Environ. Sci. Technol. 2008, 42, 8965–8970
Herbicidal Effects of Sulfamethoxazole in Lemna gibba: Using p-Aminobenzoic Acid As a Biomarker of Effect R I C H A R D A . B R A I N , * ,† ALEJANDRO J. RAMIREZ,‡ BARRY A. FULTON,† C. KEVIN CHAMBLISS,§ AND BRYAN W. BROOKS† Center for Reservoir and Aquatic Systems Research, Department of Environmental Science, Baylor University, Waco, Texas, 76798, Mass Spectrometry Core Facility, Baylor University, Waco, Texas, 76798, and Department of Chemistry and Biochemistry, Baylor University, Waco, Texas, 76798
Received June 12, 2008. Revised manuscript received September 15, 2008. Accepted September 23, 2008.
Sulfamethoxazole (SMX) is among the most frequently detected antibiotics in the environment, heavily used in both human therapy and agriculture. Like other sulfonamides, SMX disrupts the folate biosynthetic pathway in bacteria, which was recently established as identical to that of plants, raising concerns over nontarget toxicity. Consequently, Lemna gibba was exposed to SMX to evaluate phytotoxic potency and mode of action (MOA) by HPLC-MS/MS measurement of p-aminobenzoic acid (pABA) metabolite levels, a precursor to folate biosynthesis and substrate of the target enzyme dihydropteroate synthase (DHPS). pABA levels were found to increase upon exposure to SMX following an exponential rise to a maxima regression model in a concentration-dependent manner. The EC50 for pABA content was 3.36 µg/L, 20 times lower than that of fresh weight (61.6 µg/L) and 40 times lower than frond number (132 µg/L) responses. These results suggest that, as in bacteria, sulfonamide antibiotics specifically disrupt folate biosynthesis via inhibition of DHPS. Analysis of pABA concentrations appears to provide a sulfonamide-specific biomarker of effect based on MOA with exceptional diagnostic capacity and sensitivity compared to traditional morphological end points. Using the EC50 for pABA content, a potential hazard was identified for L. gibba exposed to SMX, which would not have been detected based upon traditional standardized morphological approaches.
Introduction First synthesized as a dye in the early 1900s and later realized for their antimicrobial potential (1), sulfonamides are credited as the first class of antimicrobial agents. Subsequently, a myriad of broad-spectrum synthetic compounds widely used to treat diseases and infections for both humans and livestock * Corresponding author phone: (254) 710-2625; fax: (254) 7102969; e-mail:
[email protected]. † Center for Reservoir and Aquatic Systems Research, Department of Environmental Science. ‡ Mass Spectrometry Core Facility. § Department of Chemistry and Biochemistry. 10.1021/es801611a CCC: $40.75
Published on Web 10/31/2008
2008 American Chemical Society
have been propagated. Exact estimates of sulfonamide usage are unknown, though approximately 23,000 tons of antibiotics are produced each year in the U.S. alone (2). As a consequence of their application, these compounds tend to enter the environment via two main routes: sewage effluent from human sources and agricultural runoff from animal husbandry operations (3). Sulfonamides readily contaminate both surface water and groundwater (4) due to their physiochemical properties, namely their low soil sorption coefficients (Kd), particularly sulfamethoxazole (SMX) with measured Kd values of 0.22 and 1.8 L/kg (5). Sulfonamides have been detected in wastewater treatment plant (WWTP) effluent in both the U.S. and Canada at 0.21 and 0.24 µg/L (SMX), respectively (6, 7). In surface waters receiving both agricultural and urban inputs, maximum measured concentrations are in the range 0.12-1.9 µg/L in the U.S (4, 8). and 0.007-0.408 µg/L in Canada (9) with SMX consistently displaying the greatest and most frequent detection among the sulfonamide class. Furthermore, among antibiotic classes, sulfonamides are relatively stable in the environment; the half-life of SMX in aquatic systems is 19 days (10). Hence, sulfonamides display a considerable propensity for contaminating aquatic environments. Pharmacologically, sulfonamide antibiotics act as structural analogues of the substrate p-aminobenzoic acid (pABA), inhibiting the enzyme dihydropteroate synthase (DHPS) in the folate biosynthetic pathway (Figure 1) (11, 12). In plants, this pathway is essentially the same as in bacteria and is now almost completely elucidated (13). Currently, eight of the ten enzymes have been cloned and characterized from plants, including DHPS (13). In the mitochondria, DHPS catalyzes the formation of dihydropteroate from hydroxymethyldihydropterin pyrophosphate (HMDHP-PPi; produced in the
FIGURE 1. Overview of folate biosynthesis and proposed mode of action of sulfamethoxazole in plant cells. Pteridine synthesis occurs in the cytosol whereas pABA is synthesized in the plastids; these metabolic precursors are then assembled inside the mitochondria to produce dihydropteroate (DHP), which is finally glutamylated to synthesize folates. Sulfonamide antibiotics are thought to target dihydropteroate synthase (DHPS-indicated with a bold X) by acting as a structural analog of pABA. Acronyms include: GTP (guanosine triphosphate), DHM (dihydromonapterin), DHN (dihydroneopterin), DHN-P (dihydroneopterin monophosphate), DHN-PPP (dihydroneopterin triphosphate); HMDHP (hydroxymethyldihydropterin), Glu (glutamate), and THF (tetrahydrofolate). Dashed arrows indicate multiple metabolic steps excluded for simplicity. Modified from ref 13. VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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cytosol) and pABA (produced in the chloroplast) (13, 14) (Figure 1). The expression of the same target enzyme in bacteria and plants not only exemplifies evolutionary conservation, but also inspires questions and concerns regarding the susceptibility of plants inadvertently exposed to sulfonamide antibiotics in the environment (15). Not surprisingly, sulfonamides have demonstrated phytotoxicity in higher plants (Lemna gibba), green algae, and blue-green algae with EC50s ranging from 81 to 2330 µg/L (16, 17), 146 to 7800 µg/L (18, 19), and 26.8 to 135 µg/L (19, 20), respectively. Among the sulfonamide class, SMX is consistently reported as the most phytotoxic compound (15). It should be noted to avoid confusion that sulfonamide antibiotics differ markedly from sulfonamide herbicides, which block branched chain amino acid synthesis through the inhibition of acetolactate synthase (ALS) (21). Considering the folate biosynthetic pathway equivalence, it is a reasonable postulate that the phytotoxic mode of action of sulfonamides in plants, as in bacteria, is to target DHPS. As a consequence of DHPS inhibition in L. gibba, metabolic products (folates) would be expected to decrease upon exposure to an inhibitor (SMX), whereas precursors (e.g., pABA) would be expected to increase. The methodological approach executed here was to measure pABA, a stable precursor upstream of, and substrate for, DHPS (13) via LCMS/MS. Several physiochemical attributes render routine folate analyses unsuitable for pathway-specific metabolite analyses (22-25). Since changes in metabolite concentrations provide the foundation on which all other stressor-induced effects depend, they are typically the most sensitive indicators of adverse effects. Consequently, these measures have been employed as biomarkers for a range of compounds in aquatic and terrestrial plants (26). However, as a requisite for a biomarker of effect, measured changes in pathway-specific metabolites must be mechanistically and causally linked to effects at the morphological level in order to be useful as measures of effect for hazard or risk assessment (26, 27), which currently rely largely on morphological data. An example highlighting these considerations has been demonstrated previously in L. gibba exposed to statin pharmaceuticals (28), and the present research further illustrates their importance in evaluating biologically active compounds such as antibiotics. Given the frequent detection in various environmental matrices and the uniform potency displayed among aquatic higher plants and algae, sulfonamides warrant further research concerning the mode of phytotoxic action to refine the risk assessment case. Hence, this investigation was composed of three main objectives: (1) to evaluate the mode of phytotoxic action of sulfamethoxazole in Lemna gibba by measuring the level of pABA in a concentration-dependent manner, suggesting DHPS as the target enzyme; (2) to evaluate the viability and sensitivity of pABA as a biomarker of effect specific to sulfonamides compared to traditional morphologic end points; and (3) to conduct a refined comparative hazard assessment with the biochemical and morphological data.
Experimental Section Chemicals and Reagents. All chemicals were reagent grade or better, obtained from commercial vendors and used as received. The standards sulfamethoxazole (CASRN: 723-466), pABA (CASRN: 150-13-0), and internal standard 10,11dihydrocarbamazepine (CASRN: 3564-73-6) were purchased in the highest available purity (Sigma-Aldrich, St. Louis, MO). The isotope pABA-13C6 (99% purity) was purchased from BetaChem (Leawood, KS). Distilled water was purified and deionized to 18 MΩ with a Barnstead Nanopure Diamond UV water purification system. 8966
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Experimental Procedure. Tests with Lemna gibbaG3 were conducted in accordance with a seven-day daily static renewal protocol using half-strength Hutner’s medium (29). Briefly, 21 experimental units (10 mL (60 × 15 mm) culture dishes) were used in a randomized complete block design (RCBD) with seven treatments. Because acetone was used as a carrier for SMX, at 0.01% in all SMX treatments, both solvent (0.01%) and nonsolvent controls were employed and compared for significant differences in all end points upon cessation of exposure (29). Five SMX treatment concentrations were used, including 10, 30, 100, 300, and 1000 µg/L. Each of the seven different treatments were replicated in triplicate and randomly assigned to each of 3 blocks. Two L. gibba plants, each with four fronds, were transferred to each culture dish from axenic test cultures obtained from the University of Toronto Culture Collection (UTCC; Toronto, ON, Canada). Two sets of identical trays and culture plates were employed for each test. During each static-renewal, respective SMX treatments were prepared daily from stock solutions (maintained in acetone) and plants were carefully transferred between sets of corresponding trays (29). Upon completion of the 7-day exposure period, frond number and fresh mass morphological metrics were assessed for each experimental unit prior to pABA extraction. The experiment was conducted at 23.5 °C under constant cool white fluorescent light (GE Ecolux 20W; 7,243 ( 190 lux) in a Thermo 815 growth chamber (Thermo Fisher Scientific Inc., Waltham, MA). LC-MS/MS Analysis of Sulfamethoxazole. For each culture dish, sulfamethoxazole exposure concentrations were verified using a benchtop liquid chromatograph-tandem mass spectrometer (LC-MS/MS) consisting of a Varian ProStar HPLC system coupled to a Varian model 1200 L triplequadrupole (Varian, Inc., Palo Alto, CA). Chromatographic separation of sulfamethoxazole and 10,11-dihydrocarbamazepine (internal standard) was achieved using a 15 cm × 2.1 mm (5 µm, 80 Å) Extended-C18 column (Agilent Technologies, Palo Alto, CA). A binary mobile phase gradient, consisting of 0.1% (v/v) formic acid in water (A) and methanol was applied as follows: 75% A for 4 min, to 25% A in 3 min, back to 75% A in 1 min, and equilibrated for 7 min at 75% A. Additional chromatographic parameters were as follows: injection volume, 10 µL; column temperature, 30 °C; flow rate, 350 µL/min. The mass spectrometer was operated in positive electrospray ionization mode. Detector, needle, shield, and capillary voltages were set to 1.8, 5.0, 0.4, and -0.04 kV, respectively. The MS/MS transitions monitored for quantitation purposes were m/z 254 > 156 for sulfamethoxazole and m/z 239 > 194 for the internal standard at collision energies of -12.5 and -18.5 V, respectively. Daily spiking concentrations for sulfamethoxazole exposures were determined using an internal standard calibration curve. Matrix-matched calibration samples were prepared by serial dilution of a stock solution containing 100 ng/mL of sulfamethoxazole in half-strength Hutner’s media, resulting in eight standards ranging from 3 to 30 ng/mL. A constant amount of the internal standard (5 ng) was added to both standards and samples using a 100 ng/mL stock solution of 10,11-dihydrocarbamazepine prepared in media. The response factor was calculated by dividing the peak area for sulfamethoxazole by the peak area for the internal standard, and a calibration curve was prepared by plotting a linear regression (r2 g 0.999) of the response factor versus analyte concentration for all calibrators analyzed. Instrument calibration was monitored through the use of continuous calibration verification (CCV) samples with an acceptability criterion of (15%. In a given run, one blank and one CCV sample were interspersed between every 15 samples. Sulfamethoxazole exposure concentrations were also verified in each culture dish after 24 h of exposure in the first and last batch for each concentration level.
Calculation of Exposure Values. Fresh treatment solutions were prepared and sampled daily (days 0-6) for verification via LC-MS/MS. On days 1 and 7 of the experiment, the concentrations of SMX in each 10 mL culture plate were subsequently measured after 24 h of exposure. Exposure concentrations were calculated as the average of the measured daily treatment solution preparations (for days 0-6) and the average of the treatment concentrations 24 h after exposure (for days 1 and 7). These average values were then combined to give an average exposure for the entire experimental period. This was done to ensure accuracy of exposure for each daily renewal and to take into consideration any decomposition of SMX over the daily 24 h exposure period (28), providing a more realistic exposure estimate for statistical analyses of effects. pABA Extraction. pABA extractions were modified from ref 30. Briefly, total plant fresh weight (FW) for each experimental unit was measured after dabbing plants dry on absorbent sheets. Plants were then placed in respective 7 mL borosilicate glass scintillation vials (Fisher Scientific, Fair Lawn, NJ) and thoroughly macerated in 2 mL of 100% methanol using a Tissuemiser (Fisher Scientific, Fair Lawn, NJ) set to rotate at 30,000 rpm, which was subsequently rinsed with an additional 5 mL of methanol. pABA was then extracted for 1 h from the ground tissue at room temperature (∼25°) in a flow bench. Extracts were then coarsely filtered via transfer to 16 × 125 mm borosilicate glass culture tubes (Fisher Scientific), by filtering through a funnel containing a glass wool plug. Vials were then rinsed twice with 1.5 mL of methanol and the rinsates were also filtered into test tubes containing plant extract. The filtered extract was then gently blown dry under a stream of nitrogen at 30 °C using a Zymark Turbovap LC (Zymark Corp., Hopkinton, MA). Once dry, the plant extract residue was reconstituted in 0.8 mL of methanol, and 0.1 mL of 2 M HCl was added to each culture tube and thoroughly mixed via sonication and vortexing for 2 min. Acid was added to hydrolyze the glucosidic bond of the β-Dglucopyranosyl ester (pABA-Glc) such that only total free pABA was analyzed (30). Culture tubes were then incubated for 1 h in an oven set to 80 °C. Subsequently, 0.1 mL of 2 M NaOH was added to neutralize each sample, followed by thorough mixing as above. Finally, samples were spiked with a constant amount of pABA-13C6 (200 ng) and filtered using Pall Acrodisc hydrophobic Teflon Supor membrane syringe filters (13-mm diameter; 0.45-µm pore size; VWR Scientific, Suwanee, GA) prior to LC-MS/MS analyses. pABA Analysis by LC-MS/MS. pABA extracted from plant tissue was quantitated using the same LC-MS/MS system and experimental conditions described for SMX with the following modifications. A constant retention time of 4.86 min (relative standard deviation (RSD) < 5%) for pABA and pABA-13C6 (pABA isotope) was achieved by applying the following gradient: 97% A for 7 min, to 2% A in 1 min, held for 5 min, back to 97% A in 1 min, and equilibrated for 9 min at 97% A. Two MS/MS transitions were monitored for quantitation and qualitative identification of pABA in plant extracts: m/z 138 > 77 and m/z 138 > 94, at collision energies of -18.5 and -11.5 V, respectively. Only one MS/MS transition (m/z 144 > 83, collision energy -19.0 V) was monitored for pABA-13C6. pABA concentrations were measured using an isotope dilution approach. pABA stock solutions containing 1000 or 100 ng/mL were serially diluted in water to prepare 10 calibration points ranging from 2.5 to 400 ng/mL. A constant amount of pABA-13C6 (200 ng) was added to both calibrators and samples as an internal standard using a 400 ng/mL stock solution of pABA-13C6 prepared in water. In addition to the quality control samples utilized in analyses of SMX, three matrix spikes were also prepared by adding 5, 100, and 300 ng of pABA to untreated pooled plants
TABLE 1. Re-parameterized Equations Used to Fit the Concentration-Response Relationships of Sulfamethoxazole Exposed Lemna gibba, in SigmaPlot 9.0 regression
equationa
exponential rise y ) y0+ a(1 - ((1 to a maxima (y0p/a))(x/ECx))) four parameter y ) y0 + a/(1 + ((x/ECx)b) logistic ((a/((1 - p)(y0 + a) - y0))- 1))
modeling type increase decrease
a
The variable ECx is the calculated effective concentration at which proportion p of the end point is affected, x is the actual concentration (µg/L), y is the response or change from control of the end point modeled, and a, b, and y0 are constants.
taken from cultured dishes after acquiring the fresh weight. Samples were extracted using the same extraction procedure described above. Recoveries were calculated for each matrix spike by subtracting the average pABA concentration found in unspiked samples (i.e., pABA native to the unexposed plant) from the analytical concentration of pABA measured in spiked samples and dividing this difference by the appropriate spiking level. Matrix spike recoveries for the 5, 100, and 300 ng spiking levels were 81%, 91%, and 90%, respectively. Statistical Analyses. Tests of significant differences were analyzed using proc GLM of SAS v8.2 (SAS Institute, Cary, NC) with one-way analysis of variance (ANOVA) to identify significant effects (p < 0.05). No significant differences were found between the solvent and nonsolvent controls (R ) 0.05) for any assessment end point; fresh weight (p ) 0.4585), frond number (p ) 0.1489), or pABA concentration (p ) 0.5120); therefore, the control values were pooled for comparison with SMX treatments (29). When significance was found for a treatment-control comparison, a lowest observed effects concentration (LOEC) was computed by comparing the means of each treatment to the controls using Dunnett’stest(R)0.05).Theendpointconcentration-response data were then modeled using nonlinear regression equations (Table 1) performed in Sigmaplot 9.0 (SPSS Inc. Chicago, IL). Model fit was evaluated based on the mean adjusted coefficient of determination and by graphical interpretation of the model’s fit. EC10, EC25, and EC50 values were estimated with their associated standard errors. Hazard Quotients. Under the U.S. Food and Drug Administration (USFDA) guidance for industry concerning the environmental assessment of human drug and biologics applications (31) a predicted environmental concentration (PEC) threshold value of 0.10 µg/L is suggested, based on a 1.0 µg/L effluent concentration divided by a 10-fold standard dilution factor. Furthermore, for Tier 2 assessments (acute ecotoxicity testing on a base set of aquatic organisms) it is suggested that the EC50 for the most sensitive organism be divided by a factor of 100 (31), providing the following HQFDA equation: HQFDA ) (1.0 µg/L/10)/(EC50/100). Because this approach does not account for effluent-dominated streams, which experience little to no in-stream dilution, likely representing maximum exposure scenarios for pharmaceuticals (32), HQs were additionally calculated for effluentdominated streams (HQEDS) using the following equation: HQEDS ) (1.0 µg/L) (EC50/100). Hazard quotients were also calculated based on the highest surface water measured exposure concentration (MEC) of SMX (1.9 µg/L; (8)) in conjunction with the raw EC50s for L. gibba, with no additional safety factors for comparison using the following equation: HQRAW ) (MEC/EC50). The MEC is similar to SMX surface water concentrations reported elsewhere (1.02 µg/L; (4)), and only 10-fold higher than the median reported concentration of SMX (0.15 µg/L; (8)), and is thus environmentally realistic. VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Results and Discussion Verification of SMX Exposure Concentrations; Evidence for Uptake by L. gibba. The exposure concentrations for sulfamethoxazole were accurately achieved within 7, 2, 17, 5, and 9% of the targeted 10, 30, 100, 300, and 1000 µg/L values, respectively. The relative standard deviation of SMX exposure levels (n ) 3) was
