Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes

Human biomonitoring of early genetic effects requires accurate, sensitive and, if possible, easy and not too time-consuming methodologies to assess mutations. One of the most promising methodologies at the present time is the cytokinesis block micronucleus (MN) assay (CBMN), which detects both chromosome breakage and chromosome loss in once-divided binucleated (BN) cells. Many studies have been published with this methodology, but before its extensive application is recommended, it is necessary to evaluate its strengths and limitations. Recently, Fenech et al. reviewed the advantages of the CBMN assay for biomonitoring purposes. However, up to now information present in mononucleated (MONO) cells has rarely been taken into account, although it might be complementary to that assessed in BN cells. Indeed, MONO cells should indicate damage which was present in vivo before the start of culture and BN cells may contain pre-existing micronuclei (MNi) plus lesions which are expressed as MNi during in vitro culture. To address this question, the objectives of this paper were as follows. (i) To situate the CBMN assay in a historical and mechanistic perspective. (ii) To consider whether impaired mitotic capacity in vitro may be responsible for false negative biomonitoring studies if MN in MONO cells are not taken into account in the CBMN test. The following factors were considered: division delay for repair and mitotic block, in vitro apoptosis and necrosis of damaged cells, mitotic slippage and correlation between MN expression in vitro versus in vivo. (iii) To analyse the factors which may cause a negative result in the CBMN assay in biomonitoring when exposure to specific genotoxins is evident. The specific effects of aneugens and of adaptive responses to chronic low level exposure were examined. (iv) To compare the sensitivity of MONO and BN cells in relation to the genotoxic mechanism. (v) To propose an adequate sampling scheme to study MN in both MONO and BN cells, It was concluded that a more comprehensive assessment of DNA damage may be achieved if the CBMN assay includes measures of: (i) MNi in MONO cells; (ii) MNi in BN cells; (iii) apoptotic cells; (iv) necrotic cells, It is probable that the 24 h post-phytohaemagglutinin time point may be the optimal time to assess the frequency of MNi in MONO cells, apoptotic cells and necrotic cells. It is also practical to include these measures when scoring MNi in BN cells after cytokinesis block.