A skin-like cytochrome P450 cocktail activates prohaptens to contact allergenic metabolites

Allergic contact dermatitis is a complex syndrome representing immunological responses to cutaneous exposure to protein-reactive chemicals. Although many contact sensitizers directly can elicit this disorder, others ( prohaptens) require activation. Knowledge regarding the activating mechanisms remains limited, but one possibility is metabolic activation by cytochrome P450 (CYP) enzymes in the skin. We have, after quantitative reverse transcriptase-PCR studies of the CYP content in 18 human skin samples, developed an enriched skin-like recombinant human ( rh) CYP cocktail using CYP1A1, 1B1, 2B6, 2E1, and 3A5. To validate the rhCYP cocktail, a prohaptenic conjugated diene ((5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene) was investigated using: the skin-like rhCYP cocktail, a liver-like rhCYP cocktail, single rhCYP enzymes, liver microsomes, keratinocytes, and a dendritic cell ( DC) assay. The diene was activated to sensitizing epoxides in all non-cell-based incubations including the skin-like rhCYP cocktail. An exocyclic epoxide metabolite (( 7R)-7-isopropenyl- 4-methyl-1-oxaspiro[2.5] oct-4-ene) was found to be mainly responsible for the allergenic activity of the diene. This epoxide also induced pronounced DC activation indicated by upregulation of IL-8. The skin-like rhCYP cocktail provides a simplified alternative to using skin tissue preparations in mechanistic studies of CYP-mediated skin metabolism of prohaptens and offers the future possibility of designing in vitro predictive assays for assessment of allergenic activity of prohaptens.