXR protein levels in liver. Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls. A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding. We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.     

Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury

Summary

Glutathione (GSH) plays several important roles in the protection of cells against oxidative damage, particularly following exposure to xenobiotics. Ferric nitrilotriacetate (Fe-NTA) is a potent depletor of GSH and also enhances tissue lipid peroxidation. In this study, we show the effect of Fe-NTA treatment on hepatic GSH and some of the glutathione metabolizing enzymes, oxidant generation and liver damage. The level of hepatic GSH and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase all decrease following Fe-NTA administration. In these parameters the maximum decrease occurred at 12 h following Fe-NTA treatment. In contrast, γ-glutamyl transpeptidase was increased at this time. Not surprisingly, the increase in the activity of γ-glutamyl transpeptidase and decreases in GSH, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione S-transferase were found to be dependent on the dose of Fe-NTA administered. Fe-NTA administration also enhances the production of H₂O₂ and increases hepatic lipid peroxidation. Parallel to these changes, Fe-NTA enhances liver damage as evidenced by increases in serum transaminases. Once again, the liver damage is dependent on the dose of Fe-NTA and is maximal at 12 h. Pretreatment of animals with antioxidant, butylated hydroxy anisole (BHA), protects against Fe-NTA-mediated hepatotoxicity further supporting the involvement of oxidative stress in Fe-NTA-mediated hepatic damage. In aggregate, our results indicate that Fe-NTA administration eventuates in decreased hepatic GSH, a fall in the activities of glutathione metabolizing enzymes and excessive production of oxidants, all of which are involved in the cascade of events leading to iron-mediated hepatic injury.     

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-¹³C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.     

Severe dysfunction of respiratory chain and cholesterol metabolism in Atp7b(-/-) mice as a model for Wilson disease

Wilson disease (WD) is caused by mutations of the WD gene ATP7B resulting in copper accumulation in different tissues. WD patients display hepatic and neurological disease with yet poorly understood pathomechanisms. Therefore, we studied age-dependent (3, 6, 47weeks) biochemical and bioenergetical changes in Atp7b(-/-) mice focusing on liver and brain. Mutant mice showed strongly elevated copper and iron levels. Age-dependently decreasing hepatic reduced glutathione levels along with increasing oxidized to reduced glutathione ratios in liver and brain of 47weeks old mice as well as elevated hepatic and cerebral superoxide dismutase activities in 3weeks old mutant mice highlighted oxidative stress in the investigated tissues. We could not find evidence that amino acid metabolism or beta-oxidation is impaired by deficiency of ATP7B. In contrast, sterol metabolism was severely dysregulated. In brains of 3week old mice cholesterol, 8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and lathosterol were all highly increased. These changes reversed age-dependently resulting in reduced levels of all previously increased sterol metabolites in 47weeks old mice. A similar pattern of sterol metabolite changes was found in hepatic tissue, though less pronounced. Moreover, mitochondrial energy production was severely affected. Respiratory chain complex I activity was increased in liver and brain of mutant mice, whereas complex II, III, and IV activities were reduced. In addition, aconitase activity was diminished in brains of Atp7b(-/-) mice. Summarizing, our study reveals oxidative stress along with severe dysfunction of mitochondrial energy production and of sterol metabolism in Atp7b(-/-) mice shedding new light on the pathogenesis of WD.     

Low content of hepatic reduced glutathione in patients with Wilson's disease

In five of six patients with symptomatic Wilson's disease (WD) with increased hepatic copper content, increased renal copper excretion, and decreased serum concentrations of ceruloplasmin, significantly low levels of hepatic reduced glutathione (GSH) were found. Three of these patients showed increased levels of oxidized glutathione which in part could account for the missing GSH. These changes may result from increased lipid peroxidation due to the rise of intracellular copper concentration. Furthermore, WD patients showed a 50% decrease in the activity of hepatic GSH S-transferases. From these results we conclude that the disturbance in the hepatic glutathione system of patients with symptomatic WD may contribute to the perpetuation of liver damage. These patients, additionally, may be predisposed to an increased sensitivity to drugs interacting with glutathione.     

Transfer of nitric oxide from the liver to erythrocytes--an ESR study using nitroglycerin-treated mice

Nitric oxide (NO) formation in the liver and blood of the mouse following intraperitoneal treatment with nitroglycerin (glycerol trinitrate, GTN) was determined using electron spin resonance (ESR) spectroscopy. ESR signals of heme-NO complexes were detected at maximum levels within 5 min in the liver, but increased to a maximum level about 15-30 min later in the blood. GTN is not metabolized to release NO in vitro in the blood of the mouse. The hepatic microsomes which showed the heme-NO complexes ESR signals were incubated with mouse erythrocytes, with the result that a hemoglobin-NO signal was obtained from the erythrocytes. The activities of microsomal cytochrome P-450, the hepatic level of glutathione, and the reduction rate of nitroxide radicals in the in vivo liver, measured using L-band ESR spectroscopy, were temporarily decreased following GTN administration. In conclusion, NO in the liver could be scavenged by circulating erythrocytes, which might minimize NO-induced liver damage.