Beate Miller

Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay : position of the GUM working group on the in vitro micronucleus test

In order to license a pharmaceutical or chemical, a compound has to be tested for several genotoxicity endpoints, including the induction of chromosomal aberrations in vitro. A working group within the GUM has evaluated published data on the in vitro micronucleus test with the aim of judging its suitability as a replacement for the in vitro chromosomal aberration test. After strict rejection criteria were applied, a database including 96 publications and 34 compounds was obtained. For 30 of these compounds, data on both tests were available. For 24 of the 30, concordant results in both test systems were obtained (80% correlation). The discordant results in 6 compounds can be explained by a known or suspected aneugenic potential of these compounds. Considering that cell types and test protocols were extremely heterogeneous, this correlation is rather encouraging. Comparison of the different protocols, and experience established within the working group yielded several recommendations for the routine use of the in vitro micronucleus test. Although many cell lines are suitable, those most often used in genotoxicity testing (e.g. CHL, CHO, V79, human lymphocytes, L5178Y mouse lymphoma cells) are recommended. Cytochalasin B may be used in the case of human lymphocytes; however, the possibility of its interaction with aneugenic test compounds should be considered. For continuously dividing cell lines, cytochalasin B is not recommended by the working group. Although, there seems to be flexibility in the choice of treatment and sampling times, the average generation time of the chosen cell line of choice should be taken into account when determining sampling time, and treatment of cells for at least one cell cycle duration is recommended. The use of appropriate cytotoxicity tests is strongly recommended. Although studies on some parameters of the test protocol may be useful, the introduction of the in vitro micronucleus test into genotoxicity testing and guidelines should not be delayed. Even in its present state, the in vitro micronucleus is a reliable genotoxicity test. Compared with the chromosomal aberration test, it detects aneugens more reliably, it is faster and easier to perform, and it has more statistical power and the possibility of automation.

Chapter 9 Measurement of Micronuclei by Flow Cytometry

Publisher Summary This chapter describes a technique for obtaining a suspension of micronuclei and nuclei for flow cytometric measurement of the frequency of micronuclei per main nuclei in cell cultures or human lymphocytes exposed to ionizing radiation or chemicals. The two-step method includes a treatment of the cells with salt solution I, which contains a detergent, followed by an additional treatment with solution II containing citric acid and sucrose. Both solutions contain ethidium bromide (EB) as a DNA-specific fluorescent dye; in some cases Hoechst 33258 (HO) is used additionally. By this two-step treatment the cellular membrane and the cytoplasm are destroyed and nuclei and micronuclei are released in suspension. No mechanical treatment or centrifugation steps are necessary to obtain a solution of main nuclei and micronuclei, because mechanical treatment would induce cellular or nuclear debris that could overlap the small micronuclei during flow cytometric measurement. The nuclear membrane is maintained and mitotic cells that could release chromosomes in the suspension are usually not destroyed. With this technique it is additionally possible to measure the DNA content of micronuclei and main nuclei for a simultaneous analysis of the cell-cycle distribution (main nuclei) and the DNA distribution of micronuclei. Analysis of the DNA distribution of micronuclei can give additional information on the mechanisms of action of the inducing agent.