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0013-7227/79/1045-1442$02.00/0 Endocrinology Vol. 104, No. 5 Copyright © 1979 by The Endocrine Society Printed in U.S.A.

Diethylstilbestrol and Binding to Serum Albumin and Plasma of Rat and Human*

DANIEL M. SHEEHAN AND MARK YOUNG Department of Health Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, 72079; and the Department of Biochemistry, University of Arkansas Downloaded from https://academic.oup.com/endo/article/104/5/1442/2592405 by guest on 30 September 2021 for Medical Science, Little Rock, Arkansas 72201

ABSTRACT. The equilibrium binding of with a-fetoprotein. This was verified by Scatchard plots of DES (DES) and 17/?-estradiol (E2) to plasma proteins has been char- and E2 binding to rat and human pregnancy plasma. High acterized. DES exhibits a 10- to 20-fold greater binding affinity affinity, low capacity binding was demonstrated with E2 but not index for bovine serum albumin and rat plasma than E2. As with DES. The significantly lower binding of DES suggests that expected, E2 gave high values for binding to plasma from preg- increased delivery of DES to the fetus may be at least partially nant mice or rats, reflecting the presence of a-fetoprotein. DES responsible for the transplacental toxicity and carcinogenicity of bound to these samples as it did to bovine serum albumin and DES. (Endocrinology 104: 1442, 1979) rat plasma. These results suggested that DES interacts weakly

HE INTERACTION of hormones with blood pro- Materials and Methods Tteins is a significant determinant of hormonal po- tency. Such interactions influence , , E2 was obtained from Research Plus Laboratories and availability to target tissues (See Refs. 1-3 for re- (Denville, NJ) and was used without further purification. DES, from Sigma Chemical Co. (St. Louis, MO), was essentially 100% views). Diethylstilbestrol (DES), a nonsteroidal , pure, as judged by high pressure liquid chromatography and is one of the more socially and economically important temperature-programmed gas chromatography. Labeled hor- synthetic hormones. It has been used extensively to 3 3 mones, [6,7- H]E2 (42 Ci/mmol) and [monoethyl-l- H]DES increase in cattle (4) and for treatment of (62.4 Ci/mmol), were obtained from New England Nuclear menopausal symptoms (5), postmenopausal can- (Boston, MA) and were repurified by Sephadex LH-20 chro- cer (6), and prostatic carcinoma (7). However, an asso- matography every 2 months to insure radiochemical purity (11). ciation between the administration of DES to pregnant The solvent system was benzene-methanol with ratios of 85:15 3 3 women and a marked increase in the occurrence of en- for [ H]E2 and 80:20 for [ H]DES. Sephadex (TM) G-25 (coarse) dometrial carcinoma in their offspring has been reported was obtained from Pharmacia (Sweden). All other chemicals (8). Additionally, a considerable body of evidence has were reagent grade or better. Blood was collected in heparinized tubes and centrifuged, accumulated from animal studies demonstrating a vari- and the plasma was stored in aliquots at —20 C. Rat and mouse ety of toxic (including carcinogenic) effects of DES (9, pregnancy plasma was collected from animals in the third 10). trimester of pregnancy. Human pregnancy plasma was obtained Despite these observations, the intraction of DES with from women in labor. Bovine serum albumin powder (fraction plasma proteins has not been well defined. Consequently, V; BSA) was supplied by ICN Pharmaceuticals (Irvine, CA). the possible role of such interactions in modulating the Protein determinations were carried out by the method of normal and toxic actions of DES is unclear. The results Lowry et al. (12) using BSA as the standard. described here demonstrate large differences in the bind- The binding of radiolabeled hormones to plasma or BSA was 3 ing of DES and 17/?-estradiol (E2) to serum albumin, measured by a equilibration technique (13). Briefly, [ H]E2 3 plasma and the pregnancy-associated proteins, rat a-fe- or [ H]DES (about 100,000 cpm) was added to 200 mg Sephadex toprotein (aFP), and human -estradiol-bind- G-25 (preswollen in 1 ml 0.155 M sodium phosphate, pH 7.4) in 1.5 X 4.5-cm glass vials. Other components, including protein ing globulin (TEBG). and nonradioactive carrier, were added to give a final B Received June 21,1978. volume of 2.0 ml. The partitioning of DES and E2 (5 X 10~ M Address requests for reprints to: Dr. Daniel M. Sheehan, National to about 2 X 10"10 M) in the absence of protein was independent Center for Toxicological Research, Jefferson, Arkansas 72079. of hormone concentration. At least three such determinations * This work was presented in part at the Third Annual National Research Center for Toxicological Hormone Research Symposium, were included in every experiment. After incubation for 1 h at November 15-17,1976, Little Rock, AR. 22 C with gentle shaking, the samples were incubated undis-

1442 E2 AND DES BINDING TO PLASMA PROTEINS 1443

turbed for 30 min to allow complete settling of the Sephadex. Results Duplicate aliquots (200 /xl) were then dissolved in Scintiverse (Fisher Scientific, Pittsburgh, PA), and the samples were Initial determination of the equilibrium binding of E2 counted in a Packard Tricarb liquid scintillation counter. The and DES to BSA and various plasmas were carried out amount of unbound (Su) and bound (Sb) hormone was calcu- by the gel equilibration method described by Pearlman lated via a computer program. In this assay, hormone partitions and Crepy (13). In this procedure, a fixed amount of between the external and internal volumes of the Sephadex, steroid is titrated with an increasing concentration of while protein is restricted to the external volume. The amount of hormone in the external volume above protein-free controls protein. The reciprocal of the protein concentration at is a measure of binding. The work of Pearlman and Crepy (13) which the Su:Sb ratio is unity is determined at several provides details of the theory and data reduction for this assay. input steroid concentrations (Fig. 1). The data derived

from a group of such experiments are then plotted, as Downloaded from https://academic.oup.com/endo/article/104/5/1442/2592405 by guest on 30 September 2021 shown in Fig. 2. The lines for the binding of E2 to pooled adult rat plasma, 1-month-old female rat plasma, and 10 Sb=l.O»lO M BSA show no apparent concentration dependence, and a binding affinity index (value for the reciprocal of the protein concentration at Su:Sb = 1) of about 1. These data indicate that normal plasma and BSA possess only one class of low affinity, high capacity sites for E2. DES, however, shows a significantly higher affinity (10- to 20- I/P (I/O) fold) than E2 for these samples. Additionally, the slight concentration dependence of binding suggests the possi-

Sb = I.ii x io« M bility of site heterogeneity. I/P=O.«S L/C The binding of E2 and DES to rat and mouse preg- nancy plasma was also investigated. In addition to albu- min, these plasmas are known to contain aFP, which has high affinity for E2 (14-18). Such high affinity sites are demonstrated by the significant slope of E2 binding to 71 pregnancy plasmas (Fig. 2). However, the binding of DES I/P (I/O) to these samples was identical to the DES binding to

FlG. 1. Calculation of binding affinity for E2 binding to plasma. Plasma BSA and plasma from nonpregnant animals. Thus, DES from 20-day-old female rats was titrated against fixed concentrations of does not appear to bind with high affinity to rodent aFP. 3 [ H]E2. The input E2 concentrations in panels A-E were, respectively, The low affinity binding observed with DES and BSA 1.18 x 1(T9, 1.12 x :i(T8,1.01 X 10~7,1.00 x 10"6, and 1.00 x 10~5 M. The concentration of Sb at an Su:Sb ratio of 1 as well as the value for the was further characterized by Scatchard (19) analysis (Fig. reciprocal of the protein concentration (1/P) required to achieve that 3). At least two classes of low affinity, high capacity sites 6 6 ratio is shown in each panel. on BSA [Kdi = 4 x 10~ M (ih = 3) and Kd2 = 9.5 x 10"

100

FIG. 2. The binding of E2 and DES to various plasma samples and BSA. Values of the reciprocal of the protein concen- 10 tration (1/P) and the concentration of bound steroid at Su:Sb = 1 were deter- mined, as described in Fig. 1, for each of the samples. The data are plotted on a log-log scale, according to Pearlman and Crepy (12). Pooled adult rat plasma: E2 (O), DES [not determined (N.D.)]; preg- nant mouse plasma: E2 (D), DES (•); pregnant rat plasma: E2 (A), DES (A); 28-day-old female rat plasma: E2 (V), DES (•); BSA: E2 (+), DES (X).

106 10' 10" 10* 101 1/Sb (M) 1444 SHEEHAN AND YOUNG Kntlo i 1979 Vol 104 , No 5

M (n2 = 12)] are found by graphical resolution (20). It should be noted that these studies were carried out up to the limits of DES in . The 2.0 solubility of DES under the experimental conditions was first determined in the absence of protein and was the limiting value used, since protein binding can increase the apparent solubility of hormones. It is not possible, however, to obtain a reliable Scatchard plot for E2 bind- ing to BSA because of the insolubility of E2 over a major 1.0 portion of the necessary concentration range.

The binding of DES to a representative plasma sample Downloaded from https://academic.oup.com/endo/article/104/5/1442/2592405 by guest on 30 September 2021 from nonpregnant animals is shown in Fig. 4. The curves are similar to the BSA curves with 8 X 10~8 mol DES/ mg protein and a Kd of 14.5 X 10~6 M. (The equivalent values from Fig. 3 for BSA in moles of DES per mg 8 8 protein are nj = 4 X 10~ and n2 = 17 x 10~ .) We have 8 12 16 2O 24 28 observed no apparent site heterogeneity with plasma. 6 Human pregnancy plasma also contains a high affinity Sb M(x10 ) FIG. 4. Binding of DES to rat plasma. Plasma obtained from 85-day- estrogen binder, TEBG, which is unrelated to aFP (13, old female rats was assayed at a final protein concentration of 166 /xg/ 21, 22). The results of experiments in which the binding ml (curve 1) or 333 /ig/ml (curve 2) at various input DES concentrations. of E2 and DES to human and rat pregnancy plasmas was The values for Kd and n (in moles of DES per mg protein) are: curve 1: 6 8 Kd = 14.6 X 10" M, n = 7.7 X 10" ; curve 2: Kd = 14.1 X 10"" M, n = 8.5 examined are presented as Scatchard plots in Figs. 5 and 8 6. The presence of both TEBG and aFP is indicated by x 10~ the high affinity binding of E2; no such binding is seen Discussion with DES. It has been shown in several laboratories (23-25) that the interaction of hormones with plasma proteins mod- ulates their activity. For instance, Anderson et al. (26) 2.0 ; have reported that the binding of estradiol and to serum albumin changes the relative amounts of these hormones available to intact uteri. It is of interest, there- fore, to more fully determine the binding characteristics of E2 and DES to plasma. The method chosen yields results similar to those obtained by classical equilibrium dialysis, as demon- strated by Pearlman and Crepy (13) for TEBG and in this work for aFP. Additionally, Ryan et al. (27) have directly compared equilibrium dialysis and batch Seph- adex techniques and find the two methods comparable. With several plasma samples as well as BSA, DES exhibits a 10- to 20-fold greater binding than E2. The suggestion of site heterogeneity from the slope of the DES curve (Fig. 2) is supported by the nonlinear Scat- chard plot for binding of DES to BSA (Fig. 3). Recently, Savu et al. (28) reported some data on the binding of DES to serum albumin. They found significant binding of DES but were unable to precisely determine equilib- 4 8 12 16 rium parameters. Their data, however, are consistent 6 Sb M(xlO ) with our findings. We have found that valid measure- FIG. 3. Scatchard analysis of DES binding to BSA. A final concentra- ments can be made if the protein concentration is raised tion of 66.7 jug/ml (A) or 133 jug/ml (B) BSA was titrated with increasing so that tracer quantities of DES yield Sb:Su values of 1- concentrations of DES. The resultant curves have been resolved into 4, and if the higher concentrations of DES are added so two components (20). The values for Kd and n (moles of DES per mole 6 6 that the DES is minimally diluted. Under these condi- BSA) are: A: Kdl = 4.6 X 10~ M, n, = 3.0, and Kd2 = 9.3 x 10~ M, n2 6 6 = 12.2; B: Kd, = 3.1 x 10" M, ni = 2.6, and Kd2 = 9.7 x 10~ M, n2 = 11.2. tions, the Scatchard plot can be resolved into at least two E2 AND DES BINDING TO PLASMA PROTEINS 1445 Downloaded from https://academic.oup.com/endo/article/104/5/1442/2592405 by guest on 30 September 2021

2 4 6 8 10 12 14 16 18 Sb M(xlO?) 1.4 B 6 8 10 12 14 16 18 1.2 Sb M(xlO9)

B 0.8 0.6 l.O 0.4 0.2 0.8 2 4 6 8 10 12 14 16 18 20 22 24 26 0.6 Sb M(xl0?)

FIG. 6. The binding of E2 (A) and DES (B) to rat pregnancy plasma. 0.4 Details are the same as in Fig. 5, except Kd = 2.5 x 10~9 M and bound 6 E2 = 1.1 X 10~ M. 0.2 the major low affinity binder in normal plasma (1, 3), and our data suggest that this is the case with DES as well. 2 4 6 8 10 12 14 16 9 However, we cannot determine if both hormones bind to Sb M(xlO ) the same sites, since their solubility limits prevent mean- FIG. 5. The binding of E2 (A) and DES (B) to human pregnancy ingful competition studies from being carried out. plasma. E2 binding was assayed at a final concentration of 250 jug/ml protein, while DES binding was assayed at 50 jug/ml plasma protein to Our data show a lack of high affinity binding of DES obtain Sb:Su ratios near unity. The biphasic Scatchard plot in A has to aFP and TEBG (Figs. 2, 5 and 6). Some workers have been resolved into two components ( ) by the method of Rosenthal used competition studies to show that DES either does (19). The points represent data, while the line through them is calcu- not compete with E2 for binding to aFP at all (14,16, 29) lated from the two-component model. The high affinity component has a K of 2 X 10~9 M, and a bound E value of 7.4 x 10"7 M. or is a weak competitor (15, 30, 31), while others have d 2 employed semiquantitative nonequilibrium methods to examine DES and aFP interaction (17). With TEBG, classes of high capacity, low affinity binding sites. The competition studies have suggested that DES is a weak sum of the product of the number of binding sites and binder (32, 33). This study demonstrates that DES does their Kds for the two binding sites is 10- to 20-fold higher not bind with high affinity to aFP or TEBG under than that reported for E2 binding to albumin (3). This conditions where DES binding is measured directly and same conclusion is also reached when the binding affini- at equilibrium. The dissociation constant and binding ties of E2 and DES for BSA are compared (Fig. 2) by the capacity of human and rat pregnancy plasma with E2 method of Pearlman and Crepy (13). The binding of DES that we find is consistent with published reports (14-18, to normal plasma appears to be similar to DES binding 34,35). Thus, the failure of DES to bind with high affinity to albumin (Figs. 3 and 4). For E2, albumin appears to be is not due to lack of competent aFP or TEBG. Studies 1446 SHEEHAN AND YOUNG Endo • 1979 Vol 104 • No 5 are presently underway to determine the actual equilib- 14. Soloff, M. S., J. E. Creange, and G. 0. Potts, Unique estrogen- binding properties of rat pregnancy plasma, Endocrinology 88: rium parameters for DES binding to aFP and TEBG. 427, 1971. The lowered binding of DES to aFP and TEBG may 15. Raynaud, J. P., C. Mercier-Bodard, and E. E. Baulieu, Rat estradiol result in increased delivery to target tissues of DES binding plasma protein (EBP), 18: 767, 1971. compared to E2 during pregnancy. This altered pattern 16. Liang-Tang, L. K., and M. S. Soloff, Characterization of a binding protein specific for 17 beta-estradiol and in rat pregnancy of DES delivery may be one of the causative factors in plasma, Biochim Biophys Acta 263: 753, 1972. the transplacental toxic effects of DES (9, 10). 17. Aussel, C, J. Uriel, and C. Mercier-Bodard, Rat alpha-fetoprotein: isolation, characterization and estrogen-binding properties, Bio- The present study demonstrates that DES binds more chimie 55: 1431, 1973. strongly than E2 to normal plasma and BSA and much 18. Swartz, S. K., M. S. Soloff, and J. R. Suriano, Binding of more weakly to the high affinity binding proteins found by a-fetoprotein in rat amniotic fluid, Biochim Biophys Acta 338: 480, 1974. during pregnancy in rodents and humans. These differ- Downloaded from https://academic.oup.com/endo/article/104/5/1442/2592405 by guest on 30 September 2021 19. Scatchard, G., The attraction of proteins for small molecules and ences may be partly responsible for the reported effects ions, Ann NY Acad Sci 51: 660, 1949. of DES when administered to pregnant women or rats. 20. Rosenthal, H. E., A graphic method for the determination and presentation of binding parameters in a complex system, Anal Biochem 20: 525, 1967. Acknowledgments 21. Tavernetti, R. R., W. Rosenbaum, W. G. Keely, N. P. Christy, and We thank Malcolm Bowman and Dr. Edward Helton (NCTR) for M. S. Roginsky, Evidence for the presence in human plasma of an determinations of hormone purity, and Dr. L. Doyle (UAMS) for estrogen-binding factor other than albumin: abnormal binding of supplying human pregnancy plasma. estradiol in men with hepatic cirrhosis, J Clin Endocrinol Metab 27: 92, 1967. 22. Vermeulen, A., and L. 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