|Mercer, Kelly -|
|Wynne, Rebecca -|
|Moutos, C -|
|Lumplin, Charles -|
|Suva, Larry -|
|Ronis, Martin -|
Submitted to: Society of Toxicology
Publication Type: Abstract Only
Publication Acceptance Date: November 15, 2010
Publication Date: March 1, 2011
Citation: Mercer, K., Wynne, R., Moutos, C., Lumplin, C.K., Suva, L.J., Badger, T.M., Ronis, M.J. 2011. Alcohol-induced bone loss is blocked in p47phox -/- mice lacking functional nadph oxidases. The Toxicologist. 120(S2):278. No. 1302. Interpretive Summary: Alcohol abuse is a known cause of bone loss and alcoholism is a well known risk factor for development of osteoporosis. Previous studies in our lab have suggested that alcohol-induced bone loss involves oxidative stress and that the source of oxygen radicals in bone may be a group of enzymes called NADPH oxidases. In the current study, we examined the effects of alcohol on bone loss in wild type mice (controls) and in mice where a protein cofactor p47phox which is essential for activity of most Nox enzymes is missing (p47phox -/- mice). After 4 weeks of alcohol feeding in liquid diets we observed similar blood alcohol concentrations. However whereas wild type mice lost bone, p47phox -/- did not. Analysis of bone turnover revealed that in wild type mice alcohol both inhibited bone formation and increased bone resorption by osteoclasts. In contrast, p47phox -/- mice still showed inhibition of bone formation but had no bone resorption. In bone cell cultures from wild type mice, alcohol increased expression of RANKL, a factor essential for formation of osteoclasts that eat away bone. This did not happen in cultures from p47phox -/- mice. In addition, a specific chemical inhibitor of the enzyme Nox2 blocked oxygen radical formation in bone cell cultures. These data suggest that Nox1/2 enzymes are activated by alcohol to produce oxidative stress in bone and increase bone resorption by stimulating osteoclast formation.
Technical Abstract: Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a result of knocking out the coactivator protein p47phox. C57BL/6 and p47phox -/-female mice (age 6 wk.) were pair-fed liquid diets of up to 30% EtOH for 4 wk. (blood EtOH 100-200 mg/d). In C57BL/6 mice, EtOH treatment reduced tibial cortical and trabecular bone density (P<0.05). EtOH-induced bone loss was completely prevented in p47phox -/-mice. Analysis of serum bone turnover markers revealed that EtOH treatment both increased bone resorption and decreased bone formation (P< 0.05). Resorption was unchanged in EtOH-treated p47phox -/- mice; whereas bone formation was decreased to the same level observed in wild type mice. In vitro, EtOH treatment of bone marrow derived primary osteoblast (Ob) cultures from C57BL/6 mice which express Nox1, Nox2 and Nox4, resulted in induction of expression of receptor activator of NF'B ligand (RANKL) mRNA, an essential factor for osteoclast differentiation. However, no induction of RANKL was observed in cultures from p47phox -/- mice. Furthermore, in vitro studies with the pre-osteoblastic mouse ST-2 cell line demonstrated that EtOH-induced hydrogen peroxide production was inhibited by co-treatment with either 0.5 µM DPI or 5 µM of the Nox2 inhibitor gliotoxin. These data suggest that EtOH-induced bone loss is mediated at least in part by ROS generated via EtOH activation of Nox 1/2 enzymes in bone marrow Ob/pre-Ob and increased RANKL expression resulting in increased osteoclastogenesis and bone resorption. In addition, these data demonstrate that.