Comparison of 125-Iododeoxyuridine (¹²⁵IUdR) and [³H]Thymidine ([³H]TdR) for Assessing Cell Proliferation in the Murine Local Lymph Node Assay

Abstract

The murine local lymph node assay (LLNA) evaluates the dermal sensitization potential of chemicals by assessing the cell proliferation within the auricular lymph nodes following topical application of a chemical to the ears. Traditionally, [³H]thymidine ([³H]TdR) is used to measure proliferation. Because of its long half-life and the resulting expense of disposing of [³H]TdR waste, the usefulness, reliability, selectivity, and sensitivity of 125-iododeoxyuridine (¹²⁵IUdR) was evaluated as an alternative radiolabel in the LLNA. An optimal response was obtained using the following: daily exposure on the dorsum of both ears for 3 days; 2 days of rest; on the sixth day IV injection of 2 μCi/mouse ¹²⁵IUdR; removal of nodes after 5 h. Female CBA/J mice were exposed to either vehicle or 1,6-diisocy-anatohexane (HDI), 2-hydroxyethyl acrylate (HEA), methyl salicylate (MS), potassium dichromate (PD), or dinitrochlorobenzene (DNCB); injected IV with 20 μCi [³H]TdR or 2 μCi ¹²⁵IUdR; and proliferation in the pooled auricular lymph nodes from individual mice was assessed. Stimulation indices (SI) of ≥3 indicated a positive response. Both an SI ≥ 3 and statistical significance were observed for the known sensitizers HDI and HEA using either ¹²⁵IUdR or [³H]TdR. For the nonsensitizer MS, neither an SI ≥ 3 nor statistical significance was detected using ¹²⁵IUdR or [³H]TdR. Both isotopes detected a positive response based on statistical analysis at 0.5 and 0.25% PD, while ¹²⁵IUdR detected further significance at 0.1% PD. An SI ≥ 3 was detected by both isotopes at 0.5% PD, while ¹²⁵IUdR detected an SI ≥ 3 at 0.25% PD. Statistical significance was observed for [³H]TdR at 0.025, 0.05, 0.1, and 0.25% DNCB and at 0.1 and 0.25% DNCB for ¹²⁵IUdR. An SI ≥ 3 was observed at 0.05, 0.1, and 0.25% DNCB for [³H]TdR and at 0.1 and 0.25% DNCB for ¹²⁵IUdR. ¹²⁵IUdR is a suitable, cost-effective alternative radiolabel to [³H]TdR for assessing cell proliferation in the LLNA. Key Words: Local lymph node assay, Skin sensitization, Mouse, [³H]thymidine, 125-iododeoxyuridine, Cell proliferation.