Peroxynitrite-mediated oxidative modification of low-density lipoprotein by aqueous extracts of cigarette smoke and the preventive effect of fluvastatin


      Cigarette smoking is known to promote atherosclerosis, possibly through enhanced oxidative stress. The aim of the present study was to elucidate the possible involvement of peroxynitrite in oxidative modification of low-density lipoprotein (LDL) induced by aqueous extract of cigarette smoke (CSE) and the preventive effect of fluvastain, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor with antioxidative activity, in vitro and in vivo. Modification of LDL was monitored by LDL subfraction analysis using anion-exchange HPLC, TBARS formation and 3-nitrotyrosine production. Incubation of LDL with CSE caused a marked increase in oxidative modification of LDL and nitration of tyrosine residues in the apolipoprotein B. These modifications were prevented by treatment with fluvastatin as well as Vitamin E in a concentration-related manner. Fluvastatin was equal to or more effective than Vitamin E for preventing protein nitration, but weaker for preventing oxidative modification. When CSE was injected daily into the ear vein of Watanabe heritable hyperlipidemic rabbits for 5 months, both oxidative modification and nitration of the plasma LDL noticeably occurred. These changes induced by CSE could be effectively prevented by the simultaneous oral administration of fluvastatin (10 and 30 mg/kg) or Vitamin E (150 mg/kg). Fluvastatin prevented the LDL nitration more effectively than Vitamin E. These results suggest that peroxynitrite in CSE is involved in oxidative modification of LDL and that fluvastatin can efficiently prevent LDL modification by scavenging peroxynitrite. Fluvastatin may be potentially beneficial to hypercholesterolemic patients with oxidative stress such as smoking.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Ross R.
        The pathogenesis of atherosclerosis—an update.
        N. Eng. J. Med. 1986; 314: 488-500
        • Steinberg D.
        • Parthasarathy S.
        • Carew T.E.
        • Khoo J.C.
        • Witztum J.L.
        Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity.
        N. Engl. J. Med. 1989; 320: 915-924
        • Steinberg D.
        • Lewis A.
        Conner Memorial lecture. Oxidative modification of LDL and atherogenesis.
        Circulation. 1997; 95: 1062-1071
        • O’Leary V.J.
        • Darley-Usmar V.M.
        • Russell L.J.
        • Stone D.
        Pro-oxidant effects of lipoxygenase-derived peroxides on the copper-initiated oxidation of low-density lipoprotein.
        Bichem. J. 1992; 292: 631-634
        • Hogg N.
        • Darley-Usmar V.M.
        • Wilson M.T.
        • Moncada S.
        The oxidation of alpha-tocopherol in human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide.
        FEBS Lett. 1993; 326: 199-203
      1. Parthasarathy S, Santanam N. Mechanisms of oxidation, anti-oxidants, and atherosclerosis. Curr Opin Lipidol 1994;5:371–5.

        • Patel R.P.
        • Diczfalusy U.
        • Dzeletovic S.
        • Wilson M.T.
        • Darley-Usmar V.M.
        Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.
        J. Lipid Res. 1996; 37: 2361-2371
        • Kaur H.
        • Halliwell B.
        Evidence for nitric oxide-mediated oxidative damage in chronic inflammation.
        FEBS Lett. 1994; 350: 9-12
        • Smith M.A.
        • Richey Harris P.L.
        • Sayre L.M.
        • Beckman J.S.
        • Perry G.
        Widespread peroxynitrite-mediated damage in Alzheimer’s disease.
        J. Neurosci. 1997; 17: 2653-2657
        • Squadrito G.L.
        • Pryor W.A.
        Oxidative chemistry of nitric oxide: the role of superoxide, peroxynitrite, and carbon dioxide.
        Free Radic. Biol Med. 1998; 25: 392-403
        • Marek W.R.
        • Salas E.
        Nitric oxide—biological mediator, modulator and factor of injury; its role in the pathogenesis of atherosclerosis.
        Atherosclerosis. 1995; 118: S69-S80
        • Yokode M.
        • Kita T.
        • Arai H.
        • Kawai C.
        • Narumiya S.
        • Fujiwara M.
        Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract.
        Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 2344-2348
        • Kagota S.
        • Yamaguchi Y.
        • Shinozuka K.
        • Kwon Y.M.
        • Kunitomo M.
        Cigarette smoke-modified low density lipoprotein impairs endothelium-dependent relaxation in isolated rabbit arteries.
        Gen. Pharmcol. 1996; 27: 477-481
        • Yamaguchi Y.
        • Kagota S.
        • Haginaka J.
        • Kunitomo M.
        Evidence of modified LDL in the plasma of hypercholesterolemic WHHL rabbits injected with aqueous extracts of cigarette smoke.
        Enviro. Toxicol. Pharmacol. 2000; 8: 255-260
        • Yamaguchi Y.
        • Matsuno S.
        • Kagota S.
        • Haginaka J.
        • Kunitomo M.
        Oxidants in cigarette smoke extract modify low-density lipoprotein in the plasma and facilitate atherogenesis in the aorta of Watanabe heritable hyperlipidemic rabbits.
        Atherosclerosis. 2001; 156: 109-117
        • Yamaguchi Y.
        • Kagota S.
        • Haginaka J.
        • Kunitomo M.
        Peroxynitrite-generating species: good candidate oxidants in aqueous extracts of cigarette smoke.
        Jpn. J. Pharmacol. 2000; 82: 78-81
        • Yamaguchi Y.
        • Kagota S.
        • Haginaka J.
        • Kunitomo M.
        Participation of peroxynitrite in oxidative modification of LDL by aqueous extracts of cigarette smoke.
        FEBS Lett. 2002; 512: 218-222
        • Hussein O.
        • Schlezinger S.
        • Rosenblat M.
        • Keidar S.
        • Aviram M.
        Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after fluvastatin therapy is associated with the hypocholesterolemic effect of the drug and its binding to the LDL.
        Atherosclerosis. 1997; 128: 11-18
        • Aviram M.
        • Hussein O.
        • Rosenblat M.
        • Schlezinger S.
        • Hayek T.
        • Keidar S.
        Interactions of platelets, macrophages, and lipoproteins in hypercholesterolemia: antiatherogenic effects of HMG-CoA reductase inhibitor therapy.
        J. Cardiovasc. Pharmacol. 1998; 31: 39-45
        • Rikitake Y.
        • Kawashima S.
        • Takeshita S.
        • Yamashita T.
        • Azumi H.
        • Yasuhara M.
        • et al.
        Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in choelsterol-fed rabbits.
        Atherosclerosis. 2001; 154: 87-96
        • Inoue T.
        • Hayashi M.
        • Takayanagi K.
        • Morooka S.
        Lipid-lowering therapy with fluvastatin inhibits oxidative modification of low density lipoprotein and improves vascular endothelial function in hypercholesterolemic patients.
        Atherosclerosis. 2002; 160: 369-376
        • Hatch F.T.
        • Lees R.S.
        Practical methods for plasma lipoprotein analysis.
        Adv. Lipid Res. 1968; 6: 1-68
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        Protein measurement with the folin phenol reagent.
        J. Biol. Chem. 1951; 193: 265-275
        • Kunitomo M.
        • Yamaguchi Y.
        • Matsushima K.
        • Bando Y.
        Cholesterol metabolism in serum and aorta of inbred mice fed a high-cholesterol diet.
        Japan J. Pharmacol. 1984; 34: 153-158
        • Yagi K.
        A simple fluorometric assay for lipoperoxide in blood plasma.
        Biochem. Med. 1976; 15: 212-216
        • Pryor W.A.
        • Stone K.
        Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite.
        Ann. N. Y. Acad. Sci. 1993; 686: 12-28
        • Ischiropoulos H.
        Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species.
        Arch. Biochem. Biophys. 1998; 356: 1-11
        • Hazen S.L.
        • Heinecke J.W.
        3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima.
        J. Clin. Invest. 1997; 99: 2075-2081
        • Yamamoto A.
        • Hoshi K.
        • Ichihara K.
        Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes.
        Eur. J. Pharmacol. 1998; 361: 143-149
        • Yamaguchi Y.
        • Matsuno S.
        • Kagota S.
        • Haginaka J.
        • Kunitomo M.
        Fluvastatin reduces modification of low-density lipoprotein in hyperlipidemic rabbit loaded with oxidative stress.
        Eur. J. Pharmacol. 2002; 436: 97-105
        • Balavoine G.G.
        • Geletii Y.V.
        Peroxynitrite scavenging by different antioxidants. Part I. Convenient assay.
        Nitric Oxide. 1999; 3: 40-54
        • Ishii M.
        • Shimizu S.
        • Momose K.
        • Yamamoto T.
        SIN-1-induced bytotoxicity in cultured endothelial cells involves reactive oxygen species and nitric oxide: protective effect of sepiapterin.
        J. Cardiovasc. Pharmacol. 1999; 33: 295-300
        • Morre K.P.
        • Darley-Usmar V.
        • Morrow J.
        • Roverts II, L.J.
        Formation of F2-isoprostanes during oxidation of hyman low-density liporotein and plasma by peroxynitrite.
        Circ. Res. 1995; 77: 335-341
        • Carr A.C.
        • McCall M.R.
        • Frei B.
        Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection.
        Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1716-1723
        • Hazen S.L.
        • Zhang R.
        • Shen Z.
        • Wu W.
        • Podrez E.A.
        • MacPherson J.C.
        • et al.
        Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation in vivo.
        Circ. Res. 1999; 85: 950-958
        • Petruzzelli S.
        • Puntoni R.
        • Mimotti P.
        • Pulera N.
        • Baliva F.
        • Fornai E.
        • et al.
        Plasma 3-nitrotyrosine in cigarette smokers.
        Am. J. Respir. Crit. Care Med. 1997; 156: 1907-1920