Article pubs.acs.org/est

A Quantitative Assay for Reductive Metabolism of a Pesticide in Fish Using Electrochemistry Coupled with Liquid Chromatography Tandem Mass Spectrometry Ugo Bussy, Yu-Wen Chung-Davidson, Ke Li, and Weiming Li* Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan 48824, United States ABSTRACT: This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography−mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser f ulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4aminophenol) was the only reductive metabolite identified through liquid chromatography−high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than thoose of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

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INTRODUCTION Electrochemistry associated with mass spectrometry (EC−MS) has been widely used to simulate drug metabolism catalyzed by cytochrome P450 (CYP450) in the past decades.1−10 Its application in quantitative analyses of drug metabolites, however, has been limited. The EC−MS approach is straightforward to implement; it does not require biological materials and results in clean sample matrix, and its reaction can be scaled up.11−16 These advantages have allowed EC−MS to be widely used in identification of drug metabolites.17−20 On the other hand, because drug metabolites are expected to show different responses to atmospheric-pressure ionization techniques such as electrospray and because their quantification requires a standard curve calibrated with standard compounds, the use of EC−MS in quantitative analyses has been limited. Nonetheless, Tong et al. reported the use of electrochemistry online with liquid chromatography and mass spectrometry (LC−MS) for the quantification of a drug and its unstable metabolite.21 This method can be applied in only very limited cases (quantitative electrochemical reaction with a single oxidation product). To determine the absolute concentration of the drug metabolite, the synthesis of a pure standard remains to be the most accepted method. Nitro aromatic compounds are usually found in anti-infective agents, anticancer drugs, and environmental toxins.22,23 © 2015 American Chemical Society

Electrochemistry has been extensively used to simulate metabolic reactions of various nitro aromatics.24 For example, electrochemistry online with mass spectrometry has been utilized in simulating nitro benzene reduction.25 It is wellknown that the electrochemical reduction of nitro aromatics includes three consecutive two-electron, two-proton reductions that generate successively nitroso, hydroxylamine, and amine metabolites. In fish and mammals, the same intermediates were produced via an anaerobic metabolism,26,27 while in bacteria via an aerobic metabolism.28−30 More recently, nitro aromatic xenobiotic metabolism has been simulated with electrochemical behaviors of three xenobiotics.31 A cancer drug, the nilutamide {5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl)phenyl]imidazolidine-2,4-dione}, and two pesticides were considered as nitro aromatic xenobiotic models. The two pesticides examined in this study, namely, TFM (3trifluoromethyl-4-nitrophenol) and niclosamide [5-chloro-N(2-chloro-4-nitrophenyl)-2-hydroxylbenzamide], are currently used for the population control of sea lamprey (Petromyzon marinus), a pest in the Laurentian Great Lakes. TFM was Received: Revised: Accepted: Published: 4450

November 28, 2014 February 20, 2015 March 2, 2015 March 2, 2015 DOI: 10.1021/es5057769 Environ. Sci. Technol. 2015, 49, 4450−4457

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Environmental Science & Technology

Figure 1. (A) Full 1H NMR spectra of aTFM with a close-up of the aromatic area. (B) Integration and attribution of the three 1H aromatic signals of aTFM. (C) Attribution of 1H−1H couplings.

level of reduced p-nitrobenzoic acid. Moreover, white sturgeon exhibited only a limited capacity for nitro reduction. This supports the notion that the reductive metabolism of nitroaromatic is important for TFM bioactivation and its relative selectivity toward sea lamprey and lake sturgeon. TFM is used here as a model to demonstrate that the electrochemical reactions can be scaled up to obtain pure standard compounds for quantitative LC−MS/MS analysis. The scaled up electrochemical reduction, coupled with a purification process, yielded a pure aTFM standard. TFM and its aTFM metabolite standards were used to develop an absolute quantitative LC−MS/MS method based on multiplereaction monitoring (MRM) by tandem mass spectrometry. The ability of these three fish species to reduce TFM to aTFM was evaluated in vitro using liver and gill tissue extracts incubated with TFM. The quantitative monitoring of aTFM production supports compartmental localization of the nitro reductase activity in fish liver and further defines possible mechanisms of selective toxicity of TFM toward sea lamprey.

identified after screening thousands of chemicals in 1960s for its ability to selectively kill sea lamprey with minimal effect in nontarget species such as rainbow trout (Oncorhynchus mykiss). It has been hypothesized that TFM toxicity in sea lamprey is caused by uncoupled mitochondrial oxidative phosphorylation with ATP synthesis.32−34 Later, lake sturgeons (Acipenser f ulvescens) were found to be more susceptible to TFM than trout,35,36 even though lake sturgeon can detoxify TFM through glucuronidation.35 To further understand the relative selectivity of TFM toward sea lamprey and sturgeon, we investigated TFM reductive metabolism in these animals. Structural similarities among lampricides and some human nilutamide drugs suggest the importance of the nitroaromatic substituent. Both TFM and niclosamide contain a nitro aromatic functional group. The nilutamide molecule, with a structure similar to that of TFM (containing a trifluoromethyl substituent in the ortho position of the nitroaromatic functional group), also induced uncoupling of the mitochondrial oxidative phosphorylation and ATP synthesis.37,38 This led to the hypothesis that the nitroaromatic functional group plays a predominant role in TFM metabolism and could contribute to its relative selectivity in fish. Three fish species were selected as model animals to study the reductive metabolism of TFM. Rainbow trout and lake sturgeon were selected as nontargeted animals, showing high and weak resistance to TFM exposure, respectively.36 Sea lamprey is the main target of TFM pesticide usage. The environmental degradation and metabolism of TFM have been studied in different fish species and documented.39 Even though the conjugation of the phenol ring with glucuronic acid is considered the main detoxification pathway in fish,36 reduction of the nitro aromatic functional group to the aniline metabolite (amino-TFM; aTFM or 3-trifluoromethyl-4-aminophenol) has been identified in aquatic Chnironomus tentans larvae.40 The capability of fish metabolism to reduce nitroaromatic has been investigated by Buhler and Rasmusson, focused on the reduction of p-nitrobenzoic acid by fish organ extract.41 These authors identified a significant expression of nitro reductase activity in the cytosolic fraction of liver cell extracts. In addition, pacific lamprey (Entosphenus tridentatus) exhibited the lowest while rainbow trout showed the highest

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MATERIALS AND METHODS Chemicals. Tetrabutylammonium hexafluorophosphate (NBu4PF6), 4-nitro-3-(trifluoromethyl)phenol (TFM), ammonium acetate, acetic acid, magnesium chloride, glucose 6phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), N-acetylcysteine (NAC), β-nicotinamide adenine dinucleotide phosphate sodium salt (NADP), sodium pyrophosphate tetrabasic (pyrophosphate buffer), methanol-d4, and high-performance liquid chromatography (HPLC) grade solvents were purchased from Sigma-Aldrich (St. Louis, MO). Electrochemical Synthesis and Purification of AminoTFM (aTFM). A TFM (20.6 mg in 10 mL, at a concentration of 10 mM) solution was made in anhydrous acetonitrile (1% glacial acetic acid) with tetrabutylammonium hexafluorophosphate (100 mM) as a supporting electrolyte. Electrochemical parameters were monitored by an Emstat3 portable potentiostat driven by PSTrace software from Palmsens (Ultrecht, The Netherlands). Carbon microfiber electrodes, fabricated as described by Bussy et al., were used as working and auxiliary electrodes.42−44 Reduction was conducted at −2.0 V versus a Pd/H2 pseudoreference electrode for 20 h. The reduced 4451

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et al. to enhance nitro reductase expression in fish liver cell extracts.41 Incubates were flushed with ultrapure N2 (99.998%) and kept at 25 °C while being gently shaken for 4 h; 500 μL of acetonitrile was added after the incubation to precipitate the proteins in a water/ice bath for 15 min. Finally, samples were vortexed and centrifuged for 10 min at 15000g. Supernatants were transferred to autosampler vials and stored at −20 °C until LC−MS analysis. Liquid Chromatography−High-Resolution Mass Spectrometry (LC−HRMS) Screening. Liquid chromatography− high-resolution mass spectrometry experiments were performed on an Acquity UPLC H-Class instrument coupled with a Xevo G2-S QToF system from Waters. Analysis started with the injection of 10 μL samples separated on an Acquity BEH C18 instrument (100 mm × 2.1 mm inside diameter, 1.7 μm particle size). Liquid chromatography and mass spectrometry parameters were used as previously reported.31 Quantitative Liquid Chromatography−Tandem Mass Spectrometry Assay and Method Development. Liquid chromatography−tandem mass spectrometry experiments were performed on a Xevo TQS triple-quadrupole mass spectrometer supplied by an UPLC H-Class liquid chromatograph from Waters. MS/MS parameters were optimized on a commercial TFM standard and the electrochemically synthesized aTFM metabolite. Stock solutions were made for both analytes in a methanol/water (1:1) mixture to reach a concentration of 1 mg/mL. MS/MS conditions were optimized in the negative mode of the electrospray ionization source using a concentration of 10 μg/mL. Optimizations were conducted by flow injection at a flow rate of 0.3 mL/min with a solvent composition of methanol and 10 mM ammonium acetate in water (1:1). Cone voltages were optimized to 30 V for the two molecules, while collision energies were optimized to 20 and 25 V for aTFM and TFM, respectively. The TFM concentration was measured through the 205.91 to 175.92 transition, corresponding to the neutral loss of a NO molecule. The quantitative assay of aTFM was obtained through the MS/MS transition from 175.93 to 115.92, corresponding to the neutral loss of three hydrofluoric acid molecules. Liquid chromatography conditions were optimized on a BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) with a binary gradient between methanol and water (10 mM ammonium acetate). The rate of 15% of B is kept for 3 min before being increased to 60% within 2 min. The rate of B is then increased to 99% in 1 min and kept for 2 min. The initial rate of 15% is then restored in 0.1 min and kept until the end of the experiment (10 min). For quantitative assessment, calibration curves were made of nine points from 0.1 to 1000 ng/mL. Curves were built by plotting the signal area obtained by MRM versus the standard concentration. Figure 3 shows that the TFM molecule is more sensitive than aTFM; therefore, aTFM areas were magnified by 100 for the sake of convenience. The saturation of the TFM signal at the higher concentration may be due to the formation of ion cluster ([2M − H]−). Indeed, an ion cluster was observed when TFM was ionized (negative mode of electrospray) at high concentrations at m/z 413.0280, whereas no ion cluster was observed when aTFM was ionized (data not shown).

solution was freeze-dried, solubilized in 1 mL of a water/ methanol (90:10) mixture, and purified by reverse phase semipreparative HPLC with a Luna C18 column (100 Å, 10 mm × 250 mm, 5 μm particle size) from Phenomenex (Torrance, CA). A binary gradient between water (solvent A) and methanol (solvent B) was used at a flow rate of 3 mL/min supplied by a Waters (Milford, MA) 1525 binary HPLC pump. The gradient was as follows: 95% A for 0 min, 95% A for 5 min, 10% A for 60 min, 10% A for 70 min, 95% A for 72 min, and 95% A for 100 min. The column outlet was directly hooked to a photodiode array detector (Waters PAD 2996), and 1 min fractions were then collected using a Waters fraction collector III for 100 min. Only the amino metabolite of TFM was obtained in sufficient quantity to render the NMR spectrum. The overall conversion rate of TFM to aTFM was ∼11% (2.6 mg). The product was freeze-dried overnight and dissolved in methanold4 for 1H NMR analysis (Figure 1). The sample was analyzed by 1H NMR with an Agilent (Santa Clara, CA) DirectDrive2 instrument at 500 MHz. A spectrum was compiled from 64 scans with a recovery delay of 10 s to allow quantitative analysis. Animal Sample Collections. Six prespermiating male and six preovulatory female sea lamprey were collected by agents of the U.S. Fish and Wildlife Service Marquette Biological Station (Marquette, MI) and the Department of Fisheries and Oceans Canada Sea Lamprey Control Centre (Sault Ste. Marie, ON). All test subjects were captured from the same stream on the same day to reduce variation in maturity. Standard operating procedures for transporting, maintaining, handling, anesthetizing, and euthanizing sea lamprey were approved by the Institutional Committee on Animal Use and Care of Michigan State University (AUF#05/09-088-00). Three rainbow trout (wild animals) were fished in Grand River, Grand Rapids, MI (latitude and longitude coordinates of 42.97384864563183, −85.67455172538757). Six juvenile farmraised rainbow trout were provided by the Michigan Department of Natural Resources Harrietta State Fish Hatchery and used with the approval of the Michigan State University Animal Use and Care Committee (AUF#08/12-148-00). Six one-year-old lake sturgeon were gifts from K. Scribner from the Department of Fisheries and Wildlife of Michigan State University. We were not able to determine the gender of the fish because the gonad was not developed at this stage. The body length and weight were not examined at the time of tissue collection (by the collaborators). Ethical guidelines were followed throughout the course of this research. Tissue Extractions and Protein Concentration Determination. Compartmental protein and mitochondria were extracted using the Mitochondria Isolation Kit and Compartmental Protein Extraction Kit (BioChain, Newark, CA) according to the manufacturer’s instructions. Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Incubation Conditions. Incubations were conducted in pyrophosphate buffer (0.2 M, pH 8) with glucose 6-phosphate (G6P, 10 mM), glucose-6-phosphate dehydrogenase (G6PDH, 1 unit/mL), magnesium chloride (MgCl2, 25 mM), βnicotinamide adenine dinucleotide phosphate (NADP, 1 mM), N-acetylcysteine (NAC, 2 mM), and TFM (100 μM, 20 μg/mL). Additives and buffer parameters (nature, pH, and concentrations) were used according to the method of Buhler

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RESULTS AND DISCUSSION The incubation of TFM with different fractions of liver extract was first analyzed by LC−HRMS. On the basis of our previous 4452

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Environmental Science & Technology work,31 electrochemistry was used to generate a set of metabolites associated with the reduction of the nitroaromatic functional group of the TFM molecule. Moreover, LC−HRMS showed that the cytosolic fraction contained the most concentrated nitro reductase activity in fish liver.41 Dimers such as hydrazine, azo, and azoxy or hydroxylamine metabolites were not observed when TFM was incubated with liver cell extracts. Figure 2 displays the combined chromatograms of the parent molecule (TFM) and its nitroso and amino (aTFM) metabolites. Despite the signal observed at the exact mass corresponding to the nitroso metabolite of TFM, the presence of this metabolite in liver incubates could not be confirmed because of the mismatched retention time. Its retention time of 4.8 min (the same as the TFM retention time) suggests that the signal of a possible nitroso metabolite in the liver incubates was more likely to be an artifact of TFM ionization. The absence of nitroso and hydroxylamine metabolites can be attributed to the matrix complexity and possible binding to biocomponents contained in the liver cell extracts. Moreover, dimerizations were not likely to occur during incubation because of the limited concentration of the reactive metabolites (nitroso and hydroxylamines) during enzymatic reduction compared to electrochemical conditions. Only aTFM was observed in liver incubate, showing similar retention time and exact mass when compared to those of the electrochemically generated metabolite set. None of the nitro aromatic reduction metabolites of TFM was observed in control samples (containing G6P, MgCl2, G6PDH, NADP, TFM, N-acetylcysteine, and pyrophosphate buffer). These observations demonstrate the reduction of TFM to aTFM in the presence of fish liver cell extract. Therefore, aTFM was set as the main target of subsequent electrochemical synthesis for standard production. None of the other known reductive metabolites of TFM31 were considered during quantitative LC−MS/MS assay development. Figure 1 displays the 1H spectra of the synthesized aTFM metabolite. Two signals are observed at 3.3 and 4.9 ppm and are attributed to the residual signal of the solvent (methanol).45 No significant signal is observed except in the aromatic proton area (Figure 1). Signal areas (Figure 1B) as well as coupling constants (Figure 1C) support the attribution shown in Figure 1. Moreover, high-resolution mass spectrometry (ESI−-HRMS) analysis of the same sample shows an exact m/z ([M − H]−) at 176.0355 that approximates the expected theoretical value at m/z 176.0368, and a Δm/z of 1.3 mDa confirms the raw formula of aTFM (data not shown). Integration of aromatic 1H NMR signals and the absence of an unattributed signal demonstrate the high purity of the electrochemically synthesized metabolite (Figure 1) and support its usage as a standard for quantitative assay development. Commercially available TFM standard and electrochemically produced aTFM metabolite were then used to develop a LC−MS/MS method for the absolute quantifications of both TFM and aTFM for pesticide metabolism investigation. TFM showed a better response to negative electrospray ionization, leading to a saturated standard curve at the highest concentration, while aTFM showed linearity from 0.01 to 1000 ng/mL (Figure 3). Because nitroaromatic reduction can be conducted chemically, the use of electrochemistry for metabolite synthesis is very promising for other xenobiotic metabolic reactions. In application, the electrochemistry approach bypasses multistep organic synthetic

Figure 2. Total ion chromatogram of TFM samples after electrochemical reduction (A) and control experiments (B) of incubation without liver cell extract. Chromatograms C−E represent incubations with cytosolic liver cell extracts of wild trout, sturgeon, and male sea lamprey, respectively.

processes such as carbon hydroxylations or heteroatom dealkylations.10,14 The resulting quantitative method was applied to measure aTFM in extracts from cellular fractions incubated with TFM in various fish species. Rainbow trout were used as an animal model showing resistance to TFM treatment; both farm-raised (n = 6) and wild (n = 3) animals were studied. Lake sturgeon (n = 6) and adult sea lamprey (six females and six males) were 4453

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Figure 3. Calibration curves for TFM (×) and aTFM (+) molecules, with signal area vs standard concentration. The main graph represents the method response vs the whole range of concentrations. The inserted graph zooms in on the linearity portion in the lower concentration range.

Figure 4. Concentrations of aTFM after incubation for 4 h with liver extracts from rainbow trout, preovulatory female sea lamprey, and prespermiating male sea lamprey. Error bars show standard deviations of three replicates. The control is TFM incubated without liver extract. Labels indicate significant differences: *, different from every other group; a, different from sea lamprey (male and female) and rainbow trout (farm-raised and wild); b, different from male sea lamprey, lake sturgeon, and wild and farm-raised rainbow trout; c, different from sea lamprey (male and female), lake sturgeon, and farm-raised rainbow trout; d, different from sea lamprey (male and female); e, different from sea lamprey (male and female) and wild rainbow trout; f, different from sea lamprey (male and female) and farm-raised rainbow trout; g, different from sea lamprey (male and female) and wild rainbow trout; h, different from sea lamprey (male and female); i, different from sea lamprey (male and female).

selected as models for TFM sensitive species.35,36 Fresh liver and gill tissues were fractionated (cytoplasmic, nucleic, membrane, and mitochondrial fractions) and incubated for 4 h with TFM. Same species fraction were pulled together to minimize inter-individual variation. All fractions were diluted to have the same protein concentration (2 mg/mL). Incubations were triplicate and conducted under oxygen free conditions to avoid the futile cycle that is known to quench the reduction of nitroaromatic in fish and mammals.26 Incubations were terminated by acetonitrile that precipitates proteins. After centrifugation, the remaining supernatant was analyzed by LC− MS/MS for quantitative determination of aTFM. As expected, the TFM concentration exceeded the linear range of the calibration curve and was not determined. Figure 4 presents the concentration of aTFM measured after incubation for 4 h with liver cell extracts (2 mg of protein/mL). No significant quantities of aTFM were observed when TFM was incubated with any gill fractions (data not shown). Male sea lamprey generally contained higher aTFM concentrations than females. The aTFM metabolite concentrations were 498 ± 28 and 405 ± 11 ng/mL in male and female sea lamprey cytosolic extract, respectively. The same fractions in lake sturgeon and wild rainbow trout contained 434 ± 9 and 151 ± 10 ng/mL aTFM, respectively. The aTFM concentrations in nucleic fraction were 532 ± 22, 249 ± 21, 62 ± 7, and 25 ± 5 ng/mL for male sea lamprey, female sea lamprey, lake sturgeon, and wild rainbow trout, respectively. Membrane protein incubates contained the lowest concentration of aTFM: 294 ± 17 ng/mL for male and 125 ± 2 ng/mL for female sea lamprey, 26 ± 3 ng/mL for lake sturgeon, and 13 ± 0.5 ng/mL for wild rainbow trout. Finally, in the mitochondrial fraction, the measured aTFM concentrations were 176 ± 12, 111 ± 5, 18 ± 1, and 29 ± 1 ng/mL for male sea lamprey, female sea lamprey, lake sturgeon, and wild rainbow trout, respectively. As expected, the cytosolic and nucleic fractions showed the highest level of expression of nitro reductase activity for the three species studied,41 while membrane proteins showed a limited amount of nitro reductase activity. Mitochondrial proteins showed very little enzymatic reduction of TFM to aTFM. The highest concentrations of aTFM were measured in the presence of male sea lamprey liver cell extract at 500 ng/

mL, corresponding to 5% of the initial TFM concentration (20 μg/mL). No significant decrease in TFM concentration was observed. Opposite to the reductive metabolism of the nitro aromatic functional group, the oxidative metabolism of the aromatic ring and the phenol group requires oxygen. Data in Figure 4 allow direct comparison between the species and cellular fractions studied. ANOVA and post hoc tests were performed, and all cellular fractions showed significant differences among species and between male and female sea lamprey (p < 0.0001). The highest concentrations of aTFM were observed in cytosolic and nucleic fractions of sea lamprey liver cell extracts. Also, nitro reduction activity tends to be significantly higher in male sea lamprey. Incubates of wild and farm-raised rainbow trout liver cell extracts showed concentrations of aTFM on the same order of magnitude. However, the cytosolic (p = 0.01) and mitochondrial (p < 0.05) fractions were different between the wild and farm-raised trout. From the cytosolic concentrations of aTFM, it is clear that female sea lamprey and lake sturgeon showed a similar reductive metabolism of TFM (p = 0.09), which appears to be weaker in rainbow trout. However, other liver fractions showed more similarity between sturgeons and rainbow trout than sea lamprey (Figure 4). This indicates that the ability for lake sturgeons to reduce the nitroaromatic functional group of TFM was between those of sea lamprey and rainbow trout. These observations are consistent with known species-specific sensitivity toward TFM.35 It has been shown that the LC 25 values of TFM were 1.92 and 5.9 mg/L for lake sturgeon and rainbow trout, respectively, while the LC 99.9 for sea lamprey was 1.4 mg/L.35 Nevertheless, lake sturgeon showed tolerance to TFM exposure relatively higher than that of sea lamprey, and 4454

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Future investigations in sea lamprey should be conducted at different life stage of these pest animals. The possible bioactivation of TFM through nitro aromatic reduction should also be evaluated in the future.

this may be due to their capability to detoxify TFM through glucuronidation of its phenol group.36 In addition, it should be noted that the LC 99.9 in sea lamprey was evaluated in larvae and usually under specific water conditions, especially regarding alkalinity and pH that have drastic effects on the lethal concentration.46 Notably, significant differences in aTFM concentrations in all four cellular fractions were observed between male and female sea lampreys (p < 0.0001). Male fractions consistently produced more aTFM than female fractions. The factor or factors that directly cause these differences are not well-known and need to be investigated further. The potential influence of other factors, such as life stage, size, and geographic origin, should also be considered. For example, adult sea lamprey have recently been identified as the most sensitive to TFM treatment.47 Moreover, TFM uptake related to water condition has never been evaluated. Similar sex differences in TFM susceptibility in sturgeon and trout may exist. This requires further confirmation in sexually identifiable animals because sex differences in CYP450 activities are most pronounced at later reproductive stages in fish.48 Moreover, the oxidative metabolism and possible interaction of reactive metabolites of aTFM with biomolecules such as DNA and proteins should be examined. We showed that metabolism of nitroaromatic compounds was associated with nitroaromatic reduction. The identification of aTFM metabolite in the liver samples, especially in those of sea lamprey, raises the possibility of secondary reactive metabolites through aTFM oxidative metabolism. In fact, the aTFM metabolite shows a structure auspicious to parabenzoquinonimine formation. This class of reactive oxygen metabolites is associated with many toxicological effects, mostly implicated in liver injury.18,49−52 The dehydrogenation of hydroquinoid-like molecules leads to the formation of reactive quinoid entities that can bind covalently to cellular macromolecules such as proteins and peptides or may also induce oxidative stress by generating free radicals.53 Moreover, reaction of quinoid metabolites with CYP450 reductase can result in the formation of reactive oxygen species (ROS).53 Therefore, oxidative metabolism of aTFM should be further investigated. The quantification of aTFM in fish liver incubates suggests that the reduction of TFM undergoes subsequent formation of nitroso and hydroxylamine metabolites. The prostatic cancer drug nilutamide shares strong structural similarities with TFM.31 TFM and nilutamide both uncoupled mitochondrial oxidative phosphorylation with ATP production.33,34,37 In addition, the reduction of the nitroaromatic functional group of nilutamide yielding hydroxylamine and nitroso reactive metabolites has been introduced as a plausible source of its toxic effect.38 Because of the possible bioactivation of TFM through the same nitroaromatic reductive metabolism, formation of hydroxylamine and nitroso reactive metabolites, the action of these two metabolites on mitochondrial oxidative phosphorylation uncoupling should be investigated. In fact, the formation of nitroso metabolite has been identified as a bioactivation pathway (oxidative phosphorylation uncoupler) of the antibacterial chloramphenicol on rat liver mitochondria.54 In summary, a quantitative LC−MS/MS method has been developed to measure aTFM, the main metabolite of TFM, in three animal species, and the results further define the mode of toxicity. Major differences were observed in different cellular compartments. These results demonstrate that differential reductive metabolism of TFM in these species may be associated with their selective sensitivity for this pesticide.

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AUTHOR INFORMATION

Corresponding Author

*Department of Fisheries and Wildlife, Michigan State University, Room 13, Natural Resources Building, 480 Wilson Rd., East Lansing, MI 48824. E-mail: [email protected]. Telephone: (517) 432-6705. Fax: (517) 432-1699. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Daniel Jones and Lijun Chen of the Michigan State University Mass Spectrometry Facility for helpful advice. We also thank Professor Kim Scribner of the Fisheries and Wildlife department of Michigan State University for providing lake sturgeon tissues. We thank Dr. Mohammed Boujtita and Renaud Boisseau from the CEISAM laboratory of the University of Nantes (Nantes, France) for providing carbon microfiber electrodes. Finally, we thank Dr. Mar Huertas, Dr. Chu-Yin Yeh, Tyler Buchinger, and Skye Fissette for technical assistance. This study was funded by a grant from the Great Lakes Fishery Commission.

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REFERENCES

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