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Development and Utilization of an Ex Vivo Bromodeoxyuridine Local Lymph Node Assay (LLNA) Protocol for Assessing Potential Chemical Sensitizers
Citation:
Williams, W., C. Copeland, E. Boykin, S. Quell, AND D. Lehmann. Development and Utilization of an Ex Vivo Bromodeoxyuridine Local Lymph Node Assay (LLNA) Protocol for Assessing Potential Chemical Sensitizers. JOURNAL OF APPLIED TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 35(1):29-40, (2015).
Impact/Purpose:
We developed an ex vivo BrdU-labeling procedure for the murine local lymph node assay (LLNA) eliminating the need for in vivo injections. The results of this assay correctly identified the strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate (AHCP), ammonium tetrachloroplatinate (ATCP) and cis-diamminedichloroplatinum (II) (CDDP). The assay correctly identified AHCP, ATCP and CDDP as sensitizers based on calculation of the 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.
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 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 the 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-ELISA labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate (AHCP), ammonium tetrachloroplatinate (ATCP) and cis-diamminedichloroplatinum (II) (CDDP). The assay correctly identified AHCP, ATCP and CDDP as sensitizers based on calculation of the 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.
