|Chen, Jinran -|
|Lazarenko, Oxana -|
|Shankar, Kartik -|
|Blackburn, Michael -|
|Lumpkin, Charles -|
|Ronis, Martin -|
Submitted to: Journal of Pharmacology and Experimental Therapeutics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 18, 2010
Publication Date: March 11, 2011
Citation: Chen, J., Lazarenko, O.P., Shankar, K., Blackburn, M.L., Lumpkin, C.K., Badger, T.M., Ronis, M.J. 2011. Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats. Journal of Pharmacology and Experimental Therapeutics. 336(3):734-742. Interpretive Summary: Chronic alcohol consumption causes bad quality of bone and easier bone fracture, the mechanisms of how chronic alcohol effect on bone are largely unknown and potential treatments to prevent alcohol-induced bone fracture remain unclear. We have previously demonstrated that, chronic ethanol (EtOH) treatment leads to accumulation of reactive oxygen species (ROS) in bone cells dependent on one enzyme called NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox). EtOH-induced ROS production might effect on both inhibition of bone formation and enhancing of bone resorption. Using a cycling female Sprague-Dawley rat model, we gave alcohol directly into gastrointestinal track. We found that EtOH infusion for 4 weeks reduced bone quality. Co-administration of diphenylene iodonium chloride (DPI), a Nox inhibitor, by daily injection of 1 mg/kg/d, abolished EtOH-induced inhibition of bone formation. EtOH-induced inhibition of bone formation was associated with up-regulation of mRNA levels of all three Nox subtypes 1, 2, 4 in bone. These data suggest that inhibition of Nox expression or activity may be new target for prevention or treatment of chronic EtOH-induced bone loss, and perhaps other conditions resulting in oxidative stress associated bone resorption such as aging.
Technical Abstract: Previous in vitro data suggest that ethanol (EtOH) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-beta estradiol co-administration. Moreover, co-administration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox 1, 2, 4 and the receptor activator of NF-kB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-beta estradiol, NAC and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated EtOH directly up-regulated Nox expression in osteoblasts. Pre-treatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.