- Email: [email protected]
Efficacy of the antioxidant ebselen in experimental uveitis
Free Radical Biology & Medicine, Vol. 27, Nos. 3/4, pp. 388 –391, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(99)00067-2
Original Contribution EFFICACY OF THE ANTIOXIDANT EBSELEN IN EXPERIMENTAL UVEITIS FRANCISCO BOSCH-MORELL,* JOAQU´ıN ROMA´ ,* FRANCISCO J. PUERTAS,* NURIA MAR´ıN,* MANUEL Dı´AZ-LLOPIS,†,‡ and FRANCISCO J. ROMERO* *Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine & Dentistry, University of Valencia, Valencia; †Ophthalmology Unit, University Hospital La Fe, Valencia; and ‡Ophthalmology Unit, Department of Surgery, School of Medicine, University of the Basque Country, Leioa, Spain (Received 11 November 1998; Revised 19 February 1999; Accepted 16 March 1999)
Abstract—Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues. © 1999 Elsevier Science Inc. Keywords—Free radicals, Antioxidant, Glutathione peroxidase, Malondialdehyde, Endotoxin, Glutathione, Peroxynitrite
INTRODUCTION
studies and even also in humans [5]. Its antioxidant capacity is largely attributable to its property as an enzyme mimic of the seleno enzyme glutathione (GSH) peroxidase (Gpx) [6]. Moreover, this compound has antiinflammatory properties in view of its inhibitory effects on the formation of leukotrienes in different experimental models [7–9], and more recently, it has also been reported its peroxynitrite scavenger effect [10,11]. The main goal of this article was to establish the extent of the ocular damage associated with endotoxininduced uveitis (EIU) in the rat eye. For this purpose we have studied the modifications of the antioxidant capacity of the whole eye, the accumulation of LPO products also in the whole eye, and the effect of the systemic administration of ebselen on all these parameters.
Uveitis is an inflammatory process associated with the generation of an oxidative stress situation; this is confirmed by the occurrence of lipid peroxidation (LPO) in experimental uveitis [1–3]. LPO is a process normally associated with an increase in oxygen consumption and with a decrease of antioxidants in the tissue where it takes place [4]. The generation of lipid derivatives during this process results, among others, in the chemoattraction of inflammatory cells that furthermore, once activated, might close the vicious circle of oxygen species production. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a selenoorganic compound with antioxidant properties, and of extremely low toxicity observed largely in animal Address correspondence to: Francisco J. Romero, Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine & Dentistry, University of Valencia, Av. Blasco Iban˜ez, 17, E-46010-Valencia, Spain; Tel: ⫹ (34) 96386-4646; Fax: ⫹ (34) 963864642; E-mail: [email protected]
MATERIALS AND METHODS
Thirty-two Lewis rats, about 200 g of weight, were divided into four groups. A control group that received 388
Efficacy of ebselen in uveitis
389
only the vehicles of the treated groups, another was injected with 100 g of endotoxin, diluted in 0.1-ml saline, from Escherichia coli, serotype 026 (Sigma Quı´mica, Alcobendas, Spain) into the foot pad. The third group was injected with endotoxin and received orally two doses of ebselen (100 mg/kg, dissolved in olive oil), the first simultaneously with endotoxin and the second 12-h later, and the last group received ebselen alone. Ebselen was provided by Rhone/Poulenc/Ciba Geigy/ Natterman (Cologne, Germany). Animals were sacrificed 24-h after injection of endotoxin in a 100% ether atmosphere. Eyes were enucleated and frozen in liquid nitrogen for biochemical studies. In order to determine the ebselen-dependent “glutathione peroxidase-like” activity, whole eyes were homogenized in 2% perchloric acid containing 1-mM EDTA, and the peroxidase activity studied in the neutralized supernatant. GPx activity towards H2O2 [12], GSH [13], ebselen [14], and MDA [15,16] concentrations were determined according to the references cited; except that 1 mM azide was added to the GPx activity assay system to inhibit catalase. Findings and biochemical results of differents groups were compared with the Student’s t-test. RESULTS
Figure 1 shows the histopathological findings of eyes treated with endotoxin compared with eyes from control animals; the lymphocytic infiltration observed confirms the uveitis in these animals, as previously reported [2]. GSH content of the eyes decreased 24 h after administration of endotoxin. This decrease is associated with a decrease in GPx activity and an increase in the concentration of MDA (Table 1). Oral administration of ebselen prevented the endotoxin-induced GSH depletion as well as the GPx decrease and MDA increase, whereas ebselen alone had no effect on either GSH or MDA contents. GPx activity decreases in the uveitis group respect to the controls (Table 1). The total GPx activity of ebselentreated eyes (ebselen alone or together with endotoxin) was higher than controls, whether this increase could be dependent on the presence of ebselen or its derivatives is proofed by the fact that ebselen-dependent GPx-like activity (determined as the activity present in the acidic extract) fully accounts for the increase observed (Table 1). These results clearly show that animals treated with endotoxin and ebselen together have a total GPx activity statistically higher than the endotoxin-treated, whereas the differential GPx activity (which represents the enzymatic activity of the tissue protein) in the former group is similar to control ones. It is not possible to establish if the GPx-like activity, detected in the neutralized acid
Fig. 1. Photomicrographs of hematoxylin-eosin stained sections of eyes from control (a) and endotoxin-treated (b) animals.
extract of the eye is dependent on unmodified ebselen, or if any of the metabolites formed retains some GPx-like activity and may also accumulate in the eye. Chromatographic analysis of samples from both ebselen plus endotoxin or ebselen alone allowed the recognition in the whole eye homogenates of significant amounts of unmodified ebselen (Fig. 2) and a mixture of Ebselen derivatives that are eluted from the column somewhat later (data not shown). DISCUSSION
Ebselen has been tested in several experimental models [5], even also using endotoxin as inductor of inflammation [8]. Preliminary results from our laboratory demonstrated a protective effect of ebselen on some clinical and histopathological features of endotoxin-induced uveitis in the rat [17]. The results presented confirm that ebselen effectively ameliorates biochemical parameters of experimental endotoxin-induced uveitis. Previous literature data confirmed the capacity of ebselen to inhibit lipid peroxidation [18], most probably via its GPx-like activity, but also by inhibiting leukotriene B4 formation and probably other cyclo-oxygenase products [7–9] and peroxynitrite toxicity [10,11]. The action of free radicals is basic in the
390
F. BOSCH-MORELL et al.
Table 1. Effect of the Administration of Endotoxin, Ebselen, and Both Together on GSH and MDA Content and GPx Activity in the Whole Eye Homogenate of Lewis Rats GPx (nmol/mg protein ⫻ min) Group
GSH (nmol/mg protein)
MDA (nmol/mg protein)
Total
GPx-like
Differential
Control Endotoxin Ebselen Endotoxin ⫹ ebselen
12.89 ⫾ 1.20 9.80 ⫾ 2.15* 12.47 ⫾ 1.75‡ 12.85 ⫾ 1.88‡
0.040 ⫾ 0.004 0.066 ⫾ 0.008† 0.041 ⫾ 0.007§ 0.043 ⫾ 0.009§
6.84 ⫾ 4.81 4.81 ⫾ 0.87 11.89 ⫾ 2.24§ 10.95 ⫾ 1.66†
0.30 ⫾ 0.12 0.37 ⫾ 0.33 5.04 ⫾ 1.83†,‡ 4.84 ⫾ 0.93†,‡
6.54 ⫾ 1.34 4.44 ⫾ 0.74* 6.86 ⫾ 2.45§ 6.11 ⫾ 1.76§
Results are means ⫾ standard deviation of eight different assays, of at least three different animals. *p ⬍ .005, † p ⬍ .001 both vs. control. ‡ p ⬍ .01, § p ⬍ .001 both vs. endotoxin group.
inflammatory mechanism of endotoxin-induced uveitis. This is in agreement with the increase in malondialdehyde in the eye shown in Table 1 and previously observed by others [1]. Interestingly, the novel finding of a significant decrease in GSH content, observed in eyes of endotoxin-treated rats (Table 1), confirms this inflammatory-induced oxidative challenge. The decrease in GPx activity in the eyes of these same animals could be explained by the susceptibility of this enzyme to oxidative modifications [19]. In this sense, it has been very recently proposed that GPx may act as a sensory molecule for the detection of oxidative stress [20], in view of the ability of nitric oxide and its derivatives to inactivate Gpx [21]. The results in Table 1 show a higher tissue GPx activity (differential activity) in animals treated
with endotoxin and ebselen together than in the endotoxin-treated group, suggesting that the peroxynitrite scavenging action of ebselen [10,11] may result in the protection of tissue enzyme. The suitability of a therapeutic approach with ebselen for ocular tissues is supported by: (i) the presence of unmodified ebselen in the eye homogenates after oral administration even in the group without inflammation, which clearly demonstrates that it crosses blood-aqueous and blood-retina barrier; (ii) ebselen replenishes the cellular antioxidant defenses by recovering GSH content and both recovering and protecting tissue GPx activity; and finally (iii) by its extremely low toxicity in animals and humans [5]. Acknowledgements — This work has been supported by grants PM960103 from the Direccio´n General de Ensen˜anza Superior, Madrid, and 96/1504 and 99/0568 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS), Madrid, Spain, to FJR.
Fig. 2. Chromatograms of whole eye homogenates of control rats and animals treated with endotoxin, ebselen, and both together. Arrow shows the retention time of ebselen standards. (A) Control; (B) endotoxin; (C) ebselen; (D) ebselen ⫹ endotoxin.
REFERENCES [1] Rao, N. A.; Fernandez, M. A.; Cid, L. L.; Romero, J. L.; Sevanian, A. Retinal lipid peroxidation in experimental uveitis. Arch. Ophthalmol. 105:1712–1716; 1987. [2] Rao, N. A. Role of oxygen free radicals in retinal damage associated with experimental uveitis. Trans. Am. Ophthalmol. Soc. 88:797– 850; 1990. [3] Wu, G.-S.; Sevanian, A.; Rao, N. A. Detection of retinal lipid hydroperoxides in experimental uveitis. Free Radic. Biol. Med. 12:19 –27; 1992. [4] Halliwell, B.; Gutteridge, J. M. C. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 23:1396 – 1397; 1984. [5] Sies, H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Radic. Biol. Med. 14:313–323; 1993. [6] Mu¨ller, A.; Cadenas, E.; Graf, P.; Sies, H. A novel biologically active seleno-organic compound. I. Glutathione peroxidase-like activity in-vitro and antioxidant capacity of PZ 51 (ebselen). Biochem. Pharmacol. 33:3235–3239; 1984. [7] Safayhi, H.; Tiegs, G.; Wendel, A. A novel biologically active seleno-organic compound- V. Inhibition by ebselen (PZ 51) of rat peritoneal neutrophil lipoxigenase. Biochem. Pharmacol. 34:2691–2694; 1985. [8] Wendel, A.; Tiegs, G. A novel biologically active seleno-organic compound-VI. Protection by ebselen (PZ51) against galactosamine/endotoxin-induced hepatitis in mice. Biochem. Pharmacol. 35:2115–2118; 1986.
Efficacy of ebselen in uveitis [9] Kuhl, P.; Borbe, H. O.; Ro¨mer, A.; Fischer, H.; Parnham, M. J. Selective inhibition of leukotriene B4 formation by ebselen: a novel approach to antiinflammatory therapy. Agents Actions 17: 366 –367; 1985. [10] Sata, N.; Klonowski-Stumpe, H.; Han, B.; Haussinger, D.; Niederau, C. Cytotoxicity of peroxynitrite in rat pancreatic acinar AR4-2J cells. Pancreas 15:278 –284; 1997. [11] Sies, H.; Sharov, V. S.; Klotz, L. O.; Briviba, K. Glutathione peroxidase protects against peroxynitrite-mediated oxidations–a new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272:27812–27817; 1997. [12] Lawrence, R. A.; Parkhill, L. K.; Burk, R. F. Hepatic cytosolic non-selenium dependent glutathione peroxidase activity: its nature and the effect of selenium deficiency. J. Nutr. 108:981–987; 1978. [13] Romero, F. J.; Roma´, J. Glutathione and protein kinase C in peripheral nervous tissue. Meth. Enzymol. 252:146 –153; 1995. [14] Mu¨ller, A.; Gabriel, H.; Sies, H.; Terlinden, R.; Fischer, H.; Ro¨mer, A. A novel biologically active selenoorganic compound. VII. Biotransfromation of ebselen in perfused rat liver. Biochem. Pharmacol. 37:1103–1109; 1988. [15] Richard, M. J.; Guiraud, P.; Meo, J.; Favier, A. High performance liquid chromatography separation of malondialdehyde thiobarbituric acid adduct in biological materials (plasma and human cell) using a commercially available reagent. J. Chromatogr. 577:9 – 18; 1992. [16] Romero, M. J.; Bosch-Morell, F.; Romero, B.; Rodrigo, J. M.; Serra, M. A.; Romero, F. J. Serum malondialdehyde: possible use for the clinical management of chronic hepatitis C patients. Free Radic. Biol. Med. 25:993–997; 1998.
391
[17] Dı´az-Llopis, M.; Chipont, E.; Bosch-Morell, F.; Puertas, F. J.; Hermenegildo, C.; Navea, A.; Romero, F. J. Efficacy of Ebselen (PZ-51) in experimental endotoxin-induced anterior uveitis (abst.). Invest. Ophthalmol. Vis. Sci. 34:1050; 1993. [18] Narayanaswami, V.; Sies, H. Antioxidant activity of ebselen and related selenoorganic compounds in microsomal lipid peroxidation. Free Radic. Res. Comm. 10:237–244; 1990. [19] Pigeolet, E.; Remacle, J. Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals. Free Radic. Biol. Med. 11:191–195; 1991. [20] Taniguchi, N. Inactivation of glutathione peroxidase, a possible sensory molecule, by reactive oxygen and nitrogen species. Pathophysiology 5(Suppl. 1):61; 1998. [21] Asahi, M.; Fujii, J.; Takao, T.; Kuzuya, T.; Hori, M.; Shimonishi, Y.; Taniguchi, N. The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor. J. Biol. Chem. 272:19152–19157; 1997.
ABBREVIATIONS
EIU— endotoxin-induced uveitis GPx— glutathione peroxidase GSH— glutathione LPO—lipid peroxidation MDA—malondialdehyde
PII S0891-5849(99)00067-2
Original Contribution EFFICACY OF THE ANTIOXIDANT EBSELEN IN EXPERIMENTAL UVEITIS FRANCISCO BOSCH-MORELL,* JOAQU´ıN ROMA´ ,* FRANCISCO J. PUERTAS,* NURIA MAR´ıN,* MANUEL Dı´AZ-LLOPIS,†,‡ and FRANCISCO J. ROMERO* *Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine & Dentistry, University of Valencia, Valencia; †Ophthalmology Unit, University Hospital La Fe, Valencia; and ‡Ophthalmology Unit, Department of Surgery, School of Medicine, University of the Basque Country, Leioa, Spain (Received 11 November 1998; Revised 19 February 1999; Accepted 16 March 1999)
Abstract—Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues. © 1999 Elsevier Science Inc. Keywords—Free radicals, Antioxidant, Glutathione peroxidase, Malondialdehyde, Endotoxin, Glutathione, Peroxynitrite
INTRODUCTION
studies and even also in humans [5]. Its antioxidant capacity is largely attributable to its property as an enzyme mimic of the seleno enzyme glutathione (GSH) peroxidase (Gpx) [6]. Moreover, this compound has antiinflammatory properties in view of its inhibitory effects on the formation of leukotrienes in different experimental models [7–9], and more recently, it has also been reported its peroxynitrite scavenger effect [10,11]. The main goal of this article was to establish the extent of the ocular damage associated with endotoxininduced uveitis (EIU) in the rat eye. For this purpose we have studied the modifications of the antioxidant capacity of the whole eye, the accumulation of LPO products also in the whole eye, and the effect of the systemic administration of ebselen on all these parameters.
Uveitis is an inflammatory process associated with the generation of an oxidative stress situation; this is confirmed by the occurrence of lipid peroxidation (LPO) in experimental uveitis [1–3]. LPO is a process normally associated with an increase in oxygen consumption and with a decrease of antioxidants in the tissue where it takes place [4]. The generation of lipid derivatives during this process results, among others, in the chemoattraction of inflammatory cells that furthermore, once activated, might close the vicious circle of oxygen species production. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a selenoorganic compound with antioxidant properties, and of extremely low toxicity observed largely in animal Address correspondence to: Francisco J. Romero, Experimental Toxicology & Neurotoxicology Unit, Department of Physiology, School of Medicine & Dentistry, University of Valencia, Av. Blasco Iban˜ez, 17, E-46010-Valencia, Spain; Tel: ⫹ (34) 96386-4646; Fax: ⫹ (34) 963864642; E-mail: [email protected]
MATERIALS AND METHODS
Thirty-two Lewis rats, about 200 g of weight, were divided into four groups. A control group that received 388
Efficacy of ebselen in uveitis
389
only the vehicles of the treated groups, another was injected with 100 g of endotoxin, diluted in 0.1-ml saline, from Escherichia coli, serotype 026 (Sigma Quı´mica, Alcobendas, Spain) into the foot pad. The third group was injected with endotoxin and received orally two doses of ebselen (100 mg/kg, dissolved in olive oil), the first simultaneously with endotoxin and the second 12-h later, and the last group received ebselen alone. Ebselen was provided by Rhone/Poulenc/Ciba Geigy/ Natterman (Cologne, Germany). Animals were sacrificed 24-h after injection of endotoxin in a 100% ether atmosphere. Eyes were enucleated and frozen in liquid nitrogen for biochemical studies. In order to determine the ebselen-dependent “glutathione peroxidase-like” activity, whole eyes were homogenized in 2% perchloric acid containing 1-mM EDTA, and the peroxidase activity studied in the neutralized supernatant. GPx activity towards H2O2 [12], GSH [13], ebselen [14], and MDA [15,16] concentrations were determined according to the references cited; except that 1 mM azide was added to the GPx activity assay system to inhibit catalase. Findings and biochemical results of differents groups were compared with the Student’s t-test. RESULTS
Figure 1 shows the histopathological findings of eyes treated with endotoxin compared with eyes from control animals; the lymphocytic infiltration observed confirms the uveitis in these animals, as previously reported [2]. GSH content of the eyes decreased 24 h after administration of endotoxin. This decrease is associated with a decrease in GPx activity and an increase in the concentration of MDA (Table 1). Oral administration of ebselen prevented the endotoxin-induced GSH depletion as well as the GPx decrease and MDA increase, whereas ebselen alone had no effect on either GSH or MDA contents. GPx activity decreases in the uveitis group respect to the controls (Table 1). The total GPx activity of ebselentreated eyes (ebselen alone or together with endotoxin) was higher than controls, whether this increase could be dependent on the presence of ebselen or its derivatives is proofed by the fact that ebselen-dependent GPx-like activity (determined as the activity present in the acidic extract) fully accounts for the increase observed (Table 1). These results clearly show that animals treated with endotoxin and ebselen together have a total GPx activity statistically higher than the endotoxin-treated, whereas the differential GPx activity (which represents the enzymatic activity of the tissue protein) in the former group is similar to control ones. It is not possible to establish if the GPx-like activity, detected in the neutralized acid
Fig. 1. Photomicrographs of hematoxylin-eosin stained sections of eyes from control (a) and endotoxin-treated (b) animals.
extract of the eye is dependent on unmodified ebselen, or if any of the metabolites formed retains some GPx-like activity and may also accumulate in the eye. Chromatographic analysis of samples from both ebselen plus endotoxin or ebselen alone allowed the recognition in the whole eye homogenates of significant amounts of unmodified ebselen (Fig. 2) and a mixture of Ebselen derivatives that are eluted from the column somewhat later (data not shown). DISCUSSION
Ebselen has been tested in several experimental models [5], even also using endotoxin as inductor of inflammation [8]. Preliminary results from our laboratory demonstrated a protective effect of ebselen on some clinical and histopathological features of endotoxin-induced uveitis in the rat [17]. The results presented confirm that ebselen effectively ameliorates biochemical parameters of experimental endotoxin-induced uveitis. Previous literature data confirmed the capacity of ebselen to inhibit lipid peroxidation [18], most probably via its GPx-like activity, but also by inhibiting leukotriene B4 formation and probably other cyclo-oxygenase products [7–9] and peroxynitrite toxicity [10,11]. The action of free radicals is basic in the
390
F. BOSCH-MORELL et al.
Table 1. Effect of the Administration of Endotoxin, Ebselen, and Both Together on GSH and MDA Content and GPx Activity in the Whole Eye Homogenate of Lewis Rats GPx (nmol/mg protein ⫻ min) Group
GSH (nmol/mg protein)
MDA (nmol/mg protein)
Total
GPx-like
Differential
Control Endotoxin Ebselen Endotoxin ⫹ ebselen
12.89 ⫾ 1.20 9.80 ⫾ 2.15* 12.47 ⫾ 1.75‡ 12.85 ⫾ 1.88‡
0.040 ⫾ 0.004 0.066 ⫾ 0.008† 0.041 ⫾ 0.007§ 0.043 ⫾ 0.009§
6.84 ⫾ 4.81 4.81 ⫾ 0.87 11.89 ⫾ 2.24§ 10.95 ⫾ 1.66†
0.30 ⫾ 0.12 0.37 ⫾ 0.33 5.04 ⫾ 1.83†,‡ 4.84 ⫾ 0.93†,‡
6.54 ⫾ 1.34 4.44 ⫾ 0.74* 6.86 ⫾ 2.45§ 6.11 ⫾ 1.76§
Results are means ⫾ standard deviation of eight different assays, of at least three different animals. *p ⬍ .005, † p ⬍ .001 both vs. control. ‡ p ⬍ .01, § p ⬍ .001 both vs. endotoxin group.
inflammatory mechanism of endotoxin-induced uveitis. This is in agreement with the increase in malondialdehyde in the eye shown in Table 1 and previously observed by others [1]. Interestingly, the novel finding of a significant decrease in GSH content, observed in eyes of endotoxin-treated rats (Table 1), confirms this inflammatory-induced oxidative challenge. The decrease in GPx activity in the eyes of these same animals could be explained by the susceptibility of this enzyme to oxidative modifications [19]. In this sense, it has been very recently proposed that GPx may act as a sensory molecule for the detection of oxidative stress [20], in view of the ability of nitric oxide and its derivatives to inactivate Gpx [21]. The results in Table 1 show a higher tissue GPx activity (differential activity) in animals treated
with endotoxin and ebselen together than in the endotoxin-treated group, suggesting that the peroxynitrite scavenging action of ebselen [10,11] may result in the protection of tissue enzyme. The suitability of a therapeutic approach with ebselen for ocular tissues is supported by: (i) the presence of unmodified ebselen in the eye homogenates after oral administration even in the group without inflammation, which clearly demonstrates that it crosses blood-aqueous and blood-retina barrier; (ii) ebselen replenishes the cellular antioxidant defenses by recovering GSH content and both recovering and protecting tissue GPx activity; and finally (iii) by its extremely low toxicity in animals and humans [5]. Acknowledgements — This work has been supported by grants PM960103 from the Direccio´n General de Ensen˜anza Superior, Madrid, and 96/1504 and 99/0568 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS), Madrid, Spain, to FJR.
Fig. 2. Chromatograms of whole eye homogenates of control rats and animals treated with endotoxin, ebselen, and both together. Arrow shows the retention time of ebselen standards. (A) Control; (B) endotoxin; (C) ebselen; (D) ebselen ⫹ endotoxin.
REFERENCES [1] Rao, N. A.; Fernandez, M. A.; Cid, L. L.; Romero, J. L.; Sevanian, A. Retinal lipid peroxidation in experimental uveitis. Arch. Ophthalmol. 105:1712–1716; 1987. [2] Rao, N. A. Role of oxygen free radicals in retinal damage associated with experimental uveitis. Trans. Am. Ophthalmol. Soc. 88:797– 850; 1990. [3] Wu, G.-S.; Sevanian, A.; Rao, N. A. Detection of retinal lipid hydroperoxides in experimental uveitis. Free Radic. Biol. Med. 12:19 –27; 1992. [4] Halliwell, B.; Gutteridge, J. M. C. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 23:1396 – 1397; 1984. [5] Sies, H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Radic. Biol. Med. 14:313–323; 1993. [6] Mu¨ller, A.; Cadenas, E.; Graf, P.; Sies, H. A novel biologically active seleno-organic compound. I. Glutathione peroxidase-like activity in-vitro and antioxidant capacity of PZ 51 (ebselen). Biochem. Pharmacol. 33:3235–3239; 1984. [7] Safayhi, H.; Tiegs, G.; Wendel, A. A novel biologically active seleno-organic compound- V. Inhibition by ebselen (PZ 51) of rat peritoneal neutrophil lipoxigenase. Biochem. Pharmacol. 34:2691–2694; 1985. [8] Wendel, A.; Tiegs, G. A novel biologically active seleno-organic compound-VI. Protection by ebselen (PZ51) against galactosamine/endotoxin-induced hepatitis in mice. Biochem. Pharmacol. 35:2115–2118; 1986.
Efficacy of ebselen in uveitis [9] Kuhl, P.; Borbe, H. O.; Ro¨mer, A.; Fischer, H.; Parnham, M. J. Selective inhibition of leukotriene B4 formation by ebselen: a novel approach to antiinflammatory therapy. Agents Actions 17: 366 –367; 1985. [10] Sata, N.; Klonowski-Stumpe, H.; Han, B.; Haussinger, D.; Niederau, C. Cytotoxicity of peroxynitrite in rat pancreatic acinar AR4-2J cells. Pancreas 15:278 –284; 1997. [11] Sies, H.; Sharov, V. S.; Klotz, L. O.; Briviba, K. Glutathione peroxidase protects against peroxynitrite-mediated oxidations–a new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272:27812–27817; 1997. [12] Lawrence, R. A.; Parkhill, L. K.; Burk, R. F. Hepatic cytosolic non-selenium dependent glutathione peroxidase activity: its nature and the effect of selenium deficiency. J. Nutr. 108:981–987; 1978. [13] Romero, F. J.; Roma´, J. Glutathione and protein kinase C in peripheral nervous tissue. Meth. Enzymol. 252:146 –153; 1995. [14] Mu¨ller, A.; Gabriel, H.; Sies, H.; Terlinden, R.; Fischer, H.; Ro¨mer, A. A novel biologically active selenoorganic compound. VII. Biotransfromation of ebselen in perfused rat liver. Biochem. Pharmacol. 37:1103–1109; 1988. [15] Richard, M. J.; Guiraud, P.; Meo, J.; Favier, A. High performance liquid chromatography separation of malondialdehyde thiobarbituric acid adduct in biological materials (plasma and human cell) using a commercially available reagent. J. Chromatogr. 577:9 – 18; 1992. [16] Romero, M. J.; Bosch-Morell, F.; Romero, B.; Rodrigo, J. M.; Serra, M. A.; Romero, F. J. Serum malondialdehyde: possible use for the clinical management of chronic hepatitis C patients. Free Radic. Biol. Med. 25:993–997; 1998.
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ABBREVIATIONS
EIU— endotoxin-induced uveitis GPx— glutathione peroxidase GSH— glutathione LPO—lipid peroxidation MDA—malondialdehyde