Identification of PAH-DNA depurinating adducts by means of fluorescence line narrowing spectroscopy

PAH carcinogens like benzo[a]pyrene, 7,12 dimethyl benz[a]anthracene, and most recently the extremely potent dibenzo[a,l]pyrene have been found to react at the N3 or N7 positions of adenine or at the N7 or C8 positions of guanine. These reactions, which may proceed via a radical cation mechanism or via the diolepoxide pathway, lead to loss of the base (depurination) and leave a nick or apurinic site in the DNA helix. In order to detect this type of DNA damage, depurinating adducts can be extracted from culture media or tissues and separated by means of multidimensional HPLC. For unambiguous identification the relevant HPLC fractions are collected and their fingerprint spectra recorded by means of fluorescence line narrowing spectroscopy (FLNS). The latter technique involves cooling the sample to 4 K, laser excitation into the S₁ electronic state, and time-resolved detection of the vibrationally resolved fluorescence spectrum. Following this approach depurinating adducts from the above mentioned carcinogens were determined in various in vitro systems, as well as in rat mammary gland tissue and mouse skin. Application of FLNS to the analysis of stable adducts (in intact DNA or following DNA digestion) is also discussed.