Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening

BMC Clinical Pharmacology

Clive Meredith²  Arturo Anadon¹  Graham Errington²  Carolina Garcia-Canton¹ 

[1] Department of Toxicology and Pharmacology, Universidad Complutense de Madrid, Madrid, Spain;British American Tobacco, Group Research and Development, Regents Park Road, Southampton, Hampshire SO15 8TL, UK

关键词: High content screening;    Genotoxicity;    Cigarette smoke;    In vitro;    DNA damage;    GammaH2AX;   

Others  :  1084902
DOI  :  10.1186/2050-6511-15-41

 received in 2013-10-15, accepted in 2014-07-16,  发布年份 2014

【 摘 要 】

Background

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. 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).

Methods

Whole 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.

Results

The 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.

Conclusions

Overall, 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.

【 授权许可】

   
2014 Garcia-Canton et al.; licensee BioMed Central Ltd.

【 图 表 】

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