Photo-induced Reactions of Benzo(a)Pyrene with DNA in Vitro¹

The induction of lesions in DNA by combinations of benzo(a)pyrene (B(a)P) and near-ultraviolet light (NUV) with wavelengths of 300 to 480 nm was investigated. Using centrifugal methods or agarose gel electrophoretic techniques to monitor the conversion of the superhelical double-stranded PM2 DNA to the relaxed circular form, it was possible to quantitate the rates of single-strand breakage in the DNA when analyzed in neutral or alkaline conditions. Irradiation of a B(a)P and DNA mixture with NUV resulted in a 12-fold increase in the single-strand breaks observed, compared to breaks induced by radiation alone. In oxygen, there were about 40% more single-strand breaks induced compared to breaks induced in an argon environment. Rates of the single-strand breakage observed under alkaline conditions were 1- to 2-fold greater than those observed in neutral conditions. Using a phenol extraction procedure and molecular sieve column chromatographic methods, it was possible to quantitate the amount of B(a)P that was bound to the DNA as a function of NUV fluence. In an argon-saturated medium, 12 B(a)P molecules were bound per PM2 genome per single-strand break, whereas about 16 B(a)P molecules were bound per PM2 genome per single-strand break when irradiation was performed in oxygen. From studies concerning the alkaline stability of “bound” B(a)P, it is concluded that as much as 30% of the B(a)P molecules bound in the presence of oxygen may be linked to the DNA through phosphotriester bonds. NUV-induced complexes of B(a)P and DNA that were enzymically digested to nucleoside residues and eluted through a Sephadex LH-20 column revealed several distinct peaks eluting through a 30 to 95% methanol gradient that were indicative of B(a)P-nucleoside adducts.