Imaging-based temporal dynamics of electrophile-induced NRF2 signaling in liver cells identifies adaptive versus adverse repeated exposure conditions.

The activation of stress responses upon chemical exposure is critical to restore cellular homeostasis. One of these pivotal pathways is the oxidative stress response activated by NRF2 under control of sensor KEAP1. Soft electrophiles are known to bind with KEAP1 and consequently activate NRF2. However, the understanding of the NRF2 activation dynamics and influence on cellular fate during prolonged repeated exposure conditions is still lacking. Therefore, we accurately mapped the NRF2 activation dynamics upon single or repeated exposures using HepG2 BAC GFP reporters combined with live cell confocal imaging. In a 2D set-up, a concentration-dependent NRF2 activation and subsequent SRXN1 induction upon exposure to sulforaphane or CDDO-Me was observed. The level of NRF2 activation upon sulforaphane exposure highly correlated with cytotoxicity induction. Reactivation of NRF2 upon a second exposure without a recovery period was lower compared to initial exposure, yet resulted in a strong and sustained upregulation of SRXN1. In contrast, inclusion of recovery periods did allow for strong NRF2 and SRXN1 upregulation. In 3D, increased SRXN1 upregulation was observed upon a second exposure with sulforaphane after recovery up to 6 days compared to the first exposure, while similar or lower SRXN1 induction was observed after secondary CDDO-Me treatment. In conclusion, NRF2 activation dynamics and subsequently SRXN1 induction is highly chemical, concentration and exposure duration dependent. Recovery periods allow cells to adapt and initiate a secondary NRF2 response. Together this allowed for further understanding of NRF2 dynamics and cellular fate upon repeated exposures contributing to improving chemical safety testing.