Toxicology Letters 192 (2010) Contents lists available at ScienceDirect. Toxicology Letters. journal homepage:

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1 Toxicology Letters 192 (2010) Contents lists available at ScienceDirect Toxicology Letters journal homepage: 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 , Republic of Korea b Department of Public Health, Keimyung University, Republic of Korea c College of Pharmacy, Seoul National University, Seoul , Republic of Korea article info abstract Article history: Received 25 September 2009 Received in revised form 19 October 2009 Accepted 21 October 2009 Available online 30 October 2009 Keywords: Non-radioisotopic local lymph node assay 5-Bromo-2 -deoxyuridine Flow cytometry Immunohistochemistry Non-radioisotopic local lymph node assay (LLNA) employing 5-bromo-2 -deoxyuridine (BrdU) with flow cytometry (FACS) or immunohistochemistry (IHC) is gaining attention due to a regulatory issue of using radioisotope, 3 H-thymidine, in vivo in traditional LLNA. In this study, to compare the performance of these non-radioisotopic endpoints, 7 chemicals with known sensitizing potencies were examined in 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 IHC to determine BrdU incorporated lymph node cells (LNCs). Weight and histology of treated ears were also examined to evaluate chemical-induced edema and irritation. Both FACS and IHC could successively identify the skin sensitizers from non-sensitizers. Comparison of FACS and IHC with traditional LLNA 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 successfully detect sensitizers with a good correlation to traditional LLNA. Notably, FACS showed almost equivalent sensitivity and accuracy to traditional LLNA Elsevier Ireland Ltd. All rights reserved. 1. Introduction Mouse local lymph node assay (LLNA) is a validated alternative method for testing the sensitization potential of chemicals, replacing the conventional guinea pig maximization tests (Farrell et al., 2009). This method offers important advantages over guinea pig assays for the animal welfare in terms of reduction and refinement without deteriorating the assay quality, leading to a wide acceptance of LLNA as a representative alternative method for the identification of chemicals with a skin sensitizing potential. In LLNA, the skin sensitization potential is determined by measuring lymphocyte proliferation in the draining auricular lymph nodes in response to the treated chemicals (Gerberick et al., 2007). Radiolabeling of proliferating lymphocytes using 3 H-thymidine has been commonly employed, however many countries have a strict Abbreviations: LLNA, local lymph node assay; FACS, flow cytometry; PPD, p-phenylenediamine; DNCB, 2,4-dinitrochlorobenzene; IS, isopropylalcohol; SLS, sodium lauryl sulfate; BrdU, 5-bromo-2 -deoxyuridine; IHC, immunohistochemistry; HCA, hexylcinnamaldehyde. Corresponding author at: Amorepacific Corporation R&D Center, Boradong, Giheung-gu, Yongin-si , Republic of Korea. Tel.: ; fax: address: kimlim@amorepacific.com (K.-M. Lim). regulation limiting the introduction of radioisotopes into animal in vivo, mandating a heavy ventilation, filtering and completely isolated animal housing facilities. Accordingly, traditional LLNA has not been generalized widely, raising an urgent need for the development and the validation of non-radioisotopic endpoints in LLNA. Recently, to avoid the use of 3 H-thymidine in vivo and to introduce non-radioisotopic endpoints in LLNA, many attempts have been made. These include the examination of the phenotype of proliferating lymphocyte subsets (Gerberick et al., 1999), the indirect estimation of lymphocyte proliferation by measuring intracellular ATP content of lymph nodes (Idehara et al., 2008) and the assay of ex vivo cytokines production by draining lymph node cells (LNCs) (Dearman et al., 1999, 1994; Ku et al., 2008; van den Berg et al., 2005). Of these approaches, the measurement of 5-bromo- 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 3 H-thymidine. BrdU is a non-radioisotopic analog of thymidine, which is incorporated into DNA during the S-phase of the cell cycle. It has substituted 3 H-thymidine labeling in many biological assays measuring cellular proliferation (Porstmann et al., 1985). To measure BrdU incorporation into lymph node, many antibody-based assay methods are available including flow cytometric analysis (FACS) (Suda et al., 2002), immunohistochemical staining (IHC) /$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi: /j.toxlet

2 230 K.-M. Jung et al. / Toxicology Letters 192 (2010) Table 1 Tested chemicals and their reported sensitization potency. Substance name (abbreviation) Vehicle Potency category EC3 a (%) Test concentration (%) 2,4-Dinitrochlorobenzene (DNCB) AOO Extreme , 0.1, 0.25, Phenylenediamine (PPD) AOO Strong , 1, 3 Isoeugenol AOO Moderate , 5, 10 Hexylcinnamaldehyde (HCA) AOO Moderate 9.9 5, 10, 25 Eugenol AOO Weak , 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 ). 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 ( 10 6 cells) in an auricular lymph node from treated mice.

3 K.-M. Jung et al. / Toxicology Letters 192 (2010) 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; Yamano and Shimizu, 2009). ELISA-based immunoassay method is developed first for the measurement of BrdU incorporation and accordingly a validation study has been conducted earliest (ICCVAM, 2009a). However, FACS or IHC method is also enjoying a large interest since they are highly sensitive and specific to BrdU incorporated lymphocyte population. Especially, FACS allows a rapid analysis of many extra parameters such as surface markers, cell counts or lymphocyte population identifications at one scope, providing a useful methodology and advantage for the screening and characterization of potential sensitizers. In this study, to compare the performance of FACS and IHC, 7 chemicals selected from recommended performance standard chemicals of ICCVAM 2009, p-phenylenediamine (PPD), 2,4- dinitrochlorobenzene (DNCB), isoeugenol, hexylcinnamaldehyde (HCA), eugenol, sodium lauryl sulfate (SLS) and isopropylalcohol (IS) were examined in LLNA where bilateral auricular lymph nodes were separately isolated and undergone FACS or IHC respectively. In addition, ear inflammation and lymph node changes by chemical treatment were evaluated by observing the morphology and the histology of ear and lymph node after chemical treatments. Finally, the results from the respective assay methods were compared with traditional LLNA data from published references to provide an insight into the performance of these methods in the evaluation of the chemicals with a sensitization potential. 2. Materials and methods 2.1. Chemicals and reagents 2,4-Dinitrochlorobenzene (DNCB), isopropylalcohol (IS), isoeugenol, hexylcinnamaldehyde (HCA), eugenol, sodium lauryl sulfate (SLS), p-phenylenediamine (PPD) and 5-bromo-2 -deoxyuridine (BrdU) were obtained from Sigma Aldrich (San Diego, CA). PPD, DNCB, HCA, isoeugenol, eugenol and IS was dissolved in acetone:olive oil (AOO; 4:1) and SLS was dissolved in 50% ethanol or DMF. BrdU was dissolved in phosphate-buffered saline (PBS) at a concentration of 20 mg/ml Animal housing and experimental protocol Both the animal care and the study protocol were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) of Amorepacific R&D center. Animals were kept under controlled conditions of temperature (23 ± 3 C) and relative 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 weeks old, body weight g) were purchased from Orient Bio (Seoul, Korea), and were used in all experiments. Before experiments, animals were kept for at least 1 week on laboratory solid diet (Purina Co., Korea) for acclimation. The mouse 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 l of the various concentrations of test chemicals (Table 1) or vehicle on the dorsal area of both ears daily for 3 consecutive days (Days 0, 1 and 2). On Day 4, mice were intraperitoneally administered with BrdU and mice were sacrificed after a day (Day 5). After sacrifice, ear punch biopsies (6 mm full thickness skin) were collected and weighed with a laboratory balance (Mettler Toledo, Columbus, OH) as a marker of ear swelling. Bilateral auricular lymph nodes were separately isolated, weighed and undergone lymphocyte preparation or tissue processing for FACS assay or hema-

4 232 K.-M. Jung et al. / Toxicology Letters 192 (2010) Table 2 Stimulation index obtained with FACS method. Test article Concentration (%) Stimulation index Defined potency category IS ± 0.68 Non-sensitizer SLS (50% ethanol) ± 1.08 Weak SLS (DMF) ± 0.46 Moderate ± ± 0.98 DNCB ± 0.89 Extreme ± ± ± 6.74 PPD ± 1.06 Extreme ± ± 6.96 Isoeugenol ± 1.41 Moderate ± ± 1.21 HCA ± 0.25 Moderate ± ± 0.80 Eugenol ± 0.94 Moderate ± ± 1.32 PPD, DNCB, HCA, isoeugenol, eugenol and IS were dissolved in acetone:olive oil (AOO; 4:1) and SLS was dissolved in 50% ethanol or DMF. Values are mean ± S.D. of 5 6 animals. Potency category was defined according to Gerberick et al. (2005). toxylin and eosin staining with immunohistochemistry for BrdU incorporated lymph node cells Lymph node cell counts in auricular lymph nodes and flow cytometry analysis for BrdU positive LNCs Lymph node cells (LNCs) were prepared from lymph node by disintegration through 70 m mesh (BD Biosciences, Franklin Lakes, NJ) in 1 ml PBS. The LNCs were counted using a hemacytometer after staining with trypan blue. The LNCs ( /ml) were washed once by centrifugation (300 g) for 5 min with PBS and re-suspended for fixation and permeabilization step, according to the instruction manual of BrdU Flow kits (BD Pharmingen TM, Franklin Lakes, NJ). Then LNCs were permeabilized using Cytoperm plus buffer, which contains 10% DMSO. After DNA was denatured by incubating for 1 h with DNase, LNCs were washed, and incubated with FITC conjugated anti-brdu antibody for 20 min at RT in the dark at a dilution of 1:50. Cells were washed once more and then re-suspended in 20 l 7-AAD solution to label DNA. Ten thousand 7-AAD expressing cells were gated, and the number of the cells expressing BrdU was analyzed using BD FACSCalibur TM system Immunohistochemical staining for BrdU positive LNCs For immunohistochemical staining (IHC) of incorporated BrdU, representative tissue samples were fixed in 10% neutral phosphate-buffered formalin, processed in a routine manner, embedded in paraffin, and sectioned at 5 m thickness. Sections were incubated with monoclonal mouse anti-brdu antibody (M0744, DakoCytomation, Glostrup, Denmark) as a primary antibody at a dilution of 1:150 overnight at 4 C. After rinsed three times, sections were incubated with polymer-hrp secondary antibody (Klear Mouse DAB Kit, Golden Bridge International, Mukilteo, WA) for 1 h at RT. Immunoreactivity was detected by the standard avidin biotin immunoperoxidase method. Counterstaining with Meyer s hematoxylin was then performed Table 3 Stimulation index obtained with IHC method. Test article Concentration (%) Stimulation index Defined potency category IS ± 0.16 Non-sensitizer SLS (50% ethanol) ± 0.28 Non-sensitizer SLS (DMF) ± 0.90 Moderate ± ± 2.00 DNCB ± 0.23 Extreme ± ± ± 1.76 PPD ± 0.49 Extreme ± ± 0.49 Isoeugenol ± 0.63 Moderate ± 3.29 HCA ± 0.17 Non-sensitizer ± ± 0.97 Eugenol ± 0.97 Weak ± ± 1.40 Values are mean ± S.D. of 5 6 animals. Potency category was defined according to Gerberick et al. (2005).

5 K.-M. Jung et al. / Toxicology Letters 192 (2010) Fig. 3. Flow cytometric analysis of LNCs for the BrdU incorporation. 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. (A) LNCs from auricular lymph nodes were stained with FITC conjugated anti-brdu antibody and analyzed with FACS. Nuclei were counterstained using 7-AAD. (B) Representative FACS histogram for LNCs from AOO and 3% PPD treated mice. (C) Percentage of positive BrdU incorporated LNCs. * Significant difference from vehicle group, p < 0.05, values are mean ± S.D. (N = 5 or 6). for 5 min. Thereafter they were evaluated under light microscope (Olympus, Japan) and analyzed with Image-Pro Plus (Media Cybernetics, Bethesda, MD). To demonstrate the difference of the BrdU-labeled lymphocytes of each group quantitatively, images of 9 sites were randomly selected ( pixels, magnification 400) and analyzed. The results were expressed as mean number of BrdU incorporated LNCs per group Statistics Values are shown as mean ± S.D. for all groups. The data were subjected to oneway analysis of variance followed by Duncan s multiple range test or Student t-test to determine which means were significantly different from the control (p < 0.05). Correlation between the tests was evaluated by Pearson s correlation analysis. Statistical analysis was performed using SPSS software (Chicago, IL). 3. Results 3.1. Changes in lymph node weight and lymph node cell count To compare the performance of FACS and IHC LLNA method, 7 chemicals with known potencies (Table 1) were examined in LLNA where bilateral auricular lymph nodes were separately iso-

6 234 K.-M. Jung et al. / Toxicology Letters 192 (2010) Fig. 4. Immunohistochemical analysis of LNCs for the BrdU incorporation. (A) Representative low-power microscopic photographs of IHC for BrdU incorporation in lymph nodes. The number of BrdU corporated lymphocytes (brown) increased in proportion with PPD concentration. Original magnification 12.5, bar indicates 1000 m. (B) High-power microscopic photograph of lymph node sections of the mice treated with AOO, IS, SLS, DNCB and PPD. Arrow denotes BrdU positive cell of the treated mice. Original magnification 400, bar indicates 50 m. (C) Number of LNCs immunopositive to BrdU antibody. * Significant difference from vehicle group, p < 0.05, values are mean ± S.D. (N = 5 or 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) lated and undergone FACS or IHC. Firstly, the changes in auricular lymph node weights and the number of LNCs (lymph node cells) were measured to evaluate the effects of chemicals on lymph nodes. Fig. 1A C shows the representative data from PPD, DNCB, SLS and IS. The weights of the lymph nodes of the mice treated with sensitizers, PPD (0.1%, 1% and 3%), DNCB (0.25%) and irritant, SLS (15% in 50% ethanol), were increased significantly when compared to the respective vehicle control groups (Fig. 1A and C). Meanwhile, the numbers of LNCs were significantly increased by sensitizers, PPD and DNCB while IS or SLS did not affect LNC numbers (Fig. 1B) Changes in mouse ears Biopsies of ear pad were collected after sacrifice and examined to evaluate the irritation. Fig. 2A and B shows the representative data from PPD, DNCB, SLS and IS. The weights of the ears treated with 15% SLS, 1% and 3% PPD, were increased significantly compared to respective vehicle control group (Fig. 2A), reflecting that SLS and PPD induced ear edema. These ear edema data well matched the inflammatory infiltration and epidermal hyperplasia by PPD (1% and 3%) and SLS (15%) as observed by increased inflammatory cells in the histological examination (Fig. 2B), confirming that these

7 K.-M. Jung et al. / Toxicology Letters 192 (2010) Fig. 5. Comparison of stimulation index (SI) values obtained by flow cytometry, immunohistochemistry and traditional LLNA. (A) Comparison of SI values between traditional LLNA (LLNA) and FACS measurement (FC). (B) Comparison of SI values between LLNA and IHC measurement (IHC). (C) Comparison of SI values between FC and IHC. Correlation between the tests was evaluated by Pearson s correlation analysis and regression analysis. Correlation coefficient (CC) and p values were obtained by Pearson s correlation analysis and slope, by regression analysis. Values of traditional LLNA are from the previously reported results (Betts et al., 2006; Gerberick et al., 2005; Warbrick et al., 1999). S is SLS 15%(in 50% ethanol) and D is 0.25% DNCB. chemicals had skin irritation potential at the tested doses. In contrast, topical applications of vehicle (AOO or 50% ethanol), IS 50%, PPD 0.1% or DNCB 0.25% did not alter the ear weight or histological appearance Flow cytometry analysis for BrdU incorporated LNCs To evaluate the effects of test article treatment on auricular LNC proliferation, DNA synthesis was assessed by measuring the incorporation of BrdU into LNCs with FACS using FITC conjugated anti-brdu antibody. Fig. 3A C shows the representative data from PPD, DNCB, SLS and IS. In the analysis of 10,000 LNCs with FACS, all the tested doses of PPD and DNCB increased BrdU positive LNC population significantly while IS or SLS treatment induced no significant increases in BrdU incorporated LNCs (Fig. 3A C). The stimulation index (SI) was calculated from the fold increase of the total number of positive BrdU LNCs in a lymph node compared to that of the respective vehicle control groups. Table 2 shows the SI values obtained with FACS method for the 7 test chemicals. While FACS method produced comparable results to traditional LLNA for most of the chemicals, PPD and eugenol were determined to be stronger sensitizers than traditional LLNA. SI values for 15%

8 236 K.-M. Jung et al. / Toxicology Letters 192 (2010) Table 4 Estimated EC3 values and performance of FACS and IHC in identifying sensitizers based on the reported results from T-LLNA. Category Material EC3 a (%) T-LLNA FACS IHC Sensitizer DNCB PPD 0.11 <0.10 <0.10 Isoeugenol HCA >25 Eugenol Non-Sensitizer IS False positive SLS (in DMF) Sensitivity (%) 100 (6/6) 83 (5/6) Specificity (%) 100 (1/1) 100 (1/1) a EC3 values are obtained with least square method with the linear ranges. SLS, an irritant was estimated to be 3.0 ± 1.08 with 50% ethanol as a vehicle but it showed 6.6 ± 0.98 with DMF as a vehicle, suggesting that the choice of vehicle is important for LLNA Immunohistochemical staining for BrdU incorporation in lymph nodes Auricular lymph nodes from treated mice were undergone IHC staining with anti-brdu antibody and polymer-hrp secondary antibody. Fig. 4A C shows the representative data from PPD, DNCB, SLS and IS. PPD and DNCB treatment increased both lymph node size and BrdU positive LNCs (0.25% DNCB, 90.3 ± 10.6 and 3% PPD, ± 9.71, respectively vs 19.8 ± 7.18 for AOO). The number of BrdU incorporated LNCs of the mice were also significantly increased by the treatment of SLS (34.0 ± 5.20 vs 20.8 ± 8.96 in 50% EtOH), whereas IS-treated mice showed no significant increase (26.0 ± 3.08, Fig. 4C). Table 3 shows the SI values obtained with IHC method for the 7 test chemicals. In contrast to FACS, HCA and SLS (in 50% ethanol) were determined to be non-sensitizer in IHC method Comparison of non-radioisotopic endpoints and traditional LLNA We compared the SI values obtained from FACS, IHC and the reported results from traditional LLNA with 3 H-thymidine uptake assay (Betts et al., 2006; Gerberick et al., 2005; Warbrick et al., 1999) with Pearson s correlation and regression analysis. As shown in Fig. 5A and B, both FACS and IHC methods showed well-correlated SI data to traditional LLNA as determined by strong statistical significances (p < 0.01) and linear relationship (correlation coefficients close to +1) in Pearson s correlation analysis. However, the comparison between FACS and IHC indicated that FACS method was more sensitive than IHC as determined by a high slope in regression analysis (slope = 4.11 in FACS vs IHC) (Fig. 5C). In addition, comparison between FACS and traditional LLNA showed that FACS method could detect sensitizers with equivalent sensitivities to, or higher than traditional LLNA (slope = in traditional LLNA vs FACS) (Fig. 5A). In support of this, comparison of EC3 values and definition of sensitizers obtained from these methods (Table 4) demonstrated that FACS method shows an equivalent performance to traditional LLNA. 4. Discussion In this study, we demonstrated that the skin sensitization potential could be successfully identified in LLNA by non-radioisotopic endpoints, FACS and IHC for BrdU incorporated lymph node cells (LNCs). Both FACS and IHC could successively differentiate sensitizers with comparable accuracies to traditional LLNA employing 3 H-thymidine. To our best knowledge, it is the first study to compare the performance of non-radioisotopic endpoints of LLNA in the same animals. Non-radioisotopic LLNA methods can be more generally used in many countries since the disposition of the radioactive waste, contaminated animal corpses and exhaled air are strictly regulated, limiting the widening of traditional LLNA employing radioactive 3 H-thymidine in vivo. Recently, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has presented evaluation reviews on the validation status of the LLNA: BrdU-ELISA and LLNA: BrdU-FC as a non-radioisotopic modification of the traditional LLNA and ECVAM published a draft guideline for BrdU-FC (ICCVAM, 2009a,b). In these reviews, it was concluded that these non-radioisotopic endpoints might be acceptable but require further validation studies. While the traditional LLNA assesses LNC proliferation by measuring the incorporation of radioactive 3 H- thymidine using liquid scintillation count, the LLNA:BrdU-FC and LLNA:BrdU-ELISA assess the incorporation of BrdU, an analog of thymidine (Takeyoshi et al., 2004), depending on the affinity of anti-brdu antibody. In the current study, we demonstrated that LLNA:BrdU-FC and LLNA:BrdU-IHC method give well-correlated data to traditional LLNA, supporting that these methods can replace traditional LLNA in the future. In the present study, the strong irritant, SLS in 50% ethanol was determined to be a weak sensitizer in FACS while IHC defined it as non-sensitizer. Although most non-sensitizers were concluded as negative in the traditional LLNA, false positive results could be often observed with a non-sensitizer and a strong irritant like SLS (Basketter et al., 1998). Indeed, Jacobs et al. (2006) have reported that toxic dose of non-sensitizing skin irritants can cause the death of epidermal keratinocytes and dying keratinocytes can serve as an adjuvant for stimulating dendritic cell migration and maturation in a non-antigen-dependent manner. We could confirm indeed that when SLS was applied in DMF, another accepted vehicle for LLNA, stronger and persistent irritation was induced and higher SI values (6.6 ± 0.98 in FACS and 5.0 ± 2.00 in IHC) were obtained, leading to a conclusion that SLS is a sensitizer with a moderate potency. This result indicates the importance of the vehicle selection in LLNA test and the necessity of the multiple vehicle tests for the chemicals with ambiguous results. Additionally, the LLNA results of strong irritants should be reviewed carefully to discern possible complications from strong irritation and keratinocyte cytotoxicity. While many reports suggested the utility of FACS or IHC as the new non-radioisotopic endpoints for LLNA, there has been no research comparing these two endpoints in the same animals to our best knowledge. Both FACS and IHC could successively identify the sensitizers with a good correlation to traditional LLNA. Although FACS or IHC is more time-consuming and expensive, they can be conducted non-radioisotopically, indicating that these

9 K.-M. Jung et al. / Toxicology Letters 192 (2010) methods could be more generally used in the countries having strict regulations for the in vivo use of radioisotopes. Incidentally, these countries should pay a large resource in facility, ventilation and the disposal of radioactive waste to use 3 H-thymidine, making the traditional LLNA to be more expensive assay method than FACS and IHC. Moreover, the signal to noise level, that is, the stimulation index data of FACS method were comparable or superior to traditional LLNA (Fig. 5A) and IHC method (Fig. 5C), suggesting that FACS method could be a good alternative for traditional LLNA. FACS, along with cell counting, can measure various markers in one process such as cellularity, BrdU incorporation, cell viability and surface markers with high specificity, reducing further the noise from non-lymphocytes or non-specific binding. Meanwhile, IHC determines the BrdU incorporated cells depending on antibody binding, and it could not differentiate non-lymphocytic cells, compromising the specificity and sensitivity to a certain degree. In support of this, HCA, a moderate sensitizer was determined to be negative in IHC method (Table 4). In this regard, we suggest that FACS assay appears to be a better method than IHC in the evaluation of sensitization potential of chemicals with non-radioisotopic LLNA. In conclusion, we have confirmed that the evaluation of proliferation in the auricular lymph node with FACS and IHC in LLNA could provide a good alternative screening method for contact allergens, without the use of radioisotope. And we could demonstrate that FACS could show a higher sensitivity than IHC in the measurement of BrdU incorporation suggesting that this method could be a good substitute for traditional LLNA using radioactive 3 H-thymidine. Conflict of interest The authors have declared no conflicts. 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