n-monoacetylcystine and Dermatitis--Contact

Although extracellular adenosine 5'-triphosphate (eATP) has a crucial role in the sensitization phase of contact hypersensitivity (CHS), the mechanism by which hapten causes keratinocyte cell death and ATP release is unknown. We examined the time course of cell death, reactive oxygen species (ROS) production, and ATP release in HaCaT cells and in normal human keratinocytes after exposure to nonmetal haptens, NiCl2, or irritants. Both haptens and irritants caused cell death of keratinocytes but with different time courses. N-acetylcysteine (NAC) significantly reduced only nonmetal hapten-induced cell death as assessed by propidium iodide exclusion. We examined the effects of antioxidants and pannexin (Panx) inhibitors on cell death, ROS production, and ATP release by chemical-treated HaCaT cells. Nonmetal hapten-induced cell death, but not NiCl2- or irritant-related cell death, was dependent on reactivity to thiol residues in the cells. NAC reduced cell death and ATP release, whereas antioxidants and Panx inhibitors did not inhibit cell death but significantly attenuated ATP release. Panx1 small interfering RNA (siRNA) also suppressed ATP release from hapten-exposed HaCaT cells. Intraperitoneal injection of a Panx1 inhibitor attenuated murine CHS. These findings suggest that nonmetal hapten reactivity to thiol residues causes membrane disruption of keratinocytes and ROS production that leads to ATP release through opening of Panx hemichannels.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Benzopyrans; Cell Death; Cells, Cultured; Connexins; Cystine; Dermatitis, Contact; Dinitrochlorobenzene; Disease Models, Animal; Female; Haptens; Humans; Irritants; Keratinocytes; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Nickel; Protein Structure, Quaternary; Protein Structure, Tertiary; Pyrimidinones; Reactive Oxygen Species; RNA, Small Interfering

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

Topics: Antigens, CD34; B7-2 Antigen; Blotting, Western; Cell Line; Cystine; Dendritic Cells; Dermatitis, Contact; Fetal Blood; Flow Cytometry; Heme Oxygenase-1; Humans; Intracellular Signaling Peptides and Proteins; Irritants; Kelch-Like ECH-Associated Protein 1; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Skin; Up-Regulation