Arsenic induces gender difference of estrogen receptor in AECII cells from ICR fetal mice

Accepted Manuscript Arsenic induces gender difference of estrogen receptor in AECII cells from ICR fetal mice

Wangjun Che, Mengping Yang, Yaling Cheng, Mei Wu, Yajia Lan, Hao Zhang PII: DOI: Reference:

S0887-2333(19)30061-X https://doi.org/10.1016/j.tiv.2019.01.014 TIV 4438

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

15 April 2018 10 January 2019 21 January 2019

Please cite this article as: W. Che, M. Yang, Y. Cheng, et al., Arsenic induces gender difference of estrogen receptor in AECII cells from ICR fetal mice, Toxicology in Vitro, https://doi.org/10.1016/j.tiv.2019.01.014

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ACCEPTED MANUSCRIPT Arsenic Induces Gender Difference of Estrogen Receptor in AECⅡCells from ICR Fetal Mice Wangjun Che*,†,‡ , Mengping Yang*,§,Yaling Cheng*, Mei Wu*, Yajia Lan*, Hao Zhang*,‡

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* Department of Environmental Health and Occupational Medicine, West China school of Public

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Health, Sichuan University, No. 16, Section 3, Ren Min Nan Road, Chengdu 610041, People’s

[email protected](M.W.);

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Republic of China. [email protected](H.Z.); [email protected](Y.C.); [email protected](W.C.). [email protected](Y.L.).

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†Department of Occupational Health, Kunming Center for Disease Control and Prevention, No. 4, Ziyun Road, Xishan District, Kunming, Yunnan 650228, People’s Republic of China.

§

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[email protected](W.C.)

Chongqing Center for Disease Control and Prevention, No. 8, Changjiang second Road, Yuzhong

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‡Co- Correspondence authors

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District, Chongqing 400042, People’s Republic of China. [email protected](M.P.);

Correspondence TO: [email protected](H.Z.); Tel（fax）: +86-28- 85503257

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[email protected](W.C.); Tel（fax）: +86-871- 64321455

Running title: Arsenic induced gender differences of estrogen receptor and its mediated signal pathway in AECⅡ cells from ICR fetal mouse

ACCEPTED MANUSCRIPT ABSTRACT Arsenic is a confirmed human lung carcinogen with estrogenic activity. There are gender differences in the incidence of lung cancer. Estrogen receptors (ER) play an important role in the process of the development of lung cancer. In order to understand the gender difference effects of

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ER during carcinogenesis of lung induced by arsenic, the effects of arsenic and estrogen receptor

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antagonist (ICI182780) on expression levels of estrogen receptor beta (ERβ), extracellular

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regulated protein kinase (ERK1/2) and nuclear factor κB (NF-κB/P65) in type Ⅱ alveolar epithelial cells (AECⅡ) from different sex ICR fetal mice lung were detected. Results showed

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that arsenic increased the expression levels of mRNA and protein of ERβ, ERK1/2 and

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NF-κB/P65, and ICI182780 inhibited the increase. Furthermore, there remains a gender difference in these changes. To summarize, the observations here strongly suggested that estrogen receptor

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and its mediated signal pathway molecules might have critical roles of the gender difference of

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incidence of lung cancer in arsenic induced.

Key words：Mice; TypeⅡalveolar epithelial cells; Estrogen receptor β; Sodium arsenite; Lung

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cancer；Gender difference

ACCEPTED MANUSCRIPT 1. Introduction Lung cancer has become one of the most common death causes in both male and female malignant tumors（Jemal et al.,2011）. Air pollution, smoking and estrogen level are known risk factors of occurrence and development of lung cancer. The incidence of lung cancer in women

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continues to rise while that in men is gradually declined, indicating that women are more

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sensitive to lung carcinogens than men. However, the cause of gender difference in lung cancer is

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unclear.

IARC (1980) confirmed that arsenic and its compounds are one of the causes of human lung

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cancer in 1980. Non-occupational arsenics are mainly caused by ingesting water and foods

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contaminated with arsenic. Because women have obviously higher incidence of lung cancer than men among arsenic-exposed population, estrogen and its receptors have become the focus of

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researches on gender differences in lung cancer. Gender difference in lung cancer may be related

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with estrogen levels in women (Baik et al., 2010). Clinical and experimental data have also proved that estrogen and its receptors have played an important role in the occurrence,

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development and prognosis of lung cancer (Fucic et al., 2010; Paulus et al., 2011). Estrogen

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receptors including ERα and ERβ are the core components for endogenous and exogenous estrogens to exert their biological effects in the body, and play an important role in the growth and differentiation of target organs and the normal physiological functions of tissue cells. Moreover, Estrogen are one risk factor of canceration of some tissues and organs (skin, lung, bladder, and endometrial cancer (Karagaset al.,2001; IARC.,2004; Jongen et al.,2009). Nowadays, few studies have been reported on the expression level of ERβ in lung cancer tissues and cells and its gender difference, and results obtained are also lack of consistency. It is agreed that estrogen

ACCEPTED MANUSCRIPT receptors in normal lung tissue are mainly ERβ with little or no ERα. Compared with non-cancer tissues or cell lines, ER was not detected in lung cancer tissues, but the level of ER protein expression was significantly elevated (Schwartz et al., 2005). In the lung cancer patients with positive ER, it is considered as a functional receptor in lung tissue, and its positive expression

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has a promoting effect on the occurrence and development of non-small cell lung cancer (Zeng et

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al., 2010).

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The only way to repair injured alveolar epithelial cells is the proliferation and migration of AEC Ⅱ cells and its transformation to type I alveolar epithelial cells, thus making alveolar wall

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re- epithelialization. Kim et al. (2005) proved that mice stem cells being as the origin of lung

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adenocarcinoma could differentiate into AEC Ⅱ cells in vitro. This result has also aroused more attentions on the role of AEC Ⅱ cells in the development of lung adenocarcinoma.

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ER exerts its biological effects by mediating various signal pathways and auxiliary factors.

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MAPK-ERK1/2 is an important signal pathway, of which ERK1/2 is related with cell proliferation (Duhamel et al.,2012). When a subject-receptor complex enters into the cell, the

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signal transduction is accomplished by activating Ras factor or protein kinase C and

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Raf-MEK-ERK cascade reaction (Yao et al., 2014). After activation, ERK1/2 can rapidly phosphorylate target protein and enhance its activity on the one hand. On another hand, it can enter the nucleus, promote the phosphorylation of transcription factor, and regulate downstream target gene expression (Glaros et al.,2010). Therefore, the detection of ERK1/2 gene and its protein expression level can reflect whether receptors outside the nucleus perform signal transduction through ERK1/2 pathway under sodium arsenite exposure. Nuclear factor κB（NF-κB) participates in the regulation of multiple gene transcription and is

ACCEPTED MANUSCRIPT closely related with tumor, inflammation and cell apoptosis (Vallabhapurapu et al.,2009). It has been found that estrogen can inhibit or activate NF-κB through both genomic and nongenomic signaling cascades mediated by ER receptors (Stice et al.,2008). Studies have shown that ER receptors can promote the transcriptional activity of NF-κB in ligand-dependent form in some

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cells. At the same time, ER receptor can also cooperate with NF-κB to increase the expression of

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COX-2 and inhibit cell apoptosis through non-gene pathway (Hirano et al., 2007). Detecting the

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expression level of P50/P65 protein can reflect whether NF-κB changes or not after sodium arsenite exposure. This can indirectly reflect changes in ER expression in the lung cells.

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Arsenic is an endocrine disruptor with estrogenic activity. Further study has shown that

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arsenic exposure can significantly increase the expression of estrogen receptor-related genes, steroid metabolism-related enzymes and lung cancer-related genes in the lung tissues of female

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mice on the occurrence of lung adenocarcinoma in female mice induced by arsenic (Shen et al.,

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2007). The main purpose of this study is to investigate the accumulation level of arsenic in mice lung tissue and the changes in the expression of ER, ERK1/2 and NF-κB in AECII under arsenic

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(an environmental estrogen carcinogen) exposure. In addition, the effects of arsenic on ER and

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signal transduction pathway-related indexes, which included ERK1/2, NF-κB and BP50/p65, were observed after estrogen receptor antagonist pretreatment, and the mechanism of gender difference in arsenic- induced lung cancer is preliminary explored. 2.Materials and methods 2.1. Chemicals, animals, antibodies, and reagents ICR mice purchased from Chengdu Dashuo Biotech Co. Ltd. were fed at SPF animal house at laboratory animal center of public health college of Sichuan University. DMEM/F12 medium

ACCEPTED MANUSCRIPT and fetal bovine serum were purchased from Hyclone (USA) and Gibco (USA) companies respectively. Trypsin, DNA enzyme and type I collagenase were the products of Sigma (USA). ICI182780 (ER inhibitor), TBST and FastFire qPCR PreMix were purchased from Dalian Meilun biotechnology Co. Ltd. and Tiangen Biotech (Beijing) Co. Ltd, respectively. TRIZOL and

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predyed protein marker were from Invitrogen (USA) and Thermo Scientific (USA), respectively.

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Rabbit, anti-mouse ER and second antibody HRP-labeled, goat, anti-rabbit IgG were purchased

were from Bioss (Beijing) biotechnology Co. Ltd.

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2.2. Culture and identification of cells

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from Abcam (USA) and Boster biotechnology Co. Ltd, respectively. Rabbit, anti-mouse β-actin

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The culture of AEC Ⅱ cell was done according to the method described by Zheng et al.(2010). Briefly, Two or three pregnant ICR mice ( 17~ 19 days) were sacrificed by cervical decapitation

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and sterilized with 75% alcohol, then the uterus and the placenta were removed, the fetal mice

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were separated and assigned to one of the two group according to sex, the fetal lungs of the same sex were explanted and washed 3-5 times with PBS at 4 ℃. Following the fetal lungs were

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transferred to cell culture flask to be treated separately with 0.25% trypsin and 0.1% collagenase

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type I (containing 0.04g/l DNase I) for 20 to 30 min, Then the incubation was stopped by fetal bovine serum. The lung tissue suspension was filtered with 200-mesh sieve and the crude cell suspension were centrifuged; the cell precipitation was collected and suspended with DMEM/F12 medium containing 10% fetal bovine serum and 100 U/ml penicillin and 100 U/ml streptomycin. Subsequently, the cell suspension obtained was transferred into a sterile culture bottle coated with 5mg/ml mouse IgG and cultured in 50% CO2 incubator at 37 ℃ for 45 min. The culture fluid of cells not attached to the wall was taken out and inoculated into another sterile culture bottle

ACCEPTED MANUSCRIPT coated with IgG. This operation was repeated 3 times. Finally, the suspended cells were incubated in a new culture bottle coated with 5 mg/ml mouse IgG and cultured in 50% CO2 at 37 ℃ for 24 h, then the suspension cells were abandoned, the cells attached to the bottom are pure AEC Ⅱ cells for experiments. The AEC Ⅱ cells isolated were transferred to a sterile culture bottle and

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cultured in 50% CO2 incubator at 37 ℃ for 24 h till to their attachment to the wall. The cells

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were fixed with 40 g/L paraformaldehyde and their surface antigens were closed with goat serum.

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Afterwards, the cells were incubated with rabbit, anti-mouse first antibody at 4 ℃ overnight and with HRP-labeled, goat, anti-rabbit second antibody darkly at room temperature for 1 h in turn.

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Finally, the cells were dyed with DAPI for 5 min at a dark place. The expression of SP-C protein

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was observed under fluorescence-inverted microscope and the purity of AEC Ⅱ cells were calculated.

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The animals used in this study were handled in accordance with the principles of laboratory

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animal care adopted by the Sichuan University of animal experimentation. This study was approved by the institution's Ethics Review Board.

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2.3. Grouping and treating.

For the present study, in order to detect the effects of NaAsO2 on the expression levels of ER

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β mRNA and protein, after the AEC Ⅱ cells isolated were cultured for 24 h, the AEC Ⅱ cells from male and female fetal ICR mice were treated respectively with No reagent (control group), 0.1% DMSO (DMSO group), 0.5 μmol/l NaAsO2 (low dose group), 1.25 μmol/l NaAsO2 (middle dose group) and 5 μmol/l NaAsO2 (high dose group) for 24 h at 37 ℃. But for the part of the effects of estrogen receptor antagonist, ICI182780, after the AEC Ⅱ cells isolated were cultured for 24 h, the AEC Ⅱ cells from male and female fetal ICR mice were treated respectively with no reagent (control group), 0.1% DMSO (DMSO group), 5 μmol/l

ACCEPTED MANUSCRIPT NaAsO2 (NaAsO2 group) and 5μmol/l NaAsO2 + 1.0×10-4 μmol/l ICI182780 (NaAsO2 + ICI182780 group) for 24 h at 37 ℃. 2.4. MTT assay The cytotoxicity of NaAsO2on AEC Ⅱcells of female and male fetal mice was examined by

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MTT assay according to manufacturer’s instruction (ATCC, Manassas, America) with some modifications. Briefly, the AECⅡcells purified were suspended and adjusted to the concentration

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of 106/ml, then were aliquoted into each well of a 96 well culture plate. Six repeated samples

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were set for each dose group. After 24 h incubation, the medium was replaced with fresh medium having various concentrations of NaAsO2, respectively. The cells were then incubated for another

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24 h. At the end of the experiments; cells were washed with phenol red-free minimum essential

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medium for two times. 180μl medium and 20μl MTT (0.5 mg/ml final concentration) were then added into each well. The samples were incubated for another 4 h. The medium was removed, and

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150 μl DMSO was added. The plate was shaken slightly for 2 min to facilitate the dissolution of

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the blueformazan particles. The absorbance was determined at 570 nm using a Microplate Reader. The experiment was repeated three times.

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2.5. Real-time RT- PCR

The total RNA of the cells in each group was extracted by TRIzol method. The absorbance

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(A) values of RNA at 260 nm and 280 nm were determined by nucleic acid quantitative analyzer. Synthesis of cDNA was done according to Thermo Scientific Revert Aid First strand cDNA Synthesis Kit reverse transcriptional Kit. By reference to FastFire qPCR PreMix Kit, the total system of 20 L; reaction conditions as follows: at 95 ℃ for 1 min, at 95 ℃ for 5 s, at 55 ℃ for 10 s, at 72 ℃ for 15 s; the total of 40 cycles. Being -actin as internal control, the relative expression of target gene was calculated by using 2-ΔΔCt.Three repeated samples were set for each

ACCEPTED MANUSCRIPT dose group, the experiment was repeated three times. 2.6. Western blot analysis The prepared PMSF (Beyotime, China), Western and IP cell lysate (Beyotime, China) were mixed in volume ratio of 1:9. After AECⅡcell was treated for 24 h, the cells were centrifuged

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and precipitated. The resulting supernatant was discarded, and packed cell volume was estimated. After the precipitant obtained was mixed with RIPA cell lysate and kept on ice for 30 min, the

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mixture was centrifuged at 4 ℃ and 12 000 r/min for 5 min. The resulting supernatants in each

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group were stored at -80 ℃. Total protein concentration was measured by BCA method, then it was mixed with 5 × SDS-PAGE loading buffer in volume ratio of 1:4 (the total mass of loading

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protein was 30 g, and the total loading volume was 15 L in each hole). The mixture obtained

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was heated in boiling water bath for 5 min to denature the protein and centrifuged briefly. The resulting supernatants were taken into the sample holes, and protein marker (5 µL) was added to

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the side hole. The power supply was switched to start electrophoresis. After it was completed, the

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gel was transferred to PVDF membrane (0.22 m) followed by a closure with 5% defatted milk powder at room temperature for 1 h. then the ER first antibody with a dilution ratio of 1:1000

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was added, and closed overnight at 4 ℃. Following the membrane was washed three times with TBST, then the second antibody with a dilution ratio of 1:2500 was added, after having been

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incubated for 1h at room temperature, the membrane was washed another three times with TBST. According to the ECL Light Kit (Beyotime, China), images were captured by gel imaging system. Gray scale analysis was performed applying Image J image analysis software and being -actin as internal control. Relative protein expression level was characterized by IOD ratio of each protein to internal reference protein -actin. Three repeated samples were set for each dose group; the experiment was repeated three times. 2.7. Statistical analysis

ACCEPTED MANUSCRIPT The experimental data are represented by mean ± standard deviation. The data were analyzed applying SPSS 20.0 software. Multi group comparison was done using One Way ANOVA. Comparison between every two groups was carried out by SNK (Student-Newman-Keuls) test if the variance is homogeneous and by Games-Howell test if the variance is not homogeneous. The

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comparison between male and female mice in each dose group was performed using independent

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sample T test. For all analyses, the criterion of significance was set at P < 0.05.

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3. Results 3.1. Morphological characteristics and yield

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As shown in Figure 1, after cultured for 18-24 h, the AECⅡcells purified began to stretch

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and adhere to the wall of culture bottle and fused gradually. The cells had a circular or cubic shape, and formed island growth pattern. Their nuclei were obvious.

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The count of AECⅡcells isolated from 6 fetal mice was up to (10±5) × 106/mL. The results

isolated was (87±2.5) %.

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3.2. Cytotoxicity of arsenic

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of immunofluorescence staining of surfactant protein C showed that the purity of AECⅡcells

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The cytotoxic effects of NaAsO2 on AECⅡcells at concentrations of 0. 5 – 400 μmol/L were examined by measuring cell viability via MTT assay at 24 h. As shown in Figure 2, For the AEC Ⅱcells from male and female fetal lung, the viabilities decreased gradually as the concentration of NaAsO2 increased, and a clear dose–response relationship was also identified under these doses. For the groups treated with NaAsO2, at the concentrations that are higher than 1.25 ml/ml, all the groups exhibited significantly reduced cell viabilities compared with the control. At the concentrations of 15 µmol/L of NaAsO2, the cell viabilities were about 50%. Furthermore, no

ACCEPTED MANUSCRIPT significant differences were found in the viabilities between the AECⅡcells from male fetal lung and that from female at any the same tested concentration (P > 0.05). 3.3. Relative expression of ERβ mRNA and protein induced by arsenic Figure 3 showed the expression levels of ERβ mRNA and protein in AECⅡcells.

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For the cells from female fetal mice lung, there is significantly difference in the expression

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level of ERβ mRNA at any tested concentration between the group exposed to NaAsO2 and the

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control (P < 0.05).at the same time, the expression level of ERβ protein was significantly higher in any exposure concentrations of NaAsO2 than that in the control group (P<0.05).

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But for the cells from male fetal mice lung, did not exhibit a significant impact on the

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expression level of ERβ mRNA and protein at all tested concentrations (P > 0.05). Compared to the cells form male fetal mice lung at the same concentration, the expression

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level of ERβ mRNA and protein were significantly higher at the middle and high exposure

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concentrations of NaAsO2 (P < 0.05)

3.4. Effects of the antagonist ICI182780 on expression of ERβ mRNA and protein induced by

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arsenic

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Results of the effects of the antagonist ICI182780 on expression levels of ERβ mRNA and protein induced by arsenic were summarized in Figures 4 and 5. For the cells from female fetal mice lung, there was a significant difference on the expression levels of ERβ mRNA and protein between the arsenic exposure group and the control or DMSO group (P<0.05). Furthermore, in comparison with the NaAsO2 group, the expression levels of ERβ mRNA and protein were obviously lower than the NaAsO2+ICI182780 group (P<0.05).

ACCEPTED MANUSCRIPT But for the cells from male fetal mice lung, there were no significant differences on the expression level of ERβ mRNA and protein among all tested concentrations (P > 0.05). In addition, for the NaAsO2 group, the expression level of ERβ mRNA and protein were higher in the cells from female fetal mice lung than that from the male.

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3.5. Relative expression of ERK1/2 mRNA and protein induced by arsenic

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The expression levels of ERK1/2 mRNA and protein induced by arsenic was presented in

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Figure 6 and Figure 7.

For the cells from female fetal mice lung, there is significantly difference in the expression

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levels of ERK1/2 mRNA at any tested concentration between the group exposed to NaAsO2 and

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the control (P<0.05). Compared to the control, the expression level of ERK1/2 significantly increased at the low dose (P < 0.05), and obviously decreased at the middle and high dose (P <

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0.05). But for the ERK1/2 protein (P < 0.05), increased at the low dose and decreased at the high

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dose (P < 0.05), did not change at the middle dose (P > 0.05). Similarly, for the cells from male fetal mice lung, the expression level of ERK1/2 mRNA

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and protein increased at the low dose and decreased at the high dose (P < 0.05), did not change at

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the middle dose (P > 0.05).

Compared to the cells from male fetal mice lung at the same concentration, the expression levels of ERK1/2 protein were significantly higher at all the same groups including the control (P < 0.05), so did the ERK1/2 mRNA except for the middle dose (P < 0.05). 3.6. Effects of the antagonist ICI182780 on expression of ERK1/2 mRNA and protein induced by arsenic Figures 7 and 8 illustrate the results of the effects of the antagonist ICI182780 on expression

ACCEPTED MANUSCRIPT of ERK1/2 mRNA and protein induced by arsenic. For the cells from both male and female fetal mice lung respectively, in comparison with the control or DMSO group, the expression levels of ERK1/2 protein significantly increased at the arsenic exposure group and evidently decreased at theNaAsO2+ICI182780 group (P<0.05).

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were obviously lower in the NaAsO2+ICI182780 group (P<0.05).

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Furthermore, in comparison with the NaAsO2 group, the expression levels of ERK1/2 protein

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For the cells from female fetal mice lung, compared to the control or DMSO group, the expression level of ERK1/2 mRNA significantly increased only at the arsenic exposure group

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(P<0.05). But for the male, changes happened in both arsenic exposure and NaAsO2+ ICI182780

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groups (P<0.05).

In addition, for the arsenic exposure group, the expression level of ERK1/2 mRNA and

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protein were higher in the cells from female fetal mice lung than that from the male.

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3.7. Relative expression of NF-κB P65 mRNA and protein induced by arsenic The expression levels of NF-κB P65 mRNA and protein induced in AECII cells were shown

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in Figures 9 and 10.

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For NF-κB P65 mRNA, compared to the control, the expression level of ERβ mRNA significantly increased in the cells from both male and female fetal mice lung at the middle and high dose groups (P < 0.05).at the same time, the expression levels of NF-κB P65 protein were significantly higher in any exposure concentrations of NaAsO2 than that in the control group (P<0.05). In comparison with the cells form male fetal mice lung at the same concentration, the expression levels of NF-κB P65 mRNA were significantly higher at the middle and high exposure

ACCEPTED MANUSCRIPT concentrations of NaAsO2 (P < 0.05), but for the NF-κB P65 protein, there were significantly differences between all arsenic exposure groups and the control (P < 0.05). 3.8 Effects of the antagonist ICI182780 on expression of NF-κB P65 mRNA and protein induced by arsenic

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For the cells from both male and female fetal mice lung respectively, in comparison with the

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control or DMSO group, the expression levels of NF-κB P65 mRNA and protein significantly

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increased at the arsenic exposure group (P<0.05), only the NF-κB P65 protein evidently increased in the cells from both male fetal mice lung in the NaAsO2+ICI182780 group (P<0.05).

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In addition, for the arsenic exposure group, the expression level of NF-κB P65 mRNA and

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protein were obviously higher in the cells from female fetal mice lung than that from the male (P<0.05).

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4. Discussion

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Endocrine disruptors influence the transmission of hormone signals in cells, tissues and organs mainly by binding ligands by simulating natural hormones or by inhibiting the binding of

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natural hormones and ligands (Sonnenschein et al.,1998), thus resulting in dysfunction of the

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body. Zhao et al. (2011) found that the over-expression of ER promoted the development of non-small cell lung cancer and that the inhibition of SiRNA on ER gene could inhibit the growth of cancer cells and induce their apoptosis. As an environmental estrogen substance, arsenic may enhance the expression of ER and interfere with ER signal pathway by combining with ER receptor to form arsenic-ER complex, therefore affecting the development of lung cancer. Results from this study showed that no significant difference in the expression of ER mRNA and protein was observed between the female and the male control groups under different

ACCEPTED MANUSCRIPT doses of arsenic exposure. In the absence of sodium arsenite exposure, the expression of ER in AECⅡcells in female and male fetal mice was consistent. After arsenic exposure, the expression level of ER mRNA increased in female mice in each dose group, and it was higher in female mice in middle and high dose groups than that in male mice in the same dose groups. The results

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of ER protein data were in accordance with the overall trend of mRNA expression. ERβ protein

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expression in female mice in middle and high dose groups was higher than that in the controls and

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that in male mice in the same dose group. This indicates that AECⅡcells in female fetal mice are more sensitive to arsenic, an estrogen-like environmental carcinogen, than those in male fetal

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mice. Tokar et al. (2011) performed a carcinogenic experiment on arsenic exposure in the whole

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life period of CD1 mice in 2011. The incidence of adenoma and adenocarcinoma in the female mice was dose-dependent, and the difference was significant between arsenic exposure group and

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the control group. But no significant difference was found in both of them in male mice. Result

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from cell test in this study demonstrated that ER mRNA and protein expression in male mice in each dose group was not significantly different from that in the control. This result coincides with

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that of the above animal carcinogenic experiment.

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This study also showed that the expression level of ER mRNA and protein in female mice exposed to both arsenic and ICI182780 (estrogen receptor antagonist) was lower than that in arsenic exposure group, indicating that ICI182780 blocks the up-regulation of ERβ gene and protein in AECII of female fetal mice caused by arsenic, and further determining the effect of ERβ in this regulation procedure. But no significant difference was observed in this expression in male mice between in only arsenic exposure group and combined exposure group (arsenic and ICI182780). This may be associated with delayed toxic effect of arsenic on male AECⅡcells or

ACCEPTED MANUSCRIPT resulted from insensitivity of male AECⅡcells to arsenic or metabolic mechanism of their cells. The detailed cause for this is needed to be explored. In addition, no significant difference was found in expression level of ER mRNA and protein in male mice between in each arsenic exposure group and in the control. The results of early cell experiment showed that the expression

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level of ER mRNA and protein in male AECⅡcells in arsenic exposure group was higher than

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that in the control after their exposure to arsenic for 24 h (Xiong et al.,2017). It might be that

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arsenic specifically induced the expression level of ER and ER and changed their intracellular distribution, thus altering the biological effects of the estrogen signal pathway mediated by ER

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and ER, and finally causing different expression levels of these two receptors in male AECⅡ

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cells. More and more evidences have also proved that the effect of estrogen is not mediated by a subtype receptor, but is a balance between the mediating action of ER and ER (Nemenoff and

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Winn, 2005).

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In the extra nuclear receptors, the expression level of ERK1/2 gene and protein increased in the low dose group, but it decreased in the middle and high dose groups after exposure to sodium

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arsenite for 24 h. Therefore, it can be considered that sodium arsenite exposure for 24 h can make

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ER directly bind to target genes through a non-ligand-dependent pathway, promote the transcription of target genes with the action of auxiliary co-regulatory factors. Trace arsenic exposure can realize the rapid phosphorylation of the target gene promoter by ERK1/2 signal pathway outside nucleus, change the cell cycle and promote the proliferation. But this pathway is inhibited at high dose arsenic exposure. The expression of P65 gene and protein obviously increased in middle and high dose groups, indicating that sodium arsenite exposure can promote the activation of NF-κB, and its expression enhanced with an increase in the concentration of

ACCEPTED MANUSCRIPT sodium arsenite. In view of the relationship between NF-κB and estrogen receptors, high expression of ER receptor intra nuclear further enhances the activity of NF-κB after it is activated by sodium arsenite exposure. At the same time, ER nuclear receptor cooperates with NF-κB by non-gene pathway and activates COX-2, thus eventually promoting cell proliferation

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and inhibiting cell apoptosis. The expression of NF-κB was down regulated under the block of

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estrogen receptor antagonist, suggesting that blocking ER can inhibit over-expression of NF-κB.

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Moreover, estrogen receptor antagonist shows more obvious blocking effect on female AECⅡ cells.

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It has been found that gender differences of varying degrees in the expression of ERβ,

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ERK1/2 and NF-κB/P65in AECⅡcells of mice in this study to some extent. Environmental estrogen carcinogens can enhance ERK1/2 expression by MAPK signal pathway, and further

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promote the high expression of ERβ, thus affecting cell proliferation and apoptosis. This is a

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possible pathway for the gender difference in lung cancer. This study has provided a clue to elucidate the gender difference in lung cancer caused by environmental pollutants, and the

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Acknowledgments

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specific mechanism needs to be further verified.

This study was supported by the National Natural Science Foundation of China (grant number81160340) and Kunming city health technical personnel training project & Ten, Hundred and Thousand Talent Project (grant number2016-sw(province)-65). Conflicts of interest The authors declare no conflict of interest.

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Figure Legends

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Chinese]

Fig. 1 Morphology and immunofluorescence of AEC Ⅱ cells (A: AEC Ⅱ cells cultured for 24h; B:AEC Ⅱ cells cultured for 48h; C: AECⅡcells cultured for 0h after being purified; D: AECⅡcells cultured for 24h after being purified) Fig. 2 Effects of NaAsO2 on the viabilities of the AECⅡ cells from male and female fetal mice lung（* P < 0.05 compared with the blank）

ACCEPTED MANUSCRIPT Fig. 3 Effects of NaAsO2 on the expression levels of ERβ mRNA (A) and protein (B) in AECⅡ cells from male and female fetal mice lung (Δ P < 0.05 compared with the blank; # P < 0.05 compared with the male in the same dose group) Fig. 4 ER blot in AECII cells of fetal mice lung in different treatment groups (1, 5: blank control;

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2, 3, 4: 0.5 µmol/L, 1.25µmol/L, 5µmol/L; 6: solvent control; 7: 5µmol/L; 8: arsenic +

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ICI182780)

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Fig. 5 Effects of ICI182,780 on the levels of ERβ mRNA (A) and protein (B) induced by NaAsO2 in the AECⅡ cells from male and female fetal mice lung (* P < 0.05 compared with the

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blank and solvent control group; △ P < 0.05 compared with the male in the same dose

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group; # P < 0.05 compared with the same sex of arsenic + ICI182780 group) Fig. 6 Effects of NaAsO2 on the expression levels of ERK1/2 mRNA (A) and protein (B) in AEC

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Ⅱ cells in arsenic exposure group (Δ P < 0.05 compared with the blank and solvent

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control group; # P < 0.05 compared with the same sex of arsenic + ICI182780 group) Fig. 7 ERK1/2 blot in AECⅡ cells in different treatment groups (1, 5: blank control; 2, 3, 4: 0.5

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µmol/L, 1.25µmol/L, 5µmol/L; 6: solvent control; 7: 5µmol/L; 8: arsenic + ICI182780)

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Fig. 8 Effects of ICI182,780 on the levels of ERK1/2 mRNA (A) and protein (B) induced by NaAsO2 in the AECⅡ cells from male and female fetal mice lung (* P < 0.05 compared with the blank and solvent control group; △ P < 0.05 compared with the male in the same dose group; # P < 0.05 compared with the same sex of arsenic + ICI182780 group) Fig. 9 Effects of NaAsO2 on the expression levels of NF-κB /P65 mRNA (A) and protein (B) in AECⅡ cells in arsenic exposure group (Δ P < 0.05 compared with the blank; # P < 0.05 compared with the male in the same dose group)

ACCEPTED MANUSCRIPT Fig. 10 P65 blot in AECⅡ cells in different treatment groups (1, 5: blank control; 2, 3, 4: 0.5 µmol/L, 1.25µmol/L, 5µmol/L; 6: solvent control; 7: 5µmol/L; 8: arsenic + ICI182780) Fig. 11 Effects of ICI182,780 on the levels of NF-κB /P65 mRNA (A) and protein (B) induced by NaAsO2 in AECⅡ cells from male and female fetal mice lung (* P < 0.05 compared with

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the blank and solvent control group; △ P < 0.05 compared with the male in the same dose

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group; # P < 0.05 compared with the same sex of arsenic + ICI182780 group)

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Highlights 

Arsenic induced gender differences of estrogen receptor and its mediated signal pathway in AECII cells from ICR fetal mouse. The expression of estrogen receptor beta induced by arsenic could be inhibited by estrogen

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receptor antagonist ( ICI 182780).

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AECII cells from male and female ICR fetal mouse was used in this study, respectively.

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