Skin is a uniquely susceptible target organ for allergic reactions to small molecular weight compounds (haptens). Prohaptens need metabolic bioactivation in order to become antigenic. Incubation of prohaptens with single cytochrome P450 isoenzyme (CYP) cocktails or CYP-containing microsomes are generally very cytotoxic. The S9 fraction prepared from rat liver has been tried successfully, however a disadvantage is that it contains murine liver CYPs but not human cutaneous CYPs. This led us to redesign our protocol by spiking rat liver S9 with human cutaneous CYPs (e.g. CYP 1B1) and measuring the activation of NRF2 in KeratinoSens cells. Carvoxime (Ca) is a potent prohapten known to be a strong sensitizer in the murine LLNA. It is activated by human skin CYP 1B1 that is not expressed in liver and Ca was not reactive in the KeratinoSens assay using rat liver S9. Addition of Ca to the spiked preparation as well as CYP 1B1 itself strongly activated NRF2 whereas heat inactivated preparations showed no reactivity. Several fragrances are prohaptens. The cutaneous metabolom of the fragrance creosol (Cr) was analyzed by HR-MAS NMR after topical application to a 3D human skin equivalent. 1,2 benzoquinone-hapten-protein-adduct and another up to now unidentified hapten-protein-adduct were found in the upper epidermis, formaldehyde in the dermis. Since this metabolism requires a demethylation of Cr, we studied the possible involvement of human cutaneous CYPs in the KeratinoSens assay with rat liver S9 after spiking with human cutaneous CYPs. Addition of Cr to the spiked preparation strongly activated NRF2 and heat inactivated preparations as well as preparations only with S9 showed no reactivity. Taken together this modified KeratinoSens assay allows to identify prohaptens which are bioactivated by human cutaneous CYPs.

Prohapten-activation by human cutaneous cytochrome P450 isoenzymes – identified with a modified KeratinoSens assay