Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke
Ewald Roemer1, Hans-Peter Lammerich, Lynda L Conroy
1Philip Morris Products S.A., R&D, Rue des Usines 90, 2000 Neuchatel, Switzerland. Ewald.Roemer@pmi.com
Toxicology Letters
|April 6, 2013
Summary
This study developed a novel, high-throughput assay to measure the inhibition of gap-junctional intercellular communication (GJIC) by cigarette smoke. The assay accurately quantifies GJIC inhibition, demonstrating its utility for complex mixtures.
Area of Science:
- Toxicology
- Cell Biology
- Assay Development
Background:
- Inhibition of gap-junctional intercellular communication (GJIC) is linked to tumor promotion.
- Cigarette smoke total particulate matter (TPM) is a known GJIC inhibitor.
Purpose of the Study:
- To characterize a new GJIC screening assay using automated fluorescence microscopy and a non-invasive parachute technique.
- To evaluate the assay's performance with cigarette smoke TPM.
Main Methods:
- Rat liver epithelial cells (WB-F344) were stained with Calcein AM.
- Cells were exposed to TPM from reference and single-tobacco cigarettes.
- Dye transfer to adjacent cells quantified GJIC, with TPA as positive control and DMSO as solvent control.
Main Results:
- A reproducible dose-response inhibition of GJIC by TPM was observed.
- The assay demonstrated high reproducibility (3.7%) and repeatability (6.9%) for 2R4F cigarette TPM.
- The assay successfully discriminated between different tobacco types and the reference cigarette.
Conclusions:
- The developed assay is precise and suitable for high-throughput screening of complex mixtures like cigarette smoke.
- This assay can reliably determine the GJIC inhibitory activity of various tobacco products.
