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Investigation of the phototoxicity and cytotoxicity of naproxen, a non-steroidal anti-inflammatory drug, in human fibroblasts

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Abstract

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain and inflammation associated with several diseases. Naproxen, 2-(6-methoxy-2-naphthyl) propionic acid (NAP), belongs to this pharmacological class and appears to be associated with a high incidence of both photoallergic and phototoxic reactions. In this study, using human fibroblasts, we examined the biological effects of NAP photosensitization induced by UVA, the predominant UV component of sunlight reaching the Earth’s surface. We showed that NAP or UVA alone have no cytotoxic effects at the concentrations and doses used in this study. The same result was observed when cells were pre-incubated with NAP but irradiated without NAP. In marked contrast, exposure of cells in the presence of NAP led to a drastic reduction of cell viability. These results suggest that the phototoxicity is mainly due to irradiation of extracellular NAP that damages cell membranes. Moreover, we showed that NAP itself led to a low but reproducible production of reactive oxygen species (ROS), to protein modifications by lipid peroxidationderived aldehydes, to p38 phosphorylation and to the slowing-down of DNA replication, while UVA treatment alone showed no effects. NAP photosensitization with UVA led to protein S-glutathionylation, oxidation of the proliferating cell nuclear antigen (PCNA), oxidation of cellular tryptophan, phosphorylation of Chk1 and inhibition of DNA replication. However, using small interfering RNA to down regulate Chk1 expression in cells, we showed that Chk1 is not required to slow the S-phase down. Nevertheless, inhibition of Chk1, but not of p38, sensitized the cells to the phototoxic effects of NAP. Collectively, our data suggest that the interaction of NAP with the cells triggers oxidative damage and a replication stress, which are exacerbated by UVA radiation. As oxidative and replication stress-induced genome instability are important factors in aging and tumor predisposition, it is of interest to evaluate the consequence of a non-steroidal anti-inflammatory drug, like naproxen, on genomic instability.

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Abbreviations

UVA:

ultraviolet A

NAP:

naproxen

NSAID:

non-steroidal anti-inflammatory drugs

PCNA:

proliferating cell nuclear antigen

1O2:

singlet oxygen

BSA:

bovine serum albumin

MEM:

minimum essential medium

PBS:

phosphate buffered saline

CM-H2DCFDA:

5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester

MMT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NaN3:

sodium azide

BrdU:

5-bromo-2-deoxy-uridine

HPLC:

high performance liquid chromatography

Trp:

tryptophan

Tyr:

tyrosine

Ser:

serine

MeOH:

methanol

NEM:

N-ethylmaleimide

SDS:

sodium dodecyl sulphate

Na3VO4:

sodium orthovanadate

DTT:

dithiothreitol

siRNA:

small interfering RNA

References

  1. G. T. Wondrak, M. K. Jacobson, E. L. Jacobson, Endogenous UVA-photosensitizers: mediators of skin photodamage and novel targets for skin photoprotection, Photochem. Photobiol. Sci., 2006, 5, 215–237.

    Article  CAS  PubMed  Google Scholar 

  2. S. D. Lamore, S. Azimian, D. Horn, B. L. Anglin, K. Uchida, C. M. Cabello, G. T. Wondrak, The malondialdehyde-derived fluorophore DHP-lysine is a potent sensitizer of UVA-induced photooxidative stress in human skin cells, J. Photochem. Photobiol., B, 2010, 101, 251–264.

    Article  CAS  Google Scholar 

  3. C. J. Le Coz, A. Bottlaender, J. N. Scrivener, F. Santinelli, B. J. Cribier, E. Heid, E. M. Grosshans, Photocontact dermatitis from ketoprofen and tiaprofenic acid: cross-reactivity study in 12 consecutive patients, Contact Dermatitis, 1998, 38, 245–252.

    Article  PubMed  Google Scholar 

  4. L. Matthieu, L. Meuleman, E. Van Hecke, A. Blondeel, B. Dezfoulian, L. Constandt, A. Goossens, Contact and photocontact allergy to ketoprofen. The Belgian experience, Contact Dermatitis, 2004, 50, 238–241.

    Article  CAS  PubMed  Google Scholar 

  5. J. V. Castell, M. J. Gomez-Lechon, C. Grassa, L. A. Martinez, M. A. Miranda, P. Tarrega, Involvement of drug-derived peroxides in the phototoxicity of naproxen and tiaprofenic acid, Photochem. Photobiol., 1993, 57, 486–490.

    Article  CAS  PubMed  Google Scholar 

  6. M. A. Miranda, I. Morera, F. Vargas, M. J. Gomez-Lechon, J. V. Castell, In vitro assessment of the phototoxicity of anti-inflammatory 2-arylpropionic acids, Toxicol. in Vitro, 1991, 5, 451–455.

    Article  CAS  PubMed  Google Scholar 

  7. A. J. Ridley, J. R. Whiteside, T. J. McMillan, S. L. Allinson, Cellular and sub-cellular responses to UVA in relation to carcinogenesis, Int. J. Radiat. Biol., 2009, 85, 177–195.

    Article  CAS  PubMed  Google Scholar 

  8. J. Baier, T. Maisch, M. Maier, E. Engel, M. Landthaler, W. Baumler, Singlet oxygen generation by UVA light exposure of endogenous photosensitizers, Biophys. J., 2006, 91, 1452–1459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. R. M. Tyrrell, M. Pidoux, Singlet oxygen involvement in the inactivation of cultured human fibroblasts by UVA (334 nm, 365 nm) and near-visible (405 nm) radiations, Photochem. Photobiol., 1989, 49, 407–412.

    Article  CAS  PubMed  Google Scholar 

  10. L. M. Davids, B. Kleemann, D. Kacerovska, K. Pizinger, S. H. Kidson, Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells, J. Photochem. Photobiol., B, 2008, 91, 67–76.

    Article  CAS  Google Scholar 

  11. F. Bosca, M. L. Marin, M. A. Miranda, Photoreactivity of the nonsteroidal anti-inflammatory 2-arylpropionic acids with photosensitizing side effects, Photochem. Photobiol., 2001, 74, 637–655.

    Article  CAS  PubMed  Google Scholar 

  12. L. L. Costanzo, G. De Guidi, G. Condorelli, A. Cambria, M. Fama, Molecular mechanism of naproxen photosensitization in red blood cells, J. Photochem. Photobiol., B, 1989, 3, 223–235.

    Article  CAS  Google Scholar 

  13. S. Giuffrida, G. De Guidi, P. Miano, S. Sortino, G. Condorelli, L. L. Costanzo, Molecular mechanism of drug photosensitization: VIII. Effect of inorganic ions on membrane damage photosensitized by naproxen, J. Inorg. Biochem., 1996, 63, 253–263.

    Article  CAS  PubMed  Google Scholar 

  14. L. L. Costanzo, G. De Guidi, S. Giuffrida, S. Sortino, G. Condorelli, G. Pappalardo, Antioxidant effect of copper(ii) on photosensitized lipid peroxidation, J. Inorg. Biochem., 1995, 57, 115–125.

    Article  CAS  PubMed  Google Scholar 

  15. T. Artuso, J. Bernadou, B. Meunier, J. Piette, N. Paillous, Mechanism of DNA cleavage mediated by photoexcited non-steroidal antiinflammatory drugs, Photochem. Photobiol., 1991, 54, 205–213.

    Article  CAS  PubMed  Google Scholar 

  16. N. Chouini-Lalanne, M. Defais, N. Paillous, Nonsteroidal antiinflammatory drug-photosensitized formation of pyrimidine dimer in DNA, Biochem. Pharmacol., 1998, 55, 441–446.

    Article  CAS  PubMed  Google Scholar 

  17. G. De Guidi, S. Giuffrida, G. Condorelli, L. L. Costanzo, P. Miano, S. Sortino, Molecular mechanism of drug photosensitization. IX. Effect of inorganic ions on DNA cleavage photosensitized by naproxen, Photochem. Photobiol., 1996, 63, 455–462.

    Article  PubMed  Google Scholar 

  18. S. Mouret, C. Philippe, J. Gracia-Chantegrel, A. Banyasz, S. Karpati, D. Markovitsi, T. Douki, UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?, Org. Biomol. Chem., 2010, 8, 1706–1711.

    Article  CAS  PubMed  Google Scholar 

  19. V. V. Agon, W. A. Bubb, A. Wright, C. L. Hawkins, M. J. Davies, Sensitizer-mediated photooxidation of histidine residues: evidence for the formation of reactive side-chain peroxides, Free Radical Biol. Med., 2006, 40, 698–710.

    Article  CAS  Google Scholar 

  20. M. J. Davies, The oxidative environment and protein damage, Biochim. Biophys. Acta, 2005, 1703, 93–109.

    Article  CAS  PubMed  Google Scholar 

  21. A. Catalfo, G. Bracchitta, G. De Guidi, Role of aromatic amino acid tryptophan UVA-photoproducts in the determination of drug photosensitization mechanism: a comparison between methylene blue and naproxen, Photochem. Photobiol. Sci., 2009, 8, 1467–1475.

    Article  CAS  PubMed  Google Scholar 

  22. G. Bracchitta, A. Catalfo, G. De Guidi, Photoinduced protein modifications by methylene blue and naproxen, Photochem. Photobiol. Sci., 2012, 11, 1886–1896.

    Article  CAS  PubMed  Google Scholar 

  23. A. Grzelak, B. Rychlik, G. Bartosz, Light-dependent generation of reactive oxygen species in cell culture media, Free Radical Biol. Med., 2001, 30, 1418–1425.

    Article  CAS  Google Scholar 

  24. P. M. Girard, M. Pozzebon, F. Delacote, T. Douki, V. Smirnova, E. Sage, Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells, DNA Repair, 2008, 7, 1500–1516.

    Article  CAS  PubMed  Google Scholar 

  25. K. Luby-Phelps, Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area, Int. Rev. Cytol., 2000, 192, 189–221.

    Article  CAS  PubMed  Google Scholar 

  26. O. I. Aruoma, B. Halliwell, B. M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Radical Biol. Med., 1989, 6, 593–597.

    Article  CAS  Google Scholar 

  27. J. Lee, E. A. Decker, Effects of metal chelator, sodium azide, and superoxide dismutase on the oxidative stability in riboflavin-photosensitized oil-in-water emulsion systems, J. Agric. Food Chem., 2011, 59, 6271–6276.

    Article  CAS  PubMed  Google Scholar 

  28. P. Bilski, A. G. Belanger, C. F. Chignell, Photosensitized oxidation of 2′,7′-dichlorofluorescein: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′-dichlorofluorescein, Free Radical Biol. Med., 2002, 33, 938–946.

    Article  CAS  Google Scholar 

  29. Y. Yang, R. Sharma, A. Sharma, S. Awasthi, Y. C. Awasthi, Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling, Acta Biochim. Pol., 2003, 50, 319–336.

    Article  CAS  PubMed  Google Scholar 

  30. T. T. Reed, Lipid peroxidation and neurodegenerative disease, Free Radical Biol. Med., 2011, 51, 1302–1319.

    Article  CAS  Google Scholar 

  31. D. I. Pattison, A. S. Rahmanto, M. J. Davies, Photo-oxidation of proteins, Photochem. Photobiol. Sci., 2012, 11, 38–53.

    Article  CAS  PubMed  Google Scholar 

  32. E. R. Stadtman, R. L. Levine, Free radical-mediated oxidation of free amino acids and amino acid residues in proteins, Amino Acids, 2003, 25, 207–218.

    Article  CAS  PubMed  Google Scholar 

  33. B. Montaner, P. O’Donovan, O. Reelfs, C. M. Perrett, X. Zhang, Y. Z. Xu, X. Ren, P. Macpherson, D. Frith, P. Karran, Reactive oxygen-mediated damage to a human DNA replication and repair protein, EMBO Rep., 2007, 8, 1074–1079.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. P. M. Girard, S. Francesconi, M. Pozzebon, D. Graindorge, P. Rochette, R. Drouin, E. Sage, UVA-induced damage to DNA and proteins: direct versus indirect photochemical processes, J. Phys.: Conf. Ser., 2011, 261, 012002.

    Google Scholar 

  35. S. I. Bae, R. Zhao, R. M. Snapka, PCNA damage caused by antineoplastic drugs, Biochem. Pharmacol., 2008, 76, 1653–1668.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. K. L. Schey, S. Patat, C. F. Chignell, M. Datillo, R. H. Wang, J. E. Roberts, Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John’s Wort), Photochem. Photobiol., 2000, 72, 200–203.

    Article  CAS  PubMed  Google Scholar 

  37. H. C. Reinhardt, M. B. Yaffe, Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2, Curr. Opin. Cell Biol., 2009, 21, 245–255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. J. Bartek, J. Lukas, Chk1 and Chk2 kinases in checkpoint control and cancer, Cancer Cell, 2003, 3, 421–429.

    CAS  PubMed  Google Scholar 

  39. J. Han, P. Sun, The pathways to tumor suppression via route p38, Trends Biochem. Sci., 2007, 32, 364–371.

    Article  CAS  PubMed  Google Scholar 

  40. D. Branzei, M. Foiani, The checkpoint response to replication stress, DNA Repair, 2009, 8, 1038–1046.

    Article  CAS  PubMed  Google Scholar 

  41. Q. Liu, S. Guntuku, X. S. Cui, S. Matsuoka, D. Cortez, K. Tamai, G. Luo, S. Carattini-Rivera, F. DeMayo, A. Bradley, L. A. Donehower, S. J. Elledge, Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint, Genes Dev., 2000, 14, 1448–1459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. J. S. Choi, M. J. Jin, H. K. Han, Role of monocarboxylic acid transporters in the cellular uptake of NSAIDs, J. Pharm. Pharmacol., 2005, 57, 1185–1189.

    Article  CAS  PubMed  Google Scholar 

  43. R. R. Fitzgerald, J. D. Walters, Accumulation of topical naproxen by cultured oral epithelium, J. Dent. Res., 2007, 86, 775–779.

    Article  CAS  PubMed  Google Scholar 

  44. K. Sugano, M. Kansy, P. Artursson, A. Avdeef, S. Bendels, L. Di, G. F. Ecker, B. Faller, H. Fischer, G. Gerebtzoff, H. Lennernaes, F. Senner, Coexistence of passive and carrier-mediated processes in drug transport, Nat. Rev. Drug Discovery, 2012, 9, 597–614.

    Article  CAS  Google Scholar 

  45. M. Adachi, H. Sakamoto, R. Kawamura, W. Wang, K. Imai, Y. Shinomura, Nonsteroidal anti-inflammatory drugs and oxidative stress in cancer cells, Histol. Histopathol., 2007, 22, 437–442.

    CAS  PubMed  Google Scholar 

  46. D. H. Gonzalez Maglio, M. L. Paz, A. Ferrari, F. S. Weill, J. Nieto, J. Leoni, Alterations in skin immune response throughout chronic UVB irradiation-skin cancer development and prevention by naproxen, Photochem. Photobiol., 2010, 86, 146–152.

    Article  PubMed  CAS  Google Scholar 

  47. P. Bertin, F. Lapicque, E. Payan, M. Rigaud, F. Bailleul, S. Jaeger, R. Treves, P. Netter, Sodium naproxen: concentration and effect on inflammatory response mediators in human rheumatoid synovial fluid, Eur. J. Clin. Pharmacol., 1994, 46, 3–7.

    Article  CAS  PubMed  Google Scholar 

  48. N. M. Davies, K. E. Anderson, Clinical pharmacokinetics of naproxen, Clin. Pharmacokinet., 1997, 32, 268–293.

    Article  CAS  PubMed  Google Scholar 

  49. F. A. van den Ouweland, P. C. Eenhoorn, Y. Tan, F. W. Gribnau, Transcutaneous absorption of naproxen gel, Eur. J. Clin. Pharmacol., 1989, 36, 209–211.

    Article  PubMed  Google Scholar 

  50. E. Niki, Y. Yamamoto, M. Takahashi, E. Komuro, Y. Miyama, Inhibition of oxidation of biomembranes by tocopherol, Ann. N. Y. Acad. Sci., 1989, 570, 23–31.

    Article  CAS  PubMed  Google Scholar 

  51. J. Zhang, G. T. Bowden, Activation of p38 MAP kinase and JNK pathways by UVA irradiation, Photochem. Photobiol. Sci., 2012, 11, 54–61.

    Article  PubMed  Google Scholar 

  52. P. P. Roux, J. Blenis, ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions, Microbiol. Mol. Biol. Rev., 2004, 68, 320–344.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. M. Manrique-Moreno, M. Suwalsky, F. Villena, P. Garidel, Effects of the nonsteroidal anti-inflammatory drug naproxen on human erythrocytes and on cell membrane molecular models, Biophys. Chem., 2010, 147, 53–58.

    Article  CAS  PubMed  Google Scholar 

  54. L. O. Klotz, C. Pellieux, K. Briviba, C. Pierlot, J. M. Aubry, H. Sies, Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA, Eur. J. Biochem., 1999, 260, 917–922.

    Article  CAS  PubMed  Google Scholar 

  55. Z. Guo, S. Kozlov, M. F. Lavin, M. D. Person, T. T. Paull, ATM activation by oxidative stress, Science, 2010, 330, 517–521.

    Article  CAS  PubMed  Google Scholar 

  56. R. Bermejo, M. S. Lai, M. Foiani, Preventing replication stress to maintain genome stability: resolving conflicts between replication and transcription, Mol. Cell, 2012, 45, 710–718.

    Article  CAS  PubMed  Google Scholar 

  57. A. Franchitto, P. Pichierri, Understanding the molecular basis of common fragile sites instability: role of the proteins involved in the recovery of stalled replication forks, Cell Cycle, 2011, 10, 4039–4046.

    Article  CAS  PubMed  Google Scholar 

  58. W. C. Burhans, M. Weinberger, DNA replication stress, genome instability and aging, Nucleic Acids Res., 2007, 35, 7545–7556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Dipartimento di Scienze Chimiche, Università degli Studi di Catania, 95125, Catania, Italy

    G. Bracchitta, A. Catalfo & G. De Guidi

  2. Institut Curie, Centre de Recherche, Orsay, F-91405, France

    G. Bracchitta, S. Martineau, E. Sage & P. M. Girard

  3. CNRS, UMR3348, Orsay, F-91405, France

    G. Bracchitta, S. Martineau, E. Sage & P. M. Girard

  4. INCA—Consorzio Interuniversitario Chimica per l’Ambiente, Marghera (VE), Italy

    G. De Guidi

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  1. G. Bracchitta
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  2. A. Catalfo
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  3. S. Martineau
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  4. E. Sage
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  5. G. De Guidi
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  6. P. M. Girard
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Correspondence to G. De Guidi or P. M. Girard.

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Bracchitta, G., Catalfo, A., Martineau, S. et al. Investigation of the phototoxicity and cytotoxicity of naproxen, a non-steroidal anti-inflammatory drug, in human fibroblasts. Photochem Photobiol Sci 12, 911–922 (2013). https://doi.org/10.1039/c3pp25326k

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