The pyrene-like fluorescence of adducts derived from the covalent binding of (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to ̃ 100 K. This effect is primarily associated with the strong quenching of thepyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electrontransfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (±)- anti-BPDE-poly(dA-dT). (dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (±)-reverse-BPDE [(±)-trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene]. Adduct levels as low as 10 pmol in a 400 μl sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (̃ 1 BPDE residue/20 000 bases), such an analysis requires only 100 μg of DNA. Conformationally different adducts derived from the binding of the isomeric (±)-anti-BPDE, (±)-reverse-BPDE or (±)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasiintercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.
ASJC Scopus subject areas
- Cancer Research