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

Abstract

Glutathione (GSH) plays a major role in skin detoxification processes due to its ability to conjugate electrophilic exogenous compounds with, and sometimes without, catalysis by glutathione-s-transferase (GST). GST activity has been demonstrated both in skin and in most in vitro skin equivalents but so far studies have focussed on chemical clearance (conjugate identification and rate of conjugation) and did not consider the GSH lifecycle (conjugation, recycling, synthesis). We used the model skin sensitizer 2,4-dinitrochlorobenzene (DNCB) to investigate the effects of chemical exposure on GSH lifecycle in reconstructed human epidermis (RHE). We demonstrated that the RHE model is suitable to carry out repeated cycles of 2-h exposure to DNCB over a 3-day period. After each exposure to DNCB, the level of GSH is diminished in a dose dependent manner. After a 22-h recovery period, GSH is replenished back to initial levels. Accumulation of the nuclear factor E2-related factor 2 (Nrf2) in the cytosol also occurs within the 2 h of exposure to DNCB but returns to baseline during each recovery period, demonstrating that activation of the Nrf2 signaling pathway offers a rapid response to chemical stress. The amount 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 of skin may not be fixed in time but defence mechanisms might be activated in response to exposure to exogenous compounds, resulting in their accelerated clearance.

Protein Haptenation and Its Role in Allergy

“…One of the most studied experimental skin sensitizers, 2,4-dinitro-1- chlorobenzene (DNCB), was shown to readily interact with GSH, depleting the cellular levels completely by 4 h when applied at a subtoxic concentration to HaCaT cells . It was also shown that cellular GSH levels recover rapidly after repeated subtoxic DNCB exposure in reconstituted epidermis models in vitro . Perhaps most interestingly, Bailey and colleagues have found that DNCB covalently modifies the enzyme which binds it to GSH, glutathione-s-transferase ω (GST-ω), at the active center, potentially impeding phase II metabolism .…”

“… 52 It was also shown that cellular GSH levels recover rapidly after repeated subtoxic DNCB exposure in reconstituted epidermis models in vitro . 102 Perhaps most interestingly, Bailey and colleagues have found that DNCB covalently modifies the enzyme which binds it to GSH, glutathione-s-transferase ω (GST-ω), at the active center, potentially impeding phase II metabolism. 103 This particular modification was processed by the DNCB treated HaCaT cells in this experiment and presented on the surface associated with the MHC I molecule.…”

A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin

“…the capacity of skin to generate or import cofactors on demand, could also play an important part in the defence system against exogenous chemicals. As clearly illustrated by comparing rates of glutathione conjugation with and without the addition of GSH (Supplementary data Figure C), cofactor amounts are an important influence on metabolic rates (Spriggs et al, 2016) and should be considered when studying inter-individual variability using sub cellular fractions. In addition, it was also found in the initial method development stages, that co-incubating all cofactors and drugs simultaneously induced a competition of enzymes for substrate and/or cofactors resulting in an inhibitory effect on one another compared to when incubated separately; this was particularly the case when a test chemical could undergo multiple metabolic pathways (Data not shown).…”