ToxSci Advance Access originally published online on August 18, 2008
Toxicological Sciences 2008 106(2):376-383; doi:10.1093/toxsci/kfn171
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Higher Levels of Ethyl Paraben and Butyl Paraben in Rat Amniotic Fluid than in Maternal Plasma after Subcutaneous Administration
* Department of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
Department of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, DK-2860 Søborg, Denmark
1 To whom correspondence should be addressed at Department of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Fax: +45-3545-6054. E-mail: hanne.01.frederiksen{at}rh.regionh.dk.
Received May 30, 2008; accepted July 17, 2008
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ABSTRACT |
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Parabens are a group of antimicrobial preservatives widely used in cosmetics, pharmaceuticals, and in foods. Previous in vitro and in vivo studies have shown weak estrogenic effects of some parabens. Thus, especially, exposure of fetus and infants via the mother is a matter of concern. In order to obtain more knowledge about the distribution of ethyl paraben and butyl paraben in pregnant rats and pups after perinatal exposure, the presented study was designed. The data show response and distribution of ethyl paraben and butyl paraben in maternal rat plasma, pools of amniotic fluids, placenta, whole-body fetuses, and in fetal liver after dosing of dams with 100, 200, and 400 mg/kg body weight (bw)/day from gestational day 7 to 21. After cesarean section of dams, the fluids and tissues were collected, deconjugated, and purified by solid-phase extraction, and ethyl paraben and butyl paraben were analyzed by liquid chromatography-tandem mass spectrometry. Markedly higher levels of ethyl paraben compared to butyl paraben were found in all fluids and tissues. Both ethyl paraben and butyl paraben in maternal plasma, livers, and whole-body tissues from fetus seemed to be saturated after dosing with
100 mg/kg bw/day, while both compounds were excreted into amniotic fluid in a dose-dependent manner. Significant difference was found between the level of ethyl paraben in maternal plasma and amniotic fluid after dosing with 200 mg/kg bw/day as well as between the levels of butyl paraben in maternal plasma and amniotic fluid after dosing with 100, 200, and 400 mg/kg bw/day. Key Words: ethyl paraben; butyl paraben; distribution; pregnant rat; endocrine disruptor.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Parabens, the alkyl esters of p-hydroxybenzoic acid, are a group of antimicrobial preservatives widely used in cosmetics such as skincare products, soaps and deodorants, pharmaceuticals, and in foods such as snacks, candy, and dried meat products (Elder, 1984
; Golden et al., 2005; Rastogi et al., 1995; Soni et al., 2005). Previous in vitro and in vivo studies have shown weak estrogenic effects of some parabens (Byford et al., 2002; Darbre et al., 2002, 2003, 2004; Lemini et al., 2003; Okubo et al., 2001; Pugazhendhi et al., 2005). Furthermore studies have shown effects on the male reproductive system in rats and mice (Golden et al., 2005; Lemini et al., 2004; Oishi, 2001, 2002, 2004), whereas a recent study could not confirm these effects (Hoberman et al., 2008). In rats, the most potent parabens were propyl-, butyl-, and benzyl paraben, while methyl- and ethyl paraben did not seem to have any effect on the male reproductive system (Oishi, 2001, 2002, 2004). However, it has been shown that exposure to environmental chemicals during the early development of the male reproductive system may be of crucial importance for the male reproductive health later in life (Sharpe and Skakkebaek, 1993, 2008). For that reason, especially, fetal exposure via the mother to parabens with longer alkyl chains, such as propyl- and butyl paraben, is a matter of concern. Due to the common use of parabens as preservatives in skin products, most human studies during recent years have focused on allergic symptoms and dermal uptake of parabens (Akomeah et al., 2007; El Hussein et al., 2007; Ishiwatari et al., 2007; Janjua et al., 2007, 2008). However, data on human exposure, excretion, and effects on reproductive health of parabens are limited.
In rats, rabbits, cats, and dogs, the parabens are rapidly excreted into urine, and the major metabolite for all parabens is the nonspecific p-hydroxybenzoic acid, followed by glycin, glucuronic, and sulfuric acid conjugates of p-hydroxybenzoic acid. However, the pure ester parent compound was recovered in tissues such as brain, pancreas, and spleen in dogs. In rabbits, 0.2–0.9% of the ester parent compounds were excreted unchanged in urine, but the unchanged parabens were excreted in various forms such as glucuronides and sulfate conjugates. The metabolic profile of parabens has been found to depend upon the exposure route (Golden et al., 2005; Soni et al., 2005; Ye et al., 2006a). Recently, a method for analyzing parent parabens in its pure form after deconjugation was developed, and human exposure to parabens was determined using levels of the parent parabens as the biomarker. Methyl- and propyl paraben were measured in almost all samples, but also ethyl- and butyl paraben were commonly excreted compounds into human urine (Ye et al. 2006a, 2006b). Therefore, we need to know more about the parabens, their distribution, metabolism, and possible health risk after early exposure.
To study the distribution of ethyl paraben and butyl paraben in rats after in utero exposure, we have developed a method for extraction of ethyl paraben and butyl paraben in plasma, amniotic fluid, placenta, and tissues from fetuses followed by a short and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. After exposure of pregnant rats to ethyl paraben and butyl paraben, the compounds were measured in maternal plasma, amniotic fluid, placenta, whole fetuses, and fetal livers.
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MATERIALS AND METHODS |
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Chemicals
Ethyl paraben (ethyl 4-hydroxybenzoate, CAS no. 120-47-8) and butyl paraben (p-hydroxybenzoic acid n-butyl ester, CAS no. 94-26-8) were obtained from Fluka (distributed Sigma-Aldrich, Brøndby, Denmark) and d4-methyl paraben was obtained from C/D/N Isotopes Inc. (Quebec, Canada). Phosphoric acid, glacial acetic acid, formic acid, and sodium dihydrogen phosphate dihydrate were obtained from Merck (Darmstadt, Germany). Acetonitrile, cyclohexane, methanol, ammonium acetate, and sodium sulfate were all obtained from J.T. Baker (Deventer, the Netherlands). Both Merck and J.T. Baker chemicals were distributed by Bie & Berntsen A/S (Rødovre, Denmark). Ethyl acetate was obtained from BDH Laboratories Supplies (Poole, England, and distributed by VWR international, Rødover, Denmark). 4-Methylumbelliferone, 4-methylumbelliferyl β-D-glucuronide, and 4-methylumbelliferyl sulfate were obtained from Sigma-Aldrich. β-Glucuronidase (Escherichia coli K12) and arylsulfatase were obtained from Roche Diagnostics (Mannheim, Germany). Milli-Q water was cleaned in a Millipore system (Synthesis A10). Solid-phase extraction (SPE) cartridges (Strata XL, 200 mg, 3 ml) were obtained from Phenomenex (Allerød, Denmark). All chemicals were of analytical or high-performance liquid chromatography (HPLC) grade, and all chemicals, solutions, and laboratory wares were checked for contamination with parabens before use.
Animal study
Two studies using time-mated, young adult Wistar rats (HanTac: WH; Taconic M&B, Ejby, Denmark), supplied day 3 after mating, and subcutaneous (sc) dosed from gestational day (GD) 7 to 21 with 100, 200, and 400 mg/kg body weight (bw)/day of ethyl paraben or butyl paraben, respectively, were conducted. The animals were dosed the last time on GD 21, about 90 min before they were sacrificed, and tissues were collected. The first experiment was conducted with doses of 100 and 200 mg/kg bw/day of ethyl paraben and butyl paraben dissolved in dimethyl sulfoxide/groundnut oil 1:1. Because of local toxicity at the injection sites observed in some of the animals at GD 21, the experiment was stopped. Only organs and blood from animals sacrificed at GD 21 were collected and stored for later analyses of paraben distribution. A new study was initiated using 200 and 400 mg/kg bw/day of butyl paraben and 400 mg/kg bw/day of ethyl paraben dissolved in pure groundnut oil. The animal study was mainly conducted to study end-point effects of ethyl paraben and butyl paraben on rats after in utero exposure, and a more detailed description of the animal study and effects is found in Taxvig et al. (2008). However, in the present study, four to six rats from each dose group from both studies were chosen randomly for determination of the distribution of ethyl paraben and butyl paraben in pregnant rats. During the cesarean section of dams at GD 21, the amniotic fluids from all fetuses in each dam were pooled. The fetuses were decapicitated, and blood samples were collected and pooled within litter. Maternal plasma and pools of amniotic fluid, a placenta, a decapitated fetus, and a male and a female fetal liver from each dam were stored at – 70°C until analysis for levels of ethyl paraben and butyl paraben.
Preparation of Samples, Standards, and Control Materials
Extraction of rat tissue.
The tissues of male and female liver, fetuses, and placenta were homogenized by fine cutting and mixing. Homogenates were prepared by sonication with an Ultra Turrax of 0.10 g of each tissue added 10 ml of phosphate buffer (2.2 g sodium dihydrogen phosphate and 1 ml phosphoric acid dissolved in water ad 100 ml) for control rat tissue, 50 and 100 ml phosphate buffer for tissues from rats treated with 200 and 400 mg butyl paraben/kg bw/day, respectively, and 200 ml phosphate buffer for tissues from rats treated with 400 mg ethyl paraben/kg bw/day. Homogenates were stored at – 20°C until use. After thawing, 1 ml was added 1 ml phosphate buffer, 5 ml extraction solution (5% cyclohexane in ethyl acetate), and 2 g of sodium sulfate and mixed by vortex. After 10 min, the suspensions were centrifuged at 4000 rpm for 5 min. Supernatants were removed, and extraction of pellets were repeated twice by adding 2 ml of extraction solution followed by vortex mixing and centrifugation. The supernatants from the three extraction steps were pooled, and the solvent was evaporated at 45°C under a stream of N2. The residues were resuspended in 2 ml 2M ammonium acetate buffer (pH 6.5).
Enzymatic hydrolyses.
All samples, standards, and quality controls were enzymatically hydrolyzed by deconjugation of glucuronidated and sulfatated metabolites, and the total amount of ethyl paraben and butyl paraben were determined. Maternal plasma and amniotic fluid samples were thawed and mixed, and aliquots of 10 µl each were added 1990 µl of 2M ammonium acetate buffer. Both fluid and tissue samples were added 10 µl of 0.12M phosphoric acid and 20 µl 100 ng/ml of the internal standard solution, d4-methyl paraben. As a control for the enzymatic reaction, all samples were added 20 µl of a mixture of 100 ng/ml 4-methylumbelliferyl β-D-glucuronide and 100 ng/ml 4-methylumbelliferyl sulfate, and the extent of the deconjugation product, 4-methylumbelliferone was cheeked by LC-MS/MS. Immediately prior to the incubation, all samples were added 15 µl of the enzyme mixture containing β-glucuronidase/arylsulfatase 1:2 (fresh prepared), mixed, and incubated for 90 min at 37°C in a shaking water bath. The reaction was terminated by addition of 1000 µl 3.6M phosphoric acid.
The SPE.
All samples were purified using automated SPE (Aspec XL; Gilson Inc., Middleton, WI). The SPE cartridges were preconditioned by the use of 1.0 ml acetonitrile, 1 ml Milli-Q water, and 1 ml 0.15M ammonium acetate buffer, pH 2–3. Subsequently, samples were applied to the columns, and the tubes were rinsed with 3 ml 0.15M ammonium acetate. Prior to elution, the cartridges were washed with 2 ml of 1% phosphoric acid and 2 ml of 5% methanol. The compounds were eluted with 1.5 ml acetonitrile followed by 1.5 ml ethyl acetate, and the eluates were evaporated to dryness (45°C, 5–15 psi N2). The residues of total metabolites were resuspended in 200 µl of resuspension solution (20% acetic acid and 60% acetonitrile in water).
Standard and quality controls.
For all tissues and fluids, calibration standards were prepared by spiking pools of tissue and fluid from control rats (n = 4) treated similarly to the samples with ethyl paraben, butyl paraben, and d4-methyl paraben. Stock solutions of ethyl paraben (104 µg/ml), butyl paraben (104 µg/ml), and d4-methyl paraben (100 ng/ml) were diluted by 10% of acetonitrile and added the pools just before the evaporation to a final concentration after resuspension of 0.52, 1.04, 5.2, 26, and 104 ng/ml for ethyl paraben and butyl paraben and 10 ng/ml for d4-methyl paraben. For quality control, pools of tissue and fluid from control rats (n = 4) were treated similarly to the calibration standards. Aliquots of maternal plasma and amniotic fluid were spiked before and after enzymatic hydrolyses and SPE (pre-preparation) with two different levels of ethyl paraben and butyl paraben to a final concentration of 5.2 and 52 ng/ml, respectively. Pools of the tissues were spiked before and after pre-preparation with ethyl paraben and butyl paraben to a final concentration of 52 ng/ml. Stock solutions, standards, and quality controls were stored at – 20°C until use.
Analytical Method
LC separation.
Analysis of ethyl paraben and butyl paraben were accomplished using high-performance liquid chromatography (Surveyor; ThermoFinnigan, San Jose, CA). The injection volume was 20 µl on a Synergi 4U fusion-RP 80A column (75 mm x 2.0 mm x 4 µm) (Phenomenex, Anschaffenburg, Germany). The flow rate was 350 µl/min, and the column temperature was 25°C. Solvent were A: 0.1% acetic acid in water and B: 0.1% acetic acid in acetonitrile. Solvent programming was 0.0–1.0 min, 10% B; 1.1 min, 60% B; 2.5 min, 65% B; 3.0 min, 70% B, 3.2 min, 75% B; 3.3 min, 80% B; 3.6–4.5 min, 90%; and 4.6–5.5 min, 10% B.
MS detection.
A Finnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer in combination with the X-calibur software program was used for detection and quantitation (Thermo Electron Corporation, San Jose, CA). The instrument was run in negative mode using an electrospray source (electrospray ionization source). Following interphase settings were used: spray voltage, 3000 V; sheath gas (N2) pressure, 55 psi; auxiliary gas (N2) pressure, 10 psi; capillary temperature, 350°C; collision gas (Ar) pressure, 1.0 mTorr. Ion individual settings: The tube lens offsets were 100 V for all masses, and the collision energies were 15, 15 and 16 V for ethyl paraben, butyl paraben, and d4-butyl paraben, respectively.
Statistical analyses.
All data are expressed as means ± SD. Data were tested for normal distribution and for homogeneity of variance by standardized residuals plot. When necessary, logarithmic transformations were performed. Normally distributed data were analyzed by t-test, and data not normally distributed were analyzed by nonparametric Mann-Whitney U-test. The litter was the statistical unit, and p values < 0.05 were considered statistically significant. All statistical analyses were preformed using SPSS (SPSS for Windows, version 16.0; SPSS Inc., Chicago, IL).
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RESULTS |
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Method Characteristics and Validation
The retention time of the compounds separated on the LC column were 3.30 min (d4-methyl paraben), 3.52 min (ethyl paraben), and 4.09 min (butyl paraben). In negative ion mode [M-H]–, the parent masses of ethyl paraben and butyl paraben were m/z 165.05 and 193.09 with product masses of m/z 92.00 and 92.02, respectively, and the parent mass of the internal standard, d4-methyl paraben, was m/z 155.06 with product mass ion of m/z 96.05. All the MS/MS results were calculated from peak area ratios of the internal standard d4-methyl paraben versus standard or sample concentrations. Calibration standards were measured before and after samples in every sequence. The calibration plots all had correlation coefficients (r2) > 0.99. Figure 1 shows LC-MS/MS chromatograms of a control pool of amniotic fluid spiked with 52 ng/ml of ethyl paraben and butyl paraben and other characteristic chromatograms of amniotic fluid, maternal plasma, placenta, fetus, and fetal liver all from dams dosed with ethyl paraben or butyl paraben.
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The limit of detection (LOD) with a signal/noise ratio = 3 was calculated as 3S0, where S0 is SD of the lowest standard in the calibration curve. LOD was determined in all fluids and tissues and ranged up to 0.31 and 0.25 ng/ml for ethyl paraben and butyl paraben, respectively, reflecting good method sensitivity for both compounds in all matrices.
For quality control, pools of fluids or tissue from control rats were spiked with different levels (5.2 and 52 ng/ml) of ethyl paraben and butyl paraben. Sets of quality control samples were prepared for every sequence in which each of the matrices was measured. Five replicate analyses of maternal plasma and amniotic fluid spiked with two different concentrations were used for determination of SPE recoveries and method accuracy, while each of the tissue pools were spiked once with the high level of ethyl paraben and butyl paraben. SPE recovery was calculated on the basis of measured compound when spiked before and after pre-preparation. Good SPE recoveries were obtained for ethyl paraben and butyl paraben in both maternal plasma and amniotic fluid at low and high spike levels (87–98%) and for the tissues at high spike level (74–102%) (Table 1). The method accuracy was expressed as the percentage of expected levels when spiked before pre-preparation. For ethyl paraben and butyl paraben in maternal plasma and amniotic fluid at low and high spike levels, the method accuracy ranged from 74 to 108% and for the tissues at high spike level the method accuracy ranged from 70 to 101% (Table 1). Both the SPE and spiked recoveries for butyl paraben for most of the tissues were lower than the recoveries found for ethyl paraben, while no specific differences were found for the recoveries of the compounds in maternal plasma or in amniotic fluids.
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Parabens Distributed in Fluid and Tissue
Maternal plasma and amniotic fluid were obtained from two different animal studies. No significant differences were found in the content of ethyl paraben and butyl paraben in the control groups or in the group dosed with 200 mg/kg bw/day; therefore, we decided to use the materials from both studies. Dosing of ethyl paraben and butyl paraben did not affect the rats or their offspring regarding number of fetuses, weights of dams, fetuses, or organs according to data reported by Taxvig et al. (2008).
Only very low levels of ethyl paraben and butyl paraben were measured in all matrices in controls. The highest amounts were found in the pools of amniotic fluids ranging from < LOD to 16 ng/ml for ethyl paraben and from < LOD to 36 ng/ml for butyl paraben and with mean levels of 5.8 and 9.0 ng/ml, respectively. Since none of the blank controls used to test background residues and other contamination in laboratory wares or in our LC-MS/MS instrument prior analytical runs showed any problems with paraben contamination, the traces of parabens in control rat tissues might come from the animal study itself, such as food or drinking water. However, the levels of both ethyl paraben and butyl paraben in all test groups and all matrices were significantly different from the levels in the control groups.
The mean concentrations of ethyl paraben measured in maternal plasma were 2023 ± 787 and 4110 ± 1215 ng/ml in rats dosed with 100 and 400 mg/kg bw/day, respectively, whereas the concentration of ethyl paraben in rats dosed with 200 mg/kg bw/day was lower than the two other groups (mean ± SD = 1121 ± 318 ng/ml). A significant difference between ethyl paraben in the rats dosed with 200 and 400 mg/kg bw/day was found, but no coherence between increasing dose and ethyl paraben concentration in maternal plasma was found (Fig. 2A). The concentration of ethyl paraben measured in amniotic fluid from rats dosed with 100 mg/kg bw/day was similar to the concentration in maternal plasma, but in contrast to ethyl paraben in maternal plasma, the concentration of ethyl paraben in amniotic fluids were increasing in a dose-dependent manner with mean values of 2470 ± 1189, 6111 ± 4244, and 14,118 ± 13,874 g/ml for rats dosed with 100, 200, and 400 mg ethyl paraben/kg bw/day, respectively. In dams dosed with 200 mg ethyl paraben/kg bw/day, the differences between the content in maternal plasma and amniotic fluid were significant (Fig. 2A).
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For the dams dosed with 400 mg ethyl paraben/kg bw/day, comparable levels of ethyl paraben were found in maternal plasma and in male and female fetal livers. The mean ethyl paraben concentration in these samples was around 4000 ng/ml or ng/g (Table 2), whereas the mean concentrations in placenta and amniotic fluid were approximately threefold higher, 15,315 ng/g and 14,118 ng/ml, respectively (Table 2 and Fig. 2A). However, no statistically significant difference was found due to large variations in the content of ethyl paraben in these matrices. The levels of ethyl paraben in placenta ranging from 3368 to 53,515 ng/g and in amniotic fluids from 3631 to 38,380 ng/ml, which showed that some of the rats had similar ethyl paraben levels as found in maternal plasma and the other tissues, while other had a more than 10-fold higher level of ethyl paraben in placenta and amniotic fluid than in maternal plasma and tissues (Table 2 and Fig. 2A).
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For rats dosed with butyl paraben, identical concentrations of butyl paraben were measured in maternal plasma in all three test groups, ranging from 89 to 511 ng/ml and with a mean level of about 220 ng/ml (Fig. 2B). Butyl paraben in amniotic fluid in all three test groups was significantly higher (p < 0.05 and p < 0.005) than in maternal plasma. About 4-, 10-, and 5-fold higher concentrations of butyl paraben were measured in amniotic fluids than in maternal plasma from the groups dosed with 100, 200, and 400 mg/kg bw/day, respectively. Thus, the butyl paraben concentration in amniotic fluid did not increase in a dose-dependent manner as for ethyl paraben. Furthermore, a very large variation in the content of butyl paraben in amniotic fluids was found as for ethyl paraben in amniotic fluid, ranging from the low level, also measured in maternal plasma, to level of around 40-fold higher (Fig. 2B).
Nearly similar mean levels of butyl paraben (
260–480 ng/ml) were measured in fetuses and in male and female livers from fetuses in rats dosed with 200 and 400 mg/kg bw/day (Table 2). The levels of butyl paraben in all tissues in the two dose groups (Table 2) tended to be around twofold higher than the levels of butyl paraben in maternal plasma, although this result was not statistically significant (Fig. 2A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Recently, it was shown that deconjugated parabens measured in urine could be used as biomarkers for human exposure (Ye et al. 2006a
, 2006b). In the present study, we have also measured the parent compounds ethyl paraben and butyl paraben as markers for the distribution of ethyl paraben and butyl paraben in fluids and tissues from pregnant rats after sc dosing of the test compound during gestation. We developed a method for extraction of ethyl paraben and butyl paraben in several biological matrices followed by a fast LC-MS/MS method. The method validation showed good characteristic of both ethyl paraben and butyl paraben on column and high sensitivity, good SPE recovery, and method accuracy in all matrices.
The levels of ethyl paraben in maternal plasma and in all tissues were around 10-fold higher than the levels of butyl paraben in the same matrices despite equal dosing. In amniotic fluids, the levels of ethyl paraben were around threefold higher than butyl paraben at the same doses. This indicates that the bioavailability after sc administration or the metabolism was different for the two compounds. The low recovery of butyl paraben compared to ethyl paraben is in agreement with other studies. After oral administration, it was found that the recovery of methyl, ethyl, and propyl paraben in dogs ranged from 56 to 94%, while only 40 and 48% of butyl paraben were recovered after iv administration (Jones et al., 1956). After whole-body topical application in humans with cream containing the same level of butyl paraben, diethyl phthalate, and dibutyl phthalate (DBP), only 0.32% of butyl paraben was recovered in urine compared to the phthalates for which 5.79 and 1.82%, respectively, were recovered (Janjua et al., 2008). Thus, few studies have indicated that as the length of the alkyl chain increases, the rate of excreted parent compound in urine decreases (Soni et al., 2005), probably because the more unpolar the parent paraben becomes, the more paraben will rapidly be hydrolyzed to the nonspecific main metabolite p-hydroxybenzoic acid.
In spite of the different recoveries of the two parent compounds, a similar pattern of distribution for both ethyl paraben and butyl paraben was found in all the test groups. The level of both ethyl paraben and butyl paraben in maternal plasma seems to be more or less saturated when rats were dosed with concentrations 100 mg/kg bw/day. Similar levels as found in maternal plasma were measured in male and female fetal livers and whole-body fetuses at both doses, indicating that also in these tissues a saturated level of both ethyl paraben and butyl paraben had been achieved. In contrast to maternal plasma and the tissues from fetus, ethyl paraben and butyl paraben accumulated in amniotic fluid in a dose-dependent manner (except for butyl paraben in the highest dose group). These data indicated that following excretion of these compounds in the fetus, they accumulate in the amniotic fluid, thus leading to a continued exposure of the fetus. Only placental tissue from dams dosed with ethyl paraben at the highest dose was analyzed, but these data seem to indicate that ethyl paraben may accumulate in placenta as well. A similar dose-dependent increase of compound in amniotic fluid was also shown in a previous study of rats dosed with phthalates, where strong correlations between doses of DBP and di(2-ethylhexyl) phthalate and their corresponding monoester metabolites in amniotic fluid as well as in maternal urine were found (Calafat et al., 2006). This gives matter to concern as it may be hypothesized that the fetus may have two sources for ongoing exposure to parabens: partly through the placenta and partly through the amniotic fluid that constantly surrounds the fetal body as well as serves as the source of drinking. In contrast to our results, pharmacokinetic studies of DBP in pregnant rats have shown that in general the concentration of the major metabolite, monobutyl phthalate (MBP), was higher in both maternal and fetal plasma than in amniotic fluid (Fennell et al., 2004) and that MBP and conjugates were rapidly transferred to embryonic tissues, but the concentration in maternal plasma and other maternal tissues was much higher than in placenta embryo and amniotic fluid (Saillenfait et al., 1998). In a single human study of phthalates in amniotic fluids, this trend was confirmed; the levels of MBP and other phthalate metabolites were much lower in amniotic fluid than the general level measured in human serum and urine (Silva et al., 2004). Other endocrine disruptors such as bisphenol F and trenbolone have also been analyzed in both amniotic fluid and maternal plasma, but similar to the phthalates and in contrast to ethyl paraben and butyl paraben in the presented study, the concentrations in maternal plasma were higher than in amniotic fluid (Cabaton et al., 2006; Hotchkiss et al., 2007). The pharmacokinetics of DBP in pregnant rats showed that half-lives of MBP and MBP-glucuronide in fetal plasma were about twofold longer than the half-lives in maternal plasma, while the half-lives of MBP and MBP-glucuronide in amniotic fluid were about 3- and up to 20-fold longer (64 h for the highest dose), respectively (Fennell et al., 2004). This indicates that the excretion of DBP metabolites from amniotic fluid was very slow, and an on-going exposure might lead to accumulation of metabolites in amniotic fluid. Only few pharmacokinetic studies of parabens have been conducted, and in general, the majority of parabens ( 80%) were excreted after 24 h in rabbits, dogs, and cats (Soni et al., 2005). To our knowledge, this is the first pharmacokinetic study of parabens performed in pregnant rats. A more thorough pharmacokinetic study of the distribution of parabens to maternal plasma and urine, placenta, cord blood, fetus blood, and amniotic fluid as well as distribution to other maternal organs is warranted.
In conclusion, we have developed a method for extraction of ethyl paraben and butyl paraben from rat plasma, amniotic fluid, and tissues such as placenta, liver, and whole fetuses. The analytical method for ethyl paraben and butyl paraben based on LC-MS/MS resulted in high sensitivity, good SPE recovery, and accuracy for both ethyl paraben and butyl paraben in all matrices. The study of the distribution of ethyl paraben and butyl paraben in the different matrices after dosing of pregnant rats during gestation showed markedly higher levels of ethyl paraben compared to butyl paraben in all fluids and tissues. The distribution of ethyl paraben and butyl paraben in maternal plasma, fetal livers, and whole-body fetuses seemed to be saturated, and comparable levels of the compounds were found with doses 100 mg/kg bw/day. In contrast, the concentration of both ethyl paraben and butyl paraben in amniotic fluid increased in a dose-dependent manner and were in the highest doses groups found in much higher levels than in maternal plasma or in the fetal tissues. The level of ethyl paraben in placenta and in amniotic fluid was similar in the highest dose group. This apparent accumulation of parabens in amniotic fluid is a matter of concern as this may lead to a higher exposure of the fetus to parabens than expected so far.
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FUNDING |
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Danish Research Agency (grant no. 2107-05-0006); the Lundbeck Foundation (j.nr. 124/05).
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ACKNOWLEDGMENTS |
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For skilled technical assistance, the authors thank Ole Nielsen, Department of Growth and Reproduction, Rigshospitalet, as well as Dorte Hansen, Birgitte Møller Plesning, and Lillian Sztuk from the Department of Toxicology and Risk Assessment, National Food Institute.
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