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Allergic Contact Dermatitis is a skin condition that affects up to twenty percent of the North American and Western European population. The molecular initiating event (MIE) of this type IV delayed hypersensitivity is the formation of antigenic species by covalent modification of endogenous proteins. Cysteine residues, though not the most prevalent residues in skin proteins, are prone to react with small electrophilic molecules and thus play a crucial role in protein haptenation. The role of cysteine is also very prominent in removal of electrophiles. Glutathione (GSH) is the most prominent antioxidant in cells and contains a cysteine residue, providing a free reactive thiol for GSH conjugation of electrophiles and their subsequent removal. It is also the co-factor of an important set of enzymes involved in the metabolic clearance system, glutathione S-transferases (GST). However, it remains unclear whether the reactivity of small exogenous electrophiles with intracellular GSH could be a key factor in determining the epidermal bioavailability of sensitising chemicals. GST activity has been demonstrated both in skin and in most in vitro skin equivalents but so far studies have focussed on specific chemical clearance. The GSH cycle (synthesis, conjugation, recycling) has not been comprehensively studied in the HaCaT cell line, which is often used as a surrogate model for human skin. We showed that the model sensitisers 2,4-dinitrohalobenzenes, reacting with thiols via a SNAr mechanism, depleted intracellular GSH within the first hour of exposure in HaCaT cells, in a dose dependent manner. Synthesis of de novo GSH was investigated 24 hours after treatment with a single non-toxic dose of 10 μM of three dinitrohalobenzenes. GSH concentration was found similar or slightly higher than the level measured in control cells for 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) but not for 1- bromo-2,4-dinitrobenzene (DNBB), which appeared to partially prevent GSH repletion. This data inferred that the repletion step was the differentiating factor between chemicals belonging to the same reactivity domain. The toxicity of the halogen released in the cells was most likely the reason for observing these differences and this might have been missed if only the clearance kinetics were investigated. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was concomitantly observed for all compounds within two hours, and at concentrations less than 10 μM. Nrf2 activation is the precursor event for the overproduction of Phase II metabolic enzymes such as GSTs, enzymes involved in the synthesis of GSH such as Glutamyl cysteine ligase (GCL) and Glutathione synthetase (GS) as well as GSH related enzymes acting against oxidative stress such as Glutathione Reductase (GR). HaCaT cells treated with the model aldehydes benzaldehyde and phenylacetaldehyde, which oxidised cysteine residues in chemico, did not suffer a significant depletion of GSH levels at sub-toxic concentrations. In addition, these aldehydes have been shown to react with amines via a Schiff base mechanism to form stable protein adducts. Our set of data indicated that the Schiff base mechanism was most probably favoured and that an oxidation of GSH (a reduction of the GSH:GSSG ratio), was effectively and rapidly controlled by GR activity. The volatility of aldehydes was likely to limit the bioavailability of these compounds intracellularly, possibly increasing the probability of haptenation with cell membrane proteins. Skin sensitisers are often defined by their potency (ranging from extreme to non-sensitising) rather than their chemical reactivity domain. We compared the clearance of two extreme sensitisers by the GSH pathway: DNCB and diphenylcyclopropenone (DPCP). We demonstrated that two chemicals able to react with thiols in chemico via different mechanisms (SNAr for DNCB and Michael addition for DPCP) were affecting GSH levels differently in HaCaT cells. DNCB induced GSH depletion at non-toxic concentrations, while exposure to DPCP decreased cell viability at concentrations for which GSH stock was not significantly reduced. DPCP reactivity with thiols has been suggested in cell membranes. Contrary to the results obtained for DPCP, diethyl maleate (DEM), a moderate sensitiser also reacting via Michael addition, was cleared by GSH at a rate similar to the rate of de novo GSH synthesis, masking the overall depletion of GSH. Exposure to DEM induced an overproduction of GSH after 24 hours that was more pronounced than the one observed for the more potent sensitiser DNCB, showing that the sensitising potency of chemicals could not be correlated solely to the ability to be conjugated by GSH (i.e. weak sensitisers would be cleared rapidly while potent sensitisers would not). These experiments demonstrated that the defence mechanisms in the HaCaT cell line were providing a rapid response to chemical stress. To investigate the effects of chemical exposure on GSH lifecycle in reconstructed human epidermis (RHE), we attempted repeated cycles of 2 hour exposure to DNCB over a week long period. For three consecutive treatments, each exposure to DNCB led to GSH depletion. Replenishment to basal level was observed after a 22 hour recovery period. Accumulation of Nrf2 in the cytosol also occurred within the two hours of exposure to DNCB but returned to baseline during each recovery period. The amount of GSH conjugate formed (dinitrophenyl glutathione) increased after each exposure, suggesting that the metabolic capacity of skin may be enhanced in response to exposure to exogenous compounds. In conclusion, the GSH cycle is fully active in skin and participates in the clearance of some exogenous compounds with electrophilic properties. GSH skin metabolism can also potentially be enhanced after repeated exposures to small quantities of electrophiles. Future risk assessments for skin sensitisation potential of topically applied chemicals could integrate these findings to correlate more realistically to in vivo scenarios. |
