Development and validation of a new in vitro assay designed to measure contact allergen-triggered oxidative stress in dendritic cells

Abstract

Selected contact allergens are known to induce phenotypic and functional maturation of dendritic cells (DCs). Such changes occurring in DCs have been employed as assay readouts to predict skin-sensitizing potentials of small chemicals. To respond to the urgent needs for reliable in vitro tests to identify contact allergens, we sought to develop a DC-based assay designed to detect early change(s) induced by sensitizers. Signature gene expression profiles of skin sensitization were determined by GeneChip and quantitative RT-PCR analyses of RNA samples harvested from mouse skin and XS106 DC line after exposure to dinitrofluorobenzene (DNFB). Production of reactive oxygen species (ROS) was examined indirectly by measuring the level of oxidative stress-XS106 DCs were labeled with a fluorescent dye, CM-H(2)DCFDA, exposed to test chemicals, and then examined for fluorescence signals by flow cytometer. DNFB induced abundant mRNA expression of several redox regulatory genes in both mouse skin and XS106DCs. Expression of these genes was inducible by hydrogen peroxide and blocked by a ROS inhibitor, diphenyleneiodonium. Rapid and significant ROS production was induced by 25 of the 28 tested skin sensitizers, but only by 3 of the 21 tested skin irritants. Our small-scale validation study demonstrates the practical utility of our DC-based ROS production assay to detect structurally diverse contact allergens with varying sensitizing potencies. It is tempting to speculate that ROS production in DCs may represent an early event during the sensitization phase.