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

Abstract

Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.

Differential regulation of CXC ligand 1 transcription in melanoma cell lines by poly(ADP-ribose) polymerase-1

“…Inhibition of PARP-1 activity in airway epithelial cells, on the other hand, showed reduced NF-kB activation and reduced CXCL8 expression upon H 2 O 2 induction and prevented lung inflammation in vivo (Boulares et al, 2003). Similarly, CXCL8 as well as intercellular adhesion molecule-1 expression were reduced by PARP inhibitors in HaCaT keratinocytes (Szabo et al, 2001). These data indicate that PARP-1 can act as both an inhibitor and activator of NF-kB-dependent transcription.…”

Pharmacological Modulation of Poly(ADP-ribose) Polymerase-Mediated Cell Death: Exploitation in Cancer Chemotherapy

“…Several groups have reported that inhibition of PARP activity induces protection against necrotic cell death. However, PARP inhibition does not protect cells from apoptosis (Palomba et al, 1999;Szabó et al, 2001;Tentori et al, 2001). Thus, a role of PARP activation in necrosis is consistent with the fact that the inhibition or absence of PARP provides the most remarkable protection in disease models such as stroke, myocardial infarction, or mesenteric ischemia-reperfusion injury, which are characterized predominantly by necrotic-type cell death (Miesel et al, 1995).…”

Optimalized transient transfection of chondrogenic primary cell cultures

“…For investigation of cellular viability, cells cultured in wells of 96-well microtiter plates were used and 10 μL MTT reagent (3-/4,5-dimethylthiazolyl-2/-2,5-diphenyltetrazolium bromide; 25 mg MTT/5 mL PBS) was pipetted into each well. Cells were incubated for 2 hours at 37°C and following addition of 100 μL of MTT solubilizing solution the absorption was detected at 570 nm using a Chameleon microplate reader (Hidex Ltd., Turku, Finland) on the second day of culturing [32].…”

“…Distal parts of the limb buds of Ross hybrid chicken embryos of Hamburger-Hamilton stages 22-24 were removed and primary micromass cultures of chondrifying mesenchymal cells were established from cell suspensions with a density of 15x10 6 cells/mL [17,32]. Droplets (100 mL) of the suspension were inoculated onto the bottom of plastic Petri dishes (Nunc, Naperville, IL, USA) with the day of inoculation is considered as day 0.…”