Wolf T, Luepke N P
Universität Osnabrück, Pharmakologie und Toxikologie, Germany.
Mutat Res. 1997 Nov 27;394(1-3):163-75. doi: 10.1016/s1383-5718(97)00136-8.
The formation of micronuclei (MN) is a widely used and accepted endpoint of genotoxicity testing. The micronucleus assay provides a simple and rapid indirect measure of the induction of structural or numerical chromosome aberrations. In this work we describe hen's eggs, incubated for 11 days, as ex vivo assay system for the detection of micronucleus formation in young erythrocytes (Hen's Egg Test for Micronucleus Induction, HET-MN). At this stage of development the chick embryo presents a high metabolic competency which allows an adequate activation of several types of promutagens, as previously reported by several authors. As all stages of maturing erythrocytes are present in the bloodstream of the chick embryo, we could conveniently use samples of peripheral blood for scoring micronuclei as well as for determining the ratio between mature and immature erythrocytes as a measure of an undisturbed erythropoiesis. The obtained blood smears were stained by a modified May-Gruenwald-Giemsa procedure and scored microscopically. The examinations were facilitated by using a semiautomatic image analysis system. We could demonstrate a strong increase of the micronucleus frequency after the administration of the promutagens diethylnitrosamine (DENA), 7,12-dimethyl-benz[a]anthracene (DMBA), cyclophosphamide (CP), ifosphamide (IF), mitomycin C (MMC), and the direct-acting mutagen methanesulfonic acid methyl ester (MMS) compared to the concomitant negative controls. CP was used to demonstrate a dose-response relation and the effect of using two different routes of application (air cell and albumen). Nuclear aberrations, other than MN, were demonstrated after application of high doses of CP or IF. Expanded exposure times revealed a similar effect. The HET-MN, as an ex vivo assay, is a simple, inexpensive, and rapid assay system for genotoxicity testing, positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects.
微核（MN）的形成是遗传毒性测试中一种广泛使用且被认可的终点指标。微核试验提供了一种简单快速的间接方法来检测结构或数量染色体畸变的诱导情况。在本研究中，我们描述了将孵化11天的鸡蛋作为体外检测系统，用于检测幼红细胞中的微核形成（鸡蛋微核诱导试验，HET - MN）。在这个发育阶段，鸡胚具有较高的代谢能力，这使得多种类型的前诱变剂能够充分活化，正如几位作者先前报道的那样。由于鸡胚血液中存在成熟红细胞的各个阶段，我们可以方便地使用外周血样本对微核进行计数，并确定成熟与未成熟红细胞的比例，以此作为红细胞生成未受干扰的指标。获得的血涂片采用改良的美蓝 - 吉姆萨染色法染色，并在显微镜下进行计数。使用半自动图像分析系统有助于检查。与同期阴性对照相比，我们可以证明给予前诱变剂二乙基亚硝胺（DENA）、7,12 - 二甲基苯并[a]蒽（DMBA）、环磷酰胺（CP）、异环磷酰胺（IF）、丝裂霉素C（MMC）以及直接作用诱变剂甲基磺酸甲酯（MMS）后，微核频率显著增加。使用CP来证明剂量 - 反应关系以及两种不同给药途径（气室和蛋白）的效果。在给予高剂量的CP或IF后，除了微核外还出现了核畸变。延长暴露时间显示出类似的效果。HET - MN作为一种体外试验，是一种用于遗传毒性测试的简单、廉价且快速的检测系统，介于纯体外和体内试验之间，严格符合动物保护法规和伦理要求。
