UVA-Induced DNA Single-Strand Cleavage by 1-Hydroxypyrene and Formation of Covalent Adducts between DNA and 1-Hydroxypyrene
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- 作者：Shiming Dong ; Huey-Min Hwang ; Xiaochun Shi ; Laketa Holloway ; and Hongtao Yu
- 刊名：Chemical Research in Toxicology
- 出版年：2000
- 出版时间：July 2000
- 年：2000
- 卷：13
- 期：7
- 页码：585 - 593
- 全文大小：141K
- 年卷期：v.13,no.7(July 2000)
- ISSN：1520-5010
文摘
1-Hydroxypyrene (HOP), a metabolite found in the urine of humans and laboratory animalsexposed to polycyclic aromatic hydrocarbons (PAHs), is known to be both acutely toxic andgenotoxic. It has been widely used as a biomarker for studying PAH exposure. In this research,we have found that, upon UVA irradiation, HOP causes DNA single-strand cleavages and formsHOP-DNA covalent adducts. The UVA-induced cleavage of supercoiled plasmid 
X174 DNAis dependent upon both HOP concentration and UVA dosage. A longer irradiation time or higherHOP concentration induces more DNA cleavage. Results of the photocleavage experimentscarried out in the presence of reactive oxygen species scavengers, histidine, sodium azide,mannitol, SOD, and desferal indicate that both the superoxide free radical and singlet oxygenare likely involved in causing DNA single-strand cleavage. The photocleavage is inhibited bythe presence of an excited singlet-state quencher, KI, indicating that it is an excited-statereaction. Along with light-induced DNA cleavage, HOP also forms DNA covalent adducts whilebeing degraded upon light irradiation. Light-induced degradation of 20 
M HOP follows first-order reaction kinetics in a 10% methanolic buffer (10 mM phosphate) solution in the absenceor presence of 40 M calf thymus DNA, with degradation half-lives of 20 or 15 min, respectively.The shorter degradation half-life in the presence of DNA is due to the formation of the HOP-DNA covalent adduct. The formation of the HOP-DNA covalent adduct is evidenced bycomparing the UV-vis absorption and fluorescence emission spectra of the pure HOP withthose of the HOP-DNA adduct. The covalent HOP-DNA adduct produced due to irradiationwas purified by either extensive dialysis (3 × 500 mL buffer solutions), phenol and chloroformextraction followed by ethanol precipitation, or chloroform extraction alone. The isolated HOP-DNA adduct has an absorption peak at 353 nm, which is 8 nm red-shifted compared to that offree HOP. The fluorescence emission for HOP-DNA is at least 70 times weaker than that forfree HOP in solution. In summary, the findings with HOP reveal that, in addition to metabolicactivation that eventually leads to the formation of alkylated DNA adducts or other forms ofDNA damage, HOP may be activated by light to produce DNA single-strand cleavage andcovalent DNA adducts. These DNA lesions can be sources of toxicity.
X174 DNAis dependent upon both HOP concentration and UVA dosage. A longer irradiation time or higherHOP concentration induces more DNA cleavage. Results of the photocleavage experimentscarried out in the presence of reactive oxygen species scavengers, histidine, sodium azide,mannitol, SOD, and desferal indicate that both the superoxide free radical and singlet oxygenare likely involved in causing DNA single-strand cleavage. The photocleavage is inhibited bythe presence of an excited singlet-state quencher, KI, indicating that it is an excited-statereaction. Along with light-induced DNA cleavage, HOP also forms DNA covalent adducts whilebeing degraded upon light irradiation. Light-induced degradation of 20 M HOP follows first-order reaction kinetics in a 10% methanolic buffer (10 mM phosphate) solution in the absenceor presence of 40 M calf thymus DNA, with degradation half-lives of 20 or 15 min, respectively.The shorter degradation half-life in the presence of DNA is due to the formation of the HOP-DNA covalent adduct. The formation of the HOP-DNA covalent adduct is evidenced bycomparing the UV-vis absorption and fluorescence emission spectra of the pure HOP withthose of the HOP-DNA adduct. The covalent HOP-DNA adduct produced due to irradiationwas purified by either extensive dialysis (3 × 500 mL buffer solutions), phenol and chloroformextraction followed by ethanol precipitation, or chloroform extraction alone. The isolated HOP-DNA adduct has an absorption peak at 353 nm, which is 8 nm red-shifted compared to that offree HOP. The fluorescence emission for HOP-DNA is at least 70 times weaker than that forfree HOP in solution. In summary, the findings with HOP reveal that, in addition to metabolicactivation that eventually leads to the formation of alkylated DNA adducts or other forms ofDNA damage, HOP may be activated by light to produce DNA single-strand cleavage andcovalent DNA adducts. These DNA lesions can be sources of toxicity.