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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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Environmental Science & Technology

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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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†

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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

ACS Paragon Plus Environment


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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



67

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),

239

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

244

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

259

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

263

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

289

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

290

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

295

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

297

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

300

to the corresponding exposure groups (Figure 5).

301

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

305

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

306

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

307

0.05).

308

4. DISCUSSION

309

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

310

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

311

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

312

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

313

animal studies would provide substantial evidences for accurate evaluation of

314

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

315

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

316

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

317

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

318

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



319

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

320

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

321

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

322

induce deleterious risks to the environment and human health.

323

Previous toxicological data showed contradictory findings about the endocrine

324

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

325

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

326

vivo.19 MVLN transcriptional luciferase experiment could efficiently screen

327

estrogenic responses of chemicals using MVLN cells stably transfected with luciferase

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

341

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

342

steroidogenesis assay, as a standard evaluation procedure in endocrine disruptor

343

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

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

potential risks in causing estrogenic effects through the perturbation in

358

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

359

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

362

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

363

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

364

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

365

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

368

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

Synthetic Phenolic Antioxidants Cause Perturbation in

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