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org/est Article

Paraben Exposure Related To Purine Metabolism and Other

Pathways Revealed by Mass Spectrometry-Based Metabolomics
Hongzhi Zhao,§ Yuanyuan Zheng,§ Lin Zhu, Li Xiang, Yanqiu Zhou, Jiufeng Li, Jing Fang, Shunqing Xu,
Wei Xia,* and Zongwei Cai*
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ABSTRACT: Parabens are widely used as common preservatives

in the pharmaceutical and cosmetic industries. Exposure to
parabens has been found to be associated with metabolic
Downloaded via UNIV OF GLASGOW on July 18, 2020 at 08:25:24 (UTC).

alterations of human and an increased risk of metabolic disease,

such as diabetes. However, limited information is available about
metabolic pathways related to paraben exposure. In this study,
three parabens were determined in the urine samples of 88
pregnant women by using ultrahigh-performance liquid chroma-
tography coupled with triple quadrupole mass spectrometry
(UHPLC-QqQ MS). The samples were divided into different
groups based on tertile distribution of urinary paraben
concentrations. Metabolic profiling of the 88 urine samples was
performed by using UHPLC coupled with Orbitrap high-resolution MS. Differential metabolites were screened by comparing the
profiles of urine samples from different paraben-exposure groups. The identified metabolites included purines, acylcarnitines, etc.,
revealing that metabolic pathways such as purine metabolism, fatty acid β-oxidation, and other pathways were disturbed by parabens.
Eighteen and three metabolites were correlated (Spearman correlation analysis, p < 0.05) with the exposure levels of methyparaben
and propylparaben, respectively. This is the first MS-based nontargeted metabolomics study on pregnant women with paraben
exposure. The findings reveal the potential health risk of exposure to parabens and might help one to understand the link between
paraben exposure and some metabolic diseases.

■ INTRODUCTION
Parabens, widely used as preservatives,1 are ubiquitous in the
environment and have been detected in water,2 indoor dust,3
soil,4 and food.5 Humans may be exposed to parabens through
ingestion, inhalation, and absorption through the skin.
Methylparaben (MeP) and propylparaben (PrP) were detected
in more than 95% of urine samples in the general population,
indicating a worldwide spread of paraben exposure.6,7
However, as endocrine disruptors, parabens may interfere
with metabolism and cause endocrine function disorder. Both
in vitro and in vivo studies have shown the estrogenic or
antiandrogenic activities of parabens.8,9 Parabens may interfere
with metabolism and affect human health,10 particularly in
pregnant women because of their susceptibility to risk factors.
Epidemiological studies have shown that exposure to parabens
was associated with an increased risk of gestational diabetes
mellitus,11 type 2 diabetes,12 obesity,13 and possibly inflam-
mation14,15 and may be associated with adverse birth
outcomes.16 Unfortunately, the physiological mechanisms
that link the exposure to parabens to adverse health outcomes
were still not very well understood.
A metabolomics study, which focuses on changes in
metabolites and corresponding pathways, is an attractive tool
to investigate the metabolic state of an organism and products
of environmental conditions.17 Studying human metabolites in
body fluids with this approach may reflect the final outcomes
of functional alterations caused by the environment or diseases.
Recently, mass spectrometry (MS)-based metabolomics has
been used to search for toxicity biomarkers, particularly
metabolites related to environmental pollutant exposure. A
study in Sweden found that circulating levels of dichlor-
odiphenyldichloroethylene and hexachlorobenzene were asso-
ciated with the levels of 16 metabolites by using MS-based
nontargeted metabolomics.18 In another study, five potential
biomarkers in maternal urine were identified to be related to
cadmium exposure by using ultrahigh-performance liquid
chromatography (UHPLC) coupled with quadrupole/time-
of-flight high-resolution MS.19 The identified differential
metabolites may serve as potential biomarkers for under-
Received: December 15, 2019
Revised: February 9, 2020
Accepted: February 26, 2020
Published: February 26, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.est.9b07634

3447 Environ. Sci. Technol. 2020, 54, 3447−3454
Environmental Science & Technology
standing the underlying molecular mechanisms of the toxicity
of environmental pollutants. Despite the potential of
metabolomics studies in researching the toxicity of endo-
crine-disrupting chemicals, there has been no report of an MS-
based, nontargeted metabolomics study for human exposure to
parabens so far. Because of the toxicity of parabens, we
hypothesized that the differences in paraben exposure may lead
to metabolic differences between different paraben-exposure
groups at a molecular level and conducted a metabolomics
study to test the hypothesis.
In this study, urinary concentrations of parabens of 88
pregnant women in early pregnancy were measured. Metabolic
profiling differences between the low, medium, and high
urinary paraben groups of the study population were
investigated to analyze the putative metabolites and underlying
signaling pathways disturbed by parabens. The findings might
provide a better understanding of the health risk of paraben
exposure in pregnant women.
■ MATERIALS AND METHODS
Study Population and Sample Collection. The research
protocol was approved by the ethics committees of the Tongji
Medical College, Huazhong University of Science and
Technology. Healthy pregnant women were recruited in
Wuhan, China, during 2013−2015. The procedures were in
accordance with the Declaration of Helsinki. None of the 88
participants had diabetic nephropathy. All participants
provided their written informed consent. Morning urine
samples were collected from them at gestational weeks 10−
15 after overnight fasting. Face-to-face interviews were
conducted with the participants to collect information about
their socioeconomic characteristics (e.g., maternal age and
education) and lifestyle behaviors during pregnancy (e.g.,
smoking). The prepregnancy body mass indices (PBMIs) were
calculated using self-reported prepregnancy weights and
heights.
Quantitation of Parabens. The quantitation of three
urinary parabens, including MeP, ethylparaben (EtP), and PrP,
was performed following the procedures reported in our
previous study.20 Briefly, β-glucuronidase was used to convert
the conjugated form to the free form. Liquid−liquid extraction
was used for the sample preparation, and isotope-labeled
parabens (13C6-MeP, 13C6-EtP, and 13C6-PrP) were used as the
internal standards. Quality-control samples were analyzed to
monitor the performance of the method, and blank samples
were studied to monitor possible interferences.20 Instrumental
analysis was performed on an Ultimate 3000 UHPLC system
(Dionex, Sunnyvale, CA, U.S.A.) coupled with a Thermo
Scientific TSQ Quantiva Triple Quadrupole mass spectrometer
(Thermo Scientific, San Jose, CA, U.S.A.) in multiple reaction
monitoring mode. A heated electrospray ionization (HESI)
source was performed in negative ion mode. The mobile phase,
column, and parameters were used according to our previous
study.20
Metabolic Profiling Acquisition. To prepare samples for
metabolic profiling, 100 μL of urine samples were mixed with
100 μL of methanol with 4-chlorophenylalanine (2.5 μg/mL,
C9H10ClNO2), the internal standard for checking the shift of
MS response. After 1 min vortex and 10 min centrifugation at
15 000 rpm (4 °C), the supernatant was injected to the
UHPLC-Orbitrap MS for analysis.
The metabolic profiling analyses of the 88 urine samples
were performed by UHPLC coupled with a Q Exactive Focus
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Orbitrap high-resolution mass spectrometer (Thermo Scien-
tific, Bremen, Germany) as described in a previous study with
some modifications.21 The Waters ACQUITY UPLC HSS T3
column (100 mm × 2.1 mm, 1.8 μm) was used for UHPLC
separation. The mobile phase was 0.1% formic acid in water
(A) and acetonitrile (B). The elution gradient was as followed:
0−1 min, 2% B; 1−12 min, 2−55% B; 12−13 min, 55−64%;
14−15 min, maintain 100% B; then back to 2% B in 0.2 min.
The sample injection volume was 10 μL. The HESI source was
operated in positive electrospray ionization mode. MS was in
full-scan mode with a scan range from m/z 70 to 1 000 and
resolution 70 000. Other MS parameters are listed as follows:
sheath gas flow rate, 40 arb; auxiliary gas flow rate, 10 arb;
spray voltage, 3.5 kV; capillary temperature, 320 °C; S-lens RF
level, 55; and auxiliary gas heater temperature, 320 °C.
Quality-control samples were prepared by mixing 10 μL of
each of the 88 samples and analyzed at the beginning, after
every 10 injections, and at the end of every batch to monitor
the stability of the instrument.
Statistical Analysis of Paraben Exposure. The concen-
trations of parabens that fall below the limit of detection
(LOD) were assigned a value equal to the corresponding LOD
divided by the square root of 2.22 Specific gravity (SG) was
used to adjust for the dilution of maternal urine using the
formula “SG-adjusted concentration = unadjusted concen-
tration × [(1.0145 − 1)/(SGi − 1)]”.22 SGi represents the
individual SG of urine sample, and 1.0145 is the median SG.
All statistical analyses were performed using IBM SPSS
Statistics (version 20).
To investigate the metabolome alteration related to MeP
exposure, 88 pregnant women were divided into three groups
according to the tertile distribution of MeP concentration (SG-
adjusted) in the urine samples. The metabolic profiling data set
was correspondingly categorized into three groups, namely,
low-MeP, medium-MeP, and high-MeP groups. The same
strategy was adopted to compare the differences in metabolic
profiling of different EtP exposure groups and different PrP
exposure groups, respectively.
Metabolomics Data Analysis and Identification of
Differential Metabolites. The retention time−m/z pairs
(features) were extracted from each sample profile by using
XCMS package in R language. To improve data precision, the
signal drift correction was performed by using statTarget23,
which was according to quality-control-based random forest
signal correction. The intensities of the features were
normalized to total ion current (TIC) to adjust for variations
in urine dilution.
Mann−Whitney U tests were used to compare levels of the
features among different groups, and results with p values <
0.05 were considered statistically significant. The features with
at least one significant difference in the comparison of different
groups (high-MeP vs low-MeP, medium-MeP vs low-MeP,
high-EtP vs low-EtP, medium-EtP vs low-EtP, high-PrP vs low-
PrP, and medium-PrP vs low-PrP) were considered as
candidates for further identification.
Differential metabolites in urine samples were identified
based on accurate data of m/z values, MS/MS fragments, and
retention time. All the accurate m/z values were searched in
HMDB (http://www.hmdb.ca/), metlin (https://metlin.
scripps.edu/), and mzCloud (https://www.mzcloud.org/)
databases.21 The identified metabolites were confirmed with
standards whenever commercial standards were available.
Pathway network analyses were performed on Cytoscape
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3.5.1 with MetScape. Other analyses, such as Spearman

Abbreviations: PBMI, prepregnancy body mass index; SD, standard deviation; L-MeP, low-MeP group; M-MeP, medium-MeP group; H-MeP, high-MeP group; L-EtP, low-EtP group; M-EtP, medium-
28.73 ± 3.12
20.97 ± 2.77

13.01 ± 1.07
correlation analyses between the levels of parabens and

H-PrP
metabolites, were performed on IBM SPSS Statistics version

categories according to PrP level

20.

25

22
4

4
3
RESULTS

27.96 ± 3.66
21.00 ± 3.01

13.03 ± 1.09
Participant Characteristics. The demographic character-

M-PrP
istics of the study population are listed in Table 1. The mean
age of all participants was 28.43 ± 3.19 (mean ± SD) years

24

23
5

3
3
old, with 84% being primiparous women. Of the participants,
72 (81.8%) were well-educated (more than high school). None

28.57 ± 2.84
20.57 ± 2.77

12.98 ± 1.20
of them was smoking during pregnancy. The gestational weeks

L-PrP
of sample collection were 13.01 ± 1.11 weeks (mean ± SD).
There was no significant difference between low-, medium-,

25

27
5

3
0
and high-exposure groups of pregnant women on maternal age,
PBMI, and gestational week of sample collection.

28.47 ± 2.59
20.83 ± 2.59

12.96 ± 1.34
Paraben Exposure Levels. The concentrations of urinary

H-EtP
parabens are shown in Table 2. The detection rates for MeP,

categories according to EtP level

EtP, and PrP were >85%. The geometric means of SG-adjusted

23

25
3

2
2
concentrations of MeP, EtP, and PrP were 19.45, 1.28, and
1.18 ng/mL (Table 2), respectively. MeP was the most

20.92 ± 3.29

13.01 ± 0.95
28.50 ± 3.0
abundant paraben. MeP concentrations were stratified using

M-EtP
tertiles: lowest (<6.19 ng/mL), medium (6.42−56.91 ng/mL),
and highest (≥61.15 ng/mL) tertiles. The tertiles for EtP were

25

24
<0.47, 0.47−1.36, and ≥1.36 ng/mL, and the tertiles for PrP

4
1
were 0.23, 0.24−4.08, and ≥4.17 ng/mL.

28.33 ± 2.64

13.06 ± 1.06
Identification and Comparison of Metabolites among

20.7 ± 2.62
L-EtP

EtP group; H-EtP, high-EtP group; L-PrP, low-PrP group; M-PrP, medium-PrP group; H-PrP, high-MeP group.
Differential Paraben-Exposure Groups. A total of 60
differential metabolites were identified (Table S1), including

26

23
purine derivatives, amino acids, acylcarnines, and others.

4
3
Among them, 15 metabolites were confirmed by commercial
28.43 ± 3.19
21.27 ± 2.57

12.93 ± 1.12
standards, such as hypoxanthine, xanthine, uric acid, and
H-MeP

cortisol. Detailed information on identified metabolites, such

as accurate m/z, MS/MS fragment ions, and molecular
categories according to MeP level

25

24
formula, is shown in Table S1. As an example, the MS/MS
4

3
2
spectrum of hypoxanthine is shown in Figure S1. The
Table 1. Characteristics of Study Population (Mean ± SD or Number)a

comparison results are shown in Figures 1 and 2 and Tables

28.04 ± 3.43
20.68 ± 2.83

13.02 ± 1.02
M-MeP

S2−S7.
Compared with the low-MeP group (Tables S2 and S3), the
levels of 25 and 14 identified metabolites were significantly
24

25
5

2
2

higher (fold change > 1.2 and Mann−Whitney U test p < 0.05)
in high-MeP and medium-MeP groups, respectively. Interest-
28.37 ± 3.10
20.57 ± 3.08

13.07 ± 1.22

ingly, the fold change of hypoxanthine between high-MeP and

L-MeP

low-MeP groups was 1.45 (Figure 1b and Table S2), and the
xanthine level was higher in the medium-/low-MeP groups
25

23
5

5
2

(fold change 1.64, Table S3).

Compared with low-EtP group (Tables S4 and S5), the
28.43 ± 3.19
20.82 ± 2.81

13.01 ± 1.11

levels of 1 and 12 metabolites were significantly higher (p <

total

0.05) in high-EtP and medium-EtP groups, respectively. For

example, the level of uric acid, the product of purine
74
14

10

72
6

metabolism, was higher in the medium-/low-EtP groups

(fold change 1.93) (Figure 1b and Table S5).
gestational weeks of sample collection

Compared with the low-PrP group (Tables S6 and S7), the

levels of 8 and 2 metabolites were significantly higher in high-
PrP and medium-PrP groups, respectively. For example, the
characteristics

more than high school

level of hypoxanthine in the high-PrP group was higher

less than high school

compared with the low-PrP group with fold change 1.67

maternal age (years)

(Figure 1b and Table S6).

Correlation between Differential Metabolites and
primiparous
multiparous

high school
PBMI (kg/m2)

education (n)

Paraben Concentrations. Spearman’s rank correlation

parity (n)

coefficient between the levels of metabolites in urine and the

SG-adjusted concentrations of parabens was shown in Figure 3
and Table S8. The levels of 18 identified metabolites (such as
a

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Table 2. Urinary Concentrations of Parabens (Free Plus Conjugated) in Study Populationa

tertiles
concentration (ng/mL) LOD (ng/mL) DR (%) GM (95% CI) lowest middle highest
SG-adjusted MeP 0.05 98 19.45 (12.24, 30.92) <LOD-6.19 6.42−61.15 80.79−1284.53
SG-adjusted EtP 0.01 98 1.28 (0.80, 2.03) <LOD-0.46 0.47−1.36 1.71−3341.64
SG-adjusted PrP 0.05 89 1.18 (0.71, 1.97) <LOD-0.23 0.24−4.17 6.17−243.77
a
Abbreviations: CI, confidence interval; DR, detection rate; GM, geometric mean; SG, specific gravity.

Figure 2. Box plots of differential metabolites in different tertiles of

paraben levels.

three metabolites (trimethylamine N-oxide, dihydrobiopterin,

Figure 1. Metabolites in purine metabolism pathway. (a) Purine
metabolism pathway. (b) Box plots of differential metabolites in
different tertiles of paraben levels (* indicates p < 0.05; significance
test was analyzed with Mann−Whitney U test). (c) Spearman
correlation between the levels of MeP and metabolites in purine
metabolism pathway. Abbreviations: MeP, methylparaben; EtP,
ethylparaben; PrP, propylparaben.
hypoxanthine and 7-methylxanthine) were positively correlated
with MeP concentration (p < 0.05). The levels of benzoic acid
were correlated with EtP concentration, while the levels of
3450
and 2,6-dimethylheptanoyl carnitine) were correlated with PrP
concentration (p < 0.05 for all).
Altered Pathways Related to Paraben Exposure
Levels. As shown in Figure S2, purine metabolism, tyrosine
metabolism, histidine metabolism, and glycine, serine, alanine,
and threonine metabolism pathways were disturbed in the
comparison of high-MeP vs low-MeP (Figure S2) and
medium-MeP vs low-MeP (Figure S3).
Purine metabolism, tryptophan metabolism, steroid hor-
mone biosynthesis and metabolism, and other pathways were
enriched in the network of the comparison of medium-EtP vs
low-EtP (Figure S4). The purine metabolism as well as the
urea cycle and metabolism of arginine, proline, glutamate,
aspartate, and asparagine pathways were enriched in the
network of the comparison of high-PrP vs low-PrP (Figure S5).
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Figure 3. Spearman correlations between differential metabolite and
paraben levels. The bigger point represents a stronger correlation, and
p < 0.05 for all. The point in red represents a positive correlation,
while the point in blue represents a negative corrlation.
Overall, the purine metabolism pathway was disturbed by the
exposure of MeP, EtP, and PrP.
■ DISCUSSION
To the best of our knowledge, this is the first nontargeted
metabolomics study conducted on paraben exposure to
pregnant women. Paraben-related metabolites were identified.
Purine metabolism, tryptophan metabolism, and other path-
ways were found to be associated with the exposure to MeP,
EtP, and PrP.
In this study, higher detection frequencies (>85%) of three
parabens (MeP, EtP, and PrP) were observed, suggesting that
the participants were widely exposed to parabens. MeP was the
main component among the detected parabens. The maternal
urinary concentrations of parabens were similar to the finding
in our previous study.22 Urinary paraben concentrations of our
participants were also relatively lower than those in pregnant
women from the United States (n = 207),24 northern Puerto
Rico (n = 922),25 and Norway (n = 48).26
Energy metabolism-related metabolites were observed to be
altered in urine samples of high- or medium-exposure groups,
which indicated that the population with higher paraben
exposure was under the risk of energy metabolism disorders.
Our data suggested that exposure of parabens is associated
with disturbed purine metabolism pathways, which are
important in energy metabolism and play a role in the
development of diabetes.21 Xanthine, hypoxanthine, and uric
acid increased in response to paraben exposure (Tables S2, S5,
and S6), indicating that the purine metabolism was disrupted
by paraben exposure. The higher trend was also observed in
the level of xanthine (a metabolite formed from oxidation of
hypoxanthine by xanthine oxidoreductase) in the high-MeP
group versus the low-MeP group (Figure 1b), although there
was no statistical significance (Mann−Whitney U test p >
0.05). The correlation of the levels of MeP and 7-
methylxanthine (Figure 1c and Table S8), a metabolite of
xanthine, also suggested that purine metabolism was disturbed
by parabens. What is more, the level of uric acid, the end
product of purine metabolism, was higher in the medium-EtP
group than in the low-EtP group. Because the increase of uric
acid concentration is associated with a higher risk of insulin
resistance, a feature of diabetes,27 the observed upregulation of
uric acid related to EtP exposure might support the previously
reported finding that exposure to EtP in early pregnancy was a
risk factor to gestational diabetes mellitus.11 Besides, purine
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metabolism also plays a role in the development of obesity,28
which is a possible explanation of the obesogenic potential of
parabens.13 Therefore, the alteration of the purine metabolism
pathway might be one of mechanisms of paraben exposure
being related to some metabolic diseases, such as diabetes11
and obesity.28
Apart from affecting purine compounds, exposure of
parabens may also have led to increased risks of obesity or
diabetes through altering some other energy metabolism-
related metabolites and metabolic pathways, such as
acylcarnitines, valine, and others. Acylcarnitines are inter-
mediates in the key energy metabolic pathways of fatty acid β-
oxidation.29 The elevation of acylcarnitine (such as hepta-
noylcarnitine) levels related to paraben exposure (Tables S2
and S5−S7) indicated that paraben exposure might be
associated with disturbed energy metabolism. In addition, the
disorder of acylcarnitine metabolism, involved in mitochon-
drial lipid dysregulation, may cause an imbalance in insulin
synthesis and secretion30 and play roles in the development of
diabetes.31
In this study, we also found that exposure to parabens may
lead to an increased level of valine, an amino acid involved in
energy metabolism and essential for humans, as the valine
levels were higher in the medium-EtP group than in the low-
EtP group. The upregulation of the valine level has been
reported to be related with many diseases, such as hyper-
valinemia,32 pancreatic cancer,33 and diabetes.34 In addition to
the earlier-mentioned metabolites, pyrrolidonecarboxylic acid
is important in the intracellular transport of free amino acids,
and it has been reported that the concentration of
pyrrolidonecarboxylic acid was higher in participants with
impaired fasting glucose or type 2 diabetes.35 In the present
study, the upregulation of pyrrolidonecarboxylic acid (high-
MeP vs low-MeP groups) and a correlation between levels of
pyrrolidonecarboxylic acid and MeP were also observed,
suggesting that the glutathione metabolism pathway might be
affected by MeP exposure. Therefore, the findings might also
provide a possible explanation for the association with paraben
exposure and diabetes.
Our findings agree with the results from previous studies on
human cells and animals. A visible adverse effect of paraben on
the energy metabolism of HepG2 liver cells was reported.36
Animal experiments also suggested that paraben intoxication
can result in impaired cell energy metabolism.37 Taken
together, exposure to parabens may be associated with some
energy metabolism metabolites and metabolic pathways. Our
findings may provide a possible explanation of the association
between paraben exposure and diabetes or obesity.
Exposure to paraben may be linked to increased risk of
diabetes and obesity as its exposure disturbed the levels of
some other metabolites, such as 5-hydroxytryptophan. The
present study found that the level of 5-hydroxytryptophan,
formed by hydroxylation of tryptophan, was higher in the
medium-MeP group than in the low-MeP group. It was
reported that 5-hydroxytryptophan suppresses adaptive
thermogenesis in brown adipose tissue, and its exposure has
associations with obesity.38 In addition, 5-hydroxytryptophan
was reported to be a precursor of serotonin (5-hydroxytrypt-
amine), which may be associated with an increased risk of
obesity and type 2 diabetes in rats.39 Therefore, exposure to
parabens may have contributed to increased risks of obesity or
diabetes through altering the levels of 5-hydroxytryptophan.
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Environmental Science & Technology
Exposure to paraben may be linked to inflammation as its
exposure disturbed the levels of some other metabolites, such
as cortisol. It was found that levels of cortisol and
tetrahydrocortisone were lower in the medium-EtP group
compared with the low-EtP group. The decreasing trend of
cortisol induced by EtP exposure was similar to the result of a
study of pregnant women in Korea (n = 46),40 which found
that the concentrations of urinary cortisol were lower among
pregnant women with higher EtP levels within a day before
delivery. Low production of endogenous cortisol was related to
inflammation.41 In addition, previous studies found that the
urinary MeP concentration of pregnant women in the United
States (n = 482) was positively associated with pro-
inflammatory markers (IL-6).15 A study in zebrafish reported
that genes involved in inflammation could be affected by
parabens.42 Taken together, the exposure to parabens might be
associated with inflammation through the alteration of the
levels of cortisol and others.
Besides, exposure to paraben may be associated with
oxidative stress as its exposure disturbed the levels of
pyrrolidonecarboxylic acid and other endogenous metabolites.
In the present study, the level of pyrrolidonecarboxylic acid (5-
oxoproline), an indicator for oxidative stress, was disturbed by
exposure to all three parabens, indicating that women with a
high exposure to parabens might be under oxidative stress.
Urinary paraben concentrations in pregnant women in the
United States (n = 464) were also reported to be positively
associated with oxidative stress biomarkers, 8-hydroxydeox-
yguanosine (8-OHdG) and 8-isoprostane.43 In addition, a
previous study reported that parabens could induce oxidative
stress in zebrafish.42 Oxidative stress plays important roles in
the development of many diseases, such as diabetes44 and renal
fibrosis.45 Therefore, the participants with higher exposure to
parabens may be under the risk of adverse health outcome by
inducing the metabolites that are related to oxidative stress.
In the present study, it was interesting to find that more
altered metabolites were observed in the comparison of the
medium-EtP group vs the low-EtP groups than that of the
high-EtP group vs the low-EtP group comparison pairs. This
finding is similar to the previous studies suggesting that the
dose−response curve of the toxic endocrine-disrupting
chemicals is often not linear.46,47 Studies on persistent organic
pollutants have also shown the possibility of a nonlinear
relationship between exposure to persistent organic pollutants
and type 2 diabetes,48 and the risk of disease does not increase
as the dose increases.49 The nonlinear dose−response curves
of endocrine-disrupting chemicals are often U-shaped or
inverted U-shaped.47 A possible explanation is that chronic
exposure to a high dose may activate receptors that induce
changes in different pathways that produce opposing effects on
the same end point.47 The findings in this study that more
metabolites were altered in the medium-EtP group than in the
high-EtP group might be explained in a similar way.
One of the most notable strengths of our study is the sample
collection method, i.e., urine samples were collected during the
gestational weeks (weeks 10−15) at the same stages of
pregnancy. This approach rules out interference of endogenous
metabolites fluctuation due to the progression of pregnancy,
because physiological conditions and metabolomic profiles of
pregnant women in different gestational weeks are very
different. In addition, nontargeted metabolomics with quality
control provides a credible tool for the search of paraben
exposure-related metabolites. The principal limitation in our
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study is the relatively small sample size. The findings need to
be verified with studies of more participants. Another
limitation is the collection of one-spot samples with single-
exposure measurements without different time-point samples.
Considering the short half-life of parabens, collecting samples
at different points in time might further improve the accuracy
of estimating the exposure levels of the participants. Moreover,
a one-spot urine sample only can reflect a transient
metabolomic profile. Studies about metabolomic profiles in
other pregnancy stages altered by parabens would be needed in
the future. Lastly, information about the use of personal care
products and the diet of the study population was not
collected. Different exposure pathways may lead to different
toxicokinetics, which is difficult to identify from the
biomonitoring data. The present study focuses on the
screening of the alteration associated with paraben exposure.
The different toxicokinetics caused by different exposure
pathways should be investigated in the future.
In conclusion, the alterations of urinary metabolome in
pregnant women associated with paraben exposure were
investigated in this study by using an MS-based nontargeted
metabolomics approach. The findings suggested that exposure
to parabens in early pregnancy was associated with purine
metabolism, fatty acid β-oxidation, tryptophan metabolism,
and other pathways. This study highlights the evidence for the
linkage of paraben exposures and adverse health outcomes or
health risks, such as diabetes, energy metabolism disorder,
inflammation, and others. Although further confirmation is
needed in studies of other populations of a larger scale, our
findings provide novel insights into paraben toxicity. The
potential health risk associated with the exposure to paraben
on pregnant women, maybe even the general population,
should not be ignored.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.est.9b07634.
Detailed information on identified metabolites in
maternal urine; metabolites significantly different in
the comparison of different groups (high-MeP vs low-
MeP, medium-MeP vs low-MeP, high-EtP vs low-EtP,
medium-EtP vs low-EtP, high-PrP vs low-PrP, and
medium-PrP vs low-PrP); Spearman correlation coef-
ficient between metabolites and the level of each
paraben (MeP, EtP, and PrP); MS/MS spectrum of
hypoxanthine; metabolic network of the differential
metabolites in the comparison of different groups (high-
MeP vs low-MeP, medium-MeP vs low-MeP, high-EtP
vs low-EtP, medium-EtP vs low-EtP, high-PrP vs low-
PrP, and medium-PrP vs low-PrP) (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Wei Xia − Key Laboratory of Environment and Health, Ministry
of Education & Ministry of Environmental Protection, and State
Key Laboratory of Environmental Health (Incubation), School
of Public Health, Tongji Medical College, Huazhong University
of Science and Technology, Wuhan 430074, China;
orcid.org/0000-0001-6182-9806; Phone: +86-27-
83657705; Email: xiawei@hust.edu.cn; Fax: +86-27-
83657781
https://dx.doi.org/10.1021/acs.est.9b07634
Environ. Sci. Technol. 2020, 54, 3447−3454
Environmental Science & Technology
Zongwei Cai − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China; orcid.org/
0000-0002-8724-7684; Phone: +852-34117070;
Email: zwcai@hkbu.edu.hk; Fax: +852-34117348
Authors
Hongzhi Zhao − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Yuanyuan Zheng − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Lin Zhu − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Li Xiang − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Yanqiu Zhou − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Jiufeng Li − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Jing Fang − State Key Laboratory of Environmental and
Biological Analysis, Department of Chemistry, Hong Kong
Baptist University, Hong Kong SAR, China
Shunqing Xu − Key Laboratory of Environment and Health,
Ministry of Education & Ministry of Environmental Protection,
and State Key Laboratory of Environmental Health
(Incubation), School of Public Health, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan
430074, China; orcid.org/0000-0002-7771-3821
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.est.9b07634
Author Contributions
§
H.Z. and Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by National Natural Science
Foundation of China (21437002) and the General Research
Fund (12319716) and Collaborative Research Fund (C4024-
16W) from Research Grants Council of Hong Kong. Dr.
Simon Wang at the Language Centre of HKBU has improved
the linguistic presentation of the manuscript.
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