Pharmacokinetics of Dibutylphthalate in Pregnant Rats

Timothy R. Fennell1,3, Wojciech L. Krol2, Susan C. J. Sumner1 and Rodney W. Snyder1

CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709–2137

Received July 1, 2004; accepted September 16, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Dibutylphthalate (DBP) can cause adverse effects on the developing male reproductive tract when administered late in gestation to pregnant rats. The objectives of this study were to evaluate the metabolism of DBP in female rats, and the pharmacokinetics of DBP in pregnant rats on gestational day (g.d.) 20. The identities of DBP metabolites in urine and in maternal and fetal plasma were confirmed by LC-MS/MS, as monobutylphthalate (MBP) and its glucuronide, monohydroxybutylphthalate and its glucuronide, and butanoic acid phthalate and its glucuronide. An LC-MS/MS method was developed for the quantitation of MBP and its glucuronide. MBP and MBP glucuronide were quantitated in maternal and fetal plasma, and in amniotic fluid from pregnant rats administered a single dose of DBP (50, 100, or 250 mg/kg by gavage in corn oil) on g.d. 20. The pharmacokinetics of MBP and MBP glucuronide were determined. MBP was the major metabolite in maternal and fetal plasma. With increasing dose, there was a nonlinear increase in area under the curve (AUC) for MBP, with a ten-fold increase in maternal plasma, and an eight-fold increase in fetal plasma between 50 mg/kg and 250 mg/kg. In amniotic fluid, the major metabolite initially was MBP, but by 24 h after dosing, the major metabolite was MBP glucuronide. Isomers of the MBP glucuronide were detected in amniotic fluid, suggesting acyl group migration, known to occur with acyl glucuronides. This study indicated that MBP, thought to be the active metabolite of DBP, can cross the placenta in late gestation, and that the metabolism of MBP is saturable.

Key Words: dibutylphthalate; monobutyphthalate; pharmacokinetics; pregnant rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Concern over the health effects of phthalate esters has arisen as a result of their widespread use and the potential for reproductive effects. Dibutylphthalate (DBP) is used in the production of adhesives, as a plasticizer in cellulose plastics, and as a solvent for dyes (Kavlock et al., 2002Go). The largest source of exposure to the general population is thought to come from foods, and estimated exposures are less than 10 µg/kg/day (Kavlock et al., 2002Go).

A number of studies have indicated that DBP administered to rats late in pregnancy can have adverse effects on the developing reproductive system in male rats. Ema et al. (1998)Go found a reduced anogenital distance and increased incidence of undescended testes at the mid and high doses in male fetuses from dams administered 0, 0.5, 1.0, and 2.0% DBP in the diet on gestational days (g.d.) 11–21. These exposures were estimated to be 351, 555, and 661 mg DBP/kg/day at the low, mid, and high doses, respectively.

Administration of DBP by gavage to Sprague-Dawley rats during late pregnancy (g.d. 12–21) causes adverse effects on the developing male reproductive tract (Mylchreest et al., 1999Go). In the high-dose group administered 500 mg/kg/day, hypospadias, cryptorchidism, agenesis of the prostate, epididymis, and vas deferens, degeneration of the seminiferous epithelium, interstitial cell hyperplasia of the testis, thoracic nipples, and decreased anogenital distance were observed. In the intermediate-dose group administered 250 mg/kg/day, agenesis of the epididymis was observed. In the low-dose group administered 100 mg/kg/day, the only effect observed was delayed preputial separation.

In evaluating the dose response and the potential hazard associated with the low-level exposures of humans, it is important to know how DBP is metabolized and the distribution of active metabolites to the developing fetus during pregnancy. More than 90% of an oral or iv dose of DBP is excreted within 48 h in rats, and DBP is taken up and rapidly hydrolyzed to monobutylphthalate (MBP) (Tanaka et al., 1978Go). DBP-derived radioactivity is excreted primarily in the urine. A glucuronide conjugate of MBP is a major metabolite in rats (Foster et al., 1983Go). MBP and phthalic acid are also excreted in urine (Tanaka et al., 1978Go). Side chain oxidation products have been suggested to result from {omega} and {omega}-1 oxidation (Albro and Moore, 1974Go). LC-MS/MS has recently been used to analyze the metabolites of DBP in cattle with MBP, MBP glucuronide, phthalic acid, monoethylphthalate, and monohydroxybutylphthalate detected in urine (Coldham et al., 1998Go).

The kinetics of 14C-DBP in pregnant rats have been examined following administration of 0.5 and 1.5 g/kg by gavage on g.d. 14 (Saillenfait et al., 1998Go). MBP was the major metabolite detected in plasma, embryo, placenta, and amniotic fluid, with MBP glucuronide present as a minor metabolite.

MBP is thought to be the active metabolite of DBP. MBP caused testicular atrophy in rats (Foster et al., 1981Go; Oishi and Hiraga, 1980Go) and caused postimplantation loss and an increased number of malformations when administered to pregnant rats at doses of 0, 250, 500, and 625 mg/kg by gavage between g.d. 7 and 15 (Ema et al., 1995Go). Skeletal malformations were observed when MBP was administered between g.d. 7 and 9 and g.d. 13 and 15, but not between g.d. 10 and 12 (Ema et al., 1996Go). MBP was found to be embryotoxic and produced similar effects compared to DBP, and at a similar potency, when administered to pregnant rats on g.d. 10 (Saillenfait et al., 2001Go). MBP caused a decrease in anogenital distance in male rat fetuses when administered to pregnant rats between g.d. 15 and 17 (Ema and Miyawaki, 2001Go). Exposure of pregnant rats to MBP late in pregnancy (g.d. 15–18) caused a reduction in fetal testis testosterone and a decrease in the rate of fetal testis descent (Shono et al., 2000Go).

The objectives of this study were (1) to verify that the metabolism of DBP in female rats was similar to that reported previously in male rats (Albro and Moore, 1974Go; Foster et al., 1983Go; Tanaka et al., 1978Go) and (2) to conduct a pharmacokinetic study of the major metabolites of DBP in rats during late gestation at low doses that produced effects on the development of the male reproductive tract. To accomplish this objective, we investigated the metabolism of DBP in nonpregnant and pregnant rats using liquid chromatography–mass spectrometry (LC-MS). Quantitative analysis of the two major metabolites of DBP, MBP and its glucuronide conjugate, was conducted in maternal and fetal blood and amniotic fluid collected at various times following administration of DBP to pregnant rats on g.d. 20.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Chemicals. DBP was obtained from Aldrich Chemical Co. (Milwaukee, WI). [Ring-14C]-DBP (20.8 mCi/mmol) was obtained from Wizard Laboratories (West Sacramento, CA). 13C4-Monobutylphthalate was obtained from Cambridge Isotope Laboratories (Andover, MA). Unlabeled monobutylphthalate was obtained from Chem Service Inc. (West Chester, PA). Mono-n-butyl phthalate glucuronide (MBP Gluc) was isolated from urine of rats treated with 14C-labeled DBP. HPLC-grade water, acetonitrile, and methanol were obtained from Burdick and Jackson (Muskegon, MI). Acetic acid (HPLC grade) was purchased from EM Science (Gibbstown, NJ). Formic acid (96%) was obtained from Aldrich (Milwaukee, WI). Tetraethylammonium hydroxide was obtained from SACHEM (Austin, TX). Control plasma was obtained from female rats not treated with DBP.

4-Hydroxybutylphthalate was prepared by an adaptation of the procedure of Albro et al. (1973)Go for the synthesis of mono(2-ethylhexyl)phthalate, by refluxing 2.96 g (20 mmol) of phthalic anhydride with 9.0 g (100 mmol) of 1,4-dihydroxybutane in 100 ml toluene for 4 h. The reaction mixture was extracted with 0.4 M K2CO3, acidified with HCl, and extracted with ether. The ether layer was dried with MgSO4, and the ether was evaporated under vacuum. The hydroxybutylphthalate was characterized by 1H NMR and by LC-MS.

Phthalate-butanoic acid was prepared by dissolving 1.1 g 4-hydroxybutylphthalate (5 mmol) and 0.15 g sodium carbonate in 1.5 ml of water. Potassium permanganate (1.42 g) in 27 ml of water was gradually added over 3–4 h, keeping the reaction on ice. The reaction mixture was warmed to room temperature, and the precipitated manganese dioxide was removed by filtration. The mixture was acidified with dilute sulfuric acid and extracted with ether. The ether layer was removed, dried with anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The product was characterized by LC-MS.

Animals. Female Sprague Dawley Rats (Crl:CD(SD)Br) were obtained from Charles River Laboratories (Raleigh, NC). Nonpregnant rats were delivered at 8 weeks of age and were acclimated for 7 days before use. Pregnant rats (sperm positive on g.d. 0) were shipped on g.d. 12 and dosed on g.d. 20. During the acclimation period and conduct of the experiment, the temperature was maintained at approximately 65–75°F and relative humidity at approximately 30–70%, with a 12-h photo period (7 A.M–7 P.M. for light phase). Pelleted NIH-07 rodent chow (Zeigler Bros., Gardners, PA) and filtered tap water were supplied ad libitum. This study followed Federal guidelines for the care and use of laboratory animals and was approved by the Institutional Animal Care and Use Committee at CIIT.

Treatment
Dose vehicle. Dibutylphthalate was administered as a solution in corn oil (1.0 mg/kg). This was chosen to match the mode of administration used by Mylchreest et al. (1999)Go in investigating the effects of DBP administered in pregnancy.

Pilot studies. To confirm the disposition and metabolism of DBP in female rats, 14C-DBP (specific activity 1.19 mCi/mmol) was administered to female CD rats by gavage at a dose of 100 mg/kg in corn oil (1.0 ml/kg). The weight of dose administered was used to calculate the amount of radioactivity administered. Urine, feces, and carcasses were collected at 24 h after dosing and were analyzed for radioactivity. Metabolites in urine were analyzed by reverse-phase HPLC with radiochemical detection. The major radioactive metabolite peak was isolated by solid-phase extraction and HPLC. The major metabolites were separated by HPLC and characterized by NMR and LC-MS/MS.

14C-DBP (specific activity 0.92 mCi/mmol) was administered to four pregnant CD rats (g.d. 20) by gavage (100 mg/kg in corn oil, 1.0 ml dose/kg). Two h after dosing the pregnant rats were euthanized with CO2, and maternal blood was collected by cardiac puncture. Fetuses were removed, and fetal blood was collected from the jugular vein with a heparinized capillary. Blood was pooled from fetuses for each dam, and carcasses were collected for analysis of 14C total radioactivity. Maternal liver, fat, placenta, and carcass were collected for analysis of 14C by digestion of tissue with tetraethyl ammonium hydroxide and liquid scintillation counting. Radioactivity in plasma was analyzed by HPLC with flow-through radiodetector. Metabolites in maternal and fetal plasma were characterized by HPLC and LC-MS/MS.

Pharmacokinetic studies. Three groups of 30 pregnant CD rats each were administered a single dose of 50, 100, or 250 mg/kg unlabeled DBP by gavage in corn oil (1.0 ml/kg). For each dose group, the study was conducted over 3 days, with dosing conducted between 8 and 10 A.M. on days 1 and 2, with sample collection conducted on days 1–3, at 5, 15, and 30 min and 1, 2, 4, 8, and 24 h after dosing. Groups of 3 dams were euthanized with CO2, and blood was collected by cardiac puncture in a heparinized Glasspak syringe (Becton Dickinson, Rutherford NJ). Fetuses were removed, and fetal blood was collected from the jugular vein with a heparinized capillary. Fetal blood was pooled for each litter, giving a total of three fetal blood samples per dose and time point. Maternal blood was transferred to tubes containing 50 µl sodium fluoride (4.3 g/100 ml water), and fetal blood was collected in tubes containing 20 µl sodium fluoride used to inhibit plasma esterase activity. Maternal liver, fat, and placentae were collected. Amniotic fluid from four fetuses per dam was collected. The male reproductive tracts were dissected from the male fetuses and pooled for fetuses from each dam. All samples were stored in glass tubes or vials with Teflon inserts. Plasma was prepared from each blood sample by centrifugation. Plasma and tissue samples were frozen and stored at –20°C.

Amniotic fluid from four fetuses per dam was collected. The male reproductive tracts were dissected from the male fetuses and pooled for fetuses from each dam. Of the samples collected for pharmacokinetic analysis, only the results of the analysis of DBP metabolites in maternal and fetal plasma and in amniotic fluid are presented in this report.

For generation of standard curves and methods development, samples of maternal and fetal plasma and maternal liver, fat, and placentae were collected from six untreated pregnant CD rats.

Dose solution analysis. For administration of 14C-DBP, the concentration of radioactivity was verified by dilution of aliquots of dose solution and quantitation of radioactivity by scintillation counting. The concentration of DBP in each dose solution for pharmacokinetic studies was verified by GC analysis. Aliquots of dose solution were diluted in chloroform and analyzed on an HP5890 Series II gas chromatograph with an FID detector (Hewlett-Packard, Palo Alto, CA). The column used was a HP-1, 5 m x 0.53 mm I.D. with a 2.65 µm film thickness with a 1 m x 0.53 mm deactivated silica guard column. Split injection was used at an approximate split ratio of 1:12. Gas flow rates were measured at an oven temperature of 70°C and were typically 5.2 ml/min for the carrier gas (nitrogen), 31.1 ml/min for hydrogen, and 25.4 ml/min for the make-up gas. The initial oven temperature of 70°C was held for 1.0 min, then ramped at 50°C/min to a final temperature of 270°C and held for 5.0 min. Injector temperature was 270°C and detector temperature was 275°C. Samples (1 µl) were loaded onto the column using a Hewlett Packard model 7673 autosampler.

Detection and characterization of metabolites. Urine and plasma samples obtained from rats administered 14C-DBP were analyzed by HPLC on an HP1100 workstation equipped with a binary pump, diode array detector, and a Packard TR525 flow scintillation analyzer (Packard Bioscience, Meriden, CT). For urine samples, an Ultrasphere ODS (C18, 4.6 x 250 mm, 5 µm) column (Beckman, Fullerton, CA) was used with a gradient of 0.01 M ammonium acetate, pH 2.75 and acetonitrile at a flow rate of 0.8 ml/min. From 0 to 5 min, the column was eluted with 25% acetonitrile, and from 5 to 27.5 min, a linear gradient from 25 to 80% acetonitrile was used. From 27.5 to 40 min, the column was eluted with 80% acetonitrile and then recycled to 25% acetonitrile from 40 to 50 min. For analysis of plasma samples, metabolites were eluted from a LC-Hisep Hydrophobic Phase (4.6 x 250 mm, 5 µm) column (Supelco, Bellefonte, PA) with a gradient of 180 mM ammonium acetate in acetonitrile at a flow rate of 1.0 ml/min. After elution at 15% acetonitrile for 5 min, a 15-min linear gradient was run to 60% acetonitrile. HPLC column effluent was mixed with Packard FloScint II for detection of radioactivity in plasma and urine using the flow scintillation analyzer.

The metabolite of 14C-DBP tentatively identified as MBP glucuronide was isolated from urine for further characterization using solid phase extraction. Aliquots of urine were acidified with 0.1% formic acid and applied to a C18 Sep-Pak cartridge (Waters, Milford MA). The cartridge was eluted with increasing concentrations of methanol:water containing 0.1% formic acid. The fractions eluting with 45–55% methanol containing the MBP glucuronide peak were combined and repurified using an additional Sep-Pak, which was washed with water, 45% methanol, and eluted with 50% methanol.

NMR spectra of MBP glucuronide were acquired with a 5-mm dual proton-multinuclear probe on a Varian VXR-300 spectrometer (Palo Alto, CA). Spectra were acquired with D2O (Aldrich Chemical Co., St. Louis, MO) as solvent.

For LC-MS/MS analysis of metabolites, HPLC was conducted on a Beckman ODS Ultrasphere column (C18, 4.6 x 250 mm, 5 µm) at a flow rate of 1 ml/min, with a mobile phase of 0.1% formic acid in acetonitrile/water. A gradient from 15 to 80% acetonitrile in 15 min was used. MS analysis was conducted with a PE Sciex API 3000 in negative ion mode with a turbo ion spray interface (200 µl/min). Each plasma extract was analyzed first in full scan mode (Q1). The TIC chromatograms were compared with radioactivity data. Additional MS/MS scans were performed on any detected metabolites to verify presence and confirm identity.

Quantitative Analysis of MBP and MBP Glucuronide
Standard solutions. Stock solutions of MBP (separate weighing for calibration standards and quality control samples) and of internal standard, 13C4-MBP, were prepared by dissolving accurately weighed standard compounds. Primary stock solutions of MBP (approximately 2 mg/ml each) were prepared in methanol-water (50:50), and 13C4-MBP (approximately 1.5 mg/ml) in methanol. MBP glucuronide was isolated from rat urine, freeze dried, and dissolved in water, yielding a primary stock solution at approximately 3 mg/ml. Further dilution of the primary stock solutions with water yielded secondary stock solutions at approximately 1 mg/ml (MBP and MBP glucuronide), and 0.15 mg/ml (13C4-MBP). Both analytes were combined in standard working solutions. Calibration standard and quality control working solutions were prepared by serial dilutions in water. 13C4-MBP internal standard working solution was prepared in water at 5000 ng/ml. All solutions were stored in a freezer at approximately –20°C.

Sample preparation. Calibration standards and quality control samples were prepared by combining 25 µl of blank rat plasma with 25 µl of corresponding working solution in a 2-ml silanized autosampler vial. Biological samples were prepared by combining 25 µl of plasma with 25 µl of water in a similar container. A matrix blank (25 µl of plasma and 50 µl of water) and control blank (25 µl of plasma and 25 µl of water) were prepared with each calibration curve. Calibration standards were prepared daily. Quality control samples were prepared before analysis of biological samples had started, in sufficient quantities for the whole study, and stored at –20°C.

Extraction procedure. Samples, QC standards, and calibration standard working solutions were thawed at room temperature. Aliquots of 25 ml of internal standard solution were added to all samples except matrix blanks and briefly mixed. A 425-µl aliquot of 0.1% formic acid in acetonitrile was added to each vial. The samples were vortexed for 5 min and then centrifuged for 6 min at 3000 rpm and 4°C. The supernatant aliquots of 100 µl were transferred to fresh vials with silanized inserts and immediately analyzed. A typical daily sample batch included two calibration curves, two sets of quality control samples, 5–6 solvent blanks, and 65 unknown samples.

LC-MS/MS instrumentation and analytical conditions. The liquid chromatography system consisted of two Series 200 Micro Pumps, Series 200 autosampler, and on-line Series 200 Vacuum Degasser, all from Perkin-Elmer (Norwalk, CT). Liquid chromatography was performed on a Luna Phenyl-Hexyl column (50 x 2 mm), particle size 3 µm, protected by guard column Security Guard Phenyl (4 x 2 mm) (Phenomenex, Torrance, CA), with a flow rate of 350 µl/min under ambient conditions. The mobile phases consisted of 0.05% aqueous acetic acid as solvent A and acetonitrile as solvent B. The gradient started at 30% B and increased to 95% in 3 min, where it was held for 2 min. A 5-min post-run time was used to equilibrate the column. The sample injection volume was 1 µl. When the samples were analyzed, the column effluent was diverted to waste for the first 1 min and the last 7 min using a Model C2, 6-port Valco (Houston, TX) switching valve to minimize contamination of the mass spectrometer. A Spectroflow 400 (Kratos Analytical, Ramsey, NJ) LC pump was used to deliver make-up solvent, acetonitrile-water (50:50), to the mass spectrometer at 350 µl/min when column effluent was diverted to waste. The column effluent or make-up solvent was split 50:50 before entering the source.

A PE SCIEX API 3000 triple-quadrupole mass spectrometer (Perkin-Elmer SCIEX, Concord, ON, Canada) interfaced with the liquid chromatograph via a turbo ion spray source was used for mass analysis and detection. The flow rates of curtain, nebulizing, and collision gases were at settings of 11 (nitrogen), 7 (air), and 3 (nitrogen), respectively. The turbospray gas was nitrogen at 7 l/min at 375°C. The nebulizing potential, declustering potential, and focusing potential were set at –4000 V, –25 V and –90 V, respectively. All experiments were conducted in the negative ion mode. Quantitation was performed using selected reaction monitoring (SRM) of precursor–product ion transitions at m/z 221.1 -> 77.1 for MBP, m/z 225.1 -> 79.1 for 13C4-MBP, m/z 397.1 -> 221.1 for MBP glucuronide. The collision energy was optimized to –25 eV for the determination of both MBP and 13C4-MBP and to –22 eV for the determination of MBP glucuronide. Mass calibration was performed weekly by the infusion of a propylene glycols (PPG) solution into the source. The peak widths of precursor and product ions were maintained at ~0.7 u at half-height. Data acquisition and peak integration were performed using PE Sciex Analyst 1.1 software residing on a Dell Optiflex GX 300 computer.

Data processing. All data were evaluated for accuracy of integration and manually reintegrated if necessary and then transferred to an EXCEL (Microsoft, Redmond, WA) spreadsheet for further statistical calculations. Linearity of calibration was verified by average relative response factor (ARRF) method. The method assumes that, when the variation between individual relative response factors (RRF), measured as the relative standard deviation, is less than or equal to 20%, the use of the linear model is generally appropriate, and the calibration curve can be assumed to be linear and to pass through the origin. To evaluate the linearity of the calibration curve, RRFs were calculated for individual standards from both calibration sets according to the formula:

The ARRF and the relative standard deviation were calculated, and the ARRF was then used to calculate concentrations of calibration standards, quality control samples, and unknowns, according to the formula:

The response factors of both MBP and MBP glucuronide were calculated relative to the same internal standard, 13C4-MBP. Consistent background MBP contamination was subtracted from all data before calculating response factors or concentrations for this analyte. All samples with analyte concentrations exceeding the highest standard were diluted to fall within the range and reextracted.

Pharmacokinetic analysis. All pharmacokinetic analysis was conducted using WinNonlin version 1.5 (Pharsight Corporation, Mountain View, CA). Analysis was conducted using a noncompartmental model for extravascular administration.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Characterization of Metabolites
The majority of a dose of 100 mg/kg 14C-DBP administered to virgin female rats by gavage was excreted in urine at 24 h. As a percentage of the administered dose, urinary excretion of radioactivity was 77% at 24 h. Approximately 7% of the dose was excreted in feces, and 0.008% was recovered in carcass (Table 1).


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TABLE 1 Disposition of Radioactivity in Nonpregnant Female Rats Administered [U-ring-14C] DBP

 
Administration of 100 mg/kg 14C-DBP to pregnant female rats on g.d. 20 was conducted as a pilot study for the investigation of the pharmacokinetic behavior of DBP in pregnant rats (Table 2). The concentration of DBP-derived materials was calculated from the concentration of radioactivity in fat, carcass, and plasma from the dams, and in the fetal plasma and carcass. A single time point (2 h after dosing) that was expected to be the time of peak blood concentration was used (Saillenfait et al., 1998Go). In the dams, the concentrations of DBP-derived radioactivity were similar in the plasma (329 µM) and carcass (357 µM). The concentration in fat was approximately 10-fold lower. In fetal plasma, the concentration of radioactivity was approximately half that of maternal plasma (182 µM). These two preliminary studies conducted by administration of 14C-DBP to nonpregnant and pregnant rats provided an idea of the approximate range necessary for quantitation of metabolites. Samples of urine and plasma were also used to identify the metabolites that could be analyzed in further studies.


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TABLE 2 Radioactivity in Plasma and Carcass of Pregnant CD rats on Gestational Day 20

 
Using HPLC and radioactivity detection for analysis of urine from nonpregnant rats administered 14C-DBP, two major radioactive peaks were found in urine (Fig. 1). One was identified as MBP by coelution with a standard. The other major peak was isolated and characterized by 1H (Fig. 2) and 13C NMR (not shown) as MBP glucuronide. The MBP glucuronide peak accounted for 61 ± 8% of the 14C label in urine, while 19 ± 8% was associated with the MBP peak.



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FIG. 1. HPLC chromatogram of female rat urine 24 h after dosing with 100 mg/kg 14C-DBP. Two major metabolites assigned as MBP and MBP glucuronide were detected.

 


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FIG. 2. 1H NMR spectrum of MBP glucuronide in D2O. This metabolite was isolated from urine of female rats after dosing with 100 mg/kg 14C-DBP.

 
In the separation of plasma metabolites, using a shielded hydrophobic phase column for direct injection of plasma samples, two major radioactive peaks were found on HPLC of maternal and fetal plasma (Fig. 3). These were characterized as MBP and MBP glucuronide based on coelution with standards.



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FIG. 3. Metabolites in plasma from a pregnant rat (top) and in fetal plasma (bottom) at 2 h following administration of 14C-DBP (100 mg/kg) by gavage. Two major metabolites assigned as MBP and MBP glucuronide were detected.

 
Further characterization of urinary and plasma metabolites was conducted with LC-MS (see Supplementary Data for chromatogram of maternal plasma). Metabolites were detected as follows in urine and plasma:

Phthalic acid. The earliest eluting identified metabolite, detected in urine (1.6–2.6%, based on radioactivity) and plasma (0.7%). Identification was verified by matching retention time and fragmentation pattern with a standard. A molecular ion was identified at m/z 165 (M–H). Two major fragments detected as daughter ions were m/z 121 (benzoate) and m/z 77 ().

MBP. This was the major metabolite in plasma (77.1%) and second most abundant in urine (12–31%). Identification was verified by matching retention time and fragmentation pattern with a standard, and showed little fragmentation with m/z 221 (M–H) as the predominant ion in the spectrum. Other ions were present at m/z 177 (loss of free carboxy group), m/z 149 (loss of butoxy chain), m/z 71 (side butyl chain), and m/z 121 (benzoate).

MBP glucuronide. This was the major metabolite found in urine (53–69%) and second most abundant in plasma (19%). Identity was verified by matching retention time and fragmentation pattern with a reference standard. Two peaks were found in all chromatograms, including the standard, with slightly different fragmentation pattern. Major fragments are m/z 221 (aglycone ion), m/z 175 (glucuronide ion), and m/z 113 (fragment of m/z 175).

Mono-n-hydroxybutylphthalate. Three closely eluting peaks were present in chromatograms from urine (total 8–9%) and plasma (1.7%) samples, all with identical spectra, which suggested the same metabolite with different sites of hydroxylation. Identification of mono-4-hydroxybutylphthalate (the first eluting peak) was performed by analyzing urine samples spiked with the M-4-HBP reference solution. The parent ion for each peak was m/z 237 (M–H), with daughter ions in each spectrum at m/z 89 (n-hydroxybutyl chain) and m/z 121.

Monobutanoic phthalic acid. Found in urine (0.8–0.9%) and plasma (0.6%). The parent ion was at m/z 251, and the most intense daughter ion fragment was m/z 103 (hydroxybutanoic acid side chain). The other fragment in the spectra was the phthalate ion (m/z 165).

Mono-n-hydroxybutylphthalate glucuronide. Three distinct peaks were found in urine (total 2.5–2.9%) and plasma (0.2%) with identical spectra which contained parent ions at m/z 413 (M–H), and daughter ion fragments at m/z 237, m/z 175 (glucuronide ion), and m/z 113 (fragment of m/z 175).

Monobutanoic phthalic acid glucuronide or mono-1-hydroxybutan-2-one phthalic acid glucuronide. Traces were detected in urine samples (0.5–1.0%), but were not found in plasma. There were three peaks present in each chromatogram at m/z 427, one eluting at about 10 min and two at 17.1 and 17.3 min. A daughter ion (m/z 251) was present in the spectrum for each peak, along with a glucuronide daughter ion (m/z 175) and a fragment of m/z 175 (m/z 113). The spectrum of the early eluting peak contained also a fragment at m/z 103 (hydroxybutyrate) suggesting that this could be a conjugate of the monobutanoic phthalic acid.

Attempts to measure the parent DBP in plasma and urine were not successful, using either HPLC with detection of radioactivity, or LC-MS/MS. This is consistent with the rapid hydrolysis of DBP and has been previously observed with DBP administered to rats at doses up to 875 mg/kg (NIEHS, 1995Go).

MBP and MBP Glucuronide in Plasma
Quantitative analysis of MBP and MBP glucuronide was conducted by negative ion LC-MS/MS, with 13C4-MBP added as the internal standard. A typical chromatogram of MBP, 13C4-MBP, and MBP glucuronide conducted is shown in Figure 4. MBP was quantitated by selected reaction monitoring (SRM) of the parent ion at m/z 221, and a fragment at m/z 77 (benzene ring). The 13C4 internal standard, with the labels on the carboxyl groups and at the 1 and 2 carbons of the ring, produced the expected parent ion at m/z 225 and fragment ion at m/z 79. Calibration curves were constructed for MBP over the range of 100–5000 ng/ml. MBP glucuronide was quantitated by MRM of the parent ion at m/z 397 and fragment ion at m/z 221. For quantitation of MBP Gluc, an isotope-labeled internal standard was not available. MBP glucuronide was quantitated against the 13C4-MBP internal standard, using a standard curve prepared with MBP glucuronide and 13C4-MBP. Calibration of MBP glucuronide was conducted in the range from 50 to 5000 ng/ml. For both MBP and MBP Gluc, values below the concentrations of the lowest standards (100 ng/ml for MBP and 50 ng/ml for MBP glucuronide) are not reported.



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FIG. 4. LC-MS/MS of MBP (top), 13C4-MBP (middle), and MBP glucuronide (bottom) in maternal rat plasma at 2 h following administration of unlabeled DBP (250 mg/kg) by gavage on g.d. 20. Metabolites were detected by selected reaction monitoring (SRM) of precursor–product ion transitions at m/z 221.1 -> 77.1 for MBP, m/z 225.1 -> 79.1 for 13C4-MBP, m/z 397.1 -> 221.1 for MBP glucuronide.

 
Kinetics of MBP and MBP Glucuronide in Plasma
With administration of DBP, MBP rapidly appeared in both maternal plasma and fetal plasma at all three doses administered. This is illustrated with plots of the concentration measurements of MBP and MBP glucuronide in maternal and fetal plasma from rats administered 50, 100, and 250 mg/kg DBP on g.d. 20 (Fig. 5). The parameters calculated using a noncompartmental analysis of the pharmacokinetic data are presented in Table 3. In maternal plasma samples from all three dose groups, MBP was the major metabolite present. Cmax for MBP was approximately 3- to 4-fold higher than Cmax for MBP glucuronide in maternal plasma, and AUC for MBP was approximately 2- to 4-fold higher than AUC for MBP Gluc. The half-life for MBP in maternal plasma was similar across doses at 2.75–2.94 h. The half-life for MBP glucuronide was in a similar range (2.89–3.52 h). At the highest dose, Tmax was longer for both MBP (2 h vs. 0.5 h) and MBP glucuronide (2 h vs. 1 h) in maternal plasma. The AUC for MBP increased disproportionately at 250 mg/kg. Also at 250 mg/kg, the concentration of MBP and MBP glucuronide in maternal plasma, and MBP in fetal plasma showed a decrease at 1 h, which was followed at 2 h by substantial increases.



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FIG. 5. Kinetics of MBP and MBP glucuronide in plasma from pregnant rats administered 50 (top), 100 (middle), or 250 (bottom) mg unlabeled DBP/kg body weight on g.d. 20. Values represent mean ± SD of at least three animals.

 

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TABLE 3 Pharmacokinetic Parameters for MBP and MBP Glucuronide in Maternal and Fetal Rat Plasma and Amniotic Fluid

 
In general, Tmax for MBP and MBP glucuronide in fetal plasma was achieved later (0.5–3 h) than in maternal plasma. Cmax for MBP in fetal plasma ranged from 40% of the maternal Cmax at 50 mg/kg to 65% of Cmax at 250 mg/kg. The AUC for MBP in fetal plasma ranged from 59–71% of that observed in maternal plasma. Cmax for MBP glucuronide in fetal plasma ranged from 30% of maternal plasma Cmax at 50 mg/kg to 110% at 250 mg/kg.

MBP and MBP Glucuronide in Amniotic Fluid
An LC-MS/MS chromatogram of amniotic fluid is shown in Figure 6. Three peaks were detected that corresponded to MBP glucuronide. Each peak has similar fragmentation, with M–H at m/z 397, and a daughter ion at m/z 221, corresponding to MBP. Additional peaks were visible in the MBP chromatogram that corresponded to the retention times of these MBP glucuronide peaks. These suggest that all three of these peaks arise from MBP Gluc. For quantitation, the peaks areas of all of the MBP glucuronide peaks were combined, and a concentration of the sum of all three peaks were calculated. The additional peaks observed in the MBP chromatogram are considered to arise from MBP Gluc, which gives a fragment ion at m/z 221, similar to the parent ion for MBP.



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FIG. 6. LC-MS/MS of MBP (top), 13C4-MBP (middle), and MBP glucuronide (bottom) in amniotic fluid at 8 h following administration of DBP (250 mg/kg) by gavage. Metabolites were detected by selected reaction monitoring (SRM) of precursor–product ion transitions at m/z 221.1 -> 77.1 for MBP, m/z 225.1 -> 79.1 for 13C4-MBP, m/z 397.1 -> 221.1 for MBP glucuronide. The presence of several peaks for MBP glucuronide suggests that acyl group migration may have occurred.

 
The concentration of MBP in amniotic fluid reached a maximum at 4 h in each dose group, and fell gradually, with a half-life of approximately 6 h in the 100 and 250 mg/kg groups (Fig. 7). MBP glucuronide reached a peak at 8 h in each dose group, and decreased only slightly by 24 h.



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FIG. 7. Concentration of MBP (top) and MBP glucuronide (bottom) in amniotic fluid obtained from rats administered DBP on g.d. 20. Values represent mean ± SD of at least three animals.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
The characterization of metabolites of DBP in this study essentially agrees with the previous observations of others (Foster et al., 1983Go; Saillenfait et al., 1998Go; Tanaka et al., 1978Go; Williams and Blanchfield, 1975Go). MBP was the major metabolite detected in plasma. The major metabolite of MBP was its glucuronide, which was detected in plasma and was the major metabolite in urine. A number of other metabolites of MBP were detected in urine and plasma. These indicated further hydrolysis of MBP to phthalic acid and oxidation of the butyl side chain of MBP to give monohydroxybutyl phthalate (a number of isomers) and phthalate butanoic acid. Monohydroxybutyl phthalate was detected as a glucuronide, as was monobutanoic phthalic acid. MBP has been implicated as the most likely metabolite of DBP involved in the generation of adverse effects. Since the major metabolites detected in plasma were MBP and MBP glucuronide, these were the focus for quantitative analysis.

The metabolites of DBP in cattle have been extensively characterized by ion trap LC-MS/MS by Coldham et al. (1998)Go. The fragmentation of metabolites reported previously is in general agreement with our findings. While, in cattle, the only metabolite derived from {omega} and {omega}-1 oxidation was monohydroxybutylphthalate, we observed a number of monohydroxybutylphthalate isomers, monobutanoic acid phthalate, and glucuronide conjugates.

Recently, LC-MS/MS has been used for detection and quantitation of various phthalate monoester metabolites in human urine and plasma (Blount et al., 2000aGo,bGo; Silva et al., 2003Go). The method of analysis consisted of incubation of urine with ß-glucuronidase, concentration of purification metabolites by solid phase extraction, and analysis by LC-MS/MS. Standardization was conducted with 13C4-monoalkyl phthalate standards. MBP was one of the phthalate metabolites analyzed, using the same fragment ions as reported here. The geometric mean percentage of free MBP in human urine was 5.6% (Silva et al., 2003Go), with a large interindividual variability. The major metabolite is inferred to be MBP glucuronide, based on its release by incubation with ß-glucuronidase. The fraction of free MBP in the urine was lower in humans than in the rat urine measured in this study (approximately one fourth of the total MBP in urine was free). Free MBP was also detected in human plasma (geometric mean 13.5 ng/ml). A large fraction of the measurements of free and total serum monoester phthalates indicated that free MBP was less than 10% of the total MPB (in greater than 75% of the measurements), indicating that, in most people, MBP was present in serum as its glucuronide conjugate (Silva et al., 2003Go).

In this study, we have found both MBP and MBP glucuronide in plasma from pregnant and nonpregnant rats, and in fetal rats. In contrast to the observations above in humans, where the free fraction of MBP is low, the measurements in rats indicated a mean fraction of MBP glucuronide of 29, 23, and 19% in the maternal blood, at 50, 100, and 250 mg/kg, respectively. As would be expected, the fraction of MBP glucuronide was low immediately following administration and higher at the later time points.

The amount of MBP reaching the developing reproductive system is one of the parameters of interest in conducting a risk assessment of DBP. The capability of the fetus to metabolize MBP is also of interest. MBP concentrations were generally higher in maternal plasma than fetal plasma, with Cmax ranging from 2.4- to 4-fold higher in maternal plasma. However, the AUC 0–{infty} values for maternal plasma MBP were 1.4–1.7 fold higher than fetal plasma MBP. Cmax for MBP in maternal plasma was approximately 3–4 fold higher than MBP Gluc, which is similar to the MBP:MBP glucuronide ratio found in fetal plasma of 3–4 fold. However, AUC 0–{infty} for MBP were 2.3- to 4-fold higher than MBP glucuronide in maternal plasma but 1.7- to 2.5-fold higher in fetal plasma.

With increasing dose in this study, Tmax for MBP and MBP glucuronide in maternal plasma increased, suggesting that absorption becomes rate limiting at the highest doses. There was also a nonlinear increase in AUC for MBP in maternal plasma, with a 10-fold increase between 50 mg/kg and 250 mg/kg. Similarly, there was an 8-fold increase in AUC for MBP in fetal plasma with a 5-fold increase in administered dose between 50 mg/kg and 250 mg/kg. These results suggest a saturation of the metabolism and elimination of MBP. The prolongation of Tmax at the highest dose administered and the appearance of a transient decrease in MBP concentration at 1 h are indicative of enterohepatic circulation. A change in the extent of the recirculation could contribute to the nonlinear relationship between dose administered and AUC for MBP. A physiologically based pharmacokinetic model for DBP (Keys et al., 2000Go) has incorporated enterohepatic circulation to describe pharmacokinetics of MBP following oral administration of DBP to rats (NIEHS 1994Go, 1995Go). While Keys et al. (2000)Go concluded that their enterohepatic circulation model did not describe the pharmacokinetic data as well as a combined diffusion limited–pH trapping model, this did not mean that enterohepatic circulation did not occur. The presence of secondary peaks in both iv and oral concentration time course data and the measurement of MBP in bile indicated that enterohepatic circulation was occurring.

The pharmacokinetics of 14C-DBP were investigated in pregnant rats on g.d. 14 (Saillenfait et al., 1998Go). Total radioactivity and the radioactivity associated with MBP and MBP glucuronide were quantitated in maternal plasma, whole embryo, placenta, and amniotic fluid. At a dose of 0.5 g/kg, Cmax for MBP was approximately 7-fold higher than MBP glucuronide in maternal plasma. At the higher dose of 1.5 g/kg, Cmax for MBP was approximately 10-fold higher than MBP glucuronide in maternal plasma. This contrasts with the 3- to 4-fold difference between Cmax for MBP and MBP glucuronide in maternal plasma found in this study (Table 3). Similarly, the AUC 0–{infty} for MBP in this study ranged from 2.3 times higher than for MBP glucuronide at the lowest dose, to 4 fold higher at the highest dose. At the higher doses used by Saillenfait et al. (1998)Go, AUC 0–{infty} for MBP was approximately 5-fold higher than for MBP Gluc, and Cmax for MBP was 4-fold higher in maternal plasma compared with the whole embryo. These studies together suggest that there is a saturation of the glucuronidation of MBP at increasing doses in pregnant rats.

The detection of MBP glucuronide in fetal plasma in this study has not resolved whether MBP is metabolized in the fetus, or whether MBP glucuronide crosses the placenta. The development of UDP glucuronyl transferase activity for different substrates in the developing rat liver is known to occur during late gestation (late fetal group, g.d. 16–20) or around birth (neonatal group, day 2 following birth) (Wishart, 1978Go). The category that would contain the isoform of UDP glucuronyl transferase that metabolizes MBP is not known. The rapid appearance of MBP and the delay in appearance of MBP glucuronide could indicate fetal metabolism of MBP at a much slower rate than maternal glucuronidation, if MBP glucuronide does not cross the placenta. Alternatively it could indicate that MBP glucuronide crosses the placenta at a much slower rate than MBP. The detection of metabolites of drugs in plasma, urine and amniotic fluid of the fetus does not provide definitive evidence of hepatic metabolism in the fetus (Ring et al., 1999Go).

MBP glucuronide is an acyl glucuronide. In plasma, it was identified as a single radioactive peak on HPLC, or as a single peak on LC-MS/MS. However, in amniotic fluid, it was observed as several peaks, each with the same mass spectrum, suggesting the formation of isomers. Acyl glucuronides are known to rearrange, in which the 1-O-ß-glucuronide can undergo a pH-dependent migration catalyzed by OH to the 2-O, 3-O, and 4-O positions (Bailey and Dickinson, 2003Go; Blanckaert et al., 1978Go). The potential consequences of this rearrangement are several. The rearranged forms of acyl glucuronides are resistant to ß-glucuronidase and, thus, would not be measured in an assay which required analysis of the aglycone form (Dickinson et al., 1984Go). Acyl glucuronides can form protein adducts by one of several mechanisms. A transacylation reaction can result in displacement of the glucuronide (Ruelius et al., 1986Go; van Breemen and Fenselau, 1985Go). A Schiff's base mechanism can result from ring opening of the 2-O, 3-O, and 4-O forms, yielding a transient aldehyde that can react with protein amine groups (Smith et al., 1986Go, 1990Go). Whether MBP glucuronide can react with proteins in vitro or in vivo has not been determined.

The data obtained in this study provide an improved description of DBP pharmacokinetics in pregnancy, provide a data set with measurements of MBP and MBP glucuronide in both maternal and fetal plasma, and demonstrate a nonlinear increase in AUC with increasing administered dose. The data obtained will enable refinement of the PB-PK model previously described by Keys et al. (2000)Go.


    SUPPLEMENTARY DATA
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Supplementary Data is available online.


    ACKNOWLEDGMENTS
 
The authors would like to thank the CIIT Animal Care Staff for their assistance with the study. We would like to thank Dr. Paul Foster for his guidance, and the many staff members who contributed to the in-life portion of the study: Carla Williams, Barbara Elswick, Duncan Wallace, Horace Parkinson, Dr. Katie Turner, Dr. Norman Barlow, Dr. Chris Bowman, Delorise Williams, Kim Lehmann, David Weil, Renee Hoyle-Thacker, and Susan Ross.


    NOTES
 
1 Current address: RTI International, P.O. Box 12194, Research Triangle Park, NC 27709–2194. Back

2 Current address: GlaxoSmithKline, Research Triangle Park, NC 27709. Back

3 To whom correspondence should be addressed at RTI International, P.O. Box 12194, 3040 Cornwallis Road, Research Triangle Park, NC 27709–2194. Fax: (910) 541-6499. E-mail: Fennell{at}rti.org.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
 SUPPLEMENTARY DATA
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