Insensitivity of the in vitro cytokinesis-block micronucleus assay with human lymphocytes for the detection of DNA damage present at the start of the cell culture

Gnter Speit Regina Linsenmeyer Petra Schtz Stefanie Kuehner The cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes is a well-established assay in genotoxicity testing and human biomonitoring. For both approaches, human lymphocytes are stimulated by phytohaemagglutinin (PHA) and cultured for about 72 h; 44 h after PHA stimulation, cytochalasin B (CytB) is added and micronuclei (MN) are scored in binucleated cells. The main difference between these two applications is the way lymphocytes are exposed to mutagens. In order to maximise the probability of detecting a mutagen, the OECD guideline 487 recommends starting the exposure to the test substance at 44-48 h after PHA stimulation. In human biomonitoring, blood samples are taken from subjects exposed to environmental mutagens in vivo and lymphocytes with induced DNA damage at the start of the cell culture are investigated with regard to potentially increased MN frequencies in binuclear lymphocytes. We compared the sensitivity of these two protocols by either treating lymphocyte cultures for 2 h with known DNA-damaging mutagens at the start of the culture or 42 h after PHA stimulation. The mutagens used were methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-ethylurea (ethyl nitrosourea; ENU), styrene oxide (SO), ()-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and mitomycin C (MMC). All substances induced MN under the conditions of the standard in vitro CBMN assay but only MMC clearly induced MN in lymphocytes exposed at the start of the culture. All mutagens (except MMC, a known crosslinker) were tested by the comet assay with blood cultures exposed at the start of the culture and clearly induced DNA migration. The nuclear division index (NDI) indicated that damaged lymphocytes proliferated well in these experiments. The lack of increased MN frequencies despite increased damage levels and good proliferation suggests that the CBMN assay is rather insensitive for the detection of mutagens/clastogens when damage is induced at the start of the blood cultures. Potential consequences for the interpretation of human biomonitoring studies are discussed in this article. Introduction The cytokinesis-block micronucleus assay (CBMN assay) is a well-established assay in genotoxicity testing and a frequently used method in human biomonitoring. An OECD guideline (487) is available for an appropriate performance of the in vitro micronucleus test in genotoxicity testing (1) and recommendations have been published for its use in human biomonitoring (2,3). When using human lymphocytes, the use of cytochalasin B (CytB) and the evaluation of binuclear cells are recommended to limit analysis to cells that have passed through one mitosis after exposure. It should be noted that the OECD guideline for the in vitro CBMN assay recommends exposing lymphocytes 4448 h after phytohaemagglutinin (PHA) stimulation and not at the start of the cell culture. The reason for suggesting this treatment protocol is to achieve maximum sensitivity. Cycle synchronisation will have disappeared, proliferating cells in all phases of the cell cycle are exposed and the possibility to repair induced damage before micronuclei (MN) are produced is limited. The CBMN assay is also frequently used for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. During the past years, the CBMN assay has become the most important cytogenetic assay in human biomonitoring. MN frequencies are usually measured in binuclear lymphocytes according to the standard protocol (i.e. addition of CytB after 44 h, preparation after 72 h). The in vivo or in vitro origin of MN measured under these conditions is not completely clear (4). Theoretically, MN measured in lymphocytes in the CBMN assay can already be formed in vivo during cell division in the bone marrow, in the spleen or in lymph nodes and persist until the blood sample is taken and analysed in the CBMN assay. However, because the analysis of MN in the CBMN assay is limited to binuclear cells, that is cells that have gone through a cell division in culture, it can be expected that the majority of MN seen in binuclear cells was formed during in vitro cell division (5). This assumption was confirmed by a study with blood samples from patients after cytostatic therapy. Clearly increased MN frequencies were measured in binuclear lymphocytes in the group of patients and the analysis of MN in mononuclear lymphocytes before cell division clearly indicated that MN mainly arose ex vivo during the cultivation of lymphocytes (6). The formation of MN in vitro as a consequence of mutagen exposure of human subjects would require that lymphocytes are exposed in vivo, that mutagen-induced DNA damage persists in peripheral lymphocytes and that blood samples with increased levels of DNA damage in lymphocytes are collected. DNA damage relevant for MN formation then has to persist during cell culture and MN are produced during cell division in the presence of CytB. It has already been realised in previous publications that MN in lymphocytes are a relatively insensitive indication of in vivo exposures to genotoxic chemicals because damage is effectively repaired prior to S-phase (7-9). In the context of our research into the mutagenic activity of formaldehyde (FA), we also demonstrated that FA does not induce MN in the CBMN assay when applied at the beginning of the lymphocyte culture but only if exposure is delayed and potential repair of induced damage is prevented (10). Based on these published data and the experience with the in vitro CBMN assay, we now characterised the potential of known mutagens to induce MN in the CBMN assay after exposure at the beginning of the culture or 42 h later (before the addition of CytB). We studied the effects of methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-ethylurea (ethyl nitrosourea; ENU), styrene oxide (SO), ()-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and mitomycin C (MMC) on MN frequencies and proliferation of lymphocyte cultures. We also performed the comet assay to demonstrate the DNA-damaging effect of these mutagens in blood cultures exposed at the start of the culture. Our results confirm that lymphocytes with enhanced DNA damage levels at the beginning of the cell culture rarely show increased MN frequencies in binuclear lymphocytes in the CBMN assay. Our findings may have important implications for the interpretation of CBMN assay results from biomonitoring studies of human populations exposed to DNA-damaging chemicals in vivo. Materials and methods Chemicals, blood samples and mutagen exposure MMS, EMS, ENU, SO and MMC were purchased from Sigma (Munich, Germany). BPDE was supplied by BIU (Grosshansdorf, Germany). MMS, EMS, ENU and MMC were dissolved in water, SO and BPDE were dissolved in DMSO. Aliquots of BPDE were frozen at 80C and thawed immediately before use. Samples were only thaw (...truncated)