The effect of antioxidants on the Para-Aminobenzoic acid photosensitised formation of thymine dimer and singlet molecular oxygen.

Abstract

On exposure to UV radiation DNA is damaged. The thymine cyclobutane dimer is one of the most significant of the lesions formed. This dimer is associated with the mutagenic and carcinogenic effect of UV radiation. It has been shown to cause skin cancer. Thymine dimerisation can be effected by direct irradiation or via an endogenous photosensitiser that is involved in energy transfer. This deleterious effect of UV radiation has prompted the use of sunscreens as photoprotectants. Para-aminobenzoic acid (PABA) is an active ingredient that was once widely used in sunscreen formulations. Reports of allergic reactions and staining by PABA have led to the discontinuation of its use in sunscreencontaining products. It has been shown to be absorbed by human cells and to. photosensitise the dimerisation of thymine in DNA. It can also photosensitise the formation of singlet oxygen. Singlet oxygen has been implicated in many photodynamic disorders, carcinogensis, free radical reactions and aging. Because the photochemical properties of PABA on DNA have been well established by a number of earlier researchers, it proved an appropriate choice of photosensitiser in order to determine the effect of vitamin antioxidants on the photosensitised formation ofthymine dimer and singlet oxygen. Vitamin E (a-tocopherol) and vitamin C (ascorbic acid) are two important antioxidants that have been extensively studied in free-radical reactions and cancer-related ailments. They are known to reduce the formation of thymine dimers and also quench singlet . oxygen. They are found extensively in sunscreen formulations because of their photoprotective properties. The photoprotection is due to both their absorptive and antioxidant properties. They are known to act either individually or synergistically by physical quenching or reaction with free radicals. Their abilities to reduce the PABA-photosensitised formation of thymine dimer have been studied in this work. The study involved investigating their individual abilities and the synergism by varying their concentrations, the concentrations of thymine and PABA, and the photon flux. The pH of the solutions was always maintained at 3.0. Analysis and quantification of the photoproducts was done by using reverse phase high perfonnance liquid chromatography with a photo diode array UV detector. Studies on the P ABA-photosensitised production of singlet oxygen and its quenching by the antioxidants were also carried out at pH 3.0. With this regard, irradiation time and concentrations of PABA and the two vitamins were varied. Thymine was subsequently introduced so as to investigate its effect on singlet oxygen fonnation. The presence and change in concentration of singlet oxygen was monitored by using N,N-dimethyl-4-nitrosoaniline, RNO. The change in absorbance of RNO was measured at 350 nm, which is its maximum absorption wavelength at pH 3.0. The fonnation of singlet oxygen was inferred from these measurements. PABA-photosensitised thymine dimer yield increased with an increase in irradiation time and an increase in the concentration of thymine. However, the yield decreased with an increase in the concentration of PABA. The presence of the vitamins C and E either acting alone or when combined had an effect on the yield of thymine dimer. Both the two antioxidants can increase or decrease thymine dimer yield. However, this depended on the concentrations of the reagents, the irradiation time and whether the two vitamins were in combination or alone. Therefore, both synergistic and antagonistic properties of the two vitamins were observed in the PABA-photosensitised thymine dimer fonnation. Singlet oxygen fonnation generally increased with an increase in irradiation time except for the experiments in which the two vitamins were present together. When the two vitamins were present, there was an increase in the fonnation of singlet oxygen with irradiation time for a few minutes then the fonnation remained constant despite an increase in the irradiation time. An increase in the concentration of PABA also increased the fonnation of measurable singlet oxygen. The introduction of the antioxidants either individually or together decreased the yield of singlet oxygen. The presence of thymine had different effects on the total amount of measurable singlet oxygen from those seen above. Generally, singlet oxygen formation increased with increase in concentration of thymine. Thymine in the presence of increasing concentrations of PABA showed different trends in the formation of singlet oxygen. These trends depended on the presence or absence of the antioxidants and whether the antioxidants were present individually or together. In this set of experiments, the presence of thymine resulted in an increase in the total amount of singlet oxygen formed. An exception to this is when vitamin E is introduced. In this case the presence of thymine indicated formation of a smaller amount of singlet oxygen. In both cases of the absence and presence of thymine, the trend in the formation of singlet oxygen with increase in concentration of any of the antioxidants varied depending on whether the antioxidant was alone or present with the other. From the above it is evident that both synergistic and antagonistic properties of the two vitamins are exhibited regarding their ability to quench singlet oxygen in aqueous solution. The synergistic property of the two vitamins is lost in the presence of thymine. This implies that in the experimental conditions studied, thymine can photosensitise singlet oxygen formation despite the presence of the two vitamins. It is highly likely that both chemical and physical quenching by vitamins C and E take place. Some observations suggested a possible chemical interaction between the photosensitiser (PABA) and thymine with the quenchers. More work needs to be done in this regard to test this hypothesis.