Cross-Species Comparison of Conazole Fungicide Metabolites

In vitro metabolism of conazole fungicides for interspecies comparison. .... An isocratic mobile phase (0.4 mL/min) consisting of 60% acetonitrile and...
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Environ. Sci. Technol. 2008, 42, 947–954

Cross-Species Comparison of Conazole Fungicide Metabolites Using Rat and Rainbow Trout (Onchorhynchus mykiss) Hepatic Microsomes and Purified Human CYP 3A4 CHRISTOPHER S. MAZUR* AND JOHN F. KENNEKE U.S. EPA, National Exposure Research Laboratory, Ecosystems Research Division, 960 College Station Rd., Athens, GA, 30605

Received August 16, 2007. Revised manuscript received October 25, 2007. Accepted October 31, 2007.

Ecological risk assessment frequently relies on crossspecies extrapolation to predict acute toxicity from chemical exposures. A major concern for environmental risk characterization is the degree of uncertainty in assessing xenobiotic biotransformation processes. Although inherently complex, metabolite identification is critical to risk assessment since the product(s) formed may pose a greater toxicological threat than the parent molecule. This issue is further complicated by differences observed in metabolic transformation pathways among species. Conazoles represent an important class of azole fungicides that are widely used in both pharmaceutical and agricultural applications. The antifungal property of conazoles occurs via complexation with the cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell wall synthesis. This mode of action has cause for concern regarding the potential adverse impact of conazoles on the broad spectrum of CYP-based processes within mammalian and aquatic species. In this study, in vitro metabolic profiles were determined for thirteen conazole fungicides using rat and rainbow trout (Oncorhynchus mykiss) liver microsomes and purified human CYP 3A4. Results showed that 10 out of the 13 conazoles tested demonstrated identical metabolite profiles among rat and trout microsomes, and these transformations were well conserved via both aromatic and aliphatic hydroxylation and carbonyl reduction processes. Furthermore, nearly all metabolites detected in the rat and trout microsomal assays were detected within the human CYP 3A4 assays. These results indicate a high degree of metabolic conservation among species with an equivalent isozyme activity of human CYP 3A4 being present in both the rat and trout, and provides insight into xenobiotic biotransformations needed for accurate risk assessment.

Introduction Comprehensive ecological risk assessment requires a detailed understanding of systems biology. Since it is virtually impossible to test all organisms with any one * Corresponding author fax: 706-355-8202; e-mail: mazur.chris@ epa.gov. 10.1021/es072049b

Not subject to U.S. Copyright. Publ. 2008 Am. Chem. Soc.

Published on Web 12/21/2007

chemical compound, cross-species extrapolation is often implemented to understand metabolism-induced toxicity (1). Most toxicological screening assays are currently based upon mammalian (rodent) receptors or tissues whose defined mode of action is then applied across aquatic species for environmental risk characterization (2, 3). Thus, a major complication with environmental exposure and risk assessment models is assessing the degree of conservation used for extrapolation among differing classes of vertebrate species (4). Upon uptake, a major factor in determining the toxicological fate of a xenobiotic among fish and mammalian species is often dependent on elucidating the initial biotransformation pathway(s) of the parent chemical. The liver is the primary organ responsible for the metabolism of foreign chemicals; which through enzymatic-mediated processes, generally facilitates clearance by converting toxicants to more water soluble, excretable products (5). The cytochrome P450 (CYP) superfamily comprises a broad class of phase I oxidative enzymes that catalyze many hepatic detoxification processes for a vast array of xenobiotics in vertebrate species (6, 7). In human liver, the CYP 3A subfamily alone is known to contribute to the metabolism of as many as 60% of major drug therapuetics (8). Liver microsomes, which are metabolically active subcellular tissue fractions of the endoplasmic reticulum that have been isolated via ultracentrifugation, are routinely used for laboratory in vitro metabolic assays to assess and delineate xenobiotic biotransformation pathway(s) (9, 10). Conazoles (1,2,4-triazoles) represent an important class of azole-containing fungicides that are widely used in both pharmaceutical and agricultural applications for the treatment of systemic fungal infections. As agrochemicals, conazoles are used to protect various fruit, vegetable, cereal crops, and seeds from fungal growth and are often detected in surface waters due to runoff and spray drift (11, 12). The antifungal properties of conazoles result from binding of the azole ring moiety to the heme protein of fungal CYP51 C-14 to inhibit demethylation in ergosterol biosynthesis (13). This mode of action has cause for concern regarding the potential adverse impact of conazoles on CYP-mediated biotransformation mechanisms within mammalian and aquatic species (14–16). Studies have shown that certain conazoles are considered to be hepatotumorigenic in rodent species (17, 18), and several of these fungicides are currently listed for reregistration in the Federal Register to ensure triazole chemical exposures are not exceeding human safety standards (19). In addition to the critical role CYP enzymes play in liver biotransformation of foreign chemicals, this versatile family of enzymes is responsible for a broad spectrum of physiological functions which include the biosynthesis or catabolism of steroid hormones (20, 21). Recently, the U.S. Environmental Protection Agency (U.S. EPA) has developed an Interspecies Correlation Estimation (ICE) program which aims to reduce the amount of toxicity testing by predicting toxicity values for multiple species based upon acute toxicity data from a known species (2). A strong emphasis has been placed on the characterization of fish biotransformation processes to improve understanding of the mechanisms of xenobiotic detoxification and to accurately predict bioaccumulation potential. The rainbow trout (Onchorhynchus mykiss) has historically been among the most frequently tested freshwater salmonids with respect to both toxicology and physiology research (3, 4, 22, 23). A recent report focusing on the bioaccumulation and biotransforVOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 947

FIGURE 1. Proposed LC/MS/MS fragmentation pathways for triadimefon and its metabolite. Base peaks are represented in bold in the table. mation of conazoles within rainbow trout stressed the need for metabolite identification for proper risk assessment (24). Predicting acute toxicity through cross-species extrapolation requires a detailed understanding of metabolic processes, and little information exists regarding the biotransformation of conazoles in either mammalian or aquatic species. Although inherently complex, metabolite identification through hepatic transformation is imperative for accurate ecological risk assessment as the product(s) formed may pose a greater toxicological threat than the parent molecule (25–27). Relatively few reports exist addressing the uncertainty in assessing the interaction of CYP enzymes with xenobiotics across vertebrate species. The present study was aimed to evaluate the cross-species in vitro transformation of conazole fungicides within rat and rainbow trout hepatic microsomes as well as purified human recombinant CYP 3A4 for reducing the uncertainty in ecological risk assessment.

Materials and Methods Reagents. All conazoles were obtained from the U.S. EPA National Pesticide Standard Repository, (Fort Meade, MD). Propiconazole metabolite standards were a generous gift from Syngenta (Greensboro, NC). β-Nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphate, glucose6-phosphate dehydrogenase, magnesium chloride, phosphate, and trizma buffers were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol and acetonitrile from 948

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Fisher Chemicals (Fair Lawn, NJ) were of analytical grade. Perchloric acid (60–62%) was obtained from J.T. Baker (Phillipsburg, NJ). Microsome Preparation Procedure. Frozen hepatic microsomes from male Sprague–Dawley rats at a protein concentration of 20 mg microsome protein/mL were purchased from In Vitro Technologies (Baltimore, MD). The same lot of microsomes was used for all of the studies and stored at -80 °C until use. Microsomal characterization including ethoxycoumarin-o-deethylase activity (ECOD) (28), total protein (29), and total cytochrome P-450 (30) was conducted within our laboratory and results were in agreement with the values provided by In Vitro Technologies. Juvenile rainbow trout (Lake Burton Fish Hatchery, GA) were held for a ten day period in fiberglass aquaria with carbon-filtered, recirculating, dechlorinated tap water chilled to 12 °C. Trout livers were excised upon cervical dislocation, washed, and coarsely minced in an ice-chilled 1.15% KCl solution. The KCl solution was then drained and a wet weight of liver material was recorded. The liver was transferred to a prechilled glass homogenizer where 4 mL volumes of a 0.25 M sucrose solution were added per gram of liver weight. The sample was homogenized on ice and then transferred to an ice-chilled centrifuge tube. The liver homogenate was centrifuged in a high speed centrifuge (Beckman) at 8000g for 20 min at 4 °C. Next, the supernatant was carefully decanted and centrifuged at 100000g for 60 min at 4 °C. The supernatant was then

vortexed and allowed to stand for 10 min in the heating or cooling block to ensure temperature equilibration. In a separate vial, a NADPH-regenerating system (NRS) was preincubated at 37 °C (rat) or 11 °C (trout) for 10 min with 100 mM phosphate buffer (rat) or 100 mM trizma buffer at pH 8.0 (trout) and a final reaction concentration containing 5 mM NADP, 7 mM glucose-6-phosphate, 1.25 mM MgCl2, and 1.5 units of glucose-6-dehydrogenase. The reaction was initiated by the addition of 250 µL NRS to the microsomal suspension. Rat microsome assays were conducted for 0–30 min, whereas the low temperature trout microsome assays were conducted for 0–4 h. All assays were terminated with a 100 µL addition of 60% perchloric acid and immediately placed on ice. The samples were centrifuged at 4 °C for 10 min at 10600g, and the supernatant was transferred to sealcap HPLC vials for analysis. Human CYP 3A4 Assays. Human recombinant CYP 3A4 was purchased from In Vitro Technologies (Baltimore, MD). Conazole screening assays (20–40 µM) were conducted using 10 pmol of recombinant CYP3A4 following the rat microsomal incubation procedure described above. LC/MS/MS Analytical Methods. Analysis of all conazole samples was performed on a Varian 1200L tandem mass spectrometer interfaced with a Varian 430 autosampler and a Varian 230 high-performance liquid chromatograph (HPLC) quaternary pump system equipped with an Alltech 631 column heater and photodiode array detector. All system operations were controlled by Varian MS Workstation version 6. Injections (100 µL) were made onto a Nucleosil 100 C18 column (4.6 × 100 mm, 5 µm particle diameter, Alltech, Deerfield, IL). An isocratic mobile phase (0.4 mL/min) consisting of 60% acetonitrile and 40% water was employed while maintaining column temperature at 30 °C. Samples were introduced to the mass spectrometer in positive electrospray ionization (ESI +) mode with a needle potential of 3.95 KV. The drying gas pressure was maintained at 1.3 kTorr (25 psi) with temperature settings of 175–225 °C. Capillary potential was set to 30 eV and the shield offset was 600 V. The collision gas was Ar and collision cell pressure was 2.7 mTorr (5.2 × 10-5 psi). Collision energy for fragment identification was varied from -10 to 25 eV and the detector potential was 1800 V.

Results

FIGURE 2. Representative LC/MS/MS spectra for triadimenol standard and comparative rat and trout samples. discarded and the microsome pellet was resuspended with a 1:1 volume per gram of original wet weight with a pH 8.0, 0.066 M trizma base buffer containing 0.25 M sucrose, and 0.0054 M EDTA. Trout liver microsome total protein content was determined using the Bradford assay (29) and stored at -80 °C until use. Microsome Incubation Procedure. Incubations of microsome samples with conazoles were conducted in microcentrifuge tubes placed in a heating block at 37 °C (rat) or a cooling block at 11 °C (trout). All conazole standards were dissolved in acetonitrile and stored in amber vials at 4 °C. Microsomal suspensions were prepared at a final concentration of 0.125 mg microsome protein/ml in phosphate buffer (100 mM) at pH 7.4 (rat) or trizma buffer (100 mM) at pH 8.0 (trout). Next, conazole stock solutions (acetonitrile) were added to achieve a final substrate concentration level of 20–40 µM while not exceeding 1% organic solvent. The system was

Preliminary experiments were performed to ensure that saturating substrate concentrations were used to determine the metabolic profiles for all conazoles under conditions that gave linear product formation with respect to time and microsomal protein concentration. Due to the relatively small size of the juvenile trout and low yield of liver tissue per individual, it was necessary to pool animals to ensure that the same microsome matrix was used throughout all experiments. In accordance, all rat microsomes were purchased from the same lot for quality assurance purposes. The termination of microsomal assays with perchloric acid immediately following NRS addition ensured that all components were present within the zero time point control matrix. Abiotic controls were conducted with the conazole fungicide and buffer media alone. In addition, controls were conducted with an active microsomal matrix containing no conazole to account for time dependent changes in the reaction matrix. Analysis using full-scan LC/MS was first implemented to determine the molecular mass of all metabolites observed while comparing active versus control samples. Structures of all unidentified molecular ion(s) were then investigated through selected ion fragmentation using LC/MS/MS. Triadimefon. Hepatic transformation of the conazole triadimefon resulted in the formation of a single metabolite peak showing nearly identical retention times for both rat VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Proposed LC/MS/MS fragmentation pathways for trans-bromuconazole and its metabolites. Base peaks are represented in bold in the table. and trout microsomal systems. LC/MS analysis of the parent triadimefon was indicated by the appearance of the molecular ion [M + H] + m/z 294 ion, whereas the linear formation of the metabolite peak (Td-M1) in both species resulted in an [M + H] + m/z 296 ion. Analysis of both triadimefon and Td-M1 using LC/MS/MS allowed for structural characterization (Figure 1). Triadimefon displayed a base peak of m/z 197, and two major ions: m/z 225 and m/z 69. The m/z 69 was also found to be the base peak present within the Td-M1 metabolite mass spectra, suggesting no alteration had occurred to the triazole ring. However, the m/z 225 ion observed in the parent triadimefon, which represents the remaining structure upon loss of the triazole ring, appeared two mass units higher (m/z 227) within the Td-M1 spectra. The base peak of m/z 197 for triadimefon corresponded to the loss of carbon monoxide from the m/z 225 fragment. The presence of an m/z 227 ion within the Td-M1 spectrum is likely due to the reduction of the carbonyl group, and the intense m/z 99 which was found only in the Td-M1 spectra provided further evidence of this observation. An authentic triadimenol standard representing the carbonyl reduced form of triadimefon was analyzed by LC/MS/MS, and the same fragmentation pattern (m/z 69, m/z 99, m/z 227) and retention time was observed, confirming that the Td-M1 metabolite was triadimenol. Representative LC/MS/MS spectra of the triadimefon metabolite (triadimenol) observed in both rat and trout microsome assays along with the authentic metabolite standard are depicted in Figure 2. trans-Bromuconazole. Initial LC/MS analysis conducted for the interspecies hepatic transformation of trans-bromuconazole indicated that both the rat and trout metabolized the parent molecule. Disappearance of the parent compound resulted in the concomitant linear formation of two unidentified metabolite peaks: Br-M1 and Br-M2, which dis950

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played nearly identical retention times in both the rat and trout species (Figure 3). trans-Bromuconazole demonstrated a molecular ion [M + H] + m/z 376 while both product peaks Br-M1 and Br-M2 exhibited a [M + H] + m/z 392 ion, consistent with the addition of an oxygen atom through a phase I hydroxylation. LC/MS/MS selected ion fragmentation of trans-bromuconazole [M + H] + m/z 376 ion resulted in three major ions: m/z 159, m/z 227, and m/z 307. Figure 3 shows a proposed fragmentation pathway for trans-bromuconazole. The intense m/z 227 transition ion observed suggests the loss of the bromotetrahydrofuranyl ring, whereas the m/z 307 ion indicates the loss of the 1,2,4-triazole ring structure from the parent molecule. The base peak m/z 159 ion represents a dichlorotropylium ion resulting from the rearrangement of m/z 227 ion and subsequent fragment loss. LC/MS/MS characterization of the [M + H] + m/z 392 metabolite ion was performed on the Br-M2 product peak due to low abundance of the Br-M1 (Figure 3). An initial loss of water from Br-M2 yielded the formation of an m/z 374 ion in both the rat and trout. A key base peak transition ion m/z 243 suggest a hydroxylated form of the m/z 227 rearrangement fragment observed with the parent transbromuconazole indicating the loss of the bromotetrahydrofuranyl ring. The m/z 243 transition ion then proceeded to lose water as confirmed by the presence of the m/z 225 ion. Hydroxylation of the dicholorophenyl ring was confirmed by varying the collision energy for both the parent [M + H] + m/z 376 ion and Br-M2 metabolite [M + H]+ m/z 392 ion and observing the corresponding loss of the triazole ring m/z 69 ion. We postulate that hydroxylation of the phenyl ring would likely take place at either the 3 or 5 position since the chlorine atoms are ortho and para directing. Propiconazole. Cross-species comparison of the microsomal incubation of propiconazole resulted in the elution

FIGURE 4. Proposed LC/MS/MS fragmentation pathways for propiconazole and its metabolites. Base peaks are represented in bold in the table. of three distinct metabolite peaks in both the rat and trout. All three metabolites (Pr-M1, -M2, -M3) increased with time and displayed nearly identical retention times (Figure 4). Initial LC/MS analysis of each metabolite demonstrated an intense [M + H] + m/z 358 ion signifying the addition of an oxygen atom to the parent propiconazole [M + H] + m/z 342 ion. LC/MS/MS fragmentation of propiconazole resulted in the formation of two major ions: m/z 69 and the base peak m/z 159, which represents the 1,2,4-triazole ring structure and dichlorotropylium ion, respectively. However, selected ion LC/MS/MS fragmentation on each of the metabolite ions (m/z 358) in both the rat and trout consistently resulted in a single base peak of m/z 256 at varying collision energies. Authentic metabolite standards were obtained for propiconazole hydroxylated in three differing aliphatic side-chain positions (X, Y, Z), as depicted in Figure 4, and demonstrated the same retention times as the product peaks observed with the rat and trout samples. LC/MS/MS analysis confirmed the presence of the base peak m/z 256 in the mass spectra of all three metabolite standards. The presence of the m/z 256 ion suggest a rearrangement of the dichlorophenyl and triazole ring structures resulting in the partial loss of dioxalane ring containing the aliphatic side chain. Additional Conazoles. Our initial LC/MS/MS findings demonstrated identical metabolic profiles among rat and

trout species via: aromatic and aliphatic hydroxylation reactions, and carbonyl reduction. Based upon these results, an interspecies comparison of rat and trout microsomal assays was conducted for 10 additional conazoles (Figure 5) using LC/MS molecular mass and retention time. Results from these experiments indicate that 10 out of 13 total conazoles tested demonstrated identical metabolic profiles (Table 1). The observed increase of molecular mass 16 Da indicates that phase I hydroxylation of the parent substrate was the predominant catalyzed reaction. Microsomal assays conducted with epoxiconazole and metconazole showed no activity in either the rat or trout. A slightly different hexaconazole metabolic profile was observed with the detection of Hex-M3 and M4 in trout microsome systems, whereas neither was detected in the rat. Trace level detection of metabolites within paclobutrazole and ipconazole assays resulted in differing metabolic profiles in rat versus trout. Conazole Metabolism using Human CYP 3A4. Many human hepatic CYP enzymes have been isolated and characterized and are commercially available for interspecies comparison, and several studies have indicated that the CYP 3A subfamily may play an active role in the metabolism of conazoles (12, 31–33). Metabolic studies were conducted with the group of 13 conazoles using VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Discussion

FIGURE 5. Chemical structures of conazoles used for additional in vitro analyses. purified human CYP 3A4. LC/MS analysis indicated a high degree of similarity in phase I metabolites observed in both the rat and trout microsome matrices and purified CYP 3A4 studies (Table 1). CYP 3A4 assays conducted with epoxiconazole showed no activity just as within the rat and trout microsomal matrix. Human CYP 3A4 transformation of metconazole resulted in the formation of two metabolites: (Met-M1 and M2) with the increased molecular mass 16 Da indicating hydroxylation of the parent molecule; whereas neither metabolite was detected in the rat or trout. Trace level detection of paclobutrazole and ipconazole metabolites determined in the trout and rat microsomes respectively, were both observed using human CYP 3A4. Triadimefon was the only conazole tested that was not transformed with CYP 3A4, but did undergo phase I reaction with both microsome matrices. 952

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Cross-species extrapolation is paramount to ecological risk assessment for predicting the diverse biological effects of exposure to chemicals within the environment. The technical basis for current exposure models is not sufficient to explain a dose to effect continuum without interspecies comparison, and a great deal of uncertainty regarding extrapolation among mammalian and aquatic vertebrate species has been documented (2, 34, 35). Although recent in vivo and in vitro studies suggest conazole fungicides exhibit toxic effects in both fish and mammalian systems, virtually no information exists addressing the interspecies metabolic fate of this class of chemicals (14, 36–38). Conservation of biotransformation processes among fish and mammalian systems is key to understanding the potential risk of chemicals as a consequence of environmental exposure, and significant differences between rat and fish metabolic processes have been documented (34, 35, 39). Our initial LC/MS/MS findings delineating conazole biotransformation pathways within rat and trout microsomes showed that both species exhibited similar metabolic profiles. Through detailed metabolite identification, we were able to demonstrate that the microsomal interspecies comparison was well conserved via both aromatic (i.e., bromuconazole) and aliphatic hydroxylation (i.e., propiconazole) reactions, and the carbonyl reduction of triadimefon (Figures 1, 3, 4). Results from the interspecies comparison of rat and trout using LC/MS resulted in identical metabolic profiles for 10 out of 13 conazoles tested (Table 1.) The differences observed among hexaconazole, paclobutrazole, and ipconazole assays were most likely due to different rates of metabolite formation and limits of detection for the rat and trout microsome systems. Further investigation of metabolite kinetics is needed as this type of metabolic characterization between rat and trout provides valuable insight into scenarios where metabolite formation may prove to be the toxic agent and adversely affect diverse aspects of physiology (5). One of the most abundant CYP enzymes among human liver microsomes belongs to the CYP 3A subfamily, which has been postulated to metabolize as much as 60% of all therapeutic compounds (8). Previous reports have shown that CYP 3A may be genetically expressed and/or play an active role in the metabolism of conazole compounds (12, 31–33). In an effort to improve the understanding of the degree of conservatism among vertebrate species, purified recombinant human CYP 3A4 was screened in comparison with rat and trout microsomes among the group of 13 conazoles (Table 1). LC/MS conducted on the limited sample size indicated that nearly every metabolite detected in the rat and trout microsomal assays was detected within the human CYP 3A4 assays. These results suggest a high degree of conservation among species with an equivalent isozyme activity of human CYP 3A4 being present in both the rat and trout in vitro assays. An interesting finding from this comparative study was marked by the confirmation of the reduced metabolite triadimenol from triadimefon in both the rat and trout, but not CYP 3A4, which is known mainly to catalyze oxidative processes (40). In contrast, metconazole was hydroxylated (Met-M1,-M2) within the human CYP 3A4 assays but neither metabolite was detected in either the rat or trout. It must be noted that the level of metabolite detection in an impure microsome matrix is higher in comparison to the purified human enzyme preparations, which could account for the differences observed. Epoxiconazole was unique in that it showed no biotransformation among all three systems tested. One may postulate that such findings may be the result of steric hindrance due to the presence of a fluorophenyl moiety in addition

TABLE 1. LC/MS Analysis of Additional Conazolesa rat compound

metabolite

trans-bromuconazole Br-M1 Br-M2 propiconazole Pr-M1 Pr-M2 Pr-M3 triadimefon Td-M1 myclobutanil Myc-M1 hexaconazole Hex-M1 Hex-M2 Hex-M3 Hex-M4 triticonazole Triti-M1 epoxiconazole Epx-M1 metconazole Met-M1 Met-M2 diniconazole Dini-M1 fenbuconazole Fen-M1 uniconazole Uni-M1 paclobutrazole Pac-M1 ipconazole Ip-M1 Ip-M2 Ip-M3 a

trout

human CYP 3A4

RT (min)

MS (m/z)

RT (min)

MS (m/z)

RT (min)

MS (m/z)

27.92 14.54 17.47 32.74 13.31 13.64 14.28 21.76 18.17 20.77 10.44 28.28 11.29 12.02 ND ND 20.72 11.89 23.30 ND 30.63 ND ND 31.19 14.96 24.82 21.13 23.56 12.31 18.66 ND 37.34 14.49 17.85 20.43

376 392 392 342 358 358 358 294 296 289 305 314 330 330 ND ND 318 334 330 ND 320 ND ND 326 342 337 354 292 309 294 ND 334 350 350 350

28.14 14.66 17.48 32.78 13.34 13.66 14.37 21.92 18.35 20.80 10.24 28.67 11.11 11.96 14.86 15.78 20.66 11.90 23.09 ND 30.37 ND ND 31.99 14.92 24.59 21.12 23.29 12.30 18.40 11.25 37.77 ND ND ND

376 392 392 342 358 358 358 294 294 289 305 314 330 330 330 330 318 334 330 ND 320 ND ND 326 342 337 354 292 309 294 310 334 ND ND ND

28.35 14.73 17.24 33.11 13.33 13.33 14.89 21.96 ND 21.16 10.59 28.56 11.39 12.23 15.10 ND 20.15 11.60 23.11 ND 29.62 14.30 15.84 31.45 14.99 24.64 13.97 23.19 12.30 18.31 11.02 37.14 14.37 17.81 21.12

376 392 392 342 358 358 358 294 ND 289 305 314 330 330 330 ND 318 334 330 ND 320 336 336 326 342 337 353 292 308 294 310 334 350 350 350

ND not detected.

to the (di) chlorophenyl ring found in all the other conazles tested (Figure 5). Further investigation into the phase I transformation of triadimefon, metconazole and epoxiconazole needs to be conducted. It is necessary to emphasize that the cross-species comparisons of metabolic profiles conducted within this study does not address a kinetic evaluation for the rate(s) of metabolite formation. Such an assessment would require determination of kinetic rate constants for both product formation and substrate depletion. Comparison of enzyme kinetics across mammalian and nonmammalian species often poses difficulties with respect to incubation temperature, substrate saturation, intracellular pH, and determination of specific enzyme activity. However, an increasing need for such complete metabolism information has been recognized for accurate exposure assessment of environmental contaminants across a wide variety of species within the aquatic environment. It is interesting to note, that several of the conazoles shown to undergo in vitro biotransformation in this study were determined by Konwick et al. not to bioaccumulate within rainbow trout in vivo (24). The results from this study help provide the initial metabolic framework needed to address bioaccumulation potential and toxicity of conazoles across vertebrate species. In summary, conazoles are widely used agricultural and pharmaceutical chemical agents known to inhibit specific cytochrome reactions. Evaluation of the microsomal biotransformation of such foreign chemicals across rat and trout species is critical for cross-species extrapolation and accurate risk assessment. Our results demonstrate that the in vitro

hepatic transformation of conazoles is highly conserved among rat (mammalian) and rainbow trout (aquatic) species. In addition, the enzyme activity observed within the microsomal assays was generally consistent with the metabolic profile observed using purified recombinant human CYP 3A4.

Acknowledgments The authors express appreciation to Tom Sack, Cather Brown, and John Evans for their assistance in conducting laboratory analysis. We would also like to thank Dr. Susan Richardson (U.S. EPA, Athens, GA) and Dr. Michael Bartlett from the University of Georgia for their assistance with spectra interpretation. Disclaimer: This paper has been reviewed in accordance with the U.S. Environmental Protection Agency’s peer and administrative review policies and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation of use.

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