Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a Nox4-Independent Manner.

Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.

Skeletal toxicity resulting from exposure of growing male rats to coplanar PCB 126 is associated with disruption of calcium homeostasis and the GH-IGF-1 axis and direct effects on bone formation.

Skeletal toxicity has been reported following exposure to polychlorinated biphenyl (PCB) mixtures. However, molecular mechanisms remain poorly understood. We exposed groups of male 4-5-week-old Sprague-Dawley rats to 3,3', 4, 4', 5-pentachlorobiphenyl (PCB 126), a dioxin-like coplanar PCB congener by a single i.p. injection of 5 µmol/kg in soy oil vehicle or vehicle alone. After 4 weeks, rats were euthanized. PCB exposure resulted in hypocalcemia (P < 0.05) and significant increases in serum PTH without changes in serum phosphorous. Hyperparathyroidism was accompanied by increased expression of mRNAs of vitamin D3 metabolizing cytochrome P450 enzymes CYP27B1 and CYP24 in the kidney (P < 0.05). PCB exposure also reduced body weight, serum IGF-1, and hepatic expression of mRNAs encoding the male-specific GH-pattern-regulated CYP2C11 and CYP3A2 relative to controls (P < 0.05). PCB exposure reduced long bone length, diameter, and surface area, but increased trabecular thickness and volume (P < 0.05). Serum osteocalcin (P < 0.05), a marker and a regulator of bone formation, was reduced, but PCB exposure had no effect on the bone resorption marker RatLaps. Exposure of human intestinal Caco-2 cells to 10-100 nM PCB 126 in the presence of vitamin D3 resulted in inhibition of mRNAs for the calcium transporters TRPV6 and PMCA1b (P < 0.05). In addition, PCB 126 suppressed osteoblastogenesis in primary bone marrow mesenchymal stem cell cultures which was blunted by the AhR antagonist CH-223191. These data provide novel evidence that skeletal toxicity after exposure to PCB 126 is a result of disruption of calcium homeostasis and the GH-IGF-1 axis, and involves direct AhR-mediated effects on bone formation.

Skeletal Toxicity of Coplanar Polychlorinated Biphenyl Congener 126 in the Rat Is Aryl Hydrocarbon Receptor Dependent.

Epidemiological evidence links polychlorinated biphenyls (PCBs) to skeletal toxicity, however mechanisms whereby PCBs affect bone are poorly studied. In this study, coplanar PCB 126 (5 µmol/kg) or corn oil vehicle was administered to N = 5 and 6 male and female, wild type (WT) or AhR -/- rats via intraperitoneal injection. Animals were sacrificed after 4 weeks. Bone length was measured; bone morphology was assessed by microcomputed tomography and dynamic histomorphometry. Reduced bone length was the only genotype-specific effect and only observed in males (p < .05). WT rats exposed to PCB 126 had reduced serum calcium, and smaller bones with reduced tibial length, cortical area, and medullary area relative to vehicle controls (p < .05). Reduced bone formation rate observed in dynamic histomorphometry was consistent with inhibition of endosteal and periosteal bone growth. The effects of PCB 126 were abolished in AhR -/- rats. Gene expression in bone marrow and shaft were assessed by RNA sequencing. Approximately 75% of the PCB-regulated genes appeared AhR dependent with 89 genes significantly (p < .05) regulated by both PCB 126 and knockout of the AhR gene. Novel targets significantly induced by PCB 126 included Indian hedgehog (Ihh) and connective tissue growth factor (Ctgf/Ccn2), which regulate chondrocyte proliferation and differentiation in the bone growth plate and cell-matrix interactions. These data suggest the toxic effects of PCB 126 on bone are mediated by AhR, which has direct effects on the growth plate and indirect actions related to endocrine disruption. These studies clarify important mechanisms underlying skeletal toxicity of dioxin-like PCBs and highlight potential therapeutic targets.