Tryptophan metabolites 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) increase oxidative stress and...

The aryl hydrocarbon receptor (AhR) is activated by kynurenine (Kyn), a tryptophan metabolite that accumulates with age, and this process drives osteoblast dysfunction. However, Kyn can be further metabolized, and the extent to which downstream metabolite molecules activate AhR in mesenchymal lineage cells and impact bone formation activity was unclear from previous studies. We hypothesized that Kyn metabolites activate AhR signaling and impair bone formation to drive bone loss. In the current study, tryptophan, Kyn, and 3-hydroxy-kynurenine (3HK) dose-dependently activated AhR in mesenchymal stem cell models, with 3HK being the most potent activator. Treating mesenchymal stem cells with 3HK and 3-hydroxyanthranilic acid (3HAA) dose-dependently induced DNA damage that at lower concentrations induced senescence and at higher concentrations promoted apoptotic cell death. This cell death was rescued upon scavenging reactive oxygen species with N-acetylcysteine, suggesting a mechanism of apoptosis related to increased oxidative stress. With regards to bone formation activity, the differentiation of primary bone marrow stromal cells into matrix-producing osteoblasts was blunted upon the introduction of Kyn, 3HK or 3HAA into osteogenic differentiation media, with 3HK and 3HAA inducing the greatest deficits in mineralized matrix production. In vivo administration of 3HAA to C57BL/6 mice was detrimental to whole-body bone mineral density and cortical bone mass, although trabecular bone was largely unaffected. Together, our results suggest that several intermediate metabolites in the tryptophan-Kyn pathway activate AhR and impede the differentiation of osteoblasts by inducing DNA damage, senescence and oxidative stress, which may have negative consequences for cortical bone in vivo.