Photo-induced reactions of benzo(a)pyrene with DNA in vitro.

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 centrif ugal 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 1to 2-fold greater than those observed in neutral conditions. Using a phenol extraction procedure and molecular sieve column chromato graphic methods,it was possibleto quantitatethe amountof 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.