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IMPROVED HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF 8‐METHOXYPSORALEN MONOADDUCTS and CROSS‐LINKS IN POLYNUCLEOTIDE, DNA, and CELLULAR SYSTEMS: ANALYSIS OF SPLIT‐DOSE PROTOCOLS
Author(s) -
Olack Gerard,
Gattolin Paola,
Gasparro Francis P.
Publication year - 1993
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1993.tb02953.x
Subject(s) - irradiation , dna , polynucleotide , yield (engineering) , high performance liquid chromatography , chemistry , mutagenesis , chromatography , mutant , materials science , biochemistry , physics , nuclear physics , metallurgy , gene
— The distribution of 8‐methoxypsoralen‐thymidine photoadducts from polynucleotides, calf thymus DNA and mammalian cells treated with [ 3 H]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.