title: Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke.
abstract: Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.
authors.affiliation: Philip Morris Products S.A., R&D, Rue des Usines 90, 2000 Neuchatel, Switzerland. Ewald.Roemer@pmi.com
authors.forename: Ewald
authors.initials: E
authors.lastname: Roemer
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Lammerich
authors.forename: Lynda L
authors.initials: LL
authors.lastname: Conroy
authors.forename: Dirk
authors.initials: D
authors.lastname: Weisensee
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D017629:Gap Junctions / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000187:drug effects; D008856:Microscopy, Fluorescence; D052638:Particulate Matter / Q000009:adverse effects; D010703:Phorbol Esters / Q000009:adverse effects; D051381:Rats; D012906:Smoke / Q000009:adverse effects*; D012907:Smoking / Q000009:adverse effects
chemical_list: D052638:Particulate Matter; D010703:Phorbol Esters; D012906:Smoke; C119222:phorbol-12-myristate