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

Abstract

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, 0.37 μmol/litre, 24 hr 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 (0.08 μmol/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.