Moreover, these exposure agents are not fully representative of human exposure as cells are not fully exposed to both the particulate and vapour phase components of the cigarette smoke. A number of whole smoke exposure systems are being developed to address these problems, but have only recently entered a phase where dosimetric comparisons can be made and have not yet been validated. Whole smoke exerts significant cytotoxicity and therefore precise exposure conditions need to be defined in order to detect specific genotoxic effects. Of course the real key to definition of appropriate smoke exposure systems for toxicity testing is to understand the contribution buy RAD001 of individual tobacco

smoke constituents to the genotoxic effects (both singly and in combination) and to estimate their concentration in tobacco smoke particulate and vapour phase fractions. This understanding then facilitates the design of appropriate tobacco smoke exposure systems, focusing on key drivers of genotoxicity, facilitating product comparisons and providing a scientific rationale for any observed differences in genotoxic potential. To date, there are a limited number of studies using whole mainstream cigarette smoke (WMCS) in in vitro genotoxicity assays. WMCS was first used as a smoke exposure system in the in vitro micronucleus assay ( Massey et al., 1998 and Okuwa

et al., 2010). In addition, Aufderheide et al. developed a WMCS method to evaluate the mutagenicity of cigarette smoke in various bacterial strains in the Ames test ( Aufderheide

and Gressmann, 2007 and Aufderheide and Histone demethylase Gressmann, Staurosporine concentration 2008). To date, there is no published information of this exposure system in the MLA assay. In the field of non-regulatory assays, WMCS was used by Thorne et al. to measure oxidative DNA damage in the in vitro comet assay ( Thorne et al., 2009). Studies to measure the activation of H2AX in response to DNA damage in vitro after cigarette smoke exposure have also used CSC, TPM or cigarette smoke extract (CSE) as a smoke exposure system ( Albino et al., 2004, Albino et al., 2006, Albino et al., 2009, Tanaka et al., 2007a, Tanaka et al., 2007b, Luo et al., 2004, Zhao et al., 2009, Jorgensen et al., 2010 and Darzynkiewicz et al., 2011). The in vitro γH2AX assay was originally used to measure DSBs following cigarette smoke exposure ( Albino et al., 2004). Human A549 pulmonary adenocarcinoma cells were exposed to cigarette smoke and normal human bronchial epithelial (NHBE) cells to CSC. Both cell systems showed a dose-related response in γH2AX activation. Once the relationship between smoke exposure and γH2AX activation was confirmed, Albino et al. used the assay to evaluate cigarettes with different tar deliveries. The results indicated that the increment in γH2AX intensity was proportional to the estimated tar delivery rather than the cigarette type or smoking behaviour ( Albino et al., 2009). Interestingly, when Kato et al.