Physiological Changes and Perturbation of Lipid Metabolism in …s-space.snu.ac.kr/bitstream/10371/141942/1/000000150175.pdf · 2019-11-14 · Perturbation of Lipid Metabolism in

보건학석사 학위논문

Physiological Changes and

Perturbation of Lipid Metabolism in

Offspring after Prenatal Exposure to

Bisphenol S in C57BL/6 Mice

비스페놀 S의 태중 노출에 따른 후세대

마우스에서의 생리학적 변화 및 지질대사 변화

2018년 2월

서울대학교 보건대학원

환경보건학과 환경보건전공

백 화 영

i

ABSTRACT





Hwayoung Baek

Department of Environmental Health Sciences

Graduate School of Public Health

Seoul National University, Korea

Due to the similarities of chemical structure and characteristics, bisphenol S

(BPS) has been introduced to industries as a substitute of bisphenol A (BPA),

a well-known endocrine disruptor, leading to similar concerns against

environmental health. However, the toxicity reference of BPS has not yet

been clearly established, and it has not been elucidated whether exposure of

the maternal body, such as BPA, affects the lipid metabolism of the postnatal

generation. This study aims to identify the markers of health effects,

especially for lipid metabolism in the offspring.

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BPS was given to pregnant C57BL/6 mice at 0, 21.4, 214 and 1072 ppm in

0.5% ethanol vehicle via drinking water for ten days (GD 9) before delivery.

The offspring was separated from their dams after three weeks of lactation.

Each group was sacrificed at pre-pubertal age (PND 21), pubertal (PND 56)

and adult (PND 161); body size and organ weights were recorded and lipids,

adipokine hormone, insulin and glucose in serum were measured with ELISA

kits and Enzymatic Colorimetric Assay kits. Serum lipidome was determined

by UPLC-qTOF-MS and Progenesis QI software followed by lipid annotation

and pathway analysis. Metabolites detected from serum were identified using

online database such as HMDB and KEGG.

In pre-pubertal mice, decreases of the body weights and adipose tissues

were found at the lowest dose of BPS and dose-dependence differed by

gender; the serum triglyceride (TG) decreased and LDL cholesterol and

glucose increased at middle or high dose in males, while TG and total

cholesterol decreased at low dose, and insulin and LDL increased in the

middle or high dose in females. After the pubertal period, the treatment effects

on body weights disappeared, while total fat, subcutaneous (SAT) and brown

adipose tissues (BAT) decreased in males, and total fat and SAT increased in

females at middle dose exposure in puberty; the serum total cholesterol

increased and the serum glucose and adiponectin decreased at low dose in

males while total cholesterol, TG, LDL, leptin, insulin increased at middle

dose in the females with no dose-dependence. Adult male mice showed that

the treatment effects on body weights disappeared, but decreased VAT at high

dose but not statistically significant; LDL, TC decreased and TG increased in

males at high dose. Among the lipidome, we identified more

Lysophosphatidylcholine (LPC), Phosphatidylcholin (PC), Sphingomyelin

(SM), Phosphatidylethanolamine (PE) and cholesterol ester species involved

in the metabolic pathways associated with glycerophospholipid, sphingolipid

and purine in the exposed mice.

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In conclusion, we found changes in phenotypes and lipid metabolism

among the offspring after prenatal exposure to BPS, and the changes of lipid

profile suggest potential markers of early biological effects although

confirmation through further studies is needed.

Keywords: Bisphenol S, Prenatal exposure, Lipid metabolism, Lipidomic

analysis, Biochemical analysis, UPLC-Q-TOF/MS

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Contents

ABSTRACT I

LIST OF FIGURES V

1. INTRODUCTION 6

2. MATERIALS AND METHODS 9

3. RESULTS 21

4. DISCUSSION 37

5. CONCLUSIONS 43

6. REFERENCES 44

7. SUPPLEMENTARY INFORMATION 52

v

List of Figures

Figure 1. Outline of Study. ................................................................. 10

Figure 2. Schematic Representation of the Animal Study .............. 13

Figure 3. ............................................................................................... 24

Figure 4. Biochemical indicator level in offspring mice following

prenatal BPS exposure................................................................. 28

Figure 5. Scoring plots of (A) OPLS-DA and (B) VIP-PLOT

analysis in adult high dose group. .............................................. 30

Figure 6. Fold change of each dose group by metabolites............... 32

Figure 7. An overview of the pathways those associate with BPS

exposure, Pre-puberty group ...................................................... 34

Figure 8. An overview of the pathways associate with BPS exposure

in puberty group. ......................................................................... 35

Figure 9. An overview of the pathways associate with BPS exposure

in adult group ............................................................................... 36

6

1. Introduction

Bisphenol S (BPS) is a bisphenol-based substance in which two phenolic

rings with high thermal stability are bonded by sulfur. Because of the

hazardousness of Bisphenol A (BPA), like endocrine attempt, metabolic

disorder, hypertension and early induction of Sexual maturity, it is widely

used as a substitute material. BPS is a widely used as the analogue of BPA

because of its commercial use and widespread consumers. BPS is used for the

industrial productions, such as, a constituent of thermal paper (Liao et al.,

2012) and phenolic resin, and as an electroplanting solvent (Clark et al.,

2012).Production of BPS is growing year by year, and according to the

European Chemicals Agency (2014), BPS is currently producing around 1000

to 10,000 tonnes per year.

Recent studies have already shown that BPA promotes differentiation of

adipocytes in adipose tissue, increases fat accumulation, and increases the

expression of genes involved in lipid differentiation. In this regard, animal

studies, including mice and rats, have shown that perinatal BPA exposure

results in weight gain in offspring and postpartum (Rubin et al., 2009, Sakurai

et al., 2004). And a tendency to differentiate fibroblasts into adipocytes when

administered with insulin has been reported (Masuno et al., 2002). In

particular, it has been shown that adipose tissue mass is increased by exposure

to BPA in rats at the postnatal and postnatal stages (Miyawaki et al., 2007),

and increased serum lipid (triglycerides, total cholesterol, LDL-cholesterol)

and confirmed the reduction of adiponectin (Liang et al., 2016). In the human

body, BPA is known to inhibit the release of adiponectin, which is known to

lower the likelihood of obesity-related diseases from adipose tissue and

adipocytes (Hugo et al., 2008). That is, similar effects on gene expression that

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are important for adipocyte formation, lipid accumulation and lipid

metabolism have the potential to increase health problems such as obesity and

diabetes by affecting the disturbance or energy balance of lipid tissue

metabolism.

BPS has a physical and chemical characteristic similar to that of BPA.

However, due to its structural similarity, and studies have shown that BPS is

more biodegradable than BPA, has a longer half-life, and has higher skin

permeability than BPA (Zalko et al. 2011, Danzl et al., 2009).

However, the toxicity reference of BPS is not yet registered in the EPA

IRIS, and the regulation of BPS use is currently only available as 50 μg / kg

as the specific migration limit (SML) of the EU food container use regulation

standard. To date, studies on biological effects of BPS exposure are mostly

cellular and in vivo experimental studies are limited. Animal studies on BPS

have shown that only long-term exposure to BPS with high-fat diet before and

after birth has been associated with the effects of BPS on obesity, as in BPA,

by Moral et al. (2016). In this article, we analyzed the changes in the

indicators of obesity, hyper-insulinemia, hyperglycemia, and other obesity,

including the expression of lipid metabolism gene markers in adipose tissue,

and determined the difference in obesity induction according to BPS exposure.

In this study, we measured the changes of lipid metabolism and the

physiological changes, the pathological state, and the exposure of the BPS

substance alone except the high fat diets. Especially, in the fetal period, since

it is the period during which health effects can be determined, it is a primary

goal to explore the effects of exposure to BPS during the fetal period on

growth and health after birth. According to this principle, exposure to

environmental factors during specific sensitive periods of development,

mainly in utero and immediately after birth, can interfere with maternal

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hormonal and nutritional signaling to the developing organism. And through

changed metabolic set points, resulting in a permanent or long-term change in

the structure or function of the organism including metabolic homeostasis and

endocrine and reproductive functions, can ultimately predispose an individual

to chronic diseases later in life, e.g. obesity and related metabolic disorders

(Oken and Gillman, 2003, Gluckman et al., 2005, Esterik et al., 2014).

Therefore, we observed whether there is a difference from the control group

at each major life stage (pre-pubertal, pubertal, and adult) and whether the

exposure to BPS in the fetal period causes any physiological change or

pathological condition. This change in lipid metabolism and its cause, and

whether it can distinguish between substance exposures, is an important

advance on exposure and health effects. For this, a representative clinical lipid

marker in the blood such as Triglycerides (TG), Total cholestreol (TC), Low

density lipoprotein-cholesterol (LDL-C) and hormones such as leptin,

adipokine, insulin were measured by commercialized KIT, and the lipid

concentrations and hormone levels between groups were quantitatively

compared. And after screening the lipid profile using UPLC-Q-TOF, we tried

to develop a system that can identify the substances that show the difference

between the groups and identify each relevant metabolic pathway to be used

as an exposure signature.

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2. Materials and Methods

2.1. Outline of animal experiment

A general overview of the study is as follows. In this study, we investigated

the changes of metabolic syndrome related indicators by measuring the blood

biomarkers of the second generation mouse when the pregnant mother was

exposed to BPS, and identified the effect biomarker through lipid metabolism

change and identified the lipid metabolism that may cause the biomarker

change.

Serum was assayed for lipid and lipoprotein, adipocyte secretion hormone,

peptide hormone, and blood sugar. Non-target lipid analysis was performed

using UPLC Q-TOF to determine the changes in serum levels of lipid

metabolites.

Results from UPLC-q-TOF analysis were analyzed using Progenesis QI

software. This software allows the identification of lipid metabolites that

show significant differences between exposed and unexposed groups, and

identifies and identifies these metabolized metabolites in an online database

and identifies the increase and decrease of biomarkers identified among the

groups.

10

Figure 1. Outline of Study.

11

2.2. Animals and materials

Pregnant C57BL6 mice were purchased from KOATEC Inc. (Cheongwon-

Gun, Korea). For the chemicals exposed to mice, BPS was purchased from

Sigma-Aldrich, ethanol used as a vehicle control solvent was purchased from

Sigma-Aldrich, and isoflurane used for inhalation anesthesia of mice was

purchased from JW pharma. In the biochemical analysis stage, Ultra Sensitive

Mouse Insulin ELISA Kit (Cat. # 90080), Mouse Leptin ELISA Kit (Cat. #

90030) and Mouse LDL-Cholesterol Kit (Cat. # 79980) were purchased from

Crystal Chem (Downers Grove, USA). HDL and LDL/VLDL Cholesterol

Assay Kit (Cat. # ab65390), Triglyceride Quantification Kit (Cat. # ab65336)

were purchased from abcam (UK). Mouse Adiponectin ELISA Kit (Cat. #

EZMADP-60K) were purchased from Millipore (Darmstadt, Germany).

For the pretreatment of samples, ACN (Acetonitrile), IPA (Isopropanol)

were purchased from J. T. Baker and the third distilled water obtained from

the Milli-q system were used. Two types of analytical instruments were used

Plate reader Infinite 200 PRO from Tecan Life Sciences and LC-q-TOF from

WATERS were used.

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2.3. Animal Study Design

Exposure Process

The animal experiment was carried out at the animal facilities of Institute of

Laboratory Animal Resources in Seoul National University. The experimental

protocols were reviewed and approved by the IACUC of Seoul National

University (approval no. SNU-160930-3) and every animal were treated

humanely and with regard for alleviation of suffering.

The mice were housed in standard polypropylene mouse cage and were

acclimatized for 1 week before exposed to BPS. The room where mice were

maintained was an air-conditioned at a temperature of 24 Celsius degree, a

relative humidity of 00% and a 12h light/dark cycle. A maximum of five mice

were housed in each cage. Each animal had ad libitum access to water and a

regular diet throughout the experimental period. We used glass water bottles

to ensure that related compounds did not leach from plastic water bottles.

Water and food consumption, body weight and any physical observations

were recorded once in a week.

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Figure 2. Schematic Representation of the Animal Study.

Four groups of pregnant C57Bl/6J mice were exposed to each four dose

levels of BPS including vehicle control from the end of acclimatized period.

The treatment was ended after delivery and the lactation was lasted three

weeks. After weaning, the offspring were separated from their dam and

divided into two groups for each gender (male and female) and then into three

groups for each major life points. The major life points of mice were set in

three groups: pre-pubertal (Postnatal Day 21, PND21), pubertal (PND56), and

adult (PND161) and at each point, they were sacrificed.

There are four treatment groups and exposed to BPS in their drinking water

at dose of 0, 5, 50, 250 mg/kg bw/day from gestational day 9 to 19.The

middle BPS dose used in this study is 50 mg / kg / day, which is the lowest-

observed-adverse-effect level (LOAEL) of BPA, a similar structure of BPS.

And the dose level was set to a low dose and a high dose with a difference of

5 times or 10 times.

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BPS dose was based on mean daily unadjusted water intake in C57Bl/6J

(about 7 ml/30 g mice) (Bachmanov et al., 2002) and in order to obtained

expected BPS exposure of 0; 0.2; 1.5; 50 mg/kg bw/day. BPS was dissolved

in absolute ethanol as vehicle and the final concentration of ethanol was 0.5%.

We gave the vehicle control group drinking water containing only 0.5%

ethanol.

Water intake was determined by measuring the difference in the amount of

water placed in the water bottle during the exposure period, and the levels of

BPS consumed weekly were estimated. The average BPS intake was 0, 3.86 ±

0.86, 38.25 ± 6.37, 196.04 ± 25.20mg/kg bw/day for respective expected

doses of 0, 5, 50, 250 mg/kg bw/d of BPS and was a statistically significant

difference in drinking water intake (β = 1.779, p <0.0001) depending on BPS

exposure dose.

Sample Collection

Samples of each mouse were collected after fasting for 6-8 hours at each

observation point, mice were anesthetized by inhalation using isoflurane and

biological samples were collected.

For clinical chemistry and metabolism profiling, serum was collected from

the whole blood of mice, which collected from inferior vena cava by using a 1

ml syringe. About 400 to 600 μl of whole blood was collected in the SST

tube and then clotted for 15 minutes after collection to separate into serum

and centrifuged at 12,000 rpm for 20 minutes. The supernatant (serum) was

then, transferred to the cryotube.

White Adipose Tissue (WAT) was obtained from three kinds of gonadal fat,

visceral fat and subcutaneous fat. First, the gonadal fat was extracted from the

uterine horns of the female ovaries, and the male and female gonadal females

15

were extracted from the left and right testicles. Visceral fat collected around

the organs such as the kidneys and spleen, and subcutaneous fat was collected

between the groin, retroperitoneum, and armpit. Brown Adipose Tissue (BAT)

extracted interscapular brown adipose tissue.

In addition, the brain, liver, left kidney, and spleen were excised from the

other solid tissues. All of the samples were weighed and placed in a sterilized

1.5 mL microtube, cooled with liquefied nitrogen, and stored frozen at -70 ° C

until analysis.

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2.4. Biochemical Analysis

Serum lipid, lipoprotein cholesterol levels

The levels of serum total cholesterol (TC), triglycerides (TG), LDL-

cholesterol (LDL-C) were measured using Enzymatic Colorimetric Assay kits

for TC, TG and LDL-C respectively.

Serum adipokine hormone and insulin levels

The levels of serum adiponectin, leptin and insulin levels were measured

using Enzyme-Linked Immunosorbent Assay (ELISA) kits for adiponectin,

leptin and insulin respectively.

Serum glucose level

Fasting glucose was measured using glucose meter (Accu-check; Roche,

Mannheim, Germany). The blood glucose concentration is measured in mg /

dL, and is repeated three times for each individual, and then the average was

calculated

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2.5. Metabolomics Analysis

Sample preparation for UPLC-MS

For each precipitation conditions, serum samples (50ul) were precipitated

by addition of 3 volumes of IPA precooled to -20℃.Samples were vortex

mixed for 1min. After 10min of incubation at room temperature, samples

were stored overnight at -20℃ to improve protein precipitation and then

centrifuged at 14,000g for 20min at 4℃ (Sarafian et al., 2014). The

supernatant was collected (>150ul) and transferred to an auto sampler vial and

then injected into the UPLC-MS system for analysis.

An in house quality control (QC) was prepared by pooling and mixing the

same volume of each sample (Wu et al., 2014). The QC sample provides a

representative analyte containing all the samples that would be encountered

during the entire sample sequence in an average length of one QC every 10

injections (Zhao et al., 2016).

Chromatography conditions

The mass spectrometry detection was performed by the Waters SYNAPT

G2-S MS/ACUITY UPLC System (Manchester, UK). The chromatographic

analysis was performed in a Waters ACUITY UPLC System controlled with

Masslynx (V4.1, Waters Corporation, Milford, USA).

An aliquot of 1ul of samples solution was injected into an ACUITY UPLC

CSH C18 column (2.1mm × 100mm, 1.7μm, Waters Corporation) at 55℃,

the flow rate was 0.4ml min-1. The mobile phase A consists of ACN/H2O

(60:40, v:v) mixed with 10mM ammonium formate and 0.1% formic acid and

mobile phase B IPA/ACN (90:10, v:v) mixed with 10mM ammonium formate

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and 0.1% formic acid (Sarafian et al., 2014). And a gradient was used: 0-2min,

60-57% A; 2-2.1min, 57-50% A; 2.1-12min, 50-46% A; 12-12.1min, 46-30%

A; 12.1-18min, 30-1% A; 18-18.1min, 1-60% A; 18.1-20min, 60% A. After

every sample injection, a needle wash cycle was done to remove the remnants

and prepare for the next sample.

Mass spectrometry conditions

To profile the lipid by using UPLC-Quadrupole-Time-of-Flight (Q-TOF)

Mass Spectrometry, following mass spectrometry conditions were applied. In

positive ion-mode, MS parameters were as follows: capillary voltage was set

at 2.5kV, cone voltage at 30V, source temperature at 120℃, desolvation

temperature at 400℃, desolvation gas flow at 800L/h, and cone gas flow at

20L/h. Acquisition was performed from m/z 100 to 1500.In negative ion

mode, MS parameters were as follows: capillary voltage was set at 2.5kV,

cone voltage at 30V, source temperature at 120℃, desolvation temperature at

500℃, desolvation gas flow at 800L/h, cone voltage at 25L/h.

For accurate analysis, acquisition was performed from m/z 100 to 1500. In

both ionization modes, leucine enkephalin (m/z 556.2771 in ESI+, m/z

554.2615 in ESI−) was continuously infused at 10 μL/min and used as lock-

mass correction. And the mass spectrometer was calibrated using a solution of

sodium formate before the experiment.

Data processing

The data of serum samples were extracted by the UPLC-Q-TOF/MS system.

All potential metabolites including the retention time, the exact mass, and the

MS/MS data were supplied by the chromatographic peaks in the BPI

chromatograms. MSe data were processed by the Progenesis QI software to

19

peak detection and auto-alignment. The precise molecular mass and its mass

fragments were detected by mass spectrometer (SYNAPT-G2-Si) and

determined within a reasonable degree of measurement error (<5ppm).

And then, the data of exposure and control group were investigated by

OPLS-DA analysis, through this analysis, the metabolite profile of exposure

group was gathered within the group and was significantly separated from

control group.

Pathway Analysis

Metabolites identified from online database such as human Metabolome

Database (HMDB), ChemSpider, Kyoto Encyclopedia of Genes and Genomes

(KEGG) and LIPIDMAPS were further imported into the IMPaLA: Integrated

Molecular Pathway Level Analysis website (http://impala.molgen.mpg.de/)

and the KEGG (http://www.kegg.jp/kegg/) and analyzed the pathway.

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2.6. Statistical Analysis

All data acquired from biochemical analysis were analyzed statistically.

Descriptive statistics were recorded to compare the mass and concentration of

biological parameters control versus each exposure groups. They were

presented as arithmetic means (AM) ± SD because the results were acquired

from plural mice which have same exposure group. The mass concentrations

at each PND are shown as AMs ± SD.

Since the data of subjects were normally distributed when we tested them

with Shapiro-Wilks Test, the results of each exposure group were compared to

vehicle control using the Student’s T-Test. A result was considered significant

when P≤ 0.05. All analyses were conducted using SAS software (9.4; SAS

Institute, Cary, NC, USA). SigmaPlot software (ver. 10; Systat Software, San

Jose, CA, USA) was used to visualize the results.

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3. Results

3.1. Effect of BPS on body weight, adipose tissue mass and other

organ weights

Figure 3 shows all measures at each life point that were normalized and

compared with median of vehicle group. Values are expressed as the adjusted

median value divided by the median value of Vehicle control group, and error

bar means interval from median to 75th of measured data.

In pre-pubertal male mice group, adjusted median bodyweight was showed

a tendency to decrease, especially, low-, high- dose group showed significant

decrease (0.93 ± 0.08 (p<0.01), 0.96 ± 0.07 (p<0.05)). But, there was no

significant difference in pubertal, adult group. In females, adjusted median

body weight was significantly lower in low- and mid- dose group than in

controls (0.95 ± 1.02 (p<0.01), 0.96 ±1.06 (p<0.05)) but as shown in male

group, there was no significant difference in pubertal, adult group. Total fat

mass (Tfat), Subcutaneous adipose tissue (SAT) and Brown adipose tissue

(BAT) was significantly lower in the mid - dose group of pubertal male mice

group (0.89 ± 0.07 (p<0.05), 0.7 ± 0.19 (p<0.01), 0.86 ± 0.14 (p<0.05)), but

body weight was not significantly different from controls.BAT was

significantly lower in BPS5 males than in controls (0.64 ± 0.21 (p<0.05)) with

body weight.

Unlike the male group, Total fat mass and SAT mass were significantly

higher in mid- dose group of pubertal female mice than those of controls(1.21

± 0.07 (p<0.05), 1.58 ± 0.16 (p<0.01)), but the treatment effects on body

22

weights disappeared.

Adult male mice showed that the treatment effects on body weights

disappeared, but decreased VAT at high dose but not statistically significant.

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(A) Male body weight (B) Female body weight

(C) Male total fat mass (D) Female total fat mass

(E) Male subcutaneous adipose tissue

mass (F) Female subcutaneous adipose tissue

mass

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(G) Male visceral adipose tissue mass (H) Female visceral adipose tissue mass

(I) Male brown adipose tissue mass (J) Female brown adipose tissue mass

Figure 3. Every measures at each life point were normalized and compared with

median of vehicle group using Student's t-test. The black dotted line represents the

vehicle control group, the green line represents the low dose group, the blue line

represents the medium dose group and the red line represents the high dose group.

The number of each exposure female mice group was 37, 33, 42, 17 in pre-pubertal

group, 18, 17, 24, 14 in pubertal group and 4, 13, 11, 4 in adult group. The number of

each exposure male mice group was 42, 33, 40, 25 in pre-pubertal group, 25, 18, 21,

21 in pubertal group and 18, 17, 7 in adult group. Values are expressed as the adjusted

median value divided by the median value of Vehicle control group, and error bar

means interval from median to 75th of measured data. (*: P<0.05, **: P<0.01; Vehicle:

0.5% ethanol solution; Low: 5 mg/kg bw/day BPS in 0.5% ethanol solution; Mid: 50

mg/kg bw/day BPS in 0.5% ethanol solution; High: 250 mg/kg bw/day BPS in 0.5%

ethanol solution.)

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3.2. Effect of BPS on Biochemical Parameters

In pre-pubertal male mice group, serum TG was significantly lower in mid-

dose group than in controls (0.60 ± 0.10 compared with 0.93 ± 0., p<0.05) and

LDL cholesterol and glucose increased at mid- or high dose in males (1.62 ±

0.39 compared with 1.02 ± 0.36, p<0.05, 1.29 ± 0.12 compared with 1.02 ±

0.17, p<0.01), but serum TC level was not significantly different in mid- dose

male mice group compared with controls. And an increase in insulin and

leptin but, not statistically significant (Figure 4A).

In pre-pubertal female mice group, serum TG and total cholesterol

decreased at low dose female mice group than in controls (0.51 ± 0.25

compared with 1.24 ± 0.52, p<0.05, 0.53 ± 0.10 compared with 0.90 ± 0.29,

p<0.05). And insulin increased in high dose in females (1.61 ± 0.24 compared

with 1.03 ± 0.35, p<0.01). Also, serum glucose and leptin showed an increase

but, not statistically significant (Figure 4B).

In puberty male mice group, serum TC was significantly higher in low dose

male mice group than in controls (1.19 ± 0.19 compared with 0.98 ± 0.09,

p<0.05) and, serum adiponectin level was significantly lower in low- dose

male mice group than in controls (0.75 ± 0.05 compared with 1.01 ± 0.07,

p<0.01). Serum glucose and leptin level were significantly lower in high dose

group than in controls (0.85 ± 0.14 mg/dL compared with 0.99 ± 0.13, p<0.05,

0.23 ± 0.08 compared with 0.86 ± 0.49, p<0.05) and also decrease in insulin

but, not statistically significant (Figure 4C).

In puberty female mice group, serum glucose level was significantly higher

in low dose female group than in controls (1.20 ± 0.14 compared with 1.05 ±

0.15, p<0.05) but, not statistically significant decrease in insulin. And

adiponectin level was significantly lower in low-, high dose female mice

26

group than in controls (0.68 ± 0.06 compared with 0.99 ± 0.07, p<0.01, 0.86 ±

0.05 compared with 0.99 ± 0.07, p<0.05) and TC levels were higher in high

dose female mice group than in controls (0.64 ± 0.10nmol/μL compared with

0.96 ± 0.19, p<0.05). And total cholesterol, TG, LDL, leptin, insulin

increased at mid- dose in the females with no dose-dependence.

Except for adult group, all exposed group showed an increase LDL-

cholesterol in both male and female group, especially statistically significant

in mid-, high dose of pubertal female mice group and in mid- dose pre-

pubertal male mice group.

In adult male mice group, serum LDL-cholesterol and TC were

significantly lower in high dose group than in controls (0.71 ± 0.08 compared

with 1.00 ± 0.15, p<0.05, 0.58 ± 0.05 compared with 0.95 ± 0.23, p<0.01), but,

TG was significantly higher in high dose group than in controls (2.09 ± 0.37

compared with 0.92 ± 0.30, p<0.01).

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(A) Pre-pubertal male mice group (B) Pre-pubertal female mice group

(C) Pubertal male mice group (D) Pubertal female mice group

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(E) Adult male mice group (F) Adult female mice group

Figure 4. Biochemical indicator level in offspring mice following prenatal BPS exposure. Effects of prenatal BPS exposure to 5, 50,

250 mg/kg B.W/d in (A) prepubertal male mice group, (B) prepubertal female mice group, (C) pubertal male mice group, (D) pubertal

female mice group, (E) adult male mice group, (F) adult female mice group. (TG: triglyceride; TC: total cholesterol; LDL: low density

lipoprotein cholesterol) Values are expressed as the adjusted median value divided by the median value of vehicle control group, Mean±

SD. (*: P<0.05, **: P<0.01; Vehicle: 0.5% ethanol solution; Low: 5 mg/kg bw/day BPS in 0.5% ethanol solution; Mid: 50 mg/kg bw/day

BPS in 0.5% ethanol solution; High: 250 mg/kg bw/day BPS in 0.5% ethanol solution.)

29

3.3. Multivariate Statistical Analysis on Lipid Metabolites

Typically, the trajectory analysis of score plots from principal component

analysis (PCA) and orthogonal partial least squares discriminant analysis

(OPLS-DA) was performed by EZinfo 2.0 software and, according to the

result (adult-high dose group under the ESI-mode) of OPLS-DA (Fig. 4A), we

found that the metabolic profiles in the different groups could be separated

clearly. The results of the corresponding VIP plot based on serum profiling

data showed a significant difference in ions between the BPS exposure and

control groups (Fig. 4B) and the ions furthest from the origin were regarded

as potential biomarkers responsible for the differences between the BPS

exposure and control groups. We selected the biomarkers that met a VIP

threshold of 1.0 and had a value of p < 0.05 (Student's t-test) as candidates for

the next steps.

http://pubs.rsc.org/en/content/articlehtml/2017/RA/C7RA05722A

30

Figure 5. Scoring plots of (A) OPLS-DA and (B) VIP-PLOT analysis in adult

(PND161) high dose group under the ESI-mode.

31

3.4. Detection and Identification of Biomarker Candidates

Then, they were matched to the network database such as Human

Metabolome Database (HMDB), ChemSpider, Kyoto Encyclopedia of Genes

and Genomes (KEGG) and LIPIDMAPS.

The datasets of positive and negative ionization modes contained 4, 32 and

8, 30 metabolites in each of the male and female in the pre-pubertal low dose

group, 26, 1 and 22, 5 metabolites in the pre-pubertal middle dose group and

14 and 14 metabolites in the pre-pubertal high dose group. The datasets of

positive and negative ionization modes contained 20, 5 and 14, 13 metabolites

in each of the male and female in the pubertal low dose group 3, 6 and 10, 9

metabolites in the pubertal middle dose group and 4, 1 and 16, 6 metabolites

in the pubertal high dose group. The datasets of positive and negative

ionization modes contained 34 and 12 metabolites in the adult low dose group

of male and 50 and 21 metabolites in the adult high dose group of male.

After all, endogenous lipid metabolites were tentatively identified and

summarized in supplement table1-8. And the main lipid changes of BPS

exposure group involves glycerophospholipids (GPs), phosphatidylcholine

(PC), phosphatidyl ethanolamine (PE), sphingolipids (SLs),

phosphatidylserine (PS), cholesterol ester (CE), triglycerol (TG),

diacylglycerol (DG), and ceramide (Cer).

To investigate the magnitudes of the changes in the tentatively identified

markers within the metabolic pathway, a graph comparing the relative

intensity of the marker between the two groups was shown for each life points

(Fig. 6).

32

Figure 6. Fold change of each dose group by metabolites. Metabolites were found from ten (male 5, female 5) mice per exposure

group. Metabolites which shown statistically significant difference between vehicle control group were presented (VIP value > 1.0,

p< 0.05). (C00416: LPA (0:0/18:1), LPA (0:0/18:2); C02737:PS (18:0/20:0); C00350:PE (15:0/22:1), PE (16:0/24:1), etc. (total 18); C00157: PC (14:0/18:1), PC

(14:0/18:2) etc. (total 57); C04230: LysoPC (14:0), LysoPC (16:0) etc. (total 8); C00366: Uric acid; C00836: Sphinganin; C00195: Ceramide (d18:1/12:0), Ceramide

(d18:1/22:0) etc. (total 3); C00550: SM (d14:1/20:0), SM (d16:0/26:1) etc. (total 12); C01190: Glucosylceramide (d18:1/24:1); C02686: Galactosylceramide (d18:1/22:0);

C02960: CerP (d18:0/26:0), CerP (d18:1/26:0) etc. (total 3); C06126: Galabiosylceramide (d18:1/16:0); C18043: Cholesterol sulfate;)

33

3.5. Pathway Reconstruction

The tentatively identified biomarkers were further imported into the

IMPaLA: Integrated Molecular Pathway Level Analysiswebsite

(http://impala.molgen.mpg.de/) and the KEGG (http://www.kegg.jp/kegg/).

We found that they were mainly involved in 3 classes of pathway, namely,

glycerophospholipid metabolism, sphingolipid metabolism and purine

metabolism. An overview of the pathway analysis is shown in Fig. 7-9.

The results showed that these target pathways differed by life point,

exposure dose, and sex. As for the glycerol-phospholipid metabolism, in our

study, we found a down or up regulation of the levels of glycerophospholipids

such as lysophosphatidic acid (LPA), phosphatidylserine (PS),

phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC)

diacylglycerol (DG) and phophatidylcholine (PC), in BPS exposure groups'

serum. And as for the sphingolipid metabolism, we found a down or up

regulation of the levels of sphingolipids such as sphinganin-1-phosphate,

sphinosine-1-phosphate, sphingosine, sphinganin, ceramide, sphingomyelin

(SM), glucosylceramide, galactosylceramide, ceramide-1-phosphate and

digalactosylceramide and uric acid as for purine metabolism. The relative

intensity of the marker compared to the control group for each group is shown

in Figure 6.

http://www.google.co.kr/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwjE8obXys7XAhUMgbwKHQ17AAYQFgglMAA&url=http%3A%2F%2Fimpala.molgen.mpg.de%2F&usg=AOvVaw2B4_74tU72GwM3-12b_IPw

34

Figure 7. An overview of the pathways those associate with BPS exposure, Pre-

puberty (PND21). Each metabolites were found from ten (male 5, female 5) mice

per exposure group. Metabolites which shown statistically significant difference

with vehicle group were presented. (VIP value > 1.0, p< 0.05) (C00416: LPA

(0:0/18:1), LPA (0:0/18:2); C02737:PS (18:0/20:0); C00350:PE (15:0/22:1), PE (16:0/24:1),

etc. (total 18); C00157: PC (14:0/18:1), PC (14:0/18:2) etc. (total 57); C04230: LysoPC (14:0),

LysoPC (16:0) etc. (total 8); C00366: Uric acid; C00836: Sphinganin; C00195: Ceramide

(d18:1/12:0), Ceramide (d18:1/22:0) etc. (total 3); C00550: SM (d14:1/20:0), SM (d16:0/26:1)

etc. (total 12); C01190: Glucosylceramide (d18:1/24:1); C02686: Galactosylceramide

(d18:1/22:0); C02960: CerP (d18:0/26:0), CerP (d18:1/26:0) etc. (total 3); C06126:

Galabiosylceramide (d18:1/16:0); C18043: Cholesterol sulfate;)

35

Figure 8. An overview of the pathways associate with BPS exposure in puberty

(PND56) group. Each metabolites were found from ten (male 5, female 5) mice

per exposure group. Metabolites which shown statistically significant difference

with vehicle group were presented. (VIP value > 1.0, p< 0.05) (C00416: LPA

(0:0/18:1), LPA (0:0/18:2); C02737:PS (18:0/20:0); C00350:PE (15:0/22:1), PE (16:0/24:1),

etc. (total 18); C00157: PC (14:0/18:1), PC (14:0/18:2) etc. (total 57); C04230: LysoPC (14:0),

LysoPC (16:0) etc. (total 8); C00366: Uric acid; C00836: Sphinganin; C00195: Ceramide

(d18:1/12:0), Ceramide (d18:1/22:0) etc. (total 3); C00550: SM (d14:1/20:0), SM (d16:0/26:1)

etc. (total 12); C01190: Glucosylceramide (d18:1/24:1); C02686: Galactosylceramide

(d18:1/22:0); C02960: CerP (d18:0/26:0), CerP (d18:1/26:0) etc. (total 3); C06126:

Galabiosylceramide (d18:1/16:0); C18043: Cholesterol sulfate;)

36

Figure 9. An overview of the pathways associate with BPS exposure in adult

(PND161) group. Each metabolites were found from 5 vehicle group mice, 5 low

dose mice and 4 high dose mice, only male. Metabolites which shown statistically

significant difference with vehicle group were presented. (VIP value > 1.0, p<

0.05) (C00416: LPA (0:0/18:1), LPA (0:0/18:2); C02737:PS (18:0/20:0); C00350:PE

(15:0/22:1), PE (16:0/24:1), etc. (total 18); C00157: PC (14:0/18:1), PC (14:0/18:2) etc. (total

57); C04230: LysoPC (14:0), LysoPC (16:0) etc. (total 8); C00366: Uric acid; C00836:

Sphinganin; C00195: Ceramide (d18:1/12:0), Ceramide (d18:1/22:0) etc. (total 3); C00550: SM

(d14:1/20:0), SM (d16:0/26:1) etc. (total 12); C01190: Glucosylceramide (d18:1/24:1); C02686:

Galactosylceramide (d18:1/22:0); C02960: CerP (d18:0/26:0), CerP (d18:1/26:0) etc. (total 3);

C06126: Galabiosylceramide (d18:1/16:0); C18043: Cholesterol sulfate;)

37

4. Discussion

BPA as well as BPS are considered as an environmental obesogen through

promoting adipogenesis, lipid accumulation and endocrinal disrupting

chemicals (EDCs) altering adipokine hormone release. And lipids are the

fundamental components of biological membranes and they are highly diverse

groups of molecules regarding their structure and function. Previous reports

had shown that the perturbation of lipid metabolism played a critical role in

the initiation and progression of metabolic syndrome. Thus, some

dysregulated lipids may act as important biomarkers.

In this study, we assumed that exposure to BPS early in life can program an

organism for higher susceptibility to develop obesity and related metabolic

impairment. Therefore, we measured the phenotypes such as body weight,

total fat mass, weight of each type of adipose tissue and the BPS-induced

perturbation of biochemical markers related to metabolic disorders such as TG,

TC, LDL-C, leptin, adiponectin, insulin, glucose and lipid metabolites.

Obesity usually determined by excessive accumulation of adipose tissue

and increment of weight. However, in our phenotype data result, body weight

and adipose tissue weight were decreased. According to a previous study of

Moralet al.(2016), prenatal BPS exposure (0.2; 1.5; 50 μg/kg bw/day) to mice

made no significant difference in body weight in the exposed group compared

to the control group. However, in group with high fat diet, the chronic

exposure to BPS of the male mice induced a significant increase in body

weight compared to the control group and a difference in the exposure dose

from 102 to 106times. The reason for this difference is thought to that the high-

38

fat diet was shown to have a synergistic effect in the expression of obesity by

exposure in BPS in the long term.

In pre-pubertal mid-dose group, triglycerides, which are blood lipids,

decreased with body weight loss, but LDL cholesterol was increased

compared with the control group in both male and female group.

lysophosphatidylcholines (LysoPCs) were identified in pre-pubertal and

pubertal of low-, mid-, high dose and both male and female group, which can

be found in Figure 7 and the Supplement Table S1-6. LysoPCs are products or

metabolites of phosphatidylcholines (PCs), which are structural components

of animal cell membranes. LysoPC is present at high concentrationsin

oxidized LDL - cholesterol, and formed by the reaction of phospholipase A2

and, abnormal levels of LysoPC may suggest a disturbance of lipid and

glucose homeostasis and have therefore been used as a potential diagnostic

biomarker in various diseases (Zhao et al.,2016, Zhou et al.,2015).

Considering the relationship between the biochemical indicator changes and

lipid metabolism changes, it is thought to be the significantly increased PC

and LysoPC in the pre-pubertal and pubertal group indicated that prenatal

BPS exposure can inhibit LysoPC hydrolases, then, result in the increase LDL

concentration in mice serum.

In pre-pubertal high-dose group, leptin, insulin and blood glucose are

increased. Especially, in male group, blood glucose is increased to a

statistically significant level, and female group have a statistically significant

increase in insulin. Previous study has shown that BPS treatment induced an

increase in insulin plasma level and strong increases of HOMA-IR suggest

that these mice exhibited a systemic insulin resistance (Moral et al.,2016).

Insulin resistance plays a pivotal role in many metabolic diseases such as

obesity, metabolic syndrome, and type 2 diabetes. In these metabolic

disorders, blood glucose and Free Fatty Acids (FFA) are elevated in the blood

39

and oxidative stress is elevated in exposed cells and tissues. In the case of

insulin resistance, FFA is injected into the liver. FFA accumulates in the liver

through β-oxidation or esterification of triglyceride (TG). In addition, an

excessive increase in oxidative stress due to excessive FFA itself and

activation of the inflammatory signal transduction pathway leads to direct

liver damage, and TG is accumulated in the liver as a protective mechanism.

Our results found that the levels of lysoPC and PC were significantly

increased in the pre-pubertal high dose group, which is indicative of enhanced

peroxidation and oxidative stress. Such peroxidation and oxidative stress can

further lead to apolipo-protein B proteolysis, thus impairing the secretion of

very low-density lipoproteins (VLDL) so that exports of TG from the liver

will decrease. And our lipidomic analysis results, which can be found in the

supplement table S3, are consistent with the results of clinical chemistry

indicators in which decreased serum triglycerides.

After the pubertal period, the treatment effects on body weights

disappeared regardless of exposure dose. However, pubertal mice showed

lower total fat, SAT and BAT in males than vehicle and total fat and SAT

were increased in females at medium dose exposure. After puberty, which

begins to characterize males and females, somatic differences begin to emerge,

partly due to differences in hormones. Women of Child-Bearing Age try to

accumulate body fat to prepare for pregnancy and lactation. As the secretion

of estrogen, a female hormone, increases in women, it increases the body fat

with the development of the breasts, and accumulates fat in the hips and

thighs.

We also identified important and essential structural components of

membrane lipid bilayers as potential biomarkers such as

lysophosphatidylcholine (LPC), phosphatidylcholine (PC), spingomyelin(SM),

40

phosphatidylethanolamine (PE) and cholesterol ester species in most exposed

group. The metabolic pathways associated with the observed metabolites were

glycerolphospholipid metabolism, sphingolipid metabolism and purine

metabolism. To our knowledge, only one in vitro study has investigated the

potential effect of BPS in lipid metabolism and found that BPS at low

concentrations induced pro-inflammatory phenotype by modulating metabolic

pathways which include glycolytic, glutathione (GSH), sphingomyelin (SM),

ceramide (CER), glycerophospholipids (GPs) and glycerollipids (GLs) (Zhao

et al.,2016). And lipid species of SM, CER, and GPS are metabolites that are

also shown statistical change against the control group in our study.

And previous studies have shown that one of the EDC substances, benzo

(a)pyrene disrupted phospholipid and sphingolipid metabolism, which were as

well as the metabolic pathways identified in our study.

First, as to glycerophospholipid metabolism, glycerophospholipids were

derived from the hydrolysis of phosphatidylcholine by the regulation of

phospholipase and they can play a major role in cell signaling and lipid

metabolism. Phosphatidylcholine is the major phospholipid component of all

plasma lipoprotein class, it is a glycerophospholipid in which a

phosphorylcholine moiety occupies a glycerol substitution site. As is the case

with diacylglycerols, glycerophosphocholines can have many different

combinations of fatty acids of varying lengths and saturation attached at the

C-1 and C-2 positions. The disordered of phosphatidylcholine could directly

influence the levels of low-density lipoprotein in the blood which could cause

other diseases such as hyperlipidemia-related diseases and atherosclerotic

injury.

And we also found sphingolipid metabolism turned to be abnormal in

response to prenatal BPS exposure. Sphingomyelin is a type of sphingolipid

41

found in animal cell membranes, especially in the membranous myelin sheath

which surrounds some nerve cell axons. Unlike other lipid metabolites,

sphingolipids are biosynthesized by inflammation and multiple cytokines, and

are known to be increased in various tissues of obese and diabetic animal

models (Merrill et al.,2002). In addition, since intracellular concentration is

regulated by the concentration of fatty acid, which is a biosynthetic substrate

of sphingolipid, it is sensitive to the concentration of fatty acid in blood,

which is increased by obesity (Kuller et al.,2006).

Current clinical parameters such as TG, TC, LDL-C and related hormones

do not address what really happened in mechanisms and lipid metabolism, so

it is necessary to identify early biological effect biomarkers and develop more

effective methods. So, we measure blood biomarkers in mice and observe

how metabolic syndrome related indicators change, and search for early

biological effect markers through lipid metabolism changes.

However, there are still some limitations in this study. First, it would be

ideal if BPS had been exposed during entire pregnancy, but because of the

pregnant was committed artificially and the pregnant mice were needed

acclimatized period. However, if the effect of prenatal exposure to chemical

substances during pregnancy is attributed to epigenetic changes, the genetic

expression may change according to external stress after 9 days of gestation of

the mouse and this expression will be imprinted in later generations.

According to Khalyfa et al. (2014), after exposure of high-fat diets from gd12

days, post-exposure mutations of the tissue adipocytokine gene were

identified, and the phenotype of the offspring was changed through posterior

transformation and increased risk of insulin resistance and hyperlipemia

respectively.

42

Second, even we observed early biologic effect markers and identified

involved metabolic pathways, biomarkers identified in the lipidomic analysis

were not exactly the same as the biomarkers used as ‘prognostic of diagnostic

indicators of disease or a sensitive and specific tool for risk assessment’.

Although the biomarkers used were provided potential disturbed pathways,

they did not specifically respond to exposure to BPS and therefore could not

be used as exposure biomarkers in the risk assessment of exposure BPS.

As we mentioned above, significant changes were observed in

lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine,

sphingomyelin, and cholesterol ester species and the association between

phenotypic changes and lipid metabolism changes were descripted. Therefore,

these metabolites can be suggested as early-effect biomarkers in BPS

exposure. However, further efforts are required to discover more specific

biomarkers such as gene and some key enzymes involved in the related

pathway for exposure to BPS to better understand these metabolic disorders.

43

5. Conclusions

In this study, biochemical assay such as TG, TC, LDL-C and related

hormones coupled with a UPLC-Q-TOF/MS based lipidomic analysis were

conducted in mice serum to investigate the effect of prenatal exposure in

second-generation mice.

As a result, in pre-pubertal mice, decreases of the body weights and adipose

tissues were found at the lowest dose of BPS and dose-dependence differed by

gender. After the pubertal period, the treatment effects on body weights

disappeared, while total fat, subcutaneous (SAT) decreased in males, and total

fat and SAT increased in females at middle dose exposure in puberty. So, we

found that there was a difference between males and females in fat mass. And

endogenous lipid metabolite changes were observed, mainly associated with

glycerophospholipid metabolism and sphingolipid metabolism, and the

relationship between phenotypic changes and lipid metabolism changes was

also observed.

As BPS is a widely accepted EDC, it is necessary to understand the

metabolites which are the last products of cellular adjustment processes in the

body that can describe the early event of the physical condition and is

considered as closely correlative with histopathological changes. Therefore,

through combining the lipidomic analysis data and clinical biochemistry

indicator data, we may be able to suggest the direction of detailed toxic

mechanism studies by presenting early biological effect marker for the further

studies.

44

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48

국문초록

비스페놀 S의 태중 노출에 따른

후세대 마우스에서의 생리학적 변화

및 지질대사 변화

백 화 영

서울대학교 보건대학원

환경보건학과 환경보건전공

Bisphenol S (BPS)는 Bisphenol A (BPA)의 유해성과 그 물리적,

화학적 특성의 유사성 때문에 BPA 의 대체물질로 널리 사용되고

있다. 그러나 그 구조적 유사성으로 인해 BPS 가 BPA 와 유사한

독성 영향을 갖고 있으며, BPA 보다 반감기가 길며 피부투과성이

높아 BPA 보다 유해할 수 있다는 보고가 이어지고 있다. 그러나

BPS 의 독성 참고치는 아직 확립되지 않았으며, BPS 가 BPA 와 같이

모체의 노출이 산후 세대의 지질 대사에 영향을 미치는지 의 여부

역시 아직 밝혀지지 않았다. 이 연구의 목적은 산모가 BPS 에

노출되었을 때 후성세대에서 혈중생체지표 또는 지질대사의 변화를

관찰하고, 그것을 기반으로 잠재적 영향 바이오마커를 제시하는

것이다.

49

BPS 의 각 네 가지 용량 수준 (0, 5, 50, 250, 250 mg / kg bw / day)에

노출된 네 그룹의 임신마우스가 출산한 자손을 성별 및 유년기

(출생 후 21 일, PND 21), 사춘기 (PND 56) 및 성년기 (PND 161)의

주요생애시점으로 분류하였다. 각 시점의 마우스에서 혈청과 장기를

수집하였으며. 채취된 혈청을 Enzymatic Colorimetric Assay 키트와

Enzyme-Linked Immunosorbent Assay (ELISA) 키트를 사용하여

생화학지표를 측정하였다. 또한 UPLC-Quadrupole-Time-of-Flight Mass

Spectrometry 와 Progenesis QI 소프트웨어를 사용하여 혈청을

분석하여 지질대사산물의 정성적 변화를 파악하였으며. 이렇게

혈청에서 검출된 대사산물은 HMDB 와 KEGG 데이터베이스를

사용하여 확인하였다.

BPS 에 노출시킨 결과, 유년기 (3 주령) 수컷마우스에서 체중과

지방 조직의 감소는 BPS 의 저용량 노출 군에서 발견되었고 용량

의존성은 성별에 따라 달랐다. 혈중 중성 지방 (TG)은 수컷의 중간

및 고용량 노출군에서 감소하였고, 중성지방 및 총 콜레스테롤은

저용량 노출군에서 감소하였고, 인슐린과 LDL-콜레스테롤은 암컷의

중간 또는 고용량 노출군에서 증가를 보였다.

사춘기 (8 주령)시 체중에 대한 노출 효과가 사라졌으며, 중간용량

노출군에서는 수컷의 총 지방, 피하 지방 (SAT) 및 갈색 지방 조직

(BAT)이 감소하고 반면, 암컷은 총 지방과 피하지방 (SAT)이

증가했다. 수컷의 저용량 노출군에서 총 콜레스테롤은 증가했으며

혈청 포도당 및 아디포넥틴은 감소를 보였으며, 암컷에서는 총

콜레스테롤, 중성지방, LDL-콜레스테롤, 렙틴, 인슐린이 중간용량

노출군에서 증가를 보였다.

50

성년기 시 사춘기와 마찬가지로 암수모두에서 노출량에 무관하게

체중의 차이를 보이지 않았으나, 수컷 마우스의 고용량

노출군에서는 통계적으로 유의수준은 아니지만 내장지방이

감소했으며, 혈중 총 콜레스테롤 및 LDL-콜레스테롤 수치는

감소하였고 중성지방은 높은 용량으로 증가 하였다.

지질대사체 분석에서, 대부분의 노출그룹에서 막지질 이중층의

중요하고 필수적인 구조적 구성 요소인 phosphatidylethanolamine

(PE), lysophosphatidylcholine (LPC), phosphatidylcholine (PC),

sphingomyelin (SM), 및 Cholesterol ester (CE)를 잠재적인

바이오마커로 확인했다. 관찰 된 대사 산물과 관련된 대사 경로는

glycerophospholipid 대사, sphingolipid 대사 및 purine 대사였다.

종합적으로, 대조군과 차이를 나타내는 관찰된 대사 산물과 관련된

대사 경로는 주로 3 종류의 경로, 즉, 글리세롤 인지질 대사,

스핑고지질 대사 및 purine 대사에 관여한다는 것을 발견했다.

본 연구는 BPS 에 노출된 모체가 출산한 2 세대 마우스의

혈중생체지표를 측정하여 대사증후군 관련 지표가 어떻게

변화하는지를 관찰하고, 지질 대사체 변화를 통해 early biological

effect marker 를 탐색하였다. 이를 종합적으로 고려할 때, BPS 가

모체에 노출됐을 때, 2 세대 마우스에서 표현형 변화와 지질 대사

변화 사이의 관계가 관찰되었으며, 지질대사체의 경우 주로

글리세롤 인지질 대사 및 스핑고지질 대사와 관련된 대사산물에서

변화가 나타남을 볼 수 있었다. 따라서 이러한 경로에서 변화가

나타난 대사산물을 early biological effect marker 로 설정하여

51

추가적인 연구를 진행할 경우 세부적인 독성기전연구가 가능할

것이다.

주요어: 비스페놀 S, 태중 노출, 지질대사, 지질대사체 분석,

임상화학분석, UPLC-Q-TOF/MS

52

7. Supplementary information





Table S1. Identified metabolites in low dose pre-pubertal group ............. 53

Table S2 Identified metabolites in middle dose pre-pubertal group ......... 55

Table S3 Identified metabolites in high dose pre-pubertal group ............. 56

Table S4 Identified metabolites in low dose pubertal group ..................... 58

Table S5 Identified metabolites in middle dose pubertal group ................ 60

Table S6 Identified metabolites in high dose pubertal group .................... 61

Table S7 Identified metabolites in low dose adult group ........................... 62

Table S8 Identified metabolites in high dose adult group .......................... 64

53

Table S1. Identified metabolites in low dose group, Pre-pubertal

No Metabolite name Rt m/z Formula Trend Ion

Mode Sex

Fold

Change Related pathway

1 LPA(0:0/18:1) 2.1 459.2484 C21H41O7P ↑* [M+H]+ M 1.16 glycerophospholipid

2 LPA(0:0/18:2) 1.64 455.218 C21H39O7P ↑* [M-H]- M 1.41 glycerophospholipid

3 PE(20:2/24:1) 13.31 852.653 C49H92NO8P ↑* [M-H]- M 1.27 glycerophospholipid

4 PC(16:0/18:1) 9.66 804.5733 C42H82NO8P ↑* [M-H]- M 1.16 glycerophospholipid

5 PC(20:4/P-18:1) 9.2 792.5879 C46H82NO7P ↑* [M+H]+ M 1.35 glycerophospholipid

6 SM(d18:1/18:1) 7.5 773.5834 C41H81N2O6P ↑* [M-H]- M 1.16 sphingolipid

7 LysoPC(14:0) 1.59 468.3091 C22H46NO7P ↓* [M+H]+ F 0.87 glycerophospholipid

8 LysoPC(16:0) 1.87 540.3304 C24H50NO7P ↓* [M-H]- F 0.88 glycerophospholipid

9 LysoPC(16:1) 1.54 538.3143 C24H48NO7P ↓** [M-H]- F 0.58 glycerophospholipid

10 LysoPC(P-18:0) 2.48 508.3759 C26H54NO6P ↑** [M+H]+ F 1.54 glycerophospholipid

11 PS(18:0/20:0) 10.99 856.6036 C44H86NO10P ↓* [M-H]- F 0.77 glycerophospholipid

12 LPA(0:0/18:1) 2.1 459.2484 C21H41O7P ↑** [M+H]+ F 1.23 glycerophospholipid

13 PE(22:2/15:0) 7.93 802.5575 C42H80NO8P ↓** [M-H]- F 0.77 glycerophospholipid

14 PE(22:6/18:0) 7.07 790.5399 C45H78NO8P ↓* [M-H]- F 0.8 glycerophospholipid

15 PC(14:0/18:2) 6.12 774.528 C40H76NO8P ↓** [M-H]- F 0.33 glycerophospholipid

16 PC(16:0/18:1) 10.86 760.5856 C42H82NO8P ↓* [M+H]+ F 0.82 glycerophospholipid




20 PC(16:1/16:1) 6.84 730.5371 C40H76NO8P ↓** [M+H]+ F 0.29 glycerophospholipid



23 PC(18:0/16:0) 12.13 806.5895 C42H84NO8P ↓* [M-H]- F 0.67 glycerophospholipid


















40 Ceramide

(d18:1/12:0) 13.64 1007.893 C30H59NO3 ↑* [M-H]- F 1.16 sphingolipid

41 Ceramide

(d18:1/24:0) 14.83 694.6388 C42H83NO3 ↓** [M-H]- F 0.68 sphingolipid

(*: P<0.05, **: P<0.01)

54

Table S1. Continued


Mode Sex

Fold


42 Sphinganine 3.76 284.2956 C18H39NO2 ↑** [M+H]+ F 1.71 sphingolipid

43 Uric acid 3.38 149.0104 C5H4N4O3 ↓* [M-H]- F 0.009 purine

44 PI(16:0/18:2) 5.28 833.5152 C43H79O13P ↑* [M-H]- M 1.35

45 cholest-5-en-3beta-yl 16.95 645.5607 C43H74O2 ↑** [M+H]+ M 1.53

46 N-Undecanoylglycine 3.04 242.1769 C13H25NO3 ↓* [M-H]- M 0.8

47 TG(14:1/18:0/16:1) 15.77 866.7181 C51H94O6 ↓* [M+H]+ M 0.54

48 TG(15:0/14:1/16:1) 13.64 797.6039 C48H88O6 ↓* [M-H]- M 0.84

49 TG(16:0/24:1/o-18:0) 15.77 975.8949 C61H118O5 ↓* [M-H]- M 0.82

50 CE(18:2) 17 666.6337 C45H76O2 ↑* [M+H]+ F 1.21

51 1-(11Z-eicosenoyl)-

glycero-3-phosphate 2.76 487.28 C23H45O7P ↑** [M+H]+ F 1.14

52 CDP-DG(16:0/18:0) 7.6 962.5294 C46H85N3O15P2 ↓** [M-H]- F 0.62

53 DG(17:0/0:0/17:0) 14.55 619.5267 C37H72O5 ↑* [M+H]+ F 1.3

54 DG(17:0/19:0/0:0) 15.11 647.5596 C39H76O5 ↑* [M+H]+ F 1.22

55 DG(20:2n6/0:0/22:5n3) 7.78 744.5522 C44H72O5 ↓* [M+H]+ F 0.54

56 DG(i-16:0/0:0/i-16:0) 14.02 591.494 C35H68O5 ↑* [M+H]+ F 1.38

57 Docosanamide 14.78 384.3272 C22H45NO ↑* [M+H]+ F 1.17

58 LysoPE(0:0/16:0) 1.97 452.2778 C21H44NO7P ↓** [M-H]- F 0.38

59 LysoPE(18:2/0:0) 1.72 476.2778 C23H44NO7P ↓* [M-H]- F 0.36

60 MG(0:0/16:0/0:0) 3.09 353.2668 C19H38O4 ↑* [M+H]+ F 1.51

61 MG(18:0/0:0/0:0) 3.96 381.2983 C21H42O4 ↑* [M+H]+ F 1.24

62 PC(P-16:0/P-16:0) 10.53 734.5687 C40H80NO8P ↓** [M+H]+ F 0.73

63 stearoyl sphingomyelin 9.38 775.5974 C41H83N2O6P ↓* [M-H]- F 0.83

64 TG(14:0/18:4/20:0) 15.82 918.7499 C55H98O6 ↓* [M+H]+ F 0.39

65 TG(18:0/14:1/20:4) 15.72 916.7324 C55H96O6 ↓** [M+H]+ F 0.43

66 TG(18:3/18:3/22:5) 15.67 942.7526 C61H96O6 ↓** [M+H]+ F 0.29

67 TG(18:3/20:3/22:6) 15.82 968.7687 C63H98O6 ↓** [M+H]+ F 0.26

68 TG(20:2n6/14:1/20:5) 14.17 859.6861 C56H92O6 ↓** [M-H]- F 0.72

69 TG(20:3n6/20:3n6/20:5) 16.1 975.7409 C63H100O6 ↓** [M+H]+ F 0.41

70 TG(20:4/20:4/22:6) 15.62 992.7682 C65H98O6 ↓* [M+H]+ F 0.33

71 TG(20:4/22:5/20:4) 15.95 999.7405 C65H100O6 ↓* [M+H]+ F 0.48

72 TG(20:4/22:6/22:6) 15.54 1016.767 C67H98O6 ↓** [M+H]+ F 0.32

73 TG(22:2/22:4/22:6) 16.33 999.817 C69H110O6 ↓* [M+H]+ F 0.55

74 TG(22:6/20:5/22:6) 15.21 1019.709 C67H96O6 ↓* [M+H]+ F 0.45

(*: P<0.05, **: P<0.01)

55

Table S2. Identified metabolites in middle dose group, Pre-pubertal


Mode Sex

Fold


1 LysoPC(16:1) 1.54 538.3143 C24H48NO7P ↑* [M-H]- M 1.42 glycerophospholipid

2 LysoPC(22:6) 1.49 612.3299 C30H50NO7P ↑* [M-H]- M 1.19 glycerophospholipid

3 PC(16:0/18:1) 10.86 760.5855 C42H82NO8P ↑** [M+H]+ M 1.28 glycerophospholipid




7 SM(d16:0/26:1) 14.55 815.6988 C47H95N2O6P ↑* [M+H]+ M 1.54 sphingolipid

8 CerP(d18:0/26:0) 15.01 794.6205 C44H90NO6P ↓** [M-H]- M 0.6 sphingolipid

9 Palmitic acid 1.87 255.2336 C16H32O2 ↑** [M-H]- M 1.17

10 PI(16:0/22:4) 7.17 887.5628 C47H83O13P ↑** [M+H]+ M 1.31

11 CE(18:2) 17 666.6336 C45H76O2 ↓* [M+H]+ M 0.91

12 CE(18:1) 17.38 673.5891 C45H78O2 ↓* [M+H]+ M 0.69

13 (4E)-4-Octadecenoic

acid 9.71 281.249 C18H34O2 ↑* [M-H]- M 1.18

14 3-Oxotricosanoic

acid 13.41 759.6478 C23H44O3 ↓** [M+H]+ M 0.48

15 DG(18:0/20:4/0:0) 7.17 627.5345 C41H72O5 ↑** [M+H]+ M 1.37

16 DG(8:0/20:1/0:0) 13.89 475.4131 C31H58O5 ↓** [M+H]+ M 0.7

17 Ganglioside

GM3(d18:1/22:1) 13.36 1199.7735 C63H114N2O21 ↓** [M+H]+ M 0.41

18 GPA(13:0/25:0) 12.31 774.5984 C41H81O8P ↑** [M+H]+ M 1.44

19 GPA(18:4/4:0) 1.72 481.2363 C25H41O8P ↑* [M-H]- M 1.21

20 GPGro(18:4/26:2) 3.3 859.5842 C50H87O10P ↑** [M-H]- M 4.68

21 GPSer(15:1/26:2) 12.41 900.5955 C47H86NO10P ↑* [M-H]- M 1.29

22 GPSer(17:1/22:2) 9.66 872.5623 C45H82NO10P ↑* [M-H]- M 1.2

23 GPSer(17:2/26:2) 15.01 862.5923 C49H88NO10P ↑** [M-H]- M 3.75

24 MG(18:0/0:0/0:0) 2.05 400.342 C21H42O4 ↑** [M+H]+ M 2.32

25 N-Undecanoylglycine 3.04 242.1769 C13H25NO3 ↓** [M-H]- M 0.66

26 TG(15:0/14:1/16:1) 13.64 797.6039 C48H88O6 ↓* [M-H]- M 0.62

27 TG(22:6/20:5/22:6) 15.21 1019.7086 C67H96O6 ↑** [M+H]+ M 2.02

(*: P<0.05, **: P<0.01)

56

Table S3. Identified metabolites in high dose group, Pre-pubertal


Mode Sex

Fold



2 PC(16:0/20:4) 12.56 824.603 C44H81NO8P+ ↑* [M+H]+ M 1.29 glycerophospholipid


4 PC(20:2/20:3) 11.14 836.6144 C48H86NO8P ↑* [M+H]+ M 1.45 glycerophospholipid


6 Cholesterol sulfate 3.43 465.3041 C27H46O4S ↑* [M-H]- M 1.23 sphingolipid

7 PC(16:0/P-18:1) 9.25 788.581 C42H82NO7P ↑* [M-H]- M 1.23 glycerophospholipid




11 PE(18:3/24:1) 11.19 868.6066 C47H86NO8P ↑** [M-H]- M 1.44 glycerophospholipid

12 SM(d16:0/26:1) 14.55 815.6988 C47H95N2O6P ↑* [M+H]+ M 1.21 sphingolipid

50 PC(18:0/P-18:0) 12.9 796.6159 C44H88NO7P ↑* [M+H]+ F 1.5 glycerophospholipid

51 LysoPC(22:6) 1.49 612.3299 C30H50NO7P ↑* [M-H]- F 1.16 glycerophospholipid

52 PE(18:0/22:5) 11.14 774.5446 C45H80NO8P ↑* [M-H]- F 1.23 glycerophospholipid

13 PI(16:0/22:4) 7.17 887.5628 C47H83O13P ↑** [M+H]+ M 1.37

14 Methylphosphonic

acid 19.36 130.9669 CH5O3P ↓* [M-H]- M 0.79

16 CE(18:2(9Z,12Z)) 17 666.6336 C45H76O2 ↓* [M+H]+ M 0.91

17 L-methionine S-oxide 19.08 329.0854 C5H11NO3S ↓* [M-H]- M 0.85

18 TG(16:1/17:1/17:2) 16.1 844.7354 C53H94O6 ↓* [M+H]+ M 0.51

19 DG(18:0/20:4/0:0) 7.17 627.5345 C41H72O5 ↑** [M+H]+ M 1.44

20 DG(8:0/20:1/0:0) 13.89 475.4131 C31H58O5 ↓** [M+H]+ M 0.83

21 DGDG(20:1/17:0) 13.97 981.6468 C52H96O15 ↑* [M-H]- M 1.32

22 Ganglioside

GM3(d18:1/22:1) 13.36 1199.7735 C63H114N2O21 ↓* [M+H]+ M 0.78

23 Glucosylceramide(d18

:1/24:1) 13.79 854.6692 C48H91NO8 ↑* [M-H]- M 1.76

24 GPA(13:0/25:0) 12.31 774.5984 C41H81O8P ↑** [M+H]+ M 1.3

25 GPA(24:0/26:1) 17 919.71 C53H103O8P ↓* [M-H]- M 0.69

26 GPA(26:1/18:0) 16.16 835.6192 C47H91O8P ↓* [M-H]- M 0.72

27 GPCho(18:2/19:0) 13.23 800.615 C45H86NO8P ↑* [M+H]+ M 1.54

28 GPGro(18:4/26:2) 3.3 859.5842 C50H87O10P ↑** [M-H]- M 4.73

29 GPSer(15:1/26:2) 12.41 900.5955 C47H86NO10P ↑* [M-H]- M 1.44

30 GPSer(17:2/26:2) 15.01 862.5923 C49H88NO10P ↑** [M-H]- M 4.59

31 N-Undecanoylglycine 3.04 242.1769 C13H25NO3 ↓** [M-H]- M 0.7

32 PI(16:0/20:2) 5.23 883.5302 C45H83O13P ↑* [M-H]- M 1.26

33 TG(14:0/16:0/16:1) 15.77 840.7048 C49H92O6 ↓* [M+H]+ M 0.48

34 TG(15:0/14:1/16:1) 13.64 797.6039 C48H88O6 ↓* [M-H]- M 0.73

35 TG(15:0/18:0/22:1) 15.44 947.8288 C58H110O6 ↓* [M-H]- M 0.86

36 TG(16:0/18:1/18:2) 16.77 874.7839 C55H100O6 ↓* [M+H]+ M 0.67

37 TG(16:0/24:1/o-18:0) 15.77 975.8949 C61H118O5 ↓** [M-H]- M 0.75

38 TG(16:1/16:1/18:0) 16.77 848.7661 C53H98O6 ↓* [M+H]+ M 0.65

39 TG(16:1/16:1/20:4) 16.16 868.7349 C55H94O6 ↓* [M+H]+ M 0.47

40 TG(17:0/17:1/18:3) 16.49 877.723 C55H98O6 ↓** [M+H]+ M 0.73

(*: P<0.05, **: P<0.01)

57

Table S3. Continued


Mode Sex

Fold


41 TG(17:0/17:1/20:3) 16.77 900.7996 C57H102O6 ↓* [M+H]+ M 0.62

42 TG(17:1/17:2/20:3) 16.39 896.7683 C57H98O6 ↓* [M+H]+ M 0.64

43 TG(18:0/18:1/20:2n6) 17.33 930.8438 C59H108O6 ↓** [M+H]+ M 0.51

44 TG(18:2/14:0/18:3) 13.97 845.662 C53H92O6 ↑* [M-H]- M 1.36

45 TG(18:3/20:2/20:3) 16.95 931.7765 C61H102O6 ↓* [M+H]+ M 0.71

46 TG(20:0/i-12:0/14:0) 15.95 842.7202 C49H94O6 ↓* [M+H]+ M 0.47

47 TG(20:2/20:2/20:3) 16.67 925.8016 C63H108O6 ↓* [M+H]+ M 0.65

48 TG(20:5/21:0/22:3) 13.41 1033.8802 C66H112O6 ↓* [M+H]+ M 0.87

49 TG(22:0/13:0/i-13:0) 17.05 824.7671 C51H98O6 ↓* [M+H]+ M 0.7

53 GPSer(15:1/26:2) 8.26 836.5775 C47H86NO10P ↑* [M-H]- F 1.18

54 N-Undecanoylglycine 3.04 242.1769 C13H25NO3 ↓* [M-H]- F 0.81

55 (7E)-7-Octadecenoic

acid 3.71 281.2491 C18H34O2 ↓* [M-H]- F 0.68

(*: P<0.05, **: P<0.01)

58

Table S4. Identified metabolites in low dose group, Pubertal


Mode Sex

Fold


1 LysoPC(17:0) 1.64 508.3405 C25H52NO7P ↑* [M-H]- M 1.93 Glycerophospholipid

2 LysoPC(20:4) 1.06 544.3377 C28H50NO7P ↓* [M+H]+ M 0.64 Glycerophospholipid

3 LysoPC(P-18:0) 1.49 508.3741 C26H54NO6P ↑** [M+H]+ M 3.73 Glycerophospholipid

4 LysoPC(0:0/18:0) 1.64 568.3616 C26H54NO7P ↑* [M-H]- M 1.83 Glycerophospholipid

5 SM(d18:1/23:0) 13.08 845.6715 C46H93N2O6P ↓** [M-H]- M 0.78 Sphingolipid

6 PE(15:0/22:1) 5.94 782.5688 C42H82NO8P ↓** [M+H]+ M 0.47 Glycerophospholipid

7 PE(18:1/18:0) 8.39 766.5383 C41H80NO8P ↓** [M-H]- M 0.09 Glycerophospholipid


9 PC(15:0/18:2) 6.12 742.5379 C41H78NO8P ↓** [M-H]- M 0.8 Glycerophospholipid

10 PC(16:1/22:6) 4.37 848.5432 C46H78NO8P ↓* [M-H]- M 0.33 Glycerophospholipid



13 PC(14:0/18:1) 5.84 732.5518 C40H78NO8P ↓* [M+H]+ M 0.34 Glycerophospholipid




17 PC(16:0/22:5) 6.56 808.5829 C46H82NO8P ↓** [M+H]+ M 0.31 Glycerophospholipid




21 PC(18:2/P-18:1) 7.02 768.5872 C44H82NO7P ↓* [M+H]+ M 0.67 Glycerophospholipid


23 SM(d14:1/20:0) 5.56 703.5756 C39H79N2O6P ↑* [M+H]+ M 1.29 Sphingolipid

24 Galactosylceramide

(d18:1/22:0) 13.03 828.6535 C46H89NO8 ↓* [M-H]- F 0.59 Sphingolipid

25 Glucosylceramide


26 SM(d18:1/23:0) 13.08 845.6716 C46H93N2O6P ↓* [M-H]- F 0.82 Sphingolipid

27 PE(22:6/20:0) 7.17 818.5696 C47H82NO8P ↓* [M-H]- F 0.85 Glycerophospholipid

28 PC(15:0/18:2) 6.13 742.5379 C41H78NO8P ↓* [M-H]- F 0.81 Glycerophospholipid

29 PC(18:0/22:6) 7.35 834.5995 C48H84NO8P ↓* [M+H]+ F 0.76 Glycerophospholipid

30 PC(18:0/P-18:1) 7.12 772.5834 C44H86NO7P ↓* [M+H]+ F 0.73 Glycerophospholipid

31 PC(18:1/P-18:1) 7.73 814.5989 C44H84NO7P ↑** [M-H]- F 1.92 Glycerophospholipid

32 PC(18:2/18:0) 8.21 786.6001 C44H84NO8P ↓* [M+H]+ F 0.82 Glycerophospholipid

33 PC(18:2/P-18:1) 7.02 768.5872 C44H82NO7P ↓** [M+H]+ F 0.6 Glycerophospholipid

34 Cholesterol sulfate 2.54 465.3044 C27H46O4S ↓** [M-H]- F 0.76 Sphingolipid

35

3beta-Hydroxy-16beta-

(hydroxymethyl)-5alpha-

androstan-17-one

1.54 301.2188 C20H32O3 ↑** [M-H]- M 5.63

36

3-(Hexadecyloxy)-2-

hydroxypropyl 2-

(trimethylammonio)ethyl

phosphate

1.44 482.3587 C24H52NO6P ↑** [M+H]+ M 2.62

37

1-

Oleoylglycerophosphoin

ositol

0.83 619.2876 C27H51O12P ↑** [M-H]- M 5.58

38 1-pentadecanoyl-

glycero-3-phosphate 1.77 395.2212 C18H37O7P ↑** [M-H]- M 4.27

(*: P<0.05, **: P<0.01)

59

Table S4. Continued


Mode Sex

Fold


39 3,9,15-Docosatriynoic

acid 1.77 327.2341 C22H32O2 ↑** [M-H]- M 3.91

40 DG(22:0/0:0/14:0) 13.97 647.5582 C39H76O5 ↑* [M+H]+ M 1.1

41 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓** [M-H]- M 0.78

42 LysoPE(18:2/0:0) 1.11 476.2784 C23H44NO7P ↓* [M-H]- M 0.37

43 TG(20:0/20:2n6/20:2n

6) 13.41 939.8421 C61H110O6 ↓* [M+H]+ M 0.72

44 TG(22:2/14:1/18:4) 14.4 940.7366 C57H96O6 ↓* [M+H]+ M 0.47

45

S-

aminomethyldihydroli

poamide

0.65 269.1356 C9H20N2OS2 ↓* [M+H]+ M 0.05

46 DG(21:0/21:0/0:0) 15.83 745.6085 C45H88O5 ↑* [M-H]- F 1.1

47 DG(i-19:0/0:0/i-20:0) 15.83 701.585 C42H82O5 ↑* [M-H]- F 1.11

48 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓* [M-H]- F 0.85

49 GPSer(19:0/22:4) 8.06 898.5788 C47H84NO10P ↓* [M-H]- F 0.8

50 N-Palmitoyl GABA 9 746.6028 C20H39NO3 ↓* [M+H]+ F 0.77

51 TG(18:2/14:0/18:3) 12.9 859.6601 C53H92O6 ↓* [M-H]- F 0.56

52 TG(18:3/14:1/18:4) 3.38 839.6171 C53H86O6 ↓** [M-H]- F 0.44

(*: P<0.05, **: P<0.01)

60

Table S5. Identified metabolites in middle dose group, Pubertal


Mode Sex

Fold


1 SM(d18:1/22:1) 15.29 805.622 C45H89N2O6P ↓* [M-H]- M 0.83 Sphingolipid


3 SM(d17:1/26:1) 14.96 868.7264 C48H95N2O6P ↓* [M+H]+ M 0.57 Sphingolipid

4 PE(22:6) 6.69 746.5141 C43H74NO7P ↓* [M-H]- M 0.77 glycerophospholipid

5 PC(18:1/P-18:1) 7.73 814.5988 C44H84NO7P ↑** [M-H]- M 2.27 glycerophospholipid


7 PC(18:1/P-18:1) 7.73 814.5988 C44H84NO7P ↑** [M-H]- F 2.71 glycerophospholipid

8 Cholesterol sulfate 2.53 465.3044 C27H46O4S ↓* [M-H]- F 0.8 Sphingolipid

9 CerP(d18:1/26:1) 13.08 806.6434 C46H90NO6P ↓* [M+H]+ F 0.63 Sphingolipid

10

2-linoleoyl-sn-

glycero-3-

phosphocholine

1.06 564.3303 C26H50NO7P ↑* [M-H]- M 1.24

11 DG(18:3/22:0/0:0) 5.41 719.585 C43H78O5 ↓** [M-H]- M 0.75

12 GPA(17:0/26:0) 5.18 823.6214 C46H91O8P ↓** [M-H]- M 0.51

13 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓** [M-H]- M 0.68

14 TG(18:0/14:0/16:1) 15.72 822.7505 C51H96O6 ↓* [M+H]+ M 0.65

15 TG(18:2/14:0/18:3) 12.9 859.6601 C53H92O6 ↓** [M-H]- M 0.52

16 TG(18:3/14:1/18:4) 3.38 839.617 C53H86O6 ↓** [M-H]- M 0.29

17 TG(20:0/20:2n6/20:2

n6) 13.41 939.8421 C61H110O6 ↓* [M+H]+ M 0.76

18 PI(16:0/18:2(9Z,12Z)

) 4.24 833.5165 C43H79O13P ↓* [M-H]- F 0.74

19 PI(18:0/20:4(8Z,11Z,

14Z,17Z)) 5.36 887.5608 C47H83O13P ↓* [M+H]+ F 0.72

20 DG(18:3/22:0/0:0) 5.41 719.585 C43H78O5 ↓** [M-H]- F 0.7

21 GPA(17:0/26:0) 5.18 823.6214 C46H91O8P ↓** [M-H]- F 0.4

22 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓** [M-H]- F 0.69

23 N-Palmitoyl GABA 9 746.6028 C20H39NO3 ↓* [M+H]+ F 0.79

24 TG(18:1/o-

18:0/20:3) 16.39 879.814 C59H108O5 ↓* [M+H]+ F 0.46

25 TG(18:2/14:0/18:3) 12.9 859.6601 C53H92O6 ↓** [M-H]- F 0.52

26 TG(18:3/14:1/18:4) 3.38 839.617 C53H86O6 ↓** [M-H]- F 0.24

27 TG(20:4/22:6/22:6) 14.55 1021.721

6 C67H98O6 ↓* [M+H]+ F 0.71

28 TG(22:2/14:0/20:4) 15.54 948.7991 C59H102O6 ↓* [M+H]+ F 0.52

(*: P<0.05, **: P<0.01)

61

Table S6. Identified metabolites in high dose group, Pubertal


Mode Sex

Fold


1 Galabiosylceramide(

d18:1/16:0) 9.66 862.6243 C46H90NO6P ↑* [M+H]+ M 1.41 Sphingolipid

2 LysoPC(17:0) 1.64 508.3405 C25H52NO7P ↑* [M-H]- M 2.21 Glycerophospholipid

3 LysoPC(0:0/18:0) 1.64 568.3616 C26H54NO7P ↑* [M-H]- M 2.04 Glycerophospholipid

4 Galactosylceramide(

d18:1/22:0) 13.02 828.6534 C46H89NO8 ↑* [M-H]- M 1.39 Sphingolipid



7 SM(d18:1/24:1) 12.51 857.673 C47H93N2O6P ↓** [M-H]- M 0.78 Sphingolipid

8 SM(d16:1/26:1) 12.74 813.6834 C67H98O6 ↓* [M+H]+ M 0.88 Sphingolipid

9 PE(22:2/P-18:1) 10.81 814.6283 C20H39NO3 ↑* [M+H]+ M 1.45 Glycerophospholipid

10 PE(22:6/20:0) 7.17 818.5695 C47H82NO8P ↓* [M-H]- M 0.87 Glycerophospholipid

11 PE(22:6/P-16:0) 6.69 746.5141 C43H74NO7P ↓** [M-H]- M 0.75 Glycerophospholipid

12 Ceramide

(d18:1/22:0) 13.56 666.6036 C40H79NO3 ↑* [M-H]- M 1.65 Sphingolipid


14 Cholesterol sulfate 2.53 465.3044 C27H46O4S ↓* [M-H]- M 0.75 Sphingolipid

15 CerP(d18:1/26:1) 13.08 806.6434 C47H83O13P ↑** [M+H]+ M 1.33 Sphingolipid

16 Glucosylceramide




19 PI(16:0/18:1) 4.14 857.5167 C43H81O13P ↓* [M-H]- M 0.86

20 11'-Carboxy-gamma-

tocotrienol 4.52 859.5356 C25H36O4 ↓* [M-H]- M 0.77

21 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓** [M-H]- M 0.62

22 TG(18:2/14:0/18:3) 12.9 859.6601 C53H92O6 ↓** [M-H]- M 0.58

23 TG(18:3/14:1/18:4) 3.38 839.617 C53H86O6 ↓** [M-H]- M 0.33

25 PI(18:0/20:4) 5.36 887.5608 C47H83O13P ↓* [M+H]+ F 0.75

26 GPA(20:4/25:0) 16.49 859.5863 C48H87O8P ↓** [M-H]- F 0.66

27 TG(18:2/14:0/18:3) 12.9 859.6601 C53H92O6 ↓** [M-H]- F 0.52

28 TG(18:3/14:1/18:4) 3.38 839.617 C53H86O6 ↓** [M-H]- F 0.48

(*: P<0.05, **: P<0.01)

62

Table S7. Identified metabolites in low dose group, Adult


Mode Sex

Fold



2 PE(P-16:0/22:6) 9.87 746.5109 C43H74NO7P ↑* [M-H]- M 1.21 Glycerophospholipid


4 PC(15:0/20:1) 10 812.6151 C43H84NO8P ↑* [M+H]+ M 1.28 Glycerophospholipid


6 PC(18:0/22:5) 9 836.6148 C48H86NO8P ↑* [M+H]+ M 1.22 Glycerophospholipid


8 PC(18:1/18:0) 11.37 788.6159 C44H86NO8P ↑* [M+H]+ M 1.23 Glycerophospholipid




12 PC(22:5/18:0) 11.47 880.6038 C48H86NO8P ↑** [M-H]- M 1.38 Glycerophospholipid


14 PC(22:5/16:0) 9.54 852.5734 C46H82NO8P ↑** [M-H]- M 1.49 Glycerophospholipid


16 PC(22:6/20:0) 10.81 862.6289 C50H88NO8P ↑** [M+H]+ M 1.67 Glycerophospholipid


18 PC(P-18:0/20:3) 7.55 818.6025 C46H86NO7P ↓** [M+H]+ M 0.77 Glycerophospholipid


20 SM(d18:1/23:0) 13.56 801.682 C46H93N2O6P ↓** [M+H]+ M 0.68 Sphingolipid


22 CerP(d18:1/26:0) 13.02 799.666 C44H88NO6P ↓** [M+H]+ M 0.45 Sphingolipid

23 DHAP(18:0) 1.59 459.248 C21H41O7P ↑** [M+H]+ M 1.31

24 PI(16:0/18:2) 6.02 833.5159 C43H79O13P ↓** [M-H]- M 0.55

25 PI(16:0/20:2) 7.68 861.5471 C45H83O13P ↓* [M-H]- M 0.63

26 Cysteinyldopa 9.1 281.0522 C12H16N2O6S ↑* [M+H]+ M 18.98

27 (3beta,24R)-Ergost-

4-en-3-ol 16.21 383.3687 C28H48O ↑** [M+H]+ M 1.56

28

2alpha-Methyl-

5alpha-androstane-

3,17-dione

2.63 325.2134 C20H30O2 ↓** [M+H]+ M 0.64

29

1-O-Phosphono-

alpha-L-

arabinopyranose

2.2 268.9814 C5H11O8P ↑* [M+H]+ M 19.41

30 Di-Stearoyl-3-Sn-

Phosphatidylcholine 13.23 813.684 C44H89NO8P+ ↓* [M+H]+ M 0.66

31 PE-

Cer(d14:1(4E)/18:0) 5.69 689.5578 C38H77N2O6P ↓** [M+H]+ M 0.6


33 DG(22:6/24:1/0:0) 12.08 814.6311 C49H82O5 ↑** [M+H]+ M 1.52

34 DG(i-16:0/18:0/0:0) 13.84 619.5264 C37H72O5 ↑* [M+H]+ M 1.16

35 GPEtn(13:0/26:1) 13.51 832.6054 C44H86NO8P ↑* [M-H]- M 1.27

36 GPGro(20:0/22:0) 13.74 843.6475 C48H95O10P ↓** [M-H]- M 0.84

37 GPIns(18:0/20:4) 7.35 885.5473 C47H83O13P ↓** [M-H]- M 0.74

38 GPSer(16:0/22:1) 9.66 862.5846 C44H84NO10P ↓* [M-H]- M 0.8

39 GPSer(16:0/24:4) 5.94 804.55 C46H82NO10P ↓** [M+H]+ M 0.68

40 PS(18:0/20:4) 5.69 850.4968 C44H78NO10P ↑* [M+H]+ M 15.06

(*: P<0.05, **: P<0.01)

63

Table S7. Continued


Mode Sex

Fold


41 TG(18:3/20:4/20:5) 14.68 945.6934 C61H94O6 ↑* [M+H]+ M 1.26

42 TG(20:1/18:1/20:4) 15.67 998.812 C61H106O6 ↑* [M+H]+ M 1.41

43 TG(20:5/18:2/22:6) 14.83 966.7527 C63H96O6 ↑** [M+H]+ M 1.77

44 TG(22:5/22:6/22:5) 15.49 1027.774 C69H102O6 ↑** [M+H]+ M 1.32

45 TG(22:5/18:2/22:6) 15.29 994.7837 C65H100O6 ↑* [M+H]+ M 1.68

46 TG(22:6/22:6/22:6) 14.68 1040.768 C69H98O6 ↑* [M+H]+ M 1.5

(*: P<0.05, **: P<0.01)

64

Table S8. Identified metabolites in high dose group, Adult


Mode Sex

Fold


1 LysoPC(14:0) 1.26 531.2486 C22H46NO7P ↑** [M+H]+ M 1.58 Glycerophospholipid

2 LysoPC(16:0) 6.35 478.3284 C24H50NO7P ↓** [M+H]+ M 0.75 Glycerophospholipid










12 PC(20:4/P-18:1) 9.43 836.5777 C46H82NO7P ↓* [M-H]- M 0.81 Glycerophospholipid


14 PC(22:5/16:0) 9.54 852.5734 C46H82NO8P ↑* [M-H]- M 1.22 Glycerophospholipid




18 PC(O-16:0/0:0) 1.64 504.3422 C24H52NO6P ↓* [M+H]+ M 0.73 Glycerophospholipid

19 PC(P-18:0/20:3) 7.55 818.6025 C46H86NO7P ↓** [M+H]+ M 0.79 Glycerophospholipid


21 PE(22:4/P-18:1) 10.53 800.6146 C45H80NO7P ↓* [M+H]+ M 0.79 Glycerophospholipid

22 PE(P-18:0/24:4) 9.71 790.6068 C47H86NO7P ↑* [M+H]+ M 1.19 Glycerophospholipid





27 Ceramide (d18:1/22:0) 13.46 685.6239 C40H79NO3 ↑** [M+H]+ M 1.12 Sphingolipid

28 CerP(d18:1/26:0) 13.02 799.666 C44H88NO6P ↓** [M+H]+ M 0.52 Sphingolipid

29 CE(18:1) 16.62 673.5884 C45H78O2 ↓** [M+H]+ M 0.69

30 CE(18:3) 15.95 669.5603 C45H74O2 ↓** [M+H]+ M 0.51


32 (3beta,24R)-Ergost-4-en-

3-ol 16.21 383.3687 C28H48O ↑** [M+H]+ M 1.74

33 Di-Stearoyl-3-Sn-

Phosphatidylcholine 13.23 813.684 C44H89NO8P+ ↓** [M+H]+ M 0.73

34

(5Z,8Z,11Z,14Z)-N-(2-

Hydroxyethyl)-16,16-

dimethyl-5,8,11,14-

docosatetraenamide

13.89 386.3409 C26H45NO2 ↑** [M+H]+ M 1.14

35 PE-NMe2(18:0/18:0) 7.22 820.5778 C43H86NO8P ↓** [M+H]+ M 0.71

36

1-(8Z,11Z,14Z-

eicosatrienoyl)-glycero-

3-phosphate

1.39 483.2491 C23H41O7P ↓** [M+H]+ M 0.72

37 PE-Cer(d14:1(4E)18:0) 5.69 689.5578 C38H77N2O6P ↓** [M+H]+ M 0.53

38 3-Hydroxy-11Z-

octadecenoylcarnitine 1.39 424.3401 C25H47NO5 ↓** [M+H]+ M 0.14

39 CL(16:0/18:1/18:0/18:1) 0.98 1467.998 C79H150O17P2 ↓** [M-H]- M 0.45

(*: P<0.05, **: P<0.01)

65

Table S8. Continued.


Mode Sex

Fold


40 DG(16:0/20:4/0:0) 4.62 599.5018 C39H68O5 ↓** [M+H]+ M 0.66

41 DG(17:0/19:0/0:0) 14.35 647.5583 C39H76O5 ↑** [M+H]+ M 1.08

42 DG(22:0/22:2/0:0) 14.02 697.6512 C47H88O5 ↑* [M+H]+ M 1.1

43 DG(22:6/24:1/0:0) 12.08 814.6311 C49H82O5 ↑* [M+H]+ M 1.37

44 DG(i-16:0/18:0/0:0) 13.84 619.5264 C37H72O5 ↑** [M+H]+ M 1.25

45 DG(i-16:0/18:0/0:0) 13.84 579.5339 C37H72O5 ↑** [M+H]+ M 1.23

46 DHAP(18:0) 1.59 459.248 C21H41O7P ↑* [M+H]+ M 1.36

47 GPA(20:4/26:2) 7.78 797.586 C49H85O8P ↓* [M+H]+ M 0.76

48 GPA(24:0/26:1) 17.38 919.7097 C53H103O8P ↑** [M-H]- M 1.14

49 GPEtn(13:0/26:1) 13.51 832.6054 C44H86NO8P ↑* [M-H]- M 1.25

50 GPEtn(22:6/18:0) 8.11 790.5378 C45H78NO8P ↓** [M-H]- M 0.79

51 GPGro(14:1/6:0) 14.07 1103.606 C26H49O10P ↑** [M-H]- M 1.33

52 GPIns(16:1/22:2) 6.94 909.547 C47H85O13P ↑* [M-H]- M 1.44

53 GPSer(13:0/26:1) 10.2 812.577 C45H86NO10P ↓** [M-H]- M 0.81

54 GPSer(14:0/24:1) 1.82 838.5572 C44H84NO10P ↓* [M-H]- M 0.55

55 GPSer(16:0/24:4) 5.94 804.55 C46H82NO10P ↓** [M+H]+ M 0.62

56 GPSer(17:0/21:0) 12.64 856.6041 C44H86NO10P ↑** [M-H]- M 1.29

57 GPSer(17:0/24:4) 11.8 898.5766 C47H84NO10P ↓* [M-H]- M 0.9

58 Iminoaspartic acid 18.93 195.0377 C4H5NO4 ↑** [M+H]+ M 1.17

59 MGDG(18:3/26:2) 14.02 925.6601 C53H92O10 ↓** [M-H]- M 0.67

60 MGDG(25:0/26:0) 11.98 1017.833 C60H116O10 ↑* [M-H]- M 1.16

61 Pitavastatin 13.89 439.2032 C25H24FNO4 ↑* [M+H]+ M 1.08

62 SQDG(26:1/17:0) 14.35 981.647 C52H98O12S ↓** [M-H]- M 0.72

63 SQDG(26:2/16:0) 13.56 965.6141 C51H94O12S ↑** [M-H]- M 1.43

64 SQDG(26:2/18:0) 14.02 993.6473 C53H98O12S ↓** [M-H]- M 0.67

65 TG(14:1/20:4/20:5) 13.36 907.6207 C57H90O6 ↑* [M-H]- M 1.23

66 TG(16:0/18:3/22:2) 15.95 950.8151 C59H104O6 ↑** [M+H]+ M 1.84

67 TG(16:0/22:5/22:4) 15.87 974.8198 C63H104O6 ↑* [M+H]+ M 1.37

68 TG(18:0/18:2/20:3n6) 15.82 953.7565 C59H104O6 ↑* [M+H]+ M 1.5

69 TG(20:5/18:2/22:6) 14.83 966.7527 C63H96O6 ↑** [M+H]+ M 1.67

70 TG(22:1/22:0/o-18:0) 14.12 1025.928 C65H126O5 ↑** [M+H]+ M 1.12

71 TG(24:0/16:0/18:3) 15.16 923.8464 C61H112O6 ↑* [M+H]+ M 1.22

(*: P<0.05, **: P<0.01)

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