Tiaprofenic acid-photosensitized damage to nucleic acids: A mechanistic study using complementary in vitro approaches

In order to determine whether or not tiaprofenic acid (TPA) could cause cel lular DNA damage, human fibroblasts were irradiated in the presence of the drug and subsequently examined by means of the comet assay. This led to the observation that TPA actually sensitizes cellular DNA to the subsequent ir radiation, When TPA was irradiated in the presence of supercoiled plasmid D NA, it produced large amounts of single-strand breaks (SSB); this is consis tent with the effects observed on cellular genomic DNA by the comet assay. More importantly, low concentrations of TPA, unable to produce direct SSB, caused photo-oxidative damage to DNA as revealed by the use of excision-rep air enzymes. The fact that TPA-irradiated DNA was a substrate of formamidop yrimidine glycosylase as well as endonuclease III revealed that both purine and pyrimidine bases were oxidized. This was further supported by the TPA- photosensitized oxidation of 2'-deoxyguanosine which led to a product mixtu re characteristic of mixed type-I/II mechanisms. Thymidine was less reactiv e under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucl eosides with clearly different rate constants (10(8) vs 10(7) M-1 s(-1), re spectively). As cellular RNA also contains oxidizable bases, it could be th e target of similar processes, thus interfering with the biosynthesis of pr oteins by the cells. Extraction of total RNA from TPA-irradiated human fibr oblasts, followed by gel electrophoresis and PCR analysis, confirmed this h ypothesis. Finally, photosensitization experiments with Saccharomyces cerev isiae showed that, in spite of an efficient drug-yeast interaction leading to cytotoxicity, neither intergenic recombination nor gene conversion took place, Thus, while TPA-photosensitized damage to nucleic acids can result i n genotoxicity, the risk of mutagenicity does not appear to be significant.