Two-Stage Transformation Assay for Cigarette Smoke Condensates using Murine c3h-10t1/2 Fibroblasts

We investigated whether the two-stage transformation assay can be applied in routine testing for promoter-like activity of cigarette smoke condensate (CSC) as an in vitro equivalent of an in vivo tumorigenicity assay (mouse skin painting). We adopted a published assay procedure (Frazelle et al., 1983a), using 3-methylcholanthrene (MCA, 0.37mumol/litre, 24hr treatment) as the initiator. Rigorously standardized experimental conditions, such as multiparameter-screened serum, one fixed subculture level, and a rigid preculturing schedule, were employed. Transformation was expressed as the fraction of dishes containing type II and type III foci. Compared to the positive control, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), transformation responses to CSC (three CSC batches that had different in vivo activity) were lower. Variations in dose-response relationships did not allow distinction between two of the three CSC batches, even with data pooled from seven assay repetitions over 2 years. In a second approach, to enhance the assay resolution, that is, the signal-to-noise ratio, promoter treatment twice per week was ineffective: the response and the background were both increased. Lowering the initiator concentration (0.08mumol/litre) enhanced the signal-to-noise ratio for TPA, but not for CSC. Even after standardization and enhancement of sensitivity, the two-stage transformation assay is useful primarily for qualitative assessment of promoter-like activity of weak promoters, such as CSC, rather than for quantitative comparisons.