Although it is well known that women are more susceptible to the toxic effects of ethanol (EtOH) than men, much less is known about the molecular mechanisms underlying alcohol toxicity in women especially as relates to bone. Alcohol abuse during early adulthood results in impaired bone growth and in the U.S.A. approximately 20% of women age 18-30 (4.4 million) binge drink. A resulting reduction in peak bone mass would predispose women to osteoporosis in later life. The molecular mechanisms underlying the toxic effects of EtOH on bone remain poorly understood. EtOH inhibits bone formation. Bone loss in female rats and mice fed EtOH is blocked by dietary antioxidants including N-acetylcysteine and vitamin E and by DPI an inhibitor of NADPH oxidase (NOX) enzymes. However, effects of EtOH to inhibit osteoblastogenesis and stimulate bone marrow adiposity were not blocked in p47phox -/- mice lacking active NOX1/2. We hypothesize that another source of reactive oxygen species (ROS) mediates the effects of EtOH on bone formation. Ex-vivo bone marrow cultures from NOX 4 -/- mice will be utilized to test the hypothesis that NOX4 mediates the effects EtOH-induced inhibition of mesenchymal stem cell self-renewal and differentiation into osteoblasts or adipocytes. A mouse liquid diet model will be used to test this hypothesis in vivo in mice with cell specific ablation of NOX4 in osteoblast precursors (Prx-1-Cre-lox mice). Alternatively, the role of mitochondrial derived ROS in EtOH inhibition of bone formation will be tested using the mitochondrial ROS inhibitor mitoTEMPO. At higher concentrations characteristic of binge drinking, EtOH can also increase bone resorption via induction of RANKL, a member of the TNF family expressed primarily in osteocytes, which signals through the receptor RANK to stimulate osteoclastogenesis. The role of NOX derived ROS in EtOH-induced RANKL production from osteocytes and other effects of EtOH on osteocyte function and cortical bone morphology will be determined in vivo in mice lacking NOX4 or with inactive NOX1/2 in osteocytes (NOX4 and rac-1 Dmp-1 Cere-lox mice) fed EtOH via liquid diet and in an EtOH binge drinking model. In addition, the role of excess NADH produced during EtOH metabolism in NOX activation will be tested in bone cells in vitro.

Public Health Relevance

In young women, drinking is an increasing problem and may result in long term increases on osteoporosis risk in older women as the result of impairments in the attainment of peak bone mass. Our data suggest that dietary antioxidants prevent alcohol-induced osteopenia as a result of blocking excess hydrogen peroxide (H2O2) produced as the result of activation of NADPH oxidase enzymes as a consequence of ethanol metabolism. Since alcohol-induced bone loss shares many features in common with bone loss during menopause and aging, molecular studies of alcohol actions on H2O2 signaling cascades in bone may provide fundamental insights into common molecular pathways underlying regulation of bone growth and turnover.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37AA018282-06
Application #
9126707
Study Section
Systemic Injury by Environmental Exposure (SIEE)
Program Officer
Orosz, Andras
Project Start
2016-05-15
Project End
2021-04-30
Budget Start
2016-05-15
Budget End
2017-04-30
Support Year
6
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Louisiana State Univ Hsc New Orleans
Department
Pharmacology
Type
Schools of Medicine
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112
Watt, James; Alund, Alexander W; Pulliam, Casey F et al. (2018) NOX4 Deletion in Male Mice Exacerbates the Effect of Ethanol on Trabecular Bone and Osteoblastogenesis. J Pharmacol Exp Ther 366:46-57
Alund, Alexander W; Mercer, Kelly E; Pulliam, Casey F et al. (2017) Partial Protection by Dietary Antioxidants Against Ethanol-Induced Osteopenia and Changes in Bone Morphology in Female Mice. Alcohol Clin Exp Res 41:46-56
Alund, Alexander W; Mercer, Kelly E; Suva, Larry J et al. (2016) Reactive Oxygen Species Differentially Regulate Bone Turnover in an Age-Specific Manner in Catalase Transgenic Female Mice. J Pharmacol Exp Ther 358:50-60