Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Hepatocyte synthesis of NO in response to the proinflammatory cytokines is associated with a multitude of autocrine and paracrine biochemical activities, such as inhibition of protein synthesis and ribonucleotide reductase and activation of soluble guanylyl cyclase (Nathan, 1992). However, the physiological relevance of these observations remains unknown and controversial. In particular, the potential hepatoprotective or hepatotoxic role of NO in states of oxidative injury is the subject of ongoing investigation. In an in vivo murine model of endotoxin-induced hepatocellular injury, NO synthesis was associated with inhibition of microthrombosis and oxygen-free radical-mediated hepatotoxicity (Harbrecht et al., 1992a, 1992b). In the setting of APAP-mediated oxidative stress in an in vitro model of rat hepatocytes in primary culture, we have previously demonstrated that inhibition of hepatocyte NO synthesis exacerbates injury (Kuo and Slivka, 1994). In this model, NO synthesis was induced by coincubation of hepatocytes with a combination of IL-1, interferon-, tumor necrosis factor- and endotoxin. Inhibition of NO synthesis accelerated APAP-induced depletion of intracellular reduced glutathione, the major nonprotein thiol responsible for electrophile detoxification. Interestingly, incubation of hepatocytes with APAP or benzenetriol, an exogenous source of spontaneous superoxide radical production, increased cytokine-mediated NO synthesis (Kuo and Abe, 1995; Kuo and Slivka, 1994). These observations suggest that: 1) hepatocyte production of NO may be protective in states of oxidative injury and conversely, 2) oxidative stress may increase hepatocyte NO synthesis as a protective mechanism. It is unknown whether these observations are dependent on a specific proinflammatory cytokine or rather upon the end-product itself, NO. Also, the mechanism by which oxidative stress increases NO production has not been addressed.

Using APAP as a source of oxidant injury, we hypothesized that hepatocyte NO synthesis, mediated by IL-1 alone, is hepatoprotective and that the degree of APAP-induced oxidative stress, in part, regulates NO production. Our purpose was to determine the effects of 1) IL-1-mediated NO synthesis on APAP-associated hepatotoxicity and cytochrome P450 isoenzyme activity, the major pathway for production of toxic APAP metabolites and 2) APAP-induced oxidative stress on the pathway for hepatocyte NO production. Specifically, using rat hepatocytes in primary culture, APAP incubation and IL-1-mediated NO synthesis were examined. Hepatocyte injury was determined by AST release, although the NO metabolites, nitrite and nitrate, were measured as indicators of NO synthesis. To determine whether IL-1 or NO altered the metabolic pathway for APAP, cytochrome P450 isozyme mass and enzymatic activity were measured. Finally, the metabolic pathway for NO synthesis was analyzed with respect to iNOS enzyme activity and molar content, steady-state iNOS mRNA expression, iNOS gene transcription and iNOS promoter activity to define the synthetic step that is regulated by the presence of APAP-mediated oxidative stress.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. Recombinant IL-1 was obtained from R&D Systems (Minneapolis, MN). L-Arginine, N-acetyl-L-cysteine, dithiothreitol, butylated hydroxytoluene, 7-ethoxyresorufin, phenacetin, erythromicin, aniline, SOD, BSA, sodium nitrite, nitrate reductase, methylene blue, HEPES and 8-bromo-cyclic GMP were obtained from Sigma Chemical Co. (St. Louis, MO). ODQ was purchased from ICN Pharmaceuticals, Inc. (St. Louis, MO). Williams' E media, FCS, penicillin/streptomycin, trypsin, and EDTA were purchased from Gibco Co. (Grand Island, NY). [³H]-L-arginine and Biotrak cyclic GMP enzyme immunoassay system were purchased from Amersham Inc. (Arlington Heights, IL). Collagenase D was purchased from Boehringer-Ingelheim Co. (Indianapolis, IN). Percoll was obtained from Pharmacia Co. (Uppsala, Sweden). N-methyl-L-arginine was purchased from Calbiochem (San Diego, CA). pSV--galactosidase vector was obtained from Promega Inc., Madison, WI. All other chemicals were of reagent grade. The Nco1 restriction endonuclease fragment of murine macrophage derived inducible NOS cDNA was a gift of J. Cunningham, M.D., Brigham and Women's Hospital, Boston, MA (GenBank M84373). pNOS-CAT, constructed from pCAT-Basic (Promega Corp., Madison, WI) with the rat hepatocyte iNOS promoter (GenBank X95629) fused upstream to a chloramphenicol acetyltransferase gene, was a gift from Professor W. Eberhardt, University of Basel, Basel, Switzerland.

Rat hepatocyte isolation. Male Lewis rats (200-300 g; Harlan Inc., Indianapolis, IN) fed water and food ad libitum were used for hepatocyte isolation as described by Schuetz et al. (1986). After anesthetization with sodium pentobarbital, the portal vein was cannulated. The liver was perfused with calcium-free Krebs' bicarbonate buffer followed by 150 mg collagenase D in 200 ml Krebs' bicarbonate buffer containing 1.2 mmol/liter CaCl₂ and 1.8% bovine serum albumin. All solutions were maintained at 37°C and aerated with 95% O₂/5% CO₂. The partially digested liver was excised, passed over 60-µm nylon mesh and resuspended in DPBS. Hepatocytes were purified by centrifugation through DPBS at 50 × g for 5 min. After a second centrifugation through a 30% Percoll-DPBS gradient, hepatocytes were resuspended in Williams' E media with 1 mmol/liter L-arginine, 1 µmol/liter insulin, 15 mmol/liter HEPES (pH 7.4), penicillin/streptomycin and 10% heat-inactivated low endotoxin FCS. Hepatocyte purity was assessed by leukocyte esterase staining, whereas viability was assessed by trypan blue exclusion. Preparations were routinely >90% viable and >98% pure. The cell suspension was incubated in plastic wells for 30 min to remove residual Kupffer cells. Hepatocytes were then plated at a density of 5.0 × 10⁵ cells/ml onto collagen-coated wells. After 2 hr, the media was changed to remove unattached cells. After 24 hr of incubation at 37°C in 95% O₂/5% CO₂, cells were washed twice and fresh medium was applied for experimental use.

Generation of NO in isolated rat hepatocytes. Isolated rat hepatocytes were stimulated to produce NO by incubation in Williams' E media with 10% heat inactivated FCS, 50 ng/ml IL-1, and 1 mmol/liter L-arginine. In selected instances, the competitive substrate inhibitor of NO synthase, NMA or the hepatotoxin, acetaminophen (0-10 mM), was added. After incubation for 18 hr at 37°C in 95% O₂/5% CO₂, the supernatant was aspirated and the cells washed twice with DPBS. After treatment with trypsin/EDTA, the hepatocytes were harvested for biochemical assays. IL-1 stimulation, in the presence and absence of L-NMA, was not associated with hepatocyte injury as determined by AST release and trypan blue exclusion (data not shown).

Measurement of NO. NO release from cells in culture was quantified by measurement of the NO metabolite, nitrite, using the technique of Snell and Snell (1984). To reduce nitrate to nitrite, conditioned media (200 µl) was incubated in the presence of 1.0 U nitrate reductase, 50 µM NADPH and 5 µM FAD. Sulfanilamide (1%) in 0.5N HCl (50% v/v) was then added. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-(naphthyl))ethylenediamine was added; after incubation at room temperature for 10 min, absorbance at 570 nm was compared with that of an NaNO₂ standard.

Preparation of microsomes from cultured rat hepatocytes. Samples of isolated rat hepatocytes were homogenized in four volumes of 250 mM sucrose, 0.1 M potassium phosphate, pH 7.4, 1 mM EDTA, 0.1 mM DTT. After centrifugation at 10,000 × g for 25 min, the supernatant was again centrifuged at 100,000 × g for 1 hr at 4°C. The pellet was homogenized in 0.1 M sodium pyrophosphate, pH 7.25, 23 µM butylated hydroxytoluene, 1 mM EDTA and again centrifuged at 100,000 × g for 1 hr at 4°C. The resulting microsomal preparation was suspended in 0.1 M potassium phosphate, pH 7.4, glycerol (20%, v/v), 1 mM EDTA, 0.1 mM DTT. Microsomal preparations were kept at 70°C until ready for use.

Cytochrome P450 isoform-specific enzyme activities and immunoblot analysis. Cytochrome P450 isoform-specific enzyme activities, ethoxyresorufin deethylase (CYPIA2), phenacetin deethylase (CYPIA1), aniline hydroxylase (CYPIIE1) and erythromicin demethylase (CYPIIIA), were assayed using previously published techniques (Combalbert et al., 1989; Diaz et al., 1990; Bonfils et al., 1990). Microsomes were diluted to 0.2 mg/ml in 0.1 M potassium phosphate, pH 7.4, and incubated at 37°C for 5 min in the presence of the appropriate substrate. The reaction was initiated by addition of 1 mM NADPH and allowed to proceed at 37°C. Final substrate concentrations were 5 µM ethoxyresorufin, 4 µM phenacetin, 1 mM erythromicin and 1 mM aniline. Turnover (pmol of product formed/mg cell protein/unit time) was determined from the initial phase of the reaction where substrate oxidation was linear as a function of time. Immunoblot analysis of microsomal cytochrome P450 isoform content was performed with the ECL detection system (Amersham) as previously described. Briefly, hepatocytes were washed three times in PBS and incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. Separation was performed by 12% SDS-PAGE, and then electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 milliamps. The membrane was blocked overnight at 4°C in TBS containing 3% BSA. Blocked membranes were incubated with the appropriate cytochrome P450 isozyme monoclonal antibodies (Amersham), washed three times in TBS/0.1% Tween and incubated with biotinylated sheep anti-murine IgG (Amersham) for 1 hr. After washing three additional times, membranes were then incubated with strepavidin-horseradish peroxidase conjugate. After washing, bound antibodies were detected by the ECL detection system (Amersham). Blots were scanned with a computerized laser densitometer (Hoeffer Scientific Instruments, San Francisco, CA) and the area under the curve normalized to a P450 isozyme standard.

Lipid hydroperoxidation products. Generation of lipid peroxidation products was determined by the TBARS method, as described by Sokol et al. (1993). Briefly, 8 hr after stimulation, hepatocytes were harvested, washed and resuspended in phosphate buffered saline, pH 7.5. Trichloroacetic acid (10% w/v) and butylated hydroxytoluene (2% w/v) were added to an aliquot of the cell suspension in a 10:1:4 ratio. After centrifugation (10,000 × g for 10 min), thiobarbituric acid (0.67% w/v) was added to the supernatant and the resulting solution was heated at 100°C for 15 min. The specimen was again centrifuged (1000 × g for 10 min) and the absorbance of the supernatant was determined at 532 nm. A standard curve was generated using malonaldehyde tetraethylacetal; TBARS results are expressed as MDA equivalents (nmol/mg protein).

Hepatocyte inducible NO synthase immunoblot analysis. Isolated rat hepatocytes were washed three times in PBS and incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. Separation was performed on a 12% SDS-PAGE, and then electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 milliamps. The membrane was blocked overnight at 4°C in TBS containing 3% BSA. Blocked membranes were incubated with iNOS Ab (monoclonal primary anti-murine macrophage iNOS antibody from Transduction Laboratories, Lexington, KY), washed three times in TBS/0.1% Tween and incubated with biotinylated sheep anti-murine IgG (Amersham) for 1 hr. After washing three additional times, membranes were incubated with strepavidin-horseradish peroxidase conjugate. After washing, bound antibodies were detected by the ECL detection system (Amersham). Blots were scanned with a computerized laser densitometer (Hoeffer Scientific Instruments, San Francisco, CA) and the area under the curve normalized to the murine macrophage iNOS enzyme standard.

Hepatocyte inducible NO synthase activity and iNOS mRNA expression. NO synthase activity in the cytosolic fraction was assayed by measuring the stoichiometric formation of L-[³H]citrulline from L-[³H]arginine (Evans et al., 1992; McMillan et al., 1992; Lamas et al., 1991). Activity was expressed as pmol/mg protein/min. iNOS mRNA expression was measured by Northern blot analysis (Sambrook et al., 1989). Total RNA was isolated from rat hepatocytes in primary culture by the guanidine isothiocyanate/phenol extraction technique (Oncogene Sciences XNPS 05, Oncogene Sciences Inc., Santa Cruz, CA). Ten µg of total RNA from each sample were fractionated on a 1.2% agarose gel containing 2.2 M formaldehyde. After capillary blotting onto Nytran-N+ (Schleicher & Schuell, Keene, NH), the membranes were hybridized with 2 ng/ml of labelled NOS cDNA probe in Amersham Rapid Hybridization Buffer (Amersham). The Nco1 restriction endonuclease fragment of murine macrophage-derived iNOS cDNA (gift of Dr. J. Cunningham, Brigham and Women's Hospital, Boston, MA; GeneBank M84373) was used as the probe. The membranes were washed at 55°C. Blots were exposed overnight at 70°C. Relative mRNA levels were quantitated by scanning densitometry and normalized to a  actin standard.

Measurement of reduced and oxidized glutathione. Rat hepatocyte suspensions were deproteinized with 0.4N perchloric acid containing 4 mg/dl DTPA added as an antioxidant. In selected instances, aliquots were deproteinized with perchloric acid containing DTPA and 40 mM N-ethylmaleimide. Acidified samples at 4°C were dispersed by vortexing and disrupted with a homogenizer. The suspensions were centrifuged at 4°C for 10 min at 10,000 × g. Reduced GSH and oxidized GSSG were determined after derivatization with 1-fluoro-2,4-dinitrobenzen; after 45 min of incubation in the dark, the samples were dessicated and resuspended in 50 µl 80% methanol. The samples were injected onto a Hewlett-Packard HPLC system with an Spherisorb NH₂ column (20 × 0.4 cm, 5 µm particles). The flow rate was 1.0 ml/min with solvent A as 80% methanol and solvent B as 0.5 M sodium acetate in 64% methanol. After injection of the sample, the mobile phase was held at 80% A, 20% B for 5 min followed by a 10-min linear gradient up to 1% A, 99% B until GSSG was eluted. The optical density of the eluate was measured at 365 nm using an ultraviolet detector (Reed et al., 1980).

Synthesis of S-nitroso-BSA. SNOBSA was synthesized by combining equimolar NaNO₂ and BSA in 0.5N HCl for 30 min at room temperature as previously described (Stamler et al., 1992). Using the technique of Snell and Snell (1984), the concentration of SNOBSA was measured by Hg⁺⁺ displacement of NO followed by diazotization with sulfanilamide and coupling to the chromophore N-(1-(napthyl)-ethylenediamine. Comparison with a known standard allows determination of displaceable NO, which entirely derived from SNOBSA. Before use, the S-nitrosoprotein solution was neutralized to pH 7.0 with 0.1N NaOH. Previous work has confirmed the presence of S-nitrosothiol bonds in the above species using ¹⁵N-nuclear magnetic resonance spectroscopy (Stamler et al., 1992). Synthesis of SNOBSA by this technique produced a molar ratio of SNOBSA to BSA of 0.85 ± 0.04. Based on the presence of a single free thiol on BSA, a single NO group per bovine serum albumin molecule is presumed (Stamler et al., 1992).

Nuclear run-on analysis. Nuclear run-on analysis was performed as previously described (Greenberg and Bender, 1993). Briefly, 100 µl of hepatocyte nuclei were incubated for 5 min at 30°C with 150 µCi of [-³²P]rUTP (800 Ci/mmol) in 100 µl of 10 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 300 mM KCl and 5.0 mM each of ATP, CTP and GTP. Labeled RNA was isolated by the acid-guanidine thiocyanate method. Before ethanol precipitation, labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-tris(hydroxymethyl)methyl 2-aminoethansulfonic acid (pH 7.4), 0.2% SDS and 10 mM EDTA. Target DNA was spotted onto nylon membranes with a slot blot apparatus. Target iNOS DNA was the Nco1 restriction endonuclease fragment of murine macrophage-derived iNOS cDNA. -Actin and  bacteriophage DNA served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 hr using 5 × 10⁶ cpm of labeled RNA in hybridization buffer (50% formamide, 4× SSC, 0.1% SDS, 5× Denhardt solution, 0.1 M sodium phosphate (pH 7.2) and 10 µg/ml salmon sperm DNA. After hybridization, the membranes were washed twice at room temperature in 2× SSC and 0.1% SDS, and three times at 56°C in 0.1× SSC and 0.1% SDS. The membranes were then exposed to x-ray film and scanned on a laser densitometer.

Transient transfection of rat hepatocytes and CAT assays. Hepatocytes were transfected by a modification of the DEAE-dextran procedure (Sambrook et al., 1989). After cells were washed twice with Williams' E media, 10 µg plasmid DNA containing the iNOS promoter construct were added per 10⁷ cells in 1 ml of Williams' E without serum prewarmed to 37°C and containing DEAE-dextran (250 µg/ml) and 50 mM Tris (pH 7.4). The suspension was incubated at 37°C for 45 to 60 min followed by a 1-min shock with 10% DMSO at room temperature. The cells were washed, distributed to 100-mm plates, each with about 5 × 10⁶ cells in 10 ml of complete medium, and incubated at 37°C in 5% CO₂. At least 24 hr later, the medium was changed, and IL-1 was added. Approximately 14 hr later, the cells were washed with ice-cold PBS, resuspended in 0.25 mM Tris (pH 7.8) and subjected to three cycles of freezing and thawing. Lysates were centrifuged (11,700 × g for 10 min at 4°C); the supernatant was heated at 65°C for 10 min to inactivate CAT inhibitors and then centrifuged as above. The supernatant was assayed for CAT activity using a CAT ELISA technique (Boehringer Mannheim, Indianapolis, IN). Transfection efficiency was normalized by cotransfection of a -galactosidase reporter gene with a constitutively active early SV40 promoter. All values are expressed as pg CAT/mL.

Determination of AST and total protein. AST in the cell culture supernatant was measured by the Gemstar automated colorimetric technique (Schiapparelli Biosystems Inc., Fairfield, NJ). Total protein was determined by the method of Lowry et al. (1951). All measured values were normalized to total cell protein to correct for variation in cell number from well to well. Total cell protein varied by <10% among the various treatment groups.

Statistical methods. All values are presented as mean ± S.E.M. of four experiments. Data were analyzed with the two-tailed unpaired Student's t test or analysis of variance with Newman-Keuls post hoc comparison, as appropriate. P values <0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

APAP and IL-1-mediated NO production. APAP-(0-10 mM) mediated hepatotoxicity was examined in the setting of IL-1-(50 ng/ml) induced NO synthesis (fig. 1A). In both control and IL-1-treated hepatocytes, increasing APAP concentration was associated with increased AST release into the culture medium. AST release in response to APAP was not statistically different between CONT and IL-1-treated hepatocytes. However, addition of the iNOS inhibitor, L-NMA, to IL-1-treated hepatocytes resulted in significantly increased AST release at all APAP concentrations. In the presence of 2 mM APAP or 5 mM APAP, IL-1/NMA treatment resulted in a ~2- to 2.5-fold increase in AST release when compared to that of IL-1 or control cells. Addition of L-NMA to control cells did not alter the AST response to APAP when compared to that of control cells alone (data not shown). NO production was also determined (fig. 1B). In control cells, NO synthesis was not significantly altered at any concentrations of APAP. Similarly, in IL-1/NMA-treated hepatocytes, NO production in the absence and presence of APAP was minimal and not significantly different from that of control cells. In contrast, NO production in IL-1-treated cells was 6-fold greater than that of Control cells. In the presence of both APAP and IL-1, NO production was found to increase in a APAP concentration-dependent fashion. Intracellular levels of reduced GSH were measured. In both control and IL-1-treated hepatocytes, GSH levels inversely correlated with APAP in a concentration dependent fashion. GSH content in control and IL-1-treated cells was not significantly different. In contrast, addition of NMA to IL-1 cells was associated with a 3-fold decrease in the intracellular content of GSH. In the presence of increasing concentrations of APAP, GSH content was noted to decrease further in IL-1/NMA-treated hepatocytes. At all concentrations of APAP, GSH content of IL-1/NMA-treated cells was significantly less than that of control or IL-1 hepatocytes (P < .01). In the presence and absence of APAP, addition of NMA to Control cells did not alter the intracellular GSH content (data not shown). Control, IL-1- and IL-1/NMA-treated hepatocytes were then incubated in the presence of both SNOBSA (50 µM), an exogenous NO donor and APAP (fig. 1D). Addition of SNOBSA to control and IL-1 hepatocytes did not significantly alter AST release in response to APAP (0-10 mM). However, in IL-1/NMA-treated cells, exogenous administration of the NO donor decreased the extent of AST release and resulted in an AST release profile that was not significantly different from that of control or IL-1-treated cells. In addition, at all APAP concentrations, intracellular GSH content in IL-1/NMA/SNOBSA-treated cells was not different from that of control or IL-1 hepatocytes (data not shown). These results suggest that 1) incubation of hepatocytes with APAP is associated with GSH depletion and cell injury, 2) inhibition of IL-1-induced NO synthesis exacerbates APAP-mediated GSH depletion and cell injury, 3) exogenous repletion of NO in IL-1/NMA-treated hepatocytes ameliorates the increased cell injury and GSH depletion associated with inhibition of endogenous NO synthesis and 4) APAP enhances IL-1-mediated NO synthesis.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   A, Acetaminophen and hepatocyte injury. Isolated rat hepatocytes in primary short-term culture were incubated in the presence of increasing concentrations of acetaminophen for 18 hr at 37°C in control (CONT), IL-1 (50 ng/ml) and IL-1 + L-NMA (100 µM) groups. Aspartate aminotransferase (AST) was assayed in the conditioned media. Data are presented as mean ± S.E.M. of four experiments. (Analysis of variance P = .001 for CONT, IL-1 and IL-1 + NMA groups; P < .01 IL-1 + NMA vs. CONT or IL-1 for 1, 2, 5 and 10 mM acetaminophen.) B, Acetaminophen and hepatocyte NO production. Isolated rat hepatocytes in primary short-term culture were incubated in the presence of increasing concentrations of acetaminophen for 18 hr at 37°C in control (CONT), IL-1 (50 ng/ml) and IL-1 + L-NMA (100 µM) groups. The NO metabolite, nitrite, was assayed in the conditioned media. Data are presented as mean ± S.E.M. of four experiments. (Analysis of variance P = .001 for CONT, IL-1 and IL-1 + NMA groups; P < .01 IL-1 vs. CONT or IL-1 + NMA for 0, 1, 2, 5 and 10 mM acetaminophen.) C, Acetaminophen and hepatocyte glutathione levels. Isolated rat hepatocytes in primary short-term culture were incubated in the presence of increasing concentrations of acetaminophen for 18 hr at 37°C in Control (CONT), IL-1 (50 ng/ml) and IL-1 + L-NMA (100 µM) groups. Intracellular stores of reduced glutathione (GSH) were determined as described in "Materials and Methods." Data are presented as mean ± S.E.M. of four experiments. (Analysis of variance P = .001 for CONT, IL-1 and IL-1 + NMA groups; P < .01 IL-1 + NMA vs. CONT or IL-1 for 0, 1, 2, 5 and 10 mM acetaminophen.) D, Acetaminophen, S-nitroso-BSA and hepatocyte injury. Isolated rat hepatocytes in primary short-term culture were incubated in the presence of increasing concentrations of acetaminophen for 18 hr at 37°C. S-nitroso-bovine serum albumin (SNOBSA, 50 µM) was added to control (CONT), IL-1 (50 ng/ml) and IL-1 + L-NMA (100 µM) hepatocytes. Intracellular stores of reduced glutathione (GSH) were determined as described in "Materials and Methods." Data are presented as mean ± S.E.M. of four experiments. (Analysis of variance P = .001 for CONT + SNOBSA, IL-1 + SNOBSA and IL-1 + NMA + SNOBSA groups.)

Guanylyl cyclase activity, APAP and IL-1 stimulation. To determine whether the hepatoprotective effect of IL-1 mediated NO synthesis in APAP induced injury was guanylyl cyclase-dependent, IL-1-treated hepatocytes were incubated in the presence of soluble GC inhibitors, methylene blue (50 µM) and ODQ (1 µM) (table 1). As noted previously, APAP (5 mM) was associated with increased cellular injury as evidenced by increased supernatant release of AST in control and IL-1-treated hepatocytes. In IL-1/NMA-treated cells, APAP-induced injury was significantly increased in comparison to that of control and IL-1-treated cells. In addition, in the context of APAP, IL-1-mediated NO synthesis was significantly increased. When methylene blue or ODQ was coincubated with IL-1, APAP-mediated hepatocyte injury was not different from that of control cells or cells treated with IL-1 alone. Measurement of hepatocyte cGMP content demonstrates that ODQ and methylene blue treatment resulted in inhibition of guanylyl cyclase activity. These results suggest that 1) the hepatoprotective effect of IL-1-induced NO production in the setting of APAP mediated injury and 2) the APAP-mediated augmentation in IL-1-mediated NO synthesis are both GC independent.

Cytochrome P450 activity, APAP and IL-1 stimulation. The enzymatic activities of the hepatocyte microsomal cytochrome P450 isozymes responsible for APAP metabolism were then examined in the context of IL-1, NMA and APAP coincubation (table 2). Activities of P450 isoforms IA1, IA2, IIE and IIIA were equivalent for control, IL-1-, control/NMA- and IL-1/NMA-treated hepatocytes. Addition of 5 mM APAP to control, IL-1-, and IL-1/NMA-treated hepatocytes did not alter the respective P450 isoform activities. In a similar fashion, immunoblot analysis demonstrated that P450 isozyme (IA1, IA2, IIE and IIIA) mass was not altered in the settings of IL-1, IL-1/NMA, APAP, IL-1 + APAP and IL-1/NMA + APAP treatment when compared to that of control cells (data not shown). These data indicate that the hepatoprotective effect of NO synthesis in APAP toxicity is not the result of alteration in P450 isozyme metabolism of APAP.

Effect of APAP on IL-1-stimulated NO synthesis. The dose response relationship between IL-1 and APAP with respect to NO synthesis in this cultured hepatocyte model was then determined (table 3). In the absence of IL-1, increased APAP concentration did not alter NO synthesis. However, in the presence of IL-1, APAP augmented NO production at each concentration of IL-1. Examination of supernatant AST levels revealed that IL-1-mediated NO synthesis did not increase the degree of APAP-induced hepatocellular injury at any concentration of IL-1. Using the technique of Berenbaum (1989), the dose-response relationship between IL-1 and APAP in the synthesis of NO fulfilled the criteria for multiplicative synergy.

Oxidative stress and IL-1-mediated NO production. Control and IL-1-treated hepatocytes were then incubated with 200 µM N-acetyl-L-cysteine (NAC), 500 U/ml SOD, 300 U/ml catalase (CAT), 20 µM desferrioxamine (DFO) and 100 µM BHT in the presence and absence of APAP to determine whether oxidative stress might play a role in either the augmentation of IL-1-mediated NO synthesis or the hepatoprotective capacity of endogenously synthesized NO (table 4). In the presence of 5 mM APAP, addition of NAC to control cells decreased AST release by 17%. NO synthesis in this setting was not different from that of control cells treated with 5 mM APAP alone. When NAC and 5 mM APAP were added to IL-1-treated hepatocytes, AST release was decreased by 17%; in addition, total NO synthesis was decreased by 18% in comparison to that of IL-1-treated cells incubated with 5 mM APAP alone. Interestingly, provision of NAC did not totally ablate the injury associated with 5 mM APAP; AST release in the IL-1 + NAC + 5 mM APAP group was still significantly greater than that of control, IL-1- or IL-1 + NAC-treated hepatocytes. Addition of SOD/CAT/DFO/BHT and NAC to IL-1 treated hepatocytes incubated with 5 mM APAP resulted in decreased NO synthesis to a value that was no different than that of IL-1-treated hepatocytes alone. In addition, AST release was also not significantly different from that of IL-1 or control cells incubated in the absence of APAP. These results indicate that ablation of oxidative stress associated with APAP hepatotoxicity completely prevented both hepatocyte injury and augmentation of IL-1-mediated NO synthesis. To further support the observation that inhibition of APAP-mediated oxidative stress decreases hepatocyte injury and augmentation of IL-1-induced NO synthesis, MDA production was determined as an indicator of the relative extent of lipid hydroperoxidation. In control, IL-1- and IL-1 + NAC-treated hepatocytes, malondialdehyde levels were not statistically different. When 5 mM APAP were added to control or IL-1-treated cells, MDA levels increased ~7-fold. Although addition of NAC to APAP + control and APAP + IL-1 groups resulted in a 2-fold decrease in MDA levels, overall extent of lipid hydroperoxidation was still significantly greater than that noted in control and IL-1 cells. When the combination of SOD/CAT/DFO/BHT was added with NAC to APAP + control and APAP + IL-1-treated hepatocytes, MDA production was restored to levels that were not statistically different from that of control and IL-1-treated cells. The trends in MDA levels paralleled those noted with AST release. These results suggest that inhibition of APAP-mediated lipid peroxidation was associated with decreased AST release and decreased augmentation of IL-1-induced NO synthesis.

iNOS and APAP associated oxidative stress. Cytosolic iNOS activity was then examined (table 5). In control cells, iNOS activity was not altered in the presence of NMA or 5 mM APAP. In the presence of 2 and 5 mM APAP, iNOS activity in IL-1-treated hepatocytes increased 64 and 188%, respectively. Addition of NAC to IL-1 + 5 mM APAP-treated cells resulted in decreased iNOS activity, although more than that of IL-1-treated hepatocytes alone. Addition of both NAC and SOD/CAT/DFO/BHT to IL-1 + 5 mM APAP cells further decreased iNOS activity to a level that was not different than that of IL-1-treated cells alone. Immunoblot analysis of iNOS was performed (fig. 2). In the presence of IL-1, iNOS protein was strongly expressed; no iNOS protein was detected in control or control + 5 mM APAP cells. When 5 mM APAP were added to IL-1-treated hepatocytes, iNOS protein was increased by almost 2-fold (170%), as determined by densitometric analysis. Addition of NAC and SOD/CAT/DFO/BHT to IL-1 + 5 mM APAP-treated cells decreased iNOS protein to a level that was 87% that of IL-1 + 5 mM APAP. These data indicate that ablation of oxidative stress decreased the APAP-mediated augmentation of IL-1-induced iNOS enzyme activity and total enzyme mass.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Immunoblot analysis of iNOS protein in rat hepatocytes. A, Rat hepatocytes were washed three times in phosphate buffered saline and incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. Separation was performed on a 12% SDS-PAGE, and then electrotransferred to a polyester-supported nitrocellulose membrane. The membrane was blocked overnight at 4°C in TBS (10 mM Tris-HCl at pH 7.5, 150 mM NaCl) containing 3% BSA. Blocked membranes were incubated with a monoclonal antibody directed against rat iNOS protein, washed three times in TBS/0.1% Tween, and incubated with biotinylated sheep anti-murine IgG for 1 hr. After washing again three times, membranes were then incubated with strepavidin-horse radish peroxidase conjugate. After washing, bound antibodies were detected by a chemiluminescent technique. A representative immunoblot is presented. Lane 1, Control; lane 2, IL-1 (50 ng/ml); lane 3, control + 5 mM acetaminophen; lane 4, IL-1 + 5 mM acetaminophen; lane 5, IL-1 + 5 mM acetaminophen + N-acetylcysteine + antioxidants; lane 6, iNOS protein standard. B, Densitometric analysis of the iNOS immunoblot.

iNOS expression and APAP-associated oxidative stress. Steady-state expression of iNOS mRNA was examined using Northern blot analysis (fig. 3). In control and control + 2 mM APAP-treated hepatocytes, no detectable iNOS mRNA was present. In control + 5 mM APAP-treated cells, a very faint band was present with a relative density of 0.2. In IL-1-stimulated hepatocytes, relative iNOS mRNA expression increased to 3.3. When 2, 5 and 8 mM APAP were added to IL-1-stimulated cells, the relative expression of iNOS mRNA increased to 4.7, 6.7 and 7.5, respectively. Nuclear run-on analysis was performed to determine whether or not the increase in iNOS mRNA that occurred in association with increased APAP concentrations was the result of increased iNOS transcription (fig. 4). In control and control + 5 mM APAP-treated hepatocytes, there was no detectable transcription of the iNOS gene. In IL-1-treated hepatocytes, transcription of the iNOS gene was detected with a relative density of 0.18 (with respect to -actin). In the presence of 2 mM APAP, IL-1-mediated transcription of the iNOS gene increased to 0.24. Similarly, in the presence of 5 mM APAP, IL-1-induced iNOS gene transcription increased more than 7-fold to a relative density of 1.25 in comparison to that of IL-1 stimulation alone. Additional nuclear run-on analysis was performed to determine whether increased iNOS gene transcription in the setting of concomitant IL-1 and APAP incubation was the result of oxidative stress (fig. 5). As noted previously, no detectable iNOS gene transcription was noted in control hepatocytes. Transcription of the iNOS gene in IL-1-treated cells was equivalent to that noted in IL-1 + NAC + SOD/CAT/DFO/BHT-treated hepatocytes in the presence of 2 and 5 mM APAP. As determined by densitometry, relative iNOS transcription in IL-1-stimulated cells was 2.0, although that of IL-1 + NAC + SOD/CAT/DFO/BHT-treated hepatocytes in the presence of 2 and 5 mM APAP was 1.8 and 1.8, respectively. These data suggest that APAP-mediated oxidative stress increases IL-1-induced iNOS gene transcription and that inhibition of oxidant stress in this setting decreases iNOS gene transcription to a level equivalent to that noted in the absence of APAP. To examine further the role of APAP-mediated oxidative stress on iNOS gene transcription, rat hepatocytes in primary culture were transiently transfected with a plasmid containing the murine macrophage iNOS promoter and a CAT reporter gene (table 6). In control hepatocytes with and without APAP, minimal acetylation products were detectable. In IL-1-stimulated hepatocytes, a 50-fold increase in promoter activity was noted. Addition of 5 mM APAP to IL-1-stimulated hepatocytes resulted in a 2-fold increase in promoter activity compared to that of IL-1-stimulated hepatocytes alone. Ablation of APAP-induced oxidative stress with addition of NAC + SOD/CAT/DFO/BHT to IL-1 + 5 mM APAP-treated hepatocytes resulted in a return in promoter activity to a level equivalent to that noted in IL-1-stimulated hepatocytes. These results suggest that oxidant stress mediated by APAP augments IL-1-induced NO production by a promoter specific effect on iNOS gene transcription and thereby enhances iNOS gene transcription.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of iNOS mRNA. A, Total RNA from rat hepatocytes in primary culture was isolated by the guanidine isothiocyanate/phenol extraction technique. Ten µg of total RNA from each sample were fractionated on a 1.2% agarose gel containing 2.2 M formaldehyde. After capillary blotting onto a nylon membrane, the membranes were hybridized with 2 ng/ml of labelled NOS cDNA probe. The Nco1 restriction endonuclease fragment of murine macrophage derived inducible NOS cDNA (gift of Dr. J. Cunningham, Brigham and Women's Hospital; GeneBank M84373) was used as the probe. The membranes were washed at 55°C. Blots were exposed overnight at 70°C. Lane 1, Control; lane 2, control + 2 mM acetaminophen; lane 3, control + 5 mM acetaminophen; lane 4, IL-1 (50 ng/ml); lane 5, IL-1 + 2 mM acetaminophen; lane 6, IL-1 + 5 mM acetaminophen; lane 7, IL-1 + 8 mM acetaminophen. A representative Northern blot is presented. B, Relative mRNA levels were quantitated by scanning densitometry and normalized to a  actin standard.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Nuclear run-on analysis of iNOS gene transcription. A, 100 µl of hepatocyte nuclei were incubated for 5 min. at 30°C with 150 µCi of [-32P]rUTP (800 Ci/mmol) in 100 µl of 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl and 5.0 mM each of ATP, CTP and GTP. Labeled RNA was isolated by the acid-guanidium thiocyanate method. Before ethanol precipitation, labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-Tris(hydroxymethyl)methyl2-aminoethansulfonic acid (pH 7.4), 0.2% SDS and 10 mM EDTA. Target DNA was spotted onto nylon membranes with a slot blot apparatus. Target iNOS DNA was the Nco1 restriction endonuclease fragment of murine macrophage derived iNOS cDNA. -Actin and -bacteriophage DNA served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 hr using 5 × 106 cpm of labeled RNA in hybridization buffer [50% formamide, 4× SSC, 0.1% SDS, 5× Denhardt solution, 0.1 M sodium phosphate (pH 7.2) and 10 µg/ml salmon sperm DNA]. After hybridization, the membranes were washed twice at room temperature in 2× SSC and 0.1% SDS, and three times at 56°C in 0.1× SSC and 0.1% SDS. Lane 1, Control; lane 2, IL-1 (50 ng/ml); lane 3, IL-1 + 2 mM acetaminophen; lane 4, control + 5 mM acetaminophen; lane 5, IL-1 + 5 mM acetaminophen. A representative nuclear run-on blot is presented. B, Densitometric analysis of a nuclear run-on blot.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Nuclear run-on analysis of iNOS gene transcription. A. 100 µl of hepatocyte nuclei were incubated for 5 min at 30°C with 150 µCi of [-32P]rUTP (800 Ci/mmol) in 100 µl of 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl and 5.0 mM each of ATP, CTP and GTP. Labeled RNA was isolated by the acid-guanidium thiocyanate method. Before ethanol precipitation, labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-Tris(hydroxymethyl)methyl2-aminoethansulfonic acid (pH 7.4), 0.2% SDS and 10 mM EDTA. Target DNA was spotted onto nylon membranes with a slot blot apparatus. Target iNOS DNA was the Nco1 restriction endonuclease fragment of murine macrophage-derived iNOS cDNA. -actin and -bacteriophage DNA served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 hr using 5 × 106 cpm of labeled RNA in hybridization buffer [50% formamide, 4× SSC, 0.1% SDS, 5× Denhardt solution, 0.1 M sodium phosphate (pH 7.2) and 10 µg/ml salmon sperm DNA]. After hybridization, the membranes were washed twice at room temperature in 2× SSC and 0.1% SDS, and three times at 56°C in 0.1× SSC and 0.1% SDS. Lane 1, Control; lane 2, IL-1 (50 ng/ml); lane 3, IL-1 + 2 mM acetaminophen + N-acetylcysteine + antioxidants; lane 4, IL-1 + 5 mM acetaminophen + N-acetyl-L-cysteine + antioxidants. A representative nuclear runon blot is presented. B, Densitometric analysis of a nuclear run-on blot.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The role of endogenous hepatocyte NO synthesis in the setting of oxidative stress is a subject of ongoing controversy and investigation. In our series of experiments, we demonstrate that inhibition of IL-1-induced synthesis of NO is hepatoprotective in the presence of APAP. Inhibition of NO production was associated with accelerated depletion of intracellular stores of reduced glutathione. The underlying mechanism was guanylyl cyclase-independent and was not the result of an IL-1- or NO-related perturbation in activity or total mass of the cytochrome P450 isozymes responsible for APAP metabolism. Conversely, APAP increased IL-1-induced NO production through an oxidative stress-associated mechanism involving iNOS promoter specific effects on transcription. APAP and IL-1 acted in a pattern of multiplicative synergy to increase NO synthesis. Ablation of APAP-mediated oxidative stress decreased iNOS promoter activity and transcription to a level that was not different from that of hepatocytes stimulated in the absence of APAP. In summary, within the setting of APAP-associated oxidative stress, IL-1-mediated hepatocyte synthesis of NO was not only protective, but was also augmented through a mechanism dependent on iNOS gene transcription. If considered within a larger context, hepatocyte synthesis of NO may exist as a protective mechanism against oxidative injury in proinflammatory states typified by allograft rejection, ischemia-reperfusion, sepsis and toxin-induced injury.

NO is a short-lived free radical with multiple cell- and organ-specific functions (Nathan, 1992; Nanji et al., 1995). However, within the realm of oxidative injury, the potential toxic or protective role of NO has been the subject of controversy. Several in vitro studies have demonstrated that NO can inhibit lipid oxidation and Fenton-type reactions (Mayer et al., 1995; Hogg et al., 1993b; Wink et al., 1994; Yates et al., 1992). Hogg et al. (1993b) have shown that NO can prevent metal-mediated lipid peroxidation, whereas Wink et al. (1993) have shown that NO derived from NONOates protects Chinese hamster V79 lung fibroblasts and H4 hepatoma cells from hydrogen peroxide- and hypoxanthine/xanthine oxidase-induced cell toxicity. In yet other systems, NO formation has been linked with a protective function in oxidative injury: inactivation of superoxide radicals produced by activated human leukocytes; inhibition of neutrophil adhesion to endothelial cells; inhibition of oxidative modification of low density lipoproteins; and inhibition of lipoxygenase-, cyclooxygenase- and hemoglobin-induced lipid oxidation (Clancy et al., 1992; Gauthier et al., 1994; Mayer et al., 1995). With regard to the liver, Harbrecht et al. (1992a, 1992b) have shown that inhibition of NO synthesis promotes oxygen free radical-induced damage manifest as intrahepatic thrombosis in an in vivo model of endotoxin-mediated liver injury. In the isolated, perfused rat liver, Bautista and Spitzer (1994) have demonstrated that inhibition of NO production results in enhanced superoxide formation. In a low-flow, reflow model of liver perfusion, Jones and Thurman (1996) have recently demonstrated that provision of the iNOS substrate, L-arginine, minimized reperfusion injury as measured by lactate dehydrogenase and MDA release. The authors hypothesized that NO-mediated improvement in the hepatic microcirculation resulted in washout of substrates responsible for toxic free radical generation. These observations notwithstanding, the protective effect of NO, whether derived from endogenous or exogenous sources, remains a subject of controversy. NO has been implicated as a toxic metabolic end product in a number of studies (Cazevielle et al., 1993; Wang et al., 1992; Yu et al., 1994). Under certain conditions, NO may react with superoxide to form peroxynitrite, which can theoretically rapidly decompose to form a hydroxyl-like radical by the metabolism of peroxynitrous acid (Radi et al., 1991a, 1991b). In bovine artery pulmonary endothelial cells, Phelps et al. (1995) have demonstrated that peroxynitrite depletes cellular GSH by a protein kinase C dependent mechanism. Salgo et al. (1995) have shown that peroxynitrite causes DNA nicks in the plasmid pBR322 and in a system of rat thymocytes. Yet another mechanism has been suggested in which the lipophilic antioxidant -tocopherol is oxidized in the presence of NO/O₂-resulting in promotion of lipid peroxidation (Hogg et al., 1993a).

The mechanism underlying the protective effect of NO in oxidative injury, both in the generalized case and in hepatocytes, is emerging. Under physiological conditions, Pryor and Squadrito (1995) suggest that peroxynitrite, formed via the interaction of NO with superoxide, may form an adduct with carbon dioxide that rapidly decomposes to nitrate and bicarbonate. Based on these kinetic data, the author argues against an important role for peroxynitrite in mediating oxidant injury in biologic systems. Hogg et al. (1993b) have proposed a pathway in which NO ablates metal-mediated lipid hydroperoxidation by scavenging radicals to produce the organic species, RONO₂, RONO or LNO. Based on this schema, Wink et al. (1994) suggest that NO may act as a primary antioxidant in biological systems by: 1) limiting lipid peroxidative chain propagation and 2) inhibiting lipoxygenase-mediated lipid oxidation, although secondarily limiting inflammation mediated by immunological mechanisms by blocking leukocyte adhesion (Mayer et al., 1995). Additional work has addressed the modulatory effect of NO in superoxide-dependent oxidation, hydroxylation and lipid peroxidation reactions. Using a model system measuring phophatidylcholine liposome peroxidation, Rubbo et al. (1994) demonstrated that 1) NO alone did not induce lipid peroxidation, 2) NO is a potent terminator of radical chain propagation and 3) NO inhibits peroxynitrite-dependent lipid peroxidation reactions. At high rates of NO synthesis, as would occur in the context of iNOS induction, NO mediates oxidant protective reactions. The interactions between NO and the inducible form of cyclooxygenase (COX2) suggests an alternative route for antioxidant role of NO. Work by Salvemini et al. (1993, 1994, 1995) suggest that NO may augment the synthesis of proinflammatory cyclooxygenase products and contribute to cell and tissue injury. However, in the absence of COX2, NO production may be protective (Harbrecht et al., 1994). These findings support the notion that NO may play an antioxidant role in the appropriate setting and tissue.

The effect of oxidative stress on iNOS activity and NO synthesis has not been widely examined. Using nitroblue tetrazolium as a superoxide scavenger, Mittal (1993) demonstrated a permissive role for superoxide in NO formation by neuronal NOS. In addition, Adcock et al. (1995) have demonstrated that pyrogallol, a pharmacological source of superoxide at physiological pH, was capable of iNOS mRNA induction in human A549 epithelial cells. In addition, the authors demonstrated increased NF-B DNA binding with increased oxidative stress, although the relationship between iNOS and NF-B was not elucidated. In pyrogallol-treated rat hepatocytes, we have demonstrated that increased NO generation, iNOS enzyme activity and steady-state levels of iNOS mRNA in association with increased oxidative stress (Kuo and Abe, 1995). Studies examining the promoter regions of the murine and human iNOS gene offer some initial information. Xie and Nathan (1994) have cloned the promoter region of the murine iNOS gene and found that two NF-B binding sites are present. Using a specific inhibitor of NF-B activation, these authors have demonstrated the NF-B/Rel complex is essential to the induction of iNOS by endotoxin. Similar studies by De Vera et al. (1996) concerning the human hepatocyte iNOS gene promoter have identified a number of consensus NF-B binding sequences. These results are significant given the observations that induction of NF-B relies on production of reactive oxygen species, as would be present in states of oxidative stress (Adcock et al., 1995).

Finally, consideration must be given to the mechanisms of APAP-associated oxidative stress. Although the studies of Mitchell et al. (1973) point to covalent protein binding of the reactive APAP metabolite as the causative factor underlying APAP-induced cell death, more recent studies argue that oxidative stress is an additional or alternative process that accounts for hepatocellular damage (Thomas, 1993; Brent and Rumack, 1993; Vermeulen et al., 1992). Currently, lipid peroxidation and alteration in intracellular calcium homeostasis with subsequent formation of reactive oxygen species are supported by experimental evidence as the most likely mechanism for the toxicity associated with APAP reactive metabolites. As such, using APAP as a model of toxin-induced oxidative stress, our experiments suggest that IL-1-induced NO synthesis is hepatoprotective and, conversely, that oxidative stress augments NO synthesis. Thus, under conditions where oxidative injury may be a component of the overall proinflammatory state, induction of iNOS with subsequent elaboration of NO is hepatoprotective. In addition, the presence of oxidative stress can further increase NO production as a protective mechanism against oxidative injury. The molecular pathway by which NO serves as an antioxidant remains the subject of ongoing studies, but certainly the effect of NO on oxidative stress-dependent transcription factors, such as NF-B, will certainly be an area for fruitful research.