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Resveratrol Inhibits Copper Ion-Induced and Azo Compound-Initiated Oxidative Modification of Human Low Density Lipoprotein Jian-Gang Zou', Yuan-Zhu Huang', Qi Chenz, En-Hui Wei2, Tze-chen Hsieh' and Joseph M. Wu''* ~Department of Cardiology, the First Affiliated Hospital, and ZAtherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, China 3Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA Abstract. To investigate whether resveratrol, a polyphenolic compound in red wine, affects the oxidation of human low density lipoprotein (LDL), LDL purified from normolipidemic subjects was subjected to CuZ~-induced and azo compound-initiated oxidative modification, with and without the addition of varying concentrations of resveratrol. Modification of LDL was assessed by the formation of thiobarbituric acid reactive substances (TBARS) and changes in the relative electrophoretic mobility (REM) of LDL on agarose gels. Resveratrol (50 1tM) reduced TBARS and REM of LDL during Cuz•`-induced oxidation by 70.5"/u and 42.3%, respectively (p<0.01), and prolonged the lag phase associated with the oxidative modification of LDL by copper ion or azo compound. These in vitro results suggest that resveratrol may afford protection of LDL against oxidative damage resulting from exposure to various environmental challenges, possibly by acting as a free radical scavenger. Introduction There is a wealth of epidemiological, clinical and laboratory data to suggest that oxidized LDL (OX-LDL) plays an important role in the development of atherosclerosis (AS). Oxidized and not unmodified LDL present within the intima of arteries is a characteristic feature of atherosclerotic lesions (1-4). Protection of the development of AS by dietary components such as cc-tocopherol, a-carotene, and butylated hydroxytoluene (BHT) may in part be effected by the ability of these chemicals to react with free radicals and suppress the oxidation of LDL. Probucol, a lipid-lowering drug, may exert a protective effect against development of spontaneous atherosclerosis in the Watanabe rabbit model (5), by acting as an antioxidant rather than as a lipid- regulating agent. An inverse relationship exists between the intake of natural antioxidants and the 1

incidence of coronary heart disease (6-9), further supporting the importance of LDL oxidation in AS. One noted exception to this relationship, however, is the apparent compatibility of a high fat diet with a low incidence of coronary heart disease, often referred to as the "French paradox". The observed disparity may be linked to the regular consumption of red wine, known to contain several phenolic compounds with antioxidant property (10). One such compound is the polyphenol resveratrol, which has been reported to be more active than vitamin E in inhibiting copper ion-catalyzed oxidation of LDL (11-15). In this communication, we investigated the effect of resveratrol to inhibit copper ion-induced and azo compound-initiated oxidative modification of LDL isolated from normolipidemic subjects. Resveratrol was shown to inhibit Cu"-induced and azo copound-initiated oxidation of LDL. Materials and Methods Reagents. Resveratrol, 1,1,3,3-tetramethoxypropane, thiobarbituric acid, and ascorbic acid (vitamin C) were purchased from Sigma Chemical Co. The azo compound, (2,2'-azobis(2- amidinopropane dihydrochloride], AAPH), was obtained from Polysciences Inc. Other chemicals were analytical grade. Isolation of LDL (16). LDL; with a density range of 1.019--1.063 g/ml, was prepared by sequential ultracentrifugation using blood from two healthy normolipidemic males. EDTA (0.05%) and gentamycin (80 mg/I) were added to minimize lipoprotein oxidation and microorganism contamination during preparation. Purified LDL was dialyzed at 4°C for 48 h against PBS (pH7.4) supplemented with 2 mM EDTA, sterilized by membrane filtration, and stored with EDTA under nitrogen. Protein concentration was determined by the Lowry's method (17). Cu2'-induced oxidation of LDL (18). LDL was dialyzed extensively against EDTA-free PBS (pH7.4) at 4°C, and incubated (500 µg protein/ml) at 37°C for 18 h with CuCh (final concentration, 40 µM) and resveratrol (final concentrations, 0, 25, 50, 100, 150 and 200 µM, dissolved in 1% DMSO, v/v). Aliquots were removed at the indicated times. Oxidation was stopped by the addition of 2.7 mM EDTA. The aliquots were assayed for TBARS and REM. Kinetics of Cu1 `-induced oxidation of LDL. LDL (500 µg/ml) was incubated with 40 LtM CuCl2 and 50 µM resveratrol at 37°C- At the indicated times the reaction was stopped with 2.7 mM EDTA and the amount of TBARS was determined. Azo compound-initiated oxidation of LDL (19). LDL (500 µg/ml) dialyzed extensively against EDTA-free PBS (pH7.4) at 4°C was immediately subjected to oxidation initiated by the azo compound (final concentration, 10 mM), with and without the addition of 50 µM resveratrol. 2

The oxidation was stopped with 10 mM vitamin C at the indicated time points, and the amount of TBARS was determined. TBARS determination. The amount of TBARS was determined as described (20). Briefly, oxidized LDL (25 µg/50 µ) was mixed with 1 ml 10% TCA and 1 ml 0.67% TBA. Following incubation at 95°C for 30 min, the absorbance was determined using a spectrofluorometer with excitation wave at 515 nm and emission wave at 553 nm. The results are expressed as formation of nmol equivalents of malondialdehyde (MDA), using freshly diluted 1,1,3,3- tetramethoxypropane as the standard. REM determination (20). The net negative charge of LDL was determined by agarose gel electrophoresis in 0.05M barbital buffer, pH 8.6. The stripes were stained with phosphomolybdic acid-magnesium chloride. REM was calculated as the mobility of oxidized LDL relative to that of native LDL (NLDL). Statistical analysis. The results are presented as mean±SD. The student t-test was used to analyze the difference between groups. The level of significance was set at p<0.05. Results CuZ+ -induced oxidation of LDL LDL was subjected to Cuz+-induced oxidation, with and without addition of resveratrol. The amount of TBARS in LDL increased about 6-fold during 18 h without resveratrol (from 6.27±0.57 nmol MDA/mg LDL before oxidation to 37.18±1.09 nmol MDA/mg LDL after oxidation, n=6, p<0.01). The addition of 25 pM resveratrol in the reaction mixture did not significantly decrease the MDA formation. At higher concentrations, 50, 100 and 200 µM significantly reduced the amount of TBARS by 70.5%, 78.7% and 82.1%, respectively, compared to values obtained in the absence of resveratrol (n=6, p<0.01) (FigurelA). Similarly, CuZ`-induced oxidation of LDL for 18 h increased REM of LDL from 1.0 before oxidation to 2.6 after oxidation (n=6, p<0.01), suggesting a significant structural modification of the native protein. The change in REM was significantly attenuated by resveratrol, in a concentration (Figure 1B) and time-dependent manner (Figure 2). At 50, 100 and 200 µM resveratrol, the REM was significantly reduced by 42.3%, 50% and 53.8%, respectively, compared to the control (n=6, . 3

p<0.01) (Figure IB). The time course study suggests that the REM of LDL following an 18 h oxidation with resveratrol is equivalent to that after 8 h oxidation in its absence (Figure 2), suggesting that resveratrol acts by retarding the oxidative modification of LDL. The time course of the oxidative modification of LDL effected by copper ion was also investigated. Figure 3A shows the effects of 50 pM resveratrol on the amount of TBARS generated during LDL oxidation, confirming that the polyphenolic compound caused a significant delay in LDL oxidation. Azo compound-initiated oxidation of LDL To further validate the antioxidative effect of resveratrol, LDL was also subjected to metal ion-independent oxidation initiated by the water-soluble azo compound AAPH. Addition of 50 µM resveratrol resulted in a significant prolongation of the lag associated with oxidation of LDL by AAPH, from 1 h to approximately 12 h (Figure 3B). Discussion Numerous clinical and experimental studies support the notion that oxidized LDL is a major atherogenetic lipoprotein in vivo. Potential atherogenenic effects ascribed to oxidized LDL include: chemotactic activity, facilitating the recruitment of circulating monocytes; enhanced uptake of LDL by macrophages through the scavenger receptor pathway, leading to the generation of foam cells; and cytotoxicity (1-4). The oxidation of LDL occurs to a significant degree in the subendothelial locality, in microenviroments that should be largely partitioned from the naturally occurring antioxidants. As such, the localized oxidation of LDL in the intima of arteries is believed to play a pivotal role in the development of fatty streaks, which is often regarded as amongst the earliest stage of formation of atherosclerotic lesion in vivo (1,3,4,5). A 4

variety of cell types, including the endothelial cells, smooth muscle cells, monocytes, macrophages and even lymphocytes, participate in the oxidative modification of LDL in vivo. The cell-mediated oxidative modification of LDL, however, can also be mimicked by simply incubating LDL in a serum-free medium with a sufficiently high concentration of copper or iron (21, 22). In this study, we determined the extent of LDL oxidation by copper ion and azo compound and evaluated the effect of resveratrol on LDL oxidation by the named chemicals. Results of these experiments showed that resveratrol effectively inhibited LDL modification induced by copper ion and azo compound, in a time- and concentration-dependent manner. Evidence supporting such a conclusion included observed reduction in the amount of 1vIDA formed and suppression of the change in relative electrophoretic mobility of oxidized LDL. At 100 I,tM resveratrol, the oxidation of LDL was almost complete, irrespective of the chemical used to initiate the oxidation process. A clue on the mechanism of action of resveratrol came from its effect in retarding the oxidation of LDL by AAPH. AAPH is a water-soluble azo compound which is thermally decomposed to produce peroxyl radicals, which in turn acts on LDL to result in its peroxidation. The addition of 50 µM resveratrol significantly inhibited LDL oxidation and prolonged the lag phase from 1 to 12 h, without significantly affecting the maximum TBARS attained. These results raise the possibility that resveratrol, like a-tocopherol, may act as a free-radical trap to halt the progression of LDL oxidation. Other mechanisms with which resveratrol may act in vivo include reactivation of oxidized a-tocopherol, reduction of either the formation or release of free radicals by activated marcophages, inhibition of platelet aggregation, promotion of nitric oxide release, and reaction with superoxide anions, hydroxyl radicals and lipid peroxyl radicals. In preliminary 5

studies, we have also found that resveratrol inhibited the proliferation of cultured smooth muscle cells (data not shown). Taken as a whole, the above-mentioned effects raise the intriguing possibility that resveratrol may act as a pleiotropic cellular effector, affording protection of the endothelial milieu and hence development of atherosclerosis by multiple mechanisms. These alternative mechanisms need to be further investigated in future studies. ACKNOWLEDGEMENTS This research was supported in part by the Vivian Wu-Au Memorial Fund, and an unrestricted research grant from the Philip Morris Co. to JMW. *Reprint requests should be sent to Dr. Joseph M. Wu, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. E-mail: Joseph Wu @ NYMC.Edu References 1. Steinberg, D., Parthasarathy, S., Carew, T.E et al. (1989) N. EngL J. Med. 320, 915-924. 2. Witztum, J.L. and Steinberg, D. (1991) J. Clin. Invest. 88, 1785-1792. 3. Shaikh, M., Martini, S., Quiney, JSi. et al. (1988) Atherosclerosis 69,165-172. 4. Haberland, M.E., Fong, D, and Chen, G.L. (1988) Science 241, 215-218. 5. Kita, T., Nagano, Y, Yokode, M. et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5928-5931. 6. Stampfer, M.J., Hennekens, C.H., Manson, J.E. et al. (1993) N. Engl. J. Med. 328,1444- 1449. 7. Rimm, E.B., Stampfer, M.J., Ascherio, A. et al. (1993) N_ EngL J. Med. 328,1450-1456. 8. Enstrom, J.E., Kanim, L.E. and Klein, M.A. (1992) Epidemiology 3, 194-202. 9. Diaz, M.N., Frei, B., Vita, J.A. et al. (1997) N. Engl. J. Med. 337, 408-415. 10. Renaud, S. and deLorgeril, M. (1992) Lancet 339, 1523-1526. 11. Frankel, E.N.,Waterhouse, A.L. and Kinselia, J.E. (1993) Lancet 341, 1103-1104. 12. Frankel, E.N., Kanner, J., German, J.B_ et al. (1993) Lancet 341, 454-457. 13. Pace-Asciak, C.R., Hahn, S., Diamandis, E.P. et al. (1995) Clin. Chem. Acta 235, 207-219. 14. Wilson, T., Knight,T., Beitz, D.C. et al. (1996) Life Sciences 59, PL15-21. 15. Jang, M., Cai, L., Udeani, G.O. et al. (1997) Science 275, 218-220. 16. Chen, Q., Esterbauer, H. and Jurgens, G. (1992) Biochem. J. 288, 249-254. 17. Schacterle, G.R. and Pollack, R.L. (1973) Anal. Biochem. 51, 654-655. 18. Lougheed, M. and Steinbrecher, U.P. (1996) J. Biol. Chem. 271, 11798-11805. 19. Stocker, R., Bowry, V.W. and Frei, B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1646-1650. 20. Steinberg, D. (1983) Atherosclerosis 3, 283-301. 21. Steinbrecher, U.P., Witztum, J.L., Parthasarathy, S. et al. (1987) Atherosclerosis 1, 135-143. 22. Torel, J., Cillard, J. and Cillard, P. (1986) Phytochemistry 25, 383-385. 6

0 NLDL Cu" DMSO , Resveratrol 25 µM Resveratrol 50 µM Resveratrol 100 µM Resveratrol 150 µM LDL oxidation (relative electrophoretic mobility, REM) .-- N W NLDL Cu" DMSO Resveratrol 25 µM Resveratrol 50 µM Resveratrol 100 µM Resveratrol 150 µM Resveratrol 200 pM 0 LDL oxidation (TBARS, nmol MDA/mg LDL) N N W W A vi O Vi O V, O G1 O -' ~ * ~ * ~ * ~ * ~ ~ ~ ~ ~ £LLMS09Z

LDL oxidation (relative electrophoretic mobility, REM) 0 0 in N rn 00 IJ A •r N bLLM909Z

~ ~ + LDL + Cu" ~ LDL + Cu" + resveratrol 0 0.5 1 2 4 6 8 12 24 18 16 B -~ LDL + Cu" 14 -~ LDL + Cu" 12 + 50 µM resveratrol --Wr- LDL + Cu*' 10 + 100 µM resveratrol 8 6 4 2 0 r I I 0 0.5 2 4 6 8 12 24 30 251 C --*- LDL + AAPH A LDL + AAPH + resveratrol 0 0.5 1-2 4 6 8 12 24 36 48 -- Time (h) Rgure 3