L.F. Salter

Para-aminobenzoic acid-photosensitized dimerization of thymine II. In pUC19 plasmid DNA

Abstract Para-aminobenzoic acid (PABA) photosensitizes the dimerization of contiguous thymine bases in DNA upon irradiation at wavelengths greater than 300 nm which are not absorbed by the DNA bases themselves. The kinetics and mechanism of this reaction has been studied for pUC19 plasmid DNA. The dependence of dimer yield on a variety of reaction conditions has been determined. A mechanism has been proposed to account for the major processes taking place and rate constants for the constituent reactions have been acquired from the literature, from previous studies on the simpler systems of PABA-photosensitized dimerization of free thymine base and thymidylyl-3′,5′-thymidine (TpT) and from calculation based upon a Stern—Volmer steady state analysis of the proposed mechanism and experimental data. Computer simulation of the mechanism with its associated rate constants gives dimer yields that are generally in reasonable agreement with the experimental data. Where discrepancies occur they have been explained in terms of production of other photoproducts.

Photoproducts formed by near-UV irradiation of thymine in the presence of p-aminobenzoic acid

Abstract In addition to the four isomers of cyclobutane thymine dimer, two non-dimer photoproducts of thymine (5,6-dihydrothymine and 5-hydroxymethyluracil) were detected when free thymine base was irradiated at 324 nm in the presence of p-aminobenzoic acid (PABA) at pH 7.0. The yields were found to be enhanced in the presence of phosphate buffer. Irradiation of thymidine at 324 nm in the presence of PABA lead to the formation of 5,6-dihydrothymidine.

PARA-AMINOBENZOIC ACID-PHOTOSENSITIZED DIMERIZATION OF THYMINE. I: IN DNA-RELATED MODEL SYSTEMS

Abstract Para-aminobenzoic acid (PABA) is able to photosensitize the dimerization of two adjacent thymines in aqueous solutions of free thymine base or thymidylyl-3′,5′-thymidine at wavelengths greater than 300 nm where thymine does not absorb. The dependence of dimer yield on a variety of reaction conditions when these two DNA- related systems were irradiated in the presence of PABA has been investigated. Reaction mechanisms have been proposed to account for the major processes taking place. The rate constants for the various constituent reactions have been assigned on the basis of experiment, values cited in the literature and calculations based upon these values as well as calculation based upon Stern—Volmer steady state analysis of the proposed mechanisms. Computer simulation of these mechanisms gave dimer yields which were in reasonable agreement with the experimental data.

The potential carcinogenic effect of Uvinul DS49―a common UV absorber used in cosmetics

Abstract The photosensitized production of thymine dimer at 324 nm was investigated using disodium-2,2′-dihydroxy-4,4′-dimethoxybenzophenone sulphonic acid (tradename, Uvinul DS49) as sensitizer. Yields of the dimer for various irradiation times, initial thymine concentrations and initial sensitizer concentrations were determined for the free thymine base in aqueous medium. A reaction mechanism is proposed for total thymine dimer formation together with the corresponding rate constants. The mechanism and rate constants were computer simulated to support their validity.

pH effects on p-aminobenzoic acid photosensitized dimer formation from the free thymine base

Abstract The photosensitizing properties of p -aminobenzoic acid (PABA) at pH 3.0 and pH 7.0 were investigated. At both pH 3.0 and pH 7.0, PABA potentiated the formation of the thymine dimer in aqueous solutions of thymine irradiated with light above 300 nm. At pH 3.0, efficient self-quenching at high PABA concentrations resulted in a markedly decreased dimer yield. A kinetic model is presented and the implications of photo-sensitization by sunscreen constituents are discussed.

A sensitive immunoassay technique for thymine dimer quantitation in UV-irradiated DNA

Abstract This paper describes an immunological technique for thymine dimer quantitation which is more sensitive than the high performance liquid chromatography (HPLC) method used previously in our laboratory. Calf thymus DNA was irradiated in the presence of the photosensitizer acetophenone to induce the formation of cis-syn thymine dimers exclusively. These DNA solutions were then denatured and injected into New Zealand white rabbits to raise antibodies against the thymine dimer. Blood was drawn from the rabbits at regular intervals and the crude serum was used in an immunoblotting protocol which immobilized the antigen-antibody complex on a membrane system. Subsequent detection and quantitation of the thymine dimer antigen was performed by enhanced chemiluminescence (ECL). Dilutions of the antibody used in the above protocol could be quantitatively related to the UV-irradiated DNA antigen, i.e. the thymine dimer. It is shown that this technique is 4000–8000 times more sensitive than the HPLC technique used previously. The above immunoblotting/ECL protocol was then used to test the validity of a proposed mechanism for acetophenone-photosensitized dimerization of thymine in DNA at concentrations more relevant to cellular systems, but previously undetectable by HPLC.

Thymine Dimer Formation Mediated by the Photosensitizing Properties of Pharmaceutical Constituents

Thymine dimer formation is a major photochemical lesion of UV-irradiated DNA and has been implicated as a precursor in skin cancer. Certain pharmaceutical constituents, such as the sunscreen absorbers para-aminobenzoic acid, Uvinul DS49, Eusolex 232 and the tranquillizer chlorpromazine, have the potential to photosensitise thymine dimerisation in thymine-containing systems. The yields of thymine dimer, obtained from the UV-irradiation of thymine substrates in the presence of the photosensitisers, were determined by reverse phase HPLC. Computer simulations of the experimental results were used to establish the kinetics and mechanisms of photosensitised thymine dimerisation in the various systems investigated. Initial studies involved the photosensitisation of free thymine base in aqueous medium. In this system, sunscreen agents were found to be effective photosensitisers of thymine dimerisation. The PABA photosensitisation of thymine dimer formation in the more biologically relevant system, pUC19 plasmid DNA, was investigated. The kinetic mechanism for the photosensitised dimerisation of contiguous thymines in pUC19 plasmid DNA at pH 7 is reported.

Kinetics of thymine dimerization

On the basis of computer simulation a mechanism for photosensitized thymine dimerization involving association between thymine and photosensitizer is proposed.

A computer simulation of the uracil hydrate mechanism for the photochemically induced deamination of free cytosine base in aqueous solution

Abstract The rate expression for the deamination of free cytosine base in unbuffered aqueous solution has been measured as In k = 8.301 - 75.230 kJ mol −1 / RT . Irradiation of similar solutions at 265 nm, followed by high performance liquid chromatography (HPLC) quantification of the uracil formed, allowed a quantum yield for uracil hydrate formation to be calculated as 4.9 × 10 −4 . This result was used together with literature values of other rate constants to simulate a mechanism for the formation of uracil from UV-irradiated cytosine solutions. A value of 7.4 × 10 11 s −1 for the internal conversion of cytosine singlet to ground-state cytosine gave the best agreement between the experimental results and the simulation.

The Kinetics of UV-Induced Thymine Dimerization

Thymine dimerization is the main photochemically induced lesion occurring in UV-irradiated DNA and an understanding of the mechanisms of direct and photosensitized dimerization is of biological importance. In this work the yields of thymine dimer obtained from UV irradiated solutions of thymine were evaluated using HPLC. Computer simulation of the results obtained was used to establish the kinetics and mechanisms of the processes involved. Based on the results obtained it has been confirmed that diffusion controlled and base stacking mechanisms are important for direct irradiation of thymine solutions. However, for photosensitization, association between photosensitizer and nucleic acid bases increases the effective quantum yield of dimerization. This suggests that biological macromolecules which operate as photosensitizers in cellular systems may damage DNA to a greater extent than that envisaged via diffusion controlled collisional processes.