Evaluation of non-radioactive endpoints of ex vivo local lymph node assay-BrdU to investigate select contact sensitizers

Abstract

The present study sought to verify the utility of the non-radioactive endpoints LLNA BrdU (5-bromo-2′-deoxyuridine) ex vivo incorporation and cytokine release using auricular lymph node cells isolated from BALB/c mice topically treated with a strong (formaldehyde or p-phenylene-diamine [PPD]), moderate sensitizer (cinnamal), or weak sensitizer (eugenol). Stimulation index (SI) and EC3 values were calculated for each agent. Based on the results of ex vivo LLNA-BrdU assays, EC3 values were calculated to be 0.29, 0.09, 1.91, and 16.60% for formaldehyde, PPD, cinnamal, and eugenol, respectively. These results were in good agreement with data from previous standard radioactive LLNA. Cytokine analyses indicated TH1 and TH2 cytokine involvement in the regulation of murine contact allergy and these could be utilized as endpoints in assessments of contact allergy in mice. In conclusion, the current study provided evidence that the non-radioactive endpoint LLNA BrdU ex vivo incorporation could be of use as a viable alternative approach to assess the skin sensitization potential of test compound with respect to improving animal welfare. This is of particular importance in the case of any laboratory where it might be difficult to handle and/or readily employ radioisotopes. Further studies will be required to confirm—across test agents—the reproducibility as well as the limits of utility of this new ex vivo BrdU method.