Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Nitric oxide (NO)-donating agents have received considerable attention as a current trend in the development of therapeutics (Keefer, 2000). Most available NO-donating agents are not tissue selective, i.e., they either decompose to NO spontaneously or are metabolized to NO in many tissues, thereby producing NO-mediated effects in a variety of organ systems when administered systemically (Keefer, 2000). O²-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO; Fig. 1) was created by adding a vinyl functional group to the terminal oxygen of pyrrolidine diazeniumdiolate (Saavedra et al., 1997). V-PYRRO/NO is a stable diazeniumdiolate, which could circulate freely throughout the body until it is metabolized to NO by enzymes, presumably cytochromes P450, in the liver (Saavedra et al., 1997; Stinson et al., 2001).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Structural formulae of V-PYRRO/NO and the proximal NO-donating compound, PYRRO/NO, which is generated via liver metabolism.

The liver-selective NO donation from V-PYRRO/NO has been demonstrated by a number of in vitro and in vivo studies. In the in vitro studies, the release of NO from V-PYRRO/NO, as assessed by the oxidative metabolites nitrite and nitrate of NO, was observed in parenchymal hepatocytes but not in other cell types, such as pulmonary artery smooth muscle cells, pulmonary artery endothelial cells, liver nonparenchymal cells, and murine macrophage cells (Saavedra et al., 1997). The stimulation of hepatic cGMP production by V-PYRRO/NO has also been demonstrated in hepatocyte cultures (Saavedra et al., 1997). In the in vivo studies, when administered orally, only 20% of administered V-PYRRO/NO could reach the systemic circulation, indicating a high first-pass effect through the liver and adding evidence that V-PYRRO/NO is a liver-selective compound (Stinson et al., 2001). When administered intravenously, the expected vasodilatory effects and hepatoprotective effects of V-PYRRO/NO have been confined to the liver and more general effects (such as lowering systemic blood pressure) have been minimal (Deleve and Wang, 1999; Ou et al., 1997; Saavedra et al., 1997; Ricciardi et al., 2001).

The pharmacological significance of NO donation to the liver is receiving more and more attention. NO has multiple, apparently controversial effects on the liver (Kröncke et al., 1997; Li and Billiar, 1999; Kim and Billiar, 2001). NO is reported to be an important mediator of hepatocellular injury produced by acetaminophen and other hepatotoxicants (Gardner et al., 1998). On the other hand, NO can be beneficial to the liver, either through improvement of hepatic circulation (Pastor et al., 1995; Wang et al., 1995; Deleve and Wang, 1999; Ricciardi et al., 2001) or by inhibition of TNF- or APO-1/Fas-mediated apoptosis (Dimmeler et al., 1998; Kim et al., 1997b, 2000; Ou et al., 1997), and/or by induction of cellular defense mechanisms, such as heat-shock proteins and antioxidants (Kim et al., 1997a). Thus, depending on the model and conditions of exposure, NO can either mediate or block toxicant-induced liver damage. To help further elucidate the pharmacological actions of the liver-selective NO donor V-PYRRO/NO, the present study utilized bacterial lipopolysaccharide in a D-galactosamine-sensitized mouse (GlaN/LPS) model to evaluate the hepatoprotective effects of V-PYRRO/NO in mice at the biochemical and genetic levels. We report the dramatic protective effects of V-PYRRO/NO against GlaN/LPS-induced liver injury. A cDNA microarray analysis was performed to profile the alterations in gene expression patterns associated with hepatoprotective effects of V-PYRRO/NO. After the initial gene array work pointed toward a significant effect of V-PYRRO/NO on TNF--mediated apoptosis pathways, further studies were directed at the effect of V-PYRRO/NO on this important pathway of cell death.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. V-PYRRO/NO was synthesized as previously described (Saavedra et al., 1997). LPS (from Escherichia coli serotype 0111: B4, TCA extract), D-galactosamine, and -actin antibody were purchased from Sigma Chemical Co. (St. Louis, MO). The mouse Atlas toxicology array and ApoAlert caspase-3 detection kits were purchased from CLONTECH (Palo Alto, CA). The gel shift assay kit was purchased from Promega (Madison, WI). The antibodies against TNFR1-associated death domain (TRADD; sc-7868), TNF-related apoptosis-inducing ligand (TRAIL; sc-7877), caspase-3 (sc-1225), and caspase-8 (sc-7890) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-caspase-1 and TNFR1 were obtained from BD PharMingen (San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies against rabbit, mouse, and goat IgG were purchased from Sigma Chemical Co. and enhanced chemiluminescence kits and [-³²P]dATP were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). All other chemicals were commercially available and of reagent grade.

Animals. Male Crl:CD-1 mice, weighing 25 to 30 g, were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Animals were housed in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care at the National Institute of Environmental Health Sciences at 20-22°C with a 12-h light/dark cycle for at least 1 week before treatment. Animals were allowed free access to rodent chow (Ralston Purina Co., St. Louis, MO) and tap water. All procedures involving the use of laboratory animals were reviewed and approved by the Institutional Animal Care and Use Committee.

Experimental Design. For the study of protective effects, mice were given multiple s.c. injections with 10 mg V-PYRRO/NO/kg at 1 h before and 1, 3, and 5 h after GlaN/LPS (700 mg/30 µg/kg, i.p.), and hepatotoxicity was evaluated at 1.5, 6, and 9 h after GlaN/LPS intoxication. The use of multiple s.c. injections of V-PYRRO/NO was based on data showing that V-PYRRO/NO has a relatively short plasma half-life in mice (Stinson et al., 2001). In the pilot study, multiple s.c. injections of V-PYRRO/NO produced better results than osmotic pump-delivered NO [V-PYRRO/NO (2 mg/ml) delivered via Alzet pump 2001D (Palo Alto, CA) at the rate of 8 µl/h, with a total dose of ~15 mg/kg for 24 h]. In additional studies, the multiple s.c. injections of V-PYRRO/NO also allowed us to give V-PYRRO/NO to animals at different time points after GlaN/LPS intoxication to evaluate the therapeutic effects (i.e., V-PYRRO/NO treatments were initiated at 0, 1.5, 3, and 5 h after GlaN/LPS intoxication). At the end of the experiment mice were anesthetized with CO₂, blood was collected by decapitation, and the livers were removed.

Evaluation of Hepatotoxicity. Serum alanine aminotransferase (ALT) activity was assayed as a marker of hepatotoxicity using a commercially available kit (Sigma 59-UV). In addition, a portion of the liver was fixed in 10% formalin, processed by standard histological techniques, stained with hematoxylin and eosin, and examined for morphological evaluation of liver injury.

Immunohistochemical Detection of TNFR1, Caspase-3, and Apoptosis. To localize the expression of TNFR1 and the activation of caspase-3, immunohistochemistry was performed using polyclonal antibodies against TNFR1 and caspase-3. Briefly, liver sections were deparaffinized in xylene and hydrated in a series of graded alcohol solutions, and endogenous peroxidase was blocked with 5% hydrogen peroxide. The sections were then incubated with primary antibodies against TNFR1 (1:100) or caspase-3 (1:100) at 4°C overnight, followed by incubation with goat anti-rabbit/goat IgG conjugated with horseradish peroxidase (1:200). The signals were visualized by ABC Immunostain Systems (Santa Cruz Biotechnology). Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using a commercial kit from Intergen (Gaithersburg, MD) according to the manufacturer's instructions.

Microarray Analysis. Atlas mouse toxicology/stress cDNA expression microarray and mouse 1.2 array were performed according to the manufacturer's instructions. Briefly, total RNA was subjected to DNaseI digestion (2 U/100 µg), and 5 to 10 µg of total RNA was converted to [-³²P]-dATP-labeled cDNA probe using Moloney murine leukemia virus reverse transcriptase and the Atlas mouse stress cDNA synthesis primer mix, according to the manufacturer's instructions (CLONTECH). The ³²P-labeled cDNA probe was purified using Chroma Spin-200 columns, denatured in 0.1 M NaOH, 10 mM EDTA, at 68°C for 20 min, followed by neutralization with an equal volume of 1 M NaH₂PO₄ for another 10 min. The membrane was prehybridized with Ultrahyb (Ambion, Austin, TX) for 30 to 60 min at 42°C, followed by hybridization overnight at 42°C. The arrays were washed two times in 2× standard saline citrate/0.1% SDS, 5 to 10 min each, and two times in 0.1× standard saline citrate/0.1% SDS for 15 to 30 min. The arrays were then sealed in a plastic bag and subjected to exposure to a Molecular Dynamics (Sunnyvale, CA) PhosphorImage screen or to X-ray film. The images were analyzed densitometrically using AtlasImage software (version 1.5). The gene expression intensities were normalized with the sum of 8 housekeeping genes on the array (40S ribosomal protein S29, 45-kDa calcium-binding protein, -actin, ornithine decarboxylase, myosin 1-, G3PDH, hypoxanthine-guanine phosphoribosyltransferase, and phospholipase A2).

Gel Shift Assay of NF-B. Nuclear protein was isolated from liver tissues as described (Parrish et al., 1999). Briefly, livers were homogenized in HEGD buffer [25 mM HEPES, 1.5 mM EDTA, 10% glycerol, 1 mM DL-dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitor cocktail (Calbiochem, San Diego, CA)], centrifuged at 12,000g for 10 min at 4°C, and washed two times with HEGD buffer. Nuclear proteins were extracted by incubation with 80 µl of HEGD buffer containing 0.5 M KCl on ice for 1 h. The samples were centrifuged at 12,000g for 15 min and supernatant was stored at 80°C. Gel shift assay was performed using gel shift assay systems from Promega according to the manufacturer's directions. NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3' and 3'-TCAACTCCCCTGAAAGGGTCCG-5') were labeled with [-³²P]-ATP with T4 polynucleotide kinase, and nuclear protein (10 µg) was incubated with gel shift binding buffer and 1 µl labeled NF-B for 20 min. The samples were then loaded onto Novex 6% DNA retardation gel and the gel was subjected to electrophoresis at room temperature in 0.5× Tris/borate/EDTA buffer at 300 V. Gels were then exposed to X-Omat film (Eastman Kodak, Rochester, NY) for autoradiography.

Western Blot Analysis. Livers were homogenized (1:10, w/v) in 20 mM Tris-HCl, pH 7.4, containing 1 mM NaF, 150 mM NaCl, 1% Triton X-100 and freshly added protease inhibitor cocktail (Calbiochem), and 100 pM phenylmethylsulfonyl fluoride. Cytosols were prepared by centrifugation at 15,000g for 10 min at 4°C. Protein concentrations were determined using the dye-binding assay (Bio-Rad, Hercules, CA). Total protein (30-40 pg) was subjected to electrophoresis on Tris-glycine polyacrylamide precast gels (4-20%) (Novex, San Diego, CA), followed by electrophoretic transfer to nitrocellulose membranes at 25 V for 3 h. Membranes were blocked in 5% dried milk and 0.1% bovine serum albumin in TBST (15 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.08% Tween 20) for 2 h at room temperature, followed by incubation with the primary antibody (1:1,000) in 2% milk in TBST overnight at 4°C. After four washes with TBST, the membranes were incubated in secondary antibody (1:5,000-1:10,000) for 60 to 120 min. After four to five washes with TBST, proteins were visualized using enhanced chemiluminescence or SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford, IL).

Determination of Caspase-3 Activity. Caspase-3 activity was determined using a commercially available kit from CLONTECH. The livers were homogenized in the lysis buffer and then centrifuged at 14,000g. The supernatant was then incubated with the fluorescent caspase-3 substrate DEVD-7-amino-4-trifluoromethylcoumarin for 1 h at 37°C. Samples were then read in an Aminco-Bowman spectrophotofluorometer with excitation and emission wavelengths set at 400 and 505 nm, respectively.

Statistics. Means and standard errors of individual groups (n = 3-10) were calculated. Data were analyzed using a one-way analysis of variance, followed by Duncan's multiple range test. The level of significance was set at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Hepatoprotection by V-PYRRO/NO Against GlaN/LPS-Induced Hepatotoxicity. Effects of multiple injections of V-PYRRO/NO on GlaN/LPS-induced liver injury are shown in Fig. 2. GlaN/LPS (700 mg/30 µg/kg, i.p.) produced liver injury, as indicated by a marked elevation of serum ALT activities at 6 and 9 h after GlaN/LPS administration. Multiple injections of V-PYRRO/NO alone (10 mg/kg, s.c. four times at 2-hr intervals) did not produce an elevation of ALT, but significantly reduced GlaN/LPS-induced elevation of serum ALT levels at 1.5, 6, and 9 h after GlaN/LPS administration. Consistent with the literature (Ou et al., 1997; Vos et al., 1997), the inducible nitric oxide inhibitor L-NAME (N^G-nitro-L-arginine methyl ester) increased GlaN/LPS hepatotoxicity (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of V-PYRRO/NO on GlaN/LPS-induced liver injury in mice. Mice were given GlaN/LPS (700 mg/30 µg/kg, i.p.) with or without the administration of V-PYRRO/NO (10 mg/kg, s.c. 1 h before and 1, 3, and 5 h after GlaN/LPS). Liver injury was evaluated by measuring serum ALT activity. Data are mean ± S.E.M. of 7 to 10 mice. , significantly different from controls, p < 0.05.

Microarray Analysis of Gene Expression after V-PYRRO/NO and GlaN/LPS. To define the altered gene expression pattern in livers of mice treated with GlaN/LPS or GlaN/LPS plus V-PYRRO/NO for 9 h, total RNA was isolated from mouse liver and subjected to cDNA microarray analysis. A representative microarray image is shown in Fig. 3A. In this Atlas mouse stress/toxicology array, several alterations in gene expression between GlaN/LPS alone and V-PYRRO/NO + GlaN/LPS treatments are readily visible. For example, the expression of uracil-DNA glycosylase, DNA repair protein Rad50, DNA damage-inducible protein GADD45, and oxidative stress protein A170 mRNA were more pronounced in the GlaN/LPS-alone group than in the V-PYRRO/NO + GlaN/LPS group. On the other hand, the expression of hepatic flavin-containing monooxygenase 1 and cytochrome P450 2E1 (CYP2E1) was higher in the V-PYRRO/NO + GlaN/LPS group than in the GlaN/LPS-alone group, suggesting that the suppressive effect of GlaN/LPS on certain liver drug-metabolizing enzymes was ameliorated by V-PYRRO/NO. The expression of the housekeeping genes, such as G3PDH, -actin, and ribosomal protein S29, was relatively similar. Following normalization with the housekeeping genes, the means ± S.E.M. of three separate hybridizations were calculated, and differences in selected gene expression are illustrated in Fig. 3B. The expression of DNA damage/repair-related genes, such as uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, DNA repair protein Rad50, DNA damage-associated protein GADD45, DNA excision repair protein ERCC1, and DNA ligase 1, was all markedly increased by GlaN/LPS treatment. V-PYRRO/NO treatment markedly reduced these enhanced gene expressions, supporting the biochemical and pathological evidence of the protective effect of V-PYRRO/NO on GlaN/LPS-induced hepatic damage. The expression of MDM-2, a p53-associated protein, was also increased 2.5-fold in GlaN/LPS-treated mice but not in GlaN/LPS plus V-PYRRO/NO-treated mice. V-PYRRO/NO treatment also attenuated GlaN/LPS-induced increases in the expression of the oxidative stress protein A170 and heme oxygenase 1. GlaN/LPS treatment also produced 20 to 50% decreases in the expression of liver cytochrome P450 enzymes, such as CYP2C29, CYP2E1, CYP2D9, CYP2F2, CYP7B1, and cytochrome P450 reductase, as well as the phase II drug-metabolizing enzymes, such as UDP-glucuronosyltransferase 2B5 (UGT2B5) and UDP-glucuronosyltransferase 1A1 (UGT1A1) (data not shown). In general, V-PYRRO/NO treatment ameliorated the suppressive effects of GlaN/LPS on the expression of these drug-metabolizing enzymes.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   A, representative photomicrographs of mouse toxicology array. Several alterations in gene expression between saline+GlaN/LPS and V-PYRRO/NO+GlaN/LPS treatments at 9 h are easily visible, whereas the expression of housekeeping genes such as G3PDH, -actin, and ribosomal protein S29 was relatively similar. B, quantitative analysis of hybrid intensity of selected genes 9 h after GlaN/LPS, GlaN/LPS+V-PYRRO/NO treatments compared with untreated controls. Data are means ± S.E.M. of four separate hybridizations. , significantly different from controls, p < 0.05.

Gel Shift Assay for NF-B and Western Blot Analysis of Apoptosis Proteins. Gel shift assay and Western blot analysis were performed to support the initial array analysis. The inhibitory effects of NO on NF-B activation have been reported (Matthews et al., 1996; Colasanti and Persichini, 2000). In the present study, NF-B was activated 1.5 and 6 h after GlaN/LPS intoxication, and V-PYRRO/NO treatments greatly attenuated NF-B activation (Fig. 4), indicating that the exogenous NO donor could provide a control mechanism for NF-B-inducible gene expression in response to GlaN/LPS. As shown in Fig. 5, the expression of caspase-1, caspase-8, TRADD, and TRAIL was increased 6 h after GlaN/LPS intoxication, and V-PYRRO/NO treatments essentially prevented these increases, in accord with the observed hepatoprotection. Additional mouse 1.2 array studies also indicated the suppressive effects of V-PYRRO/NO on the basal expression of NF-B and several apoptosis-related genes (Fig. 9).

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Effect of V-PYRRO/NO on GlaN/LPS-induced NF-B activation. Mice were given GlaN/LPS (700 mg/30 µg/kg, i.p.) with or without the administration of V-PYRRO/NO (10 mg/kg, s.c.) and liver nuclear extracts were used for gel shift assay. Lanes 1-4, saline control, saline + GlaN/LPS at 1.5, 6, and 9 h; lanes 5-8, V-PYRRO/NO control and V-PYRRO/NO + GlaN/LPS at 1.5, 6, and 9 h, respectively.

View larger version (79K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of apoptosis-related proteins in livers of GlaN/LPS- and GlaN/LPS+V-PYRRO/NO-treated animals. Lanes 1 and 2, control; lanes 3 and 4, GlaN/LPS; lanes 5 and 6, GlaN/LPS + V-PYRRO/NO.

Immunohistochemical Analysis of TNFR1, Caspase-3 and Hepatocellular Apoptosis. To further identify the key events associated with V-PYRRO/NO-mediated protection against GlaN/LPS hepatotoxicity, immunohistochemical analysis of TNFR1 and caspase-3 was performed 9 h after GlaN/LPS and V-PYRRO/NO administration (Fig. 6). Staining for TNFR1 was clearly intensified after GlaN/LPS treatment, and this effect was attenuated by V-PYRRO/NO treatment. GlaN/LPS administration enhanced staining for caspase-3, especially in the damaged cells, and this increase was greatly reduced after V-PYRRO/NO treatment. In addition, hepatocellular apoptosis, as determined by the TUNEL assay, was increased in the livers of mice treated with GlaN/LPS. Apoptosis was rare in the livers of mice treated with GlaN/LPS + V-PYRRO/NO. To further define the effect of V-PYRRO/NO on GlaN/LPS-induced apoptosis, the activity of caspase-3, an enzyme critical to the dedication of cells to apoptosis, was determined (Fig. 7). GlaN/LPS treatment resulted in marked activation of caspase-3 activity 6 h after administration, which reached a peak at 9 h after GlaN/LPS administration. Multiple injections of V-PYRRO/NO greatly suppressed GlaN/LPS-induced elevation in hepatic caspase-3 activity.

View larger version (89K): [in this window] [in a new window]   Fig. 6.   Immunohistochemical analysis of TNFR1 (A-D), caspase-3 (E-H), and hepatocellular apoptosis (I-L) in liver sections 9 h after treatment with GlaN/LPS or V-PYRRO/NO + GlaN/LPS. Magnification 200×. In mice treated with GlaN/LPS, immunohistochemical staining for TNFR1, caspase-3, and TUNEL was intensified and coincided with apoptosis/necrosis. V-PYRRO/NO treatment greatly ameliorated these alterations. In normal control mice or in mice treated with multiple injections of V-PYRRO/NO, the immunostaining was at the background level.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of V-PYRRO/NO on GlaN/LPS-induced caspase-3 activity. Mice were given GlaN/LPS (700 mg/30 µg/kg, i.p.) with or without the administration of V-PYRRO/NO (10 mg/kg, s.c.) 1 h before and 1, 3, and 5 h after GlaN/LPS. Data represent mean ± S.E.M. of 7 to 10 mice. , significantly different from controls, p < 0.05.

The Therapeutic Effect of V-PYRRO/NO. To examine whether V-PYRRO/NO could protect against GlaN/LPS toxicity even when administered at a point after exposure to GlaN/LPS, additional animals were given GlaN/LPS first, followed by s.c. injections of V-PYRRO/NO starting at 0, 1.5, 3, or 5 h after GlaN/LPS intoxication (Fig. 8). The elevated serum ALT levels were significantly attenuated when V-PYRRO/NO treatment started simultaneously with GlaN/LPS exposure, or at 1.5 h after GlaN/LPS intoxication, indicating that V-PYRRO/NO provides significant protective effects when given well after the hepatotoxic dose of GlaN/LPS. However, V-PYRRO/NO was ineffective when given 3 or 5 h after GlaN/LPS intoxication.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   The therapeutic effects of V-PYRRO/NO on GlaN/LPS-induced liver injury in mice. Mice were given GlaN/LPS (700 mg/30 µg/kg, i.p.) with or without the administration of V-PYRRO/NO (10 mg/kg, s.c. at 0, 1.5, 3, and 5 h after GlaN/LPS). Liver injury was evaluated by measuring serum alanine aminotransferase activity. Data are mean ± S.E.M. of 5 to 6 mice. , significantly different from controls, p < 0.05.

The Effect of V-PYRRO/NO Alone on Gene Expression. Figure 9 shows the effect of V-PYRRO/NO alone on the expression of selected genes associated with cell death pathways. The administration of V-PYRRO/NO at pharmacological doses suppressed endogenous inducible nitric oxide synthase expression. Consistent with the liver-selective release of NO (Deleve and Wang, 1999; Ou et al., 1997; Saavedra et al., 1997; Ricciardi et al., 2001), serum nitrite/nitrate levels were not increased by V-PYRRO/NO, as determined by the colorimetric Griess reaction assay (data not shown). The expression of genes encoding for tumor necrosis factor-, TNFR1, TNFR2, TNFR-associated factor 3 (TRAF3), TRAIL, granzyme A, and apoptosis protein BAD was suppressed by multiple injections of V-PYRRO/NO. The basal expression of the gene encoding for nuclear factor-B was also suppressed by V-PYRRO/NO treatment. In addition, Fas ligand receptor APO-1, Fas ligand, caspase-7, and caspase-2 were also suppressed to a lesser extent (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effect of V-PYRRO/NO alone on the expression of genes encoding TNF-mediated apoptosis pathways. Mice were given multiple injections of V-PYRRO/NO (four injections at 2-h intervals) and hepatic total RNA was extracted for synthesis of the cDNA probe. Data are means ± S.E.M. of three separate hybridizations using mouse 1.2 arrays. , significantly different from controls, p < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study demonstrated that the liver-selective NO donor, V-PYRRO/NO, was effective in protecting against the hepatotoxicity produced by GlaN/LPS in mice, as evidenced biochemically by decreased serum alanine aminotransferase activity and histologically by improved liver pathology. V-PYRRO/NO was effective even when administered 90 min after GlaN/LPS administration, suggesting the potential therapeutic effect of the liver-selective NO donor in endotoxemia as a postexposure "rescue" drug. In fact, the beneficial effect of the liver-selective NO donor, V-PYRRO/NO, is not limited to GlaN/LPS, GlaN/TNF-, or LPS models (Ou et al., 1997; Saavedra et al., 1997); V-PYRRO/NO is also effective in protecting against the liver injury produced by monocrotaline, a model for hepatic veno-occlusive disease (Deleve and Wang, 1999), by ischemia/reperfusion (Ricciardi et al., 2001).

To get better insight into the potential mechanisms by which V-PYRRO/NO protects against GlaN/LPS-induced hepatotoxicity, microarray analysis of gene expression was performed as an initial screening experiment. In the mouse toxicology array (140 genes), approximately 20% of genes were differentially expressed as a result of GlaN/LPS insult. To our knowledge, this is the first attempt to profile the altered gene expression patterns associated with GlaN/LPS-induced liver injury. The genes showing enhanced expression included those associated with oxidative stress and DNA damage/repair, and genes showing suppressed expression encoded certain drug metabolism enzymes. These results are consistent with the notion that oxidative stress (Jaeschke et al., 1999) and DNA damage (Gantner et al., 1995) are important events in GlaN/LPS hepatotoxicity. Importantly, all these alterations in gene expression were greatly ameliorated by V-PYRRO/NO treatment, consistent with the observed hepatoprotection. It has been shown recently that suppression of endogenous NO production by inducible NO synthase inhibitors aggravates LPS-induced hepatic injury, resulting in the formation of oxidative DNA damage (Akahori et al., 1999; Takemura et al., 2000), whereas administration of NO donors, such as hydroxylamine, increased plasma nitrite/nitrate level and ameliorated actinomycin D/LPS-induced liver DNA fragmentation (Akahori et al., 1999). Thus, our gene array study provided a profile of genetic events associated with GlaN/LPS-induced hepatotoxicity, and a clue toward the mechanisms of V-PYRRO/NO protection.

It has been shown that LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF- to provide signaling through the p55 receptor (TNFR1), followed by activation of mitogen-activated protein kinase signaling pathways and activation of NF-B, which coordinate the induction of many genes encoding inflammatory mediators and apoptosis mediators (De Nadai et al., 2000; Nowak et al., 2000; Guha and Mackman, 2001). In the present study, the increased expression of TNFR1 protein (immunohistochemistry) induced by GlaN/LPS and NF-B (gel shift assay) were evident by 6 and 9 h after GlaN/LPS administration, and these effects were greatly diminished by V-PYRRO/NO treatment. These findings are in agreement with the reports that exogenous NO donors can inhibit TNFR1-mediated signal transduction pathways (De Nadai et al., 2000), suppress mitogen-activated protein kinase-mediated signal transduction (Park et al., 2000), and block NF-B activation (Matthews et al., 1996; Buzard and Kasprzak, 2000; Colasanti and Persichini, 2000). Thus, the suppressive effect of V-PYRRO/NO on GlaN/LPS-induced TNFR1 expression and NF-B activation could play a role in protecting against GlaN/LPS hepatotoxicity and aberrant gene expression.

The increased expression of TNFR1 and activation of NF-B led to induction of a variety of apoptosis mediators, such as TRADD, TRAIL, and caspases, which could play important roles in GlaN/LPS-induced apoptotic cell death. In this regard, Western blot analysis confirmed the induction of TRADD, TRAIL, caspase-1, and caspase-8 by GlaN/LPS, and V-PYRRO/NO treatment suppressed these increases. These results are in agreement with the literature indicating that exogenous NO donors produce inhibitory effects on caspase-1 (corresponding to interleukin-1 converting enzyme) (Dimmeler et al., 1997; Fiorucci et al., 2000), caspase-8 (Dimmeler et al., 1998; De Nadai et al., 2000; Kim et al., 2000), and TRADD (De Nadai et al., 2000). Taken together, the inhibition of the TNF--related apoptosis mediators could be an important mechanism of the hepatoprotection by V-PYRRO/NO.

Caspase-3 is the key mediator in liver inflammation and execution of apoptosis following hepatotoxic insult (Cohen, 1997). A central mechanism of apoptosis inhibition appears to be either direct caspase-3 inhibition or inhibition of the conversion of procaspase-3 to the active form (LaCasse et al., 1998). In this regard, V-PYRRO/NO treatment decreased GlaN/LPS-induced caspase-3 staining in the liver sections and greatly suppressed increased caspase-3 activity as determined by the enzymatic assay. These findings are consistent with the reports that exogenous NO can inhibit hepatocellular apoptosis by preventing caspase-3 activity either directly through protein S-nitrosylation (Kim et al., 1997b; Rossig et al., 1999) or indirectly through a cGMP-mediated mechanism (Kim et al., 1997b). Taken together, the inhibitory effects of V-PYRRO/NO on TNF--mediated apoptosis may well contribute to protection against GlaN/LPS-induced apoptosis, as evidenced by TUNEL assay, but also may contribute to protection against GlaN/LPS-induced necrosis, as evidenced by improved histopathology and reduced serum alanine aminotransferase activity.

It should be noted that the mechanisms of the protective effects of the liver-selective NO-donating compound V-PYRRO/NO could be multifactorial. The antiapoptotic property does not exclude other mechanisms, including potential antioxidant mechanisms. V-PYRRO/NO-like diazeniumdiolate compounds, such as diethylamine/nitric oxide adduct (DEA/NO) and 3-(n-propylamino)propylamine/nitric oxide (PAPA/NO), have been shown to act as free radical scavengers to protect against H₂O₂ toxicity (Fitzhugh and Keefer, 2000; Wink et al., 2001). The role of the liver-selective NO donor, V-PYRRO/NO, in free radical scavenging and protection against oxidative stress is currently under investigation.

In summary, this study demonstrates that the liver-selective NO prodrug, V-PYRRO/NO, is effective in protecting against GlaN/LPS-induced liver injury and aberrant gene expression in mice. The liver-selective NO donor appears to block GlaN/LPS-induced DNA damage and subsequent apoptosis. This effect appeared to be due, at least in part, to the suppression of TNF--mediated apoptosis pathways.