Dibutyl Phthalate Contributes to the
Thyroid Receptor Antagonistic
Activity in Drinking Water Processes
N A L I , D O N G H O N G W A N G , Y I Q I Z H O U ,
M E I M A , J I A N L I , A N D Z I J I A N W A N G *
State Key Laboratory of Environmental Aquatic Chemistry,
Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, P.O. Box 2871 Beijing 100085, China
Received October 14, 2009. Revised manuscript received
July 6, 2010. Accepted July 9, 2010.
It has long been recognized that thyroid hormone (TH) is
essential for normal brain development in both humans and
animals, and there is growing evidence that environmental
chemicals can disrupt the thyroid system. In the present work,
we used a two-hybrid yeast assay to screen for agonistic or
antagonistic thyroid receptor (TR) mediated effects in drinking
waters. We found no TR agonistic, but TR antagonistic
activities in all samples from the drinking water processes.
were then calibrated regarding to a known TR-inhibitor, NH3,
and were expressed as the NH3 equivalents (TEQbio). The
observed TEQbio in waters ranged from 180.8 ( 24.8 to 280.2
for TR disrupting activities, the concentrations of potentially
(OCPs), phenols, and phthalates in organic extracts were
to NH3 (TEQcal) were estimated from their concentration-
dependent relationships, respectively, using the same set of
bioassays. Based on the TEQ approach, it was revealed that
dibutyl phthalate (DBP) accounted for 53.7 ( 8.2% to 105.5 (
16.7% of TEQbio. There was no effective removal of these
potential thyroid disrupting substances throughout drinking
water treatment processes.
In recent years, an increasing number of environmental
contaminants have been found to disrupt the endocrine
on estrogenic effect and far less attention has been paid to
identifying compounds with thyroid disrupting activity in
environment. However, in recent years, there has been an
increasing concern on the effects of synthetic chemicals on
in vivo and in vitro studies demonstrating that the thyroid
Thyroid hormones (THs) regulate growth, energy me-
of the central nervous system (3). Normal thyroid hormone
levels are essential for humans and the changes of normal
hormone levels can adversely affect pregnancy outcome,
phase, the neuropsychological development of a child can
be adversely affected, and may cause profound and irrevers-
ible damage to the newborn (5).
Increasing evidence from in vivo and in vitro studies
reported to affect the thyroid hormone system (6-8). This
hormone system disrupting chemicals can directly interfere
with thyroid receptors (TRs), either by decreasing normal
ligand (T3) binding or by providing additional ligands that
may bind to TRs (9). In our previous work, Li et al. (10)
developed a novel screening method for chemicals with TR
ant/agonistic properties using a yeast two-hybrid system,
and found a lot of chemicals such as polychlorinated
biphenyls (PCBs), flame retardants, phthalates, pesticides,
TR. Given that the major mechanism of TH action involves
T3 binding to TRs, resulting in tissue-specific activation/
repression of gene transcription (11), assessing the environ-
mental pollution interfering with TRs is of great importance.
Recently, EDCs are emerging as being of major concern
for water quality, as multiple EDCs have been detected in
enter drinking water sources and can be detected in human
bodies (13, 14), and endocrine system of human may be
affected (15). As it is frequently not possible to completely
over the fate of hormone disrupting chemicals during
drinking water treatment. Of these TR disrupting chemicals
mentioned above, some organochlorine pesticides (OCPs)
and phenols were detected in the drinking water in Beijing
in our previous work (17), and PAEs were reported to have
caused serious pollution in Beijing (18). Diethylhexyl ph-
thalate (DEHP) was detected in the groundwater in Beijing
up to 241.8 µg/L (19). Therefore, the aim of this study was
and try to identify the specific compounds responsible for
TR disrupting activities.
Materials and Methods
Chemicals. NH3 (>95%) was offered by Dr. Thomas S.
Scanlan. 3,3′,5-triiodo-L-thyronine (T3, 95%), desethyl amio-
darone hydrochloride (DAH, 98%), dimethyl sulfoxide (DMSO,
99.5%), dibutyl phthalate (DBP), diethylhexyl phthalate
dichlorodiphenyldichloroethane (p,p′-DDD, 99%), p,p′-
dichlorodiphenylethane (p,p′-DDE, 99.6%) were purchased
from Sigma Chemical (St. Louis, MO). Amiodarone hydro-
chloride (95%) was bought from Shanghai Pharmaceutical
(Shanghai, China). HPLC grade dichloromethane, hexane,
methanol, and tert-butyl methyl ether were purchased from
Fisher Scientific (Fair Lawn, NJ). 4-Aminophenol (4-AP,
97.5%) and 2,4-dichlorophenol (2,4-DCP, 99%) were pur-
chased from Acros Organics (Belgium).
(24 h) was conducted in May 2007 at a waterworks with a
total capacity of 1,500,000 m3/d located in Beijing, China.
Samples were collected from a reservoir that supplied the
source water located in Beijing and the effluents from the
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Environ. Sci. Technol. 2010, 44, 6863–6868
Published on Web 08/04/2010
2010 American Chemical SocietyVOL. 44, NO. 17, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY96863
coal and sand filtration, activated carbon filtration, and
Each of the water samples (20 L, 10 L for bioassay and 10
L for chemical analysis) of treatment processes mentioned
above and the source water were collected in precleaned
amber glass bottles. Prior to use the bottles were soaked
overnight by 10% nitric acid and soaked by chromic acid
solution for 30 min, then washed three times by double-
distilled water and methanol. The bottles were also washed
3 times with treatment process samples before sample
collection. An appropriate amount of methanol (2 mL/L)
was added in each sample right after sampling to suppress
possible biotic activities. Samples were stored at 4 °C prior
to treatment. All samples were treated within 48 h.
Water samples and procedure blanks (Mili-Q water,
conductivity of 18.2 Ω) were filtered with glass fiber filters
under vacuum at a flow rate of approximately 6 mL/min.
The cartridges were then kept under vacuum aspiration for
5 min to dry any residual water. The cartridges for bioassay
were washed with 5 mL of hexane/dichloromethane (7/3)
twice, 5 mL of tert-butyl methyl ether twice, 5 mL of
dichloromethane/methanol (9/1) twice, and 5 mL of metha-
nol at a flow rate of 1 mL/min; the extracts were then
with 10 mL of dichloromethane/methanol (9/1). Then the
water and evaporated to dryness in a rotary evaporator (R-
200, Buchi, France) at 40 °C to 2 mL. Then the dehydrated
extracts were blown to dryness under gentle nitrogen flow
and reconstituted in 0.1 mL of DMSO for bioassay im-
extracts were stored at -20 °C in glass vials. Details of
chemical analysis and quality control are described in the
Supporting Information (S1-S3).
Bioassay. The bioassays including agonistic activity test
and antagonistic activity test were conducted using yeast
were carried out in triplicate. Each assay group included the
sample, the positive control (T3for agonistic activity or T3+
and the procedural blank. Serial dilutions (5 µL) of test
samples were combined with 995 µL of medium containing
volume of DMSO did not exceed 1.0% of the total volume.
For determination of agonistic activities, the extracts were
tested in the absence of T3; and for antagonistic activities
they were tested in presence of 5 × 10-7mol/L of T3which
produced a submaximal stimulatory response (20). The
described by Gaido et al. (21).
The control assays of the yeast assay were performed for
the procedure blank. It can be seen in Figure 1 that all of the
induction or inhibition activities of procedure blanks were
the cell viability was determined spectrophotometrically as
a change of cell density (OD600nm) in the assay medium.
The procedural blank was tested in the same concentration
described before (10).
Causality Analysis. To identify the specific compounds
responsible for TR disrupting activities, potentially thyroid-
disrupting chemicals in the waters were quantified and
causality analysis was performed on the TEQ approach (22).
?-galactosidase expression by NH3 in presence of submaxi-
extracts were expressed according to the concentration-
dependent curve of NH3, which was then converted to its
water concentration and obtained the bioassay derived
to fit the linear part of the concentration-dependent curve
their toxic equivalents with respect to NH3 were calculated
from their concentrations in the water samples (TEQcal).
Relative potencies (REPs) of detected chemicals were cal-
culated. The contribution of each of the potential TR
antagonists to the TEQbiowas then estimated.
Results and Discussion
To choose a proper chemical as positive control, we tested
the NH3 and desethylamiodarone hydrochloride besides
amiodarone hydrochloride which had been tested in our
previous work (10). In the present study, NH3 and desethy-
lamiodarone hydrochloride (Figure 2A) both inhibited the
?-galactosidase activity induced by T3 in concentration-
dependent manners. NH3 and desethylamiodarone hydro-
chloride both inhibited less than 40% of induced activity by
T3(5 × 10-7mol/L) at concentration levels of 1 × 10-5mol/L
which are similar to amiodarone hydrochloride. NH3 was
reported to apparently interact with the TR ligand binding
domain and inhibit coactivator recruitment (23). Desethy-
FIGURE 1. Concentration-dependent relationships of thyroid
receptor (TR) disrupting effects of water extracts. The agonistic
activities (A) and antagonistic activities (B) of water extracts
were determined by the TR yeast bioassay and represented as
the percent induction and inhibition activity relative to the
maximum induced by 3,3′,5-triiodo-L-thyronine (T3, 5 × 10-7mol/
L). Values are presented as the average ( standard error (n )
3). A: source water, B: effluent of prechlorination, C: effluent of
coagulation, D: effluent of coal and sand filtration, E: effluent of
activated carbon, F: finished water after secondary chlorination.
0.5, 2, 7.8, 31.2, and 125 mean the concentration folds of the
6864 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 17, 2010
lamiodarone hydrochloride which is the metabolite of
amiodarone hydrochloride has been shown to be a non-
competitive inhibitor of TRs (24). Thus we chose NH3 as the
positive control in the present study.
In the present study, no TR agonistic activities were
observed even when the water samples were 125 times
concentrated (Figure 1A). However, all samples had TR
even when the water samples were 7.8 times concentrated
(Figure 1B.). The sample of procedure blank had no TR
disrupting activity as shown in Figure 1A and B. The TEQbio
of water sample was calculated according to the method in
the experimental section and ranged from 180.8 ( 24.8 to
280.2 ( 48.2 µg/L NH3. In the Supporting Information
(Figures S4-S6), it is shown that the drinking water extracts
have no general suppressive effects on TR gene expression
and the inhibition activities of the drinking water extracts
were specific to TR.
of OCPs, phenols, and phthalates in water extracts were
determined and their TEQcals were estimated. Concentra-
tions of OCPs and phenols in the water samples were below
DEHP, DEP, NH3, and DAH (A) and bioassay-derived NH3
equivalence of the samples’ antagonistic activities (NH3-EQbio)
(B). The chemicals determined by the yeast strain human
thyroid hormone receptor (hTR)-GRIP1 for thyroid hormone
receptor (TR) antagonistic activity. The chemicals’ antagonistic
activities are represented as the percent inhibition activities
relative to the maximum induced by 3,3′,5-triiodo-L-thyronine
(T3). Values are presented as the average ( standard error (n )
3). NH3-EQDBP denotes the toxic equivalents of DBP. DBP:
dibutyl phthalate, DEHP: diethylhexyl phthalate, DEP: diethy
phthalate, DAH: desethyl amiodarone hydrochloride. Footnotes
of A, B, C, D, E, and F are as the same as in Figure 1.
2. Concentration-dependentrelationships ofDBP,
TABLE 1. Concentrations of Chemicals in Drinking Water, Beijing, Chinaa
A (source water)
1.2 ( 0.1
3.6 ( 0.3
5.3 ( 0.5
0.6 ( 0.02
15.6 ( 0.8
7.1 ( 0.3
1.0 ( 0.1
3.4 ( 03
5.3 ( 0.5
0.7 ( 0.03
35.6 ( 0.2
9.6 ( 0.5
1.2 ( 0.1
4.5 ( 0.3
4.3 ( 0.3
5.3 ( 0.5
0.6 ( 0.02
17.5 ( 0.9
8.7 ( 0.4
1.3 ( 0.1
D (coal and sand filtration)
3.4 ( 0.3
5.3 ( 0.5
0.5 ( 0.02
28.7 ( 0.2
14.4 ( 0.7
1.0 ( 0.1
E (activated carbon)
3.4 ( 0.3
5.3 ( 0.5
0.5 ( 0.02
37.0 ( 2.0
6.9 ( 0.3
0.8 ( 0.1
F (secondary chlorination)
5.3 ( 0.5
0.6 ( 0.02
28.9 ( 1.5
8.5 ( 0.4
1.3 ( 0.1
1.0 × 10-4 b
2.0 × 10-5 b
1.10 × 10-6 b
2.00 × 10-7 b
3.80 × 10-8
2.40 × 10-6
0.2 × 10-3
0.1 × 10-3
0.3 × 10-3
0.6 × 10-3
0.6 × 10-3
0.3 × 10-3
0.9 × 10-3
1.8 × 10-3
diphenyltrichloroethane; p,p′-DDD: p,p′-dichlorodiphenyldichloroethane; o,p′-DDT: methoxychlo; DBP: dibutyl phthalate; DEHP: diethylhexyl phthalate; DEP: diethy phthalate; BBP:
benzyl butyl phthalate; RIC20: 20% relative inhibitory concentration; N.D.: not detected; N.C.: no consideration; “-”: no effect; REP: relative potencies ) RIC20 NH3/RIC20 compound x;
LODs: Limits of detection (S/N ) 3); LOQs: Limits of quantitation (S/N ) 9); RSD: relative standard deviation.bAccording to Li et al. (10).
VOL. 44, NO. 17, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 6865
types of chemicals may not account for the observed
antagonistic activities. Even the mixture toxicity should be
no TR antagonistic activities and both DBP and DEHP
demonstrated TR antagonistic activities in concentration-
dependent manners from 1 × 10-9mol/L to 1 × 10-4mol/L.
study were quite similar to those reported by Shen et al. (25)
The concentration of DBP and DEHP in water samples
ranged from 6.9 ( 0.3 to 37.0 ( 2.0 µg/L and was at least
1000-fold higher than the concentrations of OCPs and
phenols (Table 1). Through the quantitative data from
in waters for DBP ranged from 101.0 ( 5.4 to 239.9 ( 12.7
µg/L NH3 (Figure 2B), respectively. Based on the TEQ
approach, it was revealed that dibutyl phthalate (DBP)
accounted for 53.7 ( 8.2% to 105.5 ( 16.7% of TEQbio with
a correlation coefficient of r ) 0.84 (p < 0.05, Figure S3,
Supporting Information), while diethylhexyl phthalate (DEHP)
that DBP may play the major role in the TR antagonistic
activity in drinking water while DEHP also showed some
DBP and DEHP belong to a chemical family known as
phthalates which are synthetic compounds widely used as
polymer additives in plastics, polyvinyl chloride, rubber,
cellulose, and styrene production to improve their softness
can affect the thyroid system. In a TR reporter gene in vitro
assay using a recombinant Xenopus laevis cell line, DBP and
is a target of phthalate plasticisers that may cause thyroid
significantly and negatively correlated with urinary levels of
DBP and DEHP (31). Many studies found phthalates were
rapidly metabolized, and most of the phthalates and their
metabolites were cleared from the body within a few days
(32, 33). But one should consider that even transient
disruption of normal thyroid homeostasis will lead to
disastrous outcomes, especially in the developing nervous
system (5). And what is worse, DBP and DEHP can readily
cross the placenta and are developmental and reproductive
toxicants in laboratory animals (34, 35). At present, we have
no reliable information about whether the level of TR
antagonistic activity in drinking water can affect human
thyroid hormone system. Considering that T3 is always
present in vivo ranging from 13.9 to 26.4 nM (36), it is
suggested that ensuring the safe drinking water will be
Exposure to DBP and DEHP and other phthalates in the
general population is widespread (37). Recently, phthalates
were also detected in pooled breast milk samples from
American women (38) and in infant formula (39, 40). The
same thing has also happened in China, where widespread
samples has been reported in recent years (41, 42). Due to
the widespread application of phthalates, they have been
the most abundant compounds in various environmental
matrices compared to other organic contaminants in China
(43). In Beijing, 2.0 µg/L of diisobutylphthalate was found in
the finished drinking water (18). It was also reported that
DBP and DEHP were found up to 3.8 and 6.5 mg/kg in the
urban area of Beijing (44). In the present study, 15.6 µg/L
relatively high concentration of DBP and DEHP may be due
to that sampling was conducted in a dry season (May) in
Beijing. It was reported that DBP and DEHP were found up
to 8.3 and 5.9 µg/L in the dry season in raw drinking water
in Southern California whereas in wet season they were not
detected (45). It was reported that coagulation, flocculation,
and precipitation were largely ineffective for removing
dissolved organic contaminants (46). Although activated
carbon was reported to be effective for removing organic
effect for DBP and DEHP. It was reported that several
compounds were detectable in effluent from active carbon
filtration, and the removal efficiency using activated carbon
city and 76 µg/L in Hangzhou city in the finished drinking
water, which were higher than the raw water (48, 49). In
drinking water, which was little lower than the raw water
(45). Thus the high concentration of DBP and DEHP in the
finished water may be caused by contamination of source
processes for DBP and DEHP removal.
In the literature survey, only one study was conducted to
survey the TR agonistic activities in drinking water using
PC-DR-LUC method and it found no agonistic activity (50).
There was no work carried out on the survey of TR
antagonistic activities in drinking waters. Ishihara et al. who
used TR-mediated luciferase gene activation found strong
antagonistic activities in water samples from paper manu-
facturing plants (PMPs) (51). Gutleb et al. (52) reported that
sediment extracts showed TR antagonistic activities in the
presence of T3using T-screen method. In conclusion, this
water. DBP may be the major TR antagonist in the drinking
of our water sources by reducing thyroidal disruptive
pollutants from entering our waterways.
We appreciate the assistance from Dr. Thomas S. Scanlan
Research Program of China (2007CB407304), the National
Natural Science Foundation of China (20737003, 50778170),
and China MOST-U.S. EPA Collaborative Project on Safe
Supporting Information Available
Analytical details on analysis and bioassay confirmative
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