Selective fluorescence quenching of benzo[a]pyrene and a mutagenic diol expoxide derivative in mouse cells.

Abstract

The overlap of the fluorescence spectra of benzo(a)pyrene and its metabolites, including an ultimate carcinogen diol epoxide (+-)-7..beta..,8..cap alpha..-dihydroxy-9..cap alpha.., 10..cap alpha..-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, impedes the use of fluorescence in studying the impact of these important carcinogens on cell populations. It is shown that in cells certain collisional quenchers can be used to selectively quench the benzo(a)pyrene fluorescence, leaving the diol epoxide emission. Because such quenchers work only with close contact over distances much less than the cell dimensions, the preferential solubilities can be used to probe fluorophore locations. Benzo(a)pyrene and its diol epoxide derivative were added directly to mouse cells in culture: NMuLi(mouse liver epithelial cells), Balb 3T3 A31 HYF (mouse fibroblast cells), and MSV/MLV Balb 3T3 A31 HYF (Moloney sarcoma virus transformed mouse fibroblast cells). Fluorescence lifetimes of the aromatic hydrocarbons were measured, and fluorescence and flow cytometric studies were made on the effects of various quenchers added to suspensions of these cells. The collisional quencher n-octyl iodide, which is lipid soluble, quenches the benzo(a)pyrene emission by a factor of six with little change in the diol epoxide emission. At comparable molarity, the ionic quencher, KI, is relatively ineffective and does not offer selective effects, presumably because the I/sup -/more » does not penetrate the cell. At comparable molarity, methylene iodide is a relatively effective quencher for both aromatic hydrocarbons, but it offers little selectivity. The results were, in general, independent of the particular type of mouse cell strain.« less