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Distribution of bisphenol-A, triclosan and n-nonylphenol in human adipose tissue, liver and brain
Chemosphere 87 (2012) 796–802
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Distribution of bisphenol-A, triclosan and n-nonylphenol in human adipose tissue, liver and brain Tinne Geens a, Hugo Neels a, Adrian Covaci a,b,⇑ a b
Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Belgium Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Belgium
a r t i c l e
i n f o
Article history: Received 25 July 2011 Received in revised form 2 January 2012 Accepted 3 January 2012 Available online 24 January 2012 Keywords: Adipose tissue Brain Liver Human exposure Bisphenol-A Triclosan
a b s t r a c t In this study, an analytical method was optimized for the determination of bisphenol-A (BPA), triclosan (TCS) and 4-n-nonylphenol (4n-NP), environmental contaminants with potential endocrine disruptive activities, in human tissues. The method consisted of a liquid extraction step, derivatization with pentafluorobenzoylchloride followed by a clean-up on acidified silica and detection with gas chromatography coupled with mass spectrometry (GC-ECNI/MS). Recoveries ranged between 92% and 102% with a precision below 5%. Limits of quantification ranged between 0.3–0.4 ng g1, 0.045–0.06 ng g1 and 0.003– 0.004 ng g1 for BPA, TCS and 4n-NP in different tissues, respectively. The method was applied for the determination of BPA, TCS and 4n-NP in paired adipose tissue, liver and brain samples from 11 individuals. BPA could be detected in almost all tissues, with the highest concentrations found in adipose tissue (mean 3.78 ng g1), followed by liver (1.48 ng g1) and brain (0.91 ng g1). TCS showed the highest concentrations in liver (3.14 ng g1), followed by adipose tissue (0.61 ng g1), while it could be detected in only one brain sample. Levels of 4n-NP were much lower, mostly undetected, and therefore 4n-NP is considered of minor importance for human exposure. Despite the measurable concentrations in adipose tissue, these compounds seem to have a low bioaccumulation potential. The reported concentrations of free BPA in the various tissues are slight disagreement with pharmacokinetic models in humans and rats and therefore the possibility of external contamination with BPA during sample collection/storage cannot be ruled out. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Bisphenol-A (BPA), triclosan (TCS) and 4n-nonylphenol (4n-NP) are three phenolic chemicals with a substantial use in consumer products and a widespread distribution in the environment and humans (Ying et al., 2002; Vandenberg et al., 2007; Soares et al., 2008; Fang et al., 2010). BPA is mainly used as monomer for the production of polycarbonate and epoxy resins, dental composite fillings, thermal paper; TCS is used as antimicrobial agent in personal care products, while 4n-NP is a degradation product of NP ethoxylates, which are nonionic surfactants (Petrovic et al., 2004; Vandenberg et al., 2007). BPA and NP have been catalogued as endocrine disruptors and have recently been associated with the development of obesity (Grün and Blumberg, 2009; Heindel and vom Saal, 2009; Newbold et al., 2009), diabetes (Nadal et al., 2009) and effects on the central nervous system (Mizuo et al., 2010). TCS has been reported to interfere with thyroid hormone (thyroxine) metabolism in laboratory rodents (Crofton et al., 2007).
⇑ Corresponding author at: Toxicological Centre, Universiteitsplein 1, 2610 Wilrijk, Belgium. Tel.: +32 3 265 2498; fax: +32 3 265 2722. E-mail address: [email protected] (A. Covaci). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2012.01.002
These phenolic compounds have a rapid turnover in humans with a half-live shorter than 24 h. After absorption, they are rapidly converted to their glucuronides and sulfate conjugates to promote urinary excretion (Doerge et al., 2002; Jaeg et al., 2004; Wu et al., 2010). Two pharmacokinetic studies conducted by Völkel et al. (2002, 2005) showed that the oral administration of respectively 5 mg d16-BPA and 25 lg BPA to human volunteers resulted in a complete first pass metabolism to BPA-glucuronide. The BPA aglycone was not detected in blood at any time postdosing. Virtually all administered BPA was recovered as BPA-glucuronide in the urine after 24 h. In these experiments, a t1/2 of 4–5.4 h was calculated for BPA, showing its non-persistent character. The rapid biotransformation and elimination of BPA after oral exposure was confirmed by a study of Teeguarden et al. (2011) where the serum and urinary pharmacokinetics of a population were followed during a 24 h period. During this period, the investigated group has received meals enriched in canned food. Total BPA concentrations in the urine peaked 2.75 h post-meal, lagging the serum concentration Tmax with 1 h. Even during this higher dietary exposure, total BPA concentrations in serum were undetectable in 83% of the samples. Biomonitoring of BPA, TCS and 4n-NP mainly occurs in urine, serum or human milk (Calafat et al., 2005, 2008; Allmyr et al.,
T. Geens et al. / Chemosphere 87 (2012) 796–802
2006; Vandenberg et al., 2010). However, their distribution and levels in other human tissues, where they can cause effects as well, are not well documented. In this study, we developed an analytical method based on gas chromatography – mass spectrometry (GC–MS) for the analysis of BPA, TCS and 4n-NP as their pentafluorobenzoyl-derivatives (PFB) in various tissues, such as human adipose tissue, brain and liver. Afterwards we have determined these compounds in post-mortem tissues collected from eleven humans.
2. Materials and methods 2.1. Chemicals and materials
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100 lL recovery standard PBB-80 (3,30 ,5,50 -tetrabromobiphenyl, 100 pg lL1 in i-octane). For liver samples, the ACN layer was divided into two equal parts after extraction; one to determine the free compounds and the other to determine total (free and conjugated) levels. The free concentration of phenolic compounds was determined as described above. For the determination of the total concentration, the ACN layer was evaporated and afterwards treated with 50 lL b-glucuronidase/sulfatase in the presence of 0.75 mL of 1 M Naacetate (pH 4.5) and held at 40 °C for 60 min. After deconjugation, the sample was alkalinized with KOH and derivatization and cleanup was performed as described above. 2.4. Quality control
13
Individual standard solutions of BPA and C-BPA were purchased from Cambridge Isotope Laboratories (Andover, MA, USA); standards of TCS and 13C-TCS were from Wellington Laboratories (Guelph, ON, Canada) and 4n-nonylphenol (4n-NP) and D8-n-nonylphenol (D8-4n-NP) were from Dr. Ehrenstorfer GmbH (Augsburg, Germany). All used solvents, dichloromethane (DCM), n-hexane, acetonitrile (ACN) and iso-octane were of SupraSolv grade (Merck, Darmstadt, Germany). To prepare 10% acidified silica gel, 5 mL concentrated sulfuric acid was added dropwise to 50 g silica under continuous stirring. Polypropylene cartridges (3 mL, Supelco) filled with prewashed acidified silica gel (10%, w/w) were used for extract clean-up. Derivatization was performed with pentafluorobenzoylchloride (PFBCl; 99% purity, Sigma–Aldrich) which was diluted to a 5% (v/v) solution with hexane. 2.2. Samples Adipose tissue, liver and brain were collected in 2002 during autopsy from eleven patients at the University Hospital of Antwerp, of whom eight males and three females, aged between 9 and 62 years with an average age of 34 years. Tissues were homogenized by mixing and were stored in polypropylene containers at 20 °C. The polypropylene containers for storage were evaluated for contamination, but were all free of BPA, TCS and 4n-NP. 2.3. Method description A sample amount of 500 mg of adipose tissue and brain and 750 mg of liver were accurately weighed. Samples were fortified with 5 ng of each internal standard (13C-BPA; 13C-TCS and D8-4nNP) and after addition of 3 mL ACN, compounds were extracted from the tissues by ultrasonication during 30 min and vortexing for 3 min. After centrifugation, the ACN layer was transferred to another tube and was washed with 2 mL Hex to remove lipophilic impurities. After evaporation until dryness under a gentle stream of N2, the sample was subjected to a derivatization and a second clean up step. The extractive derivatization was carried out in a two phase-system of 1 mL MilliQ H2O and 2 mL hexane. A volume of 50 lL KOH 2 M was added for ionization of the target compounds and 50 lL pentafluorobenzoyl-chloride (PFBCl, 5% in hexane v/v) was used as derivatization reagent. The solution was vortexed for 2 min and, after phase-separation, the hexane layer was subjected to a clean-up by transferring it to a cartridge filled with a small layer of 10% acidified silica (w/w). Hereafter, another 2 mL hexane was added to the aqueous phase and the cycle was repeated to obtain a quantitative transfer of the derivatized compounds. The analytes were subsequently eluted from the acidified silica cartridge with 6 mL DCM. After evaporation until dryness under N2, the PFB-derivatives were reconstituted in
A calibration curve was made daily and linearity was evaluated through R2 values (for both analytes R2 > 0.999). Ratios of peak area of parent compound/internal standard in the Y-axis were plotted against ratio of masses of parent compound/internal standard in the X-axis. For examination of recovery and method precision, each tissue in triplicate was spiked with 10 ng of each compound. This was repeated on three successive days. To calculate recovery, the corresponding non-spiked tissues used for these experiments were analyzed in triplicate and subtracted from the measured concentrations. Since contamination with BPA may be a problem during sample preparation, additional precautions were taken to prevent this (Markhal et al., 2010; Twaddle et al., 2010). The use of plastic materials was avoided during sample collection, preparation and analysis and when possible additional washing steps of contact materials (such as SPE-cartridge) were introduced. In parallel with every twenty samples analyzed, four procedural blanks were added to the batch and were subjected to the complete procedure. Trace amounts of the analytes could still be detected in the blanks, however values were relatively constant. After analysis, average blank concentrations were subtracted from the sample results. Method limits of quantification (LOQ) were calculated as three times the standard deviation (99% confidence interval) of procedural blanks, taking into account the amount of sample used for the analysis. This makes that, if the concentration of the analyte, after subtraction of the mean blank value, is higher than the LOQ, that it can for 99% certainty be assigned to the sample and is not due to contamination during analysis. 2.5. Instrumental analysis The analysis of the PFB-derivatives was performed with a Hewlett–Packard (Palo Alto, CA, USA) 6890 GC coupled to a HP 5973 MS operated in electron capture negative ionization (ECNI) mode. The extract (1 lL) was injected in cold pulsed splitless mode (injector temperature 90 °C for 1.25 min, then increased at 700 °C min1 to 290 °C, pulse pressure 15.0 psi, time pulse 1.25 min, and splitless time 1.25 min). The GC column was a 30 m 0.25 mm i.d. 0.25 lm film thickness DB-5MS column (J&W Scientific, Palo Alto, CA, USA). The oven temperature program was 90 °C for 1.25 min, then by 25 °C min1 to 300 °C, kept for 8 min. Helium was used as carrier gas at constant flow (1.0 mL min1) with methane as moderating gas. Dwell times were set at 30 ms. The ion source, quadrupole, and interface temperatures were set at 170, 150, and, 300 °C, respectively, and the electron multiplier voltage was set at 2100 V. The MS operated in selected ion monitoring (SIM) mode. The retention times and the quantification and confirmation ions of the different compounds are given in Table 1.
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Table 1 Retention times and monitored ions for the investigated phenolic compounds and the corresponding labeled internal standards.
D8-4n-NP 4n-NP 13 C-TCS TCS 13 C-BPA BPA
TR (min)
Quantitation ion (m/z)
Confirmation ion (m/z)
13.89 13.93 14.85 14.85 19.46 19.46
422 414 494 482 628 616
421 415 299 287 418 406
3. Results and discussion 3.1. Method optimization and quality control The derivatization with PFBCl and clean-up of the extract on acidic silica is based on a method described earlier for the analysis of BPA and TCS in serum and urine (Geens et al., 2009). Shortly, the incorporation of five (for TCS and 4n-NP) or ten (for BPA) fluorine atoms transforms the compounds into highly electrophilic derivatives which make them suitable for GC-ECNI/MS analysis. The use of this technique improves the selectivity and sensitivity for these compounds. Moreover, the sensitivity of this method is further increased by the introduction of a clean-up step on acidified silica, which leads to very clean extracts. With this combination of thorough clean-up and derivatization with electrophilic groups detected with ECNI/MS, instrumental LOQs below 1 pg injected could be achieved for all analytes. Since small amounts of the analytes could be detected in blank samples, a method LOQ was applied (see further). A SIM-chromatogram of a spiked adipose tissue sample, a ‘‘real’’ adipose tissue sample is presented in Fig. 1. Chromatograms and baselines were similar for brain and liver tissues. The molecular ion was the most abundant and was used as quantification ion. For BPA and TCS, the ions formed after loss of one derivatization group (M-PFB) were used as confirmation ions (Table 1). Method performance for all tissues and analytes is summarized in Table 2. Recovery of the spiked compounds ranged between 92% and 102%. Within day and between day precision for all compounds is <5% for each tissue. As described earlier, trace amounts of the analytes could be detected in blank samples. This was especially the case for BPA which can come from materials used during analysis, reagents etc. Therefore method LOQs were used and were defined as three times the standard deviation of the blanks. Average levels of procedural blanks were 0.2 ng for BPA, 0.06 ng for TCS and <0.002 ng for n-NP. A chromatogram of a blank sample is presented is given in Fig. 1. BPA levels in the blank samples were relatively constant which resulted in method LOQs of 0.4 ng g1 for adipose tissue or brain and 0.3 ng g1 liver. These values are similar or lower compared to other studies that determined BPA in tissues (Fernandez et al., 2007, Jiménez-Diaz, 2010, Schaefer et al., 2000). For TCS and 4n-NP, blank issues are less prominent, however also method LOQs were applied. For TCS, a LOQ of 0.06 ng g1 for adipose tissue or brain and 0.045 ng g1 for liver was achieved, while for 4n-NP 0.004 ng g1 for adipose tissue or brain and 0.003 ng g1 for liver could be set. Concentrations in tissues below LOQ were replaced by fLOQ (f: detection frequency of a compound in a tissue) for further analysis of the data. 3.2. Concentrations in human tissues Descriptive parameters of the concentrations measured in the different tissues are summarized in Table 3 and Fig. 2. Both adipose tissue and brain are tissues with high lipid content and a minimal Phase II metabolic capacity. For these reasons, BPA, TCS and n-NP
were expected to be present in the more lipophilic, aglycone form and consequently, only the free BPA was measured in adipose tissue and brain. Contrary, the liver is a well-perfused, more hydrophilic organ with an extensive metabolization capacity and therefore both free and total compounds were measured, since an important contribution of the conjugated form was expected. This was already described for BPA in rats by Doerge et al. (2011). Two hours after intravenous administration of BPA to female rats, the tissue distribution and the contribution in terms of percentage of the aglycone form in each tissue were determined. The aglycone form contributed for respectively 96%, 95% and 2.5% to the total BPA concentration in adipose tissue, brain and liver (Doerge et al., 2011). In the present study, total and free concentrations of the determined compounds in liver were almost equal which means that most of the compounds were present in the aglycone form. Since this is in contrast with the findings of Doerge et al. (2011) for BPA, it is probable that deconjugation has taken place during sample storage and sample preparation. Several publications report the release of b-glucuronidase activity from liver and fetal/placental tissues during thawing and cutting of the samples (Moors et al., 2006; Waechter et al., 2007; Doerge et al., 2011). Since no precautions could be taken in the present study to prevent this deconjugation, it is very likely that the measured aglycone concentrations in liver are a result of this release of b-glucuronidase activity. 3.2.1. Bisphenol-A BPA was found with a high detection frequency in all tissues. BPA was detected in all adipose tissue and liver samples, while it could be detected in 8 out of 11 brain samples. BPA concentrations descended from adipose tissue (mean: 3.78 ng g1) over liver (mean: 1.30 ng g1) to brain samples (mean: 0.91 ng g1). As previously mentioned, total BPA in liver (mean: 1.48 ng g1) is only slightly higher than free BPA (mean: 1.30 ng g1), which is probably a result of deconjugation during sample preparation. Until now, few tissue data are available to compare. Fernandez et al. (2007) could determine BPA above detection limit in 11 out of 20 adipose tissue samples from women in Spain. BPA levels ranged between 1.80 and 12.01 ng g1 adipose tissue with mean ± standard deviation of 3.16 ± 4.11 ng g1, results which are in accordance with our results. Schaefer et al. (2000) could not detect BPA in 21 adipose tissue from German women, probably due to a high detection limit of 20 ng g1. Smeds and Saukko (2003) could detect BPA in only 2 out of 13 adipose tissue (14.9 and 47.2 ng g1 lipid weight). However, in the latter study, BPA seemed to be present in various samples, although it could be quantified only in two samples because of overlapping peaks. No literature data could be found about concentrations of BPA in brain or liver tissues. With a log Kow of 3.4, BPA has only a moderate potential for bioaccumulation (Staples et al., 1998; Heinonen et al., 2002). However, some studies demonstrate the preferential partition of BPA into lipohilic tissues, such as adipose tissue, over the hydrophilic compartment. In our study, we found a mean ratio of concentrations in adipose tissue:brain and liver:brain of 5.90 and 1.81, respectively. The median ratios of these concentrations are 3.68 and 1.48, respectively. The mean ratio of concentrations in adipose tissue:liver was 2.76, while the median ratio was 1.90. In an in vitro study, Csanady et al. (2002) determined the partition coefficients of BPA by incubating blood containing BPA with different tissues at 37 °C. The tissue:blood partition coeffiecients for brain, liver and fat were 1.08; 1.46 and 3.31 respectively. Adipose tissue had thus a 3.06 times higher partition coefficient than brain, that of liver was 1.43 times higher than that of brain. These in vitro derived ratios compare well with the ratios we found in the tissues. Nunez et al. (2001) also observed a preferential accumulation of BPA in brown adipose tissue over other tissues in rats exposed to
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A
Abundance
nNP-PFB TCS-PFB
320000 280000
13
C-TCS-PFB
240000 200000 D8-nNPPFB
160000
BPA-PFB
120000
13
C-BPA-PFB
80000 40000 0 14.0
B
14.5
15.0
Abundance 140000
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
13
C-TCS-PFB
120000 100000
D8-nNP-PFB
80000 TCS-PFB
60000 13
C-BPA-PFB
40000 20000
BPA-PFB
0 14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
n-NP 5000
C
Abundance
BPA
4000 3000 2000
TCS
1000
9000
6000 13.85 13.95
8000
5000
4000
7000
4000
3000
6000
3000
5000
2000
2000
4000
1000
1000
3000
0
2000
14.80
14.90
19.40
19.60
15.00
1000 0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
Fig. 1. GC-ECNI/MS chromatogram of a spiked adipose tissue sample (A), an adipose tissue (B) and a procedural blank (C).
BPA. In a study of Doerge et al. (2011), deuterated BPA was administered to female rats by intravenous route and tissue distribution was measured 2 h after dosing, thus during the distribution phase and before elimination substantially reduced the levels. The tissue/ serum ratio for BPA-aglycone was 5.0 for adipose tissue, 2.8 for brain and 0.73 for liver, while the tissue/serum ration for the conjugated-BPA was 0.032; 0.034 and 5.4 for adipose tissue, brain and liver, respectively. Liver, which is richly perfused and has a high Phase II metabolization capacity, had the lowest tissue/serum ratio for BPA-aglycone, whereas adipose tissue with a lipophilic composition, slow perfusion and a low metabolic Phase II capacity had the highest ratio. Brain which has also a low evidence for Phase II metabolism has also a higher tissue/serum ration for BPA-aglycone (Doerge et al., 2011). Comparison of human data with rodent
studies should be interpreted with care since the enterohepatic recirculation which is observed in rats, is not seen in humans. Also the administered dose in animal studies is orders of magnitude higher than the daily estimated human exposure. A significant positive Spearman correlation was observed between BPA concentration in adipose tissue and brain (r = 0.670; p = 0.024); BPA concentration in brain and free BPA in the liver (correlation coefficient r = 0.884; p = 0.001) and between free and total BPA in the liver (r = 0.682; p = 0.021). A significant negative Spearman correlation was found between age and BPA concentration in brain (r = 0.714; p = 0.014); weight and BPA concentration in adipose tissue (r = 0.715; p = 0.013) and weight and BPA concentration in the brain (r = 0.802; p = 0.003). These correlations should be interpreted with care since the limited number of
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Table 2 Recovery of spiked samples in different tissues. Relative standard deviation is given between brackets and n is the number of replicates. Mean (RSD)
Day 1 (n = 3)
Day 2 (n = 3)
Day 3 (n = 3)
Mean day 1–3 (n = 9)
Adipose tissue
BPA TCS 4n-NP
102 (1) 99 (1) 98 (2)
102 (1) 101 (1) 95 (2)
98 (1) 99 (1) 101 (1)
101 (2) 100 (1) 98 (3)
Brain
BPA TCS 4n-NP
99 (4) 100 (3) 97 (1)
102 (3) 99 (4) 94 (2)
95 (1) 100 (1) 100 (3)
99 (4) 99 (3) 97 (3)
Liver free
BPA TCS 4n-NP
98 (3) 95 (2) 92 (3)
102 (3) 98 (1) 95 (1)
97 (1) 96 (2) 96 (2)
99 (3) 96 (2) 94 (2)
Liver total
BPA TCS 4n-NP
97 (3) 95 (1) 94 (2)
102 (3) 98 (1) 96 (2)
97 (1) 100 (2) 102 (1)
99 (3) 97 (2) 98 (4)
Table 3 Descriptive parameters of BPA, TCS and 4n-NP in the different tissues. Concentrations (ng g1 ww)
a b c d e
Adipose tissue (free)
Brain (free)
Liver (free)
Liver (total)
BPA
Range Median Mean Detection frequency
1.12–12.28 2.09 3.78 11/11
0.77–3.35 1.01 1.30 11/11
0.90–2.77 1.03 1.48 11/11
TCS
Range Median Mean Detection frequency
4n-NP
Range Median Mean Detection frequency
0.008–0.208 0.023 0.046 11/11
LOQ = 0.4 ng g1. LOQ = 0.06 ng g1. LOQ = 0.045 ng g1. OQ = 0.004 ng g1. LOQ = 0.003 ng g1.
samples analyzed and therefore no conclusions can be linked to these observations. Fernandez et al. (2007) observed a significant Spearman correlation coefficient between age and BPA concentrations in adipose tissue. Such correlation was not found in the present study. 3.2.2. Triclosan TCS was detected in almost all liver (10/11), most adipose tissue (7/11) and only in one brain sample. One outlier (subject 1) was observed with concentrations of 3.92 ng g1 in adipose tissue, 0.23 ng g1 in brain and 29.03 ng g1 total TCS in liver. For calculation of mean concentrations and statistics, this outlier was removed. Contrary to BPA, TCS had the highest concentrations in liver (mean: 0.44 ng g1), followed by adipose tissue (mean: 0.28 ng g1) and brain (all
3.2.3. 4n-Nonylphenol In most subjects and tissues, concentrations for 4n-NP were <0.1 ng g1 and thus much lower levels compared to BPA and TCS. Similar to TCS, the highest concentrations of 4n-NP were found in liver, followed by adipose tissue and brain. Naassner et al. (2002) reported that the technical NP mixture does not contain 4n-NP. On the other hand, Calafat et al. (2005) mentioned nNP is a small percentage of the technical mixture and they could detect n-NP in low concentrations in 51% of 394 urine samples with a median value <0.1 ng mL1. Also Kuklenyik et al. (2003) could not detect n-NP in 30 urine samples from people who used paint products. Nonyl-phenol is a hydrophobic compound with a log Kow value of 4.48 and low solubility in water, therefore it partitions favorably to organic matter (Soares et al., 2008). Three previous papers reported the detection of NP in adipose tissue. Müller et al. (1998) detected NP in 25 adipose tissues from Switzerland with a median concentration of 37 ng g1 (range 20–84 ng g1). However, these concentrations were within the range of the background contamination found in the method blanks. They also mentioned, based on the pharmacokinetic data of their study, that adipose tissue levels are probably a factor of 50 lower than those background levels. Another study in Spain detected NP (Lopez-Espinosa et al., 2009) in all 20 samples with a median concentration of 57 ng g1 (range 10–567 ng g1). However in this study, background contamination is not mentioned. Very recently, Ferrara et al. (2011) reported NP at mean concentration of 122 ng g1 (range 10–226 ng g1) in 16 Italian patients undergoing bariatric
BPA concentration (ng/g)
T. Geens et al. / Chemosphere 87 (2012) 796–802
BPA
14 12
Fat
10
Brain
8
Liver Total
6 4 2 0
1
2
3
4
5
6
7
8
9
10
11
Subject TCS
Concentration (ng/g)
5.0 4.0
Fat
3.0
Brain Liver Total
2.0 1.0 0.0
1
2
3
4
5
6
7
8
9
10
11
Subject
Concentration (ng/g)
nNP 0.6 0.5
Fat
0.4
Brain
0.3
Liver Total
0.2 0.1 0
1
2
3
4
5
6
7
8
9
10
11
Subject Fig. 2. Individual concentrations (ng/g tissue) of BPA, TCS, and 4n-NP in adipose tissue, brain and liver. ‘‘Liver total’’ represents the sum of concentrations of conjugated and unconjugated compounds. For subject 1, total TCS was respectively 29.0 ng g1 and total 4n-NP in liver was 1.40 ng g1 (outside scale of Y-axis).
surgery. In these studies, no details were reported about the isomeric composition of NP. Given the high concentrations, it may be assumed that the sum of branched and unbranched NP was measured and not only 4n-NP. Indeed, we have observed in our ion chromatograms several peaks with the same ratios between ions m/z 414 and 415 (specific for NP). They eluted earlier than 4n-NP and thus we presumed that they were branched NPs. In most cases, they had higher signals than 4n-NP. Since we did not had any other standards than 4n-NP, these (presumably) NP peaks were not quantified. 3.3. Critical discussion of limitations and potential sources of errors Overall, concentrations of the studied phenolic compounds are relatively low, even in adipose tissue. The bioaccumulation of BPA was recently suggested (Stahlhut et al., 2009), but seems not very plausible given these low concentrations and its low persistence characteristics. The detectable levels of free BPA in adipose tissue and brain may seem in contrast with the findings of different studies showing the fast biotransformation and elimination after oral exposure in humans (Völkel et al., 2002, 2005; Teeguarden et al., 2011) and after oral and intravenous exposure in rhesus monkeys (Doerge et al., 2010). After oral administration of 100 lg/kg bw/d, the circulating concentration in serum of free BPA was less than 1% in both humans and monkeys, which reflects
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the extensive Phase II metabolism in gut and liver. Five minutes after intravenous administration in monkeys, only 29% of BPA was still present as aglycone. BPA aglycone showed a large volume of distribution probably reflecting the distribution of lipophilic parent compound out of the circulation into tissues where it is further conjugated before elimination. However, similar elimination half-times for BPA aglycone and total BPA suggested a fast return to the central compartment from the tissues (Doerge et al., 2010). To obtain the BPA levels measured in this study, the current pharmacokinetic models in humans and monkeys (Völkel et al., 2002, 2005; Doerge et al., 2010; Teeguarden et al., 2011) predict that this can only occur after an oral exposure of approximately 200–1300 lg/kg bw/d. This exposure is orders of magnitude higher than the estimated exposure based on urinary measurements of conjugated BPA (Doerge et al., 2010). However, the hypothesis that BPA can distribute to human tissues after non-oral exposure (such as dermal exposure), before biotransformation has occurred, need further investigation (Teeguarden et al., 2011). Since the results of BPA found in this study partly disagree with the fast elimination of free BPA, it cannot be ruled out that the measured concentrations of BPA are a result of external contamination during sample collection and storage. Despite the great care taking to avoid contamination during chemical analysis, the collection of tissue samples in a hospital setting is in general less controlled. The external contamination with BPA in hospital settings was recently described by Vandentorren et al. (2011). In that study, high free BPA levels were observed in urine of women who gave birth by a caesarean section. These high values of free BPA were dedicated to contamination with BPA from exogenous sources during sample collection. The authors suggested that contamination occurred from medical devices either from catheterization or urine probes when biomonitoring at delivery (Vandentorren et al., 2011). Only the aglycone compounds were measured in brain and adipose, while in liver both free and total compounds were measured. As described earlier, it is highly plausible that free BPA in the tissues is a result of deconjugation of the conjugated metabolites by the release of b-glucuronidase. This hypothesis is backed up by the similarly high proportion of free TCS over the total TCS observed in the liver. Furthermore, the free BPA concentrations measured in adipose and brain were very different from those measured in liver, which exclude partly the possibility that samples were contaminated with BPA during sample collection/ storage. Due to this possible deconjugation, it is of limited value to determine both total and free BPA. Moreover this makes more difficult to interpret whether the measured free concentrations are a result of internal exposure, external contamination or deconjugation. Tissue concentrations of 4n-NP were extremely low since, as earlier described, 4n-NP is a minor component of the NP-mixture and therefore it is unimportant for biomonitoring studies.
Acknowledgements Tinne Geens acknowledges the Funds for Scientific Research Flanders (FWO) for a PhD fellowship, while Adrian Covaci acknowledges FWO for a postdoctoral fellowship. References Allmyr, M., Adolfsson-Erici, M., McLachlan, M.S., Sandborgh-Englund, G., 2006. Triclosan in plasma and milk from Swedish nursing mothers and their exposure via personal care products. Sci. Total Environ. 372, 87–93. Bennett, E.R., Ross, P.S., Huff, D., Alaee, M., Letcher, R.J., 2009. Chlorinated and brominated organic contaminants and metabolites in the plasma and diet of a captive killer whale (Orcinus orca). Marin. Pollut. Bull. 58, 1078–1083.
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Distribution of bisphenol-A, triclosan and n-nonylphenol in human adipose tissue, liver and brain Tinne Geens a, Hugo Neels a, Adrian Covaci a,b,⇑ a b
Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Belgium Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Belgium
a r t i c l e
i n f o
Article history: Received 25 July 2011 Received in revised form 2 January 2012 Accepted 3 January 2012 Available online 24 January 2012 Keywords: Adipose tissue Brain Liver Human exposure Bisphenol-A Triclosan
a b s t r a c t In this study, an analytical method was optimized for the determination of bisphenol-A (BPA), triclosan (TCS) and 4-n-nonylphenol (4n-NP), environmental contaminants with potential endocrine disruptive activities, in human tissues. The method consisted of a liquid extraction step, derivatization with pentafluorobenzoylchloride followed by a clean-up on acidified silica and detection with gas chromatography coupled with mass spectrometry (GC-ECNI/MS). Recoveries ranged between 92% and 102% with a precision below 5%. Limits of quantification ranged between 0.3–0.4 ng g1, 0.045–0.06 ng g1 and 0.003– 0.004 ng g1 for BPA, TCS and 4n-NP in different tissues, respectively. The method was applied for the determination of BPA, TCS and 4n-NP in paired adipose tissue, liver and brain samples from 11 individuals. BPA could be detected in almost all tissues, with the highest concentrations found in adipose tissue (mean 3.78 ng g1), followed by liver (1.48 ng g1) and brain (0.91 ng g1). TCS showed the highest concentrations in liver (3.14 ng g1), followed by adipose tissue (0.61 ng g1), while it could be detected in only one brain sample. Levels of 4n-NP were much lower, mostly undetected, and therefore 4n-NP is considered of minor importance for human exposure. Despite the measurable concentrations in adipose tissue, these compounds seem to have a low bioaccumulation potential. The reported concentrations of free BPA in the various tissues are slight disagreement with pharmacokinetic models in humans and rats and therefore the possibility of external contamination with BPA during sample collection/storage cannot be ruled out. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Bisphenol-A (BPA), triclosan (TCS) and 4n-nonylphenol (4n-NP) are three phenolic chemicals with a substantial use in consumer products and a widespread distribution in the environment and humans (Ying et al., 2002; Vandenberg et al., 2007; Soares et al., 2008; Fang et al., 2010). BPA is mainly used as monomer for the production of polycarbonate and epoxy resins, dental composite fillings, thermal paper; TCS is used as antimicrobial agent in personal care products, while 4n-NP is a degradation product of NP ethoxylates, which are nonionic surfactants (Petrovic et al., 2004; Vandenberg et al., 2007). BPA and NP have been catalogued as endocrine disruptors and have recently been associated with the development of obesity (Grün and Blumberg, 2009; Heindel and vom Saal, 2009; Newbold et al., 2009), diabetes (Nadal et al., 2009) and effects on the central nervous system (Mizuo et al., 2010). TCS has been reported to interfere with thyroid hormone (thyroxine) metabolism in laboratory rodents (Crofton et al., 2007).
⇑ Corresponding author at: Toxicological Centre, Universiteitsplein 1, 2610 Wilrijk, Belgium. Tel.: +32 3 265 2498; fax: +32 3 265 2722. E-mail address: [email protected] (A. Covaci). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2012.01.002
These phenolic compounds have a rapid turnover in humans with a half-live shorter than 24 h. After absorption, they are rapidly converted to their glucuronides and sulfate conjugates to promote urinary excretion (Doerge et al., 2002; Jaeg et al., 2004; Wu et al., 2010). Two pharmacokinetic studies conducted by Völkel et al. (2002, 2005) showed that the oral administration of respectively 5 mg d16-BPA and 25 lg BPA to human volunteers resulted in a complete first pass metabolism to BPA-glucuronide. The BPA aglycone was not detected in blood at any time postdosing. Virtually all administered BPA was recovered as BPA-glucuronide in the urine after 24 h. In these experiments, a t1/2 of 4–5.4 h was calculated for BPA, showing its non-persistent character. The rapid biotransformation and elimination of BPA after oral exposure was confirmed by a study of Teeguarden et al. (2011) where the serum and urinary pharmacokinetics of a population were followed during a 24 h period. During this period, the investigated group has received meals enriched in canned food. Total BPA concentrations in the urine peaked 2.75 h post-meal, lagging the serum concentration Tmax with 1 h. Even during this higher dietary exposure, total BPA concentrations in serum were undetectable in 83% of the samples. Biomonitoring of BPA, TCS and 4n-NP mainly occurs in urine, serum or human milk (Calafat et al., 2005, 2008; Allmyr et al.,
T. Geens et al. / Chemosphere 87 (2012) 796–802
2006; Vandenberg et al., 2010). However, their distribution and levels in other human tissues, where they can cause effects as well, are not well documented. In this study, we developed an analytical method based on gas chromatography – mass spectrometry (GC–MS) for the analysis of BPA, TCS and 4n-NP as their pentafluorobenzoyl-derivatives (PFB) in various tissues, such as human adipose tissue, brain and liver. Afterwards we have determined these compounds in post-mortem tissues collected from eleven humans.
2. Materials and methods 2.1. Chemicals and materials
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100 lL recovery standard PBB-80 (3,30 ,5,50 -tetrabromobiphenyl, 100 pg lL1 in i-octane). For liver samples, the ACN layer was divided into two equal parts after extraction; one to determine the free compounds and the other to determine total (free and conjugated) levels. The free concentration of phenolic compounds was determined as described above. For the determination of the total concentration, the ACN layer was evaporated and afterwards treated with 50 lL b-glucuronidase/sulfatase in the presence of 0.75 mL of 1 M Naacetate (pH 4.5) and held at 40 °C for 60 min. After deconjugation, the sample was alkalinized with KOH and derivatization and cleanup was performed as described above. 2.4. Quality control
13
Individual standard solutions of BPA and C-BPA were purchased from Cambridge Isotope Laboratories (Andover, MA, USA); standards of TCS and 13C-TCS were from Wellington Laboratories (Guelph, ON, Canada) and 4n-nonylphenol (4n-NP) and D8-n-nonylphenol (D8-4n-NP) were from Dr. Ehrenstorfer GmbH (Augsburg, Germany). All used solvents, dichloromethane (DCM), n-hexane, acetonitrile (ACN) and iso-octane were of SupraSolv grade (Merck, Darmstadt, Germany). To prepare 10% acidified silica gel, 5 mL concentrated sulfuric acid was added dropwise to 50 g silica under continuous stirring. Polypropylene cartridges (3 mL, Supelco) filled with prewashed acidified silica gel (10%, w/w) were used for extract clean-up. Derivatization was performed with pentafluorobenzoylchloride (PFBCl; 99% purity, Sigma–Aldrich) which was diluted to a 5% (v/v) solution with hexane. 2.2. Samples Adipose tissue, liver and brain were collected in 2002 during autopsy from eleven patients at the University Hospital of Antwerp, of whom eight males and three females, aged between 9 and 62 years with an average age of 34 years. Tissues were homogenized by mixing and were stored in polypropylene containers at 20 °C. The polypropylene containers for storage were evaluated for contamination, but were all free of BPA, TCS and 4n-NP. 2.3. Method description A sample amount of 500 mg of adipose tissue and brain and 750 mg of liver were accurately weighed. Samples were fortified with 5 ng of each internal standard (13C-BPA; 13C-TCS and D8-4nNP) and after addition of 3 mL ACN, compounds were extracted from the tissues by ultrasonication during 30 min and vortexing for 3 min. After centrifugation, the ACN layer was transferred to another tube and was washed with 2 mL Hex to remove lipophilic impurities. After evaporation until dryness under a gentle stream of N2, the sample was subjected to a derivatization and a second clean up step. The extractive derivatization was carried out in a two phase-system of 1 mL MilliQ H2O and 2 mL hexane. A volume of 50 lL KOH 2 M was added for ionization of the target compounds and 50 lL pentafluorobenzoyl-chloride (PFBCl, 5% in hexane v/v) was used as derivatization reagent. The solution was vortexed for 2 min and, after phase-separation, the hexane layer was subjected to a clean-up by transferring it to a cartridge filled with a small layer of 10% acidified silica (w/w). Hereafter, another 2 mL hexane was added to the aqueous phase and the cycle was repeated to obtain a quantitative transfer of the derivatized compounds. The analytes were subsequently eluted from the acidified silica cartridge with 6 mL DCM. After evaporation until dryness under N2, the PFB-derivatives were reconstituted in
A calibration curve was made daily and linearity was evaluated through R2 values (for both analytes R2 > 0.999). Ratios of peak area of parent compound/internal standard in the Y-axis were plotted against ratio of masses of parent compound/internal standard in the X-axis. For examination of recovery and method precision, each tissue in triplicate was spiked with 10 ng of each compound. This was repeated on three successive days. To calculate recovery, the corresponding non-spiked tissues used for these experiments were analyzed in triplicate and subtracted from the measured concentrations. Since contamination with BPA may be a problem during sample preparation, additional precautions were taken to prevent this (Markhal et al., 2010; Twaddle et al., 2010). The use of plastic materials was avoided during sample collection, preparation and analysis and when possible additional washing steps of contact materials (such as SPE-cartridge) were introduced. In parallel with every twenty samples analyzed, four procedural blanks were added to the batch and were subjected to the complete procedure. Trace amounts of the analytes could still be detected in the blanks, however values were relatively constant. After analysis, average blank concentrations were subtracted from the sample results. Method limits of quantification (LOQ) were calculated as three times the standard deviation (99% confidence interval) of procedural blanks, taking into account the amount of sample used for the analysis. This makes that, if the concentration of the analyte, after subtraction of the mean blank value, is higher than the LOQ, that it can for 99% certainty be assigned to the sample and is not due to contamination during analysis. 2.5. Instrumental analysis The analysis of the PFB-derivatives was performed with a Hewlett–Packard (Palo Alto, CA, USA) 6890 GC coupled to a HP 5973 MS operated in electron capture negative ionization (ECNI) mode. The extract (1 lL) was injected in cold pulsed splitless mode (injector temperature 90 °C for 1.25 min, then increased at 700 °C min1 to 290 °C, pulse pressure 15.0 psi, time pulse 1.25 min, and splitless time 1.25 min). The GC column was a 30 m 0.25 mm i.d. 0.25 lm film thickness DB-5MS column (J&W Scientific, Palo Alto, CA, USA). The oven temperature program was 90 °C for 1.25 min, then by 25 °C min1 to 300 °C, kept for 8 min. Helium was used as carrier gas at constant flow (1.0 mL min1) with methane as moderating gas. Dwell times were set at 30 ms. The ion source, quadrupole, and interface temperatures were set at 170, 150, and, 300 °C, respectively, and the electron multiplier voltage was set at 2100 V. The MS operated in selected ion monitoring (SIM) mode. The retention times and the quantification and confirmation ions of the different compounds are given in Table 1.
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Table 1 Retention times and monitored ions for the investigated phenolic compounds and the corresponding labeled internal standards.
D8-4n-NP 4n-NP 13 C-TCS TCS 13 C-BPA BPA
TR (min)
Quantitation ion (m/z)
Confirmation ion (m/z)
13.89 13.93 14.85 14.85 19.46 19.46
422 414 494 482 628 616
421 415 299 287 418 406
3. Results and discussion 3.1. Method optimization and quality control The derivatization with PFBCl and clean-up of the extract on acidic silica is based on a method described earlier for the analysis of BPA and TCS in serum and urine (Geens et al., 2009). Shortly, the incorporation of five (for TCS and 4n-NP) or ten (for BPA) fluorine atoms transforms the compounds into highly electrophilic derivatives which make them suitable for GC-ECNI/MS analysis. The use of this technique improves the selectivity and sensitivity for these compounds. Moreover, the sensitivity of this method is further increased by the introduction of a clean-up step on acidified silica, which leads to very clean extracts. With this combination of thorough clean-up and derivatization with electrophilic groups detected with ECNI/MS, instrumental LOQs below 1 pg injected could be achieved for all analytes. Since small amounts of the analytes could be detected in blank samples, a method LOQ was applied (see further). A SIM-chromatogram of a spiked adipose tissue sample, a ‘‘real’’ adipose tissue sample is presented in Fig. 1. Chromatograms and baselines were similar for brain and liver tissues. The molecular ion was the most abundant and was used as quantification ion. For BPA and TCS, the ions formed after loss of one derivatization group (M-PFB) were used as confirmation ions (Table 1). Method performance for all tissues and analytes is summarized in Table 2. Recovery of the spiked compounds ranged between 92% and 102%. Within day and between day precision for all compounds is <5% for each tissue. As described earlier, trace amounts of the analytes could be detected in blank samples. This was especially the case for BPA which can come from materials used during analysis, reagents etc. Therefore method LOQs were used and were defined as three times the standard deviation of the blanks. Average levels of procedural blanks were 0.2 ng for BPA, 0.06 ng for TCS and <0.002 ng for n-NP. A chromatogram of a blank sample is presented is given in Fig. 1. BPA levels in the blank samples were relatively constant which resulted in method LOQs of 0.4 ng g1 for adipose tissue or brain and 0.3 ng g1 liver. These values are similar or lower compared to other studies that determined BPA in tissues (Fernandez et al., 2007, Jiménez-Diaz, 2010, Schaefer et al., 2000). For TCS and 4n-NP, blank issues are less prominent, however also method LOQs were applied. For TCS, a LOQ of 0.06 ng g1 for adipose tissue or brain and 0.045 ng g1 for liver was achieved, while for 4n-NP 0.004 ng g1 for adipose tissue or brain and 0.003 ng g1 for liver could be set. Concentrations in tissues below LOQ were replaced by fLOQ (f: detection frequency of a compound in a tissue) for further analysis of the data. 3.2. Concentrations in human tissues Descriptive parameters of the concentrations measured in the different tissues are summarized in Table 3 and Fig. 2. Both adipose tissue and brain are tissues with high lipid content and a minimal Phase II metabolic capacity. For these reasons, BPA, TCS and n-NP
were expected to be present in the more lipophilic, aglycone form and consequently, only the free BPA was measured in adipose tissue and brain. Contrary, the liver is a well-perfused, more hydrophilic organ with an extensive metabolization capacity and therefore both free and total compounds were measured, since an important contribution of the conjugated form was expected. This was already described for BPA in rats by Doerge et al. (2011). Two hours after intravenous administration of BPA to female rats, the tissue distribution and the contribution in terms of percentage of the aglycone form in each tissue were determined. The aglycone form contributed for respectively 96%, 95% and 2.5% to the total BPA concentration in adipose tissue, brain and liver (Doerge et al., 2011). In the present study, total and free concentrations of the determined compounds in liver were almost equal which means that most of the compounds were present in the aglycone form. Since this is in contrast with the findings of Doerge et al. (2011) for BPA, it is probable that deconjugation has taken place during sample storage and sample preparation. Several publications report the release of b-glucuronidase activity from liver and fetal/placental tissues during thawing and cutting of the samples (Moors et al., 2006; Waechter et al., 2007; Doerge et al., 2011). Since no precautions could be taken in the present study to prevent this deconjugation, it is very likely that the measured aglycone concentrations in liver are a result of this release of b-glucuronidase activity. 3.2.1. Bisphenol-A BPA was found with a high detection frequency in all tissues. BPA was detected in all adipose tissue and liver samples, while it could be detected in 8 out of 11 brain samples. BPA concentrations descended from adipose tissue (mean: 3.78 ng g1) over liver (mean: 1.30 ng g1) to brain samples (mean: 0.91 ng g1). As previously mentioned, total BPA in liver (mean: 1.48 ng g1) is only slightly higher than free BPA (mean: 1.30 ng g1), which is probably a result of deconjugation during sample preparation. Until now, few tissue data are available to compare. Fernandez et al. (2007) could determine BPA above detection limit in 11 out of 20 adipose tissue samples from women in Spain. BPA levels ranged between 1.80 and 12.01 ng g1 adipose tissue with mean ± standard deviation of 3.16 ± 4.11 ng g1, results which are in accordance with our results. Schaefer et al. (2000) could not detect BPA in 21 adipose tissue from German women, probably due to a high detection limit of 20 ng g1. Smeds and Saukko (2003) could detect BPA in only 2 out of 13 adipose tissue (14.9 and 47.2 ng g1 lipid weight). However, in the latter study, BPA seemed to be present in various samples, although it could be quantified only in two samples because of overlapping peaks. No literature data could be found about concentrations of BPA in brain or liver tissues. With a log Kow of 3.4, BPA has only a moderate potential for bioaccumulation (Staples et al., 1998; Heinonen et al., 2002). However, some studies demonstrate the preferential partition of BPA into lipohilic tissues, such as adipose tissue, over the hydrophilic compartment. In our study, we found a mean ratio of concentrations in adipose tissue:brain and liver:brain of 5.90 and 1.81, respectively. The median ratios of these concentrations are 3.68 and 1.48, respectively. The mean ratio of concentrations in adipose tissue:liver was 2.76, while the median ratio was 1.90. In an in vitro study, Csanady et al. (2002) determined the partition coefficients of BPA by incubating blood containing BPA with different tissues at 37 °C. The tissue:blood partition coeffiecients for brain, liver and fat were 1.08; 1.46 and 3.31 respectively. Adipose tissue had thus a 3.06 times higher partition coefficient than brain, that of liver was 1.43 times higher than that of brain. These in vitro derived ratios compare well with the ratios we found in the tissues. Nunez et al. (2001) also observed a preferential accumulation of BPA in brown adipose tissue over other tissues in rats exposed to
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A
Abundance
nNP-PFB TCS-PFB
320000 280000
13
C-TCS-PFB
240000 200000 D8-nNPPFB
160000
BPA-PFB
120000
13
C-BPA-PFB
80000 40000 0 14.0
B
14.5
15.0
Abundance 140000
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
13
C-TCS-PFB
120000 100000
D8-nNP-PFB
80000 TCS-PFB
60000 13
C-BPA-PFB
40000 20000
BPA-PFB
0 14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
n-NP 5000
C
Abundance
BPA
4000 3000 2000
TCS
1000
9000
6000 13.85 13.95
8000
5000
4000
7000
4000
3000
6000
3000
5000
2000
2000
4000
1000
1000
3000
0
2000
14.80
14.90
19.40
19.60
15.00
1000 0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
Fig. 1. GC-ECNI/MS chromatogram of a spiked adipose tissue sample (A), an adipose tissue (B) and a procedural blank (C).
BPA. In a study of Doerge et al. (2011), deuterated BPA was administered to female rats by intravenous route and tissue distribution was measured 2 h after dosing, thus during the distribution phase and before elimination substantially reduced the levels. The tissue/ serum ratio for BPA-aglycone was 5.0 for adipose tissue, 2.8 for brain and 0.73 for liver, while the tissue/serum ration for the conjugated-BPA was 0.032; 0.034 and 5.4 for adipose tissue, brain and liver, respectively. Liver, which is richly perfused and has a high Phase II metabolization capacity, had the lowest tissue/serum ratio for BPA-aglycone, whereas adipose tissue with a lipophilic composition, slow perfusion and a low metabolic Phase II capacity had the highest ratio. Brain which has also a low evidence for Phase II metabolism has also a higher tissue/serum ration for BPA-aglycone (Doerge et al., 2011). Comparison of human data with rodent
studies should be interpreted with care since the enterohepatic recirculation which is observed in rats, is not seen in humans. Also the administered dose in animal studies is orders of magnitude higher than the daily estimated human exposure. A significant positive Spearman correlation was observed between BPA concentration in adipose tissue and brain (r = 0.670; p = 0.024); BPA concentration in brain and free BPA in the liver (correlation coefficient r = 0.884; p = 0.001) and between free and total BPA in the liver (r = 0.682; p = 0.021). A significant negative Spearman correlation was found between age and BPA concentration in brain (r = 0.714; p = 0.014); weight and BPA concentration in adipose tissue (r = 0.715; p = 0.013) and weight and BPA concentration in the brain (r = 0.802; p = 0.003). These correlations should be interpreted with care since the limited number of
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Table 2 Recovery of spiked samples in different tissues. Relative standard deviation is given between brackets and n is the number of replicates. Mean (RSD)
Day 1 (n = 3)
Day 2 (n = 3)
Day 3 (n = 3)
Mean day 1–3 (n = 9)
Adipose tissue
BPA TCS 4n-NP
102 (1) 99 (1) 98 (2)
102 (1) 101 (1) 95 (2)
98 (1) 99 (1) 101 (1)
101 (2) 100 (1) 98 (3)
Brain
BPA TCS 4n-NP
99 (4) 100 (3) 97 (1)
102 (3) 99 (4) 94 (2)
95 (1) 100 (1) 100 (3)
99 (4) 99 (3) 97 (3)
Liver free
BPA TCS 4n-NP
98 (3) 95 (2) 92 (3)
102 (3) 98 (1) 95 (1)
97 (1) 96 (2) 96 (2)
99 (3) 96 (2) 94 (2)
Liver total
BPA TCS 4n-NP
97 (3) 95 (1) 94 (2)
102 (3) 98 (1) 96 (2)
97 (1) 100 (2) 102 (1)
99 (3) 97 (2) 98 (4)
Table 3 Descriptive parameters of BPA, TCS and 4n-NP in the different tissues. Concentrations (ng g1 ww)
a b c d e
Adipose tissue (free)
Brain (free)
Liver (free)
Liver (total)
BPA
Range Median Mean Detection frequency
1.12–12.28 2.09 3.78 11/11
0.77–3.35 1.01 1.30 11/11
0.90–2.77 1.03 1.48 11/11
TCS
Range Median Mean Detection frequency
4n-NP
Range Median Mean Detection frequency
0.008–0.208 0.023 0.046 11/11
LOQ = 0.4 ng g1. LOQ = 0.06 ng g1. LOQ = 0.045 ng g1. OQ = 0.004 ng g1. LOQ = 0.003 ng g1.
samples analyzed and therefore no conclusions can be linked to these observations. Fernandez et al. (2007) observed a significant Spearman correlation coefficient between age and BPA concentrations in adipose tissue. Such correlation was not found in the present study. 3.2.2. Triclosan TCS was detected in almost all liver (10/11), most adipose tissue (7/11) and only in one brain sample. One outlier (subject 1) was observed with concentrations of 3.92 ng g1 in adipose tissue, 0.23 ng g1 in brain and 29.03 ng g1 total TCS in liver. For calculation of mean concentrations and statistics, this outlier was removed. Contrary to BPA, TCS had the highest concentrations in liver (mean: 0.44 ng g1), followed by adipose tissue (mean: 0.28 ng g1) and brain (all
3.2.3. 4n-Nonylphenol In most subjects and tissues, concentrations for 4n-NP were <0.1 ng g1 and thus much lower levels compared to BPA and TCS. Similar to TCS, the highest concentrations of 4n-NP were found in liver, followed by adipose tissue and brain. Naassner et al. (2002) reported that the technical NP mixture does not contain 4n-NP. On the other hand, Calafat et al. (2005) mentioned nNP is a small percentage of the technical mixture and they could detect n-NP in low concentrations in 51% of 394 urine samples with a median value <0.1 ng mL1. Also Kuklenyik et al. (2003) could not detect n-NP in 30 urine samples from people who used paint products. Nonyl-phenol is a hydrophobic compound with a log Kow value of 4.48 and low solubility in water, therefore it partitions favorably to organic matter (Soares et al., 2008). Three previous papers reported the detection of NP in adipose tissue. Müller et al. (1998) detected NP in 25 adipose tissues from Switzerland with a median concentration of 37 ng g1 (range 20–84 ng g1). However, these concentrations were within the range of the background contamination found in the method blanks. They also mentioned, based on the pharmacokinetic data of their study, that adipose tissue levels are probably a factor of 50 lower than those background levels. Another study in Spain detected NP (Lopez-Espinosa et al., 2009) in all 20 samples with a median concentration of 57 ng g1 (range 10–567 ng g1). However in this study, background contamination is not mentioned. Very recently, Ferrara et al. (2011) reported NP at mean concentration of 122 ng g1 (range 10–226 ng g1) in 16 Italian patients undergoing bariatric
BPA concentration (ng/g)
T. Geens et al. / Chemosphere 87 (2012) 796–802
BPA
14 12
Fat
10
Brain
8
Liver Total
6 4 2 0
1
2
3
4
5
6
7
8
9
10
11
Subject TCS
Concentration (ng/g)
5.0 4.0
Fat
3.0
Brain Liver Total
2.0 1.0 0.0
1
2
3
4
5
6
7
8
9
10
11
Subject
Concentration (ng/g)
nNP 0.6 0.5
Fat
0.4
Brain
0.3
Liver Total
0.2 0.1 0
1
2
3
4
5
6
7
8
9
10
11
Subject Fig. 2. Individual concentrations (ng/g tissue) of BPA, TCS, and 4n-NP in adipose tissue, brain and liver. ‘‘Liver total’’ represents the sum of concentrations of conjugated and unconjugated compounds. For subject 1, total TCS was respectively 29.0 ng g1 and total 4n-NP in liver was 1.40 ng g1 (outside scale of Y-axis).
surgery. In these studies, no details were reported about the isomeric composition of NP. Given the high concentrations, it may be assumed that the sum of branched and unbranched NP was measured and not only 4n-NP. Indeed, we have observed in our ion chromatograms several peaks with the same ratios between ions m/z 414 and 415 (specific for NP). They eluted earlier than 4n-NP and thus we presumed that they were branched NPs. In most cases, they had higher signals than 4n-NP. Since we did not had any other standards than 4n-NP, these (presumably) NP peaks were not quantified. 3.3. Critical discussion of limitations and potential sources of errors Overall, concentrations of the studied phenolic compounds are relatively low, even in adipose tissue. The bioaccumulation of BPA was recently suggested (Stahlhut et al., 2009), but seems not very plausible given these low concentrations and its low persistence characteristics. The detectable levels of free BPA in adipose tissue and brain may seem in contrast with the findings of different studies showing the fast biotransformation and elimination after oral exposure in humans (Völkel et al., 2002, 2005; Teeguarden et al., 2011) and after oral and intravenous exposure in rhesus monkeys (Doerge et al., 2010). After oral administration of 100 lg/kg bw/d, the circulating concentration in serum of free BPA was less than 1% in both humans and monkeys, which reflects
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the extensive Phase II metabolism in gut and liver. Five minutes after intravenous administration in monkeys, only 29% of BPA was still present as aglycone. BPA aglycone showed a large volume of distribution probably reflecting the distribution of lipophilic parent compound out of the circulation into tissues where it is further conjugated before elimination. However, similar elimination half-times for BPA aglycone and total BPA suggested a fast return to the central compartment from the tissues (Doerge et al., 2010). To obtain the BPA levels measured in this study, the current pharmacokinetic models in humans and monkeys (Völkel et al., 2002, 2005; Doerge et al., 2010; Teeguarden et al., 2011) predict that this can only occur after an oral exposure of approximately 200–1300 lg/kg bw/d. This exposure is orders of magnitude higher than the estimated exposure based on urinary measurements of conjugated BPA (Doerge et al., 2010). However, the hypothesis that BPA can distribute to human tissues after non-oral exposure (such as dermal exposure), before biotransformation has occurred, need further investigation (Teeguarden et al., 2011). Since the results of BPA found in this study partly disagree with the fast elimination of free BPA, it cannot be ruled out that the measured concentrations of BPA are a result of external contamination during sample collection and storage. Despite the great care taking to avoid contamination during chemical analysis, the collection of tissue samples in a hospital setting is in general less controlled. The external contamination with BPA in hospital settings was recently described by Vandentorren et al. (2011). In that study, high free BPA levels were observed in urine of women who gave birth by a caesarean section. These high values of free BPA were dedicated to contamination with BPA from exogenous sources during sample collection. The authors suggested that contamination occurred from medical devices either from catheterization or urine probes when biomonitoring at delivery (Vandentorren et al., 2011). Only the aglycone compounds were measured in brain and adipose, while in liver both free and total compounds were measured. As described earlier, it is highly plausible that free BPA in the tissues is a result of deconjugation of the conjugated metabolites by the release of b-glucuronidase. This hypothesis is backed up by the similarly high proportion of free TCS over the total TCS observed in the liver. Furthermore, the free BPA concentrations measured in adipose and brain were very different from those measured in liver, which exclude partly the possibility that samples were contaminated with BPA during sample collection/ storage. Due to this possible deconjugation, it is of limited value to determine both total and free BPA. Moreover this makes more difficult to interpret whether the measured free concentrations are a result of internal exposure, external contamination or deconjugation. Tissue concentrations of 4n-NP were extremely low since, as earlier described, 4n-NP is a minor component of the NP-mixture and therefore it is unimportant for biomonitoring studies.
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