Further Fractionations of Cigarette Smoke Condensate for Bioassays: Weak Acid and Neutral Subfractions and Combinations of Active

Cigarette smoke condensate (CSC) was fractionated for biological experiments examining tumorigenic activity on mouse skin. The active fractions were combined in various ways in order to determine their additive effects. Steam distillation of the ether-soluble weak acid fraction (WA_E) provided two subfractions which were submitted for assay separately and in combination. The totaI neutral fraction was separated by solvent partition between cyclohexane and 80 % aqueous methanol as in a previous study and each subfraction was further fractionated: the part insoluble in aqueous methanol (MIN) was subjected to a 200-tube countercurrent distribution between cyclohexane and nitromethane to provide five subfractions; and the part soluble in aqueous methanol (MSN) was separated into two subfractions by partition between nitromethane and carbon disulfide. Reconstituted MIN and MSN fractions were prepared by recombination of their subfractions. Finally, the total neutrals were separated into five fractions by chromatography on silicic acid as done in a previous study in order to obtain the active fraction eluted by methanol and soluble in aqueous methanol (MM_W). This was subjected to gel filtration using benzene as eluant. The five subfractions of MM_W were submitted for assay separately and in combination. In addition to these fractions, four combinations of active fractions were prepared: WA_E with MM_W; WA_E with the polynuclear aromatic hydrocarbon (PAH) containing neutral fractions; MMW with the PAH-containing neutrals, i. e. all of the active neutral fractions; and WA_E with MM_W and the PAH-containing neutrals. The last comprises essentially all of the fractions shown to possess tumorigenic activity for mouseback skin in previous work.