ToxSci Advance Access originally published online on August 14, 2008
Toxicological Sciences 2008 106(1):55-63; doi:10.1093/toxsci/kfn167
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Pharmacokinetics and Dosimetry of the Antiandrogen Vinclozolin after Oral Administration in the Rat
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* Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory
Sección Externa de Toxicología
Sección Externa de Farmacología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Col. San Pedro Zacatenco, México D.F. CP 07360, México
National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed at Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav-IPN), Sección Externa de Toxicología, Av. IPN No. 2508, Col. San Pedro Zacatenco, México D.F. CP 07360, México. Fax: +52-55-57473395. E-mail: asierra{at}cinvestav.mx.
Received June 9, 2008; accepted August 2, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Vinclozolin (V) is a fungicide with antiandrogenic properties. To determine the pharmacokinetics and dosimetry of V, adult male rats were administered an oral dose of V (100 mg/kg) in corn oil and sacrificed over time after dosing. V and its metabolites were analyzed in serum and tissues by high performance liquid chromatography/diode array detector/mass spectrometer. V, 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), and five other metabolites were detected in serum and tissues. One metabolite was identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (M5). The mean serum concentration data for V were fitted to a one-compartment model for kinetic analysis. At 2 h, V serum concentration peaked; whereas only trace levels were detected at 24 h (t1/2 elim = 3.6 h). V was detected in all tissues and preferentially accumulated in fat. M1 serum levels increased until 8 h, being at least 2-fold higher than those of V at this time, and then declined with a t1/2 = 3.3 h. M5 was the main metabolite in serum and tissues. Serum M5 levels were 5-fold higher than V and 2-fold greater than M1 at all times. At 48 h, M5 remained the main metabolite (t1/2 elim = 13.1 h). Liver and kidney exhibited the highest levels of M5, V, and M1. M2 and 3,5-dichloroaniline had the lowest levels of V metabolites in serum and tissues. V is well absorbed, extensively metabolized and widely distributed. M5, the most abundant V metabolite, may be used as an exposure biomarker for pharmacokinetic modeling. These results may clarify the relationship between toxicity and tissue dose of V and its metabolites.
Key Words: vinclozolin; antiandrogenic; dicarboximides; biomarkers.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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There is concern for the potential adverse effects of environmental contaminants on the endocrine system of humans and wildlife (Orlando and Guillette, 2007
; Tanabe, 2002; Yang et al., 2006). Disorders of the immune, hormonal, and reproductive systems and neurobehavioral functions may result from exposure to these contaminants (Yang et al., 2006). The dicarboximide fungicide vinclozolin (V) is an example of an environmental contaminant that can alter the endocrine system. V causes antiandrogenic effects in male rats (Crews et al., 2007; Gray et al., 1994; Rider et al., 2008). There is no information whether this effect of V occurs in developing human males.
Administration of V to pregnant rats at specific gestational stages results in a significant degree of morphological feminization and demasculinization of male offspring (Gray et al., 1994). V exposure in peripubertal rats from day 22 to day 54–56 at doses of 10, 30, and 100 mg/kg/d causes delayed pubertal maturation at the high doses and increased serum levels of luteinizing hormone (at all doses), testosterone (high dose only), and 5
-androstane, 3, 17β-diol (high dose only) without affecting testis size or fertility (Monosson et al., 1999). These effects appeared to be predominantly due to V and its metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) (Kelce et al., 1994, 1997). M1 and M2 bind to the mammalian androgen receptor (AR) and act as AR antagonists. They interfere with androgen-dependent gene expression in vivo and in vitro by inhibiting AR-binding to DNA (Gray et al., 1994; Molina-Molina et al., 2006; Monosson et al., 1999; Wolf et al., 2000). M2 is 50-fold more potent than M1 and only twofold less potent than the pharmacologic inhibitor of AR, hydroxyflutamide (Wolf et al., 2000). Effects of V in vivo reflect a dose-response relationship and temporal association that support its mechanism of action. However, there is no data on the dosimetry of V, M1, and M2 in organs affected to assess this relationship.
V is transformed by chemical hydrolysis, photolysis, or metabolism by mammals and bacterial systems (Schick et al., 1999; Sierra-Santoyo et al., 2004; Szeto et al., 1989). Some specific metabolic pathways have been proposed, however, they have not been well characterized (Vanni et al., 2000). Overall, information about the biotransformation, distribution, and accumulation of V and its metabolites is very limited. The serum of rats administered repeated oral doses of V was analyzed for V, M1, and M2 (Kelce et al., 1994; Monosson et al., 1999). M1 was the major metabolite and only trace levels of V and M2 were detected. M2, M1, and V serum levels reported in the rats were well below their Ki for interaction with the AR (Kelce et al., 1994). In fathead minnows, V was the main chemical detected in tissues after a 21-day exposure and females accumulated significantly more V than males (Makynen et al., 2000). The metabolites 3-(3,5-dichlorophenyl)-5-methyl-5(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione (M4) and 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (M5) were detected in liver extracts in hens following acute oral exposure to V (Dean et al., 1988). In the fungus Cunninghamella elegans, isomers of M5 were detected as metabolic products of V (Pothuluri et al., 2000). In preliminary studies from our laboratory, we detected a metabolite that could correspond to M5 in addition to M1 and M2 in serum of a rat administered an acute dose of V (Sierra-Santoyo et al., 2004, 2006). More recently, Bursztyka et al. (2008) described V metabolism in rat liver slices and in urine of rats administered an oral dose of V. M5 was the major metabolite detected in the rat liver slices and in urine, this metabolite was mainly present as glucuronide conjugates. In addition, they also identified M4 and the 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3,4-dihydroxy-butanoic acid, a dihydroxylated metabolite of M1 (M6).
The objective of this study was to determine the dosimetry and pharmacokinetics of V and metabolites in the adult male Long-Evans rat following an acute oral dose of V. The resulting data can be used in the development of dose-response models.
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MATERIALS AND METHODS |
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Reagents.
Vinclozolin (96% purity, lot 80818) was purchased from Crescent Chemical Co. (Augsburg, Germany). M1 and M2 were kind gifts from Elizabeth Makynen (U.S. EPA, Duluth, MN). High performance liquid chromatography (HPLC)-grade water, acetonitrile, and methanol were purchased from Burdick and Jackson, Inc. (Muskegon, MI) and M3 was obtained from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of reagent grade and the highest purity available.
Animals.
Male Long-Evans rats were purchased from Charles River Breeding Laboratories (Raleigh, NC) and housed in a facility accredited by the American Association for the Accreditation of Laboratory Animal Care. The animals were housed in clear plastic cages with laboratory-grade pine shavings as bedding. Animals were maintained on Purina Rat Chow (RMH 5001, St Louis, MO) and tap water ad libitum, with a 12:12-h (light/dark) photoperiod. The room temperature ranged from 20 to 24°C, and the relative humidity ranged from 40 to 50%.
Treatment of animals.
When the animals were approximately 70 days old, they were administered an oral dose of V (100 mg/kg) dissolved in corn oil (2.5 ml/kg). Animals were sacrificed by cardiac puncture under CO2-induced anesthesia at selected time points (1, 2, 3, 4, 6, 8, 12, 24, and 48 h). Serum was obtained from blood and organs and tissues were removed. Serum, organs and tissues were stored at –70°C until processed for analysis.
Extraction of V and its metabolites.
To 100 µl of rat serum, 400 µl of 0.1M monobasic potassium phosphate buffer (PB) pH 3.3 was added, whereas 100 mg of tissues were homogenized in 0.8 ml of 0.1 M PB pH 3.3 using a Polytron homogenizer at 20,000 rpm. Previously, we had determined that addition of PB to serum stabilized V from nonenzymatic hydrolysis to M1 and M2 (Sierra-Santoyo et al., 2004). After addition of 5 ml of acetonitrile, samples were vortexed for 1 min and centrifuged at 1600 x g for 10 min at 4°C. Extracts were dried using anhydrous sodium sulfate and the solvent was evaporated under N2. Extracts were stored at –20°C and dissolved in 200 µl of acetonitrile immediately before HPLC/diode array detector (DAD) analysis.
Liquid chromatography analysis of V and metabolites.
V and its metabolites were analyzed by injecting extracts into an Agilent Model 1100 liquid chromatograph as described previously (Sierra-Santoyo et al., 2004). Concentrations of V, M1, M2, and M3 were determined using calibration graphs of each metabolite. The M5 calibration graph was prepared from the exhaustive in vitro metabolism of M2 by rat liver microsomes (> 90%). The M2 enzyme assay was carried out in a final volume of 1 ml at 37°C for 1 h. Incubation medium contained 100 mM KH2PO4 and 5 mM MgCl2 at pH 7.4, 2 mg liver microsomal protein and 50 µM M2. The reaction was commenced by addition of 1 mM nicotinamide adenine dinucleotide phosphate (reduced) and terminated by the addition of 5 ml of acetonitrile to the incubation medium and placed on ice. The terminated reactions were vortexed for 1 min and centrifuged (1650 x g) at 4°C for 10 min. The supernatant was dried using anhydrous sodium sulfate and evaporated under a stream of N2 at room temperature. Residues were dissolved in 200 µl of acetonitrile and stored at 4°C before chromatographic analysis. Concentrations of the metabolites M7, M8, and M9 were estimated using the M5 calibration graph. The response corresponding to the peak area of M5 was linear (r > 0.9989) in the range of 4–120 ng of M5 (Sierra-Santoyo et al., 2006).
M5 metabolite identification was characterized by high-performance liquid chromatohgraphy mass spectrometry (HPLC/mass spectrometer) analysis using an Agilent 1100 series liquid chromatography/mass spectrometer detector VL ion trap mass spectrometer equipped with a UV-Vis Diode-Array detector (Agilent Technologies, Santa Clara, CA). A Nucleosil (Deerfield, IL) 100-5 C18 AB column (5 µm particle size and 4.6 x 250 mm I.D.) and a Perisorb (Upchurch Scientific, Oak Harbor, WA) RP-18 guard column (20 x 1.5 mm I.D., 30–50 µm particle size) were used. The mass spectrometer was operated in positive and negative electrospray ionization (ESI) mode; dry temp, 350°C; nebulizer, 60 psi; dry gas, 11.5 ml/min; range scan, 50–400 m/z; maximum accumulation time, 300,000 µs; ICC Target, 30,000; averages, eight spectra; charge control, on. System solvents consisted of 20 mM ammonium acetate pH 3.3 (A) and methanol:acetonitrile:acetic acid (70:30:0.1%) (B). Initial solvent conditions were set at 40% A:60% B flowing at a rate of 0.7 ml/min at room temperature. After sample injection (20 µl), there was a 20-min linear gradient change to 30% A:70% B, followed by a 5-min linear gradient change to 25% A:75% B. Initial conditions were then re-established by a step gradient and the column was equilibrated for 5 min.
Recovery of V and its metabolites.
Recovery was evaluated in organ or tissue samples of rat spiked with 10 µg/g of V, M1, M2, and M3. These samples were processed for analysis as described above. Recovery was evaluated by the % of the mass of chemical detected in the spiked samples. Precision of the assay was evaluated by calculating the relative standard deviation (coefficient of variation). The recovery and precision values obtained for tissues are shown in the Table 1. Limits of quantification were defined as the lowest concentration of the calibration curve which could be measured with a precision and accuracy below 30% (Baker, 1998). Limits of quantification were 0.075, 0.035, 0.050, 0.070, and 0.047 µg/g for V, M1, M2, M3, and M5, respectively.
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Pharmacokinetic parameters.
Pharmacokinetic parameters were determined from the serum V and its metabolites concentration-time curves. The mean serum concentration data for V and its metabolites were fitted to a one-compartment model for kinetic analysis using the WinNonlin software (Scientific Consulting, Inc., Cary, NC). The absorption rate constant (Kabs) and the half-life (t1/2) for V, the elimination rate constant (Kelim), the terminal half-life, the maximal concentration in serum after oral administration (Cmax) and time to reach Cmax (Tmax) and the area under the curve (AUC) for every metabolite for an interval from 0 to 48 h were obtained from the observed data (Gibaldi and Perrier, 1975
). Statistical calculations were performed using SigmaStat Ver. 3.1.1 (Systat Software, Inc., Richmond, CA). |
RESULTS |
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Serum Disposition of Vinclozolin and Metabolites
The disposition of V was determined following an acute oral dose (100 mg/kg) suspended in corn oil by analyzing serum and tissues collected at different times after dosing. The HPLC elution profile of the acetonitrile-extractable metabolites from serum and tissues showed at least seven peaks in the first 4 h after administration (Fig. 1). The metabolites M3, M1, and M2 eluted at 10.0, 11.8, and 14.6 min, respectively. The other detected V metabolites, denoted M5, M7, M8, and M9, eluted approximately at 7.0, 9.0, 6.6, and 6.0 min, respectively (Figs. 1c and 1d).
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The mean serum concentrations of V and its metabolites determined after single oral administration of 100 mg/kg of V are shown in Figure 2. Values for the pharmacokinetic parameters for V and its metabolites are shown in Table 2. V was rapidly absorbed after oral administration with a Kabs of 1.046/h. The half-life of absorption (t1/2 abs) was calculated to be 0.66 h. The Cmax for the 100 mg/kg dose was 16.8 µM. The value for Tmax in serum was 2 h. V exhibited a monophasic decline and a good fit of the observed data to a one-compartment model was obtained. The elimination half-life (t1/2 elim) of V was 3.6 h, and it was practically undetectable at 24 and 48 h.
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M5 serum levels represented at least 40% of total V metabolites from 1 to 12 h; and from 24 h after administration this percentage increased to 80% (Fig. 2a). The Cmax of M5 was 75.3 µM and was observed at 8 h after V oral administration. The AUC for M5 was 1742.4 µM-h and was the highest value of all metabolites including V. The t1/2elim was 13.1 h.
M1, a product of nonenzymatic hydrolysis of V, was easily detected in serum within 24 h after V administration. M1 levels ranged from 6 to 31% of total V metabolites over the time course of 48 h. The Cmax of M1 was 42.5 µM and was observed at 8 h after V oral administration. The AUC for M1 was 460.9 µM h and the t1/2elim was 3.3 h.
The metabolite denoted as M8 represented between 9 and 29% of total metabolites detected in rat serum until 48 h after V dosage. The Cmax of M8 was 29.7 µM and was reached 3 h after V administration. The AUC was 357.7 µM h and its t1/2elim was 9.9 h. On the other hand, M9 metabolite was detected only within 24 h after V dosage. M9 serum levels were no greater than 9.2 µM. Its maximum peak was observed at 2 h and its t1/2 elim was 11.5 h. Serum levels of the metabolites M2, another product of V nonenzymatic hydrolysis, M3 and M7 showed concentrations lower than 2 µM within 6 h after V dosage. Thereafter, only trace levels of these three metabolites were observed using HPLC/DAD (Fig. 2b).
Tissue Disposition of Vinclozolin and Metabolites
Vinclozolin was efficiently distributed to all tissues analyzed. The tissue levels of V and its metabolites at 2 and 8 h after dosage are shown in Table 3. V was preferentially accumulated in fat; the concentrations in this tissue were the highest found at 2 and 8 h (49.7 and 182.8 µg/g, respectively), representing several-fold more than those observed in other tissues analyzed. The tissue concentrations of V were highest in fat and lowest in testis at both 2 and 8 h. The tissue concentrations of V were higher at 2 than at 8 h except in prostate, where at 8 h V concentration was 80% higher than at 2 h, and fat, where it was 3.7-fold higher at 8 h.
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M5 was the main metabolite of V detected in analyzed tissues; its concentration was higher at 8 than at 2 h. The concentration of M5 exceeded the concentration of V in all tissues examined at 2 and 8 h except fat. The highest levels of M5 were observed in liver and kidney followed by brain, testis and prostate. M5 levels in liver and kidney were at least 9-fold higher than those observed in fat, whereas in other tissues M5 levels were at least 2.5-fold higher.
M1 and M8 were consistently detected in serum and tissues at 2- and 8-h postdosing. The only exception was in brain, where M8 was not detected at 8 h. In serum, liver, kidney and testes, M1 levels were greater at 8 than at 2 h. At 8 h, M1 levels were only exceeded by those of M5. Levels of M8 were similar to those of M1 in serum and tissues at 2 h, but decreased by 8 h.
M2, M3, M7, and M9 were also detected in tissues; however, they were detected in very low levels in most of the organs at the analyzed times (Table 3).
Identification of M5
As M5 was the main metabolite of V detected in serum and tissues of treated rats, we focused on identifying this metabolite using HPLC/MSD, in addition to the identification of M1, M2, and M3 metabolites by tret and UV spectra (data not shown). Several peaks were separated by HPLC and detected by UV at 254 nm, but only one was detected in the mass spectrometer detector (Fig. 3a). On the basis of tret and spectroscopic analysis, the peak eluted at 9.1 min corresponded to M5. Data observed for this peak were consistent to those observed in the product of enzyme reaction from M2 metabolism by rat liver microsomes. The UV spectrum of M5 is shown in Figure 3b. The negative ESI data for this peak showed molecular ions [M]– at m/z 292.2 (base peak) and fragment ions at m/z 232.4 [M-C2O2H5]– and 160.3 [M-C5H9O4]– (Fig. 3c). The positive ESI mass spectra of this peak may be sodium adducted molecules [M + Na]+ at m/z 316, protonated molecules at m/z 294, and fragment ions at m/z 276 [MH-H2O]+ and 161.9 [M-C5H9O4]+, corresponding to protonated 3,5-dichloroaniline (Fig. 3d). All of these ions contained two chlorine atoms and an odd number of nitrogen. On the basis of these spectroscopic analysis results, the peak at 9.1 min was identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (M5) which has a MW of 293 (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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V is antiandrogenic in rats (Gray et al., 1994
). The effects in neonatal, pubertal and adult rats and other species have been explained on the basis of Androgen receptor competitive antagonism described for V, M1, and M2 by inhibiting the expression of androgen-dependent genes in ways that mediate subsequent phenotypic alterations in male rats (Kelce et al., 1997; Monosson et al., 1999; Rider et al., 2008; Wong et al., 1995). A range of effects have been attributed to V following studies in several animal species and experimental models (Bisenius et al., 2006; Crews et al., 2007; Makynen et al., 2000; Monosson et al., 1999; Mu et al., 2006; Nagaosa et al., 2007), effects of which are most likely impacted by the pharmacokinetics of V. However, the limited information available on the disposition and kinetics of V and its metabolites makes it difficult to extrapolate toxicological findings in animal models for human risk assessment.
The present study describes the disposition and kinetic parameters for V in the rat, its products of nonenzymatic hydrolysis M1 and M2, and some of its metabolic products denominated M3, M5, M7, M8, and M9. A proposed metabolic pathway of V in the rat is summarized in Figure 4. V is a non-ionizable lipophilic (log Kow 3.0) molecule. These properties favor that V be rapidly absorbed (t1/2 abs = 0.66 h), distributed throughout the animal and transformed by both nonenzymatic hydrolysis and oxidative processes. The products of this transformation were detected rapidly in rat serum and tissues after a single oral administration. In a previous study, our laboratory reported that V was rapidly hydrolyzed in vitro in rat serum at 37°C (t1/2 = 0.5 h). M1 represented approximately 80% of total product, whereas M2 represented approximately 20% after 2 h (Sierra-Santoyo et al., 2004). Similarly, following oral administration of V, serum levels of M1 were greater than those of M2 at all time points. Therefore, the presence of M1 and M2 in serum of rats administered V may be the result of nonenzymatic hydrolysis of V in serum. In addition, V may be taken up by tissues, nonenzymatically hydrolyzed, and the metabolites released to the blood.
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The results obtained from the in vitro studies by Kelce et al. (1994)
showed that V and metabolites M1 and M2 antagonized the binding of androgen to the rat AR. The Ki values reported were > 700, 92, and 9.7 µM, for V, M1, and M2, respectively. The maximum serum levels detected for V, M1, and M2 in this study after a single administration of V (100 mg/kg) were below the Ki values (Fig. 2). The maximum M1 concentration represented 44% of the Ki value, whereas for V and M2, their maximum concentration represented no more than 3% of the Ki value. These results suggest that M1 could be the main metabolite involved in the interaction with the AR, producing antiandrogenic effects described by V exposure. However, M2 participation cannot be discounted because the maximum M2 serum levels found in this study (0.3 µM) could also be antagonistic. Euling et al. (2002) modeled the interaction of M2 with AR at different physiological levels of 5-dihydrotestosterone (DHT) and AR. They concluded that at physiological levels of DHT, M2 would be antagonistic at concentrations of 200 nM or higher during critical windows for male urogenital tract, external genitalia, and pubertal development. Together these results suggest that the antiandrogenic effects described may be a cumulative effect of parent compound and its metabolites produced during the first hours following V repeated administration as described in several studies (Bayley et al., 2002; Gray et al., 1994; Kelce et al., 1997; Monosson et al., 1999; Uzumcu et al., 2004). This proposal is on the basis that V and M1 were rapidly eliminated from rat serum. Their half-life times were the shortest (3.6 and 3.3 h, respectively) of all metabolites and at 24 h after administration V, M2, and M1 serum levels were very low or close to the detection limits as shown in this study and by Monosson et al. (1999) after V repeated administration at 10, 30, and 100 mg/kg/day. There is no available information about the antiandrogenic properties of V metabolites other than M1 and M2. Therefore, more definitive studies are needed to determine the serum levels of V and its metabolites after repeated doses of V at levels that are associated with the anti-androgenic effects of V. V accumulated in fat and its metabolites were detected in practically all tissues. Surprisingly, in testis, prostate and brain, organs with high AR levels and where several functions are regulated by androgens, low levels of V, and its metabolites were observed until 8 h after administration of V with the exception of M5. In the case of M2, a more potent antiandrogen than V and M1, levels of this metabolite in these organs were below detection limits (Table 3). The presence in tissues of other metabolites derived from V suggests they are products of enzyme reactions within the tissue or they are taken up from serum. Liver and kidney, organs with high capacity of metabolism of xenobiotics, showed the highest levels of V metabolites.
M5 was identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide. It was the main metabolite detected in serum and tissues after a single oral administration of V (Fig. 2, Table 3). These results indicate that V is efficiently metabolized to M5 in the rat, and are in agreement to those reported in hens (Dean et al., 1988) and another rat strain (Bursztyka et al., 2008). The metabolites M7, M8, and M9, could correspond to metabolites proposed in another report (Vanni et al., 2000). Preliminary results suggest that M7 could correspond to N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3, 5-dichlorophenyl-1-carbamic acid. The definitive identification and stability of M7 as well as the remaining metabolites is a subject of further investigation.
M5 may be formed by epoxidation of the vinyl group in M2, followed by an epoxide hydrolase reaction as was proposed to occur in C. elegans (Pothuluri et al., 2000). M2 can be produced by simple hydrolysis of V. Another and more probable metabolic pathway is that V undergoes similar reactions in the vinyl group and then the oxazolidine ring is opened by hydration and decarboxylation reactions to produce M5. This proposal is supported by the finding of Bursztyka et al. (2008), whom reported the intermediary metabolite M4 as a precursor of M5. Although cytochrome P450 (CYP) would be expected to catalyze V biotransformation, there is no direct information demonstrating this occurs. V does modify hepatic CYP expression in a complex pattern of induction, suppression and inhibition in mammals and birds (Ronis et al., 1994). Therefore, repeated doses of V may modify its own metabolism which could cause significant changes in its metabolism and pharmacokinetics. Further research is needed on the involvement of CYP in V metabolism and how repeated doses of V may modify its own metabolism.
In the only human study, carried out in adults occupationally exposed to V, there was no evidence of antiandrogenic or other reproductive effects (Zober et al., 1995). Urinary levels of M3 exceeded an equivalent of the V acceptable daily intake. Not only is M3 a metabolite of V, but also of other dicarboximides such as procymidone, iprodione, and chlozolinate (Wittke et al., 2001). Thus, this metabolite is not specific for V exposure studies. The results of the present study indicate that M3 was detected in very low levels in serum and tissues, which makes it difficult to associate its in vivo levels with exposure to V. Furthermore, its presence in biological samples could be due to nonenzymatic hydrolysis of V and metabolites from the acidic conditions used during analysis (Sierra-Santoyo et al., 2004). On the basis of the results of this study, M5 could be a successful biomarker of exposure to V in dosimetry studies to establish relationships between V residues and associated effects in tissues. M5 was detected in all tissues examined, with levels higher at 8 than at 2 h exposure to V. This suggests that V in liver is metabolized to M5, which is then released to the blood, distributed and taken up by the tissues. Another possibility is that V in each tissue is metabolized to M5 and then released to the blood for excretion. Glucuronide conjugates of M5 represent the main metabolites of V in urine and may be determined directly or after hydrolysis (Bursztyka et al., 2008). Recently, it has been proposed that M1 is dihydroxylated in its vinyl group (forming M6) and converted to M5 via the metabolite M4 (Bursztyka et al., 2008). However, this pathway may not be feasible, because of what is observed in the interconversion of V and M1 (Szeto et al., 1989). The reaction from V to M1 involves the hydrolysis of the oxazolidine ring, and is favorable at physiologic pH. The reaction from M1 to V, which would involve formation of the oxazolidine ring, is favorable at acidic pH. Thus, the reaction from M6 to M4, as proposed by Bursztyka et al. (2008), which would require formation of the oxazolidine ring, may not be favorable at physiologic pH.
V is well absorbed and widely distributed following oral administration in the rat. V is accumulated mainly in adipose tissue and its metabolism in vivo is nonenzymatic (M1 and M2) and enzymatic (M3, M5, M7, M8, and M9). M5 is the main metabolite present in all tissues. It is also a stable metabolite and may be used as a biomarker of exposure in pharmacokinetics studies of V. In contrast, M2 and M3 exhibited the lowest levels of V metabolites in serum and tissues. There is no toxicological information about M5, M7, M8, and M9. Therefore, toxicological characterizations of these metabolites are needed for a better understanding of the adverse effects associated with exposure to V.
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National Research Council (CR828790 [GenBank] ) and the U.S. Environmental Protection Agency supported A. Sierra-Santoyo.
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ACKNOWLEDGMENTS |
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We thank Brenda C. Edwards for her technical assistance. This article has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the view and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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