Withdrawal of estrogen represents the primary factor determining post menopausal bone loss and has been associated with negative indicators of bone quality including the apoptotic death of osteocytes in vivo. While hormone replacement therapy in the form of Estrogen or Selective Estrogen Receptor Modulators (SERMs) demonstrates clear estrogen receptor (ER)-mediated benefits to bone mass, less is known regarding the mechanism of action of these compounds in the maintenance of bone cell populations. We have investigated the potential antioxidant effects of estrogen, estrogen derivatives and the SERMs Raloxifene and LY117018 in the prevention of oxidative stress induced apoptosis in the osteocyte like cell line MLO-Y4. Treatment of MLO-Y4 with 0.3 mM H(2)O(2) induced apoptosis that was significantly inhibited (p< or =0.002) when the cells were pre-treated for 1 h with either 17beta-estradiol, Raloxifene or LY117018 (10 nM). The stereoisomer 17alpha-estradiol also prevented H(2)O(2) induced apoptosis in MLO-Y4. Importantly, pre-treatment of ER-negative HEK293 cells with either 1 microM, 100 nM or 10 nM 17beta-estradiol, Raloxifene or LY117018 significantly inhibited H(2)O(2) induced apoptosis in these cells (p< or =4.2x10(-5)) indicating an estrogen receptor-independent effect of these compounds. Comparisons of 17beta-estradiol and similar molecules containing the putative free radical scavenger C3-OH moiety on the steroid A-ring (17alpha-estradiol, 17alpha-ethinylestradiol; 10 nM) with structurally related molecules lacking the C3-OH grouping (Mestranol and Quinestrol; 10 nM) demonstrated that only compounds containing the C3-OH moiety showed anti-apoptotic behavior in these studies (p< or =0.0033). Similarly the identification of the presence of reactive oxygen species (ROS) in cells as evidenced by the free radical indicator 2'7'-dichlorodihydrofluorescein diacetate demonstrated that 17beta-estradiol, SERMs and related molecules with C3-OH moiety were capable of blocking ROS generated in cells by H(2)O(2) (p< or =0.002) while Mestranol and Quinestrol showed no such blockade. It is possible that the loss of osteocytes during estrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated oxidant molecules. These data suggest that the osteocyte protective effects of estrogen and SERMs may operate through a common receptor-independent mechanism which may be related to the antioxidant activity of these molecules.