Biochemical and Morphological Effects of Cigarette Smoke Condensate and Its Fractions on Normal Human Bronchial Epithelial Cells in Vitro

We investigated the effect of cigarette smoke condensate (CSC), two basic fractions (BIa and BIb) of CSC, the ethanol-extracted weakly acidic fraction (WAe), and the methanol-extracted neutral fraction (Nmeoh) on the clonal growth rate, plasminogen activator (PA) activity, cross-linked envelope (CLE) formation, and ornithine decarboxylase activity, epidermal growth factor (EGF) binding, thiol levels, and DNA single strand breaks in cultured human bronchial cells. Neither CSC nor any of the fractions were mitogenic over the range 0.01-100 micrograms/ml. All were growth inhibitory at higher concentrations. The 40% growth inhibitory concentrations for CSC, BIa, BIb, WAe, and Nmeoh were 10, 10, 10, 3, and 1 micrograms/ml, respectively. Effects on CLE formation, morphology, PA, and ornithine decarboxylase activities, EGF binding, and thiol levels were evaluated using 40% growth inhibitory concentrations. We found that CSC and all fractions caused an increased formation of CLEs, from a baseline of 0.5% in the untreated cells to a maximum increase of 25% induced by Nmeoh. A squamous morphological change was observed within 1 h after exposure to Nmeoh, WAe, and CSC. The BIa and BIb fractions had little effect. Only Nmeoh increased PA significantly, from 2.5 +/- 0.4 to 5.1 +/- 0.3 units/mg cellular protein. CSC and the WAe and Nmeoh (Nmeoh greater than WAe greater than CSC) fractions caused a decrease in EGF binding, in each case reaching a maximum effect after a 10-12-h incubation. This effect on EGF binding was further characterized in the case of Nmeoh. In untreated normal human bronchial epithelial cells, by Scatchard analysis the kd was 2.0 nM and there were 1.2 X 10(5) receptors/cell. In cells incubated in medium containing Nmeoh (3 micrograms/ml) the kd was 3.2 nM and there were 1.1 X 10(5) receptors/cell. Thus, inhibition of EGF binding by Nmeoh was due primarily to a decrease in the affinity. At the 40% growth inhibitory concentrations neither CSC nor any of the fractions significantly affected intracellular thiol levels. While a 3-h incubation in medium containing CSC caused significant DNA single strand breaks only at a concentration of 100 micrograms/ml, Nmeoh caused a marked effect at 5 micrograms/ml. Neither CSC nor any of the fractions had an effect on ornithine decarboxylase activity. Due to the effects of the Nmeoh fraction on growth, morphology, EGF binding, PA activity, and formation of single strand breaks we consider it to be the most likely portion of CSC to contain compounds with actions similar to those of the phorbol ester, indole alkaloid, and polyacetate tumor promoters.