Developmental impacts of Nrf2 activation by dimethyl fumarate (DMF) in the developing zebrafish (Danio rerio) embryo

• Published in: Free radical biology & medicine 2023-01, Vol.194, p.284-297
• Main Authors: Marques, Emily S., Severance, Emily G., Min, Bellis, Arsenault, Paige, Conlin, Sarah M., Timme-Laragy, Alicia R.
• Format: Article
• Language: English
• Subjects: Animals; Antioxidants - pharmacology; Danio rerio; Developmental toxicity; Dimethyl fumarate (DMF); Dimethyl Fumarate - pharmacology; Glutathionylation; Humans; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Nrf2; Oxidative Stress; Zebrafish - genetics; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2022.12.013

Dimethyl fumarate (DMF) is pharmaceutical activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates of many cellular antioxidant response pathways, and has been used to treat inflammatory diseases such as multiple sclerosis. However, DMF has been shown to produce adverse effects on offspring in animal studies and as such is not recommended for use during pregnancy. The goal of this work is to better understand how these adverse effects are initiated and the role of DMF-induced Nrf2 activation during three critical windows of development in embryonic zebrafish (Danio rerio): pharyngula, hatching, and protruding-mouth stages. To evaluate Nrf2 activation, wildtype zebrafish, and mutant zebrafish (nrf2afh318/fh318) embryos with a loss of function mutation in Nrf2a, the co-ortholog to human Nrf2, were treated for 6 h with DMF (0–20 μM) beginning at the pharyngula, hatching, or protruding-mouth stage and assessed for survival and morphology. Nrf2a mutant fish had an increase in survival, however, morphology studies demonstrated Nrf2a mutant fish had more severe deformities occurring with exposures during the hatching stage. To verify Nrf2 cellular localization and downstream impacts on protein-S-glutathionylation in situ, a concentration below the LOAEL was chosen (7 μM) for immunohistochemistry and S-glutathionylation. Embryos were imaged via epifluorescence microscopy studies, the Nrf2a protein in the body tissue was decreased with DMF only when exposed at the hatching stage, while total protein S-glutathionylation was modulated by Nrf2a activity and DMF during the pharyngula and protruding-mouth stage. The pancreatic islet and liver were further analyzed via confocal microscopy. Pancreatic islets and liver also had tissue specific differences with Nrf2a protein expression and protein S-glutathionylation. This work demonstrates how critical windows of exposure and Nrf2a activity may influence toxicity of DMF and highlights tissue-specific changes in Nrf2a protein levels and S-glutathionylation in pancreatic islet and liver during embryonic development. [Display omitted] •DMF adverse effects on zebrafish morphology were modulated with Nrf2a activity.•Changes at developmental windows with DMF correlated with Nrf2 and glutathionylation.•DMF exposure decreased Nrf2 protein and increased glutathionylation in the islet.•Unlike the islet, liver Nrf2 and glutathionylation results suggests Nrf2 is protective.