Hydrogen peroxide, but not superoxide, stimulates bone resorption in mouse calvariae

Reactive oxygen species such as superoxide and hydrogen peroxide have been implicated as regulatory factors in the control of osteoclastic bone resorption. While superoxide radicals have been suggested to be the main bone resorbing species in organ culture and in vivo, hydrogen peroxide (H2O2) has recently been shown to activate isolated osteoclasts in vitro. In this study, we investigated the effects of hydrogen peroxide and superoxide on bone resorption in mouse calvarial organ cultures. Hydrogen peroxide stimulated bone resorption in a concentration-dependent manner in calvarial organ cultures with a maximal effect at 1 mumol/L (45Ca release; treated/control = 1.6 +/- 0.07; p < 0.001 from control). Bone resorption induced by H2O2 was significantly inhibited by catalase to 1.2 +/- 0.05; p < 0.02. In contrast, the combination of xanthine and xanthine oxidase, which generates superoxide anions, failed to stimulate bone resorption, except in the presence of superoxide dismutase (SOD), which resulted in a modest increase in bone resorption to a treated/control ratio of 1.2 +/- 0.05; p < 0.02. Analysis of calvarial bones which were exposed to H2O2 showed a significant increase in osteoclast numbers suggesting that H2O2 may be capable of stimulating osteoclast formation in addition to enhancing activity of mature osteoclasts. Our data are consistent with previous work, which has shown that H2O2 is a bone resorbing factor with effects on both osteoclast formation and in activity of mature osteoclasts. The experiments with SOD further suggest that the enhancement of bone resorption previously noted with superoxide generating systems may be due in part to generation of H2O2.