Carolina Garcia-Canton

Metabolic characterization of cell systems used in in vitro toxicology testing: lung cell system BEAS-2B as a working example.

The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.

γH2AX as a novel endpoint to detect DNA damage: Applications for the assessment of the in vitro genotoxicity of cigarette smoke

Histone H2AX is rapidly phosphorylated to become γH2AX after exposure to DNA-damaging agents that cause double-strand DNA breaks (DSBs). γH2AX can be detected and quantified by numerous methods, giving a direct correlation with the number of DSBs. This relationship has made γH2AX an increasingly utilised endpoint in multiple scientific fields since its discovery in 1998. Applications include its use in pre-clinical drug assessment, as a biomarker of DNA damage and in in vitro mechanistic studies. Here, we review current in vitro regulatory and non-regulatory genotoxicity assays proposing the γH2AX assay as a potential complement to the current test battery. Additionally, we evaluate the use of the γH2AX assay to measure DSBs in vitro in tobacco product testing.

Assessment of the in vitro γH2AX assay by High Content Screening as a novel genotoxicity test.

The γH2AX assay is widely used as a marker of DNA damage in multiple scientific fields such as cancer biomarker, clinical studies and radiation biology. In particular, the in vitro γH2AX assay has been suggested as a novel in vitro genotoxicity test with potential as a pre-screening tool. However, to date, limited assessments have been carried out to evaluate the sensitivity, specificity and accuracy of the in vitro γH2AX assay. In this study, the microscopy-based system combining automated cellular image acquisition with software quantification for High Content Screening (HCS) has been used for the first time to evaluate the in vitro γH2AX assay. A panel of well-characterised genotoxic and non-genotoxic compounds was selected to assess the performance of the in vitro γH2AX assay in the human bronchial epithelial cell line BEAS-2B. The results obtained during this preliminary assessment indicate that the in vitro γH2AX assay has a high accuracy (86%) as a result of high sensitivity and specificity (86-92% and 80-88% respectively). Our data highlight the potential for γH2AX detection in HCS as a complement to the current regulatory genotoxicity battery of in vitro assays. We therefore recommend more comprehensive assessments to confirm the performance of the in vitro γH2AX assay by HCS with a more extensive set of compounds.

Optimisation of the in vitro gamma-H2AX assay to detect pro-toxicants

Figure 2: γH2AX frequency (solid lines) and RCC (dashed lines) after 3 hr and after 3 hr followed by a 24 hr recovery period exposures to [A] BaP with S9 mix; [B] BaP without S9 mix; [C] AB1 with S9 mix; [D] AB1 without S9 mix; [E] 2AAF with S9 mix; [F] 2AAF without S9 mix. RCC error bars (n=3) are not included in the graphs for clarity. * Indicates minimum concentration showing a significant increase in γH2AX frequency compared to the vehicle-treated control.

Assessment of the in vitro gamma-H2AX assay by High Content Screening (HCS) as a novel genotoxicity test: application for the evaluation of single toxicants and mixtures

Figure 2: γH2AX frequency (solid lines) and RCC (dashed lines) after 3 and 24 hr exposure to [A] direct acting agent ethyl methanesulfonate; [B] ROS generator bleomycin sulfate; [C] topoisomerase inhibitor etoposide. RCC error bars (n=3) are not included in the graphs for clarity. * Indicates minimum concentration showing a significant increase in γH2AX frequency compared to the vehicle-treated control.