Environ. Sci. Technol. 2008, 42, 106–112
NMR Investigation of Enzymatic Coupling of Sulfonamide Antimicrobials with Humic Substances H E I D I M . B I A L K †,| A N D J O E L A . P E D E R S E N * ,†,‡,§ Molecular and Environmental Toxicology Center, Department of Soil Science, and Environmental Chemistry and Technology Program, University of Wisconsin, Madison, Wisconsin 53706
Received April 02, 2007. Revised manuscript received August 29, 2007. Accepted September 12, 2007.
Phenoloxidases mediate the oxidative transformation of soil phenolic constituents, contributing to the formation of humic substances and the chemical incorporation of some xenobiotic organic compounds into natural organic matter. We previously demonstrated phenoloxidase-mediated covalent coupling of sulfonamide antimicrobials with model humic constituents. Here, we investigate fungal peroxidase-mediated covalent coupling of 13C-sulfamethazine and 15N-sulfapyridine to humic substances. 1H–13C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance spectroscopy provided an initial indication of peroxidase-mediated covalent binding of 13Csulfamethazine to humic acid. To confirm the role of the sulfonamide anilinic nitrogen in coupling to humic acid and to determine the nature of the covalent linkage, we incubated 15Nsulfapyridine with humic acid and peroxidase and examined reaction products in 1H–15N heteronuclear multiple bond (HMBC) experiments. The HMBC spectra revealed the presence of Michael adducts (i.e., anilinohydroquinones, anilinoquinones) and possibly other covalent linkages. No evidence for Schiff base formation was observed. Analogous experiments with the modelhumicconstituentcatecholprovidedcorroboratingevidence for these assignments. Michael adducts are expected to exhibit greater environmental stability than imine linkages that can form between sulfonamides and 2,6-dimethoxyphenols. Because the free anilinic nitrogen is required for the bioactivity of sulfonamide antimicrobials, nucleophilic addition occurring through this moiety could result in the biochemical inactivation of these compounds.
Introduction The presence of antibiotics in surface waters, groundwater, and soil (1–4) has prompted concern about possible impacts on microbial community composition and promotion of antibiotic resistance (5). Sulfonamide antimicrobial agents * Corresponding author address: Department of Soil Science, University of Wisconsin, 1525 Observatory Drive, Madison, WI 537061299; phone: (608) 263-4971; fax: (608) 265-2595; e-mail:
[email protected]. † Molecular and Environmental Toxicology. ‡ Department of Soil Science. § Environmental Chemistry and Technology Program. | Present address: U.S. Department of Agriculture, U.S. Dairy Forage Research Center, 1925 Linden Dr., West Madison, WI 53706. 106
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are of particular interest because of their widespread application in livestock production (e.g., ∼409 000 kg of sulfathiazole and 363 000 kg of sulfamethazine are used annually in swine production; ref 6) and in human medicine. These synthetic, bacteriostatic sulfanilamide derivatives rank among the most frequently detected pharmaceuticals in surface waters (1). A significant fraction of administered sulfonamides are excreted in their active form or as acetylated metabolites that are readily reactivated (7). These factors provide impetus for better understanding the fate of these compounds in soil and subsurface environments. Chemical incorporation (covalent coupling) of xenobiotic compounds into natural organic matter (NOM) can be an important process for some contaminants and can result in the immobilization and reduced bioactivity of the toxicant. Covalent coupling reactions appear especially important for contaminants containing phenolic and aromatic amine moieties (e.g., chlorinated phenols, reduced TNT metabolites; refs 8–13), and are often mediated by phenoloxidases (14–17). Phenoloxidases (e.g., peroxidases, laccases, tyrosinases) are secreted extracellularly by a variety of microorganizms, released during cell lysis, and exuded by plant roots (18–20). Covalent incorporation of substituted phenols and anilines into NOM is accomplished by either creating sites within NOM for nucleophilic attack by the xenobiotic compound (12, 14, 15, 17), or by promoting radical–radical coupling between the xenobiotic compound and NOM constituents (1, 2, 21). We previously demonstrated significant phenoloxidasemediated transformation of sulfonamides in the presence of specific model humic constituents (14, 15). Sulfonamides do not appear to be substrates for these enzymes (14, 15). Instead, phenoloxidases oxidize model humic constituents to create sites susceptible for reactions with sulfonamides (e.g., nucleophilic attack by the anilinic nitrogen) (14, 15). Of the substituted phenols we previously examined, the extent of sulfonamide antimicrobial transformation was largest in incubations with 2,6-dimethoxy phenols and o-dihydroxyphenols regardless of the enzyme employed (i.e., peroxidases, laccase, tyrosinase). We demonstrated that in reactions mediated by Arthromyces ramosus peroxidase (ARP) and fungal laccase, sulfonamides can form Schiff bases and Michael adducts with model humic constituents (14, 15). ARP-mediated reaction of sulfamethazine with the 2,6dimethoxyphenol syringic acid produced an unprotonated imine quinone (Schiff base) (14). Laccase-mediated reaction of the structurally similar antimicrobial sulfapyridine with the o-dihydroxyphenol protocatechuic acid yielded Michael adducts with anilinoquinone and anilinohydroquinone linkages (15). While these studies shed light on the types of covalent linkages sulfonamides may form with NOM in soils and sediments, further investigation was required to demonstrate that sulfonamides covalently couple with humic substances and to determine the types of adducts formed. The objectives of this study were to determine whether phenoloxidases mediate covalent coupling of sulfonamide antimicrobials with humic substances and to identify the dominant covalent linkages formed. To achieve these objectives, we incubated stable isotope-labeled sulfonamide antimicrobials with soil humic acid and ARP. The use of 15Nand 13C-labeled sulfonamides facilitated identification of the covalent linkages formed between these antimicrobials and humic acid constituents by multidimensional nuclear magnetic resonance (NMR) spectroscopy. To our knowledge, this report provides the first direct spectroscopic evidence of covalent bond formation between sulfonamide antimicrobi10.1021/es070779d CCC: $40.75
2008 American Chemical Society
Published on Web 11/22/2007
als and soil humic substances, a process that may immobilize and detoxify these bacteriostatic agents.
Materials and Methods Chemicals. We purchased [phenyl-13C6]-sulfamethazine from Cambridge Isotope Laboratories (Andover, MA). DMSO-d6 (99% purity) and Dowex cation-exchange resin were obtained from Sigma Aldrich (St. Louis, MO). We synthesized 15Nsulfapyridine as previously described (15). Elliot soil humic acid (1S102H), isolated from Elliot silt loam, a typical example of fertile prairie soils in Iowa, Illinois, and Indiana, was purchased from the International Humic Substances Society (St. Paul, MN). Information on the properties of this humic acid can be obtained at www.ihss.gatech.edu/. Phenoloxidases. A. ramosus peroxidase (peroxidases; donor, H2O2 oxidoreductase E.C. 1.11.1.7) had a manufacturerreported Reinheitzahl of 2.5 and an activity of 45 U · mg-1 (Sigma). One unit of peroxidase activity was defined as the amount of enzyme required to form 1.0 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 and 20 °C. The enzyme was stored in a desiccator at -20 °C; fresh solutions were prepared for each experiment. Peroxidases promote one-electron oxidation of substrates using peroxide as the electron acceptor. Peroxidase-mediated oxidation of phenols yields phenoxy radicals that can undergo polymerization reactions (22). The multicopper oxidase laccase (p-diphenol; oxygen oxidoreductase E.C. 1.10.3.2) catalyzes the oxidation of phenolic compounds and some aromatic amines to free radicals (23). Laccase from the white rot fungus Trametes versicolor had a manufacturer-reported activity of 23.7 U · mg-1 (Novo Nordisk, Princeton, NJ). Laccase activity was defined as the amount of enzyme needed to cause a 1.0 min-1 change in absorbance at 468 nm in 3.4 mL of 1 mM 2,6dimethoxyphenol in citrate-phosphate buffer (pH 3.8). Reactions of Sulfonamide Antimicrobials with Humic Substances. We evaluated the covalent coupling of sulfonamide antimicrobials to soil humic acid in the presence and absence of ARP. Incubations of 13C-sulfamethazine or 15Nsulfapyridine with humic acid were conducted in a manner similar to that employed by Thorn et al. (10). Briefly, Elliot humic acid (8 mg) was added to 10 mL of distilled, deionized water (ddH2O; NANOpure Ultrapure Water System, Barnstead International, Dubuque, IA). The solution pH was adjusted to 6.0 with 1 N NaOH and remained stable throughout the incubation period. After dissolution of the humic acid, [phenyl-13C6]-sulfamethazine or 15N-sulfapyridine were added to final concentrations of 3.0 or 5.0 mM, respectively. Samples containing enzyme received ARP and H2O2 to achieve initial concentrations of 1.6 U mL-1 and 0.5 mM, respectively. Solutions were allowed to stir for 3 h. Acid washed, silanized, 50-mL amber glass vials with Teflon-lined screw caps were employed as reaction vessels. After the 3-h incubation, solutions were passed through a Dowex MSC-1 cationexchange resin to remove the enzyme (10). Unreacted sulfonamide and sulfonamide–humic adducts were eluted with ddH2O. Eluants were freeze-dried and dissolved in ∼1.5 mL of DMSO-d6 for NMR analysis. For incubations lacking ARP and humic acid, controls containing 13C-sulfamethazine in 0.1 mM acetate buffer (pH 5.6) were analyzed to account for any degradation of the parent compound during the incubation period. Incubation solutions containing humic substances and sulfonamide antimicrobials lacked microbial activity measurable by heterotrophic plate counts (Oxoid, Lenexa, KS). For the enzyme-mediated reactions, 13Csulfamethazine or 15N-sulfapyridine, humic acid, and H2O2 and 13C-sulfamethazine or 15N-sulfapyridine, ARP, and H2O2 controls were run in parallel. Over the pH range employed in these experiments (5.6–6.0), sulfamethazine and sulfapyridine speciation was dominated by the neutral species (24).
A minor zwitterionic species in tautomeric equilibrium with the neutral species contributes
