Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB

Abstract

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC₅₀ values of 2.3 ± 0.7 μM and 42.4 ± 20.9 μM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

Keywords: DNCB; DNFB; TRPA1; contact dermatitis; electrophiles; nitrobenzene; skin sensitizer.