Relative Potency of Xenobiotic Estrogens in an Acute in Vivo Mammalian Assay S.R. Milligan, A. V. Balasubramanian, and J.C. Kalita Physiology, Biomedical Sciences Division, King's College, London, United Kingdom
based on the assumption that estimates of The in vii effcts ofxenoestrogens are ofinterest in relation to their potential health risk and/or potency from early responses would mini- beneficial efficts on humans and animals. However, the apparent in yim potency ofthe examined mize complications arising from metabolism, response can be confounded by a short half-ife, and the metabolism ofestogens is very dependent dearance, rapid dissociation from receptors, on the nature of conversion and/or inactivation. To minimize such variables, we examined the etc. The early response chosen was the rapid estrogenic potency of a range of xenoestrogens in an acute in vivo assay-the stimulation of increase in uterine vascular permeability increased uterine vascular permeability in ovariectomized mice 4 hr after subcutaneous administra. [apparent within 15-30 min, with very large tion. While estradiol (E2) and estriol (F3; a relatilyweak natural estrogen) readily induced vascu- responses (three- to fourfold increase) within lar responses [median effiv dose (ED50) 10-9 mol], much higher amounts of xenoestrogens 3-4 hr] of estrogen exposure (5,6). This were required. Bisphenol A was about 10,000-fold less potent than E4 and E, and octylphenol response has the advantage that it can be and nonylphenol were about 100,000-fold less potent; dioctyl phthalat, beyl buty ptalate, readily quantified by monitoring the extrava- dibutyl phtbalate, and trichlorinated biphenol produced no effect. Coumestrol was the most active sation of intravenously administered [1251] phytoestrogen, with an ED50 between 10-6 and 10-7 mol; genistein wa about 10-fold less potent labeled human serum albumin (7). than coumestrol, and neither daidzein nor formononetin produced any marked efflect, even at doses up to 1O5mol. All increases in vascular permeability could be blocked by the pure anti- Methods estrogen ICI 182,780. There was no evidence that any ofthe compounds could act as an aniesro- Animals. Female Swiss albino mice (A. Tuck gen in his assay or that they could exert ynegistc ef in.combion. These results indicate & Son Ltd., Battlesbridge, Essex, U.K.), that even short-term exposure to most of the xenobiotic estogens can induce typical estrogenic approximately 3 months ofage and weighing efFecs in vivo, but their esuogenic potency is very weak even when assessed in an acute response. 25-35 g, were maintained under constant Key wori: environmental estrogens, phytoestrogens, uteru, vasular permeability, xenobiotic. conditions of lighting (lights on from 0600 Environ Health Prect 106:23-26 (1998). [Online 2 January 1998] hr to 1800 hr) and temperature (21 ± PC) hatt:p//ehpnatl.niehs.nib.gol/docs/I998/106p23-26milgan/abseract.btmI and fed on a pelleted diet (Economy Rodent Maintenance, Essex, UK) ad libitum. All experiments were performed on ovariec- There is considerable public and scientific on the complex in vivo pharmacokinetics tomized animals. Ovariectomies were per- interest in the potential effects of the expo- associated with uptake, distribution, formed under tribromoethanol anesthesia at sure of humans and other animals to envi- metabolism, and excretion. least 2 weeks before the start of each experi- ronmental estrogens. These nonsteroidal There have been virtually no systemat- ment. All test compounds were administered estrogenic compounds essentially fall into ic, detailed studies of the nature and time by sc injection in a volume of0.1 ml. two categories: they are derived from either course of the estrogenic effects of these Chemicals. 17,-Estradiol (E2), genistein dietary sources or industrial sources. The chemicals in vivo in mammals. As a first (4',5,7-trihydroxyisoflavone), and daidzein recent surge ofinterest in this area has large- stage to assessing the in vivo estrogenic (4',7-dihydroxyisoflavone) were obtained ly come from the use of very sensitive in activity, this study was undertaken to from Sigma Chemical Co. Ltd. (Dorset, vitro assays, which have allowed the identifi- establish the relative potency of the com- U.K.). Coumestrol (3,9-dihydroxy-6H-benzo- cation of weak estrogenic activity in many pounds in an acute in vivo mammalian furo-[3,2-c]-[l]benzopyran-6-one) and for- industrially derived chemicals (1). This has assay. In vivo, the complex pattern of estro- mononetin (7-hydroxy4'-methoxyisoflavone) been coupled with natural and experimental genic action on the uterus can be dissociat- were obtained from Acros Organics observations of estrogenic effects in aquatic ed into a group of early responses (e.g., (Springfield, NJ) and Extrasynthese (Genay, animals (e.g., fish) living under conditions increased vascular permeability, uterine France), respectively. 4-Nonylphenol, 4-tert- of continuous exposure to the compounds edema, uterine eosinophilia) and a group octylphenol, bisphenol A [2,2-bis(4 hydrox- (2). The possible health benefits ofdiets rich of later responses (a host of biochemical yphenyl)propane, dioctyl phthalate, benzyl in phytoestrogens has provided an addition- changes leading to cell division, differentia- butyl phthalate, and dibutyl phthalate were al and alternative focus ofattention (3). tion, and uterine growth) (5). obtained from Aldrich Chemical Co. Ltd. While the existence of dietary and envi- Estimates of estrogenic potency are (Gillingham, U.K.). Trichlorinated biphenol ronmental compounds with estrogenic markedly affected depending on which of (TCB; 3,4,3',4'-tetra-chlorobiphenyl) was a activity is clear, speculation linking them to these two groups of responses are used as gift from P. Darbre (University of Reading, epidemiological data on fertility, reproduc- the baseline. Thus while estriol and estradiol Reading, U.K.). Stock solutions ofthe chemi- tive abnormalities, and disease is severely are relatively similar when assessed in terms cals were normally dissolved in ethyl alcohol hampered by our very poor understanding of the ability of single subcutaneous (sc) or DMSO. The injection vehicle was either of the in vivo biological activity of these injections to induce early uterine responses, arachis oil (phytoestrogens) or saline (others) compounds. In vitro assays have established such single exposures to estriol are consider- that all the compounds are relatively weak ably less effective than estradiol in produc- Address correspondence to S.R. Milligan, estrogens, with both binding affinity to the ing sustained effects due to the rapid disso- Physiology, Biomedical Sciences Division, King's its College, Strand, London, WC2R 2LS, United estrogen receptor and in vitro biological ciation ofestriol from receptor (5). Kingdom. activity usually <1/1,000 of estradiol (4). The rationale behind this study was to We are grateful to the Medical Research Council for However, it is difficult to extrapolate from use one of the early specific estrogen-stim- support. J.C.K., a Commonwealth scholar from such in vitro data to the in vivo situation in ulated responses as the basis for comparing India, was supported by the British Council. which the biological activity is dependent relative estrogenic potencies. This was Received 10 June 1997; accepted 1 1 September 1997.
Environmental Health Perspectives . Volume 106, Number 1, January 1998 23 Articles * Milligan et al.
containing 25% DMSO or alcohol. The pure |* Control * Estriol A Octylphenol | Control M Esiriol A Genistein antiestrogen ICI 182,780 (a gift from A. A 17,B-Estradiol * BisphenolA 0 Nonylphenol IA* 17p-Estradiol Coumestrol 0 Daidzein Wakeling, Zeneca Pharmaceuticals, 30 30 Macclesfield, U.K.) was dissolved in alcohol to give a stock solution of 10 mg/ml ethanol cc 25 Q 25 and administered in an arachis oil vehide. LU .20 Vascular permeability changes in 20 :zU response to test treatment. A quantitive 00 index of the permeability of the uterine vas- L. 15 IC. 15 culature 4 hr after sc injection of the test U compound was obtained from the leakage of =0 10 o 10 radiolabeled albumin from the circulation .E L. U (7,8). In brief, 3.5 hr after the injection of CD the test substance, 50 pl 0.5 ,Ci [1251]- U 5 A- 5 labeled human serum albumin was injected 3 into the jugular vein of mice anesthetised 0 E~MMARW" with tribromoethanol. Thirty minutes later, -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 .4 a blood sample was drawn from the subor- Log of injected doses (mol) Loo of injected doses (mol) bital canthal sinus with a heparinized capil- Figure 1. The dose-response effects of 1713-estradi- Figure 2 The dose-response effects of 17f3-estradi- lary pipette and the animals were killed by ol, estriol, and the environmental estrogens bisphe- ol, estriol, and the phytoestrogens coumestrol, cervical dislocation. The blood sample was nol A, octylphenol, and nonylphenol on uterine vas- genistein, and daidzein on uterine vascular perme- centrifuged to provide a 100 pi plasma sam- cular permeability 4 hr after subcutaneous adminis- ability 4 hr after subcutaneous administration in ple. The uteri and a sample of thigh musde tration in ovariectomized mice. Saline controls were ovariectomized mice. Saline controls were included were removed, briefly washed in saline, and included for each experimental data point Vascular for each experimental data point. Vascular perme- weighed. The radioactivity in the uterus, permeability was determined by the extravascular ability was determined by the extravascular accu- accumulation of [12511-albumin and was expressed mulation of [12511-albumin and was expressed as plasma sample, and musde was determined. as extravascular albumin volume (EAV; see extravascular albumin volume (EAV; see Methods). Previous studies on the uterus (8,9,10) have Methods). The data are expressed as mean ± stan- The data are expressed as mean ± standard error suggested that the blood volume of the dard error (n = 6-12). Where no error bars are (n = 6-12). Where no error bars are shown, the uterus is very much smaller than the albumin shown, the errors were smaller than the symbol. errors were smaller than the symbol. Statistical sig- space (after a circulation time of30 min) and Statistical significance was determined by compari- nificance was determined by comparison with the son with the controls. controls. that the extravascular albumin space is the *p<0.05; **p".01; lp<0.001. major determinant of the total tissue albu- *p<0.05; **p
24 Volume 106, Number 1, January 1998 * Environmental Health Perspectives Articles * Relative potency of xenobiotic estrogens
These observations, coupled with the present Table 1. The ability of the antiestrogen ICI 182,780 to inhibit estrogen-induced increases in uterine vascular observations, suggest that in vitro potencies of permeability xenobiotic estrogens may not be an accurate Compound Uterine Uterine vascular Muscle vascular guide to in vivo potencies, and estimates of in administered Number weight permeability permeability vivo potency will depend on the assay used. (mol) of mice (mg) ratio (%) ratio (%) The in vivo estrogenic potency ofxenobi- Control 12 13.00 ± 1.25 4.26 + 0.51 0.64 ± 0.08 otic estrogens has received rather superficial ICI 182,780 6 8.47 ± 0.01 4.20 ± 0.27 0.47 ± 0.04 attention, with results largely based on uter- 173-Estradiol 10-12 6 16.82 ± 0.02 3.53 ± 0.39 0.5 ± 0.04 ine growth after daily sc injections (16) or 17p-Estradiol 10-12+ ICI 6 10.05 ± 0.01 3.40 ± 0.12 0.44 ± 0.06 17,B-Estradiol 10-10 6 18.72 ± 0.01 17.63 ± 2.04 0.47 ± 0.05 after administration by gavage (17). 17p-Estradiol 10-10+ ICI 6 13.23 ± 1.32 7.83 ± 1.73* 0.61 ± 0.03 However, the relative potency of estrogenic 17fi-Estradiol 10-9 6 17.38 ± 1.83 25.79 ± 1.90 0.42 ± 0.02 compounds depends on a number of factors, 17j3-Estradiol 10-9+ ICI 6 16.12 ± 0.01 22.19 ± 2.50 0.46 ± 0.05 including the route of administration and Coumestrol 10-7 6 19.58 ± 1.85 28.40 ± 2.98 0.50 ± 0.01 the nature of the response monitored. The Coumestrol 10-7 + ICI 6 15.10 ± 0.01 4.31 ± 0.36* 0.57 ± 0.04 particular problem of using uterine growth Genistein 10-6 8 19.56 ± 1.11 25.43 ± 1.92 0.60 ± 0.08 Genistein 10-6 + ICI 6 13.16 ± 0.09 5.92 ± 0.54* 0.55 ± 0.04 respotnses to assess estrogenic potency is well Estriol 10-9 6 26.5 + 0.02 18.27 ± 2.70 0.54 ± 0.04 illustrated by comparing the effects ofE2and Estriol 10-9 + ICI 6 16.32 ± 0.02 12.8 ± 1.46* 0.51 ± 0.04 E3. while single sc injections ofeither ofthese Nonylphenol 10-4 6 18.80 ± 0.01 16.74 ± 3.06 0.47 ± 0.05 hormones are very effective in inducing rapid Nonylphenol 10-4 + ICI 6 15.08 ± 0.03 4.53 ± 1.17* 0.65 ± 0.11 uterine responses (that occur within a few Octylphenol 10-4 6 12.60 ± 1.52 18.45 ± 3.49 0.53 ± 0.06 hours of administration), E3 is unable to Octylphenol 10-4 + ICI 6 13.07 ± 0.01 5.7 ± 0.10* 0.44 ± 0.04 Bisphenol A 10-5 6 16.50 ± 1.20 15.18 ± 2.40 0.42 ± 0.02 mimic E2 in terms of the uterine growth Bisphenol A 10-5 + ICI 6 8.15 ± 0.03 3.67 ± 0.48* 0.45 ± 0.02 response measured at 24 hr after injection Ovariectomized mice were subjected to daily subcutaneous (sc) injections of 100 pg ICI 182,780 for 4 days. One hour after (5). Such differences in estrogenic responses the fourth injection, the test compound was administered sc. Saline controls were included for each experimental data led to E3 being described as a weak estrogen point. Vascular permeability was determined by the extravascular accumulation of [12511-albumin and was expressed as a (18). However, the apparent low potency of ratio (mean ± standard error) of the 112511 counts per minute per milligram of tissue to [12511 counts per minute per microliter single injections of E3 to induce uterine of plasma. Thigh muscle was the control tissue. growth hides the biological reality of the *Significantly lower than the test compound alone Ip<0.OO estrogenic response. E3 is fully effective in producing the complete ranige of estrogenic dibutyl phthalate (10-4 mol), or benzyl butyl combinations tested in terms of inducing an responses when it is given as rapidly repeated phthalate (10-4 mol), and uterine vascular increase in vascular permeability. In control injections (5) or as slow-release implants permeability was determined. There was no uteri, the index of vascular permeability was (195. These apparent differences between E3 evidence that any of the environmental estro- 4.2 ± 0.51 pil/mg (mean ± SE); the combina- and E2 in terms of their potency to induce gens (at the doses given) produced any signif- tion of nonylphenol with octylphenol (both acute and sustained responses highlight the icant (p<0.05) inhibitory effect on the estradi- at 10-6 mol) and bisphenol A (10-7 mol) pro- difficulty of interpreting assessments of estro- ol-stimulated increase in vascular permeabili- duced an index of 2.91 ± 0.27 Pl/mg; for genic potency by just single in vivo assay ty. The index of vascular permeability was dibutyl phthalate with benzyl butyl phthalate methods. In contrast to the long-term nature 12.9 ± 2.5 .l/mg (mean ± SE) for E2 (10o and dioctyl phthalate (all at 10-3 mol), the of uterine growth assays, the increase in uter- mol) alone, while the combination ofE2 with index of vascular permeability was 2.79 ± ine vascular permeability is an acute, rapid, the other estrogenic compounds gave values 0.54 pl/mg. easily monitored response of the uterus to of 10.3 ± 1.5 for nonylphenol and octylphe- estrogenic stimulation (7). The estrogen nol (each at a dose of 10-6 mol), 14 ± 2.8 for Discussion receptor-mediated nature of this response bisphenol A (10-8 mol), and 11.4 ± 1.6 for The availability of very sensitive in vitro was confirmed in the present study by the dioctyl phthalate, 12.1 ± 1.8 for dibutyl bioassays for estrogens has led to the identifi- ability of the pure antiestrogen ICI 182,780 phthalate, and 14.9 ± 1.1 (pd/mg for benzyl cation of estrogenic activity in many indus- to block the responses induced by E2' E3, butyl phthalate (all at doses of 10- mol). trial (11,12) and plant-derived compounds and other compounds. The present stuJy Experiment 4-The effects ofcombina- (13). However, even in vitro assays, the estro- provides a direct indication of the acute in ions ofa variety ofenvironmentl estrogens genic activity of these compounds is usually vivo potencies of a variety of dietary and on uterine vascularpermeability. The effect less than 1/1,000-1/100,000 that of E2 industrial estrogenic compounds on an of low doses of different compounds given in (1,4,11-13). Our data is in general agree- important target site for estrogens in female combination was examined to determine ment with published data on the in vitro mammals. The results indicate that, even whether there was any evidence of synergistic potency of the xenobiotic estrogens, empha- when measured in terms of their ability to interactions in the induction of increases in sizing that these compounds can only exert induce acute responses, the activity of such uterine vascular permeability. Ovariectomized significant estrogenic activity when present compounds is very low in comparison with mice were injected sc with one of the follow- in very large quantities. Bisphenol A appears established estrogens like E2 and E3. ing combinations: 1) nonylphenol (10-6 mol), to be one of the more potent xenobiotic The relative potency of any of the envi- octylphenol (10-6 mol), and bisphenol A (10-7 estrogens (14,15); a recent in vivo study in ronmental estrogens compared to E3 and E2 mol); and 2) dibutyl phalate (10-4 mol), rats examining prolactin secretion (14) sug- in this acute assay is particularly interesting. dioctyl phalate (10-4 mol), and benzyl butyl gested that bisphenol A was only 100-500- While E3 is a relatively weak estrogen in phalate (104 mol). There was no evidence of fold less active than E2, while bisphenol A terms of any long-term growth responses to any significant (p>0.05) postive or negative potency in vitro was 1,000-5,000-fold less acute exposures, it is almost as effective as synergy or negative effects between the com- than E2. In the present study, bisphenol A E2 in inducing rapid estrogenic effects in pounds (at the doses used) in any of the was about 10,000-fold less potent than E2. the uterus. However, even in this rapid in
Environmental Health Perspectives * Volume 106, Number 1, January 1998 25 Articles * Milligan et al. vivo assay that allows the estrogenic activity C, eds). Princeton, NJ:Princeton Scientific Publishing, 12. Routledge EJ, Sumpter JP. Estrogenic activity of of the relatively weak E3 to be exposed, the 1992;295-309. sufactants and some of their degradation products 2. Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, assessed using a recombinant yeast screen. Environ potency of all the weak environmental Sumpter JP. Estrogenic effects of effluents from Toxicol Chem 15:241-258(1996). estrogens was considerably lower. This sewage treatment works. Chem Ecol 8:275-285 13. Markiewicz L, Garey J, Adlercreutz H, Gurpide E. In would tend to emphasize that prolonged in (1994). vitro bioassays of nonsteroidal phytoestrogens. J 3. Knight DC, Eden JA. A review of the clinical effects of Steroid Biochem Mol Biol 45(5):399-405 (1993). vivo estrogenic responses to such com- phytoestrogens. Obstet Gynecol 87:897-904(1996). 14. Steinmetz R, Brown NG, Allen DL, Bigsby RM, Ben- pounds are unlikely to occur unless the 4. White R, Jobling S, Hoare SA, Sumpter JP, Parker Jonathan N. The environmental estrogen bisphenol A exposures are both considerable and MG. Environmentally persistent alkylphenolic com- stimulates prolactin release in vitro and in vivo. pounds are estrogenic. Endocrinology 98:220-230 Endocrinology. 138:1780-1786 (1997). extended; this is currently under investiga- (1994). 15. Nagel SC, vom Saal FS, Thayer KA, Dhar MG, tion. However, the possibility that acute 5. Martin L, Pollard JW, Fagg B. Oestriol, oestradiol-173 Boechler M, Welshons WV. Relative binding affinity- exposures to these compounds may induce and the proliferation and death of uterine cells. J serum modified access (RBA-SMA) assay predicts significant, although short-term, estrogenic Endocrinol 69:103-115 (1976). the relative in vivo bioactivity of the xenoestrogens 6. Cohen PE, Milligan SR. Silastic implants for delivery bisphenol A and octylphenol. Environ Health responses should not be underemphasized. of oestradiol to mice. J Reprod Fertil 99:219-223 Perspect 105:70-76(1997). Rapid responses to estrogens are an impor- (1993). 16. El Samannoudy FA, Sjareja AM, Ghannudi SA, Gillaly tant feature of a number of reproductive 7. Arvidson NG. Early oestrogen-induced changes in GA, El Mougy SA. Adverse effects of phytoestrogens: uterine albumin exchange in mice. Acta Physiol effect of 1-sitosterol treatment on follicular develop- processes, such as control of the cell cycle Scand 100:325-331 (1977). ment, ovarian structure and uterus in the immature (5) and the sensitivity of the uterus to an 8. Milligan SR. The sensitivity of the uterus of the mouse female sheep. Cell Mol Biol 26:255-266(1980). implanting blastocyst (20); exogenous and rat to intraluminal instillation. J Reprod Fertil 17. Laws SC, Carey SA, Hart DW, Cooper RL. Lindane 79:251-259 (1987). does not alter the estrogen receptor or the estrogen- estrogenic agents may be able to either 9. Milligan SR, Mirembe FM. Time course of changes in dependent induction of progesterone receptors in mimic endogenous estrogens and/or pro- uterine vascular permeability associated with the sexually immature or ovariectomized adult rats. vide a disturbance to the sequence of development of the decidual cell reaction in ovariec- Toxicology 92 (11-3):127-142 (1994). tomized, steroid-treated rats. J Reprod Fertil 70:1-6 18. Huggins C, Jansen EV. Depression of oestrone-induced endogenous estrogenic stimulation essential (1984). uterine growth by phenolic oestrogens with oxygenat- for the normal pattern of uterine responses. 10. Milligan SR, Mirembe FM. Intraluminally injected oil ed functions at positions at 6 or 16: the impeded oestro- induces changes in vascular permeability in the 'sensi- gens. J Exp Med 102:335-346 (1955). tised' and 'non-sensitised" uterus of the mouse. J 19. Rudali G, Apiou F, Muel B. Mammary cancer pro- REFERENCES Reprod Fertil 74:95-104 (1985). duced in mice with oestriol. Eur J Cancer 11:39-41 11. Jobling S, Reynolds T, White R, Parker MG, Sumpter (1975). 1. Soto AM, Lin TM, Justica H, Silvia RM, Sonnenschein JP. A variety of environmentally persistent chemi- 20. Milligan SR, Cohen PE, Finn CA. The minimum C. An 'in culture' bioassay to assess the estrogenici- cals, including some phthalate plasticizers, are requirements for oestradiol to induce uterine sensi- ty of xenobiotics (E-Screen). In: Chemically-induced weakly estrogenic. Environ Health Perspect tivity for implantation and decidualization in mice. Alterations in Sexual and Functional Development: 103:582-587 (1995). Hum Reprod 1502-1506(1995). The Wildlife/Human Connection (Colborn T, Clements
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26 Volume 106, Number 1, January 1998 * Environmental Health Perspectives