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

Ülker, Özge Cemiloğlu; Ateş, İlker; Atak, Aysegul; Karakaya, Asuman

doi:10.3109/1547691x.2012.684157

Cited by 13 publications

(12 citation statements)

References 23 publications

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“…The LLNA:BrdU-ELISA could be further improved by eliminating the need for in vivo injections. Until now, attempts to develop an assay in which harvested lymph node cells are labeled ex vivo with BrdU have been limited by the small number of chemicals and concentrations tested and, in some cases, the failure to test appropriate control chemicals (Piccotti et al, 2006;Suda et al, 2002;Ulker et al, 2011Ulker et al, , 2013. Here, we addressed these limitations by evaluating an ex vivo BrdU labeling procedure for the LLNA using a panel of known sensitizers with varying potencies and control chemicals.…”

Section: Discussionmentioning

confidence: 99%

“…In 1999, the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) recommended the development and utilization of alternative LLNA endpoints (ICCVAM, 1999). One area selected for improvement involves the development of an LLNA protocol that does not require the use of radioisotope (Idehara et al, 2008;Jung et al, 2010;Kojima et al, 2011;Piccotti et al, 2006;Suda et al, 2002;Takeyoshi et al, 2001Takeyoshi et al, , 2003Takeyoshi et al, , 2005Ulker et al, 2013). A promising approach is to replace 3HTdR with its non-radioactive analog, 5-bromo-2′-deoxyuridine (BrdU).…”

Section: Introductionmentioning

confidence: 99%

“…injections may have altered cytokine production in mice (Ulker et al, 2011). Consequently, some efforts have been devoted to developing an ex vivo BrdU labeling protocol that would eliminate the need for in vivo injections (Piccotti et al, 2006;Suda et al, 2002;Ulker et al, 2011Ulker et al, , 2013. While these new methods show promise, additional development is necessary to confirm the predictive potential of an ex vivo BrdU labeling procedure.…”

Section: Introductionmentioning

confidence: 99%

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Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

Williams

Copeland

Boykin

et al. 2014

J of Applied Toxicology

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The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [(3) H]methyl thymidine. A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non-sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU-enzyme-linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis-diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

Williams

Copeland

Boykin

et al. 2014

J of Applied Toxicology

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“…The results of the in vivo and ex vivo assays were compatible both with one another and with standard radioactive LLNA results for dinitrochlorobenzene. Therefore, we concluded that the ex vivo BrdU incorporation method could be used as a viable alternative approach to assess the skin sensitization potential of the test compound in terms of improving animal welfare [9,11,12]. …”

Section: Introductionmentioning

confidence: 99%

“…The use of this method is difficult in any laboratory because this radioisotope-based method (RI) requires special facilities and handling procedures. Several authors have conducted investigations for the development of an alternative non-RI method for performing LLNA [5,6,7,8,9,10,11,12]. …”

Section: Introductionmentioning

confidence: 99%

Investigation of Allergenicity of Some Cosmetic Mixtures by Using ex vivo Local Lymph Node Assay-BrdU Endpoints

Ülker

Kaymak²,

Karakaya³

2014

Int Arch Allergy Immunol

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Background: Balsam of Peru and fragrance mix are commonly used in cosmetic products. Allergy to fragrance is the most common cause of cosmetic contact dermatitis. Methods: In the present study, ex vivo local lymph node assay-5-bromo-2′-deoxyuridine (LLNA-BrdU) was used to evaluate the dermal sensitization potential of these cosmetic mixtures. The stimulation index values and estimated concentration (EC3) values were calculated and the potency classification was found for each mixture. At the same time, in order to measure the irritant effect without having to use additional animals, a combination of ex vivo LLNA-BrdU and the irritancy assay was conducted. Results: Th1 [interleukin (IL)-2, interferon-γ] and Th2 cytokine (IL-4, IL-5) releases from lymph node cell culture were investigated as non-radioactive endpoints. According to the results of ex vivo LLNA-BrdU assays, EC3 values were found to be 3.09% (moderate) for balsam of Peru and 4.44% (moderate) for fragrance mix. Cytokine analysis results indicate that both Th1 and Th2 cytokines are involved in the regulation of murine contact allergy and can be considered as useful endpoints. Conclusion: In conclusion, according to our results, fragrance mix and balsam of Peru can be considered as moderate sensitizers; however, in high concentrations, both of them have irritation properties. The cytokines investigated can be considered as the endpoints of the ex vivo LLNA-BrdU assay.

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Use of the LLNA:BrdU-ELISA for Skin Sensitization Hazard Assessment

Lehmann

2018

Methods in Molecular Biology

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Allergic contact dermatitis (ACD) continues to be an occupational and environmental health issue. Consequently, there is a need to employ predictive tests to reduce the incidence of skin sensitization leading to clinical manifestations of ACD. For more than a decade, the murine local lymph node assay (LLNA) has been the method of choice for the identification of skin sensitizers. While the original LLNA protocol has been extensively evaluated and subjected to exhaustive validation, the use of radioisotope (i.e., tritiated thymidine; 3HTdR) has discouraged utilization of this powerful assay in some countries. To promote further utilization of this method, the original LLNA protocol was refined to use 5-bromo-2'-deoxyuridine, a nonradioactive analog of 3HTdR. The LLNA:BrdU-ELISA has been reviewed, validated, and approved for use internationally, and its performance is regarded as equivalent to the traditional LLNA. Here, we provide guidance on how to perform and interpret data from this assay.

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Evaluation of non-radioactive endpoints ofex vivolocal lymph node assay-BrdU to investigate select contact sensitizers

Cited by 13 publications

References 23 publications

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

Investigation of Allergenicity of Some Cosmetic Mixtures by Using ex vivo Local Lymph Node Assay-BrdU Endpoints

Use of the LLNA:BrdU-ELISA for Skin Sensitization Hazard Assessment

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