RE: 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 (Mutagenesis, 27, 743–747, 2012)

0 Univeristt Ulm, Institut fr Humangenetik , D-89069 Ulm, Germany *To whom correspondence should be addressed. Tel:+49-731-50065440; Email: On behalf of co-authors. In their letter to the Editor, Michael Fenech and Micheline Kirsch-Volders express their concerns about the design of a study we published in Mutagenesis (1) and challenge our conclusions. In our opinion, most of their criticisms are not justified and here we take the opportunity to detail why. - 1. Fenech and Kirsch-Volders criticise the title of our publication. However, our results actually demonstrate the insensitivity of the CBMN assay for the detection of DNA damage present at the start of the lymphocyte culture. The insensitivity of this protocol in comparison with an in vitro protocol that is recommended by the OECD guideline 487 (2) is shown for six different chemical mutagens including the cross-linking agent mitomycin C.Adifference in sensitivity can also be shown for ionising radiation (Figure1). Ionising radiation, radiomimetic chemicals and cross-linking agents do induce micronuclei (MN) when lymphocytes are exposed before the start of the culture (because they induce DNA double-strand breaks or other poorly repairable lesions), whereas most of the so-called S-phase dependent mutagens (i.e. those which induce excision-repairable lesions) do not. We discuss the insensitivity of the CBMN assay as typically used for human biomonitoring, for mutagens causing excision-repairable damage, which is the majority of genotoxic agents to which humans are exposed. It is not evident how Fenech and Kirsch-Volders can conclude that there is overwhelming evidence that this assay, as usually conducted, is highly sensitive. Our results, which are appropriately discussed, demonstrate the relative insensitivity of the biomonitoring protocol in comparison with the in vitro protocol recommended by the OECD guideline 487 for the sensitive detection of genotoxic compounds (2). However, based on the MN data we presented (1), even this sensitive protocol needs high levels of DNA damage to induce a significant increase in the frequency of micronucleated binucleated cells (BNC). 2. Fenech and Kirsch-Volders question the originality of our observations and stress the necessity of the ARA-C protocol to detect agents that predominantly induce excision-repairable lesions. However, as clearly stated in our publication, it is the first attempt to directly compare two protocols of the in vitro cytokinesis-block micronucleus assay (CBMN assay). The results presented are new and have important implications for the interpretation of human biomonitoring studies. The comment regarding the formation of MN is rather confusing and needs clarification. There should be no doubt that the CBMN assay detects MN formed as a consequence of chromatid-type aberrations and chromosome-type aberrations (i.e. acentric fragments) produced within one cell cycle after exposure. This is the basis for the detection of all types of clastogens in the in vitro CBMN assay according to OECD guideline 487 (2). If acentric chromosome fragments were necessary for the formation of MN, the majority of clastogens would not be detected. Interestingly, the schematic diagram presented by Fenech and Neville (3) shows a chromatid break as the cause of MN formation in the presence ofARA-C. It is correct that in vitro studies have shown that ARA-C enhances the formation of MN by agents inducing excisionrepairable lesions and these studies are discussed in our publication (1). However, these studies also demonstrate the insensitivity of the CBMN assay in the absence of ARAC. With one exception, human biomonitoring studies were all performed without ARA-C and positive results after in vivo exposure to agents inducing excision-repairable lesions cannot easily be explained. The only biomonitoring study that used this approach (in vitro ARA-C treatment for the first 16h of culture) failed to demonstrate any significant effect on MN frequencies in BNC using blood from individuals potentially exposed to genotoxic pollutants and/or tobacco smoke (4). If Fenech and Kirsch-Volders state that the proper protocol to detect in vitro excision-repairable DNA lesions and agents that predominantly induce them is the ARA-C protocol, does this mean that all biomonitoring studies investigating such effects without ARA-C are inappropriately performed? 3. We agree that a key question is the comparability of exposure in vitro versus in vivo. However, the plausible assumption is that the types of DNA damage induced in vitro and in vivo are the same and it has never been shown that damage levels induced in vivo are higher than those induced in vitro under controlled experimental conditions. Furthermore, the DNA repair mechanisms involved in the removal (excision) of lesions induced in vitro should be equally sensitive towards DNA damage induced in vivo and present in lymphocytes at the start of the culture. We also agree that many studies reported associations between exposure to chemical mutagens and increased MN frequencies in human biomonitoring. But these associations do not prove a causal relationship between DNA damage induced in vivo and the frequency of MN inBNC. Exposures to environmental and occupational chemicals should not be equated with exposures resulting from chemotherapy. Chemotherapy includes exposure to high doses of strong mutagens (including cross-linking agents) and systemic cytotoxic effects that frequently lead to reduced 2Gy - 0h 2Gy - 45h proliferation of cultured lymphocytes (5). Positive findings in chemotherapy patients do not explain positive findings after environmental and occupational exposure. There is no doubt that chemotherapy may induce MN in the CBMN assay and this has never been questioned. However, radiomimetic chemicals and cross-linking agents are rare among environmental mutagens. The majority of mutagens in our environment and at the workplace produce excision-repairable lesions, which are not readily detected by the standard CBMN assay in human biomonitoring. 4. We agree that the CBMN assay is used to address different questions. Its use as an indicator of exposure is still one of the main applications. Our discussion only refers to the frequent use of the CBMN assay in human biomonitoring in an attempt to detect genotoxic effects in cultured lymphocytes after occupational and environmental exposure to genotoxic chemicals. Only these studies and the plausibility and reliability of their results are the subject of our concern. The use of the CBMN assay after exposure to ionizing radiation or as an indicator of genomic instability and potential cancer risks (early effects) is not considered and should be discussed separately. In fact, Bonassi etal. (6) provided preliminary evidence that MN in peripheral blood lymphocytes is predictive of cancer risk. However, in this study, occupational exposure to mutagens or smoking status did not significantl (...truncated)