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DNA cleavage, photoinduced by benzophenone-based sunscreens.

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The topical application of sunscreens is widely practised to protect healthy and photosensitive skins from the sun. The benzophenone-derived sunscreens, e.g. 2-hydroxy-4-methoxy benzophenone-5-sulphonic acid (or benzophenone-4) and 2-hydroxy-4-methoxy benzophenone (or benzophenone-3), were ranked as the second and third most frequently used sunscreens, respectively, by the United States Food and Drug Administration (FDA) in 1996. These sunscreens are categorised as being 'safe' and 'effective'. However, it is well known that the parent compound, benzophenone, undergoes rapid hydrogen abstraction reactions on irradiation and is an extremely powerful radical generator. In addition, benzophenone has been shown to be a potent photosensitizer of thymine dimers in deoxyribose nucleic acid (DNA). More astounding to the sunscreen industry is the recent discovery that a group of non-steroidal anti- inflammatory drugs (NSAIDs) having the benzophenone backbone, e.g. ketoprofen, not only form thymine dimers when irradiated with DNA in vitro, but also photosensitize double stranded supercoiled DNA making it prone to single-strand break formation. Both these lesions, if unrepaired, may contribute to mutagenesis, carcinogenesis, inherited disease and eventually cell death. The purpose of this investigation was to determine if a group of benzophenone-derived sunscreen agents has the ability to photosensitize the cleavage of DNA, whereby supercoiled DNA is converted to the relaxed circular and linear forms. The group of UV absorbers investigated in this study included benzophenone-4, benzophenone-3 , 2,4 dihydroxybenzophenone (or benzophenone-l), 2,2'-dihydroxy-4,4'-dimethoxy benzophenone sulphonic acid (or trade name Uvinul DS49) and 2-phenylbenzimidazole-5-sulphonic acid (or trade name Eusolex 232). For comparison the parent compound benzophenone and the NSAID ketoprofen, a well-known photocleaver, were also studied. Buffered aqueous solutions of the benzophenones were irradiated in the presence of DNA at wavelengths greater than 300 nm with an Osram 500 W/2 high-pressure mercury lamp in conjunction with a 10 mm thick Pyrex filter. The irradiated samples were analysed for DNA cleavage by agarose gel electrophoresis and for DNA binding by fluorescence spectroscopy. The photostability of the UV absorbers was also investigated. In addition, computational studies were conducted to obtain the lowest energy geometrical structures of these UV absorbers and hence determine if intercalation of these UV absorbers with DNA was possible. From the photostability experiments conducted, it is apparent that the benzophenone-based UV absorbers were stable to photodecomposition when irradiated with UV light. They behaved in a manner different from their parent compound benzophenone, and from ketoprofen, where substantial photodegradation occurred upon UV irradiation. This is indicative of the rapid photoreactivity of the benzophenone backbone. The relative photostability of the UV absorbers was not anticipated and was attributed to the substituents present on the benzophenone backbone. The agarose gel electrophoresis experiments however clearly showed that benzophenone, ketoprofen, benzophenone-l, Uvinul DS49 and Eusolex 232 cleave ?X174 DNA when irradiated with UV light at wavelengths greater than 300 nm, while benzophenone-3 and benzophenone-4 did not. For these UV absorbers with the exception of benzophenone-3 and benzophenone-4, the number of single strand breaks in the DNA increased compared to when it was irradiated in their absence. In addition, the supercoiled DNA was converted to the relaxed circular and linear forms, the latter of which was undetected in the absence of the UV absorbers. Binding of benzophenone, ketoprofen, benzophenone-l and Uvinul DS49 to calf thymus DNA was also detected by the fluorescence spectroscopy technique. However, this was not observed for Eusolex 232, benzophenone-3 and benzophenone-4, since they did not compete with ethidium bromide for DNA binding sites. Where DNA cleavage did occur, the mechanism of this interaction had to be determined hence the motivation for the computational studies. From computational studies using PM3 semi- empirical calculations, it was determined that the benzophenone-based UV absorbers investigated, apart from Eusolex 232, displayed non-planar geometrical structures. This indicated that DNA intercalation of these sunscreen agents with DNA would at best be very limited, since only one half of the molecule could possibly interact with the bases of DNA. For benzophenone, ketoprofen, benzophenone-l and Uvinul DS49, photosensitised type I and type II processes involving triplet energy transfer reactions has been identified in literature as being responsible for DNA cleavage. It was determined by ab initio calculations that Eusolex 232 exists in a planar structure unlike the other UV absorbers mentioned above that were non- planar. It was concluded that although Eusolex 232 has the ability to intercalate with the base pairs of DNA, it does not do so, as shown by its lack of binding to calf thymus DNA by the fluorescence spectroscopy study. Literature alludes to photooxidation by singlet oxygen in single stranded DNA via the type II reaction and type I electron transfer reactions in double stranded DNA as the mechanism responsible for DNA cleavage induced by Eusolex 232.


Thesis (M.Sc.)-University of Natal, Durban, 2003.


Theses--Chemistry., DNA., Scission (Chemistry), Chemical reactions., Photochemistry., Sunscreens (Cosmetics), Ultraviolet radiation., Skin--Cancer.