Abstract
Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, 3H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220 + and CD3e +. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.
Original language | English |
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Pages (from-to) | 255-263 |
Number of pages | 9 |
Journal | Toxicology Letters |
Volume | 209 |
Issue number | 3 |
DOIs | |
State | Published - 25 Mar 2012 |
Bibliographical note
Funding Information:This research was supported by grants ( 2010-10182KFDA508 and 2011-11182KFDA575 ) from Korean Food & Drug Administration .
Keywords
- 5-Bromo-2'-deoxyuridine
- B cell
- Cytokine
- Flow cytometry
- IL-2
- Non-radioisotopic local lymph node assay