A- ** * -~~
A Variety of Environmentally Persistent
Chemicals, Including Some Phthalate Plasticizers,
Are Weakly Estrogenic
~~~~~~~22Susan Jobling,1 Tracey Reynolds,’ Roger White, Malcolm G. Parker, and
John P. Sumpter1
1Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, UB8
3PH UK; 2Laboratory of Molecular Endocrinology, Imperial Cancer Research Fund,
London, WC2A 3PX UK
Sewage, a couple mie of organic and
inorgan icas, is conidered o be a
major c ni A
randomscr~e* of 20-orga mnmaechemi catsp snn liqu efira rkeead
tha hal apere bl. fintrc wihthe
estradiol receptorAThs was demonstrated
by their abilty to inhibit bindg of 17B-
estradiol toh*e fish estrogen receptor.
Furter: stdis usin mainnal estogen
srens:in vbw reeae thtte o pithain......:,......
.din-b. alate (DBP)d fd* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. .. .. ... ....
estrogen ocy.:eo Phth it sesiuseEd
in the p ctis:: &oifriohu ti i d-ing PVC are amn th ms comn
inutra c;emicals. Their ..bi..q~uity ’in the
environment and tendiency to hioconcen-
trate in animl ft are wel known Nitsher
BBP nor DBP wre abe to ac as antago-
nists, indiating that, inthe presence of
iwSoul be d;Ri bn
be etiologicuget in sevra huoa dis-
eae, inldn&iorders of the maerpro-
ductive trctad breast and testicua canl-
cer. Th urn idinWhtsm lta....A.. .....+:.
lateconipoaden some Idadiie are
:wealdyes agein s’irwnesa.betsup-w
ported b- f- r tu ies on,;i-;.-..r...................... ...nvieseor"t~ :any concusions cn e made
regarding their possible role in thie develop-
~~~~~~~~~~~’-P si ..... .... ....mentof hese con irion.K workibuyl
benytl ph a, butlad y ole,
din-utlptblae pithala*e, esrgnci-
ty ewg-2wrs ’HeAchy Perpt 103:.58......7....3
Over the last 50 years, large amounts of
some estrogenic man-made chemicals have
been released into the environment (1).
These chemicals include classical environ-
mental estrogens, such as o,p’-DDT and its
metabolites, methoxychlor, and many of
the polychlorinated biphenyls (PCBs).
More recently, chemicals originating from
the plastics and detergent industries, such
as alkylphenols (2,3) and bisphenol-A (4),
have been discovered to be estrogenic.
Evidence suggests that in many instances
the presence of these chemicals has had
deleterious effects on exposed wildlife pop-
ulations (5,6). Estrogens influence many
developmental and physiological responses
in target cells by regulating the activity of
specific genes. Their action is mediated by
a soluble intracellular receptor that func-
tions as a transcription factor (7) .
Estrogens have been shown to have multi-
ple sites of activity and exert biological
actions on the reproductive tract and the
mammary gland. They also influence the
neuroendocrine system (8) and have skele-
tal effects (9,10). Untimely exposure to
natural or synthetic estrogens can adversely
affect human health, particularly with
regard to the reproductive cycle and repro-
ductive function. In addition to decreased
sperm counts in men and increased inci-
dence of disorders of the male reproductive
tract (11,12), recent epidemiological stud-
ies suggest that cumulative exposure to
estrogenic chemicals is related to the inci-
dence of reproductive cancers (13).
As many of the estrogenic xenobiotics
discovered to date have an anthropogenic
source, the highest concentrations would be
expected to occur near urbanized or indus-
trial areas. Sewage is considered to be a
major input source of organic contaminants
into the environment. The release of liquid
effluents into the rivers and oceans, the dis-
posal of dry sludge onto the land, and the
release of volatile organics into the atmos-
phere all contribute to this source of pollu-
tion. This fact, coupled with the report that
sewage effluents are estrogenic (14),
increases the possibility that there may be
other estrogenic chemicals in the environ-
ment not yet discovered.
Extensive information exists on the
occurrence and concentrations of organic
micropollutants in raw, potable, and waste
waters (15,16), yet only about 3,000 man-
made organic compounds have been identi-
fied out of a probable 60,000 (17). The
sources of these compounds range from
domestic and industrial effluents and
leachates from solid waste disposal sites to
agricultural or urban run-off and atmospher-
ic fall-out. The range of compounds found
includes aliphatic and aromatic hydrocar-
bons, polycyclic aromatic hydrocarbons
(PAHs), halogenated hydrocarbons,
organochlorine pesticides, PCBs, and phtha-
late esters (18-21), all of which are present
in various environments at highly variable
concentrations. For example, phthalates are
present in waters at concentrations ranging
from nanograms to milligrams per liter. The
reasons for the reported variability in con-
centrations in the aquatic environment
include the use of different methodologies
for analysis, geographical variation, and vari-
ations in the source of the water sample
(e.g., influent, effluent, river).
The estrogenic activity of environmen-
tal chemicals has nearly always been dis-
covered because an estrogenic effect, either
in vivo or in vitro, has occurred upon
exposure to the chemical. With the excep-
tion of studies conducted by Soto et al.
(22), no systematic screening of chemicals
has been reported. Because the estrogenic
activities of various widely used industrial
chemicals continue to be discovered, it
seems likely that additional chemicals also
exhibit activity. Our interest in the aquatic
environment led us to test some of the
major chemicals present in sewage effluent
to determine whether any of these chemi-
cals are estrogenic.
Materials and Methods
Chemicals tested. We searched the scien-
tific literature (using the Institute of
Scientific Information database and also
government reports, both published and
unpublished) in order to discover what
chemicals had been reported to be present
in sewage effluents and at what concentra-
tions. None of these reports quantified all
of the chemicals present in effluent; many
of them tended to focus on one group of
chemicals rather than the whole range like-
ly to be present. It is not known how many
chemicals are present in effluent, although
the number is probably high.
Based on this literature search, we
made a list (Table 1) of selected man-made
chemicals present in sewage effluent. We
do not claim that this table is representative
of all sewage effluents, but the chemicals
listed are likely to be present at significant
concentrations in most effluents (see
Discussion for fuller explanation of this
point).
Fish studies. Because of their document-
ed presence in the aquatic environment, the
initial examination for estrogenicity was
carried out by measuring direct binding of
the chemicals to the fish estrogen receptor.
This initial screening process was both
rapid and economical and was carried out
using a cytosolic extract from the liver of
rainbow trout; it is well documented that
estradiol receptor-binding sites are present
here in both male and female fish (23).
Livers were removed from rainbow trout,
Address correspondence to S. Jobling, Department
of Biology and Biochemistry, Brunel University,
Uxbridge, Middlesex, UB8 3PH UK.
A portion of this work was financially supported
by the Department of the Environment, UK.
Received 23 March 1995; accepted 5 December
1994.
Environmental Health Perspectives582

Table 1. Compounds tested for estrogenic activity and their primary uses
Compound
Bis(2-ethylhexyl)phthalate (DEHP)
Benzophenone
Butylated hydroxyanisole (BHA)
Butyl benzyl phthalate (BBP)
n-Butylbenzene
p-tert-Butylbenzoic acid
Caffeine
Cholesterol
p-Cresol (4-methylphenol)
Butylated hydroxytoluene (BHT)
Di-n-butyl phthalate (DBP)
2,4 Dichlorophenol
3,4 Dimethylphenol
Bis-(2-ethylhexyl)adipate (DEHA)
p-Hydroxybenzoic acid
2-Methylphenol
Musk xylene
Musk ketone
4-Nitrotoluene
p-Toluene
frozen immediately in liquid nitrogen, and
subsequently stored at -800C until required.
They were then thawed and homogenized
on ice in 2.5 volumes of buffer (50 mM
TrisHCl, 0.1 mM EDTA, 10 mM sodium
molybdate, and 1 mM monothioglycerol,
pH 7.4). The homogenate was centrifuged
at 10,000g for 30 min at 20C to yield a
crude nuclear pellet and a crude cytosolic
supernatant. The cytosol was then incubat-
ed on ice for 30 min in the presence of dex-
tran-coated charcoal to remove any endoge-
nous steroids and then spun at 50,000g for
1 hr at 20C. The final supernatant was care-
fully aspirated, decanted, and a saturation
analysis was carried out on this cytosolic
extract to establish the concentration of
[2,3,7,-3H] 17f-estradiol (86 Ci/mmol) that
saturated the receptor preparation (generally
between 2 and 10 nM). Thereafter, cytosol
samples with a protein content of 2-5
mg/ml were incubated in triplicate with a
saturating concentration of 5 nM tritiated
17f9-estradiol, both alone and in the pres-
ence of competing ligands at a wide range of
concentrations (up to 1 mM). We removed
the unbound fraction by addition of char-
coal and specific binding was quantified [as
described by Pottinger (23)]. These experi-
ments were repeated at least three times.
Mammalian studies. Apart from their
presence in waters, many of the com-
Primary uses/sources
Plasticizer
Manufacture of insecticides and antihistamines;
fixative in strong perfumes (soaps, shampoo)
Antioxidant, especially in foods
Plasticizer, especially in the production of
vinyl floor tiles, adhesives and synthetic leather
Petrochemical origin
Plastics industry; corrosion inhibitor; in
polyester manufacture in dyeing
Drinks and pharmaceuticals
Excreted steroid, emulsifying agent
To produce antioxidants; UV stabilizer
Antioxidant in food, petrol products, rubbers,
plastics, and soaps
Plasticizer in food packaging, PVC, cellulosics
and certain elastomers; insect repellant
Fungicide and germicide products
Disinfectant/microbicide
Manufacture of plastics (PVC)
Cosmetic, food, and pharmaceutical preservative
Herbicide products, phenolic resins
Scent
Scent
Manufacture of dyes
Industrial solvent
pounds identified as putative environmen-
tal estrogens originate either from the diet
or from the human usage of plastics and
cosmetics, and therefore humans could be
exposed to them via many other routes. In
view of this potential for human exposure,
we tested several of the compounds fur-
ther using mammalian-based assays
employing two human breast cancer cell
lines in vitro, ZR-75 and MCF7.
Human breast cancer ZR-75 cells were
grown initially in phenol red-free
Dulbecco’s Modified Eagles Medium
(DMEM) supplemented with 10% (v/v)
charcoal-stripped fetal calf serum containing
no hormone for 7 days. They were then
transferred into medium containing no hor-
mone (NH), 10 nM 1713-estradiol (E2), 10
5 M octylphenol (OP), or 10-5M of each of
the environmental pollutants n-butylben-
zene, di-n-butyl phthalate (DBP), butylben-
zyl phthalate (BBP), 4-nitrotoluene, bis(2-
ethylhexyl)adipate (DEHA), 2,4-
dichlorophenol, benzophenone, butylated
hydroxyanisole (BHA), butylated hydroxy-
toluene (BHT), or bis(2-ethylhexyl)phtha-
late (DEHP). Cells were cultured for a fur-
ther 10 days and counted on days 0, 3, 6, 8,
and 10. All experiments were carried out in
duplicate and repeated twice.
To determine whether the estrogenic
compounds stimulated transcriptional
activity of the estrogen receptor directly, we
examined their effects on transiently trans-
fected MCF7 cells using the reporter plas-
mids pTKLUC and pERE-TKLUC.
MCF7 cells were plated to 80% confluence
in phenol red-free DMEM and 10% char-
coal-stripped fetal calf serum and transfect-
ed using the calcium phosphate coprecipi-
tation method, as previously described
(24). The reporter plasmid pTKLUC con-
tains the herpes simplex virus thymidine
kinase (TK) promoter from -105 to +55
inserted in the Bgl II site of the luciferase
reporter plasmid pGL2-Basic (Promega).
pERE-TKLUC contains a single copy of
the vitellogenin A2 estrogen response ele-
ment (ERE) inserted upstream of the TK
promoter in pTKLUC. The transfected
DNA included the reporter (0.8 pg) and an
internal control plasmid (pJ7LacZ; 0.2 jig).
After transfection, cells were maintained
with no hormone, E2, OP, BBP, DBP,
DEHP, BHA, or BHT at the concentra-
tions indicated. After 24 hr, the cells were
harvested, and extracts were assayed for
luciferase (25) and 9-galactosidase
(Galactolight, Tropix Inc, Bedford,
Massachusetts) activities. We used 9-galac-
tosidase to correct for differences in trans-
fection efficiency. All experiments were car-
ried out in duplicate and repeated at least
twice.
We also examined the possibility that
some of these chemicals might act as antag-
onists in the presence of 17f-estradiol. In
these experiments, MCF7 cells were trans-
fected with pERE-TKLUC and pJ7LacZ,
and then incubated with 10- M 17f3-
estradiol alone, simultaneously with DPB
or BBP, or simultaneously with the anti-
estrogens 4-hydroxytamoxifen (4-OHT) or
ICI 182780. Both the phthalates and the
antiestrogens were added at the concentra-
tions indicated. The experiment was carried
out in duplicate and repeated three times.
Results
Many of the compounds tested in this ini-
tial screen reduced the binding of the tritiat-
ed natural estrogen, 179-estradiol, to the
receptor. BBP, DBP, DEHP, DEHA, ben-
zophenone, n-butylbenzene, 4-nitrotoluene,
BHA, and 2,4-dichlorophenol reduced the
binding of tritiated 17f3-estradiol to the
receptor, although whether this inhibitory
effect was due to direct competition was not
determined. Concentrations as high as 1
mM may have approached the limits of sol-
ubility of some chemicals in the solvent sys-
tem used, as suggested by the observation
that some of the curves appeared to flatten.
In these cases, higher concentrations were
not tested and hence full displacement
curves were not obtained. No accurate esti-
mations of the affinities of these chemicals
for the receptor could be obtained because
Volume 103, Number 6, June 1995 583

A - *~~~~ *~~~
--
in most cases the displacement curves were
not parallel to that of 17f9-estradiol (Fig. 1).
Musk ketone, musk xylene, p-toluene,
BHT, caffeine, cholesterol, p-hydroxyben-
zoic acid, p-tert butylbenzoic acid, 3,4-
dimethylphenol, and 2-methylphenol did
not impair binding of tritiated estradiol to
the estradiol receptor (results not shown).
When the compounds were tested for
their mitogenic effects on cell growth at 10-
5 M, the three most potent were BBP,
DBP, and BHA (Fig. 2). Many of the other
compounds were either inactive or only
weakly active at concentrations in excess of
10-4 M. The growth responses to these
chemicals were all less than the maximal
responses shown by the natural estrogen
17f-estradiol and the environmental estro-
gen OP, which we have tested in this sys-
tem previously (26).
When tested for their ability to stimu-
late the transcriptional activity of the
estrogen receptor directly (Fig. 3), BBP
stimulated transcription at concentrations
in the range 10-6 to 10-4 M. DBP, and to a
lesser extent BHA, also stimulated tran-
scription at concentrations between 10-5
and 10- M (Fig. 3). Two closely related
compounds, DEHP (a phthalate) and
BHT (an antioxidant), did not stimulate
transcription to any appreciable degreeuntil concentrations in excess of 10- M
were reached. At these high concentra-
tions, the response to these latter two
chemicals was less than 15% of the maxi-
mum response obtained with estradiol
(results not shown).
OP stimulated transcription of the
reporter gene (LUC) to a similar extent as
17f3-estradiol (albeit at a concentration
1000-fold greater) and was used for com-
parison because it is a recognized environ-
mental estrogen (26). No ligand-dependent
transactivation was detected with any of the
compounds in transfections using the
reporter plasmid pTKLUC, which lacks the
consensus ERE (results not shown).
Of the 20 compounds initially tested
(Table 1), the action of the two most potent
compounds (the phthalates) was compared
with the action of two antiestrogens (4-
OHT and ICI 182780). The compounds
were tested for their ability to inhibit tran-
scription of the reporter caused by the pres-
ence of 17g-estradiol at concentrations of
10- M (Fig. 4) and 10-8 M (data not
shown). In view of the relative binding
affinities of the phthalates for the receptor
(Fig. 1), the lower concentration of 179-
estradiol used would allow competition by
the compounds in binding to the receptor.
In contrast to the two antiestrogens, which
inhibited the response in a dose-dependent
manner, DBP and BBP increased the tran-
scriptional activity of the receptor in the
presence of 10-11 M 17g-estradiol (Fig. 4).
100
50
o
._
._i.E
O _ _
00 1 1 1 1 5 4 310’ 10 10o’ 107 10’ 10, 104 103
U.
a4IL10
ISo
a.
0
100
50
0
100
50
A
0 %w
Agricultural IE*onI;. _ 1|
109 101 10 104 105 lO 10-Mnlo-"lo’10- o,0 107’ lo’ 10-5 lo’ 10 3 lo-u
Molar Concentration
Figure 1. Inhibitory effects of organic chemicals present in sewage effluent on the binding of tritiated
17-estradiol to the rainbow trout estrogen receptor. Butylbenzyl phthalate (BBP), di-n-butylphthalate
(DBP), bis(2-ethylhexyl)phthalate (DEHP), bis(2-ethylhexyl)adipate (DEHA), benzophenone (BP), n-butyl-
benzene (BB), 4-nitrotoluene (NT), butylated hydroxyanisole (BHA), and 2,4,dichlorophenol (DCP) reduced
the binding of tritiated 171-estradiol to the receptor. Musk ketone, musk xylene, p-toluene, butylated
hydroxytoluene (BHT), caffeine, cholesterol (CHO), p-hydroxybenzoic acid, p-tert butylbenzoic acid
(BBA), 3,4-dimethylphenol, and 2-methylphenol did not impair binding of tritiated estradiol to the estradiol
receptor (most results not shown). All experiments were repeated three times. The error bars are too
small and are therefore not shown on the figure.
4 6
Day
10
Figure 2. Mitogenic effects of various environmental chemicals on breast cancer cells. Cells were
exposed to no hormone (NH), 10 nM 17B-estradiol (E2), 10-5 M octylphenol (OP), or 10-5 M of each of the
environmental pollutants n-butylbenzene, di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), 4-nitro-
toluene, bis(2-ethylhexyl) adipate, 2,4-dichlorophenol, benzophenone, butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), or bis(2-ethylhexyl)phthalate. Cells were cultured for 10 days and count-
ed on days 0, 3, 6, 8, and 10. Only those compounds that enhanced cell growth are shown. All other com-
pounds did not enhance breast cancer cell growth at this concentration to any significant degree. All
experiments were carried out in duplicate and repeated twice. Similar results were observed in a repli-
cate experiment, although the number of cells used per well at the beginning of the experiment differed.
Mean values are presented from a single experiment.
Environmental Health Perspectives
lo-lo 10-9 104 10-7 to4 lo’s 104 10,3
584

A - ~.e.
-
. - - 9.
W,;>
i...
100
..
60
40
20
IU IV WU IV- IU WI IV IH IU I- IVU IV HU IU IU HU HU
Molar Concentration
Figure 3. Stimulation of transcriptional activity of the estrogen receptor by environmental chemicals. Cells
were maintained with no hormone (NH), 17B-estradiol (E2), octylphenol (OP), butylbenzyl phthalate (BBP),
di-n-butylphthalate (DBP), bis(2-ethylhexyl)phthalate (DEHP), butylated hydroxyanisole (BHA) or butylat-
ed hydroxytoluene (BHT) at the concentrations indicated. Transcriptional activity of the estrogen receptor
in the presence of environmental chemicals is expressed as a percentage of the maximum response
induced by 179-estradiol, and is presented as mean +/- SEM.
40
0
Figure 4. Estrogi
incubated with 1
phthalate (BBP)
the phthalates a
sented and the e
Discussion
Microbial deg
in sewage rest
ucts, many c
Some of these
intermediates
while others v
we do not kn(
and we are lef
the compoun
identified. Th
cals may repr
chemicals pr
tested, 9 redux
17g-estradiol
city, and it has been estimated that the
mean human intake of BHA averages 0.13
mg/kg body weight/day (27). Our studies
indicate that BHA is six or more orders of
magnitude less potent than 179-estradiol,
and hence causes stimulatory effects on
both the transcriptional activity of the
human estrogen receptor and the growth
of breast cancer cells in vitro only at con-
centrations of 10-5 M (2-3 ppm) and
above. However, it is impossible to make
predictions on its activity in vivo because
no such studies have been carried out.
BHA may bioconcentrate to a low degree
in humans, although it is not certain
whether the lack of full recovery of BHA
from urine after ingestion is due to bioac-
cumulation of intact BHA or its metabo-
lites or to unknown routes of biotransfor-
mation (28). Although it is reported to be
present in some sewage effluents, BHA is
not as ubiquitous as its chemical cousin
BHT, which was found to be even less
estrogenic than BHA.
In contrast, phthalates are the most
abundant man-made chemicals in the envi-
ronment (29). They are produced industri-
ally in large quantities, mainly to impart
flexibility into plastics, and can leach out
of these materials into water, soil, or food
over time. BBP is also used in the produc-
tion of vinyl floor tiles, adhesives, and syn-
thetic leather; DBP is more common as a
plasticizer in food-packaging materials,
PVC, the cellulosics, and certain types of
elastomers (30-32). Thousands of tons of
plastics are disposed of annually in landfill
sites, thus enabling phthalate esters to
migrate into groundwaters via the soil. The
ubiquity of these compounds in the aque-
ous environment is well known, and their
presence is reported in river, waste, and
lo-11 10-8 i 0-7 -4 ~~~~~~~~~~~drinking
waters as well as in fish and sedi-
lo-,llo-10’ 1o- 10- 10-5 10 ments (33-39). Commonly detected
Molar Concentration species include DBP, dimethyl phthalate,
diethyl phthalate, DEHP, di-n-octylphtha-
enic phthalates act as agonists, not antagonists, in the presence of estradiol. Cells were late, BBP, and DEHA (16).
10- M 171-estradiol alone, simultaneously with di-n-butylphthalate (DPB) or butylbenzyl We, no testeI, or simultaneously with the antiestrogens 4-hydroxytoluene (4-OHT) or ICI 182780. Both We have not tested many of these
nd the antiestrogens were added at the concentrations indicated. Mean values are pre- phthalates to determine whether any of
error bars represent the SEM. them are estrogenic (we have tested only
those phthalates listed in Table 1).
This initial screening process isolated a However, our results indicate that a com-
,radation of chemicals present subset of chemicals that were likely to be prehensive survey of the estrogenic activi-
ults in a wide range of prod- able to bind to the estrogen receptor, but it ties (if any) of all commonly used phtha-
)f which are unidentified. was not possible to determine whether lates would be justified. The general popu-
e products will be transient these chemicals were agonists or antago- nation may be exposed to these compounds
in the degradation process, nists. Using more specific tests, designed to via their diet, either from food contamina-
vill be more persistent. Thus, assess whether any of these chemicals were tion, or from food or drinks directly conta-
Dw exactly what is in effluent, estrogenic , we showed that three of these minated by plastic wraps containing
t with the task of testing only compounds had significant effects on phthalates, or from polluted drinking
Ids that have been positively transactivation of the estrogen receptor and water (31,34,40). In most cases, the great-
lis group of identified chemi- breast cancer cell growth. est exposure is from food. Levels of DBP
,esent only 20% of the total BHA is commonly used as an antioxi- in foods range from 50 to 500 pg/kg in the
esent. Of the 20 chemicals dant, particularly in foods. Therefore, its United States (41). A 1987 study in the
iced the binding of tritiated route of exposure to humans is likely to be UK estimated that the average intake of
to the fish estrogen receptor. mainly via ingestion. It has a low oral toxi- DBP of food packaged in cellulose film
Volume 103, Number 6, June 1995
120
Yb
U
0a.a
00
E
=
E
a.
30
20
10
._a
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io
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585

A -
was 230 pg/day (42. Indeed, up to 14 mg
DBP/kg was found in chocolate bars and
potato snacks wrapped in printed
polypropylene films (43).
In our studies, the phthalates DBP and
BBP were estrogenic in vitro at concentra-
tions between 10-6 and 10-4 M. However,
these figures cannot be used to predict
estrogenic activity in vivo. Because they are
lipophilic, all phthalates have a tendency to
accumulate in fatty tissues and can be
absorbed through human skin very effi-
ciently. However, once they are absorbed
or ingested, they may be metabolically
cleared from the body; little is known
about the absorption and metabolism of
phthalates. The oral toxicities of phthalate
compounds in humans are generally low
(30), although at high concentrations, they
are testicular toxicants. It has been suggest-
ed that the concentration of these com-
pounds (particularly DBP) in the cellular
fraction of sperm from adult men is nega-
tively correlated with either sperm density
or the total numbers of sperm (29).
Indeed, when administered to rats in high
doses phthalates are embryofetal toxicants
as well as testicular toxicants (44-48). In
the female rat, the primary effect on repro-
duction is spontaneous abortion and
decreased litter size. Recent studies on the
embryolethality of BBP have shown that
this effect is correlated with a lowering of
plasma progesterone levels (49), and it is
possible that this is a consequence of an
estrogenic effect.
It is well established that, upon binding
to 17g-estradiol, the estrogen receptor
binds to DNA as a homodimer and acti-
vates transcription of estrogen-responsive
gene products by means of two distinct
activational regions on the estrogen recep-
tor, AF1 in the N-terminal domain, which
is estrogen independent, and AF2 in the
estrogen-binding domain, which is active
only in the presence of estrogen (50-53).
The environmental estrogen OP mimics
this action exactly; it binds to the estrogen
receptor in the same region as 17g-estradi-
ol and induces full activation (26). In con-
trast, the antiestrogen/partial agonist
tamoxifen promotes DNA binding but
fails to induce the activity of AF2 and
hence causes only a submaximal effect due
to the constitutive activity ofAF1 (54-56).
Because none of the active compounds
listed in Table 1 could induce full activa-
tion, at least at the concentrations used,
the possibility that they may also be
antiestrogenic was considered. Indeed, the
potential for harmful effects of these chem-
icals on humans or animals will depend
not only on their agonistic activity, but
also on their potential to act as antagonists
in the presence of other environmental
estrogens and/or endogenous estrogens.
Antiestrogens such as tamoxifen and ICI
182780 inhibit the action of estrogens by
competing with 17g-estradiol for the estro-
gen receptor. In contrast, many of the
halogenated aromatic compounds and
dioxins such as TCDD have been shown
to be antiestrogenic in human breast can-
cer cells (54, but their action is mediated
by the Ah receptor rather than the estrogen
receptor. Similarly, the antiestrogenic
action of dietary estrogens, such as some
phytoestrogens, is thought to be controlled
by a nonestrogen receptor-mediated mech-
anism (58). Synthetic antiestrogens, which
do act through the estrogen receptor, have
been used in the treatment of estrogen-
responsive breast cancers for several years
(59). Antiestrogenic activity may be delete-
rious if it blocks the action of estrogen dur-
ing sexual differentiation or puberty. Our
results demonstrate that in vitro the phtha-
late compounds are acting as agonists only
and do not act as antiestrogens at any con-
centration throughout their active range.
Therefore, we suggest that rather than
being contra-active, they would enhance
the effects of endogenous estrogens if they
were present.
Nothing is known about either the
acute in vivo estrogenic effects or the possi-
ble chronic effects of phthalates on humans
or wildlife if administered at low concen-
trations over long periods of time. Prior to
this report, none of the chemicals we tested
had ever been described as estrogenic. The
fact that almost 50% of the compounds
initially tested were found to inhibit the
binding of tritiated estradiol to the fish
estrogen receptor is provocative. More sur-
prising is the fact that almost 30% of these
"inhibitory" chemicals can have significant
effects on transactivation of the receptor
and breast cancer cell growth.
The possible implications of this sce-
nario to man and wildlife will depend
entirely on the estrogenic potencies of
these chemicals in vivo; to a large extent
this will depend on the processes of meta-
bolic transformation and bioaccumulation.
In addition, the effects of simultaneous
exposure to a variety of estrogenic chemi-
cals should be investigated. Since all of the
estrogenic chemicals discovered to date are
lipophilic, they probably co-exist in fat and
body fluids of exposed individuals. Much
of the current literature suggests that envi-
ronmental estrogens may act cumulatively
and that measuring the total estrogenic
burden due to environmental contami-
nants may have more relevance than assess-
ing exposure by measuring levels of indi-
vidual estrogens alone (60,61). Estrogen-
responsive sites such as the reproductive
tract or neuroendocrine centers are highly
sensitive and hence it is possible that expo-
sure to many weakly active compounds
either persistently at low concentrations, or
acutely in high concentrations, may alter
the natural hormonal balance.
In conclusion, we have discovered that
a surprisingly large proportion of environ-
mentally persistent chemicals are weakly
estrogenic and thus have introduced the
possibility that there may be hundreds, or
even thousands, of chemicals in the envi-
ronment which possess some estrogenic
activity. Although the chemicals we tested
possess some common structural features
(such as a benzene ring), there is no obvi-
ous part of their molecular structure that
might be expected to enable binding to the
estrogen receptor, and hence one cannot
easily deduce which chemicals are and
which are not estrogenic. Aquatic organ-
isms are probably exposed to these weakly
estrogenic chemicals largely, if not exclu-
sively, via water. However, terrestrial ani-
mals (including humans) are probably
exposed via many routes. The concentra-
tions required to induce effects in vivo are
essentially unknown, particularly when an
organism is exposed simultaneously to a
cocktail of estrogenic chemicals. Even if
the combined effect of exposure to a num-
ber of chemicals is additive, there is no evi-
dence to suggest that the total concentra-
tion of estrogenic chemicals in humans or
animals is high enough to cause any effects
on estrogen-responsive tissues. However,
no studies have been carried out to exam-
ine this possibility.
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