Estrogen and tamoxifen metabolites protect smooth muscle cell membrane phospholipids against peroxidation and inhibit cell growth

The goal of this study was to test the hypothesis that antioxidant estrogen s, by a mechanism independent of the estrogen receptor, protect phospholipi ds residing in the plasma membrane of vascular smooth muscle cells from per oxidation and peroxidation-induced cell growth and migration. Peroxidation of membrane phospholipids was assessed by HPLC analysis of phospholipids ex tracted from rat aortic vascular smooth muscle cells prelabeled with cis-pa rinaric acid (a fatty acid that is susceptible to peroxidation, which quenc hes its fluorescent properties). Incubation of cells for 2 hours with the p eroxyl radical donor 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) caused pe roxidation of all measured membrane phospholipids. This effect was attenuat ed by pretreating cells for 15 minutes with 50 to 5000 ng/mL of 2-hydroxyes tradiol (strong antioxidant but weak estrogen-receptor ligand) or 4-hydroxy tamoxifen (strong antioxidant and potent estrogen-receptor ligand), but not by estrone or droloxifene (both weak antioxidants but potent estrogen-rece ptor ligands). Moreover, pretreatment of cells for 20 hours with physiologi cal concentrations (0.3 ng/mL) of 2-hydroxyestradiol or pharmacologically r elevant concentrations of 4-hydroxytamoxifen (40 ng/mL) also decreased AMVN -induced phospholipid peroxidation, Both 2-hydroxyestradiol and 4-hydroxyta moxifen were as effective as 2,2,5,7,8-pentamethyl-6-hydrochromane (an anti oxidant homolog of vitamin E) in attenuating AMVN-induced peroxidation of m embrane phospholipids. Also, physiological concentrations of 2-hydroxyestra diol, but not estrone, and pharmacologically relevant concentrations of 4-h ydroxytamoxifen attenuated AMVM-induced DNA synthesis, cell proliferation, and cell migration. These studies demonstrate in vascular smooth muscle cel ls that antioxidant estrogens via a non-estrogen receptor-dependent mechani sm attenuate peroxidation of membrane phospholipids and peroxidation-induce d cell growth and migration.