Probing the microenvironment of benzo[a]pyrene diol epoxide-DNA adducts by triplet excited state quenching methods

Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25 +/- 1 degrees C were investigated: covalent adducts derived from the reaction of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BaPDE) and of (+/-)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of KTO2 approximately equal to 10(9) M-1 X S-1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of KTO2 is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of KTO2 approximately equal to 10(8) M-1 X S-1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The KTO2 values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1 X 10(-4)-3 X 10(-3) M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of KTO2 for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.