Koeper L-M, Schulz A, Ahr H J, Vohr H-W
Bayer HealthCare AG, Toxicology, Aprather Weg, D-42096, Wuppertal, Germany.
Toxicology. 2007 Dec 5;242(1-3):144-52. doi: 10.1016/j.tox.2007.09.019.
Animal testing causes ethical problems and in view of EU regulations (e.g. EU-Guideline (76/768/EEC, February 2003)) or REACH the development of reliable in vitro assays has become even more important. Up to now, we use the modified local lymph node assay (IMDS) for toxicological hazard identification of sensitizing and irritant properties of chemicals in accordance with OECD Guideline 429. In this study, we investigated whether analyses of cell signaling pathways can provide a methodology for the detection of sensitizing compounds in vitro. Murine and human skin explants as well as reconstituted skin models (epidermal model EST-1000 and full-thickness model AST-2000) were exposed to sensitizing (oxazolone and DNFB) or irritant compounds (SDS and TritonX-100). Phosphorylation of MAP-kinases (p38, ERK1/2 and JNK1/2), STAT1 and PLCgamma were determined by cytometric bead array (CBA). In skin explants, all three MAP-kinases were exclusively activated after exposure to sensitizing compounds. For the reconstituted skin models phosphorylations of p38 and JNK1/2 were obtained after stimulation with allergens, whereas treatments with irritant compounds led to ERK1/2 activation. Activation of PLCgamma and STAT1 were never detected. In conclusion, MAP-kinase activation provides a promising in vitro tool for the discrimination between sensitizers and irritants.
动物实验引发了伦理问题，鉴于欧盟法规（如欧盟指南（76/768/EEC，2003年2月））或《化学品注册、评估、授权和限制法规》（REACH），开发可靠的体外检测方法变得更加重要。到目前为止，我们根据经合组织指南429，使用改良的局部淋巴结检测法（IMDS）对化学品的致敏和刺激特性进行毒理学危害鉴定。在本研究中，我们调查了细胞信号通路分析是否能提供一种体外检测致敏化合物的方法。将小鼠和人类皮肤外植体以及重组皮肤模型（表皮模型EST - 1000和全层模型AST - 2000）暴露于致敏剂（恶唑酮和二硝基氟苯）或刺激化合物（十二烷基硫酸钠和曲拉通X - 100）。通过细胞计数珠阵列（CBA）测定丝裂原活化蛋白激酶（p38、ERK1/2和JNK1/2）、信号转导和转录激活因子1（STAT1）以及磷脂酶Cγ（PLCγ）的磷酸化情况。在皮肤外植体中，暴露于致敏化合物后，所有三种丝裂原活化蛋白激酶均被特异性激活。对于重组皮肤模型，用变应原刺激后可检测到p38和JNK1/2的磷酸化，而用刺激化合物处理则导致ERK1/2活化。从未检测到PLCγ和STAT1的活化。总之，丝裂原活化蛋白激酶的活化提供了一种有前景的体外工具，可用于区分致敏剂和刺激剂。
