Investigation into the metabolomic effects of the non-canonical amino acid azetidine-2-carboxylic acid in neuroblastoma cells

Azetidine-2-carboxylic acid (A2C) is a non-canonical amino acid (NCAA) produced by several plant species, including Beta vulgaris cultivars such as sugar beet. A2C can be incorporated into proteins in place of proline. The presence of A2C in myelin basic protein has been implicated in the pathogenesis of multiple sclerosis. Human exposure to A2C could arise from the use of fodder beets and sugar beet by-products as livestock feed. A2C is pro-inflammatory, pro-apoptotic, and induces mitochondrial dysfunction. Animal studies have examined A2C-related skeletal development anomalies linked to its incorporation into collagen. In murine models, A2C induces morphological alterations in oligodendrocytes, the myelin-producing cells. In the present study, we employed an untargeted metabolomics approach to characterise global changes in neuroblastoma cells following A2C exposure. Using spectral libraries for metabolite identification, and pathway mapping to assess cellular changes, we observed a global reduction in protein synthesis and depletion of major intracellular antioxidants following A2C exposure. This is consistent with ER stress triggered by the accumulation of aberrant A2C-containing proteins and a disruption of redox homeostasis. These findings provide mechanistic insight into A2C-induced cytotoxicity and advance our understanding of the potential role of NCAAs as environmental triggers of human disease.