Cigarette‐smoke‐induced cellular transformation in vitro using the Bhas 42 cell transformation assay

In vitro cell transformation assays detect transformed cells that have acquired the characteristics of malignant cells and thus mimic one stage of the in vivo multi-step carcinogenesis model. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. A short-term cell transformation assay using v-Ha-ras transfected Balb/c 3T3 cells (Bhas 42 cells), the Bhas assay, which has not been used with cigarette smoke before, is capable of detecting, initiating, and promoting activities of chemical carcinogens. As the particulate phase of cigarette smoke (total particulate matter, or TPM) is known to induce tumors in vivo in the mouse skin painting assay, we investigated the responsiveness of the Bhas assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard reference cigarette (3R4F). DMSO-dissolved TPM induced a dose-dependent increase of type III foci in the Bhas cell transformation assay. A significant increase in focus formation was observed in the promotion assay at sub-toxic doses between 5 and 60 microg TPM/ml (~16-fold). This novel in vitro assay using Bhas cells, which are regarded as initiated in the two-stage paradigm of carcinogenesis, is able to detect cell transformation induced by cigarette smoke condensate in a dose-dependent manner with a high dynamic range compared to the solvent control.