* Institute for Risk Assessment Sciences (IRAS), Utrecht University, Yalelaan 2, 3584 CM Utrecht, the Netherlands; Biodetection Systems B.V., 1031 CM Amsterdam, the Netherlands;
National Institute of Public Health and the Environment (RIVM), 3720 BA Bilthoven, the Netherlands
Received April 30, 2004; accepted July 26, 2004
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ABSTRACT |
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Key Words: displacement; serum proteins; estrogen; charcoal assay.
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INTRODUCTION |
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Furthermore, it has been reported that mixtures of compounds can also influence the availability of the individual compounds. Both Danzo (1997) and Déchaud et al. (1999)
found that xenoestrogens can displace the endogenous steroids dihydrotestosterone (DHT), testosterone, and estradiol, from SHBG. This displacement might disrupt the normal hormone balance, forming a pathway of endocrine disruption alternative to the receptor-binding pathway. However, SHBG is only a minor binding protein for estrogens in serum. Whether the displacement of endogenous steroids by xenobiotics can occur in whole (human) serum, or on other serum proteins, is still a question. Villeneuve et al. (2002)
found no displacement of estradiol or testosterone from carp serum proteins by polybrominated diphenyl ethers (PBDEs). In contrast, Ishihara et al. (2003)
found interference of endocrine disrupting compounds such as diethystilbestrol and pentachlorophenol with the binding of a thyroid hormone with the plasma protein transthyretin.
The possibility that xenobiotics can compete for binding sites on serum proteins and cause an increase of the free concentration of very potent endogenous estrogens is of great interest for both the in vitro assay results and the prediction of risk to humans. Humans contain a large reservoir of bound endogenous estrogens and naturally have many of the binding places on the serum proteins already occupied by endogenous compounds. Exposure to a mixture of xenobiotics could therefore theoretically lead to a ready displacement of endogenous estrogens.
Competition may also occur in in vitro tests. Most in vitro estrogenicity assays apply a small percentage of serum in the culture medium of the cells. This serum is usually stripped with charcoal, to remove the majority of the endogenous estrogens. Our hypothesis, which formed the basis for this study, is that a small amount of protein-bound estradiol, remaining after the stripping procedure, can be displaced by a test compound in the in vitro assay. This could cause an estrogenic response that is then not directly attributable to the test compound. The high dosages necessary for most reported xenoestrogens to produce an effect support this possibility of displacement: a high dose may saturate the serum proteins, leading to competition.
We have investigated this hypothesis. First, we have determined how much estradiol remains in serum after the common stripping procedure with charcoal. Further, a mathematical exposure model was formulated to predict the occurrence of competition and displacement, and this model was validated with a series of competition experiments in 98.5% serum, where free concentrations were measured using nd-SPME, a method developed by Vaes et al. (1996). Lastly, experimental and calculation results were compared for the occurrence of competition in an in vitro estrogenicity assay with 5% serum. The combination of modeling and experimental work is extremely useful for generation and exploration of hypotheses and for interpretation of the outcome of experiments.
Besides its application in the stripping of serum, charcoal is also commonly used to separate bound and free ligands in protein binding studies as performed in this work. These studies mainly include receptor-binding studies [e.g., Campbell and Clark (1984); EORTC (1973)
; Skovgaard Poulsen (1982)
], but charcoal is also applied as a separation method in binding studies with serum proteins [e.g., Danzo (1997)
; Tollefsen (2002)
; Villeneuve et al. (2002)
], including studies on competition. Pitfalls and sources of variability of this method have already been reported (Campbell and Clark, 1984
; Peck and Clark, 1977
; Pettersson et al., 1985
; Skovgaard Poulsen, 1981
; Thorpe, 1987
), and the absorption time of the charcoal appears to be critical (Laidley and Thomas, 1994
; Peck and Clark, 1977
). As the affinities for serum proteins are generally much lower than those for receptors, dissociation rates will be faster for these proteins. Thus, the charcoal will start to adsorb dissociated ligands sooner in the case of serum proteins than in the case of receptors. As we had doubts concerning the efficiency and removal rate in the charcoal assay, we verified whether only the free fraction is removed by charcoal in serum protein solutions.
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MATERIALS AND METHODS |
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Cell culture. Human embryonal kidney 293 (HEK293) cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and cultured as described by Lemmen et al. (2002).
DCC-stripping of serum. A dextran-coated charcoal (DCC) slurry with 0.5% charcoal (Norit SA-3; Boom, Meppel, the Netherlands) and 0.05% dextran (Sigma) in 50 mM Tris buffer (pH 7.4) was prepared and left to stir overnight at 4°C. Fetal calf serum was heated to 56°C for 30 min in a water bath. A volume of slurry similar to the volume of serum to be stripped was centrifuged for 10 min at 1500 x g (Megafuge 1.0 of Heraeus Sepatech, Osterode/Harz, Germany). The supernatant was discarded and the FCS was added to the remaining pellet. This slurry was gently shaken for 45 min in a water bath of 45°C, and subsequently centrifuged for 20 min at 1500 x g. The supernatant FCS was then transferred to fresh DCC pellets and the stripping procedure was repeated. The resulting supernatant FCS was filtered over a 0.2 µm filter.
Analytical procedure. [3H]estradiol in water samples and PA SPME fibers was extracted with scintillation liquid and analyzed as described in Heringa et al. (2002). Total [3H]estradiol concentrations were derived from the water samples by calibration with a series of known amounts of [3H]estradiol dissolved in scintillation liquid. Free concentrations were derived from the concentrations in the fiber (Cf, determined using the same calibration series) using equation 1.
![]() | (1) |
Octylphenol was extracted from PDMS SPME fibers and water samples with ethylacetate. The ethylacetate extracts were derivatized with MTBSTFA and spiked with two internal standards (PCB #138 and TCP) and then analyzed on a Varian GC-MS as in Mol et al. (2000). Total concentrations were derived from the water samples by calibration with a series of known concentrations of octylphenol. Free aqueous concentrations were derived from fiber measurements by a separate calibration series of fiber concentrations sampled in known free aqueous concentrations.
Mathematical model for competition. A mathematical model was formulated to predict potential competition in serum, to identify the critical factors, and to support our experimental set-up. The essence of this model was formed by the mass balances given in equation 2 (see Appendix A for their derivation). In this equation, A, S, E, and X are albumin, SHBG, estradiol, and competitor concentrations, respectively, with the subscripts free and total indicating free or total concentrations. Two letters together, such as EA, denote the concentration of the complex of the specified compound with the specified protein, in this case estradiol bound to albumin. Ka is the affinity constant, with the capital letters in the subscript indicating for which compound and protein this constant is applicable. The model included both albumin and sex-hormone binding globulin (SHBG) as binding proteins, as practically all binding of estradiol (and probably also xenoestrogens) in serum is accountable to these two proteins (Dunn et al., 1981).
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Applicability of charcoal assay for albumin or serum binding. In this experiment, we tried to determine at what absorption time the charcoal starts adsorbing more than the free fraction of ligand in serum protein solutions. A solution of 1.4 x 109 M [3H]estradiol (2.627 TBq/mmol; >90% radiochemical purity) and 5.01 x 104 M BSA (Sigma; 98% pure, essentially fatty-acid free, essentially -globulin free) in 50 mM Tris buffer (pH 7.4) and one of 7.0 x 109 M [3H]estradiol in 100% DCC-FCS was prepared and left to equilibrate at 4°C for 2 h and overnight, respectively.
Twenty-four 1.8-ml vials, containing a pellet from 1.6 ml DCC-slurry, were filled with 1.6 ml of the BSA solution and shaken. After adsorption times ranging from 30 s to 27 h, duplicates of the vials were centrifuged at 1500 x g for 2 min, after which 100 µl of supernatant was quickly transferred to a counting vial and analyzed. Two empty vials were also filled with 1.6 ml BSA solution (controls), from which 100 µl was counted and the rest was subjected to 33 min of nd-SPME (Heringa et al., 2002). These experimental procedures were performed at room temperature (20° ± 2°C). Total concentrations of [3H]estradiol in the stripped samples were divided by those of the controls and set out against the DCC adsorption time. From the fiber measurements, the free fraction of [3H]estradiol in the controls was calculated.
A similar procedure was followed for the FCS, apart from the batch of [3H]estradiol (2.627 TBq/mmol; >95% radiochemical purity) and the DCC adsorption times, which ranged from 20 s to 7 h. Further, three controls were prepared instead of two, and the centrifugation time was only 1 min. The experiment with FCS was repeated at 4°C, to study the effect of temperature on adsorption speed.
Calibration and validation of competition model. Three competitive binding assays in 98.5% serum were performed with three competitors for estradiol, to calibrate and validate the competition model. For each assay, 11.7 µl of [3H]estradiol stock solution (2.627 TBq/mmol,; >97% radiochemical purity) was added to 23.0 ml of FCS. After mixing, 1.576 ml of this mixture was transferred to 1.8-ml vials and 16 µl of a dilution of the competitor in ethanol was added to obtain total competitor concentrations of 1.0 x 109, 1.0 x 108, 1.0 x 107, 1.0 x 106, 1.0 x 105, and 1.0 x 104 M in duplicate, and a [3H]estradiol concentration of 7.2 x 109 M (physiological human concentration according to Saeki et al. (1991)). After equilibration for at least 2 h, 100 µL was sampled from each of the 12 vials and counted, and the remaining mixtures were subjected to nd-SPME for 2 h as in Heringa et al. (2002)
. The competitors studied were bisphenol-A (Sigma), ß-endosulfan (98.7%, Riedel-de Haën), and non-labeled 17ß-estradiol (98%, Sigma). The free fraction of [3H]estradiol was plotted against the nominal concentration of the competitor. The model was manually fitted to the data of the experiment with non-labeled estradiol, by adjusting only Ka,SE, Atotal, and Stotal, because the values for these parameters were uncertain. The resulting calibrated model was then compared to the data of the experiments with bisphenol-A and endosulfan for validation.
Competition of octylphenol in in vitro assay with cells. This experiment was performed to determine whether the response of octylphenol in an in vitro estrogenicity assay could be caused by displacement of a low concentration of estradiol, for example the estradiol remaining in serum after stripping. The in vitro estrogenicity assay chosen as an example assay was the estrogenicity reporter gene assay with ERß-stably transfected 293 HEK cells as described by Lemmen et al. (2002). The cells were plated in four 24-well tissue culture plates (Corning Inc., Corning, NY) with 80,000 cells and 200 µl suspension per well. After 48 h, the medium was replaced by 2.5 ml/well of fresh phenol-redfree medium containing 5% DCC-FCS and the desired concentrations of [3H]estradiol (3.52 TBq/mmol; >94% radiochemical purity) and/or octylphenol. Doseresponse curves of estradiol alone, octylphenol alone, and octylphenol with a low amount of estradiol were created. A concentration of 1 x 1011 M estradiol was chosen for the combination assay, as this was the lowest concentration at which the free fraction could be detected.
Plate-lids containing SPME fibers were then placed on all plates and the plates were placed in the incubator. Exactly 24 h later, the lids with fibers were removed and the fibers were processed as described above to measure free concentrations of estradiol and octylphenol. Samples of the culture medium were taken and analyzed as described above to obtain total estradiol and octylphenol concentrations. From these two parameters, the free fractions were calculated.
The estrogenic response (luciferase activity) of the cells in plates IVVII was then analyzed as described by Lemmen et al. (2002). Free fractions of [3H]estradiol were plotted against the nominal octylphenol or estradiol concentration and compared with the prediction of the competition model. Values for the parameters needed in the model were taken from Table 1, except for Stotal and Ka,SE, which were obtained from the model validation. For Atotal, the minimum and maximum values from Table 1 were both modeled, as this parameter is uncertain and at the same time very sensitive for the outcome.
Cell responses (E) were plotted against the logarithm of the nominal concentration and equation 3 (a four-parameter logistic equation, allowing for a variable slope) was fitted through the data points to obtain the EC50 value of the doseresponse curves.
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Precautions. National guidelines for handling and disposing of radiolabeled compounds and genetically modified cell cultures were followed.
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RESULTS |
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DISCUSSION |
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These results indicate that roughly 1% of the initially present estradiol remains in the serum, which corresponds to the results of Vignon et al. (1980). As the physiologic concentration of unconjugated estradiol in serum of fetal calves is 50130 pM (Peterson et al., 1975
), stripped FCS will contain 0.51.3 pM of unconjugated estradiol. If all of this remaining estradiol were displaced from serum proteins by other compounds, this would not be enough to produce a distinct estrogenic response in luciferase reporter gene assays (Lemmen et al., 2002
), the E-SCREEN (Soto et al., 1995
), or the YES (Arnold et al., 1996
).
However, serum also contains a 1000-fold larger concentration of sulfated estradiol: 170 nM in FCS (Fairclough et al., 1976). Sulfated estradiol can readily be taken up by cells and transformed to the unconjugated form, producing estrogenic effects (Vignon et al., 1980
). The stripping efficiency of these conjugates from serum with DCC could be much less than that of unconjugated estradiol, as Vignon et al. (1980)
found that 1015% of sulfated estrone (another endogenous estrogen) remains after the general stripping procedure. Assuming that sulfated estradiol is stripped with the same efficiency, 1725 nM of estradiol sulfates would remain in DCC-stripped FCS. In fact, Vignon et al. (1980)
measured 22.5 nM of total estrogen in DCC-stripped FCS. If only 1% of these sulfates were displaced from proteins and hydrolyzed by cells, that 1% would already be enough to cause an estrogenic effect in all three assay-types mentioned above.
Applicability of Charcoal Assay for Albumin or Serum Binding
Laidley and Thomas (1994) found a comparable effect of time on absorption of [3H]testosterone from diluted trout plasma: remaining testosterone decreased by a factor of two when the charcoal adsorption time was increased from 1 min to 2 min. However, they did not measure the total or free amount of testosterone in the control (i.e., before charcoal adsorption), as we did, so it is unclear how much of the bound fraction was adsorbed in their experiment.
Our data clearly show that the DCC treatment for removing the free fraction in binding studies with serum proteins requires caution. Further evaluation is necessary for definititive conclusions, including studies with proteins that have different dissociation rates. For example, the low affinity for albumin (8.9 x 104 M1), in comparison with the estrogen receptor (110 x 109 M1 (Kuiper et al., 1997, 1998
)), leads to a faster release of albumin-bound estradiol than receptor-bound estradiol. It would also be interesting to verify whether the results of serum binding assays using charcoal deviate from the same studies using a different separation technique. In the meantime, we prefer nd-SPME for binding and competition studies.
Calibration and Validation of Competition Model
The albumin binding appears to be the most significant factor in the occurrence of competition, which is logical, because estradiol is easily displaced from this protein as a consequence of its relatively low affinity for it; in addition, albumin has a large portion of the estradiol bound to it. This means that the possible displacement of endogenous hormones from SHBG by xenobiotics (Danzo, 1997; Déchaud et al., 1999
; Meulenberg, 2002
) has no significant consequence for the overall displacement in serum: the small amount of estradiol that can be released by SHBG will instantly be bound by the vast number of available albumin molecules.
The model appears to predict the free fractions quite well, but the turning point (where competition starts) was difficult to validate, because of the concentration limits imposed by the solubility of the competitors. The discrepancy between the model output and the experimental data could be caused by a possible substantial deviation of the estimates for the affinity constants of bisphenol-A and endosulfan for albumin (see Table 1) from the true values. Furthermore, the discrepancy could be related to the presence of other binding proteins in the serum, which are not included in the model. Serum is known to contain other binding proteins, such as 1-acid glycoprotein and corticosteroid-binding globulin (CBG) (Englebienne, 1984
). Moreover, FCS contains
-fetoprotein (Abe et al., 1976
), which is similar to albumin (Dobrila, 1996
). Lastly, the competitors may be able to bind to more than one binding site on albumin, whereas only one binding site is assumed. Further validation and also further expansion of the model, including other binding proteins and ligands, would be valuable.
The measurements show that estrogenic effects due to competition in 100% serum, as in the in vivo situation, are not likely to occur below a total xenoestrogen concentration of 1 x 104 M in the serum. This is similar to the finding of Milligan et al. (1998) that displacement from human and fish plasma will occur only at very high concentrations of estrogenic agents. It is doubtful whether such a concentration will be reached, as literature values for human blood levels of xenoestrogens are, for example, 9 nM bisphenol-A (Ikezuki et al., 2002
), 0.4146 nM p,p'-DDE (Krstevska-Konstantinova et al., 2001
), and 2.8 nM hexachlorobenzene (Charlier et al., 2003
). However, considering that all the endogenous compounds also occupy binding sites in vivo, the sum of all the exogenous compounds might rise to a high enough level to cause competition. Competition in vivo is therefore still of interest in the field of endocrine disruption.
Competition of Octylphenol in In Vitro Assay with Cells
Again, the model predictions on the concentration of octylphenol at which competition occurs and the actual measurements differ. An explanation additional to those discussed above, is that it is imaginable that octylphenol can bind to the fatty acid binding sites of albumin, as the structure of octylphenol is quite similar to that of a fatty acid. If this indeed happens, then saturation of the estradiol binding sites will occur at a higher concentration of octylphenol than the model predicts.
We do not know the reason for the underestimation of the free fractions of estradiol in Figure 5. As the model predicts the free fractions of octylphenol quite well (results not shown), the lack of evaporation, cell membrane partitioning, or binding to other proteins by octylphenol in the model does not seem to matter much. Also, in the validation experiment, the estradiol-data (Fig. 2) could be predicted quite nicely, giving the impression that the model also describes well the relevant processes for estradiol. In Figure 3, however, the measured free fraction of [3H]estradiol is different in the two plots, which theoretically should be the same. Additionally, repetition of the experiment leading to Figure 5 resulted in measured free fractions of estradiol that varied per culture plate, sometimes leading to a good prediction of the free fraction of estradiol by the model. Therefore, the cause of the underestimation in Figure 5 must be sought in the measurements.
As there appears to be more sulfated than unconjugated estradiol in stripped serum, the question arises of whether the sulfates might still be subjected to competition at relevant doses of xenoestrogen. Inclusion of estradiol sulfate in the competition model, with an albumin affinity constant of 5 x 105 M1 (Rosenthal et al., 1975) and a very low SHBG affinity constant [1 x 102 M1, as SHBG does not appear to be involved in the conjugate binding (Rosenthal et al., 1975
)], shows that displacement of the sulfates will not occur at lower competitor concentration than the displacement of unconjugated estradiol (Fig. 7A). However, Figure 7B shows that when concentrations of octylphenol are added that give an increasing cell response, then the free concentration of the sulfates also increases to levels at which estradiol gives an increasing response, This could indicate that the response seen at 1 x 106 to 1 x 105 M of octylphenol might be due (in part) to released estradiol sulfate. However, as this is the outcome of the competition model that has been shown to predict the start of displacement too early for all our experiments, caution is necessary with these calculation outcomes.
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APPENDIX A. MODEL DERIVATION |
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![]() | (A.1) |
![]() | (A.2) |
![]() | (A.3) |
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![]() | (A.4) |
![]() | (A.5) |
![]() | (A.6) |
Lfree is calculated first by the modeling software, using the lower formula of equation A.6 (for example by the GUESS...ROOTS function in Berkeley Madonna). With the obtained Lfree, the upper formula of equation I.6 can be used to calculate Ka or any other desired parameter.
In general, if there are n different ligands Li, i = 1, ..., n, which bind with binding association constant Ka,ij to m different proteins Pj, j = 1,..., m, the mass balances become:
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![]() | (A.8) |
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REFERENCES |
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Rowland, M., and Tozer, T. N. (1995). Clinical Pharmacokinetics, Willimas & Wilkins, Media (Philadelphia).
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APPENDIX B. COMPETITION MODEL SCRIPT |
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METHOD RK4
RENAME TIME = logXtot ; is log of total competitor concentration (M)
RENAME STARTTIME = logXtot0
RENAME STOPTIME = logXtotf
RENAME DTMIN = dmin
RENAME DTMAX = dmax
RENAME DTOUT = dout
logXtot0 = 10.0
logXtotf = 0.0
dmin = 1.e6
dmax = 1.
dout = 0.01
Ka,AE = 8.9e+4 ; is affinity constant of estradiol for albumin (M-1)
Ka,AX = 8.9e+4 ; is affinity constant of competitor for albumin (M-1), ;default is estradiol
Ka,SE = 1.0e+8 ; is affinity constant of estradiol for SHBG (M-1)
Ka,SX = 1.0e+8 ; is affinity constant of competitor for SHBG (M-1), ;default is estradiol
Atot = 1.8e5 ; is total albumin concentration (M)
Stot = 3.0e8 ; is total SHBG concentration (M)
Etot = 7e9 ; is total estradiol concentration (M)
ffE = Ef/Etot ; is free fraction of estradiol
ffX = Xf/Xtot ; is free fraction of competitor
ffA = Af/Atot ; is free fraction of albumin
ffS = Sf/Stot ; is free fraction of SHBG
fbE = (EtotEf)/Etot ; is bound fraction of estradiol
fbX = (XtotXf)/Xtot ; is bound fraction of competitor
Xtot = 10.00**logXtot
Af = (1.0(Ka,AE*Ef+Ka,AX*Xf)/(1.0+Ka,AE*Ef+Ka,AX*Xf))*Atot ; is free conc. of albumin (M)
Sf = (1.0(Ka,SE*Ef+Ka,SX*Xf)/(1.0+Ka,SE*Ef+Ka,SX*Xf))*Stot ;is free conc. of SHBG (M)
GUESS Ef = Etot/2. ; is free concentration of estradiol (M)
GUESS Xf = Xtot/2. ; is free concentration of competitor (M)
ROOTS Ef = (1.0+Ka,AE*Atot/(1.0+Ka,AE*Ef+Ka.AX*Xf)+Ka,SE*Stot/(1.0+Ka,SE*Ef+Ka,SX*Xf))*EfEtot
ROOTS Xf = (1.0+Ka,AX*Atot(1.0+Ka,AE*Ef+Ka,AX*Xf)+Ka,SX*Stot/(1.0+Ka,SE*Ef+Ka,SX*Xf))*XfXtot
LIMIT Ef> = 0.
LIMIT Ef< = Etot
LIMIT Xf> = 0.
LIMIT Xf< = Xtot
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at IRAS, Yalelaan 2, 3584 CM Utrecht, the Netherlands. Fax: +31-302535077. E-mail: j.hermens{at}iras.uu.nl.
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