Peroxynitrite Production, DNA Breakage, and Poly(ADP-ribose) Polymerase Activation in a Mouse Model of Oxazolone-Induced Contact Hypersensitivity Éva.

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Presentation transcript:

Peroxynitrite Production, DNA Breakage, and Poly(ADP-ribose) Polymerase Activation in a Mouse Model of Oxazolone-Induced Contact Hypersensitivity Éva Szabó, László Virág, Edina Bakondi, László Gyüre, György Haskó, Péter Bai, János Hunyadi, Pál Gergely, Csaba Szabó Journal of Investigative Dermatology Volume 117, Issue 1, Pages 74-80 (July 2001) DOI: 10.1046/j.0022-202x.2001.01388.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Tyrosine nitration, DNA breakage, and PARP activation in CHS. Ears were obtained from sensitized mice 24 h after the second challenge with oxazolone. Paraffin-embedded sections were used for nitrotyrosine staining (A, B) and for the detection of DNA breaks (C, D). PARP activity was visualized by in situ PARP assay in frozen sections (E, F). For all three reactions control ears from nonsensitized mice (A, C, E) were stained in parallel with CHS ears (B, D, F). Keratinocytes from CHS stain positive for nitrotyrosine (B), display massive DNA breakage (D), and show PARP activation (F). Infiltrating cells of microabscessi are negative for nitrotyrosine but many of them contain DNA breaks. Insert shows negative staining of a CHS section stained with normal rabbit serum instead of rabbit polyclonal antinitrotyrosine antibody. (Scale bar, 6 µm.) Journal of Investigative Dermatology 2001 117, 74-80DOI: (10.1046/j.0022-202x.2001.01388.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Peroxynitrite-induced PARP activation in the skin and in HaCaT cells. Peroxynitrite (400 nmol in 200 µl) was applied to the skin of mice. After 30 min, skin samples were excised and frozen in cryoembedding medium. Cryosections were stained by in situ PARP enzymehistochemistry. Intense PARP activation was found in the peroxynitrite-treated skin (B) but not in the control (vehicle-treated) skin (A) (scale bar, 6 µm). HaCaT cells were treated with the indicated concentrations of peroxynitrite for 30 min and PARP activity was then determined by 3H-NAD incorporation (C). Peroxynitrite induced a significant (**p <0.01) PARP activation. Pretreatment of cells with 3 mM PARP inhibitor, 3-AB, abolished PARP activation (##p <0.01). Journal of Investigative Dermatology 2001 117, 74-80DOI: (10.1046/j.0022-202x.2001.01388.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Antiproliferative and cytotoxic effect of peroxynitrite in HaCaT cells. Proliferation (left panel) and viability (right panel) of HaCaT cells have been determined after treatment of cells with the indicated concentrations of peroxynitrite. Treatment was carried out in the presence or absence of 3-AB (3 mM). Asterisks indicate significant (*p <0.05, **p <0.01) protection provided by the PARP inhibitor. Journal of Investigative Dermatology 2001 117, 74-80DOI: (10.1046/j.0022-202x.2001.01388.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Peroxynitrite-induced caspase activation and DNA fragmentation in HaCaT cells. HaCaT cells were treated with increasing concentrations of peroxynitrite in the presence or absence of 3 mM 3-AB. After 6 h, caspase activity (left panel) and DNA fragmentation were determined. Asterisks indicate significantly (*p <0.05, **p <0.01) elevated caspase activity or DNA fragmentation. Hash marks indicate significant difference between 3-AB-treated and untreated cells. Journal of Investigative Dermatology 2001 117, 74-80DOI: (10.1046/j.0022-202x.2001.01388.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of PARP inhibition on ICAM-1 expression and IL-8 production of immune-stimulated HaCaT cells. (A) ICAM-1 expression; (B) IL-8 production. HaCaT cells were stimulated for 24 h with 30 ng per ml human recombinant interferon-γ to induce ICAM-1 expression or with human recombinant IL-1β to induce IL-8 secretion. Induction was carried out in the presence or absence of 3-AB. 3-AB (3 mM) caused significant (*p <0.05, **p <0.01) inhibition of both ICAM-1 expression and IL-8 secretion. Journal of Investigative Dermatology 2001 117, 74-80DOI: (10.1046/j.0022-202x.2001.01388.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions