# I 

# Reseamrh

Articles

Environmental Xenobiotics 

# May Disrupt Normal 

# Endocrine Function by Interfering with the Binding of 

# Physiological Ligands to 

# Steroid Receptors and 

# Binding Proteins
Benjamin J. Danzo Departments of Obstetrics and Gynecology and Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-2633 USA

The diuo ofthe re utive em of

### inma and female an i te wild ha attributed to environmental cemicls (xenobioii. The ects seen"Irror alteratio might anticipate ifthe steroidhormone-dependent processes that 

### regulate these 

### system 

### impaired.To determinewhther nobiics (p t at a conc ioof 10 pM) el action throh seroid-m 

### iated.piwys, we med their 

### abi.:-inhibit the biss [3Hlphysi#ikigical ligands (presAt. at concentati.is:`f 7NM).to- nM and, receptors 

### ratndrogen-lsingprotein 

### (ABRPE)' .huin set 

### hwI:ine-bindingg 

### (hSHBG). Th 7- and -i



### dsomso

### haaichoroyloean,cons o 

### dichiorodip trichloroethane (DDT; p,p'-DDT, 

### pip'-DDE; o' -DDT), dicidrn., atrazine, 

### anm 

### tchlorophenol, caused a sai si inhibition of

### specific

### binding of 

### [3PH]5o to the andro 

### recerthat 

### rsanedfrom 

### 100%(pip'-DDE) to 25% (d ). 

### Methox 

# toteD 

# taeecpob1t6UL0ad7% Thed bind. 

# of-

.. 

### H]... c-D



## o,p'.DD...B....~~~~~~~~~~~~~~~~~...

...... ....[..



## ...

.....
"t .o As§p ~ishibitd 70% by dat Muoiner of ist lb.}$ y................isomer..............................

.. ............. .................. .. .... .....

.... ..... reduce binding significantl Metorchlor, pp.01 atraie, and nonyhenol ri



### ~.

.. R 

### [3H]5a-DHT

### binding to. .P

### byjApo 

### ately'40% Nonylphenol .reduced the bind 

### [3HJ5a-DHT to hSHBG by 70% H aclr n reduced 

### [3H]5a-DHT bind hSHBGby20%, 

### but tie e o wit ASP did n

. 

### op-Df penthla.l reutd' 

### arfa2%ihbhidn.Ife-Hl5a-DHT:.b

-.... ....A ..I:. a:. :: ::..::.i tohSHB$semCiii f f 

### :HlFd:rt.hidibinding

........ id ". ;

... .... ..I.

......... that was staitcally idnia :to tat causdb th nlWdn_AFtiM where 

# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....................,f_....

..

resulted indower ormorerapidd ion rates.4 mk age gprotein, 

### gen receptor, estrogen receptor, 

### sex

### hormonebindin globulin, xenobiotic. 

### Environa Perpect105:294-301 (1997)

During 

### the past 50 ormoreyears, vast quantities 

### of

### diverse 

### synthetic chemicals 

### (xenobiotics) have entered the environment 

### because ofefforts to increase 

### agricultural productivity and because of

### modern industrial processes. 

### These chemicals 

### include 

### herbicides, insecti

### cides, fungicides, styrenes, 

### polychlorinated biphenyls, andpenta- to

### nonylphenols (1-3). Some

### xenobioticshavebeen 

### shown to

### disrupt normal endocrine 

### functions, which leads to 

### aberrant 

### development of female and 

### male 

### reproductive tissues 

### and results in 

### decreased 

### fertilityor

### sterility 

### (1-3). An

### earlydocumented case 

### concerning 

### the 

### effects of these 

### endocrine-disrupting agents on 

### reproduction

was 

### the report 

### that 

### newly hatched 

### herring 

### gull chicks 

### collected in

### the

### LakeOntarioarea, a 

### region 

### highly contaminated with the pesti

### cide 

### DDT, had altered 

### reproductive systems 

### (4). Male chicks had 

### oviducts and 

### resembling ovaries 

### andthe

### oviduct

### s' female 

### birds 

### developed abnormally ( 

### reduction in 

### penis size 

### and serum terone 

### levels in

### juvenile alligators po 

### Lake

### Apopka in 

### Florida has been at to 

### the contamination 

### of the lake DDT

### metabolite

### p,p'-DDE (5). A

### number 

### ofexperimental stud force the concept 

### that environment 

### biotics can 

### have detrimental 

### effects reproductive health 

### of animals Circumstantial evidence is 

### accurt that 

### environmental xenobiotics 

### ma) rupting reproductive processes 

### in males 

### (10-13). Sharp (14) has po 

### that these 

### disorders in 

### male reprc 

### function arise 

### from in utero exp( 

### male 

### embryos to 

### environmental es 

### Indeed, many

### ofthese effects resemble those

u

### be that have been 

### identified in 

### the male 

### off
### uns one spring 

### of humans and animals that were 

### aSwere 

### treated during pregnancy 

### with 

### the potent 

### rt their 

### synthetic estrogen 

### diethylstilbestrol (DES), 

## iagof 

### (15-18). 

## Many ofthe environmental repro

### arogen ductive toxicants are 

### thought to 

### be estrogen 

### obuini agonists or 

### antagonists 

### (1). However, the 

### henyl- report 

### of Kelce et 

### al. 

### (19), 

### indicating that d pen- some

### xenobiotics may

### be acting as 

### androgen I-DHT antagonists, 

### and our 

### data (see 

### below), indi

### ycr, cating 

### that many common 

### xenobiotics 

### prefbiding 

### erentially inhibit steroid 

### binding to 

### the 

### FI%t 

### androgen receptor, require 

### that the estro

### 11dnot gen-mimetic 

### concept ofxenobiotic action 

### be 

### iduced expanded to

### hormonemimetics. 

### hag 

### of Although many

### xenobiotics in the envi

### lag to ronment 

### have 

### onlyweak hormonal activity 

### YE and 

### (20), their 

### lipophilic nature 

### and 

### long halfiindlng lives 

### (21-22) allow them to 

### accumulate and 

### roteins persist in 

### fatty tissues 

### of the 

### body, thus 

### others 

### increasing their concentration 

### and 

### bioavail

### andin- 

### ability. Although it appears 

### that xenobiotics 

### Healsi have 

### hormonal effects at concentrations 

### that are 

### orders of 

### magnitude higher than 

### that 

### required of 

### physiological hormones,
our

### wide exposure to 

### large numbers of

### these 

### gonads compounds may

### have additive effects. This ystemof 

### concept is 

### strengthened 

### by the observation 

### (4). The that mixtures 

### of xenobiotics have 

### been testos- 

### shown to 

### have synergistic 

### effects in in vitro 

### opulating and in vivo

### model systems 

### (23). 

### ttributed The

### endocrine-disruptingeffects ofmany 

### by the xenobiotics can

### be 

### interpreted as 

### interference

ies rein- 

### :al xenos on 

### the

; 

### (6-9). 

### iulating
y

### be dishuman stulated )ductive


### sure 

### of ;trogens. Address correspondence to B.J. Danzo, Vanderbilt

University School of Medicine, Room D-2304 MCN, 1161 21st Avenue South, Nashville, TN 37232-2633. The technical assistance ofArna Banerjee with portions of these studies is greatly appreciated. This work was supported in part by a Pilot Project grant from the Vanderbilt Center in Molecular Toxicology, which is supported by NIEHS grant ES000267. Received 20 September 1996; accepted 11 December 1996.

Volume 105, Number

### 3, March 1997 * EnvironmentalHealth 

### Perspectives 294Articles * Xenobiotics and steroid 

### receptors/binding proteins

with the steroidal regulation of the normal 

### development and function of the male 

### and female reproductive tracts. The initial step in the mechanism of action 

### of steroid hor
### mones is binding 

### ofthe 

### steroid to its receptor or 

### binding protein 

### (24). To 

### determine whether xenobiotics might act through 

### steroid receptor or 

### steroid 

### binding protein 

### pathways, we 

### examined the 

### ability ofselected xenobiotics to inhibit the 

### binding of 

### [3H]

### 171-estradiol to the estrogen receptor
or to inhibit the 

### binding of 

### [PH]5a-dihydrotestosterone 

### (5a-DHT) to the 

### androgen receptor, 

### androgen-binding protein (ABP), 

### and sex

### hormone-binding globulin (SHBG). Our 

### data indicate that xenobiotics interact 

### selectively and 

### specifically with 

### steroid receptors 

### and 

### binding proteins. 

### Thus, the 

### endocrine-disrupting effects ofenvironmen

### tal xenobiotics may occur 

### through multiple steroid

### signalingpathways. 

### Materials and Methods Selection ofXenobiotics Xenobiotics were 

### selected based on 

### publisheddata that 

### they

### had 

### endocrine-disrupting, estrogenic, or 

### antiandrogenic effects or 

### that 

### they bind to estrogen or 

### androgen receptors (1,6,19,25). All 

### xenobiotics were 

### prepared as 1.0 mM 

### stock 

### solutions in 

### absolute ethanol and used 

### directly or 

### diluted in

### absolute ethanol 

### foruse. 

### Sources 

### ofReceptors 

### and

### Binding Proteins Because 

### large quantities 

### of

### the receptors 

### and 

### binding proteins were 

### required to 

### conduct these 

### studies, we 

### obtained tissue 

### and serum containing 

### these proteins 

### from commercial and other sources. 

### Cytosols 

### prepared from frozen young 

### rabbit uteri 

### (PelFreez, Rogers, 

### AR) and frozen prostates 

### from 

### 21-day old 

### Sprague-Dawley rats 

### (Harlan 

### Bioproducts for Science, 

### Indianapolis, IN) were the
sources 

### of estrogen 

### and 

### androgen receptor, 

### respectively. The tissueswere 

### storedat -80°C 

### until used. To prepare uterine 

### cytosol, the 

### frozen tissues were 

### weighed and 

### pulverized, and TE 

### buffer (10 mM 

### Tris-HCl, pH 7.5, 1.0 mM 

### EDTA) 1:10 wt/vol was 

### added to the tissue 

### fragments. The 

### sample was 

### then 

### homogenized using a 

### Polytron 

### (Brinkman Instruments, 

### Westbury, NY). The homogenate was 

### then 

### centrifuged for 1 hr at 

### 249,000g; the supernatant is 

### referred to as 

### cytosol. Frozen prostates were 

### weighed and 

### homogenized (1:4 

### wt/vol) in TE 

### buffer containing 1.0 mM 

### phenylmethylsulfanylfluoride and 1 

### mg/ml antipain; 

### cytosol was pre

### pared as 

### for the uterus. 

### Cytosol prepared fromadult 

### Sprague-Dawleyrat 

### epididymides homogenized in TE 

### buffer 

### (1:4 wt/vol) was the source of ABP. Postpartum human serum obtained from Vanderbilt Hospital 

### (Nashville, TN) was 

### thesourceofSHBG.


### Sources 

### of

### Xenobiotics andSteroids Hexachlorocyclohexane [1

## a,2c,30(a) ,4a, 

### 5a,61-hexachlorocyclohexane],

- and y-isomers; 

### p,p'-DDT; methoxychlor [1,1,1- 

### trichloro-2,2 

### bis(p-methoxyphenyl)ethane]; dieldrin; and pentachlorophenol were 

### obtained from Sigma (St. Louis, MO). 

### Nonylphenol was obtained 

### from Aldrich (St. Louis, MO). Atrazine 

### [6-chloro-N

### ethyl-N'-(1-methyl-ethyl)-1,3,5-triazine-2,4- diamine], p,p'- DDE, 

### and op'-DDT, were 

### from Chem Service 

### (Westchester, PA). 

### Unlabeled estradiol 

### and 

### 5ac-dihydrotestosterone were 

### from Steraloids 

### (Wilton, NH), 

### and 

### [3H]17f-estradiol (87 

### Ci/mmole) and 

### [3H]5ax-DHT (130 Ci/mmole) were 

### from Dupont/NEN 

### (Boston, MA). 

### BindingAssays Charcoal assay. 

### Binding to the estrogen receptor was assessed 

### by incubating uterine 

### cytosol with 7 nM 

### [3H] 171-estradiol. 

### Binding to 

### the 

### androgen receptor, ABP, 

### and SHBG was 

### assessed 

### by incubating rat prostate 

### cytosol, epididymal cytosol, or 

### human serum, 

### respectively, with 7nM 

### [3H]5a-DHT. Samples were 

### incubated (at 40C 

### overnight for receptors, or 

### for 4-6 hr 

### forABP 

### and SHBG) 

### with 7 nM 

### [3H]ligand alone 

### (totalbinding) or

### togetherwith a 100- 

### foldmolar excess ofthe 

### corresponding unla

### beled 

### ligand to 

### determine 

### nonspecific binding. All assay 

### tubes were 

### brought up to a 

### finalvolume of0.5 ml

### withTE. 

### Xenobiotics
were 

### used as 

### inhibitors at a concentration 

### of 100 

### gM. This concentration is in 

### the range 

### ofthose 

### used

### byothers 

### for inhibition studies with 

### xenobiotics (19,23). When the 

### ability of xenobiotics to 

### inhibit the 

### binding of 

### [3H]steroids to their 

### binding proteins was 

### studied, 10 

### gl ofethanol 

### (the same 

### volume inwhich 

### thexenobiotics were

### dissolved) was 

### added to 

### the total 

### and 

### nonspecific binding tubes. 

### Thus, 2% 

### ethanol was present ineach 0.5 ml 

### incubation. This amount 

### ofethanol decreased total 

### binding to 

### the receptors 

### by 2-3% 

### and decreased total 

### binding to ABP 

### and SHBG 

### by about 10% 

### compared to 

### samples that 

### contained no 

### ethanol. The spe

### cific 

### binding that 

### occurred in the presence 

### ofethanol was set at 100% in 

### those experiments in 

### which data are 

### plotted as a percentage 

### ofcontrol. 

### After the incubations were 

### concluded, 

### binding of 

### [3H]ligands to 

### their 

### binding proteins was 

### conducted using 

### either the charcoal assay 

### (26) or sucrose 

### gradient 

### ultracentrifugation (26). The charcoal assay was 

### performed by adding 0.5 ml 

### of a 

### dextran 

### (0.05%)-coated charcoal 

### (0.5%) suspension in TP to each sample tube. The tubes were then agitated on a vortex mixer for 6 sec and then either centrifuged immediately for 5 min at 1,500g (ABP and SHBG 

### samples) or incubated on ice 

### for 10 

### min and then centrifuged (receptor sam

### ples). The difference in postcharcoal incubation time is to take into account the rapid dissociation of ligand fromABP and SHBG 

### (27) and its 

### slower dissociation from the 

### receptors (26). After centrifugation, the 

### supernatants were 

### decanted into scintillation vials and the 

### radioactivity in the samples was counted. Sucrose 

### gradient analysis. For sucrose gradient analysis, samples were incubated
as described above and extracted with pellets from 1 ml ofcharcoal suspension (28), and 300 

## gl aliquots of each sample were applied to low ionic strength (0.01 M KCI) 5-20% sucrose 

### gradients and 

### processed as previouslydescribed (28). Competition 

### studies. 

### Samples (androgen and estrogen receptors, ABP, SHBG)
were 

### incubated with a saturating concentration 

### of 

### [3H]physiological ligand (7 nM) and increasing concentrations of unlabeled 

### physiological ligand orxenobiotic competitor. After 

### incubation at 

### 4°C 

### overnight (receptors) or 

### for 4 hr (ABP and SHBG), the amount 

### of bound radioactivity was determined using the charcoal assay

### procedure. The 

### data were 

### plotted as percentage 

### of control 

### (specific binding in the 

### absence of competitor) versus the 

### log of the competitor concentration. 

### The concentration 

### of competitor 

### that inhibited 

### specific binding 

### by 50% is 

### the 

### IC50. To 

### determine the relative 

### binding affinity (RBA) 

### ofthe xenobiotics as 

### compared to 

### the 

### physiological ligand, the IC50 

### for the

### physiological 

### ligand
was 

### divided 

### by the IC50 

### of the xenobiotic 

### and the

### dividendwas 

### multipliedby 100. Determination 

### ofdissociation 

### halftime. 

### Samples were 

### incubated with 7 nM 

### [3H]physiological ligand alone or with a 

### 100-fold molar excess 

### ofunlabeled 

### physiological ligand (to 

### determine 

### nonspecific 

### binding) at 40C 

### overnight (receptors) or 

### for 4-6 hr 

### (ABP, SHBG). At the 

### conclusion of 

### the 

### incubation, unlabeled 

### physiological ligand (35 

## 9M) or 

### xenobiotic (100 

### ,uM) was 

### added to 

### each 

### sample. At various times 

### after these 

### additions, the amount 

### of

### bound radioactivity was 

### determined using the 

### charcoal assay 

### procedure. The 

### data were 

### plotted as 

### specific radioactivity bound as a percentage 

### ofcontrol versus time. Aregression line was 

### fitted to the points 

### and the dissociation 

### half-time 

## (t1l2) was 

### calculated
as

### previouslydescribed 

### (27). 

### Statistical

### Analysis The 

### datawere 

### analyzedbyone-way

### analysis ofvariance followed by Duncan's multipleEnvironmentalHealth

### Perspectives * Volume 

### 105, Number

### 3, March 1997 295Articles * Danzo

range test 

### (29). Results were considered sig

### nificant

### whenp<0.05. The dataarepresent

### edas 

### themean

### and the 

### standarderror

### ofthe
mean. 

### Results Studies on 

### the EstrogenReceptor
To screen 

### xenobiotics for possible interaction 

### with the estrogen receptor, we 

### determined their 

### ability to inhibit 

### 7 nM 

### [3H] 17f-estradiol binding to the 

### estrogen receptor 

### present in uterine cytosol 

### using the 

### charcoal assay 

### procedure. Figure 1 

### shows
our 

### results. 

### Specific estradiol 

### binding to 

### cytosol 

### occurred, which was 

### completely 

### inhibited by 700 nM 

### unlabeled estradiol. 

### Unlabeled (700 nM) 

### 5a-DHT inhibited 

### binding by about 25%. 

### Of the 

### xenobiotics tested (present at 100 

### ,uM), 

### methoxychlor, p,p'-DDT, 

### p,p'-DDE, and 

### pentachlorophenol 

### inhibitedestradiol binding to 

### asmall (10%) 

### but 

### statisticallysignificant extent. In contrast, 

### o,p'-DDT and

### nonylphenol caused
a 

### dramatic decrease in 

### [3H] 17f-estradiol binding: 60 

### and 75%, 

### respectively. Estradiol 

### binding was not 

### significantly decreased 

### by the y- or 

### &-isomer of

### hexachlorocyclohexane, 

### dieldrin, oratrazine. Further 

### evaluation 

### ofxenobiotic 

### effects
on 

### estradiol 

### binding to uterine 

### cytosol was 

### conducted using sucrose 

### gradient analysis (Fig. 2). 

### Whenuterine 

### cytosolwas 

### incubated with 7 nM 

### [3H]

## 170-estradiol alone, a 

### single 

### peak ofmacromolecular-bound 

### radioactivity
was 

### detected. The 

### peakhad a 

### sedimentation 

### coefficient ofabout 10 S, in 

### reference to a 

### 4.6 S 

### bovine serum

### albumin 

### (BSA) 

### sedimentation

### standard, which

### corresponds to 

### thatof the estrogen receptor on 

### low ionic 

### strength, 5-20% sucrose

### gradients 

### (28). Incubation in 

### the presence 

### of 700 nM 

### unlabeled estradiol eliminated the

### peak. These

### dataconfirm that the 

### estradiol 

### binding detected in uterine 

### cytosol is to 

### the estrogen receptor. As in 

### the 

### charcoal assay, 

### nonylphenol was a 

### highly effective inhibitorof 

### [3H] 

### 17p-estradiol binding to 

### the receptor 

### and 

### pentachlorophenol reduced 

### [3H] 17,-estradiol binding, but

### p,p'- DDT 

### and

### p,p'-DDE had little 

### detectable effect on

### peak

### heights. The 

### ability of 

### selected xenobiotics to
cause 

### dissociation of 

### [3H]173-estradiol from its receptor 

### compared to 

### the 

### physiological 

### ligand was 

### tested. The addition 

### ofunlabeled estradiol 

### (35 

### jiM) to 

### the estrogen receptor preparation 

### that 

### had been 

### equilibrated with 7 nM 

### [3H]

## 17p-estradiol resulted in a time

### dependent decrease in 

### the amount 

### of 

### radioactivity associated with the receptor. 

### The t

### 1/2 ofdissociation 

### (Table 1) was 

### calculated 

### (27) to 

### be 

### about 40 

### hr, which is in agreement 

### with 

### literature values 

### (26). Pentachlorophenol (100 

### jiM) resulted in a 

### dissociation rate that was 

### indistinguishable from that caused by estradiol 

### (Table 1). When 100 

### jiMnonylphenolwas used, dissociation 

### of estradiol from its 

### receptor was morerapid 

### (t112 approx7

### hr; Table 1). 

### Studies on 

### theAndrogen 

### Receptor In contrast to 

### the minor 

### effects ofmost of 

### the

### xenobiotics tested in 

### inhibiting estradiol 

### binding to its receptor (Fig. 1), manyxenobiotics inhibited 

### [3H]5a-DHT binding to 

### prostate 

### cytosol to a 

### statistically 

### significant extent 

### (Fig. 3). The most 

### effective 

### inhibitors
were 

### the DDT 

### derivatives, p,p'-DDE, o,p'- 

### DDT, and 

### p,p'-DDT, which 

### inhibited 

### binding by 100, 90, and 80%, 

### respectively. The 

### hexachlorocyclohexane isomers, 

### which 

### inhibited binding 

### by about 50%, were 

### the next most 

### potent inhibitors of 

### [3H]5aDHT 

### binding. Nonylphenol, which

140

120

100

-

C. 80

60

40

20

0 ATR PEN NUN Figure 1. Inhibition of [3H117j-estradiol binding to the estrogen receptor (estrogen receptor source, uterine cytosol) as determined by the charcoal assay procedure. Abbreviations: *E, specific binding of 

### [3HJ17P-estradiol; E, unlabeled estradiol; DHT, unlabeled 5a-dihydrotestosterone; Hg, hexachlorocyclohexane-y, Hd, hexachlorocyclohexane-8; ME, methoxychlor; pDDT, p,p'-DDT; DDE; p,p'-DDE; oDDT, o,p'- DDT; DIE; dieldrin; ATR, atrazine; PEN, pentachlorophenol; NON, nonylphenol. The data are plotted at control (%) in which the amount of specific binding of [3HJ17p-estradiol

= 100%. The mean and the standard

error of the mean are plotted. The numbers in parentheses above the bars indicate the number of experiments inwhich samples were assayed intriplicate.

1,400

1,400 _X !! *ENN

1 00

Fraction number

Figure 2. Sucrose gradient analysis of estradiol binding to the estrogen receptor. Abbreviations: *E, sample incubated with 13H]17,B-estradiol alone prior to sucrose gradient analysis; E, unlabeled estradiol;
pDDT, p,p'-DDT; PENT, pentachlorophenol; DDE, p,p'-DDE; NON, nonylphenol; BSA, bovine serum albumin.

Volume 105, Number3, March 1997 * EnvironmentalHealthPerspectives

t t UEIl Hg Hd ME pUDID DUE oDDT DIE



### 296Articles

- Xenobiotics and steroid receptors/binding proteins



### reduced 

### [3H] 171-estradiol binding to the 

### estrogen receptor 

### by 75%, 

### had no 

### effect on 

### [3H] 5a-DHT 

### binding to androgen receptor (Fig. 3), nor 

### did 

### methoxychlor or 

### estradiol. 

### Dieldrin, atrazine, 

### and 

### pentachlorophenol resulted in a 

### statistically significant 30-40% reduction in 

### [3H]5a-DHTbinding (Fig. 3). Two considerations 

### prompted us to examine 

### [3H]5a-DHT binding to prostate cytosol 

### by sucrose 

### gradient analysis. First
was 

### the report that 

### the prostate contains a 

### steroid-binding protein other than the androgen receptor 

### that is 

### fairly nonspecific and not 

### readily saturable 

### (30). This protein 

### sediments on low ionic 

### strength, 5-20%
sucrose 

### gradients at 

### approximately 3.7 S 

### (30) in contrast to 

### the 

### androgen receptor, which 

### sediments at 

### approximately 8-10 S 

### (28). The 

### second consideration was our observation that in some experiments, p,p'- DDE present at a concentration of 100 

### gM inhibited 

### [3H]55a-DHT binding to prostate cytosol to agreater extent than did 0.7-,uM 5a-DHT. These data suggested that 

### bindingmoieties 

### other than the 

### androgen receptor might be present in some prostate cytosol preparations. Figure 4A 

### shows the results of sucrose 

### gradient analysis 

### of 

### [3H]5ac-DHTbinding to cytosol 

### made from a

### batch of 15 

### prostates. A 

### single symmetrical peak ofbound 

### [3H]50aDHT was detected that had a sedimentation 

### coefficient of 8-10 S (Fig. 4A). This peak, which represents the 

### androgen receptor, was proceeded by exponentially decreasing amounts of 

### radioactivity representing unbound

### ligand thatwas not removed

### bythe charcoal extraction 

### procedure. The 8-10 S

Table 1. The effect ofvarious compounds onthe half-time of dissociation of [3H]physiological ligand from its receptor orbindingprotein. Compound Protein E DHT NON PEN ME Hd DDE
ER 40 hr

- 7 hr 40 hr

- AR

- 15hr

- - - - 15hr ABP

- 7mi

- - - 70 min

- SHBG

- 85 min 85 min 5 min ND

- - Abbreviations: E, 

### 17p-estradiol; DHT, 5ax-dihydrotestosterone; NON, nonylphenol; PEN, pentachlorophenol; ME, methoxychlor; Hd, hexachlorocyclohexane-6; DDE, o,p'-DDE; ER, estrogen receptor; AR, androgen receptor; ABP, androgen-binding protein; SHBG, sexhormone-binding globulin. Blank spaces indicatethatthese comparisons were notmade. ND, no detectable dissociation during the time studied.

160

140

120

100

.C., -3
g0 80

60

40

20

0 *DHT E DHT Hg Hd ME pDDT DDE oDDT DIE ATR PEN NON Figure 3. Inhibition of 

### [3H]5ax-DHT binding to the androgen receptor (androgen receptor source, rat prostate cytosol) as determined bythe charcoal assayprocedure. Abbreviations: *DHT, specific binding of 

### [3H]Sa-DHT to the receptor; 

### E, estradiol; DHT, 

### 5a-dihydrotestosterone; Hg, 

### hexachlorocyclohexe-y, Hd, hexachlorocyclohexane-6; ME, methoxychlor; pDDT, p,p'-DDT; DDE, p,p'-DDE; oDDT, o,p'-DDT; DIE, Dieldrin; ATR, atrazine; PEN, pentachlorophenol; NON, nonylphenol. Specific binding of 

### [3H]5a-DHT = 100%. The mean and the standard error ofthe mean are plotted. The numbers above the bars indicate the number of experiments inwhichthe samples were assayed induplicate. 

### peak was 

### eliminated when the sample was 

### incubated with 

### [3H]5a-DHT and 0.7 ,uM unlabeled 5a-DHT (Fig. 4A) and it was greadyreducedwhen sampleswere incubated with 100 ,uM p,p'-DDE or 100-,uM p,p'- DDT. No [3H]5a-DHTbindingwas detected in the 4 S region of the gradient where 

### specific or nonspecific 

### binding to moieties otherthanthereceptorwouldbepresent. 

### Analysis of a 

### batch of cytosol 

### prepared from 15 other prostates yielded the results shown in Figure 4B. In this case, macromol

### ecular-bound 

### [3H] 5a-DHTwas 

### detected in both the 4 S and 8 S regions ofthe gradient. Unlabeled 5a-DHT (0.7 jM) eliminated 

### [3H]5a-DHT binding to the androgen receptor present in the 8-10 S region and decreased, but did not eliminate, 

### [3H]5aDHT binding to 

### the 4-5 S component(s). In 

### contrast, p,p'-DDE (100 

### jiM) eliminated 

### [3H]5a-DHT binding to components in both regions of the gradient 

### (Fig. 4B). Whether the 

### 4-5 S component represents the 3.7 S low

### affinity, high capacityprostate 

### binding protein 

### reported by others (30) is not 

### known. The presence 

### ofthis unknown protein 

### provides an 

### explanation for our observation that 100 jiM

### p,p'-DDE reduced 

### binding of 

### [3H]55a-DHT to some 

### prostate cytosol preparations to a greater extent than did 0.7 jiM unlabeled 

### 5oc-DHT. The 

### ability of

### p,p'-DDE to cause dissociation of 

### [3H]55a-DHT from the prostate 

### androgen receptor was 

### determined as 

### described above for the estrogen receptor. Our data 

### (Table 1) 

### indicate that unlabeled 

### 5at-DHT and

### p,p'-DDE resulted in a simi

### larpattern 

### ofdissociation of 

### [3H]5ct-DHT from the 

### androgen receptor 

### and a t1/2 

### of greater 

### than 15 

### hr, a

### value that is in agreementwith the 

### literature (31). 

### Studies on

### Androgen-binding Protein Charcoal assay 

### analysis of 

### [3H]5ac-DHT binding to rat 

### epididymal cytosol containing ABP 

### indicated that 

### several xenobiotics resulted in a 

### statistically significant reduction in the amount of 

### [3H]5a-DHTbound toABP 

### (Fig. 5). The most 

### effectivexenobiotic 

### inhibitor was 

### the 6-isomer 

### of hexa

### chlorocyclohexane, which caused a 70% decrease in bound 

### [3H]5ac-DHT. In contrast, the inhibition 

### caused 

### by the 

### y-isomer of

### hexachlorocyclohexane did not 

### reach statistical 

### significance. 

### Methoxychlor, p,p'- DDT, and atrazine all 

### reduced 

### [3H]5aDHT 

### binding toABP 

### by 40-50% 

### (Fig. 5). p,p'-DDE reduced

### binding toABP

### by 20%. 

### Estradiol 

### inhibited 

### [3H]5ac-DHT binding toABP

### byabout 60%. Sucrose 

### gradient analysis indicated that macromolecular-bound 

### [3H]5ac-DHT in 

### epididymal cytosol sedimented at 4.6 S as

EnvironmentalHealth 

### Perspectives * Volume 105, Number3, March 1997 297Articles

- Danzo



### would be 

### expected for ABP 

### (32). This 

### binding was 

### completely inhibited 

### by unla

### beled 5a-DHT, reduced 

### by hexachlorocyclohexane-6, but not 

### affected 

### by hexa

### chlorocyclohexane-,y (Fig. 6). 

### When the 

### 6-isomer 

### ofhexachlorocyclohexane was 

### used in 

### dissociation 

### studies, we 

### noted that 

### the dissociation rate (t

### 1/2 approx 70 min) was 

### slower 

### than the t

## 1/2 ofabout 7 min 

### obtainedwhen unlabeled 

### 5a-DHTwas 

### used 

### (Table 1). This latter

### value is similar to whatwe

### havepreviouslyreported 

### (27,32). 

### Studies on 

### Sex

### Honnone-binding Globulin When the 

### binding of 

### [3H]5a-DHT to 

### human postpartum serum containing SHBG was 

### examined using 

### the charcoal assay

### procedure, we 

### noted that the 

### xenobiotics 

### hexachlorocyclohexane-6 and 

### y, o,p'- DDT, 

### pentachlorophenol, and 

### nonylphenol caused a 

### statistically significant reduction in 

### specific 

### binding (Fig. 7). Nonylphenol resulted in a 70% 

### decrease in 

### [3H]5a-DHT binding, whereas the other 

### xenobiotic competitors 

### reduced 

### binding 

### by about 20-30%. SHBG was 

### the 

### least responsive protein to 

### the 

### inhibitory effects of xenobiotics on 

### 5a-DHT 

### binding. 

### Methoxychlor, 

### p,p'-DDT, p,p'-DDE, dieldrin, 

### and atrazine were 

### ineffective at 

### inhibiting 

### [3H]5a-DHT binding to SHBG. 

### Estradiol 

### inhibited 

### [3H] 5a-DHT 

### binding to SHBG

### by 85% 

### (Fig. 7). When the 

### binding of 

### [3H]5a-DHT to
serum components was 

### examined

### bysucrose 

### gradient 

### ultracentrifugation, labelwas 

### bound to a moiety 

### sedimenting at 

### 4.6 

### S, 

### which is 

### the appropriate 

### sedimentation 

### coefficient for SHBG 

### (33). As 

### expected, unlabeled 5aDHT 

### and 

### nonylphenol inhibited 

### [3H]5aDHT 

### binding to SHBG, 

### whereas atrazine 

### didnot 

### (Fig. 8). When dissociation 

### studies were 

### conduct

### ed, the addition of unlabeled 

### 5a-DHT to SHBG 

### samples that had been 

### equilibrated with 

### [3H] 5a-DHT resulted inat

### 1/2 ofdissociation 

### oflabel 

### of

### approximately 85 min, a 

### t1/2 similar to 

### literature values 

### (27). 

### Nonylphenol resulted ina

### similar

### dissociation 

### half-time, but

### pentachlorophenol resultedina more 

### rapid dissociation rate 

### (t

### 1/2 approx 5 

### min). 

### Methoxychlor, which had no 

### effect on 

### steroid

### binding to

### hSHBG, didnot 

### effect the dissociation 

### of 

### [3H]5a-DHT from SHBG 

### duringthe time

### periodstudied 

### (Table 

### 1). Concentration 

### of

### LigandCausing a 

### 50% 

### Inhibition 

### of

### Binding to 

### Receptors 

### and

### BindingProteins To 

### further evaluate the 

### affinity ofxenobiotics 

### for steroid receptors 

### and 

### binding proteins, competition 

### studies were 

### conducted using 

### multiple concentrations 

### of competi600

500


## *1

E

aB 400

300

200

100

0 0 2 4 6 8 10 12 14 16 18 20 22 24 Fractionnumber



# I

,.

=

0 2 4 6 8 10 12 14 16 18 20 22 Fractionnumber Figure 4. Analysis of [3H]5x-DHTbindingtothe androgen receptorusing sucrose gradientultracentrifugation. Abbreviations: *DHT, sample incubated with 

### PH]5a-DHT alone prior to sucrose gradient analysis; DHT, 5a-DHT; DDE, p,p'-DDE; pDDT, p,p'-DDT. (A) Cytosol from one batchofprostateswas incubatedwith 

### [3HI5a-DHT alone, with unlabeled DHT, orwith the indicatedxenobiotics. (B) Cytosol from another batch ofprostateswas incubated as inA.

tors. As 

### indicated in 

### Table 2, 

### nanomolar concentrations 

### ofunlabeled

### physiological ligandwere 

### able to 

### reduce the 

### binding of [H] 

### ligand to its 

### corresponding receptor or

### binding

### protein

### by 50%. Incontrast, 

### micromolar concentrations 

### ofxenobiotics were 

### required to cause a 50% 

### decrease in 

### binding of 

### [3H] physiological ligands. The concentration 

### of 

### p,p'-DDE that we 

### found to 

### be effective in 

### causing a 50% 

### inhibition of 

### [3H] 5a-DHT 

### binding to 

### the 

### androgen receptor 

### (6.8 

### gM) is 

### similar to 

### the 5 ,uM 

### reported byKelce et 

### al. 

### (19) in 

### their studies on 

### the 

### androgen receptor. Our 

### values 

### for the IC50 

### of op'- DDT 

### for the estrogen receptor is in 

### the range 

### of the values 

### reported by others 

### (23), using

### different

### xenobiotics, for this receptor. We are not aware 

### of

### published data on 

### the inhibition 

### of

### physiological ligandbinding to ABP or SHBG

### byxenobiotics. 

### Discussion
Our experiments 

### demonstrate that xenobiotics interact in a 

### specific and 

### differential manner 

### with the estrogen receptor, 

### androgenreceptor, ABP, 

### and SHBG. 

### Several xenobiotics were more 

### capable of

### inhibiting 

### [3H]5a-DHT binding to 

### the 

### androgen receptor 

### than to any

### ofthe other proteins 

### studied. This 

### findingstronglysuggests 

### that 

### multiple xenobiotics acting 

### through the 

### androgen receptor-mediated 

### pathway may 

### be 

### capable of 

### disrupting physiological processes 

### regulated by this 

### pathway. Whether these 

### compounds would act 

### in vivo as 

### agonists or 

### antagonists of 

### androgen action remains to 

### be determined. The 

### study ofKelce et 

### al. 

### (19) reports 

### that 

### p,p'-DDE inhibits 

### androgenbinding to 

### the 

### androgen receptor, 

### androgen-induced tran

### scriptional activity, 

### and normal male pre

### pubertal development. Our 

### data indicate that

### p,p'-DDE was 

### the most potent 

### ofthe xenobiotics 

### tested at 

### inhibiting 

### [3H]5aDHT 

### binding to 

### the 

### androgen receptor. Our 

### study indicates that 

### p,p'-DDT and 

### o,p'-DDT are 

### also potent 

### inhibitors of 

### [3H]55a-DHT binding to 

### the 

### androgen

Volume 105, Number3, March 1997 

### * EnvironmentalHealth

### Perspectives 298E

0

40

20

Figure 5 didymid 

### 3HJ5cz-E clohexa atrazine standari inwhich

2,500

2,000

* E 1,500

E

0 Q 1,WO0

500

Figure 6 cytosol drotesto

recepto be estr that 

### it androgi 

### Bot chloroc 

### [3H]5c receptc 

### ABP, 

### b ospecif 

### produc Articles XXenobiotics and steroid 

### receptors/binding proteins

110

100

80

60 

### 1-l";;,binding to 

### human SHBG. On 

### theother 

### hand, 

# nonyiphenol was a 

### better 

### competitor

ABP. 

### This 

### finding is consistent 

### with our



### ~~~ 

### bservation 

### (Fig. 5) and reo 

### thelier ~~~~~ature 

### (27) that human SHBG 

### binds estra-

.......... ... ...... 

### .;...diol better than ABP in rat 

### epididym al 

### ...-, 

# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~cytosol. Whether the 

### effects ofxenobiotics

on 

### androgen 

### binding to human ABP are more 

### similar to 

### those on 

### binding to rat 

## ~~~~~~~~~~~~ABP or to 

### human SHBG remains to 

### be

M;:= s,tt 

### f'tJ

Lit sL

U 

### u!xdetermined. ABP 

### and SHBG 

### wereln 

### ~~~~~~~~~~~~~~considered to 

### be 

### simply steroid transport

1 sM:: 

## ~~~~~~~~~~~~~proteinsin 

### the 

### testis-epididymis and in 

### the

s~~~:. 

### ~ ~ .general circulation, 

### respectively. Evidence

now exists 

### demonstrating cell-surface receptors 

### for 

### these proteins 

### (37-38). ABP 

### E~~ ~~ ~ ~ ~ ~~~ 

### ~~~~ad SHBG 

### bind to their 

### receptors and acti- 

### ~~~~~~~vate

### adenylyl cyclase (39)~. Xenobiotics that 

### bind to 

### these 

### proteins could prevent 

### them from

### binding to 

### their receptors 

### and/or

### they 

## ~~~~~~~~could inhibit normal 

### signal transduction, ~~~~thus

### disrupting the action 

### ofthese

### proteins. *DHT E DHT Hg Hd ME pDDT DDE oDDT DIE ATR PEN NON 

### Of the xenobiotics 

### tested, fewer inter

## LInhibition of

# [3HI5a-DHTbindingto

### androgen-binding protein (ABP; 

## source, cytosol 

### from rat

### epi- 

# atdwt

the and rogen receptor 

# orhABPNonyl

es) by xenobiotics determined by charcoal assay. Abbreviations: *DHT, specific binding of 

# pheandolgwa

# arhihlypoetihbtor
o f 

## B.Nnl )HT; E, estradiol; DHT, 5az-dihydrotestosterone; Hg, hexachlorocyclohexane-y, Hd, 

## hexachlorocy- phnlwsaigypoetniborf ine-a; ME, methoxychior; pDDT, p,p'-DDT; DDE, p,p'-DDE; 

### oDDT, o,p'-DDT; DIE, dieldrin; ATR, 

### [3H]

## 17f3-estradiol binding to the estrogen i;PEN, 

### pentachiorophenol; NON, 

### nonyiphenol. Specific binding of

### *DHT

= 100%. The mean and the receptor and its inhibition was not statisti- derror of the mean are 

### plotted. The numbers above the bars indicate the number of 

### experiments 

### cally different than that caused 

### by the ithe samples were assayed intriplicate. known estrogen mimetic, 

### o,p'-DDT. Nonylphenol has been shown to induce 

### proliferation of 

### human breast cancer 

### cells 0 DHT 

### and to 

### trigger mitotic 

### activity in 

### the rat 0 lid ~ 

### endometrium 

### (40). 

### Nonylphenol has 

### been

shown to 

### inhibit 

### [3H] 17j3-estradiol 

### binding to 

### the 

### estrogen receptor present in 

### rainbow trout 

### and to 

### stimulate 

### vitellogenin gene 

### expression in this 

### species (41). Because 

### nonylphenol does not 

### inhibit 

### [3H]5a- DHT 

### binding to 

### the 

### androgen receptor, it may cause 

### the in vitro 

### effects 

### solely

### through the 

### estrogen receptor. 

### However, since 

### the 

### experiments 

### reported used 

### human or 

### fetal bovine 

### (40,41) serum, both 

### ofwhich con- tain SHBG 

## (42), in

## cultures with the 

### cells, 0 4 8 8 10 12 14 16 18 20 22 24 some 

### effects of

## nonyiphenol acting 

### through Fractionnumber SHBG cannot 

### be 

### discounted. In vivo 

### endocrine-disrupting effects of

### nonylphenol S. Sucrose gradient analysis of 

### [3H]5ax-DHT binding to 

### androgen-binding protein (ABP; source, in 

### species that possess SHBG are 

### likely to rat 

### epididymides). Abbreviations: 

### *DHT, cytosol incubated with 

### [3HJ5ac-DHT alone; DHT, 

### 5az-dihy- be mediated 

### through both the estrogen )sterone; 

### Hd, hexachlorocyclohexane-8; Hg, 

### hexachlorocyclohexane-'y. receptor and SHBG. We 

### also 

### studied the 

### ability of xenobiir. 

## o,p'-DDT is

### usually considered to 

### differ 

### considerably in 

### the 

### oligosaccharides otics to 

### influence the half-time ofdissocia

### ogenic 

### (34), but our 

### data suggests attached to 

### them 

### (36). Differences in tion 

### of 

## [3H]ligand from the 

### proteins. The could also be 

### expected to act in an 

### steroid-binding between rat ABP 

### and data that we 

### obtained indicate that while 

### enic or

### antiandrogenic manner. 

### human SHBG 

### have 

### been observed 

### (27). In some 

### xenobiotics 

### resulted in 

### dissociation 

### th the 6- 

### and 

### y-isomers of hexa- addition to 

### differential 

### binding of hexa- rates 

### identical to 

### those caused

### by the

### physi- ,yclohexane are 

### poetihibitors of 

### chlorocyclohexane isomers to ABP 

### and 

### ological ligand, others 

### resulted in more X-DHT 

### binding to the 

### androgen SHBG, other 

### binding differences between 

### rapid or in slower rates. These data suggest )r. 

### These 

### compounds bind to rat 

### the

### proteins were 

### noted. 

### Methoxychlor and that some 

### xenobiotics have a 

### three-dimeniut not to human 

### SHBG, in a stere- 

### p,p'-DDT caused 

### approximately a 40% tional structure that 

### permits access to the Pic manner. ABP and SHBG are reduction in 

### binding to 

### ABP, but had no 

### binding site

### equivalent to that ofthe natur- ,ts of the- sam gene (35),bt they saitclysinfcn-niior feto lseri,wietecnfraino te

EnvironmentalHealth Perspectives * Volume 105, Number3, March 1997 

### 299Articles

- Danzo

100 l
~ -4 1 1 -01 800

20 .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

80

'DHT E DHT Hg Hd ME pDDT DDE oDDT DIE ATR PEN NON Figure 7. The effect of xenobiotics on binding of [3H]5a-DHT to human sex hormone-binding globulin (hSHBG; source, human serum). Abbreviations: *DHT, [3H]5a-DHT, E, estradiol; DHT, 5a-dihydrotestosterone; Hg, hexachlorocyclohexane-y Hd, 

### hexachiorocyclohexane-8 ME, methoxychlor; pDDT, pp'-DDT; DDE, p,p'-DDE; oDDT, o,p'-DDT; DIE, dieldrin;ATR, atrazine; PEN, 

### pentachiorophenol; NON, nonyiphenol. Specific binding of *DHT = 100%. The mean and standard error of the mean are plotted. The numbers above the bars indicate the number ofexperiments inwhichthe sampleswere assayed intriplicate.

3,500

3,000 DHT

A~~~~~~~~~~~~~~~~~~~~~ T

1,500

1,000

500

0 5 10 15 20 25 Fractionnumber Figure 8. Sucrose gradient analysis of [3HJ5a-DHT binding to human sex hormone-binding globulin (hSHBG; source, human serum). Abbreviations: *DHT, serum incubated with [3HJ5a-DHT alone prior to gradientanaylysis; DHT, 5a-dihydrotestosterone; ATR, atrazine; NON, nonylphenol.

Table Z Relative binding affinity (RBA) and

## IC50ofxenobiotics forsteriod receptors andbindingproteins Competitor Protein DHT E DDE oDDT Hg Hd ER

- 100 (2.7nM)

- 0.007 (40

### gM) AR 100(1.1 nM)

- 0.016 (6.8 IM)

- - ABP 100 (9nM)

- - - - 0.025(38

### gM) SHBG 100 (1.2nM)

- - - 0.0007 (160 M)

- The numbers inparentheses indicate themolar concentrations ofcompetitors thatreduced specific binding by 50% 

### (IC50); blanks indicate that these comparisons were not made. Abbreviations: DHT, 5a-dihydrotestosterone; E, estradiol; DDE, pp'-DDE; oDDT, op'-DDT; Hg, 

### hexachlorocyclohexane-y, Hd, hexachlorocyclohexane-6; ER, estrogen receptor; AR, androgen receptor; ABP, androgen-binding protein; SHBG, sexhormone-binding globulin. xenobiotics 

### facilitates orhinders this access. 

### Alternatively, it is possible that 

### those xeno

### biotics that result in a more 

### rapid dissociation rate 

### than 

### the 

### physiological ligand, for 

### example, nonylphenol with the estrogen receptor 

### and pentachlorophenol with SHBG, may 

### be 

### forming adducts with the 

### binding site preventing reassociation 

### ofthe 

### labeled 

### ligand or 

### causing 

### denaturation of 

### theproteins. 

### Both scenarioswould 

### result in
an apparent increase in 

### dissociation rate. 

### If 

### this is 

### the case, 

### the 

### effects must 

### be protein

### specific 

### because pentachlorophenol does not increase 

### the dissociation rate of 

### [3H] 

### 17J-estradiol from the estrogen receptor, nor 

### does 

### nonylphenol increase 

### the rate 

### of dissociation of 

### [3H]5a-DHT from SHBG. 

### Hexachlorocyclohexane-6 results in
a 

### slower rate 

### of dissociation of 

### [3H]5ccDHT 

### from ABP 

### than 

### does unlabeled 5aDHT, a 

### phenomenon that 

### would be 

### incompatiblewithdenaturation 

### ofthe protein

### bythexenobiotic. 

### Althoughwe cannot 

### discount the 

### possibility that the apparent 

### inhibition 

### ofbindingcausedbysomexeno

### biotics is 

### actually the result 

### of the 

### denaturation or 

### adduct 

### formation, the multiple point competition 

### data are consistent 

### with
our contention 

### that several xenobiotics compete

### with

### physiological ligands for sites
on

### steroid receptors 

### andbindingproteins. Our 

### data indicate that some 

### xenobiotics, 

### especially DDT congeners, can inter

### fere with the 

### binding ofnatural 

### ligands to two or more 

### binding moieties, 

### thus 

### ampli

### fying their potential 

### endocrine-disrupting 

### effects. These

### data

### indicating that a

### xenobiotic may

### affect

### multiple signalingpathways 

### coupled with 

### data 

### showing synergistic 

### effects of

### multiple xenobiotics- on estrogen responsive genes (23) 

### should reinforce the concept 

### that environmental 

### xenobiotics, though present at 

### low concentrations, may pose a

### threat to

### humanhealth.

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