Abstract
BackgroundThe genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI).MethodsWhole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out.ResultsThe results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected.ConclusionsOverall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks.
Highlights
The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures
The aim of this study was to optimise the novel in vitro γH2AX assay by High Content Screening (HCS) that we had previously developed [14], in order to adapt it for the assessment of aerosols and to evaluate the genotoxic effect of two reference cigarettes in human lungderived BEAS-2B cells at the air-liquid interface (ALI)
The results show that the in vitro γH2AX by HCS can be used as a high throughput tool to assess the genotoxic effect of Whole mainstream cigarette smoke (WMCS) in cultures exposed at the ALI
Summary
The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. Johnson and colleagues published a thorough review on the in vitro systems used to evaluate the toxicity of cigarette smoke [4]. The authors highlighted that the majority of tobacco-related in vitro toxicology studies are carried out in non-human cell models which are poorly validated for tobacco product comparison. They concluded that better methods are needed, especially in relation to regulation and health claims. In the field of in vitro genotoxicity, the authors described that the evaluation of cigarette smoke has been carried out using mainly cigarette smoke condensate (CSC). The in vitro genotoxicity data has been mainly obtained from animal-derived cell systems which are functionally very different from human-derived cells
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