Formation of micronuclei in incubated hen's eggs as a measure of genotoxicity

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.