Synthetic Phenolic Antioxidants Cause Perturbation in

Dec 13, 2017 - In the present study, four commonly used SPAs, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl hyd...
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Synthetic Phenolic Antioxidants Cause Perturbation in Steroidogenesis In Vitro and In Vivo Xiaoxi Yang, Wenting Song, Na Liu, Zhendong Sun, Ruirui Liu, Qian S Liu, Qunfang Zhou, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05057 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Synthetic Phenolic Antioxidants Cause Perturbation in

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Steroidogenesis In Vitro and In Vivo

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Xiaoxi Yang,†,‡ Wenting Song,†,§ Na Liu,†,ǁ Zhendong Sun,†,‡ Ruirui Liu†, Qian S.

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Liu,†,‡ Qunfang Zhou,*,†,‡ Guibin Jiang†,‡

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

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100085, China

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State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

College of Resources and Environment, University of Chinese Academy of Sciences,

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Beijing 100049, China

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§College

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ǁSchool

of Medicine, Henan Polytechnic University, Jiaozuo 454000, China

of Life Science, Shanxi University, Taiyuan 030006, China

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*Correspondence to:

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Dr. Qunfang Zhou, State Key Laboratory of Environmental Chemistry and

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Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy

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of Sciences, Beijing 100085, China.

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E-mail: [email protected] 1

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Abstract

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Synthetic phenolic antioxidants (SPAs) are closely correlated with human life

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due to their extensive usages, and increasing concerns have been raised on their

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biosafety. The previous controversial findings caused continuous debates on their

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potential endocrine disrupting effects. In the present study, four commonly used SPAs,

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including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),

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tert-butyl hydroquinone (TBHQ) and 2,2'-methylenebis(6-tert-butyl-4-methylphenol)

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(AO2246), were investigated for their estrogenic effects, and the results from in vitro

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screening assays showed SPAs themselves had negligible estrogen receptor binding

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affinities. Nevertheless, significant increase in E2 secretion was observed in H295R

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cells treated with SPAs, especially for BHA. The transcriptional levels of

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steroidogenic enzymes, including StAR, 3βHSD, CYP11B1, and CYP11B2 were

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up-regulated via the mediation of protein kinase A (PKA) signaling pathway. In vivo

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experiment confirmed that waterborne exposure to BHA disturbed E2 and testosterone

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(T) levels in zebrafish gonad, thus causing potential estrogenic effects through the

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regulation of hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). Accordingly,

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this study has provided new insights for SPA-induced endocrine disrupting effects.

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Considering the allowable maximum level of individual BHA or in combination with

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TBHQ and BHT in foodstuffs (200 mg kg-1), the perturbation in steroidogenesis

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observed for relatively low concentrations of SPAs would need more public attention.

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Keywords: Synthetic phenolic antioxidant, estrogenic effects, steroidogenesis, E2 2

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secretion, hypothalamic-pituitary-gonadal-liver axis (HPGL-axis)

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Table of Contents

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1. INTRODUCTION

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Synthetic phenolic antioxidants (SPAs) are extensively used in foods, food

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packaging, cosmetics, pharmaceuticals, rubbers, and plastics to prevent products from

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oxidation or deterioration due to their low cost, easy availability, and high antioxidative

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capability.1 Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and

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tert-butyl hydroquinone (TBHQ) are the frequently used forms in daily life, wherein,

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TBHQ is also the major metabolite of BHA. Many countries, such as USA, Korea and

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Europe, have approved their usages as food additives, and the working concentrations

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in food can reach to 0.01% or 0.02%.2 According to Council Directive No 95/2/ECAs,

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the allowable maximum level of individual BHA or in combination with TBHQ and

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BHT

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2,2'-methylenebis(6-tert-butyl-4-methylphenol) (AO2246), another important form of

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SPAs, it is widely applied as additives in polymer and rubber industries, with a

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production of about 3,500 ton yr-1 world-wide.5 The widespread usages of these

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emerging chemicals have drawn increasing concerns on their potential exposure risks.

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Currently available investigations on SPAs pollution have revealed that BHA, BHT

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and AO2246 could be found in diverse environmental samples, such as municipal

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sewage sludge, house dust, sewage influent, surface water and groundwater.6-9 The

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evaluation of the potential deleterious threaten to the environment and human beings

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would thus be very important for the unintended exposure of SPAs.

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in

foodstuffs

is

200

mg

kg 1.3,4 -

As

for

Diverse toxicological effects have been reported for SPAs at authorized levels in 5

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daily products.10 For example, previous in vitro and in vivo studies showed BHA

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could cause DNA repair failure, genotoxicity, oxidative stress, carcinogenicity,

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reproductive toxicities, and endocrine disrupting effects.3,11-13 As a potential endocrine

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disruptor, BHA was reported to exert controversial effects according to the

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toxicological data from different test systems. Studies based on MCF-7 proliferation

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revealed that BHA was estrogenic and capable to bind to estrogen receptors (ER).14,15

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Nevertheless, uterotrophic assay and Hershberger assay showed that BHA exposure

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decreased uterine weights of immature female rats, suggesting its anti-estrogenic

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activity in vivo.12 BHA could also influence hormone homeostasis, as demonstrated

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by BHA-decreased testosterone production in Leydig cells and rat serum.11,16

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Similarly, BHT was reported as the potential tumor promoter, carcinogen or endocrine

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disruptor,17,18 but was less estrogenic than BHA according to cell proliferation

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assays.19 TBHQ was found to exert multiple toxicological effects, such as causing

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chromosome aberration, enhancing carcinogenic effects triggered by other chemicals,

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and inducing cell death through apoptosis or caspase activation.20 As for AO2246,

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reproductive toxicities, including degeneration in sperms, and vacuolation in sertoli

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cells were observed in rats after chronic exposure to this chemical,21 weak toxicity, no

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teratogenic or carcinogenic effects were detected for its acute exposure though.5

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Apparently, previous toxicological data on SPAs usually focused on single compound,

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and the conclusions remain ambiguous due to the inconsistent experimental models in

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different studies. Considering the direct human exposure to SPA additives through 6

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food consumption or other potential exposure routes in daily life, it was worthy of

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more efforts to figure out the potential deleterious risks of SPAs based on

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comprehensive test systems, and compare toxicity differences of the compounds with

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similar chemical structures for the purpose of finding safe alternatives or surrogates.

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Among all sorts of toxicological effects, growing awareness has been gained on

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endocrine disrupting effects of chemicals in human and wildlife, which would result

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in developmental malformations, interference with reproduction, increased cancer

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risks, and disturbances in the immune and nervous system function. The direct

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binding of chemicals with hormone receptors and their disturbance in endogenous

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hormone generation are thus of high concern.22 In this regard, MVLN transcriptional

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activity assay and H295R steroidogenesis assay offer very promising in vitro

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alternatives for the corresponding high throughput screening, and have been

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successfully applied for the evaluation of emerging chemicals.23-25 As a popular in

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vivo model, zebrafish (Danio rerio) has been found useful in studying endocrine

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disrupting effects of chemicals,26,27 and its sex steroid system regulated by

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hypothalamic-pituitary-gonadal-liver axis (HPGL-axis) is very vulnerable to

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exogenous chemical exposure.28 When compared to individual test, the combination

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of in vitro screening assay and in vivo fish test could provide more convincing

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toxicological data on emerging chemicals, and would be of great importance in their

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safety assessment.

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Regarding the endocrine disrupting effects of SPAs, there is insufficient data to 7

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draw a conclusion due to contradictory findings in prior studies. In the present study,

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four commonly-used SPAs, i.e. BHA, BHT, TBHQ, and AO2246, were screened for

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their potential endocrine disrupting effects by studying their binding affinities for ERs

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and influences on steroidogenesis. BHA with the highest capability in inducing E2

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secretion was subsequently selected as a representative SPA compound for the studies

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on steroidogenic transcriptional responses in vitro, and steroid hormone production

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involved in zebrafish HPGL-axis. This study provided new findings that SPAs

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induced endocrine disrupting effect through the perturbation in steroidogenesis, and

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their unintended exposure or dietary uptake in food additive forms would cause

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potential deleterious risks to the environment and human health.

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2. MATERIALS AND METHODS

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2.1. Chemicals. SPAs, including BHA, BHT, TBHQ, and AO2246, were all

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purchased from TCI (Tokyo, Japan), and their purities were higher than 95%.

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17β-estradiol

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sulfonamide (H89, ≥ 98%) and forskolin (≥ 98%) were purchased from Sigma (St.

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Louis, MO, USA). The stock solution of each chemical was prepared by dissolving

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the corresponding compound in dimethyl sulfoxide (DMSO, Sigma), and stored at 4

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°C till use.

(E2,

98%),

N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline

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2.2. MVLN cell experiments. MVLN cell line was cultured in DMEM

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(Hyclone, USA) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin

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and 1% Lglutamine (Gibco, USA) at 37 °C with 5% CO2. The cell culture medium 8

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was replaced by phenol red free DMEM containing 5% charcoal-treated FBS

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(Biological Industries, Israel), 1% penicillin-streptomycin and 1% Lglutamine 24 h

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before chemical stimulation.

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After the evaluation of the cytotoxicities for SPAs using Alamar Blue assay (see

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Supplementary Material),29 their estrogenic activities were screened using MVLN

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cells. Briefly, cells were plated in white 96-well microtiter plates (Perkin Elmer, USA)

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at the density of 4×104 cells/well, and cultured for 24 h. The exposure concentration

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ranges of BHA, BHT, and TBHQ were the same (10-4, 10-3, 10-2, 10-1, 100, and 101

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µM), and that of AO2246 was from 10-5 to 100 µM. Meanwhile, 17β-estradiol (E2)

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(10-7~10-3 µM) was tested as the positive control, and the solvent control group was 0.5%

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(v/v) DMSO. All treatments were conducted for 48 h in three replicates. Luciferase

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activity was measured with Luciferase Assay System (Promega Corp., USA). The

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maximum effect of E2 (10-3 µM) was set as 100%, and the relative response of each

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sample was normalized as the percentage of maximum induction of luciferase activity.

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2.3. H295R cell experiments. The H295R cell line was purchased from

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ATCC (CRL-2128, ATCC, USA), and maintained in DMEM/F12 medium

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supplemented with 1% penicillin-streptomycin, 1% insulin–transferrin–selenium-G,

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and 2.5% Nu-Serum (BD Bioscience, USA) in a CO2 incubator at 37 °C.

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Based on the cell viability assays (see Supplementary Materials), the

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steroidogenic effects of SPAs at non-cytotoxic levels (cell viability > 80%) were

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analyzed in H295R cells. Briefly, cells were plated in 6-well plates at a density of 9

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1×106 cells/well for 24 h. Then the stimulation of BHA (0, 1, 10, and 100 µM), BHT (0,

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1, 10, and 100 µM), TBHQ (0, 0.01, 0.1 and 1 µM), and AO2246 (0, 0.01, 0.1 and 1

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µM) was performed for 48 h. 0.1% DMSO and 10 µM forskolin were set as the solvent

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control and the positive control, respectively. Three replicates were tested for each

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treatment. After the exposure was terminated, the culture medium was collected for

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the measurement of steroid hormones, including E2, testosterone (T), progesterone,

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cortisol, and aldosterone. The cells from BHA exposure experiments were harvested

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for target gene transcription analysis.

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Using BHA as a representative compound, time-dependent effect of BHA on the

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induction of steroid hormone secretion was examined. Namely, H295R cells were

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exposed to 100 µM BHA for 6, 14, 24, and 48 h. The solvent controls of 0.1% DMSO

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were set at the corresponding time points. The culture medium was collected and

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submitted to E2 analysis.

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The role of protein kinase A (PKA) signaling pathway was investigated by

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co-exposure of H295R with BHA (0, 1, 10 and 100 µM) and H89 (10 µM) for 48 h,

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and the cell medium was submitted to the measurement of E2 secretion levels.

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2.4. In vivo test based on zebrafish. Adult male zebrafish (5-month-old,

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AB strain, Institute of Hydrobiology, Chinese Academy of Sciences) were maintained

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in 5-L glass tanks which contained 4 L of charcoal filtered water at 28 ± 0.5 ℃ with a 14

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h:10 h light–dark cycle. Fish were fed twice daily with live brine shrimp (Jiayin

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Biology Feedstuff Co., Ltd., Tianjin, China). Before chemical exposure, fish were 10

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acclimated for one week. After preliminary acute toxicity test, non-lethal

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concentrations of BHA (1, 2 and 5 µM) were used to expose experimental fish for 21

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days. The solvent control was 0.01% DMSO. Total 8 fish in one tank were set in each

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exposure group, and each treatment was performed in duplicate. The exposure

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solution in each tank was daily renewed by half. The actual exposure concentrations of

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BHA were monitored, and the results showed they were in the range from 88% to 91%

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of nominal concentrations (Table S1), indicating the relatively stable exposure system

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was constructed for in vivo test. After the exposure was terminated, all fish samples

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from each group were transferred to icy water for cold shock. The gonad tissues were

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dissected, weighted, snap-frozen in liquid nitrogen, and stocked at -80 °C till analysis.

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2.5. Steroid hormone measurement. Total 5 steroid hormones, including

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E2, T, progesterone, cortisol, and aldosterone were measured in H295R culture

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medium using radioimmunoassay kits (Beijing North Institute of Biological

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Technology) according to manufacturer’s instructions. The detection limits of E2, T,

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cortisol, aldosterone and progesterone were 5 pg/mL, 0.1 ng/mL, 10 ng/mL, 62.5

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pg/mL and 0.2 ng/mL, respectively.

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Zebrafish gonad tissue samples from the exposure experiments were

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homogenized in cold phosphate buffered saline (PBS) at the dilution ratio of 10. The

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homogenates were centrifuged, and the supernatants were submitted to the

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measurement of E2 and T levels using Fish E2 and T ELISA Kits (Tongwei

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Biotechnology Co., Ltd. Shanghai) according to manufacturer’s instructions. The 11

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detection limits of E2 and T were 2 pmol/L, and 10 pg/mL, respectively. The

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concentrations of E2 and T in gonad samples were calculated based on the wet weights

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of tissues.

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2.6. RNA isolation and quantitative PCR analysis. For H295R cell

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samples and zebrafish tissues, total RNA was directly extracted using Trizol reagent

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(Gibco), and purified according to the manufacturer’s instruction. The RNA

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concentrations were measured by NanoDrop (Thermo Scientific, USA), and their

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qualities, including the purity and integrity, were guaranteed by A260/280 ratio in the

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range of 1.8-2.0, and the proper profiles of 28S, 18S and 5.8S rRNA bands in 1% agar

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gel. Total 1 µg RNA was submitted to the synthesis of the first-strand complementary

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DNA (cDNA) for each sample using iscriptTM cDNA Synthesis kit (BioRad, USA) in

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ABI PCR system (Applied Biosystems, USA). Quantitative PCR was performed on

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Roche 480 Real-Time PCR system in 384-well PCR plates (Roche, USA). PCR

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reaction mixtures (10 µL) contained 0.5 µL of cDNA template, 0.5 µL of forward

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primers, 0.5 µL of reverse primers, 3.5 µL of nuclease-free water, and 5 µL of

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SYBRGreen qPCR MasterMix (BioRad, USA). The thermal cycling was 95 °C for 30

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s, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. As for H295R cells, the

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transcriptional levels of total 9 target proteins, including StAR, CYP11A1, 3βHSD,

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CYP11B1, CYP11B2, CYP17, CYP21, 17βHSD, and CYP19 were tested. As for

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zebrafish, 8 genes including FSHR, LHR, CYP19A1, StAR, 3βHSD, 17βHSD,

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CYP11B2, and CYP17A1 were evaluated in gonad samples. Beta-actin was selected 12

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as the housekeeping gene for both species in quantitative PCR analysis, as it showed

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consistent expression in different treatments. The corresponding primer sequences

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listed in Table S2 were designed using NCBI and Primer3web (4.0.0). The selected

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template regions of each target mRNA for primer design covered the introns, which

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would eliminate the potential interference from trace level contamination of genomic

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DNA (gDNA) in quantitative PCR, and avoid the unintended RNA loss due to DNase

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usage. The linearity (r2 > 0.998) was evaluated for each quantitative PCR reaction,

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and the reaction efficiencies were in the range of 90-110% (Table S2). The negative

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reactions of no template controls were performed by adding 0.5 µL of RT-PCR grade

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water (Promega, USA), instead of cDNA template, in quantitative PCR assays for the

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target genes, and no detectable amplicons were observed after 40 cycles, showing no

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cross contamination in the assay system. Triplicates were performed for each assay,

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and the relative mRNA expression of targeted genes was normalized to the

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housekeeping gene using 2−△△CT method.30

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2.7. Statistical analysis. The results were statistically analyzed by SPSS

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(18.0), and graphed using GraphPad Prism 5. All data were shown as mean ± SD.

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Differences in different exposure groups were evaluated by one-way analysis of

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variance (ANOVA) and Tukey’s test. Statistical significance was denoted when p is

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less than 0.05 or 0.01. Cell experiments were performed independently for at least 3

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times. The statistical comparison of steroidogenesis in vivo was performed for fish

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individuals in exposure groups with those in solvent control. 13

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3. RESULTS

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3.1. Screening of estrogenic effects for SPAs. Based on MVLN

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luciferase activity assay, the positive control, E2 apparently elevated luciferase activity

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(19%) when the exposure concentration was higher than 10-6 µM (Figure S1),

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showing the feasibility and high sensitivity of the screening assay. Nevertheless,

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non-cytotoxic exposure levels of BHA, BHT, TBHQ, and AO2246 (Figure S2) did not

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significantly alter luciferase activities in MVLN (p > 0.05, Figure S1), suggesting

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these chemicals had negligible estrogenic effects. No ER binding was observed for

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SPAs based on Hithunter® ER binding assay (Figure S3), confirming the above

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results.

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3.2. Effects of SPAs on steroidogenesis in H295R cells. The

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secretion levels of steroid hormones, including E2, T, cortisol, aldosterone, and

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progesterone were monitored in H295R culture medium after 48 h treatments of SPAs

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at non-cytotoxic levels (Figure S4). The positive control, 10 µM forskolin confirmed

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the feasibility of H295R model for steroidogenesis assay as the five tested steroid

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hormones were significantly increased (p < 0.05, Figure S5). In view of SPAs (Figure

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1), it was found that BHA significantly induced E2 elevation in a dose dependent

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manner (p < 0.01). E2 levels were up-regulated by 8.4-, 27.6- and 72.1-folds in 1, 10,

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and 100 µM BHA exposure groups, respectively (Figure 1A). As for the rest hormones,

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the increase trend (1.1-2.5 folds) was observed in 100 µM BHA (Figure 1B-E). In

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regard of the other three SPAs, 100 µM BHT exposure caused the second strongest 14

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effect on E2 secretion (increased by 30 folds), and the increasing trend was also

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observed in low concentrations (0.01-1 µM) of TBHQ exposure groups (Figure 1A).

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Regarding AO2246 exposure groups, the inverted dose response curve was apparently

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observed for E2 secretion (Figure 1A), suggesting some other exposure stress, other

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than the steroidogenic effect, could occur in cells exposed to 1 µM AO2246, as it was

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on the verge of being cytotoxic (89.4% cell viability, Figure S4). No obvious changes

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were detected in T, cortisol, aldosterone or progesterone secretion upon the treatments

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of BHT, TBHQ, or AO2246 (Figure 1B-E).

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As significant increase of E2 level was observed in BHA exposure groups, the

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time course for E2 production was examined in H295R cells exposed to 100 µM BHA.

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As shown in Figure 2, BHA exposure caused significant increase in E2 secretion (p
0.05).

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PKA signaling pathway was one of the most important pathways involved in

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steroidogenesis,31 its blockage could attenuate chemical-induced perturbation in

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steroid hormone secretion from H295R cells.32 In this study, H89 (10 µM) was used

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for the co-exposure experiment of H295R with different concentrations of BHA (1, 10,

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and 100 µM), and E2 secretion in each treatment was monitored. The results in Figure

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4 showed that E2 concentrations in co-exposure groups were significantly lower than

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those in BHA exposure groups at the corresponding exposure levels (p < 0.01). This

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finding indicated that H89 could efficiently block BHA induced E2 production in

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H295R cells, and PKA signaling pathway was crucially involved in the perturbation

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of steroidogenesis due to BHA exposure.

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3.4. Perturbation in steroidogenesis in vivo due to BHA exposure.

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Zebrafish experiment was designed to evaluate if SPA perturbed steroidogenesis could

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happen in vivo. After 21 d exposure to BHA, E2 and T were measured in zebrafish

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gonad samples. As shown in Figure 5, E2 concentrations were significantly increased

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by 14%, 33%, and 30% in 1, 2, and 5 µM BHA exposure groups, respectively (p < 16

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0.05). Similarly, the dose-dependent up-regulation was found for T levels in BHA

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treated zebrafish gonad samples, and the increments were 15%, 20%, and 22% relative

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to the corresponding exposure groups (Figure 5).

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Total 8 steroidogenic genes were monitored for their transcriptional changes in

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zebrafish gonads upon BHA exposure, and the results were depicted in Figure 6.

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Significant up-regulation was observed in LHR, CYP19A1, and CYP11B2 (p < 0.05).

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LHR and CYP19A1 showed relatively higher responses to BHA exposure than the

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others. In contrast, the down-regulation was observed for FSHR after BHA exposure,

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while no significant changes were detected in StAR or 17βHSD expression levels (p >

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0.05).

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4. DISCUSSION

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Considering the widespread application of SPAs, high concerns are rising on

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their biosafety, especially for those commonly used forms, like BHA, BHT, TBHQ,

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and AO2246. Nevertheless, whether they are potential endocrine disrupting chemicals

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remain ambiguous. The combination of both in vitro screening assays and in vivo

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animal studies would provide substantial evidences for accurate evaluation of

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chemical toxicities. In this study, MVLN cell experiments and Hithunter assays

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showed that four SPAs, including BHA, BHT, TBHQ, and AO2246, did not cause

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estrogenic effects through the direct binding to ERα, but H295R tests proved that

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three of the tested SPAs significantly increased E2 secretion through the perturbation

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in steroidogenesis of the cells. In vivo experiments further confirmed that the 17

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exposure of BHA, a representative SPA compound, could disturb steroid hormone

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secretion in zebrafish gonad. Accordingly, these results have provided new evidences

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for SPAs as the potential endocrine disruptors, and their exposure could potentially

322

induce deleterious risks to the environment and human health.

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Previous toxicological data showed contradictory findings about the endocrine

324

disrupting effects of SPAs. For example, BHA could present a weak estrogenic effect

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and anti-androgenic properties in vitro, while exhibit anti-estrogenic properties in

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vivo.19 MVLN transcriptional luciferase experiment could efficiently screen

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estrogenic responses of chemicals using MVLN cells stably transfected with luciferase

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reporter gene and estrogen receptor element,33 and HitHunter assay provided direct

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data on the binding affinities of chemicals for estrogen receptors.34 The in vitro

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screening based on these two assays in this study indicated that four tested SPAs did

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not show any detectable ER binding affinities or significant estrogenic effects at the

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tested concentrations (≤ 10 µM, p > 0.05). BHA exposure induced an elevation in

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luciferase activity when the concentration was increased to 10 µM though (Figure

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S1A). This finding was comparable to previous results from the luciferase assays of

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two U2-OS cell lines transfected with ERα and ERβ, wherein the lowest effect

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concentrations of BHA were 5.9 µM and 8.4 µM, respectively.35 Considering BHA, as

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a weak estrogen, gave maximal cell proliferation (66.8%) at the concentration of 50

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µM based on E-SCREEN assay,15 the estrogenic effects of BHA itself could be

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negligible (7.5%) when the concentration was controlled within 10 µM. 18

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The interferences on biosynthesis and secretion of natural steroid hormones are

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one of the important targets for endocrine disrupting chemicals.36 H295R

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steroidogenesis assay, as a standard evaluation procedure in endocrine disruptor

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screening program, allows the direct measurement of alterations in cell hormone

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production due to chemical stimulation.37 Recently, Li et al.16 reported the

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co-exposure of BHA (50 µM) and 22R-OH-cholesterol led to the decrease of T

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production in rat immature Leydig cells, suggesting SPAs might be involved in

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steroidogenesis. In this study, through the monitoring of 5 steroid hormones secreted

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from SPA-exposed H295R, it was found that E2 secretion responded most sensitively

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to SPA exposure when compared with the other tested hormones. Among 4

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compounds, BHA induced E2 secretion most efficiently in both dose- and time-

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dependent manners (Figure 1A and Figure 2). BHA exposures at the levels of 1 µM

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and 10 µM (48 h) increased E2 secretion in H295R cells by 8.4 and 27.6 folds (Figure

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1A), and the absolute E2 levels in these two groups were 225 pg/mL and 736 pg/mL

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(i.e. 0.83 nM, and 2.7 nM), respectively. These levels of E2 would nearly obtain the

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maximum transcriptional luciferase activity (Figure S1) and ERα binding affinity

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(Figure S3) according to MVLN experiment and Hithunter assay. So, BHA could pose

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potential risks in causing estrogenic effects through the perturbation in

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steroidogenesis, the estrogenic effects of 1 and 10 µM BHA itself were negligible

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though (Figure S1A). The new finding on SPA-increased E2 biosynthesis implicated

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the potential disturbance of SPAs on the endocrine system in organisms. 19

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Environmental Science & Technology

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In steroidogenesis (Figure S7), cholesterol was firstly transferred to the inner

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mitochondrial membrane by StAR, converted to progesterone by CYP11A1, and

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subsequently metabolized through multistep process to aldosterone, cortisol, T and E2,

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respectively, under the regulation of diverse enzymes, including 3βHSD, CYP21,

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CYP11B1, CYP11B2, CYP17, 17βHSD, and CYP19 etc.24 As demonstrated

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previously,38 the significant up-regulation of target gene transcriptional levels induced

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by 10 µM forskolin (Figure S6), was consistent with increased steroid hormone

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secretion (Figure S5), showing the crucial correlation between the expression of key

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steroidogenic genes and hormone levels. To understand how SPAs disturbed

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steroidogenesis, BHA was selected as a representative compound for the study of its

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function on these target genes. The results showed the transcription levels of StAR

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and 3βHSD were significantly increased in a dose-dependent manner (Figure 3, p