Inhibition of Superoxide and Nitric Oxide Release and Protection from Reoxygenation Injury by Ebselen in Rat Kupffer Cells JI-FENGWANG, PAVEL KOMAROV, HELMUT SIES AND HERBERT DE GROOT Institut fur Physiologische Chemie I, Heinrich-Heine- Universitat Dusseldorf, 0-4000 Dusseldorf, Germany

Luminol chemiluminescence in phorbolester- in cytotoxicity against hepatic endothelial and parenactivated cultured rat liver Kupffer cells was chymal cells (3). strongly inhibited by the selenoorganic compound Ebselen (2-phenyl-l,2-benzisoselenazol-3[2H]one) is ebselen (IC60= 2 pmol/L). Ebselen (2-phenyl-1,2- a selenoorganic compound exhibiting both glutathione benzisoselenazol-3[2H]one)also diminished reduction peroxidase activity and antioxidant capacity (4-8). In of ferricytochrome c (IC60= 10 pmol/L), indicating a isolated hepatocytes, ebselen was capable of inhibiting suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreatedKupffer cells, ebselen ADP-iron-induced lipid peroxidation (9)and protecting proved to be a potent inhibitor of the conversion of against diquat-induced cytotoxicity (lo), depending on oxyhemoglobin to methemoglobin (IC60= 3 pmolb) as intracellular glutathione or N-acetylcysteine levels. An a measure of nitric oxide formation. The sulfur- antiinflammatory effect of ebselen against galaccontaining analog (2-phenyl-1,2-benzisothiazol-tosamine/endotoxin-induced hepatitis in mice has been 3[2H]one) and the ebselen derivative, methylsele- attributed to inhibition of lipoxygenase and/or isomernobenzanilide, were inactive. These results indicate ization of lipoxygenase products (11).It has also been that ebselen is a potent inhibitor of NADPH oxidase in shown that ebselen inhibits superoxide anion radical Kupffer cells, as has been reported for other macro- production of guinea pig polymorphonuclear leukocytes phages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective (12), alveolar macrophages (131, a partially purified inhibition of nitric oxide synthase of macrophages. In NADPH oxidase preparation from human granulocytes line with its inhibitory effects on the release of reactive (14) and the chemiluminescence produced by zymosanoxygen species by macrophages, complemented by its stimulated mouse peritoneal macrophages (15). In this study, we demonstrate that ebselen is not only antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells an inhibitor of 0,- formation by the activated Kupffer (IC60- 5 pmol/L). (HEPATOLOGY 1992;15:1112-1116.) cells but is also an even more profound inhibitor of NO

Kupffer cells, the resident macrophages of the liver, (e.g., on actirelease superoxide anion radicals (02-) vation by 12-0-tetradecanoylphorbol 13-acetate [TPAI [l]),and these cells also release nitric oxide (NO) after pretreatment with lipopolysaccharides (LPSs) (2). Whereas 0,- is generated by NADPH oxidase, NO formation is catalyzed by NO synthase. Both of these reactive metabolites are considered important for the bactericidal and tumoricidal activities and are involved Received August 12, 1991; accepted January 16, 1992. This study was supported by the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe “Leberschadigung,” Str 92/4-1 and the National Foundation for Cancer Research, Bethesda, MD. J.-F. Wang (on leave from the Beijing College of Traditional Chinese Medicine, Heping Jie Beikou, Beijing, China) is the recipient of a fellowship of the JungStiftung fur Wissenschaft und Forschung, Hamburg. P . Komarov is on leave from the National Research Center for Biologically Active Compounds (Moscow, USSR). H. de Groot is a Heisenberg-Stipendiat (Gr 815/3-1). Address reprint requests to: Dr. Herbert de Groot, Klinische Forschergruppe Leberschadigung, Institut fur Physiologische Chemie I, Heinrich-HeineUniversitat Dusseldorf, Moorenstrape 5, D-4000 Dusseldorf, Germany. 31/1/36632

formation. It is further shown that these characteristics of ebselen are of potential importance for the prevention of reoxygenation (reperfusion) injury of the liver. The effects of ebselen are compared with its sulfur analog (2-phenyl-1,2-benzisothiazol-3[2Hlone)and to the methylated derivative (methylselenobenzanilide)to substantiate the role of selenium in its activity. MATERIALS AND METHODS Catalase, collagenase H, luminol (5-amino-2,3-dihydro-1,4phthalazinedione), superoxide dismutase (SOD)and RPMI 1640 medium were purchased from Boehringer Mannheim (Mannheim, Germany). L-Arginine, NG-nitro-L-arginine, bovine hemoglobin, cytochrome c (type 1111, LPSs and TPA were from Sigma Chemical Co. (Deisenhofen, Germany). FCS was from Gibco (Eggenstein, Germany), and desferal was from Ciba-Geigy (Basel, Switzerland). Ebselen, its sulfur analog (2-phenyl-1,2-benzisothiazol-3[2Hlone) and the ebselen derivative, methylselenobenzanilide, were kind gifts from Dr. E. Graf, A. Nattermann and Cie, Rhone-Poulenc (Cologne, Germany). Isolation and Cultivation of Rat Liver Kupffer Cells. Kupffer cells were isolated from male Wistar rats weighing 180 to 220 gm as described previously (16). Liver cell suspension

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prepared by collagenase perfusion was freed of hepatocytes by centrifugation twice at 50 g for 5 min. The supernatant was then centrifuged at 300 g for 15 min. The nonparenchymal cell pellet was resuspended in serum-free RPMI 1640 medium containing 2 mmol/L L-glutamine and 0.1 mg/ml gentamicin. Kupffer cells were separated by their ability to adhere to uncoated plastic. Kupffer cells were kept in culture in RPMI 1640medium supplemented with 20% heat-inactivatedFCS in a humidified 5%C0,/95% air atmosphere at 37” C. The purity of the Kupffer cell cultures was better than 98%, as judged by positive peroxidase staining and phagocytosis of latex particles. Kupffer cells were pretreated with LPS by adding this compound at a concentration of 0.5 pg/ml to the RPMI 1640 medium 12 hr before starting the experiments. Incubations. Experiments were performed with day 2 Kupffer cell cultures (1to 2 x lo5 cells/culture tube, 5.5 cm2). Cells were washed three times with HBSS (pH 7.4).Subsequently, Krebs-Henseleit hydrogen carbonate buffer (pH 7.4), supplemented with 10 mmol/L glucose and 20 mmoVL Hepes (with or without 84 Fmol/L trypan blue), was added to the culture tubes. Activation by TPA was achieved by adding the compound at a final concentration of 100 nM to the incubation and medium. Ebselen, 2-phenyl-1,2-benzisothiazol-3(2H)one methylselenobenzanilide were added dissolved in DMSO. In the experiments, the final concentration of DMSO was 0.5% (vol/vol). Control incubations contained 0.5% DMSO alone. In the hypoxialreoxygenation experiments, KrebsHenseleit hydrogen carbonate buffer, equilibrated with 95% N,/5% CO,, was added to the culture tubes. The tubes were sealed with silicon stoppers in which two steel cannules were inserted for gas inlet and outlet and for sample withdrawal. The tubes were flushed with 95% N,/5% CO, for 3 to 5 min and further incubated at 37” C. Cultures were reoxygenated by flushingwith 95% 0,/5% CO, for 3 to 5 min and then returning the tubes to the incubator under aerobic conditions. Aerobic control cultures were treated in the same way as described previously, with the exception that 95% N,/5% CO, was replaced by 95% 0,/5% CO,. Superoxide Anion Radical Production. Superoxide anion radical production was determined as SOD-inhibitable reduction of ferricytochrome c (17,18).Eighty micromoles per liter of ferricytochrome c with or without SOD and catalase (20 pglml each) were added to the culture tubes. At different time points, aliquots of the culture medium were transferred to Eppendorf cups, kept on ice and then centrifuged at 8,000 g for 5 min to remove cell debris. The supernatants were transferred to cuvettes for ferrocytochrome c determination at 550 nm. NO Production. NO production was determined by the spectrophotometric measurement of the conversion of oxyhemoglobin to methemoglobin (19).Krebs-Henseleit hydrogen carbonate buffer was supplemented with 1 mmol/L L-arginine, 8 pmol/L oxyhemoglobin, 40 pg/ml catalase and 20 pg/ml SOD. After incubation for 0.5 to 1 hr difference spectra vs. samples without exogenous L-arginine but with 0.5 mmol/L NG-nitroL-arginine were recorded. The oxyhemoglobin-methemoglobin conversion (i.e., NO formation) was estimated by the changes in absorbance at 578 vs. 592 nm using the extinction coefficient of 12.1 mmol/L cm. Cell Viability. Cell viability was evaluated as the percentage of cells taking up the vital dye trypan blue (16).

15

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FIG. 1. Effect of ebselen on luminol chemiluminescence in TPAactivated Kupffer cells. (A) Luminol (Lum, 50 FmoVL), TPA (100 nM), L-arginine (L-arg,0.5 mmol/L) and ebselen (10 pmol/L) were added a t the times indicated. (B) L-Arginine (1mmol/L) and ebselen (10 pmol/L) had been added a t 0 time; luminol (Lum, 50 kmol/L) and TPA (100 nM) were added at the times given. Control indicates the time course of chemiluminescence without the addition of ebselen.

nescence (Fig. l), indicating the formation of reactive oxygen species. The TPA-induced increase in luminol chemiluminescence was almost doubled by the further addition of L-arginine. Independent of the presence of L-arginine, luminol chemiluminescence was markedly inhibited by ebselen (Figs. 1and 2, Table 1).Already at 1 pmoVL ebselen, about 40% inhibition occurred, and inhibition was almost complete at 10 KmoVL ebselen. Using SOD-inhibitable cytochrome c reduction as a rather specific assay for 0 2 - the , formation of 44 nmol 0,-/106 cells x hr was detected after the addition of TPA. Again, ebselen provided marked inhibition (Fig. 2, Table 1).In contrast to luminol chemiluminescence, however, 10 p,mol/L ebselen were necessary to produce a 50% inhibitory effect, and complete inhibition was RESULTS achieved at 50 pmol/L ebselen. 0,-release by the Effects of Ebselen on the Release of Reactive Oxygen TPA-activated Kupffer cells was independent of the Species.After the addition of TPA, an almost imme- presence of L-arginine (not shown). diate, marked increase occurred in luminol chemilumiAfter pretreatment with LPSs, Kupffer cells released

HEPATOLOGY

WANG ET AL.

1114

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FIG. 3. Effects of ebselen, its sulfur analog (2-phenyl-1,2benzisothiazol-3[2H]one) and methylselenobenzanilide on loss of viability induced by hypoxiakeoxygenation in Kupffer cells. Kupffer cells in primary culture were first incubated hypoxically for 4 hr and then reoxygenated. Ebselen and its derivatives were added prior to reoxygenation. Viability of the cells was estimated by the uptake of trypan blue. Values presented are the means t S.E.M. of 3 t o 5 separate experiments.

at 50 pmol/L of the compound. The maximal inhibitory effect achieved by ebselen was close to 70%. The sulfur-containing ebselen analog (2-phenyl-1,2FIG. 2. (A) Inhibition of luminol chemiluminescence, (B) 0,release and (C) NO formation by ebselen in Kupffer cells. Luminol benzisothiazol-3[2H]one) and its methyl derivative chemiluminescence and 0,- release were determined in cells activated (methylselenobenzanilide) at a concentration of 10 by TPA (100 nM), whereas NO formation was measured in cells pmoVL produced only slight inhibitory effects on TPApretreated for 12 h r with LPSs (0.5 bg/ml). 0,- release was measured as the SOD-inhibitable reduction of ferricytochrome c and NO induced luminol chemiluminescence and 0, release formation by the conversion of oxyhemoglobin to methemoglobin. In and on LPS-induced NO formation (Table 1). the experiments where luminol chemiluminescence and NO formation Effects of Ebselen on Reoxygenation Injury. Kupffer were followed, the incubation buffer had been supplemented with 0.5 cells can be activated to release 0, by hypoxia, followed mmol/L L-arginine. Values presented are the means * S.E.M. of 3 to by resupply of oxygen (20). This hypoxiaheoxygenation4 separate experiments. induced 0,- release was also inhibited by ebselen (Table 2). As in TPA-activated cells, 10 pmol/L ebselen were necessary to achieve 50% inhibition, and inhibition was significant amounts of NO in the presence of L-arginine almost complete at 50 pmol/L ebselen. Kupffer cells released small but significant amounts of (Fig. 2, Table 1). NO formation by the Kupffer cells was strongly inhibited by ebselen as well. Half-maximal NO already in aerobic controls (Table 2). NO release was inhibition occurred around 3 pmol/L ebselen. In con- not increased within the first 4 hr after reoxygenation. trast to chemiluminescence and 0,- release, NO for- However, 12 h r after reoxygenation, almost a doubling mation could not be completely blocked by ebselen, even occurred in the rate of NO formation (not shown). ~

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INHIBITION OF SUPEROXIDE AND NITRIC OXIDE RELEASE BY EBSELEN

TABLE 1. Effects of ebselen, its sulfur analog (2-phenyl-l,2-benzisothiazol-3[2Hlone) and methylselenobenzanilide (10 pmol/L) on luminol chemiluminescence, 0,- release and NO formation in Kupffer cells

Control Ebselen Sulfur analog Methylselenobenzanilide

Luminol chemiluminescence cps x lo8 (% of control)

0,- release

13.7 ? 5 (100) 1.4 t 2 (10) 6.5 c 3 (47) 10.1 t 4 (74)

44 ? 5 (100) 22 ? 3 (50) 43 ir 4 (98) 40 t 5 (91)

NO release

nmoU106cells x hr (% of control)

32 ? 3 (100) 10 t l(31) 22 ? 2 (69) 31 t l(97)

Luminol chemiluminescence and 0,-release were determined in TPA-activated cells. NO formation was measured in LPS-pretreated cells (compare with Fig. 2).Values presented are the means t S.E.M. of 3 to 5 separate experiments. The numbers in parentheses are the percentages with respect to the controls.

TABLE 2. Trypan blue uptake, 0,- release and NO f o r m a t i o n on hypoxia-reoxygenation in Kupffer cells Inhibitor

Trypan blue uptake (%)

0% -

Condition

Aerobic control Reoxygenation Reoxygenation Reoxygenation Reoxygenation Reoxygenation Reoxygenation Reoxygenation

SOD Catalase Desferal Ebselen Sulfur analog Methylselenobenzanilide

10 t 2 55 ? 8 18 t 6 26 t 7 38 t 3 21 & 7 53 t 4 48 t 5

5.0 t 2 15.0 k 2 0.0 -a ND' 8.3 t 3 13.2 t 2 15.3 t 2

(nmol/hr x

NO

lo6 cells) 6.2 t 0.2 6.4 t 0.3 -a -a 6.2 t 0.2 1.9 t 0.2 ND ND

Kupffer cells in primary culture were first incubated under hypoxic conditions for 4 hr and then reoxygenated. SOD (20 kg/ml), catalase (20 pg/ml), desferal (0.1rnmom), ebselen (10 FrnoVL), the ebselen sulfur analog 2-phenyl-l,2-benzisothiazol-3(2H)one (10 Fmolb), methylselenobenzanilide (10 pmol/L) and L-arginine (1 mmol/L) were added prior to reoxygenation. Trypan blue uptake, 0,-and NO formation were determined 10, 1 and 4 h r after reoxygenation, respectively. Values are means t S.E.M. of 3 to 5 separate experiments. "SODand catalase are essential constituents in the assays. *ND = not determined.

O,-/H,O,released by Kupffer cells activated by hypoxidreoxygenation resulted in their self-destruction, as indicated by the protective effects of SOD, catalase and desferal (Table 2) (20). Comparable protection from reoxygenation injury was also obtained at 10 pmol/L = 5 pmoVL). NG-Nitroebselen (Fig. 3, Table 2) L-arginine, a competitive inhibitor of NO formation, was without effect on reoxygenation injury (not shown). The sulfur analog or methylselenobenzanilide was without significant effect on 0,- release and did not or only slightly prevent reoxygenation injury (Fig. 3, Table 2). DISCUSSION NADPH Oxidase and NO Synthase. Inhibition of 0,-

release by NADPH oxidase by ebselen has been described for alveolar (13) and peritoneal macrophages (15)and granulocytes (12). It also applies to Kupffer cells (Fig. 2, Table 1). In line with the inhibition of 0,release, luminol chemiluminescence was inhibited by ebselen (Figs. 1 and 2, Table 1).However, a marked difference was seen in the concentration that provided half-maximal inhibition of 0,- release and luminol chemiluminescence. Whereas 0, - release was decreased by 50% in the presence of 10 pmolL ebselen, the decrease in luminol chemiluminescence was already half-maximal at 2 pmolL ebselen. Luminol chemilumi-

nescence is usually considered to result from reaction of 0,-, H,O,, OH, '0, and HOCI with luminol (21-24). Recently, however, we demonstrated (25) that luminol chemiluminescence in TPA-activated Kupffer cells results from a cooperative action of O,-/H,O, formed by NADPH oxidase and NO released by NO synthase, presumably caused by a common intermediate such as the peroxynitrite anion (ONOO-) or sequential oxidation of luminol. Thus an explanation for the marked inhibitory effect of ebselen on luminol chemiluminescence is that it not only inhibits NADPH oxidase but also inhibits NO synthase. In addition, NO formation by LPS-pretreated Kupffer cells, where maximum amounts of NO are released but no 0,- is formed, was inhibited by ebselen at a high potency (Fig. 2, Table 1).Inhibition of NADPH oxidase by ebselen has been suggested as resulting from direct reaction of ebselen with critical thiol/disulfide groups (14).Such a reaction may also explain the inhibition of NO synthase. Like its effect on 0,- release, the effect of ebselen on NO formation depends on the presence of the selenium atom in the molecule and is lost upon its methylation. Reoxygenation Zeury. In accordance with its inhibitory effects on NADPH oxidase, ebselen provided marked protection against reoxygenation injury of Kupffer cells (Fig. 3). As demonstrated (201, this injury mainly results from release of 0,-/H,O,. However, the

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WANG ET AL.

fact that the concentration providing 50% inhibition of reoxygenation injury ( 5 pmol/L) was lower than the respective concentration for the 50% inhibition of 0,formation (10 pmol/L) suggests that part of the protection may also result from other reactivities of ebselen (e.g., antioxidant properties). A contribution of NO to reoxygenation injury of the Kupffer cells is unlikely, because no protective effect of NG-nitro-L-arginine was seen. Further, no increase in NO formation occurred at 4 hr after reoxygenation (Table 21, a time at which significant cell injury had already occurred (Fig. 3). In the intact liver, however, a late increase in NO formation by Kupffer cells may contribute to injury of neighboring cells like endothelial cells or hepatocytes, in line with a suggestion by Billiar et al. (26). In conclusion, ebselen is a potent inhibitor not only of NADPH oxidase but also of NO synthase. These characteristics are of potential significance for protection from reperfusion injury of the liver, where increasing evidence suggests that Kupffer cell activation is a decisive event (16, 20, 27, 28). It should also be of importance for the prevention of other pathological processes in which activation of macrophages or granulocytes is involved. REFERENCES 1. Dieter P, Schultze-Specking A, Decker K. Ca2 requirement of prostanoid but not of superoxide production by rat Kupffer cells. Eur J Biochem 1988;177:61-67. 2. Billiar TR, Curran RD, Ferrari FK, Williams DL, Simmons RL. Kupffer cell: hepatocyte cocultures release nitric oxide in response to bacterial endotoxin. J Surg Res 1990;48:349-353. 3 Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990;192:245-261. 4. Muller A, Cadenas E, Graf P, Sies H. A novel biologically active selenoorganic compound. I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (ebselen). Biochem Pharmacol 1984;33:3235-3239. 5. Wendel A, Fausel M, Safayhi H, Tiegs G, Otter R. .4 novel biologically active seleno-organic compound. 11. Activity of PZ 51 in relation to glutathione peroxidase. Biochem Pharmacol 1984; 33:3241-3245. 6 . Narayanaswami V, Sies H. Antioxidant activity of ebselen and related selenoorganic compounds in microsomal lipid peroxidation. Free Rad Res Commun 1990;10:237-244. 7 Narayanaswami V, Sies H. Oxidative damage to mitochondria and protection by ebselen and other antioxidants. Biochem Pharmacol 1990;40:1623-1629. 8 Schoneich C, Narayanaswami V, Asmus KD, Sies H. Reactivity of ebselen and related selenoorganic compounds with 1,2dichloroethane radical cations and halogenated peroxyl radicals. Arch Biochem Biophys 1990;282:18-25. 9. Muller A, Gabriel H, Sies H. A novel biologically active selenoorganic compound. IV. Protective glutathione-dependent effect of PZ 5 1 (ebselen) against ADP-Fe-induced lipid peroxidation in isolated hepatocytes. Biochem Pharmacol 1985;34:1185-1189. 10. Cotgreave IA,Sandy MS, Berggren M, Moldeus PW, Smith MT. N-acetylcysteine and glutathione-dependent protective effect of +

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PZ 51 (ebselen) against diquat-induced cytotoxicity in isolated hepatocytes. Biochem Pharmacol 1987;36:2899-2904. 11. Wendel A, Tiegs G. A novel biologically active seleno-organic compound. VI. Protection by ebselen (PZ 51) against galactosamine/endotoxin-induced hepatitis in mice. Biochem Pharmacol 1986;35:2115-2118. 12. Ichikawa S, Omura K, Katayama T, Okamura N, Ohtsuka T, Ishibashi S, Masayasu H. Inhibition of superoxide anion production in guinea pig polymorphonuclear leukocytes by a selenoorganic compound, ebselen. J Pharmacobio-Dyn 1987;10:595-597. 13. Leurs R, Timmerman H, Bast A. Inhibition of superoxide anion radical production by ebselen (PZ 51) and its sulfur analogue (PZ 25) in guinea pig alveolar macrophages. Biochem Intl 1989;18: 295-299. 14. Cotgreave L4, Duddy SK, Kass GEN, Thompson D, Moldeus P. Studies on the anti-inflammatory activity of ebselen. Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C? Biochem Pharmacol 1989;38:649-656. 15. Parnham MJ, Kindt S. A novel biologically active seleno-organic compound. 111. Effects of PZ 51 (ebselen) on glutathione peroxidase and secretory activities of mouse macrophages. Biochem Pharmacol 1984;33:3247-3250. 16. Rymsa B, Becker HD, Lauchart W, de Groot H. Hypoxidreoxygenation injury in liver: Kupffer cells are much more vulnerable to reoxygenation than to hypoxia. Res Commun Chem Pathol Pharmacol 1990;68:263-266. 17. Johnston RB. Measurement of 0,- secreted by monocytes and macrophages. Meth Enzymol 1984;105:365-370. 18. Arthur MJP, Kowalski-Saunders P, Gurney S, Tolcher R, Bull FG, Wright R. Reduction of ferricytochrome c may underestimate superoxide production by monocytes. J Immunol Meth 1987;98: 63-69. 19. Feelisch M, Noack EA. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur J Pharmacol 1987;139:19-30. 20. Rymsa B, Wang J F , de Groot H. 0, release by activated Kupffer cells upon hypoxidreoxygenation. Am J Physiol 1991;261:G602G607. 21. Allen RC, Loose LD. Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem Biophys Res Commun 1976;69:245-252. 22. Stevens P, Hong D. The role of myeloperoxidase and superoxide anion in the luminol- and lucigenin-dependent chemiluminescence of human neutrophils. Microchem J 1984;30:135-146. 23. Muller-Peddinghaus R. In vitro determination of phagocyte activity by luminol- and lucigenin-amplified chemiluminescence. Int J Immunopharmacol 1984;6:455-466. 24. Brestel EP. Co-oxidation of luminol by hypochlorite and hydrogen peroxide. Implications for neutrophil chemiluminescence. Biochem Biophys Res Commun 1985;126:482-488. 25. Wang J F , Komarov P, Sies H, de Groot H. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbolester-activated Kupffer cells. Biochem J 1991; 279~311-314. 26. Billiar TR, Curran RD, West MA, Hoffmann K, Simmons RL. Kupffer cell cytotoxicity to hepatocytes in coculture requires L-arginine. Arch Surg 1989;124:1416-1421. 27. Jaeschke H, Farhood A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am J Physiol 1991;260:G355-G362. 28. Caldwell-Kenkel JC, Currin RT, Tanaka Y, Thurman RG, Lemasters JJ. Kupffer cell activation and endothelial cell damage after storage of rat liver: Effects of reperfusion. HEPATOLOGY 1991;13:83-95. ~

Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.

Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC5...
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