title: Development and validation of a new in vitro assay designed to measure contact allergen-triggered oxidative stress in dendritic cells.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine, Toledo, OH 43614-5806, USA.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Miyazawa
authors.forename: Akira
authors.initials: A
authors.lastname: Takashima
mesh_terms: D000485:Allergens / Q000494:pharmacology*; D000818:Animals; D003713:Dendritic Cells / Q000187:drug effects* / Q000378:metabolism; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D020869:Gene Expression Profiling; D051379:Mice; D008810:Mice, Inbred C57BL; D051267:NF-E2-Related Factor 2 / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species; D020218:Response Elements
chemical_list: D000485:Allergens; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D017382:Reactive Oxygen Species; D004139:Dinitrofluorobenzene