Toxicology Letters 192 (2010) 229–237 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Comparison of flow cytometry and immunohistochemistry in non-radioisotopic murine lymph node assay using bromodeoxyuridine Kyoung-Mi Jung a, Il-Hong Bae a, Bae-Hwan Kim b, Wang-Ki Kim a, Jin-Ho Chung c, Young-Ho Park a, Kyung-Min Lim a,∗ a Amorepacific Corporation R&D Center, Yongin-si 446-729, Republic of Korea b Department of Public Health, Keimyung University, Republic of Korea c College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea article info abstract Article history: Non-radioisotopic local lymph node assay (LLNA) employing 5-bromo-2-deoxyuridine (BrdU) with flow Received 25 September 2009 cytometry (FACS) or immunohistochemistry (IHC) is gaining attention due to a regulatory issue of using Received in revised form 19 October 2009 radioisotope, 3H-thymidine, in vivo in traditional LLNA. In this study, to compare the performance of Accepted 21 October 2009 these non-radioisotopic endpoints, 7 chemicals with known sensitizing potencies were examined in Available online 30 October 2009 LLNA. Mice were topically treated with chemicals or vehicle on both ears for 3 days. After intraperitoneal injection of BrdU, bilateral lymph nodes were isolated separately and undergone respectively, FACS or Keywords: IHC to determine BrdU incorporated lymph node cells (LNCs). Weight and histology of treated ears were Non-radioisotopic local lymph node assay 5-Bromo-2-deoxyuridine also examined to evaluate chemical-induced edema and irritation. Both FACS and IHC could successively Flow cytometry identify the skin sensitizers from non-sensitizers. Comparison of FACS and IHC with traditional LLNA Immunohistochemistry revealed that FACS has a higher sensitivity although both assays produced comparable sensitivity and performance to traditional LLNA. In conclusion, non-radioisotopic LLNA using FACS and IHC can suc- cessfully detect sensitizers with a good correlation to traditional LLNA. Notably, FACS showed almost equivalent sensitivity and accuracy to traditional LLNA. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction regulation limiting the introduction of radioisotopes into animal in vivo, mandating a heavy ventilation, filtering and completely iso- Mouse local lymph node assay (LLNA) is a validated alternative lated animal housing facilities. Accordingly, traditional LLNA has method for testing the sensitization potential of chemicals, replac- not been generalized widely, raising an urgent need for the devel- ing the conventional guinea pig maximization tests (Farrell et al., opment and the validation of non-radioisotopic endpoints in LLNA. 2009). This method offers important advantages over guinea pig Recently, to avoid the use of 3H-thymidine in vivo and to intro- assays for the animal welfare in terms of reduction and refine- duce non-radioisotopic endpoints in LLNA, many attempts have ment without deteriorating the assay quality, leading to a wide been made. These include the examination of the phenotype of pro- acceptance of LLNA as a representative alternative method for the liferating lymphocyte subsets (Gerberick et al., 1999), the indirect identification of chemicals with a skin sensitizing potential. estimation of lymphocyte proliferation by measuring intracellu- In LLNA, the skin sensitization potential is determined by mea- lar ATP content of lymph nodes (Idehara et al., 2008) and the suring lymphocyte proliferation in the draining auricular lymph assay of ex vivo cytokines production by draining lymph node cells nodes in response to the treated chemicals (Gerberick et al., 2007). (LNCs) (Dearman et al., 1999, 1994; Ku et al., 2008; van den Berg Radiolabeling of proliferating lymphocytes using 3H-thymidine has et al., 2005). Of these approaches, the measurement of 5-bromo- been commonly employed, however many countries have a strict 2-deoxyuridine (BrdU) incorporation into DNA (Takeyoshi et al., 2001) is gathering a huge interest, owing to its similarity to the conventional LLNA method employing 3H-thymidine. Abbreviations: LLNA, local lymph node assay; FACS, flow cytometry; PPD, BrdU is a non-radioisotopic analog of thymidine, which is p-phenylenediamine; DNCB, 2,4-dinitrochlorobenzene; IS, isopropylalcohol; SLS, incorporated into DNA during the S-phase of the cell cycle. It sodium lauryl sulfate; BrdU, 5-bromo-2-deoxyuridine; IHC, immunohistochem- has substituted 3H-thymidine labeling in many biological assays istry; HCA, hexylcinnamaldehyde. ∗ measuring cellular proliferation (Porstmann et al., 1985). To mea- Corresponding author at: Amorepacific Corporation R&D Center, 314-1 Bora- sure BrdU incorporation into lymph node, many antibody-based dong, Giheung-gu, Yongin-si 446-729, Republic of Korea. Tel.: +82 31 280 5904; fax: +82 31 281 8390. assay methods are available including flow cytometric analysis E-mail address: kimlim@amorepacific.com (K.-M. Lim). (FACS) (Suda et al., 2002), immunohistochemical staining (IHC) 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.10.024 230 K.-M. Jung et al. / Toxicology Letters 192 (2010) 229–237 Table 1 Tested chemicals and their reported sensitization potency. Substance name (abbreviation) Vehicle Potency category EC3a (%) Test concentration (%) 2,4-Dinitrochlorobenzene (DNCB) AOO Extreme 0.04 0.01, 0.1, 0.25, 0.5 4-Phenylenediamine (PPD) AOO Strong 0.11 0.1, 1, 3 Isoeugenol AOO Moderate 1.5 2.5, 5, 10 Hexylcinnamaldehyde (HCA) AOO Moderate 9.9 5, 10, 25 Eugenol AOO Weak 10.1 5, 10, 25 Isopropylalcohol (IS) AOO Negative – 50 Sodium lauryl sulfate (SLS) 50% ethanol /DMF Moderate (false positive) 8.1 5, 10, 15 a Values from ICCVAM 2009, Recommended Performance Standards: Murine Local Lymph Node Assay (2009, NIH Publication No. 09-7357). Fig. 1. Changes in auricular lymph node weights and the number of LNCs (lymph node cells). (A) Representative photograph of auricular lymph node from the treated mouse. Bar indicates 5 mm. (B) After mice were treated with 0.1, 1 and 3% PPD, 0.25% DNCB, 15% SLS or 50% IS for 3 days, auricular lymph node was collected on Day 5 and weighed. *Significant difference from vehicle group, p < 0.05, values are mean ± S.D. (N = 5 or 6). (C) The number of LNC (×106 cells) in an auricular lymph node from treated mice. K.-M. Jung et al. / Toxicology Letters 192 (2010) 229–237 231 Fig. 2. Swelling and inflammation of ear. (A) After mice were treated with 0.1%, 1% and 3% PPD, 0.25% DNCB, 15% SLS or 50% IS for 3 days, 6 mm ear biopsy was obtained on Day 5 and weighed to evaluate ear swelling. *Significant difference from vehicle group, p < 0.05, values are mean ± S.D. (N = 5 or 6). (B) Representative microscopic photograph of H&E-stained ear tissue from treated mice. Original magnification ×100, bar indicates 200 ␮m. (Boussiquet-Leroux et al., 1995) and ELISA (Takeyoshi et al., 2003; 2. Materials and methods Yamano and Shimizu, 2009). ELISA-based immunoassay method is developed first for the measurement of BrdU incorporation 2.1. Chemicals and reagents and accordingly a validation study has been conducted earliest 2,4-Dinitrochlorobenzene (DNCB), isopropylalcohol (IS), isoeugenol, hexylcin- (ICCVAM, 2009a). However, FACS or IHC method is also enjoy- namaldehyde (HCA), eugenol, sodium lauryl sulfate (SLS), p-phenylenediamine ing a large interest since they are highly sensitive and specific to (PPD) and 5-bromo-2-deoxyuridine (BrdU) were obtained from Sigma–Aldrich (San BrdU incorporated lymphocyte population. Especially, FACS allows Diego, CA). PPD, DNCB, HCA, isoeugenol, eugenol and IS was dissolved in ace- tone:olive oil (AOO; 4:1) and SLS was dissolved in 50% ethanol or DMF. BrdU was a rapid analysis of many extra parameters such as surface markers, dissolved in phosphate-buffered saline (PBS) at a concentration of 20 mg/ml. cell counts or lymphocyte population identifications at one scope, providing a useful methodology and advantage for the screening 2.2. Animal housing and experimental protocol and characterization of potential sensitizers. In this study, to compare the performance of FACS and IHC, Both the animal care and the study protocol were conducted in accordance with 7 chemicals selected from recommended performance standard Institutional Animal Care and Use Committee (IACUC) of Amorepacific R&D center. ◦ chemicals of ICCVAM 2009, p-phenylenediamine (PPD), 2,4- Animals were kept under controlled conditions of temperature (23 ± 3 C) and rel- ± ◦ dinitrochlorobenzene (DNCB), isoeugenol, hexylcinnamaldehyde ative humidity (50 10 C) with alternating 12 h light and dark cycle. Throughout the study, animals had ad libitum access to tab water. Female BALB/c mice (7–8 (HCA), eugenol, sodium lauryl sulfate (SLS) and isopropylalcohol weeks old, body weight 18–22 g) were purchased from Orient Bio (Seoul, Korea), (IS) were examined in LLNA where bilateral auricular lymph nodes and were used in all experiments. Before experiments, animals were kept for at were separately isolated and undergone FACS or IHC respectively. least 1 week on laboratory solid diet (Purina Co., Korea) for acclimation. The mouse In addition, ear inflammation and lymph node changes by chemi- lymph node assay was performed according to the method of Takeyoshi et al. (2003) with minor modifications. Groups of mice (N = 5 or 6) were treated with 25 ␮lof cal treatment were evaluated by observing the morphology and the the various concentrations of test chemicals (Table 1) or vehicle on the dorsal area histology of ear and lymph node after chemical treatments. Finally, of both ears daily for 3 consecutive days (Days 0, 1 and 2). On Day 4, mice were the results from the respective assay methods were compared intraperitoneally administered with BrdU and mice were sacrificed after a day (Day with traditional LLNA data from published references to provide 5). After sacrifice, ear punch biopsies (6 mm full thickness skin) were collected and an insight into the performance of these methods in the evaluation weighed with a laboratory balance (Mettler Toledo, Columbus, OH) as a marker of ear swelling.