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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (3): 279-289    DOI: 10.3724/zdxbyxb-2021-0182
    
Inflammatory state and autism-like behavioral phenotype of offspring induced by maternal exposure to low-dose chemical mixtures during pregnancy in mice
SONG Linyang1(),WEN Wenzhi2,LIU Jing1,JIN Xiaobao3,YANG Junhua1,3,*()
1. Department of Anatomy, School of Biosciences & Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China;
2. Department of Biopharmaceutics, School of Biosciences & Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China;
3. Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou 510006, China
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Abstract  

Objective:To investigate the effects of maternal exposure to 13 chemicals mixture (CM) during pregnancy on pregnancy outcome and health status of maternal/offspring mice. Methods:C57BL/6 pregnant mice were given drinking water containing carbaryl?0.0075?mg/kg, dimethoate?0.001?mg/kg, glyphosate?0.5?mg/kg, methomyl 0.0025?mg/kg, methyl parathion 0.003?mg/kg, triadimefon 0.03?mg/kg, aspartame 40?mg/kg, sodium benzoate?5?mg/kg, calcium disodium ethylene diamine tetra-acetate?2.5?mg/kg, ethylparaben 10?mg/kg, butylparaben 0.5?mg/kg, bisphenol A 0.004?mg/kg and acacia gum 34?mg/kg. The effects of CM exposure on pregnancy outcome, health status of dams/offspring, levels of circulating inflammatory cytokines in dams/offspring and emotional related behaviors of offspring were evaluated. Results: CM exposure during pregnancy had no significant effect on pregnancy outcome, liver function, body weight of the dams in late pregnancy and uterine/ovarian weight after delivery, however, it led to an increase in maternal serum IFN-γ level （P<0.05）. CM exposure during pregnancy had no significant effect on the liver function of offspring, but increased the serum IFN-γ, prefrontal cortex IFN-γ, IL-6 and TNF-α and hippocampus IFN-γ levels in the offspring（allP<0.01）. In addition, the offspring of CM group showed significant abnormal emotion-related (autism-like) behaviors in adulthood, especially in male offspring.Conclusion: Low dose CM exposure during pregnancy may induce inflammation status in dams/offspring, and lead to autism-like behaviors in offspring, indicating the potential effects of low dose CM exposure on human maternal and infant health.

Key words： Environmental toxicant      Maternal and infant health      Development      Immunity;      Autism      Mouse     
Received: 31 March 2021      Published: 16 August 2021
CLC:  R15  
  R114  
Corresponding Authors: YANG Junhua     E-mail: 1011986184@qq.com;jhyang2018@gdpu.edu.cn
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SONG Linyang
WEN Wenzhi
LIU Jing
JIN Xiaobao
YANG Junhua
Cite this article:

SONG Linyang,WEN Wenzhi,LIU Jing,JIN Xiaobao,YANG Junhua. Inflammatory state and autism-like behavioral phenotype of offspring induced by maternal exposure to low-dose chemical mixtures during pregnancy in mice. J Zhejiang Univ (Med Sci), 2021, 50(3): 279-289.

URL:

http://www.zjujournals.com/med/10.3724/zdxbyxb-2021-0182     OR     http://www.zjujournals.com/med/Y2021/V50/I3/279


孕期多种低剂量化学物质暴露诱发子代炎症状态和自闭症样行为的实验研究

目的：研究孕期暴露于13种混合化学物质的日常饮食对妊娠结局和母代/子代小鼠健康状态的影响。方法：C57BL/6孕鼠饮用含13种混合化学物质的饮用水，日暴露剂量分别为：西维因0.0075?mg/kg、乐果0.001?mg/kg、草甘膦0.5?mg/kg、灭多威0.0025?mg/kg、甲基对硫磷0.003?mg/kg、三唑酮0.03?mg/kg、阿斯巴甜40?mg/kg、苯甲酸钠5?mg/kg、乙二胺四乙酸2.5?mg/kg、对羟基苯甲酸乙酯10?mg/kg、对羟基苯甲酸丁酯0.5?mg/kg、双酚A?0.004?mg/kg、阿拉伯树胶34?mg/kg。评估混合化学物质暴露后的妊娠结局、母代/子代健康状态、母代/子代循环血炎症因子水平和子代的情绪相关行为。结果：孕期混合化学物质暴露对妊娠结局和母鼠肝功能、孕晚期体重及分娩后子宫/卵巢质量无显著影响，但母体血清中γ干扰素水平升高（P<0.05）。孕期混合化学物质暴露对子鼠肝功能无显著影响，但可增加子鼠血清中γ干扰素的水平，前额皮质中γ干扰素、白介素-6和肿瘤坏死因子α的水平，以及海马中γ干扰素的水平（均P<0.01）。另外，混合化学物质暴露导致子鼠在成年期表现出显著的自闭症样情绪相关行为异常，其中雄性小鼠更为严重。结论：孕期混合化学物质暴露可诱发母鼠和子鼠炎症状态，并导致子鼠自闭症样行为。


关键词： 环境毒物,  母婴健康,  发育,  免疫,  自闭症,  小鼠 
4(mg/mL)×V1(mL)20(mL)=40(mg/kg)×BW(kg)V2(mL)
 

组别

n

体重

子宫质量

卵巢质量

对照组

6

59.8±7.9

2.7±0.4

0.102±0.015

实验组

6

62.1±7.7

2.4±0.6

0.098±0.014

t值

—

–0.511

0.866

0.465

P值

—

>0.05

>0.05

>0.05
Table 1 Comparison of body weight before delivery and uterus/ovary weight after delivery of maternal mice between two groups

组 别

n

AST（U/L）

ALT（U/L）

IFN-γ（pg/mL）

IL-6（pg/mL）

IL-12p70（pg/mL）

TNF-α（pg/mL）

MCP-1（pg/mL）

对照组

6

170±30

18.8±2.5

52±10

239±30

164±22

81±11

42±7

实验组

6

173±24

19.1±2.5

70±12

248±29

155±19

89±15

44±6

t值

—

–0.144

–0.260

–2.778

–0.473

0.782

–1.109

–0.281

P值

—

>0.05

>0.05

<0.05

>0.05

>0.05

>0.05

>0.05
Table 2 Comparison of liver function and serum cytokines in maternal mice between two groups

组 别

n

每窝只数

身长（cm）

体重（g）

对照组

6

7.00±0.89

3.05±0.09

1.46±0.09

实验组

6

7.17±1.17

3.03±0.14

1.47±0.08

t值

—

–0.277

0.215

–0.178

P值

—

>0.05

>0.05

>0.05
Table 3 Comparison of pregnancy outcomes between two groups

组 别

性别

n

天冬氨酸转氨酶

丙氨酸转氨酶

对照组

雄性

6

186±24

22.9±2.8

雌性

6

190±29

23.6±3.0

实验组

雄性

6

187±27

22.7±2.7

雌性

6

189±22

23.1±2.6
Table 4 Comparison of offspring liver function between two groups

组 别

性别

n

IFN-γ

IL-6

IL-12p70

TNF-α

MCP-1

对照组

雄性

6

40.30±7.17

14.70±2.26

31.50±9.91

63.51±7.40

20.41±2.77

雌性

6

40.89±6.68

15.41±2.88

36.27±7.38

67.33±9.96

18.08±2.23

实验组

雄性

6

52.19±6.54*

15.06±1.50

32.96±10.93

69.01±9.98

18.91±2.99

雌性

6

52.44±10.17*

15.04±1.85

30.58±9.76

67.05±12.40

18.32±3.46

F1值

—

—

13.609

0.000

0.291

0.400

0.285

P1值

—

—

<0.01

>0.05

>0.05

>0.05

>0.05

F2值

—

—

0.017

0.153

0.093

0.051

1.524

P2值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05

F3值

—

—

0.003

0.179

0.835

0.491

0.541

P3值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05
Table 5 Comparison of serum levels of cytokines in offsprings between two groups

组 别

性别

n

IFN-γ

IL-6

IL-12p70

TNF-α

MCP-1

对照组

雄性

6

5.59±0.98

5.53±0.82

7.24±1.66

1.99±0.44

17.27±2.62

雌性

6

5.96±0.75

5.61±1.19

7.40±1.41

1.92±0.47

17.02±2.39

实验组

雄性

6

7.34±1.04**

7.04±1.48*

7.81±1.27

2.87±0.40**

17.37±2.25

雌性

6

7.41±1.16*

7.17±1.31*

7.12±1.64

2.63±0.57**

17.20±2.05

F1值

—

—

15.476

9.490

0.055

16.712

0.021

P1值

—

—

<0.01

<0.01

>0.05

<0.01

>0.05

F2值

—

—

0.295

0.044

0.189

0.643

0.048

P2值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05

F3值

—

—

0.147

0.003

0.469

0.173

0.002

P3值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05
Table 6 Comparison of cytokines levels in prefrontal cortex of offsprings between two groups

组别

性别

n

IFN-γ

IL-6

IL-12p70

TNF-α

MCP-1

对照组

雄性

6

9.65±2.05

5.40±1.17

18.99±3.00

1.91±0.26

22.82±3.32

雌性

6

10.65±2.13

5.63±1.47

18.50±2.90

1.98±0.40

23.80±5.13

实验组

雄性

6

13.75±3.05*

5.55±0.92

17.54±3.97

1.95±0.29

23.76±3.71

雌性

6

14.71±2.85*

5.81±1.51

17.39±3.39

1.88±0.38

22.43±5.27

F1值

—

—

14.920

0.094

0.872

0.039

0.014

P1值

—

—

<0.01

>0.05

>0.05

>0.05

>0.05

F2值

—

—

0.864

0.215

0.054

0.000

0.009

P2值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05

F3值

—

—

0.000

0.001

0.016

0.295

0.402

P3值

—

—

>0.05

>0.05

>0.05

>0.05

>0.05
Table 7 Comparison of cytokines levels in hippocampus of offsprings between two groups
Figure 1 Correlation analysis of serum levels of IFN-γ in the offspring of experimental group (=12)

组别

性别

n

嗅陌生小鼠时间（s）

嗅新小鼠时间（s）

中央区停留时间（s）

进入中央区次数

漂浮不动时间（s）

不动时间（s）

对照组

雄性

6

162±31

173±34

54±20

15±4

114±17

144±20

雌性

6

188±28

203±24

72±14#

22±4##

143±20#

174±20#

实验组

雄性

6

103±32**

110±36**

31±12*

9±3*

198±22**

234±20**

雌性

6

140±24*#

154±25*#

51±13*#

14±3**#

176±17**

205±21*#

F1值

—

—

20.178

21.170

12.101

23.982

56.260

54.314

P1值

—

—

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

F2值

—

—

6.953

9.139

9.574

16.809

0.186

0.001

P2值

—

—

<0.05

<0.01

<0.01

<0.01

>0.05

>0.05

F3值

—

—

0.225

0.337

0.011

1.297

10.616

13.063

P3值

—

—

>0.05

>0.05

>0.05

>0.05

<0.01

<0.01
Table 8 Results of behavioral tests in offsprings between two groups
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[14] LI Enshu, YE Xiaoqun, FANG Li, YE Yinghui. Effect of oxygen concentration on outcome of in-vitro fertilization-embryo transfer[J]. J Zhejiang Univ (Med Sci), 2017, 46(3): 290-294.
[15] DOU Linlin,YANG Guohui,MO Weiming. Localization of gestational age reference table and its application in prenatal screening[J]. J Zhejiang Univ (Med Sci), 2017, 46(1): 59-65.