In vitro primary human lymphocyte flow cytometry based micronucleus assay: simultaneous assessment of cell proliferation, apoptosis and MN frequency

In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug development.