Improved high-performance liquid chromatographic analysis of 8-methoxypsoralen monoadducts and cross-links in polynucleotide, DNA, and cellular systems: analysis of split-dose protocols.
The distribution of 8-methoxypsoralen-thymidine photoadducts from polynucleotides, calf thymus DNA and mammalian cells treated with [3H]8-methoxypsoralen under a variety of irradiation conditions was determined using high-performance liquid chromatography and scintillation analysis. The split-dose protocol, with samples treated with 8-methoxypsoralen and low doses of long-wavelength UV radiation to generate monoadducts, washed to remove unreacted 8-methoxypsoralen, then irradiated further to convert the monoadducts to cross-links, was examined. The photoadduct distribution in the first step is dependent upon the UVA dose and the wavelength of the radiation, but it is relatively independent of 8-methoxypsoralen concentration. Low fluence and longer wavelengths generate mainly 4',5'-monoadducts, whereas higher fluences and shorter wavelengths yield more cross-links. The second irradiation step converts the 4',5'-monoadducts to cross-links as well as to 3,4-monoadducts. The overall yield of cross-links after the second irradiation step is not dependent upon the wavelength used in the first step. Cellular studies demonstrated that the split-dose protocol is applicable to mammalian systems. These results may affect the interpretation of mutagenesis studies based on the split-dose protocol, because the second step can convert 4',5'-monoadducts to both 3,4-monoadducts, the expected cross-links. Therefore, interpretations that link increases in mutagenicity after the second step in a split-dose study solely to cross-link formation may need re-examination.
Olack
G
G
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510.
Gattolin
P
P
Gasparro
F P
FP
D000818:Animals; D002417:Cattle; D002477:Cells / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000528:radiation effects; D006801:Humans; D066298:In Vitro Techniques; D008730:Methoxsalen / Q000032:analysis*; D051379:Mice; D010777:Photochemistry; D011067:Poly dA-dT / Q000528:radiation effects; D014466:Ultraviolet Rays
D011067:Poly dA-dT; D004247:DNA; D008730:Methoxsalen