An investigation of acetone-photosensitised DNA kinetics.

Abstract

Ultraviolet (UV) radiation is a potent DNA-damaging agent and a known inducer of mutations and skin cancer. The increasing incidence of skin cancer has emphasised the importance of understanding the mechanistic processes involved in the interaction of UV radiation with DNA. One of the most significant photoproducts, induced by UV light, in the DNA molecule is the cis-syn cyclobutane pyrimidine dimer. These dimers, particularly the cytosinecontaining dimers, have been implicated in the mutagenic and carcinogenic effects of sunlight. Dimerisation of contiguous pyrimidine residues in DNA can result from direct irradiation (A = 295-310 nm) or photosensitised irradiation (A > 300 nm) by endogenous photosensitisers. Direct irradiation of DNA produces a wide range of photoproducts, whereas triplet photosensitisation of DNA by acetone produces only thymine, cytosine and cytosinethymine dimers. Thus, acetone photosensitisation of DNA can be used in the elucidation of the mechanistic processes involved in the formation of photoproducts from the direct irradiation of DNA. Calf thymus DNA was irradiated in the presence of acetone at wavelengths greater than 300 nm, using a high pressure mercury lamp. Experimental conditions investigated were irradiation time, acetone concentration and DNA concentration. Irradiated DNA samples were degraded by hot acid hydrolysis to excise the dimers. The yields of thymine and cytosine-thymine dimers were able to be quantitated by reverse phase high performance liquid chromatography with DV detection.Independent kinetic mechanisms were proposed for thymine and cytosine-thymine dimerisation in calf thymus DNA. Rate constants were assigned from experimentally determined values, values cited in literature and values calculated from Stern-Volmer steady state analysis of the proposed mechanisms. Verification of the proposed kinetic mechanisms was achieved by the comparison of experimental dimer yields with those calculated from the computer simulation of the proposed kinetic mechanism. The computer program CAKE (Computer Analysis of Kinetic Equations) was used to obtain the simulated data. Good agreement between the experimental and simulated data was taken as corroboration of the proposed kinetic mechanism. A section of this work was concerned with the application of spectroradiometry to determine the amount of light intensity absorbed by irradiated solutions. The modification, calibration and operation of a Macam SR 9010 spectroradiometer to achieve this aim is discussed.