Citation:

Lehmann, D., C. Copeland, E. Boykin, S. Quell, L. Copeland, AND W. Williams. Development of an ex vivo BrdU labeling procedure for the murine LLNA. Presented at Society of Toxicology, March 10 - 14, 2013.

Impact/Purpose:

The murine local lymph node assay is commonly used to identify sensitizers. Traditionally, the assay utilizes radioisotope to measure proliferation of T cells in local lymph nodes as an indication of sensitization. The purpose of this work was to develop a novel, ex vivo labeling proceedure using bromodeoxyuridine.

Description:

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 [3H]methyl thymidine (3HTdR). A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To eliminate the need for animal injections, we developed an ex vivo labeling procedure. BALB/c mice were dosed topically on the dorsum of both ears for 3 consecutive days with vehicle or one of 3 concentrations of eugenol (EUG), cinnamaldehyde (CA), hexyl cinnaminic aldehyde (HCA), nickel sulfate (NiS), isopropanol (IP) or lactic acid (LA). On day 5, lymph nodes were harvested, and single-cell suspensions were labeled ex vivo with 3HTdR or BrdU. Lymphocyte proliferation was determined by scintillation counting or BrdU ELISA, respectively. Concentration-dependent increases in ear thickness and lymphocyte proliferation were observed for EUG, CA and HCA, but none of the doses tested resulted in excessive skin irritation. The results of both the ex vivo 3HTdR and ex vivo BrdU assays correctly identified EUG, CA and HCA as dermal sensitizers according to the criteria outlined in the Globally Harmonized System for Classification and Labeling of Chemicals. As anticipated, non-sensitizers IP and LA did not induce a positive threshold response in either assay. Furthermore, a positive threshold response was not obtained for the false negative chemical NiS in either assay. The results of both ex vivo assays are in close agreement with those of the in vivo BrdU labeling procedure. We conclude that the ex vivo BrdU labeling method offers predictive capacity comparable to the other previously established LLNA protocols while eliminating animal injections and the use of radioisotope. This abstract does not represent EPA policy.

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