title: Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers.
abstract: The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [(3) H]methyl thymidine. A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non-sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU-enzyme-linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis-diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
authors.affiliation: Cardiopulmonary and Immunotoxicology Branch, Environmental Public Health Division, National Health, and Environmental Effects Laboratory (NHEERL), US Environmental Protection Agency, Research Triangle Park, NC, USA.
authors.forename: W C
authors.initials: WC
authors.lastname: Williams
authors.forename: C
authors.initials: C
authors.lastname: Copeland
authors.forename: E
authors.initials: E
authors.lastname: Boykin
authors.forename: S J
authors.initials: SJ
authors.lastname: Quell
authors.forename: D M
authors.initials: DM
authors.lastname: Lehmann
mesh_terms: D000485:Allergens / Q000633:toxicity*; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D017449:Dermatitis, Allergic Contact / Q000209:etiology* / Q000276:immunology; D023381:Endpoint Determination; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000187:drug effects* / Q000276:immunology; D018655:Lymphocyte Count; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D018675:Toxicity Tests
chemical_list: D000485:Allergens; D001973:Bromodeoxyuridine
keywords: 5-bromo-2′-deoxyuridine (BrdU); Local lymph node assay; ex vivo; platinum; skin sensitization