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Dive into the research topics where Daniel Hernández is active.

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Featured researches published by Daniel Hernández.


Journal of Photochemistry and Photobiology B-biology | 1995

Evaluation of ketoprofen (R,S and R/S) phototoxicity by a battery of in vitro assays.

Francisco Bosca; Germano Carganico; JoséV. Castell; María José Gómez-Lechón; Daniel Hernández; David Mauleón; Luis Arboledas Martínez; Miguel A. Miranda

Abstract The various enantiomers of ketoprofen (S and R) and its racemic form ( R S ) exhibited comparable phototoxicities when examined by the following in vitro test systems: (a) effects of pre-irradiated drugs on cultured hepatocytes; (b) co-irradiation of drugs with hepatocytes or fibroblasts; (c) photohaemolysis sensitized by the various ketoprofen steroisomers; (d) drug-photosensitized formation of linoleic acid hydroperoxides. Inhibition of photohaemolysis and photodynamic lipid peroxidation by butylated hydroxyanisole and reduced glutathione suggests that the phototoxicity of ketoprofen is associated with a radical chain (type I) peroxidation of membrane lipids, leading to cell lysis In view of the above results it could be advantageous to use the pharmacologically active S(+) enantiomer instead of the R/S form, since the lower doses required would result in a diminished phototoxic potential.


Toxicology in Vitro | 1995

Photobinding of drugs to cells as an indicator of potential photoallergy.

Miguel A. Miranda; José V. Castell; María José Gómez-Lechón; Daniel Hernández; Luis Arboledas Martínez

The photobinding of drugs to cells has been investigated as a possible indicator of the photoallergic potential. Tiaprofenic acid (TA), carprofen (CP), benoxaprofen (BP) and ibuprofen (IP) were selected as test substances and human fibroblasts as model biological system. Radioactive labelling of the drugs was achieved through alkaline exchange of the alpha-carboxyl hydrogens by tritium, using (3)H(2)O as solvent. When the labelled compounds were co-irradiated with fibroblasts, TA gave rise to the highest amount of radioactivity covalently bound to cells. This also occurred, albeit to a lower extent, with CP and BP; by contrast, no effect was observed for IP. The observed rank order is in agreement with the available in vivo data. The time course of the process was determined for TA, distinguishing between covalent and non-covalent binding. The results, together with gas Chromatographie analyses of the irradiation mixtures and binding studies with TA and its major photoproduct decarboxytiaprofenic acid (decarboxy-TA) in the dark, indicated that most of the observed photobinding might be due to the photoproduct rather than to the parent drug. It is conceivable that hydrogen abstraction by the excited ketone could take place with proteins as reaction partners. This process would lead to the generation of a radical pair, whose coupling would result in the formation of a covalent bond. The contribution of this mechanistic pathway to the in vivo photobinding has to be considered when a lipophilic photoproduct is formed which, as for decarboxy-TA, still contains an active chromophore.


Photochemistry and Photobiology | 2000

Tiaprofenic Acid-photosensitized Damage to Nucleic Acids: A Mechanistic Study Using Complementary in vitro Approaches

Catherine Agapakis-Causse; Francisco Bosca; José V. Castell; Daniel Hernández; M. L. Marín; Laurent Marrot; Miguel A. Miranda

Abstract 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 (108 vs 107 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.


Photochemistry and Photobiology | 1994

Molecular basis of drug phototoxicity: photosensitized cell damage by the major photoproduct of tiaprofenic acid.

José V. Castell; María José Gómez-Lechón; Daniel Hernández; Luis A. Martinez; Miguel A. Miranda

Abstract 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.


Journal of Photochemistry and Photobiology B-biology | 2000

Photoreactivity of tiaprofenic acid and suprofen using pig skin as an ex vivo model.

Zaideth Sarabia; Daniel Hernández; José V. Castell; Gerard M. J. Beijersbergen van Henegouwen

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.


Photochemistry and Photobiology | 1998

Photobinding of Tiaprofenic Acid and Suprofen to Proteins and Cells: A Combined Study Using Radiolabeling, Antibodies and Laser Flash Photolysis of Model Bichromophores

José V. Castell; Daniel Hernández; María José Gómez-Lechón; A. Lahoz; Miguel A. Miranda; Isabel M. Morera; Julia Pérez-Prieto; Zaideth Sarabia

Drug photoallergy is a matter of current concern. It involves the formation of drug‐protein photoadducts (pho‐toantigens) 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 labeling and drug‐directed antibodies. To ascertain whether preassociation with the protein may play a role in photoreactivity, two model bichro‐mophoric 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 photo‐bind 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.


Toxicology in Vitro | 1997

Mechanisms of photosensitization by drugs : Involvement of tyrosines in the photomodification of proteins mediated by tiaprofenic acid in vitro

Miguel A. Miranda; José V. Castell; Zaideth Sarabia; Daniel Hernández; I. Puertes; Isabel M. Morera; María José Gómez-Lechón

The photosensitizing potential of drugs must be related to their photoreactivity towards the target biomolecules. In this context, a representative photosensitizing drug (tiaprofenic acid) was co-irradiated with a model protein, bovine serum albumin (BSA). This led to a significant degree of protein crosslinking and to the formation of trace amounts of drug-BSA photoadducts. Amino acid analysis of the hydrolysed (HC1) protein showed that His and Tyr undergo a dramatic decrease (approx. 90%) as a consequence of drug-mediated photodynamic processes. When the drug was irradiated in the presence of the pure amino acids, extensive phototransformation of the latter was observed. Other photosensitizing drugs gave rise to similar processes when irradiated in the presence of BSA or the isolated amino acids. In conclusion, histidine and tyrosine appear to be key sites for the photosensitized damage to proteins. Photodegradation of the isolated amino acids in vitro may be an indicator of the photosensitizing potential of drugs.


Toxicology in Vitro | 1999

An In Vitro Approach to Drug Photoallergy: Use of Drug-directed Antibodies to Assess Photobinding of Non-steroidal Anti-inflammatories to Skin Cells

Miguel A. Miranda; Zaideth Sarabia; Daniel Hernández; Isabel M. Morera; M.J. Gómez-Lechón; J.V. Castell

Photobinding of drugs to biomolecules constitutes the early key event in the onset of photoallergy. This process generally involves excitation of the drug to an excited triplet state, which in turn can interact with cell constituents leading, in the case of proteins, to the formation of covalent photoadducts. The resulting photoantigens may trigger an immune response. In the present communication, we report the use of drug-directed antibodies to detect photoadduct formation in skin cells. This has been exemplified with tiaprofenic acid and suprofen as model compounds (two well known photoallergens) and human fibroblasts as representative skin cells. Upon irradiation of cells in the presence of these non-steroidal anti-inflammatories, time-dependent photoadduct formation was observed. This occurred predominantly at the cell membrane level. Most interestingly, the immunogenicity of cell photoadducts could be demonstrated by injection of Balb/c mouse fibroblasts into immunologically identical syngenic animals, where they triggered an immune response, as evidenced by the formation of specific antibodies and sensitized T-cells.


Chemical Research in Toxicology | 1998

Drug-Photosensitized Protein Modification: Identification of the Reactive Sites and Elucidation of the Reaction Mechanisms with Tiaprofenic Acid/Albumin as Model System†

Miguel A. Miranda; José V. Castell; Daniel Hernández; María José Gómez-Lechón; Francisco Bosca; Isabel M. Morera; Zaideth Sarabia


Chemical Research in Toxicology | 2001

Antibodies directed to drug epitopes to investigate the structure of drug-protein photoadducts. Recognition of a common photobound substructure in tiaprofenic acid/ketoprofen cross-photoreactivity.

Agustín Lahoz; Daniel Hernández; Miguel A. Miranda; Julia Pérez-Prieto; Isabel M. Morera; José V. Castell

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Miguel A. Miranda

Polytechnic University of Valencia

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Isabel M. Morera

Polytechnic University of Valencia

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Zaideth Sarabia

Polytechnic University of Valencia

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Francisco Bosca

Polytechnic University of Valencia

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Luis Arboledas Martínez

Spanish National Research Council

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A. Lahoz

University of Valencia

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Agustín Lahoz

Polytechnic University of Valencia

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