Dextrorphan

EPC-KI, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran6-yl-hydrogen phosphate] potassium salt, is a novel antioxidant. In this study, we investigated a reduction of oxidative neuronal cell damage with EPC-KI by immunohistochemical analysis for 8-hydroxy-2'deoxyguanosine (8-OHdG) in rat brain with 60 min transient middle cerebral artery occlusion, in association with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and staining for total and active caspase-3. Treatment with EPC-Kl (20 mg kg^sup -1^ i.v.) significantly reduced infarct size (p

Keywords: EPC-KI; ischemia; 8-OHdG,- TUNEL; caspase-3

INTRODUCTION

L-ascorbic acid (vitamin C) and a-tocopherol (vitamin E), two endogenous antioxidants, are important members of nonenzymatic antioxidant defense systems. However, as vitamin E is insoluble in water, its water-soluble analogs are synthesized in order to improve its absorption. EPCK1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl2-(4,8,12-trimethyltridecly)-2H-1 -benzopyran-6-yl-hydrogen phosphate] potassium salt, is a novel phosphate ester of vitamin C and vitamin E, which is soluble in both water and lipid1. It was reported2 that EPC-Kl could act as a scavenger of hydroxyl radicals and an inhibitor of lipid peroxidation. Hydroxyl radical is highly reactive, and oxidizes cellular lipids, protein and nucleic acids, leading to cell death3,4. Under physiological condition, superoxide and hydrogen peroxide are constantly scavenged by superoxide dismutase (SOD), glutathione peroxidase, and catalase. Under condition of ischemia, however, an overproduction of oxygen free radicals perturbs the antioxidative defense mechanisms, and hydroxyl radicals are generated. Studies have shown that radical scavengers and inhibitors of lipid peroxidation can ameliorate ischemic neuronal damages-13. Indeed, previous study14 demonstrated that EPC-K1 reduced ischemic brain damage. However, the mechanism of its neuroprotective effect had been unclear.

CONCLUSION

The current study demonstrated that the ameliorative effect of EPC-K1 on ischemic brain injury was associated with reduction of 8-OHdG-, TUNEL-, total and active caspase-3-positive cells.

ACKNOWLEDGEMENTS

This work was partly supported by Grants-in-Aid for Scientific Research on 12877211 and 12470141 from the Ministry of Educaiton, Japan, and grants from Japan Brain Foundation, Takeda Medical Research Foundation, and Japan Heart Association.

REFERENCES

1 Ogata K, Yamamoto K, Tsuruoka H. Chemical structure and physicochemical properties of L-ascorbic acid 2-[3,4-dihydro2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1 -benzopyran6-yl hydrogen phosphate] potassium salt (EPC-Kl). lyakuhin Kenkyu 1990; 211: 222-228

2 Mori A, Edamatsu R, Kohno M, Ohmor S. A new hydroxyl radical scavenger: EPC-KI. Neuroscience 1989; 15: 371-376

3 Floyd RA, West MS, Eneff K, Schneider JE. Methylene blue plus light mediates 8-hydroxyguanine formation in DNA. Arch Biochem Biophys 1989; 273: 106-111

4 Oliver CN, Starke-Reed PE, Stadtman ER. Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain. Proc Natl Acad Sci USA 1990; 87: 5144-5147

5 Pahlmark K, Siesjo BK. Effects of the spin trap-alpha-phenyl-tertbutylnitrone (PBN) in transient forebrain in the rat. Acta Physiol Scand 1996; 157: 41-51

6 Takasago T, Peters EE, Graham DL, Masayasu H, Macrae IM. Neuroprotective efficacy of ebseren, an anti-oxidant with antiinflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. Brj Pharmacol 1997; 122: 1251-1256

7 Umemura K, Wada K, Umemura T, Mizuno A, Nakashima M. Effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model. Eur J Pharmacol 1994. 251: 69-74

8 Zhao Q, Pahlmark K, Smith ML, Siesjo BK. Delayed treatment with the a-phenyl-tent-butylnitrone (PBN) reduces infarct size following transient middle cerebral artery occlusion in rats. Acta Physiol Scand 1994; 152: 349-350

9 Abe K, Yuki S, Kogure K. Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988; 19: 480-485

10 Nishi H, Watanabe T, Sakurai H, Yuki S, Ishibashi A. Effect of MCI186 on brain edema in rats. Stroke 1989; 20: 1236-1240

11 Patt A, Horesh IR, Berger EM, Harken AH, Repine JE. Iron depletion or chelation reduces ischemia/reperfusion-induced edema in gerbil brains. J Pediatr Surg 1990; 25: 224-228

12 Cao X, Phyllis JW. a-Phenyl-tert-butylnitrone reduces cortical infarct and edema in rats subjected to focal ischemia. Brain Res 1994; 644: 267-272

13 Clemens JA, Sauders RD, Ho PP, Phebus LA, Panetta JA. The antioxidant LY231 617 reduces global ischemic neuronal injury in rats. Stroke 1993; 24: 716-723

14 Hyroyuki T, Kazunao K, Yasuhiko I, Kazuo U. Hydroxyl radical generation after the third hour following ischemia contributes to brain damage. EurJ Pharmacol 1998; 352: 165-169

15 Hayashi T, Abe K, Itoyama Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. I Cereb Blood Flow Metab 1998; 18: 887-895

16 Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokumi S. Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: Independence of tubular necrosis and sulfohydryl depletion. Lab Invest 1997; 77: 145-155

17 Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T. Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 1997; 76: 365-374

18 Rasper D, Vaillancourt J, Hadano S, Houtzager V, Seiden I, Keen S, Tawa P, Xanthoudakis S, Nasir J, Martindale D, Koop B, Peterson E, Thornberry N, Huang JQ, Macpherson D, Black S, Hornung F. Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. Cell Death Differ 1998; 5: 271-288

19 Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucl Acids Res 1984; 12: 2137-2145

20 Devasagayam TPA, Steeken S, Obendorf MSW, Schultz WA, Sies H. Formation of 8-hydroxy (deoxy)-guanosine and generation of strand breaks at guanine residues in DNA by singlet oxygen. Biochemistry 1991; 30: 6283-6289

21 Floyd RA, Carney JM. Free radical damage to protein and DNA: Mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann Neurol 1992; 32: S22-S27

22 Hayashi T, Sakurai M, Itoyama Y, Abe K. Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion. Brain Res 1999; 832: 159-163

23 Du C, Hu R, Csernansky CA, Liu XZ, Hsu CY, Choi DW. Additive neuroprotective effects of dextrorphan and cycloheximide in rats subjected to transient focal cerebral ischemia. Brain Res 1996; 718: 233-236

24 Li Y, Sharov VG, Jiang N, Zaloga C, Sabba HN, Chopp M. Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. Am J Pathol 1995; 146: 1045-1051

25 Kitagawa H, Hayashi T, Mitsumoto Y, Koga N, Itoyama Y, Abe K. Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats. Stroke 1998; 29: 1417-1422

W.R. Zhang, T. Hayashi, C. Sasaki, K. Sato, 1. Nagano, Y. Manabe and K. Abe Department of Neurology, Okayama University School of Medicine and Dentistry, Okayama, Japan

Correspondence and reprint requests to: Prof. Abe, MD, PhD, Department of Neurology, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700-8558, Japan.

[zhang@cc.okayama-u.ac.jp] Accepted for publication December 2000.

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