Effect of Repeated Daily Dosing with 2,4-Dinitrochlorobenzene on Glutathione Biosynthesis and Nrf2 Activation in Reconstructed Human Epidermis

Glutathione (GSH) plays a major role in skin detoxification processes due to its ability to conjugate electrophilic exogenouscompounds with, and sometimes without, catalysis by glutathione-s-transferase (GST). GST activity has been demonstratedboth in skin and in most in vitro skin equivalents but so far studies have focussed on chemical clearance (conjugateidentification and rate of conjugation) and did not consider the GSH lifecycle (conjugation, recycling, synthesis). We usedthe model skin sensitizer 2,4-dinitrochlorobenzene (DNCB) to investigate the effects of chemical exposure on GSH lifecyclein reconstructed human epidermis (RHE). We demonstrated that the RHE model is suitable to carry out repeated cycles of2-h exposure to DNCB over a 3-day period. After each exposure to DNCB, the level of GSH is diminished in a dose dependentmanner. After a 22-h recovery period, GSH is replenished back to initial levels. Accumulation of the nuclear factorE2-related factor 2 (Nrf2) in the cytosol also occurs within the 2h of exposure to DNCB but returns to baseline during eachrecovery period, demonstrating that activation of the Nrf2 signaling pathway offers a rapid response to chemical stress. Theamount of dinitrophenyl-glutathione (DNP-SG) formed with DNCB (1) increased between the first and second exposure and(2) reached a plateau between the second and third exposure. Collectively, these data suggest that the metabolic capacity ofskin may not be fixed in time but defence mechanisms might be activated in response to exposure to exogenouscompounds, resulting in their accelerated clearance.