UVA-induced DNA single-strand cleavage by 1-hydroxypyrene and formation ofcovalent adducts between DNA and 1-hydroxypyrene

1-Hydroxypyrene (HOP), a metabolite found in the urine of humans and labora tory animals exposed to polycyclic aromatic hydrocarbons (PAHs), is known t o be both acutely toxic and genotoxic. It has been widely used as a biomark er for studying PAH exposure. In this research, we have found that, upon UV A irradiation, HOP causes DNA single-strand cleavages and forms HOP-DNA cov alent adducts. The UVA-induced cleavage of supercoiled plasmid Phi X174 DNA is dependent upon both HOP concentration and WA dosage. A longer irradiati on time or higher HOP concentration induces more DNA cleavage. Results of t he photocleavage experiments carried out in the presence of reactive oxygen species scavengers, histidine, sodium azide, mannitol, SOD, and desferal i ndicate that both the superoxide free radical and singlet oxygen are likely involved in causing DNA single-strand cleavage. The photocleavage is inhib ited by the presence of an excited singlet-state quencher, KI, indicating t hat it is an excited-state reaction. Along with light-induced DNA cleavage, HOP also forms DNA covalent adducts while being degraded upon light irradi ation. Light-induced degradation of 20 mu M HOP follows first-order reactio n kinetics in a 10% methanolic buffer (10 mM phosphate) solution in the abs ence or presence of 40 mu M calf thymus DNA, with degradation half-lives of 20 or 15 min, respectively. The shorter degradation half-life in the prese nce of DNA is due to the formation of the HOP-DNA covalent adduct. The form ation of the HOP-DNA covalent adduct is evidenced by comparing the UV-vis a bsorption and fluorescence emission spectra of the pure HOP with those of t he HOP-DNA adduct. The covalent HOP-DNA adduct produced due to irradiation was purified by either extensive dialysis (3 x 500 mL buffer solutions), ph enol and chloroform extraction followed by ethanol precipitation, or chloro form extraction alone. The isolated HOP-DNA adduct has an absorption peak a t 353 nm, which is 8 nm red-shifted compared to that of free HOP. The fluor escence emission for HOP-DNA is at least 70 times weaker than that for free HOP in solution. In summary, the findings with HOP reveal that, in additio n to metabolic activation that eventually leads to the formation of alkylat ed DNA adducts or other forms of DNA damage, HOP may be activated by light to produce DNA single-strand cleavage and covalent DNA adducts. These DNA l esions can be sources of toxicity.