Accepted Manuscript Title: Associations of cadmium, bisphenol A and polychlorinated biphenyl co-exposure in utero with placental gene expression and neonatal outcomes Author: Xijin Xu Yin Mei Chiung Fangfang Lu Shaoshan Qiu Minhui Ji Xia Huo PII: DOI: Reference:
S0890-6238(15)00016-7 http://dx.doi.org/doi:10.1016/j.reprotox.2015.02.004 RTX 7090
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
21-5-2014 2-2-2015 6-2-2015
Please cite this article as: Xu X, Chiung YM, Lu F, Qiu S, Ji M, Huo X, Associations of cadmium, bisphenol A and polychlorinated biphenyl co-exposure in utero with placental gene expression and neonatal outcomes, Reproductive Toxicology (2015), http://dx.doi.org/10.1016/j.reprotox.2015.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Associations of cadmium, bisphenol A and polychlorinated biphenyl co-exposure in
2
utero with placental gene expression and neonatal outcomes
3
Xijin Xua,b, Yin Mei Chiunga, Fangfang Lua, Shaoshan Qiua, Minhui Jia, Xia Huo a,* a
Laboratory of Environmental Medicine and Developmental Toxicology, and Provincial Key
ip t
4
Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University
6
Medical College, Shantou, Guangdong, China
Department of Cell Biology and Genetics, Shantou University Medical College, Shantou
us
8
b
Guangdong, China
an
7
cr
5
9 Abbreviations:
11
BPA, bisphenol A; Cd, cadmium; E-waste, electronic waste; GnRH, gonadotropin releasing
12
hormone; IGF, insulin-like growth factor; LRc, leptin receptor; PCBs, polychlorinated
13
biphenyls; UCB, umbilical cord blood.
te
d
M
10
Ac ce p
14 15
* Corresponding author:
16
Xia Huo
17
Laboratory of Environmental Medicine and Developmental Toxicology, Shantou University
18
Medical College
19
22 Xinling Rd., Shantou 515041, Guangdong, China
20
Tel: + 886 754 8890 0307
21
Fax: + 886 754 8856 7562
22
E-mail address: [email protected]
23 1
Page 1 of 30
Highlights
25
• Birth weight was negatively correlated with placental Cd and cord blood PCB levels.
26
• Positive associations were found between KISS1 expression and Cd, BPA, and PCBs.
27
• Gender effects on the expressions of KISS1 and leptin receptor were observed.
28
• Birth weight was negatively associated with KISS1 expression.
Ac ce p
te
d
M
an
us
cr
ip t
24
2
Page 2 of 30
29
ABSTRACT In utero co-exposure to endocrine disrupting compounds can perturb fetal development.
31
However, the effect of co-exposure on pivotal regulatory genes has seldom been investigated.
32
We explored the effects of in utero co-exposure to cadmium (Cd), bisphenol A (BPA) and
33
polychlorinated biphenyls (PCBs) on master regulator genes. We recruited 284 healthy
34
pregnant women, of whom 262 provided both cord blood and placenta samples, and 200 had
35
all measurements taken. Placental Cd, cord blood BPA and total PCBs in the exposed group
36
were higher than a reference group. KISS1 expression level in placental tissue was three-fold
37
higher in the exposed group than in the reference, and was positively associated with all
38
toxicants. Leptin and leptin receptor expression were also significantly higher, but were only
39
associated with BPA. From our findings, we conclude that lower birth weight is correlated
40
with Cd and PCBs, and may result from the increased KISS1 mRNA expression.
M
an
us
cr
ip t
30
Keywords:
42
Prenatal exposure; Birth outcomes; KISS1 expression; Endocrine disrupting compounds
Ac ce p
te
d
41
3
Page 3 of 30
43
1. Introduction Prenatal exposure to environmental pollutants has become a problem in many countries.
45
However, the role of key regulatory genes affecting fetal growth due to in utero exposure has
46
been rarely analyzed. With the rapid development of the electronic industry, electronic-waste
47
(e-waste) has become the most rapidly growing waste world-wide [1]. Among the various
48
techniques for recovery and recycling, informal processing of e-waste in developing countries
49
results in heavy environmental pollution that causes serious health problems [2, 3]. Guiyu,
50
China, is one of the largest e-waste destinations and recycling areas in the world. Children who
51
live there display elevated blood levels of pollutants, including heavy metals and organic
52
toxicants, and adverse health effects such as DNA damage and decreased pulmonary function
53
[4-7].
M
an
us
cr
ip t
44
Recently, adverse birth outcomes related to cadmium (Cd) and polychlorinated benzenes
55
(PCBs) have been found in neonates born in Guiyu [8, 9]. Overall, a significantly lower birth
56
weight is found in e-waste exposed groups [10], and correlates with the level of lead (Pb) in
57
cord blood. Regarding genomic effects, e-waste-exposed people have shorter telomere length
58
and lower S100P levels [11, 12], and higher expression of insulin-like growth factor (IGF)
59
genes [13].
te
Ac ce p
60
d
54
However, most of these studies are based on the observation of exposure to unitary
61
toxicants or a limited number of pollutants. In reality, circumstances involve co-exposure to
62
multiple chemicals and elements, making cause and effect extremely difficult to decipher. In
63
this study we attempted to survey a recurrent scenario by simultaneously monitoring internal
64
concentrations of diverse contaminants, including Cd, bisphenol A (BPA), and PCBs.
65
The effects on fetal development following exposure to single endocrine disrupting
66
compounds (EDCs) are reported. Cd affects human development via decreasing growth 4
Page 4 of 30
67
hormones, or altering hormones in the placenta, such as leptin and its receptor, which may
68
regulate fetal organogenesis and development [14, 15]. BPA can mimic estrogenic activity,
69
impacting various
70
gonadotropin-releasing hormone (GnRH) in pups [16]. Co-exposure to BPA and Cd at
71
environmentally relevant levels is also capable of interrupting endocrines [17]. PCBs can
72
mimic the biological action of thyroid hormones, and cause disorder of other endocrines [18].
73
Kisspeptins and leptin act through specific receptors to regulate IGF-dependent fetal growth
74
[19]. Moreover, Kisspeptins are master regulators of the gonadotropic axis, and are activated
75
in the fetus during late gestation [20]. As vital upstream regulators, Kisspeptins integrate
76
central and peripheral signals via GnRH release [21]. Kisspeptin neurons in the arcuate
77
nucleus co-express the leptin receptor [22], and have emerged as one of the major conduits in
78
relaying information, on body metabolic status, to GnRH neurons. Leptin in turn affects
79
KISS1 neurons, and impacts the reproductive system through regulating KISS1 expression
80
[23].
to
induce
KISS1
and
hypothalamic
te
d
M
an
us
cr
ip t
dopaminergic processes
Gene expression of IGF-1 in placental tissue has been shown to correlate with cord blood
82
polycyclic aromatic hydrocarbon (PAH) and polybrominated diphenyl ethers (PBDEs) levels
83
[13]. In this study, we evaluated the effects of multiple toxicant exposure on upstream genes
84
related to fetal development in utero. The levels of placental cadmium (Cd), cord blood BPA,
85
and PCBs were measured to characterize the complexity of in utero exposure. In order to
86
estimate the severity of health consequences, we compared the birth outcomes in an e-waste
87
exposed area with a reference area. To explore toxic effects on growth regulation, the
88
expression KISS1, leptin, and leptin receptor (LRc) genes in placenta were quantified. This
89
study firstly reported the effects on fetal growth-regulating genes and neonatal outcomes
90
following in utero co-exposure of heavy metals and chemicals.
91
2. Material and methods
Ac ce p
81
5
Page 5 of 30
92
2.1.
Subjects This prospective study was approved by the institutional review board and Human Ethics
94
Committee of Shantou University Medical College. We interviewed 284 healthy pregnant
95
women from September 2010 to September 2011. After obtaining informed consent and assent,
96
we collected placenta samples and cord blood samples from 262 mother-infant pairs. Of them,
97
192 pairs were recruited in an e-waste recycling town, Guiyu, Shantou City, and 70 from the
98
reference area, Haojiang, China. Haojiang is approximately 45 km away from Guiyu, and is
99
famous for its tourism and fishing. Its density of population and traffic, residential lifestyle,
100
cultural background and socioeconomic status are comparable to those of Guiyu [24]. Both
101
the nutritional status of the mothers and their dietary intake were judged as normal. Neonatal
102
anthropometric measurements were recorded by an obstetrician. The general flow chart for
103
the survey is shown as scheme 1.
Ac ce p
te
d
M
an
us
cr
ip t
93
104 105
Scheme 1. General flow chart for survey on contaminants and gene expression in placenta. 6
Page 6 of 30
106
2.2.
Placental sample preparation and Cd measurement. Placenta tissue was cut immediately after delivery. A piece was excised from the central
108
region, and then stored at -70 ºC until analysis. Fresh 0.1 g samples were rinsed in saline,
109
dried at 110 ºC, and then digested in 0.4 mL ultrapure concentrated nitric acid (80 ºC). The
110
dissolved samples were diluted with an equal volume of 30% hydrogen peroxide. After
111
shaking (0 ºC), ultrapure water was added to make the total volume 3 mL. Cadmium was
112
measured by graphite furnace atomic absorption spectrometry (GFAAS) as previously
113
described [4].
114
2.3.
us
cr
ip t
107
an
Measurement of BPA
BPA was measured by gas chromatography-mass spectrometry (GC/MS). Following
116
extraction of 20 mL cord blood serum with 2 mL dichloromethane, each extract was blown to
117
dryness under nitrogen. The dried extract was incubated in 50 μL N, O-bis (trimethylsilyl)
118
trifluoroacetamide for 30 min at 50 ºC for derivatization. BPA derivatives were dissolved
119
with acetonitrile and analyzed on a DB-5MS capillary column coupled with a mass
120
spectrometer (Agilent 5975C and Agilent 7890A, Agilent Technologies, USA), using helium
121
as the carrier gas (1.0 mL/min). The sample was injected in split less mode, at 250 ºC, and
122
subjected to the following oven temperature program: initial temperature 120 ºC, held for 3
123
min, then ramped at 20 ºC/min to 220 ºC, and held for 7 min. Ions of the BPA derivatives
124
were qualitatively observed at m/z 372, and quantified at m/z 357, in selected ion monitoring
125
mode (SIM) with electron impact ionization.
126
2.4.
Ac ce p
te
d
M
115
Analysis of PCB congeners
127
A standard PCB mixture (EC-5197), containing common PCB congeners CB 28, 52, 101,
128
138, 153, 180, and 209 (Cambridge Isotope Laboratories, USA), was used. All solvents and 7
Page 7 of 30
serum samples prepared were as previously described [9]. After extraction by solid-phase,
130
total lipids (TL) were calculated [25] by using the following formula that includes cholesterol
131
(CHOL) and triglycerides (TG): TL (g/l) = 1.12 CHOL + 1.33 TG + 1.48. Samples were
132
analyzed by GC/MS with similar equipment and columns as for BPA. The column
133
temperature was raised from 110 ºC to 200 ºC at a rate of 15 ºC/min, ramped from 200 ºC to
134
245 ºC at the rate of 5 ºC/min, held for 1 min, and then ramped to 290 ºC at the rate of 30
135
ºC/min. Using electron capture negative ionization, with a quadruple ion source, GC/MS
136
interface temperatures were set to 150 ºC, 230 ºC and 280 ºC, respectively. The target ions, in
137
SIM mode, were employed for quantification.
138
2.5.
cr
us
an
Gene expression analyses
We used placental tissue to monitor regulatory genes. Total RNA was isolated from 100
M
139
ip t
129
mg placental tissue using Trizol reagent (Invitrogen) according to the manufacturer’s
141
instructions. The quantity of RNA extracted was determined by OD260 measurements and
142
1.5-2.0 μg of total RNA was reverse-transcribed to cDNA by random primers as previously
143
described [13]. Real time PCR was conducted according to the manufacturer’s instructions
144
(Takara Biotech, Dalian, China) and SYBR Green was used to detect synthesized products by
145
using primers based on the human KISS1, leptin and LRc genes. Primers were designed by
146
SANGON Biotechnology Limited Company, Shanghai, China and the sequences for the
147
leptin gene were F:5'-CCTGACCTTATCCAAGATGG-3' and R:
148
5'-GAGTAGCCTGAAGCTTCCAG-3', for the LRc gene were F:
149
5'-GCCAACAACTGTGGTCTCTC-3' and R: 5'-AGAGAAGCACTTGGTGACTG-3';
150
KISS1 primers were F: 5'-ACTCACTGGTTTCTTGGCAGC-3' and R:
151
5'-ACCTTTTCTAATGGCTCCCCA-3 to give a 70 bp band; human β-actin primers were F:
152
5'-TGACGTGGACATCCGCAAAG-3' and R: 5'-CTGGAAGGTGGACAGCGAGG-3'.
153
β-actin was used as the loading control based on the previous studies conducted in human
Ac ce p
te
d
140
8
Page 8 of 30
placenta samples [26, 27]. The sample was denatured at 95 ºC for 30 sec. PCR for the leptin
155
gene was carried out using 40 cycles of 5 sec denaturation at 95 ºC, 30 sec annealing at 60 ºC,
156
then extension at 70 ºC, for 50 sec. The leptin and LRc genes gave bands of 216 bp and 238
157
bp. According to the dissociation curve, the Tm of the KISS1 gene product was 82 ºC.
158
Real-time PCR was performed under conditions of 30 sec at 95 ºC, then 40 cycles of
159
amplification (15 sec at 95 ºC, 30 sec at 60 ºC, 40 sec at 72 ºC). In each run, standard curves
160
and a negative control were included. Each sample was run with the internal control gene
161
(β-actin) and the mRNA expression levels for all genes were calculated by the 2-ΔCt method.
162
All samples were amplified in triplicate under the same conditions.
163
2.6.
an
us
cr
ip t
154
Questionnaires
An interview questionnaire was used to attain basic information, including maternal age,
165
diet during pregnancy (milk product consumption), education (years), medication during
166
gestation, parity of the fetus (1st, 2nd, or > 3rd), Apgar score (done by a trained nurse based on
167
examining the baby's breathing effort, heart rate, muscle tone, reflexes, and skin color at 10
168
minutes after birth), reproductive history, residential area and duration, smoking and drinking
169
habits, cooking fuel, and socio-economic status. The questionnaire included factors that might
170
influence toxicant levels in the placenta (whether their job was related to e-waste, the
171
husband's occupational history, house as an e-waste recycling workshop), as previously
172
described [28].
173
2.7.
Ac ce p
te
d
M
164
Statistical analyses
174
Non-parametric analyses were used for data with skewed distributions. Spearman rank
175
correlation analysis was used to evaluate the relationships between EDCs, birth length, birth
176
weight, KISS1, leptin and LRc expression. All analyses were conducted with SPSS statistical
177
software (version 13.0, SPSS, Inc., Chicago, IL, USA). To verify the influence of multiple 9
Page 9 of 30
independent variables on contaminants and biomedical effects, a multiple regression model
179
was used. The influence of an independent variable on another continuous dependent variable
180
was assessed by a linear regression model. All examination results that changed by age,
181
gender, and smoking were adjusted by direct methods.
182
3. Results
183
3.1.
cr
ip t
178
us
Descriptive statistics
Umbilical cord blood (UCB) samples and placental tissue were collected from 262 out of
185
284 participants; 22 were excluded for missing questionnaire data or without biological
186
samples. Levels of toxicant in the samples, as well as birth-related indices from the
187
completed questionnaires, were compared, and involved 192 women from the e-waste
188
recycling town and 70 women from the reference area. Because of insufficient sample
189
quantity, only 60 samples for KISS1 and 56 samples for leptin (receptor) gene expression in
190
the reference group were analyzed. Descriptive statistics are summarized in Table 1. The
191
backgrounds of two groups show no significant difference except house as an e-waste
192
recycling workshop. The exposed group had a longer gestational age and birth length (p
0.05 0.05 >0.05
S0890-6238(15)00016-7 http://dx.doi.org/doi:10.1016/j.reprotox.2015.02.004 RTX 7090
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
21-5-2014 2-2-2015 6-2-2015
Please cite this article as: Xu X, Chiung YM, Lu F, Qiu S, Ji M, Huo X, Associations of cadmium, bisphenol A and polychlorinated biphenyl co-exposure in utero with placental gene expression and neonatal outcomes, Reproductive Toxicology (2015), http://dx.doi.org/10.1016/j.reprotox.2015.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Associations of cadmium, bisphenol A and polychlorinated biphenyl co-exposure in
2
utero with placental gene expression and neonatal outcomes
3
Xijin Xua,b, Yin Mei Chiunga, Fangfang Lua, Shaoshan Qiua, Minhui Jia, Xia Huo a,* a
Laboratory of Environmental Medicine and Developmental Toxicology, and Provincial Key
ip t
4
Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University
6
Medical College, Shantou, Guangdong, China
Department of Cell Biology and Genetics, Shantou University Medical College, Shantou
us
8
b
Guangdong, China
an
7
cr
5
9 Abbreviations:
11
BPA, bisphenol A; Cd, cadmium; E-waste, electronic waste; GnRH, gonadotropin releasing
12
hormone; IGF, insulin-like growth factor; LRc, leptin receptor; PCBs, polychlorinated
13
biphenyls; UCB, umbilical cord blood.
te
d
M
10
Ac ce p
14 15
* Corresponding author:
16
Xia Huo
17
Laboratory of Environmental Medicine and Developmental Toxicology, Shantou University
18
Medical College
19
22 Xinling Rd., Shantou 515041, Guangdong, China
20
Tel: + 886 754 8890 0307
21
Fax: + 886 754 8856 7562
22
E-mail address: [email protected]
23 1
Page 1 of 30
Highlights
25
• Birth weight was negatively correlated with placental Cd and cord blood PCB levels.
26
• Positive associations were found between KISS1 expression and Cd, BPA, and PCBs.
27
• Gender effects on the expressions of KISS1 and leptin receptor were observed.
28
• Birth weight was negatively associated with KISS1 expression.
Ac ce p
te
d
M
an
us
cr
ip t
24
2
Page 2 of 30
29
ABSTRACT In utero co-exposure to endocrine disrupting compounds can perturb fetal development.
31
However, the effect of co-exposure on pivotal regulatory genes has seldom been investigated.
32
We explored the effects of in utero co-exposure to cadmium (Cd), bisphenol A (BPA) and
33
polychlorinated biphenyls (PCBs) on master regulator genes. We recruited 284 healthy
34
pregnant women, of whom 262 provided both cord blood and placenta samples, and 200 had
35
all measurements taken. Placental Cd, cord blood BPA and total PCBs in the exposed group
36
were higher than a reference group. KISS1 expression level in placental tissue was three-fold
37
higher in the exposed group than in the reference, and was positively associated with all
38
toxicants. Leptin and leptin receptor expression were also significantly higher, but were only
39
associated with BPA. From our findings, we conclude that lower birth weight is correlated
40
with Cd and PCBs, and may result from the increased KISS1 mRNA expression.
M
an
us
cr
ip t
30
Keywords:
42
Prenatal exposure; Birth outcomes; KISS1 expression; Endocrine disrupting compounds
Ac ce p
te
d
41
3
Page 3 of 30
43
1. Introduction Prenatal exposure to environmental pollutants has become a problem in many countries.
45
However, the role of key regulatory genes affecting fetal growth due to in utero exposure has
46
been rarely analyzed. With the rapid development of the electronic industry, electronic-waste
47
(e-waste) has become the most rapidly growing waste world-wide [1]. Among the various
48
techniques for recovery and recycling, informal processing of e-waste in developing countries
49
results in heavy environmental pollution that causes serious health problems [2, 3]. Guiyu,
50
China, is one of the largest e-waste destinations and recycling areas in the world. Children who
51
live there display elevated blood levels of pollutants, including heavy metals and organic
52
toxicants, and adverse health effects such as DNA damage and decreased pulmonary function
53
[4-7].
M
an
us
cr
ip t
44
Recently, adverse birth outcomes related to cadmium (Cd) and polychlorinated benzenes
55
(PCBs) have been found in neonates born in Guiyu [8, 9]. Overall, a significantly lower birth
56
weight is found in e-waste exposed groups [10], and correlates with the level of lead (Pb) in
57
cord blood. Regarding genomic effects, e-waste-exposed people have shorter telomere length
58
and lower S100P levels [11, 12], and higher expression of insulin-like growth factor (IGF)
59
genes [13].
te
Ac ce p
60
d
54
However, most of these studies are based on the observation of exposure to unitary
61
toxicants or a limited number of pollutants. In reality, circumstances involve co-exposure to
62
multiple chemicals and elements, making cause and effect extremely difficult to decipher. In
63
this study we attempted to survey a recurrent scenario by simultaneously monitoring internal
64
concentrations of diverse contaminants, including Cd, bisphenol A (BPA), and PCBs.
65
The effects on fetal development following exposure to single endocrine disrupting
66
compounds (EDCs) are reported. Cd affects human development via decreasing growth 4
Page 4 of 30
67
hormones, or altering hormones in the placenta, such as leptin and its receptor, which may
68
regulate fetal organogenesis and development [14, 15]. BPA can mimic estrogenic activity,
69
impacting various
70
gonadotropin-releasing hormone (GnRH) in pups [16]. Co-exposure to BPA and Cd at
71
environmentally relevant levels is also capable of interrupting endocrines [17]. PCBs can
72
mimic the biological action of thyroid hormones, and cause disorder of other endocrines [18].
73
Kisspeptins and leptin act through specific receptors to regulate IGF-dependent fetal growth
74
[19]. Moreover, Kisspeptins are master regulators of the gonadotropic axis, and are activated
75
in the fetus during late gestation [20]. As vital upstream regulators, Kisspeptins integrate
76
central and peripheral signals via GnRH release [21]. Kisspeptin neurons in the arcuate
77
nucleus co-express the leptin receptor [22], and have emerged as one of the major conduits in
78
relaying information, on body metabolic status, to GnRH neurons. Leptin in turn affects
79
KISS1 neurons, and impacts the reproductive system through regulating KISS1 expression
80
[23].
to
induce
KISS1
and
hypothalamic
te
d
M
an
us
cr
ip t
dopaminergic processes
Gene expression of IGF-1 in placental tissue has been shown to correlate with cord blood
82
polycyclic aromatic hydrocarbon (PAH) and polybrominated diphenyl ethers (PBDEs) levels
83
[13]. In this study, we evaluated the effects of multiple toxicant exposure on upstream genes
84
related to fetal development in utero. The levels of placental cadmium (Cd), cord blood BPA,
85
and PCBs were measured to characterize the complexity of in utero exposure. In order to
86
estimate the severity of health consequences, we compared the birth outcomes in an e-waste
87
exposed area with a reference area. To explore toxic effects on growth regulation, the
88
expression KISS1, leptin, and leptin receptor (LRc) genes in placenta were quantified. This
89
study firstly reported the effects on fetal growth-regulating genes and neonatal outcomes
90
following in utero co-exposure of heavy metals and chemicals.
91
2. Material and methods
Ac ce p
81
5
Page 5 of 30
92
2.1.
Subjects This prospective study was approved by the institutional review board and Human Ethics
94
Committee of Shantou University Medical College. We interviewed 284 healthy pregnant
95
women from September 2010 to September 2011. After obtaining informed consent and assent,
96
we collected placenta samples and cord blood samples from 262 mother-infant pairs. Of them,
97
192 pairs were recruited in an e-waste recycling town, Guiyu, Shantou City, and 70 from the
98
reference area, Haojiang, China. Haojiang is approximately 45 km away from Guiyu, and is
99
famous for its tourism and fishing. Its density of population and traffic, residential lifestyle,
100
cultural background and socioeconomic status are comparable to those of Guiyu [24]. Both
101
the nutritional status of the mothers and their dietary intake were judged as normal. Neonatal
102
anthropometric measurements were recorded by an obstetrician. The general flow chart for
103
the survey is shown as scheme 1.
Ac ce p
te
d
M
an
us
cr
ip t
93
104 105
Scheme 1. General flow chart for survey on contaminants and gene expression in placenta. 6
Page 6 of 30
106
2.2.
Placental sample preparation and Cd measurement. Placenta tissue was cut immediately after delivery. A piece was excised from the central
108
region, and then stored at -70 ºC until analysis. Fresh 0.1 g samples were rinsed in saline,
109
dried at 110 ºC, and then digested in 0.4 mL ultrapure concentrated nitric acid (80 ºC). The
110
dissolved samples were diluted with an equal volume of 30% hydrogen peroxide. After
111
shaking (0 ºC), ultrapure water was added to make the total volume 3 mL. Cadmium was
112
measured by graphite furnace atomic absorption spectrometry (GFAAS) as previously
113
described [4].
114
2.3.
us
cr
ip t
107
an
Measurement of BPA
BPA was measured by gas chromatography-mass spectrometry (GC/MS). Following
116
extraction of 20 mL cord blood serum with 2 mL dichloromethane, each extract was blown to
117
dryness under nitrogen. The dried extract was incubated in 50 μL N, O-bis (trimethylsilyl)
118
trifluoroacetamide for 30 min at 50 ºC for derivatization. BPA derivatives were dissolved
119
with acetonitrile and analyzed on a DB-5MS capillary column coupled with a mass
120
spectrometer (Agilent 5975C and Agilent 7890A, Agilent Technologies, USA), using helium
121
as the carrier gas (1.0 mL/min). The sample was injected in split less mode, at 250 ºC, and
122
subjected to the following oven temperature program: initial temperature 120 ºC, held for 3
123
min, then ramped at 20 ºC/min to 220 ºC, and held for 7 min. Ions of the BPA derivatives
124
were qualitatively observed at m/z 372, and quantified at m/z 357, in selected ion monitoring
125
mode (SIM) with electron impact ionization.
126
2.4.
Ac ce p
te
d
M
115
Analysis of PCB congeners
127
A standard PCB mixture (EC-5197), containing common PCB congeners CB 28, 52, 101,
128
138, 153, 180, and 209 (Cambridge Isotope Laboratories, USA), was used. All solvents and 7
Page 7 of 30
serum samples prepared were as previously described [9]. After extraction by solid-phase,
130
total lipids (TL) were calculated [25] by using the following formula that includes cholesterol
131
(CHOL) and triglycerides (TG): TL (g/l) = 1.12 CHOL + 1.33 TG + 1.48. Samples were
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analyzed by GC/MS with similar equipment and columns as for BPA. The column
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temperature was raised from 110 ºC to 200 ºC at a rate of 15 ºC/min, ramped from 200 ºC to
134
245 ºC at the rate of 5 ºC/min, held for 1 min, and then ramped to 290 ºC at the rate of 30
135
ºC/min. Using electron capture negative ionization, with a quadruple ion source, GC/MS
136
interface temperatures were set to 150 ºC, 230 ºC and 280 ºC, respectively. The target ions, in
137
SIM mode, were employed for quantification.
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2.5.
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Gene expression analyses
We used placental tissue to monitor regulatory genes. Total RNA was isolated from 100
M
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ip t
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mg placental tissue using Trizol reagent (Invitrogen) according to the manufacturer’s
141
instructions. The quantity of RNA extracted was determined by OD260 measurements and
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1.5-2.0 μg of total RNA was reverse-transcribed to cDNA by random primers as previously
143
described [13]. Real time PCR was conducted according to the manufacturer’s instructions
144
(Takara Biotech, Dalian, China) and SYBR Green was used to detect synthesized products by
145
using primers based on the human KISS1, leptin and LRc genes. Primers were designed by
146
SANGON Biotechnology Limited Company, Shanghai, China and the sequences for the
147
leptin gene were F:5'-CCTGACCTTATCCAAGATGG-3' and R:
148
5'-GAGTAGCCTGAAGCTTCCAG-3', for the LRc gene were F:
149
5'-GCCAACAACTGTGGTCTCTC-3' and R: 5'-AGAGAAGCACTTGGTGACTG-3';
150
KISS1 primers were F: 5'-ACTCACTGGTTTCTTGGCAGC-3' and R:
151
5'-ACCTTTTCTAATGGCTCCCCA-3 to give a 70 bp band; human β-actin primers were F:
152
5'-TGACGTGGACATCCGCAAAG-3' and R: 5'-CTGGAAGGTGGACAGCGAGG-3'.
153
β-actin was used as the loading control based on the previous studies conducted in human
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placenta samples [26, 27]. The sample was denatured at 95 ºC for 30 sec. PCR for the leptin
155
gene was carried out using 40 cycles of 5 sec denaturation at 95 ºC, 30 sec annealing at 60 ºC,
156
then extension at 70 ºC, for 50 sec. The leptin and LRc genes gave bands of 216 bp and 238
157
bp. According to the dissociation curve, the Tm of the KISS1 gene product was 82 ºC.
158
Real-time PCR was performed under conditions of 30 sec at 95 ºC, then 40 cycles of
159
amplification (15 sec at 95 ºC, 30 sec at 60 ºC, 40 sec at 72 ºC). In each run, standard curves
160
and a negative control were included. Each sample was run with the internal control gene
161
(β-actin) and the mRNA expression levels for all genes were calculated by the 2-ΔCt method.
162
All samples were amplified in triplicate under the same conditions.
163
2.6.
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Questionnaires
An interview questionnaire was used to attain basic information, including maternal age,
165
diet during pregnancy (milk product consumption), education (years), medication during
166
gestation, parity of the fetus (1st, 2nd, or > 3rd), Apgar score (done by a trained nurse based on
167
examining the baby's breathing effort, heart rate, muscle tone, reflexes, and skin color at 10
168
minutes after birth), reproductive history, residential area and duration, smoking and drinking
169
habits, cooking fuel, and socio-economic status. The questionnaire included factors that might
170
influence toxicant levels in the placenta (whether their job was related to e-waste, the
171
husband's occupational history, house as an e-waste recycling workshop), as previously
172
described [28].
173
2.7.
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M
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Statistical analyses
174
Non-parametric analyses were used for data with skewed distributions. Spearman rank
175
correlation analysis was used to evaluate the relationships between EDCs, birth length, birth
176
weight, KISS1, leptin and LRc expression. All analyses were conducted with SPSS statistical
177
software (version 13.0, SPSS, Inc., Chicago, IL, USA). To verify the influence of multiple 9
Page 9 of 30
independent variables on contaminants and biomedical effects, a multiple regression model
179
was used. The influence of an independent variable on another continuous dependent variable
180
was assessed by a linear regression model. All examination results that changed by age,
181
gender, and smoking were adjusted by direct methods.
182
3. Results
183
3.1.
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us
Descriptive statistics
Umbilical cord blood (UCB) samples and placental tissue were collected from 262 out of
185
284 participants; 22 were excluded for missing questionnaire data or without biological
186
samples. Levels of toxicant in the samples, as well as birth-related indices from the
187
completed questionnaires, were compared, and involved 192 women from the e-waste
188
recycling town and 70 women from the reference area. Because of insufficient sample
189
quantity, only 60 samples for KISS1 and 56 samples for leptin (receptor) gene expression in
190
the reference group were analyzed. Descriptive statistics are summarized in Table 1. The
191
backgrounds of two groups show no significant difference except house as an e-waste
192
recycling workshop. The exposed group had a longer gestational age and birth length (p
0.05 0.05 >0.05
