FLUORESCENCE SPECTROSCOPY OF BENZO[a]PYRENE DIOL EPOXIDE‐DNA ADDUCTS. CONFORMATION‐SPECIFIC EMISSION SPECTRA

Abstract

The fluorescence characteristics of adducts derived from the covalent binding of the highly tumorigenic (+) and the non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to native calf thymus DNA are significantly different from one another both at room temperature and at 77 K. The ratio R of fluorescence intensities of the (0,0) band I (situated near 380 nm) and vibronic band V (near 400 nm) of the pyrene ring system in the BPDE-DNA adducts and of the tetraol (BPT) hydrolysis product of BPDE is very sensitive to the polarity of the solvent, thus mimicking the well known behavior of pyrene itself (A. Nakajima, 1971, Bull. Chem. Soc. Jpn. 44, 3272). The fluorescence excitation and emission spectra of the (+)-BPDE-DNA adducts are relatively sharp and only slightly red-shifted (2-3 nm) with respect to those of BPT in aqueous buffer solution, and R = 1.07 when the fluorescence is excited at the maximum of the absorption spectrum; this compares with R = 1.17 for BPT in water, R = 0.75 in ether, and R = 0.84 for noncovalently intercalated BPT. These results suggest that the pyrene ring system in the covalent (+)-BPDE-DNA adducts is located in an environment which is relatively exposed to the aqueous environment, while physically intercalated BPT molecules are located at hydrophobic binding sites. The fluorescence characteristics of the (-)-BPDE-DNA adducts are more heterogeneous and thus more complex than those of the (+)-adducts. The R ratio depends rather strongly on the wavelength of excitation; a minor, more highly fluorescent and relatively solvent-accessible form of adducts exhibits an R ratio of 1.01. The major, less solvent accessible form is characterized by a larger red shift in the absorption spectrum (approximately 10 nm) and emission spectrum (approximately 6 nm for the (0,0) band) relative to BPT, and an R ratio of 1.07. These characteristics suggest that the local environments of the pyrenyl residues in the (-)-BPDE-DNA adducts are significantly different from those of BPT bound noncovalently to DNA by the intercalation mechanism. Fluorescence methods, particularly at low temperatures where the bands are better resolved and the fluorescence yields are significantly greater than at room temperature, can also be used to distinguish covalent DNA adducts derived from the binding of (+)-BPDE and (-)-BPDE to native double-stranded DNA.