Reseamrh I

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 of the re utive em of inma and female an i te wild ha

attributed to environmental cemicls (xenobioii. The ects seen"Irror alteratio

might anticipate if the steroid hormone-dependent processes that regulate these system

impaired.To determine whther 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 -idsomso 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 recer that rsaned from 100%(pip'-DDE) to 25% (d ). Methox

toteD taeecpob1t6UL0ad7% Thed bind. of-.. H]... c-D "t .o o,p'.DD...B....~~~~~~~~~~~~~~~~~... ...... ....[..........

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 byjA po ately'40% Nonylphenol .reduced the bind

[3HJ5a-DHT to hSHBG by 70% H aclr n reduced [3H]5a-DHT bind

hSHBG by 20%, but tie e o wit ASP did n . op-Df

penthla.l reutd' arfa2%ihbhidn.Ife-Hl5a-DHT:.b -.... ....A ..I:. a:. :: ::..::.i

to hSHB$semCiii f f :HlFd:rt.hidibinding

that was staitcally ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~........... idnia :to tat........ causdb id ". ; ...th.... ..I.nlWdn_AFtiM ......... ....... .. . ,f_.... .. where

resulted in dower or more rapid d ion rates.4 mk age g protein,

gen receptor, estrogen receptor, sex hormonebindin globulin, xenobiotic. Environa

Perpect 105:294-301 (1997)

During the past 50 or more years, vast quantities of diverse synthetic chemicals (xenobiotics) have entered the environment because

of efforts to increase agricultural productivity

and because of modern industrial processes.

These chemicals include herbicides, insecticides, fungicides, styrenes, polychlorinated

biphenyls, and penta- to nonylphenols (1-3).

Some xenobiotics have been shown to disrupt

normal endocrine functions, which leads to

aberrant development of female and male

reproductive tissues and results in decreased

fertility or sterility (1-3). An early documented case concerning the effects of these

endocrine-disrupting agents on reproduction

was the report that newly hatched herring

gull chicks collected in the Lake Ontario area,

a region highly contaminated with the pesticide DDT, had altered reproductive systems

(4). Male chicks had oviducts and

resembling ovaries and the 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 of experimental 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 of these effects resemble those

u be that have been identified in the male offuns one spring of humans and animals that were

aS were treated during pregnancy with the potent

rt their synthetic estrogen diethylstilbestrol (DES),

iag of (15-18). Many of the environmental reproarogen 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), indiycr, cating that many common xenobiotics prefbiding erentially inhibit steroid binding to the

FI%t androgen receptor, require that the estro11d not gen-mimetic concept of xenobiotic action be

iduced expanded to hormone mimetics.

hag of Although many xenobiotics in the envilag to ronment have only weak 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 bioavailandin- 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

ystem of 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-disrupting effects of many

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 of Arna 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.

294 Volume 105, Number 3, March 1997 * Environmental Health Perspectives
Articles * 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 hormones is binding of the 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 of selected 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 of environmental xenobiotics may occur through multiple

steroid signaling pathways.

Materials and Methods

Selection of Xenobiotics

Xenobiotics were selected based on published data 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 for use.

Sources of Receptors 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 tissues were stored at -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 prepared as for the uterus. Cytosol prepared

from adult Sprague-Dawley rat epididymides

homogenized in TE buffer (1:4 wt/vol) was

the source of ABP. Postpartum human

serum obtained from Vanderbilt Hospital

(Nashville, TN) was the source of SHBG.

Sources ofXenobiotics and Steroids

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-Nethyl-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).

Binding Assays

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

for ABP and SHBG) with 7 nM [3H]ligand

alone (total binding) or together with a 100-

fold molar excess of the corresponding unlabeled ligand to determine nonspecific binding. All assay tubes were brought up to a

final volume of 0.5 ml with TE. Xenobiotics

were used as inhibitors at a concentration of

100 gM. This concentration is in the range

of those used by others 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 of ethanol (the same volume

in which the xenobiotics were dissolved) was

added to the total and nonspecific binding

tubes. Thus, 2% ethanol was present in each

0.5 ml incubation. This amount of ethanol

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 specific binding that occurred in the presence

of ethanol was set at 100% in those experiments in which data are plotted as a percentage of control.

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 samples). The difference in postcharcoal incubation time is to take into account the

rapid dissociation of ligand from ABP 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 of charcoal 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

previously described (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 or xenobiotic 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) of the xenobiotics as compared to the physiological

ligand, the IC50 for the physiological ligand

was divided by the IC50 of the xenobiotic

and the dividend was multiplied by 100.

Determination of dissociation halftime. Samples were incubated with 7 nM

[3H]physiological ligand alone or with a

100-fold molar excess of unlabeled 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 of control versus time. A regression line was fitted to the points and the

dissociation half-time (t1l2) was calculated

as previously described (27).

Statistical Analysis

The data were analyzed by one-way analysis

of variance followed by Duncan's multipleEnvironmental Health Perspectives * Volume 105, Number 3, March 1997 295
Articles * Danzo

range test (29). Results were considered significant when p<0.05. The data are presented as the mean and the standard error of the

mean.

Results

Studies on the Estrogen Receptor

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 inhibited estradiol binding to a small

(10%) but statistically significant 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, or atrazine.

Further evaluation of xenobiotic effects

on estradiol binding to uterine cytosol was

conducted using sucrose gradient analysis

(Fig. 2). When uterine cytosol was incubated

with 7 nM [3H] 170-estradiol alone, a single

peak of macromolecular-bound radioactivity

was detected. The peak had a sedimentation

coefficient of about 10 S, in reference to a

4.6 S bovine serum albumin (BSA) sedimentation standard, which corresponds to that of

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 data confirm that

the estradiol binding detected in uterine

cytosol is to the estrogen receptor. As in the

charcoal assay, nonylphenol was a highly

effective inhibitor of [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 of unlabeled

estradiol (35 jiM) to the estrogen receptor

preparation that had been equilibrated with

7 nM [3H] 17p-estradiol resulted in a timedependent decrease in the amount of

radioactivity associated with the receptor.

The t 1/2 of dissociation (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 jiM nonylphenol was used, dissociation of estradiol from its receptor was

more rapid (t112 approx 7 hr; Table 1).

Studies on the Androgen Receptor

In contrast to the minor effects of most of

the xenobiotics tested in inhibiting estradiol

binding to its receptor (Fig. 1), many xenobiotics 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 in which samples were assayed in triplicate.

1,400

1,400 _X *ENN !!

100

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, Number 3, March 1997 * Environmental Health Perspectives

t tUEIl Hg Hd ME pUDID DUE oDDT DIE

296
Articles - 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-DHT binding (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 a greater extent than did

0.7-,uM 5a-DHT. These data suggested

that binding moieties 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-DHT binding to

cytosol made from a batch of 15 prostates. A

single symmetrical peak of bound [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 that was not removed by the

charcoal extraction procedure. The 8-10 S

Table 1. The effect of various compounds on the half-time of dissociation of [3H]physiological ligand from its

receptor or binding protein.

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, sex hormone-binding globulin.

Blank spaces indicate that these comparisons were not made.

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 by the charcoal assay procedure. 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 of the mean are plotted. The numbers above the bars indicate the

number of experiments in which the samples were assayed in duplicate.

peak was eliminated when the sample was

incubated with [3H]5a-DHT and 0.7 ,uM

unlabeled 5a-DHT (Fig. 4A) and it was

gready reduced when samples were incubated

with 100 ,uM p,p'-DDE or 100-,uM p,p'-

DDT. No [3H]5a-DHT binding was detected in the 4 S region of the gradient where

specific or nonspecific binding to moieties

other than the receptor would be present.

Analysis of a batch of cytosol prepared

from 15 other prostates yielded the results

shown in Figure 4B. In this case, macromolecular-bound [3H] 5a-DHT was detected in

both the 4 S and 8 S regions of the 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 capacity prostate

binding protein reported by others (30) is

not known. The presence of this 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 similar pattern of dissociation of [3H]5ct-DHT

from the androgen receptor and a t 1/2 of

greater than 15 hr, a value that is in agreement with 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-DHT bound

to ABP (Fig. 5). The most effective xenobiotic inhibitor was the 6-isomer of hexachlorocyclohexane, 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 to ABP by 40-50% (Fig. 5).

p,p'-DDE reduced binding to ABP by 20%.

Estradiol inhibited [3H]5ac-DHT binding

to ABP by about 60%.

Sucrose gradient analysis indicated that

macromolecular-bound [3H]5ac-DHT in

epididymal cytosol sedimented at 4.6 S as

Environmental Health Perspectives * Volume 105, Number 3, March 1997 297
Articles - Danzo

would be expected for ABP (32). This

binding was completely inhibited by unlabeled 5a-DHT, reduced by hexachlorocyclohexane-6, but not affected by hexachlorocyclohexane-,y (Fig. 6).

When the 6-isomer of hexachlorocyclohexane was used in dissociation studies, we

noted that the dissociation rate (t 1/2 approx

70 min) was slower than the t 1/2 of about 7

min obtained when unlabeled 5a-DHT was

used (Table 1). This latter value is similar to

what we have previously reported (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 by sucrose

gradient ultracentrifugation, label was 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

did not (Fig. 8).

When dissociation studies were conducted, the addition of unlabeled 5a-DHT to

SHBG samples that had been equilibrated

with [3H] 5a-DHT resulted in a t 1/2 of dissociation of label of approximately 85 min, a

t1/2 similar to literature values (27).

Nonylphenol resulted in a similar dissociation

half-time, but pentachlorophenol resulted in a

more rapid dissociation rate (t 1/2 approx 5

min). Methoxychlor, which had no effect on

steroid binding to hSHBG, did not effect the

dissociation of [3H]5a-DHT from SHBG

during the time period studied (Table 1).

Concentration of Ligand Causing a

50% Inhibition of Binding

to Receptors and Binding Proteins

To further evaluate the affinity of xenobiotics 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

Fraction number

I,.

=

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

Fraction number

Figure 4. Analysis of [3H]5x-DHT binding to the androgen receptor using sucrose gradient ultracentrifugation. 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 batch of prostates was incubated with

[3HI5a-DHT alone, with unlabeled DHT, or with the indicated xenobiotics. (B) Cytosol from another batch

of prostates was incubated as in A.

tors. As indicated in Table 2, nanomolar

concentrations of unlabeled physiological ligand were able to reduce the binding of [H]

ligand to its corresponding receptor or binding protein by 50%. In contrast, micromolar

concentrations of xenobiotics 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 by Kelce 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 ligand binding to

ABP or SHBG by xenobiotics.

Discussion

Our experiments demonstrate that xenobiotics interact in a specific and differential

manner with the estrogen receptor, androgen receptor, ABP, and SHBG.

Several xenobiotics were more capable

of inhibiting [3H]5a-DHT binding to the

androgen receptor than to any of the other

proteins studied. This finding strongly suggests 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 of Kelce et al. (19) reports that

p,p'-DDE inhibits androgen binding to the

androgen receptor, androgen-induced transcriptional activity, and normal male prepubertal development. Our data indicate

that p,p'-DDE was the most potent of the

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

298 Volume 105, Number 3, March 1997 * Environmental Health Perspectives
E

0

40

20

Figure 5

didymid

3HJ5cz-E

clohexa

atrazine

standari

in which

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

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ABP, b

ospecif

produc

Articles X Xenobiotics and steroid receptors/binding proteins

110

100

80

60

1-l";;,binding to human SHBG.the other On

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 of xenobiotics

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 LitsLU u!xdetermined. ABP and SHBGwereln

~~~~~~~~~~~~~~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 of these proteins.

*DHT E DHT Hg Hd ME pDDT DDE oDDT DIE ATR PEN NON Of the xenobiotics tested, fewer interLInhibition of [3HI5a-DHT binding to androgen-binding protein (ABP; source, cytosol from rat epi- atdwtthe and rogen receptor orhABPNonyl

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

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 in triplicate. 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

0lid ~ 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 of which 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 Fraction number 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 of dissociaogenic (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 of the natur-

,ts of the- sam gene (35),bt they saitclysinfcn-niior feto lseri,wietecnfraino te

Environmental Health Perspectives * Volume 105, Number 3, March 1997 299
Articles - Danzo

100 l ~ 1-4 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 of experiments in which the samples were assayed in triplicate.

3,500

3,000 DHT

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

1,500

1,000

500

0 5 10 15 20 25

Fraction number

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

gradient anaylysis; DHT, 5a-dihydrotestosterone; ATR, atrazine; NON, nonylphenol.

Table Z Relative binding affinity (RBA) and IC50 of xenobiotics for steriod receptors and binding proteins

Competitor

Protein DHT E DDE oDDT Hg Hd

ER - 100 (2.7 nM) - 0.007 (40 gM)

AR 100 (1.1 nM) - 0.016 (6.8 IM) - -

ABP 100 (9 nM) - - - - 0.025(38 gM)

SHBG 100 (1.2 nM) - - - 0.0007 (160 M) -

The numbers in parentheses indicate the molar concentrations of competitors that reduced 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, sex hormone-binding globulin.

xenobiotics facilitates or hinders this access.

Alternatively, it is possible that those xenobiotics 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 of the

labeled ligand or causing denaturation of

the proteins. Both scenarios would result in

an apparent increase in dissociation rate. If

this is the case, the effects must be proteinspecific 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

incompatible with denaturation of the protein by the xenobiotic. Although we cannot

discount the possibility that the apparent

inhibition of binding caused by some xenobiotics 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 and binding proteins.

Our data indicate that some xenobiotics, especially DDT congeners, can interfere with the binding of natural ligands to

two or more binding moieties, thus amplifying their potential endocrine-disrupting

effects. These data indicating that a xenobiotic may affect multiple signaling pathways

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 human health.

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