DMD

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

Drug Metabolism and Disposition Fast Forward
First published on October 7, 2005; DOI: 10.1124/dmd.105.006890

0090-9556/06/3401-16-18$20.00
DMD 34:16-18, 2006

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.105.006890v1
34/1/16    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vyas, P. M.
Right arrow Articles by Svensson, C. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vyas, P. M.
Right arrow Articles by Svensson, C. K.
SHORT COMMUNICATION

ROLE OF HUMAN CYCLOOXYGENASE-2 IN THE BIOACTIVATION OF DAPSONE AND SULFAMETHOXAZOLE

Piyush M. Vyas, Sanjoy Roychowdhury, and Craig K. Svensson

Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa

(Received August 15, 2005; accepted October 4, 2005)


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
Sulfamethoxazole (SMX) and dapsone (4,4'-diaminodiphenylsulfone, DDS) are believed to mediate their adverse effects subsequent to bioactivation to their respective arylhydroxylamine and arylnitroso metabolites, resulting in covalent adduct formation with intracellular proteins. Various bioactivating enzymes, such as cytochromes P450 and myeloperoxidase, have been shown to be capable of catalyzing the N-oxidation of these compounds. We assessed the role of human cyclooxygenase-2 (COX-2) in the metabolism and subsequent adduct formation of DDS and SMX using recombinant human COX-2. Using an adduct-specific enzyme-linked immunosorbent assay, we found that the complete enzyme system gave rise to covalent adducts. However, the nonspecific COX inhibitor indomethacin did not reduce the amount of covalent adduct formed. Formation of the arylhydroxylamine metabolites was demonstrated via high performance liquid chromatography coupled with UV absorption. Metabolite formation was found to be secondary to the H2O2 in the incubation mixture and was not enzyme-mediated. Hence, COX-2 does not play a direct role in the bioactivation of these parent drugs to their arylhydroxylamine metabolites.

Administration of sulfonamide antimicrobials such as sulfamethoxazole (SMX) and the sulfone dapsone (4,4'diaminodiphenylsulfone, DDS) has been associated in humans with hypersensitivity reactions that include fever, skin eruptions, hepatotoxicity, and blood dyscrasias (Dujovne et al., 1967Go; Rieder et al., 1989Go). The mechanism of sulfonamide hypersensitivity is not well understood, but has been hypothesized to be secondary to the generation of the reactive oxidative metabolites such as SMX-hydroxylamine (SMX-NOH) and DDS-hydroxylamine (DDS-NOH) and their respective nitroso derivatives (Svensson, 2003Go). The arylhydroxylamine metabolites of DDS and SMX, unlike the parent sulfonamides, are cytotoxic to a variety of cells in vitro and have been shown to generate reactive oxygen species (Rieder et al., 1995Go; Reilly et al., 2000Go; Vyas et al., 2005Go). The parent drugs and their arylhydroxylamine metabolites have been demonstrated to haptenize cellular proteins, which may lead to immune-mediated cutaneous reactions (Manchanda et al., 2002Go; Naisbitt et al., 2002Go; Roychowdhury et al., 2005Go). Thus, the bioactivation of these arylamine xenobiotics to their respective arylhydroxylamine metabolites may be the first and most important step in the initiation of these reactions.

Previous studies have demonstrated the ability of various oxidizing enzymes, including CYP2C9, CYP2E1, and CYP3A4, as well as myeloperoxidase to bioactivate arylamines in vitro (Cribb et al., 1990Go, 1995Go; Mitra et al., 1995Go; Cashman et al., 1999Go; Winter et al., 2000Go). Cyclooxygenases (COXs), or prostaglandin H synthase, have also been shown to bioactivate heterocyclic amines to their hydroxylamine metabolites (Liu and Levy, 1998Go). Procainamide, an arylamine antiarrhythmic agent, has also been found to be oxidized to its arylhydroxylamine and arylnitroso metabolites by COX-2 (Goebel et al., 1999Go). The N-oxidation of 4-chloroaniline has also been reported to be mediated by COXs (Golly and Hlavica, 1985Go). Based upon these observations, we tested the hypothesis that COX-2 may bioactivate SMX and DDS, resulting in protein haptenation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
Materials. Unless specified otherwise, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific Co. (Pittsburgh, PA). DDS and SMX hydroxylamine metabolites were synthesized as described previously (Vyas et al., 2005Go). Human recombinant COX-2 was obtained from Cayman Chemical (Ann Arbor, MI). Rabbit antiserum was raised against SMX- and DDS-keyhole limpet hemocyanine conjugates, and specificity was assessed as described previously (Reilly et al., 2000Go). Goat anti-rabbit antibody conjugated with alkaline phosphatase was purchased from Invitrogen (Carlsbad, CA). Microtiter ELISA plates (96-well) were obtained from Rainin Instruments (Woburn, MA).

Adduct Formation of DDS by Human Recombinant COX-2. An incubation mixture containing COX-2 (100 units), arachidonic acid (1 mM), hematin (1 µM), EDTA (5 mM), and H2O2 (1 mM) in Tris-HCl buffer (50 mM, pH 8.00), with and without INDO or DDS (100 µM each), was incubated for 1 h at 37°C. A control incubation containing only buffer was also included to account for nonspecific binding to the microtiter plate. After 1 h of incubation, the reaction mixture was left overnight at 4°C for complete adhesion of protein to the microtiter plate. In addition, a DDS-bovine serum albumin adduct was added to a set of wells at this time to serve as a positive control for adduct detection. After 24 h, covalent adducts were determined by an adduct-specific ELISA as described previously (Reilly et al., 2000Go).

Determination of COX-2-mediated Arylhydroxylamine Formation of DDS and SMX via High Performance Liquid Chromatography (HPLC). DDS or SMX (800 µM) was added to the incubation mixture described above, with and without COX-2 (100 units), for 1 h at 37°C. Ascorbic acid (1 mM) was included in all incubations to stabilize the arylhydroxylamine formed. After 1 h, the reaction was terminated by addition of 3 ml of ethyl acetate and the arylhydroxylamine metabolites determined as described previously (Reilly et al., 2000Go). As a positive control to assure the catalytic activity of COX-2 under these incubation conditions, reactive oxygen species generation was determined via the oxidation of the fluorescent dye 2',7'-dichlorodihydrofluorescein (5 µM), as we have previously described (Vyas et al., 2005Go).

Determination of H2O2-Mediated Arylhydroxylamine Formation of SMX or DDS via HPLC. SMX or DDS (800 µM) was incubated in Tris-HCl buffer (50 mM, pH 8.00) and ascorbic acid (1 mM) with increasing concentrations of H2O2, ranging from 0.01 µM to 10 mM. The incubation mixtures were kept for 1 h at 37°C. After 1 h, the metabolites formed were extracted and quantified via HPLC.

Statistical Analysis. Data are presented as mean (S.D.). Statistical comparisons between two groups were made using either Student's t test (parametric method) for normalized data or Friedman's rank sum test (nonparametric method) for the data that did not pass the normality test. For the comparison between more than two groups, analysis of variance and the Holm-Sidak method for multiple pairwise comparisons was used. A value of p < 0.05 was considered to be significant.


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
Antimicrobial sulfonamides, such as SMX, and the sulfone DDS are important drugs for the treatment of Pneumocystis carinii pneumonia, especially in AIDS patients (Goldie et al., 2002Go). However, in this patient population, these drugs are commonly associated with minor to severe cutaneous drug reactions, which are believed to be secondary to their metabolism to reactive arylhydroxylamine and arylnitroso derivatives (Svensson, 2003Go). Because COX-2 is induced in the presence of various forms of environmental and pathological stress (Maier et al., 1990Go; Buckman et al., 1998Go), we hypothesized that the increased frequency of these reactions observed in AIDS patients may be secondary to elevated levels of these reactive metabolites formed as a result of COX-2 induction. As an initial test of this hypothesis, we sought to determine whether COX-2 was capable of generating these reactive metabolites.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Bioactivation and subsequent adduct formation of DDS by human recombinant COX-2. DDS (100 µM) was incubated in a mixture containing COX-2 (100 units), arachidonic acid (1 mM), hematin (1 µM), EDTA (5 mM), and H2O2 (1 mM) in Tris-HCl buffer (50 mM, pH 8.00) with and without INDO (100 µM) for 1 h at 37°C. Controls containing buffer, COX-2, and COX-2 + INDO were used to determine the nonspecific binding of anti-DDS rabbit sera. Covalent adducts were determined using adduct-specific ELISA as described under Materials and Methods. Data presented represent the mean (S.D.) optical density of six replicates. Data were analyzed statistically using analysis of variance and the Holm-Sidak test for multiple pairwise comparisons. *, p < 0.05 compared to buffer control, COX-2, and COX-2 + INDO. DDS-bovine serum albumin (DDS-BSA) was used as positive control.

 
Using an adduct-specific ELISA, we found that addition of DDS to an incubation mixture containing COX-2, hematin, EDTA, arachidonic acid, and H2O2 resulted in covalent adduct formation (Fig. 1). Importantly, however, a nonselective inhibitor for COX-1 and COX-2 (INDO) did not attenuate the formation of drug/metabolite-protein adducts. In addition, use of lower concentrations of H2O2 in the incubation mixture did not give rise to detectable covalent adducts. Various controls ruled out nonspecific binding of the primary antisera or secondary antibody as causing artifactual results. We confirmed the catalytic activity of COX-2 in this incubation mixture using reactive oxygen species generation as a positive control, as described under Materials and Methods. There was a 2.3-fold increase in the fluorescence of incubations with COX-2 as compared to incubations without COX-2 (data not shown), confirming the catalytic activity of the enzyme.

Formation of the arylhydroxylamine metabolites of DDS and SMX in the incubation mixture was confirmed via HPLC (Fig. 2). However, removal of the enzyme itself from the incubation gave rise to similar amounts of arylhydroxylamine metabolite (Fig. 2). This observation suggested that some other component in the incubation mixture was resulting in the chemical oxidation of DDS and SMX. Since removal of H2O2 from the incubation mixture prevented the formation of the arylhydroxylamine (data not shown), we suspected that we were observing a chemical oxidation of the arylamines. Indeed, we found that H2O2 alone gave rise to a concentration-dependent oxidation of SMX and DDS (Fig. 3).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2. Determination of COX-2-mediated arylhydroxylamine formation of DDS and SMX by HPLC. SMX or DDS (800 µM) was incubated in a mixture containing arachidonic acid (1 mM), hematin (1 µM), EDTA (5 mM), ascorbic acid (1 mM), and H2O2 (1 mM) in Tris-HCl buffer (50 mM, pH 8.00), with and without COX-2 (100 units), for 1 h at 37°C. After 1 h, the formed SMX-NOH or DDS-NOH was extracted and quantified via HPLC as described under Materials and Methods. Data presented represent the mean (S.D.) amount of SMX-NOH formed for nine replicates of each condition. Data were analyzed statistically using Student's t test, with no differences between incubation conditions observed.

 


View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3. Determination of H2O2-mediated arylhydroxylamine formation of DDS and SMX by HPLC. DDS and SMX (800 µM) were incubated with increasing concentrations of H2O2 with ascorbic acid (1 mM) in Tris-HCl buffer (50 mM, pH 8.00) for 1 h at 37°C. After 1 h, the formed hydroxylamine metabolites were extracted and quantified via HPLC as described under Materials and Methods. Data presented represent the mean (S.D.) amount of DDS-NOH and SMX-NOH formed for nine replicates of each condition.

 
Our results indicate that enzymatic oxidation of SMX and DDS by COX-2 is negligible, but that chemical oxidation via H2O2 may occur. These results are consistent with the report of Rubin and Curnette (1989Go), who demonstrated that H2O2 was able to oxidize procainamide, giving rise to an arylhydroxylamine metabolite. Additionally, Goebel et al. (1999Go) found that the covalent binding of procainamide arising from an incubation mixture almost identical to that used in the present study was markedly attenuated when H2O2 was removed from the incubation. However, these investigators found that in addition to H2O2, hematin was required to obtain similar levels of covalent binding in the absence of COX-2. In contrast, we did not find hematin to be an essential component for the N-oxidation of SMX or DDS (data not shown).

Taken together, these data suggest that COX-2 is unlikely to play a significant role in mediating the formation of reactive metabolites of sulfonamides. Indeed, we have recently found that the protein haptenation observed when SMX and DDS are incubated with normal human keratinocytes is not altered by the addition of nonspecific and specific inhibitors of COX (Wurster et al., 2004Go). In addition, incubation of keratinocytes with proinflammatory cytokines, which results in the induction of COX-2, does not enhance the covalent binding of SMX or DDS in these cells (F. D. Khan, S. Roychowdhury, P. M. Vyas, and C. K. Svensson, unpublished observations). These observations indicate that induction of COX-2 in the presence of environmental or pathological stress is unlikely to play a role in the increased frequency of adverse reactions to sulfonamides in AIDS patients.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants AI41395 and GM63821 to C.K.S.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.006890.

ABBREVIATIONS: SMX, sulfamethoxazole; DDS, 4,4'-diaminodiphenylsulfone, dapsone; DDS-NOH, dapsone hydroxylamine; INDO, indomethacin; SMX-NOH, sulfamethoxazole hydroxylamine; COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography.

Address correspondence to: Dr. Craig K. Svensson, Division of Pharmaceutics, College of Pharmacy, The University of Iowa, 115 S. Grand Avenue, S213 PHAR, Iowa City, IA 52242. E-mail: craig-svensson{at}uiowa.edu


   References
Top
 Abstract
 Materials and Methods
 Results and Discussion
 References
 


Buckman S, Gresham A, Hale P, Hruza G, Anast J, Masferrer J, and Pentland A (1998) COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 19: 723–729.[Abstract/Free Full Text]

Cashman JR, Xiong YN, Xu L, and Janowsky A (1999) N-oxygenation of amphetamine and methamphetamine by the human flavin-containing monooxygenase (form 3): role in bioactivation and detoxication. J Pharmacol Exp Ther 288: 1251–1260.[Abstract/Free Full Text]

Cribb AE, Miller M, Tesoro A, and Spielberg SP (1990) Peroxidase-dependent oxidation of sulfonamides by monocytes and neutrophils from humans and dogs. Mol Pharmacol 38: 744–751.[Abstract]

Cribb AE, Spielberg SP, and Griffin GP (1995) N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine in human and rat hepatic microsomes. Drug Metab Dispos 23: 406–414.[Abstract]

Dujovne D, Chan C, and Zimmerman H (1967) Sulfonamide hepatic injury. N Engl J Med 377: 785–788.

Goebel C, Vogel C, Wulferink M, Mittmann S, Sachs B, Schraa S, Abel J, Degen G, Uetrecht J, and Leichmann E (1999) Procainamide, a drug causing lupus, induces prostaglandin H synthase-2 and formation of T cell-sensitizing drug metabolites in mouse macrophages. Chem Res Toxicol 12: 488–500.[CrossRef][Medline]

Goldie SJ, Kaplan JE, Losina E, Weinstein MC, Paltiel AD, Seage GR, Craven DE, Kimmel AD, Zhang H, Cohen CJ, et al. (2002) Prophylaxis for human immunodeficiency virus-related Pneumocystis carinii pneumonia: using simulation modeling to inform clinical guidelines. Arch Intern Med 162: 921–928.[Abstract/Free Full Text]

Golly I and Hlavica P (1985) N-Oxidation of 4-chloroaniline by prostaglandin synthase. Redox cycling of radical intermediate(s). Biochem J 226: 803–809.[Medline]

Liu Y and Levy G (1998) Activation of heterocyclic amines by combinations of prostaglandin H synthase-1 and -2 with N-acetyltransferase 1 and 2. Cancer Lett 133: 115–123.[CrossRef][Medline]

Maier J, Hla T, and Maciag T (1990) Cyclooxygenase is an immediate early gene induced by interleukin-1 in human endothelial cells. J Biol Chem 265: 10805–10808.[Abstract/Free Full Text]

Manchanda T, Hess D, Dale L, Ferguson SG, and Rieder MJ (2002) Haptenation of sulfonamide reactive metabolites to cellular proteins. Mol Pharmacol 62: 1011–1026.[Abstract/Free Full Text]

Mitra AK, Thummel KE, Kalhorn TF, Kharasch ED, Unadkat JD, and Slattery JT (1995) Metabolism of dapsone to its hydroxylamine by CYP2E1 in vitro and in vivo. Clin Pharmacol Ther 58: 556–566.[CrossRef][Medline]

Naisbitt DJ, Farrell J, Gordon SF, Maggs JL, Burkhart C, Pichler WJ, Pirmohamed M, and Park BK (2002) Covalent binding of the nitroso metabolite of sulfamethoxazole leads to toxicity and major histocompatibility complex-restricted antigen presentation. Mol Pharmacol 62: 628–637.[Abstract/Free Full Text]

Reilly TP, Lash LH, Doll MA, Hein DW, Woster PM, and Svensson CK (2000) A role for bioactivation and covalent binding within epidermal keratinocytes in sulfonamide-induced cutaneous drug reactions. J Investig Dermatol 114: 1164–1173.[CrossRef][Medline]

Rieder MJ, Krause R, and Bird IA (1995) Time-course of toxicity of reactive sulfonamide metabolites. Toxicology 95: 141–146.[CrossRef][Medline]

Rieder MJ, Uetrecht J, Shear NH, Cannon M, Miller M, and Spielberg SP (1989) Diagnosis of sulfonamide hypersensitivity reactions by in-vitro "rechallenge" with hydroxylamine metabolites. Ann Intern Med 110: 286–289.[CrossRef][Medline]

Roychowdhury S, Vyas PM, Reilly TP, Gaspari AA, and Svensson CK (2005) Characterization of the formation and localization of sulfamethoxazole and dapsone-associated drug-protein adducts in human epidermal keratinocytes. J Pharmacol Exp Ther 314: 43–52.[Abstract/Free Full Text]

Rubin RL and Curnette JT (1989) Metabolism of procainamide to the cytotoxic hydroxylamine by neutrophils activated in vitro. J Clin Investig 83: 1336–1343.[Medline]

Svensson CK (2003) Do arylhydroxylamine metabolites mediate the idiosyncratic reactions associated with sulfonamides and sulfones? Chem Res Toxicol 16: 1034–1043.

Vyas PM, Roychowdhury S, Woster PM, and Svensson CK (2005) Reactive oxygen species generation and its role in the differential cytotoxicity of the arylhydroxylamine metabolites of sulfamethoxazole and dapsone in normal human epidermal keratinocytes. Biochem Pharmacol 70: 275–286.[CrossRef][Medline]

Winter H, Wang Y, and Unadkat J (2000) CYP 2C8/9 mediate dapsone N-hydroxylation at clinical concentrations of dapsone. Drug Metab Dispos 28: 865–868.[Abstract/Free Full Text]

Wurster W, Nemes R, Lamba J, Schuetz EG, Blaisdell J, Goldstein JA, Reilly TP, and Svensson CK (2004) Bioactivation of sulfamethoxazole (SMX) and dapsone (DDS) in normal human epidermal keratinocytes (NHEK) results in the formation of drug-protein adducts. Allergologie 4: 169.


This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
J. P. Sanderson, F. J. Hollis, J. L. Maggs, S. E. Clarke, D. J. Naisbitt, and B. K. Park
Nonenzymatic Formation of a Novel Hydroxylated Sulfamethoxazole Derivative in Human Liver Microsomes: Implications for Bioanalysis of Sulfamethoxazole Metabolites
Drug Metab. Dispos., December 1, 2008; 36(12): 2424 - 2428.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. P. Sanderson, D. J. Naisbitt, J. Farrell, C. A. Ashby, M. J. Tucker, M. J. Rieder, M. Pirmohamed, S. E. Clarke, and B. K. Park
Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling
J. Immunol., May 1, 2007; 178(9): 5533 - 5542.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
P. M. Vyas, S. Roychowdhury, F. D. Khan, T. E. Prisinzano, J. Lamba, E. G. Schuetz, J. Blaisdell, J. A. Goldstein, K. L. Munson, R. N. Hines, et al.
Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: I. Expression and Role of Cytochromes P450
J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 488 - 496.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
P. M. Vyas, S. Roychowdhury, S. B. Koukouritaki, R. N. Hines, S. K. Krueger, D. E. Williams, W. M. Nauseef, and C. K. Svensson
Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: II. Expression and Role of Flavin-Containing Monooxygenases and Peroxidases
J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 497 - 505.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.105.006890v1
34/1/16    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vyas, P. M.
Right arrow Articles by Svensson, C. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vyas, P. M.
Right arrow Articles by Svensson, C. K.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition