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The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated. The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis. Formation of single strand breaks was compared for each group of compounds. Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E. coli endonuclease III (revealing oxidized pyrimidines) and E. coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage. Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases. No pyrimidine dimers were observed. Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites. The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism. Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin. In the case of carprofen, a weak nucleoside degradation was obtained. The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin. In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer. However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

Carprofen is a non-steroidal antiinflammatory drug with marked photosensitising properties. In order to elucidate the mechanisms underlying the phenomenon of drug-protein photobinding, mixtures of the drug and human serum albumin were irradiated under different experimental conditions. After irradiation and subsequent gel-filtration chromatography of the photomixture, the eluting protein fraction was analysed by means of fluorescence spectroscopy. The formation of drug-protein photoadducts could be evidenced by the characteristic emission properties of the carbazole chromophore. The photobinding of the drug to human serum albumin appears to involve the formation of aryl radicals resulting from carbon-halogen photocleavage. This mechanistic interpretation is supported by the observed variations in the intensity of the fluorescence spectra, which can be correlated with the lower quantum yield emission of chlorocarbazoles as compared to non-halogenated analogues. The results from laser flash photolysis studies are also in agreement with this proposal.

The skin is repeatedly exposed to solar ultraviolet radiation. Photoreaction of drugs in the body may result in phototoxic or photoallergic side effects. Non-steroidal anti-inflammatory drugs, such as tiaprofenic acid (TPA) and the closely related isomer suprofen (SUP) are frequently associated with photosensitive disorders; they may mediate photosensitised damage to lipids, proteins and nucleic acids. Using ex vivo pig skin as a model, we investigated the photodegradation of TPA and SUP, and photobinding of these drugs to protein by means of HPLC analysis and drug-directed antibodies. Both with keratinocytes, which were first isolated from the pig skin and thereafter exposed to UVA and with keratinocytes which were isolated from pig skin after the skin was UVA exposed, time-dependent photodegradation of TPA and SUP was found, beside photoadduct formation to protein. The results of this work show that: (a) TPA and SUP were photodecomposed with similar efficiency; major photoproducts detected were decarboxytiaprofenic acid (DTPA) and decarboxysuprofen (DSUP), respectively. (b) Both drugs form photoadducts, as concluded from recognition by drug-specific antibodies. Pig skin appears to be a good model for studying the skin photosensitising potential of drugs.

The phototoxic fluoroquinolones ofloxacin, lomefloxacin, norfloxacin, ciprofloxacin and BAYy 3118 have ionizable groups with pKa values close to neutrality. Different ionic species of these fluoroquinolones, therefore, partition in various compartments and organelles of living cells according to their ionic equilibria. While all these fluoroquinolones accumulate in lysosomes, they more or less stain the rest of the cytoplasm of living HS 68 fibroblasts. As a result, photosensitized damage to other cytoplasmic sites than lysosomes can also be expected. Using microfluorometry and rhodamine 123 (Rh 123) as a specific fluorescent probe which is released from mitochondria by light absorption, we show that under ultraviolet A (UVA) irradiation norfloxacin and ciprofloxacin readily damage mitochondrial membranes. as evidenced by the UVA dose-dependent strongly accelerated release of Rh 123 from mitochondria in cells treated with norfloxacin and ciprofloxacin. Damages are already noticeable at UVA doses as low as 2 J/cm2. By contrast, no such photoinduced damage can be observed with ofloxacin, lomefloxacin and BAYy 3118, the latter being the most phototoxic derivative towards HS 68 fibroblasts. The initial photodamage induced by norfloxacin and ciprofloxacin can then propagate after the irradiation as shown by the strongly increased rate of release of Rh 123 from mitochondria of cells that have been incubated with these two fluoroquinolones and left in the dark after a pre-irradiation with 18 J/cm2 of UVA. Interestingly, the same pre-irradiation after cells have been treated with BAYy 3118 and lomefloxacin induces similar post-irradiation effects, although they have no apparent immediate photosensitizing action on mitochondria.

In order to determine whether or not tiaprofenic acid (TPA) could cause cellular 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 irradiation. When TPA was irradiated in the presence of supercoiled plasmid DNA, it produced large amounts of single-strand breaks (SSB); this is consistent 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-repair enzymes. The fact that TPA-irradiated DNA was a substrate of formamidopyrimidine 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 mixture characteristic of mixed type-I/II mechanisms. Thymidine was less reactive under similar conditions, but it also decomposed to give a typical type-I product pattern. Accordingly, the TPA triplet was quenched by the two nucleosides with clearly different rate constants (10(8) vs 10(7) M-1 s-1, respectively). As cellular RNA also contains oxidizable bases, it could be the target of similar processes, thus interfering with the biosynthesis of proteins by the cells. Extraction of total RNA from TPA-irradiated human fibroblasts, followed by gel electrophoresis and PCR analysis, confirmed this hypothesis. Finally, photosensitization experiments with Saccharomyces cerevisiae 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 in genotoxicity, the risk of mutagenicity does not appear to be significant.

The fluoroquinolone antibiotics are widely used despite their strong phototoxicity under solar UV irradiation. Although they are known as good photodynamic photosensitizers, other factors than production of activated oxygen species may play a role in the effectiveness of the phototoxic effect. Subcellular localization is one of the important parameters that may determine this strength. Using microspectrofluorometry, it is shown that norfloxacin, ofloxacin, lomefloxacin, ciproflaxin and BAYy3118 are readily incorporated into lysosomes of HS68 human skin fibroblasts although weak staining of the whole cytoplasm also occurs especially with norfloxacin. Consistent with their photoinstability in solutions, the fluoroquinolones under study are readily photobleached by UVA in the HS68 fibroblasts. The BAYy3118 derivative that has the fastest bleaching rate also shows the strongest phototoxicity toward HS68 fibroblasts. Photosensitization with these fluoroquinolones induces lysosomal membrane damage as shown by the increased rate of leakage of the lysosomal probe lucifer yellow as compared to that observed with untreated cells.

Drug photoallergy is a matter of current concern. It involves the formation of drug-protein photoadducts (photoantigens) that may ultimately trigger an immunological response. Tyrosine residues appear to be key binding sites in proteins. The present work has investigated the photobinding of tiaprofenic and (TPA) and the closely related isomer suprofen (SUP) to proteins and cells by means of radioactive labelling and drug-directed antibodies. To ascertain whether preassociation with the protein may play a role in photoreactivity, two model bichromophoric compounds (TPA-Tyr and SUP-Tyr) have been prepared and studied by laser flash photolysis. The results of this work show that (a) TPA and SUP photobind to proteins with similar efficiencies, (b) both drugs form photoadducts that share a basic common structure, as they are recognized by the same antibody and (c) drug-protein preassociation must play a key role in photoreactivity, as indicated by the dramatic decrease in the triplet state lifetimes of the model bichromophores compared to the parent drugs.

Ultraviolet-A radiation has weak effects on the release of inflammatory mediators by skin cells due to the poor overlap between UVA wavelengths and the absorption spectra of the relevant chromophores of key biomolecules. However, this situation could be very different in the presence of a photosensitizing drug. To investigate this issue, we have irradiated human skin cells (keratinocytes and fibroblasts) in the presence of fenofibric acid (the active phototoxic metabolite of fenofibrate). The results of this research show a dual effect on the production/release of inflammatory mediators: the synthesis of the proinflammatory cytokine interleukin-6 becomes strongly inhibited at photosensitizer concentrations that clearly stimulate the production of prostaglandins (PGE2) by skin cells. We have found evidences showing that the de novo synthesis of cytokines is inhibited in photosensitized cells due to the fact that cellular mRNA is degraded. Interestingly, when the medium taken from irradiated cultures is added to nonexposed cells, a significant stimulation of cytokine synthesis is observed that can be inhibited by anti-PGE2 antibodies. These observations may be relevant in vivo, where prostaglandins released by photosensitized skin cells could stimulate cytokine synthesis by underlying, nonirradiated cells.

Certain drugs can photosensitive the formation of protein modifications, which are thought to be responsible for the occurrence of photoallergy. In the present work, the UV irradiation of serum albumin in the presence of tiaprofenic acid has been studied as a model system for drug-photosensitized protein modifications. The photolysates evidenced that His, Tyr, and Trp are the reactive sites of the protein. The experimental results strongly suggest that formal hydrogen abstraction from the OH or NH groups of Tyr or Trp by the excited drug is the key photochemical process. Competition between cage escape and in cage recombination of the resulting radical pairs governs the final outcome: protein photo-cross-linking versus drug-protein adduct formation. These findings are highly relevant to understand the process of photohapten formation, the first event in the onset of photoallergy.

The photoreactivity of the photosensitizing nonsteroidal anti-inflammatory drug tiaprofenic acid (TA) and its photoproduct decarboxytiaprofenic acid (DTA) was studied both in the presence and in the absence of bovine serum albumin (BSA). The photoproduct DTA was found to be photostable in buffered aqueous solution at pH 7.4, but photodecomposed when BSA was present in the reaction medium. Both TA and DTA underwent irreversible photobinding to BSA in an almost quantitative way, as evidenced by radioactivity measurements using labeled (3H) compounds. In the case of TA, it has been proven that photobinding is mainly attributable to the phototoreactivity of in situ-generated DTA. The photodegradation and photobinding of TA were also investigated in the epidermis in vivo. Rats were exposed to UVA after application of TA to their shaven dorsal skin. During the initial periods of irradiation, the amount of TA decreased sharply, and the yield of the corresponding photoproduct (DTA) reached a maximum. Prolonged irradiation led to photodegradation of DTA. In vivo photobinding was studied using 3H-TA. Photobinding took place slowly at the beginning, but its rate increased sharply after complete photoconversion of TA, when the photoproduct DTA reached the maximum concentration. Thereafter, the decrease of DTA was more pronounced than that of TA. This indicates that-also in vivo-DTA rather than TA is responsible for the photobinding to biomacromolecules in the viable layer of the epidermis. Overall, the above results suggest that irradiation of TA in buffered aqueous solution, in the presence of proteins, is a reasonable model system to study the photodegradation and photobinding behavior of this drug in vivo. From the qualitative point of view, the main conclusion is that DTA plays a key role both in vivo and in vitro: it is the major photoproduct, it undergoes further photodegradation upon prolonged irradiation, and it appears to be responsible for the photobinding process.

Tiaprofenic acid is a photosensitizing nonsteroidal anti-inflammatory drug, whose major photoproduct (decarboxytiaprofenic acid) is also a potent photosensitizer. Because of the lack of the carboxylate moiety, this photoproduct is more lipophilic and might bind more efficiently to cell membranes, thereby causing phototoxic damage. To verify the feasibility of this hypothesis, we have prepared the 3H-labeled analogs of tiaprofenic acid and its photoproduct and examined the binding, persistence and phototoxicity of the photoproduct using poorly metabolizing (fibroblasts) and actively metabolizing cells (hepatocytes). The photoproduct of tiaprofenic acid accumulates in both cell types as it is formed. Upon removal of the photoproduct from the culture medium, it rapidly disappears from hepatocytes but not from fibroblasts. Consequently, irradiation of fibroblasts previously incubated with the photoproduct and kept in culture in the dark for 20 h results in generalized cell damage while this effect is not observed in hepatocytes. Because of its long persistence in poorly metabolizing skin cells and its reluctance to photobleaching, the formation of this photoproduct in skin may be of relevance to explain the in vivo phototoxicity of tiaprofenic acid.

The photochemistry of the photosensitizing nonsteroidal antiinflammatory drugs tiaprofenic acid and suprofen involves the intermediacy of short-lived species (i.e. radicals). The data obtained in the present work strongly suggest that such intermediates may be responsible for the phototoxicity of 2-arylpropionic acids by inducing photodynamic lipid peroxidation at drug concentrations likely to be reached in the skin. This has been investigated using linoleic acid as a model lipid and determining the amount of hydroperoxides by measuring the spectrophotometric absorption at 233 nm, associated with the formation of dienic hydroperoxides. The major photoproducts of tiaprofenic acid and suprofen are derivatives bearing an ethyl side chain. Photoproducts of this type, due to the lack of polar moieties, are highly lipophilic and likely to accumulate in the lipid bilayer of cell membranes. Taking into account their ability to induce photodynamic lipid peroxidation and their marked photostability, it is conceivable that such photoproducts can participate in many catalytic cycles, playing a significant role in the mechanism of photosensitization by tiaprofenic acid and suprofen.