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

Abstract

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.

Figure 1

Schematic representation of a single RM20S® syringe combined with British American Tobacco’s exposure chamber (UK patent publication WO 03/100417/ A1) (not to scale). The RM20S® can smoke up to eight cigarettes simultaneously. [A] Cigarette holder with cigarette in place; [B] 150 mL glass syringe where cigarette smoke dilution in air is prepared; [C] Plunger; [D] Exposure chamber containing porous membrane inserts with cells seeded on top at the ALI [E] Transwell® insert representation. Figure adapted from [26].

Figure 2

Test dilutions t-test boxplots. Expected PPM (red dot), 95% confidence interval from PPM results (blue line). The asterisk (*) indicates outliers while hash (#) indicates dilutions producing a significant different PPM than expected.

Figure 3

Scatterplot of repeatability (r) (white circle) and reproducibility (R) (red square).

Figure 4

γH2AX frequency mean ± SD after 3 h exposure to WMCS from reference cigarette. [A] 3R4F, [B] M4A. Circle (-●-) represents WMCS results, square (-■-) represents positive control etoposide (1 mM final), triangles (-▼- and -▲-) represents negative controls, air and incubator controls respectively and dotted red line represents the 1.5-fold increase over the air control indicating the threshold of genotoxic response. [C] γH2AX fold-induction for both reference cigarettes 3R4F (blue) and M4A (red), dotted line indicates genotoxic level (>1.5-fold γH2AX response).