Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity

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Abstract

Drug induced allergies are believed to be induced by conjugates consisting of biological macromolecules and active metabolites. The present study investigated whether guinea pig glutathione S-transferase (gpGST), a protein that binds with sulfanilamide (SA) and sulfamethoxazole (SMX), could be detected in the liver cytosol fraction of guinea pigs that intraperitoneally received SA or SMX, and whether gpGST is a carrier protein. We synthesized three nitroso compounds, i.e., 4-nitroso-sulfanilamide (SA-NO), 4-nitrososulfamethoxazole (SMX-NO) and fluorescent-labeled nitroso compound (DNSBA-NO), and examined binding quantities of nitroso compounds to gpGST purified from untreated female guinea pigs. Furthermore, the concentrations of IgG in serum antibody for nitroso compounds were estimated using ELISA. When guinea pigs were sensitized using the three nitroso compounds, the dose dependent skin reactions were confirmed with each compound. In addition, sensitized guinea pigs using each nitroso compound showed positive skin reactions at an elicitation test performed using gpGST alone. The results confirmed synthesis of antibody against gpGST due to hapten sensitization. Therefore, when a nitroso compound binds with gpGST in the body of guinea pigs, nitroso-gpGST acts as a neoantigen, which induces synthesis of autoantibody. Thus, gpGST appears to be one of the carrier proteins that induce sulfa drug-induced allergies. Immunization of guinea pigs with active metabolite of drugs may give information for predicting the occurrence of delayed type hypersensitivity in human.

Highlights

► Active metabolite of Sulfanilamide or Sulfamethoxazole is nitroso compound. ► Guinea pig glutathione S-transferase is a protein binded with the nitroso compound. ► Immunization of guinea pigs with the nitroso compound may give informations. ► Nitroso-glutathione S-transferase acts as a neoantigen in the body of guinea pigs.

Introduction

Sulfamethoxazole (SMX) has been frequently used to treat carinii pneumonia in HIV (human immunodeficiency virus) patients. However, SMX has been documented to occasionally induce severe skin disorders, such as morbilliform rash and delayed type hypersensitivity (DTH) [1], [2], [3]. Many studies have also reported that SMX-NO, an active metabolite of SMX, is involved in DTH [4], [5], [6], [7], [8]. With regard to carrier proteins that bind to active metabolites, studies have documented 30- or 40-kDa SMX-binding proteins in the serum of patients with allergic predispositions who took SMX [2], [9]. One study found antibodies against rat native microsomal proteins (55, 80 and 96 kDa) in the serum of a patient with a past history of SMX-induced DTH [5], [10]. Other study found the covalent adduct formation by SMX-hydroxylamine in normal human epidermal keratinocytes [11]. Furthermore, recent studies found that SMX-induced DTH was due to interactions between SMX-NO and T cells [12], [13], [14], [15], [16], [17].

As part of research to elucidate the onset mechanism of sulfa drug induced allergy, we examined the metabolic activation of sulfanilamide (SA), the base compound of sulfonamide, and clarified that the active metabolites of SA are hydroxylamine (SA-NHOH) and nitroso (SA-NO) [18], [19]. Using fluorescent-labeled compounds (DNSBA-HA and DNSBA-NO), the correlation between the intensity of guinea pig skin reactions and the synthesis of glutathione conjugate were clarified. The results suggest that DNSBA-NO is the ultimate active metabolite and that its binding site for proteins is the same for glutathione. We have previously isolated and identified physiologically important proteins and enzymes as binding proteins, such as ubiquitin, fatty acid binding protein, retinol dehydrogenase and glutathione S-transferase, from the liver of rats and guinea pigs that reacted with DNSBA-HA [20]. These binding proteins may be carrier proteins involved in sulfa drug-induced skin allergies.

Hence, the present report focused on guinea pig glutathione S-transferase (gpGST), which binds to DNSBA-NO, and determined whether gpGST is a carrier protein. In other words, in order to clarify whether the binding proteins that were detected in vitro could be detected in vivo, we intraperitoneally administered SA or SMX to guinea pigs and then used hapten antibodies to isolate and identify drug-binding proteins. Using the active metabolites of SA and SMX (SA-NO and SMX-NO), we examined guinea pig skin reactions and the synthesis of antibodies against gpGST, autologous guinea pig proteins.

Section snippets

Animals and chemicals

Adult female Hartley guinea pigs (6 weeks, 350–400 g) and adult male rabbit (2–3 kg) were purchased from Kyudo Co. Ltd (Saga, Japan). Animals were allowed free access to water and food (CG-7and CR-3, Tokyo Clea, Japan), and were separately housed under control conditions at a temperature of 24 °C and lighting from 06:30 to 18:30 daily.

Sulfanilamide (SA), sulfamethoxazole (SMX), hydrogen peroxide (H2O2), bovine serum albumin (BSA), 1-chloro-2,4-dinitrobenzene (DNCB), glutathione reduced form (GSH)

Identification of SA or SMX binding protein in liver cytosol fraction of the guinea pigs treated by SA or SMX

The purified SA or SMX binding protein was examined by SDS-PAGE. The positive immunostain by anti (SA-KLH) rabbit serum or anti (SMX-KLH) rabbit serum was detected in the protein band stained with CBB. The SDS-PAGE of SMX-binding protein purified is shown in Fig. 4. The SDS-PAGE of SA-binding protein was also purified the same as that of SMX-binding protein (data not shown).

Each immuno positive band in SDS-PAGE gel was separated and blotted on a PVDF membrane. N-terminal amino acid sequences of

Discussion

In a previous report [20], we used a fluorescent-labeled model compound for SA hydroxylamine to purify and identify binding proteins from the liver cytosol and microsomal fractions of rats and guinea pigs. From the rat liver cytosol fraction, ubiquitin (10 kDa) and fatty acid binding protein (30 kDa), and from the rat liver microsomes retinol dehydrogenase (35 kDa) were isolated. From the guinea pig liver cytosol fraction glutathione S-transferase M (gpGST M) was also isolated as subunits size of

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