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Food Safety and Toxicology

Evaluation of Estrogenic Activity of Novel Bisphenol A Alternatives, Four Bio-inspired Bisguaiacol F Specimens, by in vitro Assays Ying Peng, Kaleigh H Nicastro, Thomas H. Epps, III, and Changqing Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03746 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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Journal of Agricultural and Food Chemistry

Evaluation of Estrogenic Activity of Novel Bisphenol A Alternatives, Four Bio-inspired Bisguaiacol F Specimens, by in vitro Assays Ying Peng,1 Kaleigh H. Nicastro,2 Thomas H. Epps, III,2,3 Changqing Wu1* 1. Department of Animal and Food Science, University of Delaware, Newark, Delaware 19716, United States 2. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States 3. Department of Materials Science & Engineering, University of Delaware, Newark, Delaware 19716, United States * Changqing Wu1, (Tel: (302) 831-3029; Fax: (302) 831-2822; E-mail: [email protected])

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Abstract

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Alternatives to Bisphenol A (BPA), such as lignin-inspired bisguaiacol F (BGF), are of

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interest for food contact materials due to increasing evidence of estrogenic activity (EA) and

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exposure-correlated harmful effects of BPA and its analogues. BGF has similar thermal and

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mechanical properties to BPA, but contains additional methoxy substituents that may

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significantly reduce its endocrine disruption potential. In this study, the EA of four BGF

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samples with different regioisomer ratios was quantified relative to 17-estradiol at ten

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concentrations by using two in vitro assays: MCF-7 cell proliferation and VM7Luc4E2

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transactivation (TA). The results suggest BGF mixtures with higher molar ratios of p,p′-BGF

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and o,p′-BGF regioisomers exhibited lower EA than BPA, while BGF samples containing higher

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molar ratios of m,p′-BGF had no detectable EA over a wide range of test concentrations. These

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findings suggest the potential of BGF as a viable alternative to BPA for use in more

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environmentally friendly materials.

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Keywords: Estrogenic activity, Bisphenol A, Bisguaiacol F, BG1Luc, MCF-7, In vitro assays, VM7Luc4E2

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Introduction

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Extensive evidence on the increased risk of adverse health effects of Bisphenol A (BPA)

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has resulted in heightened public awareness to question the safe use of BPA in food contact

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materials (FCMs) along with increased scientific interest to develop safe alternatives to BPA.

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BPA is a vital monomer in the production of many polymer-based materials, including epoxy

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resins and polycarbonate plastics used in the manufacturing of many consumer products, such as

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drinking bottles, food can liners, food packaging, baby toys, and thermal printer paper.1 Due to

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the prevelance of BPA, BPA is present at low concentrations in our environment, including in

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foodstuffs, dental composites, dust, and bodies of water.2

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exposure to BPA is through ingestion, as BPA can leach into food from plastic packaging

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materials.3 Liao et al. reported that 75% of tested food samples had bisphenols at concentrations

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ranging from 0.10 ng/g fresh weight to 1130 ng/g fresh weight when measured by high-

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performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS).4

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concentration of BPA was higher in canned food than in other fresh foods because bisphenols are

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used commonly in epoxy coatings for metal cans.4,5

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The primary source of human

The

Public and scientific interest in the impacts of BPA has risen due to universal human

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exposure and the increasing evidence of its adverse health effects.

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metabolization of BPA in human bodies6,7, high BPA concentrations in human fluids were

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reported extensively.8 According to the Center for Disease Control and Prevention (CDC), in

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2008, ~93% of 2517 people selected for BPA screening had measurable quantities of the total

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BPA (including unconjugated BPA plus its primary conjugated metabolites, BPA-glucuronide 3 ACS Paragon Plus Environment

Despite of rapid


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and BPA-sulfate) in their urine samples.9 BPA at similar concentrations had been detected in

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colostrum, amniotic fluid, and umbilical cord blood.8

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urine BPA exposure concentration for women (16–49 years) was 2–3 µg/L (3.2–8.8 nM), with

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maximum concentrations of 16 µg/L (70.4 nM).1 In 2011-2012, the median urine BPA exposure

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concentration for women decreased to 1 µg/L (4.4 nM), with a maximum concentration of

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11 µg/L (48.4 nM).1 Similar BPA exposure levels were found in children (6-18 years); from

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2003 to 2012, the 95th percentile BPA concentration in children decreased significantly from

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16 µg/L to 9 µg/L.1 The decreasing trend of BPA in urine may be the result of amendments to

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food additive regulations that prohibit the use of BPA-containing polymers in food contact

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materials, along with changes in consumer behavior due to increased public education about the

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risks of BPA.1 The US Food & Drug Administration (FDA) claims that BPA is safe at the

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current levels presenting in foods;10 however it is not clear if the levels of BPA in human fluids

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will increase with the continued use of BPA in FCMs? On February 2018, the scientists from the

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Endocrine Society opposed the FDA’s statement on BPA safety and asserted that BPA-based

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polymers are not suitable for use in FCMs.11 Thus, the safe use of BPA is still contested, such

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that new and safe alternatives to BPA are necessary.

For example, in 2003-2004, the median

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One mechanism of BPA endocrine disruption is through estrogenic activity (EA),12 in

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which the compound mimics the actions of naturally occurring estrogens such as 17-estradiol

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(E2). BPA binds to estrogen receptors (ER) α and β to activate estrogen signaling pathways,

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causing endocrine disruptor activity.13 Endocrine mediated toxicity results in a wide variety of

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adverse health effects including diabetes, obesity, reproductive disorders, breast cancer, birth

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defects, chronic respiratory diseases, and cardiovascular diseases.14 For example, between 2010

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and 2017, 427 National Institute of Environmental Health Sciences (NIEHS) funded publications 4 ACS Paragon Plus Environment

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reported on the health effects of BPA.15 In 2011, Shankar and Teppala reported a positive

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correlation between high concentrations of BPA in urine and diabetes mellitus independent of

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traditional diabetes risk factors.16 According to Bhandari et al. and Carwile and Michels, BPA

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can distress neural circuits that regulate feeding behavior or change differentiation pathways of

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adipocytes, promoting obesity in children and adults.17,18 Galloway et al. reported higher daily

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BPA excretions were associated with higher total testosterone concentrations in men, increasing

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the risk of male sexual dysfunction.19 Shafei et al. provided evidence that BPA increased the

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incidence and susceptibility to neoplastic transformations among types of cancers through

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various mechanisms.13 According to Melzer et al., urinary BPA concentrations were higher in

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those persons with severe coronary artery stenosis.20

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disruptive effects of BPA, BPA was classified as an endocrine-disrupting chemical (EDC),21 and

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NIEHS suggested people avoiding using BPA-containing products, especially in food contact

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materials to reduce the exposure to BPA.22

In short, because of the endocrine

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Consumer products, such as canned goods and polycarbonate bottles, previously

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manufactured using BPA are now produced using BPA-free formulations; however, BPA-free

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does not mean EA-free. For example, Bittner reported that three BPA free Tritan™ resins,

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which claim to be EA-free, leached chemicals that had significant EA levels as measured by

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reporter gene assays.12 Moreover, the leaching of EA compounds in Tritan™ resins increased

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with exposure to UV radiation (e.g. sunlight).12 Because plastics have numerous desirable

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properties, such as high strength-to-weight ratios, high transparency, high durability, and low

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manufacturing costs,23 it is difficult for other materials such as stainless steel and glass to replace

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BPA-derived plastics, especially for single-use food storage products. Therefore, a sustainably-



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sourced and less toxic BPA alternative is desirable for manufacturing plastic containers for

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foodstuffs.

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One potential BPA alternative is bisguaiacol F (BGF), a lignin-inspired monomer with

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competitive thermal and mechanical properties to BPA in epoxy resins.24–26 Similar to the

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general synthesis of BPA, BGF is produced through an acid-catalyzed electrophilic aromatic

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substitution reaction between two compounds (in the case of BGF, lignin-derived vanillyl

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alcohol and guaiacol).24,25 For BGF, this reaction results in three regioisomers: p,p′-BGF, m,p′-

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BGF, and o,p′-BGF, as shown in Figure 1, and the ratio of regioisomers can be manipulated

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through judicious choice of catalysts and reaction conditions.27 BGF-derived epoxy resins have

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similar thermal stability and mechanical strength to BPA-derived epoxy resins, suggesting BGF

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is a promising BPA alternative.24,25 Hong et al. predicted that the p,p′-BGF regioisomer is

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possibly an ER binder by using molecular docking simulations with a decision forest method.28

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However, limited bioassay data were available to demonstrate the endocrine disruption potential

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of ER binding, and no data were available on the other BGF regioisomers. Therefore, more

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extensive risk assessment and toxicological tests must be performed before promoting BGF as a

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less endocrine disrupting alternative to BPA.29

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The EA of a test substance can be evaluated through in vitro bioassays and in vivo assays.

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Since 1998, the EPA has promoted the use of in vitro bioassays as suitable screening tools for

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suspected estrogenic chemicals because in vitro bioassays are more time efficient, more effective,

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and less expensive than in vivo assays.30 The suggested in vitro assays use human-derived cell

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lines or estrogen receptors; therefore, there are fewer discrepancies in comparison to in vivo

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results,31 and no animal ethics or welfare issues are present. The major in vitro bioassays used

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include binding assays, reporter-gene assays, and cell-proliferation assays.32 Binding assays are


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fast, cell-free screening methods that measure the binding affinity of test compounds to estrogen

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receptors. However, the binding event does not indicate whether the test compound upregulates

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or downregulates the estrogen pathway.31 Therefore, other methods are needed to verify the

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results. One such method relies on cell proliferation assays that quantify whole-cell-level EA by

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using the proliferative effect of estrogens on the MCF-7 human breast cancer cell line as the

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endpoint.33 Reporter gene assays, such as the VM7Luc4E2 transactivation (TA) test (previously

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known as BG1LucERTA bioassay), detect receptor-mediated gene expression.32 The name

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changed to the VM7Luc4E2 TA test because the DNA analysis (Short Tandem Repeat, STR)

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revealed that the original cell line was not BG1 cells, but a variant of human breast cancer

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(MCF-7) cells.34 VM7Luc4E2 cells were developed by stably transfecting MCF-7 cells with

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plasmid vectors containing the firefly luciferase gene under hormone-inducible control of

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estrogen response elements.35 The VM7Luc4E2 TA test was authorized by the Organization of

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Economic Cooperation and Development (OECD) in 2012 as a bioassay screening method for

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ER agonists and antagonists.36 The EPA Endocrine Disruptor Screening Program also uses the

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VM7Luc4E2 TA test to screen estrogenic chemicals. The National Center for Toxicological

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Research (NCTR) developed an Estrogenic Activity Database (EADB) which includes EA data

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for over 8200 chemicals measured by three types of in vitro assays and in vivo assays in 11

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different species, but the EADB does not include data for the BGF regioisomers.37

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In this study, the EA of four BGF regioisomer mixtures was quantified by the MCF-7 cell

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proliferation assay and the VM7Luc4E2 TA test at ten relevant concentrations (from 1013 M to

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104 M). A risk assessment of BGF regioisomers, specifically the endocrine disruption potential

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via ER-mediated responses, was performed to determine if specific BGF regioisomers possessed

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reduced or undetectable EA levels in comparison to BPA. BGF samples with little to no EA 7 ACS Paragon Plus Environment


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would be most desirable for replacing BPA in consumer products, especially for the packaging

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of food and drink products.

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Materials and Methods

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Materials and supplies

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MCF-7 cells were purchased from American Type Culture Collection (ATCC No.

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HTB-22). A recombinant human ovarian carcinoma cell line (VM7Luc4E2, recently renamed

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from BG1Luc4E2 cells) was kindly provided by Dr. Michael Denison, University of California,

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Davis. Both cell lines were grown and maintained in polystyrene T-75 or T-25 flasks (Corning,

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Inc.). Media and media supplements (Dulbecco's Modified Eagle Medium (DMEM), phenol

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red-free DMEM, fetal bovine serum (FBS), charcoal-stripped FBS, penicillin-streptomycin, and

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dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific. -MEM was purchased

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from Sigma (St. Louis, MO, USA). Cells were seeded into 96-well flat bottom polystyrene

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plates (Corning™ Costar™, Corning, Inc.). Promega Luciferase Assay Systems and cell lysis

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buffer 5X were purchased from Promega (Madison, WI, USA). Cell proliferation assays and

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luciferase assays were measured by a microplate spectrophotometer (Synergy 2, Bio-Tek,

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instruments, Winooski, VT).

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Tested compounds

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Four BGF samples with different regioisomer ratios were synthesized in-house. The

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syntheses were based on mimics of commercially relevant manufacturing protocols25. The

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chemical structures of the regioisomers and the mixture molar compositions are shown in

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Figure 1. BGF samples are numbered on the basis of descending molar concentration of the p,p′-

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BGF regioisomer. In brief, BGF1 contained 92.9 mol% of p,p′-BGF, 6.6 mol% m,p′-BGF, and

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0.5 mol% o,p′-BGF. BGF2 was comprised of 75.7 mol% of p,p′-BGF, 23.9 mol% m,p′-BGF, and 8 ACS Paragon Plus Environment

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0.4 mol% o,p′-BGF.

BGF3 contained 68.4 mol% of p,p′-BGF, 31.1 mol% m,p′-BGF, and

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0.5 mol% o,p′-BGF. BGF4 was 30.1 mol% of p,p′-BGF, 7.9 mol% m,p′-BGF, and 62.0 mol%

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o,p′-BGF.

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99.5 mol%, and 98.4 mol%, respectively, as determined by proton nuclear magnetic resonance

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(1H NMR) spectroscopy.

The purities of BGF1, BGF2, BGF3, and BGF4 were 99.6 mol%, 99.5 mol%,

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E2 served as an EA positive control. The EA of the test compounds was defined as the

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normalized relative maximum %E2 (%RME2) when compared to the maximum agonist effect

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produced by E2. BPA also was tested to determine if the EA of the BGF mixtures was

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statistically different from BPA and therefore a potential alternative. DMSO at a 0.1% (v/v)

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final concentration was used to the dissolve E2, BPA, and BGF samples and served as the

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vehicle control (VC).

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MCF-7 cell proliferation assay

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MCF-7 cell proliferation assays modified from the original assay proposed by Soto et al.

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were used to quantify the estrogen activity of the test compounds based on the proliferative

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effect of estrogens on MCF-7 cells.33 In brief, MCF-7 cells were maintained in phenol red

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DMEM with 10% FBS. For the cell proliferation assay, MCF-7 cells (3,500 cells/well) were

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seeded into 96-well flat bottom polystyrene plates with EA free culture medium (phenol red-free

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DMEM with 5% charcoal stripped FBS). After two days, cells were treated with fresh EA free

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culture medium containing the test chemicals (E2, BPA, BGF1, BGF2, BGF3, and BGF4) at ten

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different concentrations ranging from 1013 M to 104 M in estrogenic activity free culture

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medium (phenol red-free DMEM with 5% charcoal stripped FBS) for six days. E2 was tested at

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concentrations ranging from 10−15 M to 10−4 M to determine the maximum EA and the half

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maximal effective concentration (EC50). The medium containing a given concentration of test 9 ACS Paragon Plus Environment


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chemicals was refreshed every two days, and after six days of exposure, an MTT

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(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to test cell

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proliferation according to the manufacturer’s protocol. The cell proliferation rate was quantified

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by measuring the absorbance at 570 nm using a microplate spectrophotometer. Each test was

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repeated in three independent trials and in triplicate for each trial. The cell viability and the

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relative maximum %E2 (%RME2) were calculated through the following equations:

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Cell viability = 100% ×

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%RME2 = 100% × 𝑀𝐴𝑋𝑂𝐷 𝑜𝑓 𝐸2

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in which OD of tested is the optical density of the test compound, OD of b is the optical density

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of the blank well, OD of NT is the optical density of the no treatment group, OD of VC is the

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optical density of the vehicle control, and MAXOD of E2 is the maximum optical density of E2.38

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VM7Luc4E2 TA test

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𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡𝑒𝑑 ― 𝑂𝐷 𝑜𝑓 𝑏 𝑂𝐷 𝑜𝑓 𝑁𝑇 ― 𝑂𝐷 𝑜𝑓 𝑏

𝑂𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡𝑒𝑑 ― 𝑂𝐷 𝑜𝑓 𝑉𝐶 ― 𝑂𝐷 𝑜𝑓 𝑉𝐶

,

,

VM7Luc4E2 TA testing was based on the recombinant variant MCF-7 cell line

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developed by Rogers and Denison in 2000.34,35

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VM7Luc4E2 cells were maintained in α-MEM containing 10% FBS, then passaged in phenol

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red-free DMEM with low glucose and 10% charcoal-stripped FBS for six days to remove the

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estrogen background. During the six days, the medium was changed daily. At Day 6, cells were

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seeded into 96-well plates at 750,000 cells/mL and 100 µL/well for 24 h in phenol red-free

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DMEM with low glucose and 10% charcoal-stripped FBS. Then, the cells were treated with test

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chemicals at seven different concentrations ranging from 1011 M to 105 M for 22 h. Each test

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was repeated in three independent trials and in triplicate for each trial.

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concentrations ranging from 10−14 M to 10−6 M to determine the maximum EA and EC50. All

To perform the VM7Luc4E2 TA test,


E2 was tested at

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chemical compounds were dissolved in 100% DMSO. The final concentration of DMSO in the

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culture medium was 0.1% v/v, which served as the VC. E2 served as positive control and was

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used to calculate the normalized EA (%RME2). Optical microscopy confirmed cell viability

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after exposure to the test compounds. The cell culture medium was aspirated, and then the cells

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were lysed in cell lysis buffer for 30 min. Next, the luminescence value of each well was

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measured by a microplate luminometer (Synergy 2, Bio-Tek) with the Promega Luciferase Assay

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System.12 The EA of test chemicals was expressed as the relative maximum %E2 (%RME2)

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calculated from the following equation:

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%RME2 = 100% ×

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in which RLU of tested is the relative luminescence units of the test compounds, RLU of VC is

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the relative luminescence units of the vehicle control, and MAXRLU of E2 is the maximum

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relative luminescence units of E2.12

𝑅𝐿𝑈 of 𝑡𝑒𝑠𝑡𝑒𝑑 ― 𝑅𝐿𝑈 𝑜𝑓 𝑉𝐶 𝑀𝐴𝑋𝑅𝐿𝑈 𝑜𝑓 𝐸2 ― 𝑅𝐿𝑈 𝑜𝑓 𝑉𝐶

,

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Statistical analysis

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All data were reported as mean ± s.d. of at least three independent trials run in triplicate.

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One-way ANOVA followed by the Dunnett’s method was used to compare BGF samples to BPA

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using the statistical software JMP 13.0. Comparison of the two bioassays was performed with

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Student’s T-test using JMP 13.0. A P value less than 0.05 was considered statistically significant.

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The EC50 of test compounds was calculated using the statistical software GraphPad Prism. The

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EA classification was based on the value of EC50:12 “+++” indicates strongly active (EC50 < 1.0

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× 10-9 M), “++” indicates moderately active (1.0 × 109 M 1.0 × 10-7 M), and “--” indicates undetectable EA at the test

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concentrations. “NA” means unavailable data.



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Results

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MCF-7 Cell proliferation assay

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MCF-7 cells were treated with various concentrations of E2 ranging from 10−15 M to

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10−4 M for six days to determine the maximum EA and EC50 of E2. The maximum EA of E2

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was obtained at 10-8 M, and the EC50 of E2 was 1.9 × 10-12 M (Figure 2). The results were

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repeatable and in good agreement with literature values.38

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screening assay, E2 served as a positive control.

In the following chemical EA

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BPA, BGF1, BGF2, BGF3, and BGF4 were tested at concentrations between 10−13 M and

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10−4 M to determine the effects of test compound concentration on MCF-7 cell proliferation.

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The chemicals were considered to have undetectable EA levels when the %RME2 was less than

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~25%.38 The findings for BPA and the BGF samples indicated that the maximum EA of BPA

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and BGF1 was obtained at 10-6 M, while that of BGF4 was obtained at 10-7 M. BGF2 and BGF3

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had undetectable EA when measured by MCF-7 cell proliferation assay. The maximum %RME2

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of BPA, BGF1, BGF2, BGF3, and BGF4 were 66.5%, 44.6%, 5.5%, 14.7%, and 24.6%,

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respectively. The maximum %RME2 of BGF1 and BGF4 were 21.9% and 41.9% less than the

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maximum %RME2 of BPA, respectively. BGF2 and BGF3 had significantly less EA than BPA

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at concentrations between 10-11 M and 10-6 M (Dunnett’s method, BPA group served as control,

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P < 0.05) (Figure 2). The EC50 value of BPA, BGF1, and BGF4 was 2.5 × 10-8 M, 1.8 × 10-9 M,

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and 2.5 × 10-10 M, respectively (Table 1). The EC50 value of BGF4 was 100 times lower than

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that of BPA. The estrogenic activity of BPA was dose-dependent and positively correlated.

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However, BPA exhibited cytotoxicity at 10-4 M as the cell viability of MCF-7 cells was only

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61.7% when compared to the control group (Figure 3). BGF3 and BGF4 also decreased the cell

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proliferation by 22.7% and 24.8% respectively but these reductions were smaller than that noted 12 ACS Paragon Plus Environment

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for BPA at 10-4 M BGF1 and BGF2 did not decrease the cell proliferation at any test

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concentration (Figure 3).

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VM7Luc4E2 TA test

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VM7Luc4E2 cells were treated with concentrations of E2 ranging from 10−14 M to 10−6

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M for 22 h to determine the maximum EA and EC50. The maximum EA of E2 was obtained at

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10−6 M, while the %RME2 was not significantly different in the range 10-9 M to 10-9 M. Results

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from our three independent trials were in good agreement and had a standard deviation less than

256

0.095 or a Coefficient of Variation (CV) less than 10%. In the following chemical EA screening

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assay, E2 served as a positive control.

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EA results for the test compounds obtained through the VM7Luc4E2 TA test were

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similar to the EA results of the MCF-7 assay, but there were several different dose-responses of

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E2 at 10-6 M, and BPA and BGF4 at 10-5 M. The results of the VM7Luc4E2 TA test indicated

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that the maximum EA of BPA, BGF1, and BGF4 was obtained at 1 M. BGF2 and BGF3 had

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nearly undetectable EA as measured by the VM7Luc4E2 TA test, which was in agreement with

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the results of the MCF-7 cell proliferation assay. The maximum %RME2 of BPA, BGF1, BGF2,

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BGF3, and BGF4 were 94.9%, 47.2%, 26.1%, 26.5%, and 63.3% respectively (Figure 4). BGF2

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and BGF3 had significantly less EA than BPA in the concentration range from 1 M to 1 M

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(Dunnett’s method, BPA group served as control, P < 0.05). The EC50 of BPA, BGF1, and

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BGF4 was 2.6 × 107 M, 3.6 × 108 M, and 7.9 × 109 M, respectively (Table 1). The EC50 of

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BPA was higher than the EC50 of BGF1 and BGF4, and the maximum %RME2 of BGF1 and

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BGF4 was less than the %RME2 of BPA (Dunnett’s method, BPA group served as control, P
0.05, Figure 5) for E2,

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BPA, and the BGF samples, at all concentrations except for E2 at 1 M, and BPA and BGF4 at

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1 M.

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The EC50 values for E2 and BPA obtained from both the MCF-7 cell proliferation assay

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and the VM7Luc4E2 TA test were in good agreement with the Meta-Analysis data from Yang

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et al. (Table 1).38 Based on the standard of EA classification as defined above, E2 was strongly

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EA (+++), while BPA, BGF1, and BGF4 were moderately active (++). BGF2 and BGF3 had

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undetectable EA (--) (Table 1).

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Discussion

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In this study, the test concentrations of potential compounds spanned several orders of

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magnitude, including the median exposure concentration of total BPA in human urine (8.8 × 10-

287

9

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effect on the proliferation of MCF-7 cells at concentrations ranging from 109 M to 106 M. The

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EC50 of BPA obtained from the two in vitro assays in this study was 2.5 × 108 M and

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2.6 × 107 M, which was in agreement with the Meta-Analysis data from Yang et al. (Table 1).38

291

BGF1 and BGF4 significantly increased the cell proliferation rate of MCF-7 cells at 1 M,

292

1 M, and 1 M in comparison to the non-treatment group (negative control), but the cell

M), for greater safety and cytotoxicity evaluation.1 In comparison to E2, BPA had a similar


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293

proliferation rate was still lower than BPA at the same concentrations (P < 0.05, Figure 3).

294

Additionally, the maximum %RME2 of BGF2 and BGF3, 5.5% and 14.7% respectively, were

295

over four times lower than the %RME2 of BPA (66.5%) based on the MCF-7 cell proliferation

296

assay. The findings of the VM7Luc4E2 TA test were consistent with the results of the MCF-7

297

cell proliferation assay, which determined that the maximum %RME2 of BPA, BGF2, and BGF3

298

were 94.9%, 26.1%, and 26.5%, respectively. In short, all bisguaiacol samples had lower EA

299

when compared with BPA, and BGF2 and BGF3 had undetectable EA based on the findings of

300

both in vitro assays.

301

Although there is no significant difference in %RME2 between the MCF-7 cell

302

proliferation assay and the VM7Luc4E2 TA test (Figure 5), several different dose-response

303

trends were found in E2 at 1 M, and BPA and BGF4 at 1 M; the discrepancies were most

304

likely as a result of the cytotoxicity of E2, BPA, and BGF4 at these concentrations (Figure 3).

305

Zhang et al. also reported the cytotoxic effects of BPA on MCF-7 cells at 1 M, which was in

306

agreement with our findings.39 To the best of our knowledge, the cytotoxic effects of E2 on

307

MCF-7 cells at 1 M have not been reported previously. MCF-7 cell proliferation assays could

308

detect test compound cytotoxicity because cells were exposed to 10-4 M concentrations of the test

309

compounds for six days. The cytotoxicity of E2 and BPA at higher concentrations decreased the

310

value of %RME2 and therefore influenced the EA potential of E2 and BPA.

311

VM7Luc4E2 cells were only treated with test chemicals up to 1 M for 22 h. This time period

312

was too short to detect the cytotoxicity but long enough to test for chemicals interactions with

313

ER-α at the gene transcription level.

However,

314

Previous work evaluating the EA potential of BGF did not specify the regioisomer ratio

315

studied and therefore the results were not directly applicable to ‘real’ systems.40 The four BGF 15 ACS Paragon Plus Environment


Page 16 of 31

316

samples in this study were mixtures of three regioisomers at different molar ratios (Figure 1).

317

Hydroxyl and methoxy groups at different positions on the aromatic rings contributed to the

318

different EA measured between the BGF samples. The symmetrical chemical structure of p,p′-

319

BGF was most similar in structure to BPA. This similarity possibly explains why BGF1, which

320

contained the highest percentage of p,p′-BGF, exhibited similar EA to BPA. The results were in

321

a good agreement with the software prediction made by Hong et al. that p,p′-BGF was an ER-

322

binder.28 BGF4 containing the highest percentage of o,p′-BGF also exhibited detectable EA.

323

However, BGF2 and BGF3 had higher percentages of m,p′-BGF in comparison to BGF1 and

324

BGF4 and had undetectable EA, suggesting that m,p′-BGF had little EA relative to the other BGF

325

regioisomers (p,p′-BGF and o,p′-BGF) and BPA. Higher percentages of m,p′-BGF in BGF

326

samples might have contributed to their lower EA, but the exact structure-activity relationship

327

remains the subject of further investigation. As the stability of final polymers is a critical part of

328

FCMs, leaching potential of the BGF molecules from cured BGF-containing polymers will be

329

assessed in future investigations.

330

Similar to other bioassays, the MCF-7 cell proliferation assay and the reporter gene assay

331

may result in large standard deviation, such as the result of BGF3 measured by VM7Luc4E2 TA

332

test. However, the in vitro assays were still more suitable than animal testing for screening the

333

EA of potential chemicals with the advantage of low cost and simple procedures. In this study, a

334

cell proliferation assay and a reporter gene assay were chosen to quantify the EA of BGF

335

regioisomers compared to E2 and BPA on different cell response levels. The results of MCF-7

336

cell proliferation assay reflected the response of human cells to estrogen or estrogen-like

337

compounds at the whole cell level through proliferation rate analysis.

338

proliferation rates directly related to the estrogenic activity. However, cell proliferation is a 16 ACS Paragon Plus Environment

Abnormal, faster

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339

complex process with many proteins working together inside one cell. This assay does not

340

directly relate to the ER binding affinity of test compounds. Fortunately, this limitation was

341

overcome by including the VM7Luc4E2 TA test in the EA analysis because the VM7Luc4E2 TA

342

test positively correlates with the binding affinity of compounds to ER-α.

343

VM7Luc4E2 TA test reflected the response of human cells to estrogen or its analogs at the RNA

344

level through the luciferase gene expression rate.35

345

compounds resulted in higher luciferase gene expression levels in the VM7Luc4E2 cells.

346

However, the VM7Luc4E2 TA test could not detect the cytotoxicity of test compounds because

347

the chemical treatment time of cells was less than one day. The MCF-7 cell proliferation assay

348

remedied this limitation as MCF-7 cells were treated with test chemicals at long enough times to

349

evaluate cytotoxicity.

350

comprehensive evaluation of the EAs of test compounds at different cell response levels.

The results of

The increased binding affinity of test

Therefore, utilizing these two complementary methods enabled

351

In this study, two in vitro assays were used to evaluate the EA of bio-based BGF, a

352

potential BPA alternative, at the whole-cell level and RNA-expression level. Four BGF samples

353

containing different ratios of regioisomers exhibited different EA in comparison to BPA. BGF

354

samples containing higher molar ratios of m,p′-BGF (BGF2 and BGF3) had undetectable EA.

355

Furthermore, samples containing higher ratios of p,p′-BGF and o,p′-BGF, BGF1 and BGF4

356

respectively, had similar EC50 and the same EA classification as BPA, but lower EA by two in

357

vitro assays (Figure 1 and 4) . Therefore, BGF is a potentially less toxic and sustainable

358

alternative to BPA for applications in consumer products, especially for food and drink

359

packaging supported by in vitro bio-assays.



360

Abbreviations

361

BPA: Bisphenol A, FCMs: food contact materials, EA: estrogenic activity, BGF: Bisguaiacol F,

362

ER: estrogen receptor, E2: 17-estradiol, %RME2: the relative maximum %E2, EC50: half

363

maximal effective concentration,

364

bromide, OD: optical density values, MAXOD: maximum optical density values, VC: vehicle

365

control, DMSO: dimethyl sulfoxide, FBS: Fetal bovine serum, DMEM: Dulbecco modified eagle

366

medium, α-MEM: Minimum Essential Medium Eagle (MEM) Alpha, RLU: Relative

367

luminescence units, MAXRLU: maximum relative luminescence units, CV: Coefficient of

368

Variance, HPLC-MS/MS: high-performance liquid chromatography tandem-mass spectrometry,

369

1H

370

Environmental Health Sciences, EDC: endocrine-disrupting chemical, OD of b: the optical

371

density of the blank well, OD of NT: the optical density of the no treatment group.

372

Acknowledgments

373

The authors appreciate Dr. Michael Denison at the University of California, Davis for providing

374

the VM7Luc4E2 cells.

375

Funding Sources

376

This research was supported by a grant from the National Science Foundation (Award No.

377

DMR-1506623).

378

Notes

379

The authors declare no competing financial interest.

380

ORCID

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

NMR: proton nuclear magnetic resonance, TA: transactivation, NIEHS: National Institute of


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381

Kaleigh H. Nicastro: 0000-0003-4587-0053

382

Thomas H. Epps, III: 0000-0002-2513-0966

383



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Szafran, A. T.; Stossi, F.; Mancini, M. G.; Walker, C. L.; Mancini, M. A. Characterizing Properties of Non-Estrogenic Substituted Bisphenol Analogs Using High Throughput Microscopy and Image Analysis. PLoS One 2017, 12 (7), e0180141.

506 507



508 509

FIGURE CAPTIONS

510

regioisomers present in the bisguaiacol (BGF) samples.

511

basis of descending molar concentration of the p,p′-BGF regioisomer.

512

Figure 2.

513

as %RME2 quantified by MCF-7 cell proliferation assays (Data reported as mean ± s.d. of at

514

least three independent trials run in triplicate, %RME2 indicates the relative maximum %E2). “*”

515

indicates P < 0.05 when %RME2 of test compounds was compared to BPA at the same

516

concentration.

517

Figure 3. The effects of E2, BPA, BGF1, BGF2, BGF3, and BGF4 on the cell viability of MCF-7

518

cells as measured by MTT assays (Data reported as mean ± s.d. of three independent trials run in

519

triplicate). Cell viabilities decreased when MCF-7 cells were treated with E2, BPA, BGF3, and

520

BGF4 at concentrations above 10-6 M. Figure 4. Comparison of relative maximum %E2

521

(%RME2) of tested compounds based on concentration-response curves for VM7Luc4E2 cells

522

(Data reported as mean ± s.d. of at least three independent trials run in triplicate). “*” indicates

523

P < 0.05 when the %RME2 of test compounds was compared to BPA at the same concentration.

524

Figure 5. Comparison of MCF-7 cell proliferation assay data obtained from the VM7Luc4E2

525

TA test for the cell response to test compounds (Data reported as mean ± s.d. of at least three

526

independent trials run in triplicate). “*” indicates P < 0.05 when the %RME2 of the MCF-7 cell

527

proliferation assay was compared to the VM7Luc4E2 TA test at the same concentration by

528

Student’s T-test.

Figure 1. The chemical structure of the test compounds and the molar compositions (mol%) of BGF samples are numbered on the

The estrogenic activity of E2, BPA, BGF1, BGF2, BGF3, and BGF4 expressed


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Table 1. EC501 and EA Classification of Test Compounds Obtained from Meta-Analysis Data, MCF-7 Cell Proliferation Assays, and VM7Luc4E2 Transactivation Tests. E2

BPA

BGF1

BGF2

BGF3

BGF4

Meta EC50 (M)2

8.7 x 10-11

5.0 x 10--7

NA

NA

NA

NA

MCF-7 EC50 (M)

1.9 x 10-12

2.5 x 10-8

1.8 x 10-9

--

--

2.5 x 10-10

VM7Luc4E2 EC50 (M)

5.3 x 10-12

2.6 x 10-7

3.6 x 10-8

--

--

7.9 x 10-9

EA classification

+++

++

++

--

--

++

1:

EC50 of test compounds was calculated using GraphPad Prism. The EA classification was based on the value of EC50: “+++” indicates strongly active (EC50 < 1.0 ×10-9 M), “++” indicates moderately active (1.0 × 10-9 M 1.0 × 10-7 M), “--” indicates undetectable EA. “NA” means unavailable data. 2: Meta-Analysis data from the previous work of Yang et al.38



Figure 1


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