Abstract
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 approximately 100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer 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 microliters 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 (approximately 1 BPDE residue/20,000 bases), such an analysis requires only 100 micrograms 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 quasi-intercalated 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.
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