Loss of functional NADPH oxidase 2 protects against alcohol-induced bone resorption in female p47phox-/- mice.

BACKGROUND: In bone, NADPH oxidase (NOX)-derived reactive oxygen species (ROS) superoxide and/or hydrogen peroxide are an important stimulus for osteoclast differentiation and activity. Previously, we have demonstrated that chronic ethanol (EtOH) consumption generates excess NOX-dependent ROS in osteoblasts, which functions to stimulate nuclear factor kappa-ß receptor ligand (RANKL)-RANK signaling, thus increasing osteoclastogenesis and activity. This activity can be blocked by co-administration of EtOH with the pan-NOX inhibitor diphenylene idonium (DPI). METHODS: To test whether EtOH-induced bone loss is dependent on a functional NOX2 enzyme, 6-week-old female C57BL/6J-Ncf1/p47phox(-/-) (p47phox KO) and wild-type (WT) mice were pair-fed EtOH diets for 40 days. Bone loss was assessed by 3-point bending, micro-computed tomography and static histomorphometric analysis. Additionally, ST2 cultured cells were co-treated with EtOH and NOX inhibitors, DPI, gliotoxin, and plumbagin, after which changes in ROS production, and in RANKL and NOX mRNA expression were analyzed. RESULTS: In WT mice, EtOH treatment significantly reduced bone density and mechanical strength, and increased total osteoclast number and activity. In EtOH-treated p47phox KO mice, bone density and mechanical strength were completely preserved. EtOH p47phox KO mice had no changes in osteoclast numbers or activity, and no elevations in serum CTX or RANKL gene expression (p < 0.05). In both WT and p47phox KO mice, EtOH feeding reduced biochemical markers of bone formation (p < 0.05). In vitro EtOH exposure of ST2 cells increased ROS, which was blocked by pretreating with DPI or the NOX2 inhibitor gliotoxin. EtOH-induced RANKL and NOX2 gene expression were inhibited by the NOX4-specific inhibitor plumbagin. CONCLUSIONS: These data suggest that NOX2-derived ROS is necessary for EtOH-induced bone resorption. In osteoblasts, NOX2 and NOX4 appear to work in tandem to increase RANKL expression, whereas EtOH-mediated inhibition of bone formation occurs via a NOX2-independent mechanism.

Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice.

Chronic alcohol abuse results in decreased bone mineral density (BMD), which can lead to increased fracture risk. In contrast, low levels of alcohol have been associated with increased BMD in epidemiological studies. Alcohol's toxic skeletal effects have been suggested to involve impaired vitamin D/calcium homeostasis. Therefore, dietary vitamin D supplementation may be beneficial in reducing bone loss associated with chronic alcohol consumption. Six-week-old female C57BL/6J mice were pair-fed ethanol (EtOH)-containing liquid diets (10 or 36% total calories) for 78 days. EtOH exposure at 10% calories had no effects on any measured bone or serum parameter. EtOH consumption at 36% of calories reduced BMD and bone strength (P<0.05), decreased osteoblastogenesis, increased osteoclastogenesis, suppressed 1,25-hydroxyvitamin D3 [1,25(OH)2D3] serum concentrations (P<0.05), and increased apoptosis in bone cells compared with pair-fed controls. In a second study, female mice were pair-fed 30% EtOH diets with or without dietary supplementation with vitamin D3 (cholecalciferol; VitD) for 40 days. VitD supplementation in the EtOH diet protected against cortical bone loss, normalized alcohol-induced hypocalcaemia, and suppressed EtOH-induced expression of receptor of nuclear factor-κB ligand mRNA in bone. In vitro, pretreatment of 1,25(OH)2D3 in osteoblastic cells inhibited EtOH-induced apoptosis. In EtOH/VitD mice circulating 1,25(OH)2D3 was lower compared with mice receiving EtOH alone (P<0.05), suggesting increased sensitivity to feedback control of VitD metabolism in the kidney. These findings suggest dietary VitD supplementation may prevent skeletal toxicity in chronic drinkers by normalizing calcium homeostasis, preventing apoptosis, and suppressing EtOH-induced increases in bone resorption.

Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/ß-catenin signaling pathway.

Although alcohol effects within the liver have been extensively studied, the complex mechanisms by which alcohol causes liver cancer are not well understood. It has been suggested that ethanol (EtOH) metabolism promotes tumor growth by increasing hepatocyte proliferation. In this study, we developed a mouse model of tumor promotion by chronic EtOH consumption in which EtOH feeding began 46 days after injection of the chemical carcinogen diethylnitrosamine (DEN) and continued for 16 weeks. With a final EtOH concentration of 28% of total calories, we observed a significant increase in the total number of preneoplastic foci and liver tumors per mouse in the EtOH+DEN group compared with corresponding pair-fed (PF)+DEN and chow+DEN control groups. We also observed a 4-fold increase in hepatocyte proliferation (P < 0.05) and increased cytoplasmic staining of active-ß-catenin in nontumor liver sections from EtOH+DEN mice compared with PF+DEN controls. In a rat model of alcohol-induced liver disease, we found increased hepatocyte proliferation (P < 0.05); depletion of retinol and retinoic acid stores (P < 0.05); increased expression of cytosolic and nuclear expression of ß-catenin (P < 0.05) and phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß), P < 0.05; significant upregulation in Wnt7a mRNA expression; and increased expression of several ß-catenin targets, including, glutamine synthetase (GS), cyclin D1, Wnt1 inducible signaling pathways protein (WISP1), and matrix metalloproteinase-7(MMP7), P < 0.05. These data suggest that chronic EtOH consumption activates the Wnt/ß-catenin signaling pathways to increase hepatocyte proliferation, thus promoting tumorigenesis following an initiating insult to the liver.