UVA-ketoprofen-induced hemoglobin radicals detected by immuno-spin trapping[para]

Photochemistry and Photobiology, Jun 2003 by He, Yu-Ying, Ramirez, Dario C, Detweiler, Charles D, Mason, Ronald P, Chignell, Colin F

ABSTRACT

Ketoprofen (3-benzoyl-[alpha]-methylbenzeneacetic acid, KP) is a widely used nonsteroidal anti-inflammatory drug (NSAID) that causes both phototoxicity and photoallergy. Here, we investigated the formation of hemoglobin radicals, in both purified hemoglobin and red blood cells (RBC), induced by ultraviolet A (UVA)-KP by using "immuno-spin trapping," a novel approach that combines the specificity of spin trapping with the sensitivity of antigen-antibody interactions. The methemoglobin (metHb) radicals react covalently with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form nitroxyl radical adducts that are oxidized to the corresponding nitrone adducts, which in turn are specifically recognized by antiserum against DMPO nitrone. We found that the formation of nitrone adducts in metHb depended on the UVA dose, the KP concentration and the presence of DMPO, as determined by enzyme-linked immunosorbent assay and Western blotting. Adduct formation decreased when irradiation was carried out in the presence of catalase or nitrogen, suggesting that H^sub 2^O^sub 2^ plays a key role in KP-UVA-induced metHb radical formation. KP in the dark did not generate metHb radical-derived nitrone adducts, whereas UVA alone resulted in the formation of metHb radical-derived nitrone adducts that increased with UVA dose from 4 to 10 J/cm^sup 2^. However, KP (25 and 200 [mu]M) plus UVA (4 and 10 J/cm^sup 2^) resulted in a significant increase in the formation of metHb radical-derived nitrone adducts as compared with UVA or KP alone, indicating that KP photosensitized the production of the metHb radicals in the presence of UVA. In contrast, no metHb radical-derived nitrone adduct was detected in the absence of DMPO, even though KP and UVA were present. We also detected the hemoglobin radical formation in RBC as well as in hemolysates. The endogenous antioxidants and exogenous reduced glutathione inhibited the protein radical formation. These studies have shown that the immuno-spin-trapping technique can be used to detect radical damage in proteins as a result of photosensitizing reactions. The successful detection of protein radical formation caused by KP photosensitization could help further understand the photoallergic effect of this NSAID.

INTRODUCTION

The nonsteroidal anti-inflammatory drug ketoprofen (KP) (Scheme 1) is an arylpropionic acid derivative widely used for the treatment of rheumatic diseases (1). Recent studies have shown that KP causes photoallergic dermatitis and phototoxic effects (2-4). Photoallergic effects, in particular, are presumed to result from damage to proteins, which then act as allergens.

When ultraviolet (UV) or visible light damages proteins, they often form free radical intermediates that can, in principle, be detected using electron spin resonance (ESR) spin trapping. However, recently, an antiserum directed against the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was developed and validated (5). This new approach, immuno-spin trapping, which combines the specificity of spin trapping with the sensitivity of antigen-antibody interactions, has several advantages over ESR spin trapping (for discussion see Detweiler et al. [5] and Ramirez et al. [6]). To test the ability of immuno-spin trapping to detect light-induced protein damage, we used a system containing KP, hemoglobin or erythrocytes and DMPO.

We chose KP as a photosensitizer because it is known to induce hemolysis of erythrocytes upon UVA irradiation. Furthermore, KP acts via a mechanism that does not photogenerate KP-derived radical intermediates that are trapped by DMPO (7). Thus, when KP is photolysed in the presence of protein and DMPO, the DMPO will not prevent KP or its photoproducts from generating protein-derived radicals (7,8).

As a test protein we focused on hemoglobin, which has several advantages. It is abundant in erythrocytes and is known to form free radicals when damaged by H^sub 2^O^sub 2^, as detected by ESR spectroscopy (9). Its free radical products are known to react with DMPO; DMPO has been shown to form radical adducts with sperm whale (10) and human metmyoglobins (metMb) (11,12). These adducts are known to be recognized by the anti-DMPO serum (5).

To date, there have been no reports on protein radical production induced by KP photosensitization. In the present work we report that photoirradiated KP reacts with hemoglobin in various forms to produce radicals that can be detected by the immuno-spin-trapping technique. We have also tested KP as a source of protein damage in intact cells.

MATERIALS AND METHODS

Materials. KP, human methemoglobin (metHb), oxyhemoglobin (oxyHb), horse skeletal muscle (metMb), diethylenentriamine pentaacetic acid (DTPA), superoxide dismutase (SOD), and reduced glutathione (GSH) were purchased from Sigma Chemical Co. (St. Louis, MO). Beef liver catalase was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The spin-trap DMPO from Aldrich Chemical Co. (Chicago, IL) was vacuum distilled and stored at -20[degrees]C until use. All buffers were treated with Chelex 100(R) (Bio-Rad Laboratories, Hercules, CA) at 4[degrees]C for 24 h followed by addition of 100 [mu]M DTPA to avoid possible transition metal-catalyzed reactions.