Evaluation of Estrogenic Activity of Novel Bisphenol A Alternatives

Subscriber access provided by University of Sunderland

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

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])

1 ACS Paragon Plus Environment


1

Abstract

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

environmentally friendly materials.

14 15

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

16 17 18 19


Page 2 of 31

Page 3 of 31


20 21 22

Introduction

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

The primary source of human

The

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

38

exposure and the increasing evidence of its adverse health effects.

39

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

40

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

41

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

42

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

Despite of rapid


43

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

44

colostrum, amniotic fluid, and umbilical cord blood.8

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

that new and safe alternatives to BPA are necessary.

For example, in 2003-2004, the median

59

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

60

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

61

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

62

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

63

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

64

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

65

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

Page 4 of 31

Page 5 of 31


66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

incidence and susceptibility to neoplastic transformations among types of cancers through

74

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

75

those persons with severe coronary artery stenosis.20

76

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

77

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

78

materials to reduce the exposure to BPA.22

In short, because of the endocrine

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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



88

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

89

foodstuffs.

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

less endocrine disrupting alternative to BPA.29

104

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

105

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

106

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

107

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

108

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

109

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

110

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


Page 6 of 31

Page 7 of 31


111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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


134

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

135

of food and drink products.

136

Materials and Methods

137

Materials and supplies

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

instruments, Winooski, VT).

150

Tested compounds

151

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

152

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

153

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

154

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

155

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

156

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

Page 8 of 31

Page 9 of 31


157

0.4 mol% o,p′-BGF.

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

158

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%

159

o,p′-BGF.

160

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

161

(1H NMR) spectroscopy.

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

162

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

163

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

164

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

165

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

166

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

167

vehicle control (VC).

168

MCF-7 cell proliferation assay

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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


Page 10 of 31

180

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

181

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

182

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

183

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

184

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

185

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

186

Cell viability = 100% ×

187

%RME2 = 100% × 𝑀𝐴𝑋𝑂𝐷 𝑜𝑓 𝐸2

188

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

189

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

190

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

191

VM7Luc4E2 TA test

192

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

193

developed by Rogers and Denison in 2000.34,35

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

Page 11 of 31


202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

calculated from the following equation:

210

%RME2 = 100% ×

211

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

212

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

213

relative luminescence units of E2.12

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

,

214 215

Statistical analysis

216

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

217

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

218

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

219

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

220

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

221

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

222

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

224

concentrations. “NA” means unavailable data.



225

Results

226

MCF-7 Cell proliferation assay

227

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

228

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

229

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

230

repeatable and in good agreement with literature values.38

231

screening assay, E2 served as a positive control.

In the following chemical EA

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

Page 12 of 31

Page 13 of 31


248

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

249

concentration (Figure 3).

250 251

VM7Luc4E2 TA test

252

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

253

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

254

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

255

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

257

assay, E2 served as a positive control.

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

277

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

278

1 M.

279

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

280

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

281

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

282

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

283

undetectable EA (--) (Table 1).

284

Discussion

285

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

286

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

287

9

288

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

289

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

290

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


Page 14 of 31

Page 15 of 31


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

Page 17 of 31


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


Page 18 of 31

Page 19 of 31


381

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

382

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

383



384 385 386 387

References (1)

Environmental Protection Agency. Bisphenol A (BPA) https://www.epa.gov/sites/production/files/2017-08/documents/ace3_bpa_updates_8-417.pdf (accessed Feb 12, 2018).

388 389 390 391

(2)

Geens, T.; Aerts, D.; Berthot, C.; Bourguignon, J. P.; Goeyens, L.; Lecomte, P.; MaghuinRogister, G.; Pironnet, A. M.; Pussemier, L.; Scippo, M. L.; et al. A Review of Dietary and Non-Dietary Exposure to Bisphenol-A. Food Chem. Toxicol. 2012, 50 (10), 3725– 3740.

392 393

(3)

Geens, T.; Goeyens, L.; Covaci, A. Are Potential Sources for Human Exposure to Bisphenol-A Overlooked? Int. J. Hyg. Environ. Health 2011, 214 (5), 339–347.

394 395 396

(4)

Liao, C.; Kannan, K. Concentrations and Profiles of Bisphenol A and Other Bisphenol Analogues in Foodstuffs from the United States and Their Implications for Human Exposure. J. Agric. Food Chem. 2013, 61 (19), 4655–4662.

397 398 399

(5)

Noonan, G. O.; Ackerman, L. K.; Begley, T. H. Concentration of Bisphenol A in Highly Consumed Canned Foods on the U.S. Market. J. Agric. Food Chem. 2011, 59 (13), 7178– 7185.

400 401 402

(6)

Teeguarden, J.; Hanson-Drury, S.; Fisher, J. W.; Doerge, D. R. Are Typical Human Serum BPA Concentrations Measurable and Sufficient to Be Estrogenic in the General Population? Food Chem. Toxicol. 2013, 62, 949–963.

403 404 405 406

(7)

Teeguarden, J. G.; Twaddle, N. C.; Churchwell, M. I.; Yang, X.; Fisher, J. W.; Seryak, L. M.; Doerge, D. R. 24-Hour Human Urine and Serum Profiles of Bisphenol A: Evidence against Sublingual Absorption Following Ingestion in Soup. Toxicol. Appl. Pharmacol. 2015, 288 (2), 131–142.

407 408

(8)

Vandenberg, L. N.; Hauser, R.; Marcus, M.; Olea, N.; Welshons, W. V. Human Exposure to Bisphenol A (BPA). Reprod. Toxicol. 2007, 24 (2), 139–177.

409 410 411

(9)

Calafat, A.M.; Ye, X., Wong, Y.L.; Reidy, J.A.; Needham, L.L. Exposure of the U.S. Population to Bisphenol A and 4-tertiary-Octylphenol:2003-2004. Environ. Heal. Perpect. 2008, 116, 39-44.

412 413 414

(10)

Lin, F.; Ph, D.; Hfs-, C.; Lin, F. S.; Keefe, D. M. 2014 Updated Safety Assessment of Bisphenol A (BPA) for Use in Food Contact Applications. Dep. Heal. Hum. Serv. 2014, 8–12.

415 416 417 418

(11)

Jenni Glenn Gingery. Endocrine Society experts express concern with FDA statement on BPA safety | Endocrine Society https://www.endocrine.org/news-room/2018/endocrinesociety-experts-express-concern-with-fda-statement-on-bpa-safety (accessed Apr 13, 2018).

419 420 421

(12)

Bittner, G. D.; Denison, M. S.; Yang, C. Z.; Stoner, M. A.; He, G. Chemicals Having Estrogenic Activity Can Be Released from Some Bisphenol A-Free, Hard and Clear, Thermoplastic Resins. Environ. Health 2014, 13, 103.

422

(13)

Shafei, A.; Ramzy, M. M.; Hegazy, A. I.; Husseny, A. K.; EL-hadary, U. G.; Taha, M. M.; 20 ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31


Mosa, A. A. The Molecular Mechanisms of Action of the Endocrine Disrupting Chemical Bisphenol A in the Development of Cancer. Gene 2018, 647, 235–243.

423 424 425 426 427

(14)

Rezg, R.; El-Fazaa, S.; Gharbi, N.; Mornagui, B. Bisphenol A and Human Chronic Diseases: Current Evidences, Possible Mechanisms, and Future Perspectives. Environ. Int. 2014, 64, 83–90.

428 429 430

(15)

NIH. NIEHS-supported Bisphenol A Research Articles https://www.niehs.nih.gov/research/programs/endocrine/bpa_initiatives/bparelated/index.cfm (accessed Mar 2, 2018).

431 432

(16)

Shankar, A.; Teppala, S. Relationship between Urinary Bisphenol A Levels and Diabetes Mellitus. J. Clin. Endocrinol. Metab. 2011, 96 (12), 3822–3826.

433 434

(17)

Bhandari, R.; Xiao, J.; Shankar, A. Urinary Bisphenol A and Obesity in US Children. Am. J. Epidemiol. 2013, 177 (11), 1263–1270.

435 436

(18)

Carwile, J. L.; Michels, K. B. Urinary Bisphenol A and Obesity: NHANES 2003–2006. Environ. Res. 2011, 111 (6), 825–830.

437 438 439 440

(19)

Galloway, T.; Cipelli, R.; Guralnik, J.; Ferrucci, L.; Bandinelli, S.; Corsi, A. M.; Money, C.; McCormack, P.; Melzer, D. Daily Bisphenol A Excretion and Associations with Sex Hormone Concentrations: Results from the InCHIANTI Adult Population Study. Environ. Health Perspect. 2010, 118 (11), 1603–1608.

441 442 443

(20)

Melzer, D.; Gates, P.; Osborn, N. J.; Henley, W. E.; Cipelli, R.; Young, A.; Money, C.; McCormack, P.; Schofield, P.; Mosedale, D.; et al. Urinary Bisphenol A Concentration and Angiography-Defined Coronary Artery Stenosis. PLoS One 2012, 7 (8), e43378.

444 445 446

(21)

Gore, A. C.; Chappell, V. A.; Fenton, S. E.; Flaws, J. A.; Nadal, A.; Prins, G. S.; Toppari, J.; Zoeller, R. T. EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals. Endocr. Rev. 2015, 36 (6), E1–E150.

447 448

(22)

Bisphenol A (BPA) https://www.niehs.nih.gov/health/topics/agents/sya-bpa/ (accessed Jun 29, 2018).

449 450

(23)

Garnish, E. W. Epoxide Resins as Adhesives: Past and Present. Br. Polym. J. 1979, 11 (2), 72–80.

451 452 453

(24)

Reno, K. H.; Stanzione, J. F., III; Wool, R. P.; Sadler, J. M.; La Scala, J. J.; Hernandez, E. D. Bisphenol Alternative Derived from Renewable Substituted Phenolics and Their Industrial Application. U.S. Patent 20170210689 A1, May 27, 2015.

454 455 456

(25)

Hernandez, E. D.; Bassett, A. W.; Sadler, J. M.; La Scala, J. J.; Stanzione, J. F. Synthesis and Characterization of Bio-Based Epoxy Resins Derived from Vanillyl Alcohol. ACS Sustain. Chem. Eng. 2016, 4 (8), 4328–4339.

457 458 459 460

(26)

Curia, S.; Biundo, A.; Fischer, I.; Braunschmid, V.; Gübitz, G. M.; Stanzione, J. F. Towards Sustainable High-Performance Thermoplastics: Synthesis, Characterization, and Enzymatic Hydrolysis of Bisguaiacol-Based Polyesters. ChemSusChem 2018, 11 (15), 2529–2539.



461 462 463 464

(27)

Van de Vyver, S.; Helsen, S.; Geboers, J.; Yu, F.; Thomas, J.; Smet, M.; Dehaen, W.; Román-Leshkov, Y.; Hermans, I.; Sels, B. F. Mechanistic Insights into the Kinetic and Regiochemical Control of the Thiol-Promoted Catalytic Synthesis of Diphenolic Acid. ACS Catal. 2012, 2 (12), 2700–2704.

465 466 467 468

(28)

Hong, H.; Harvey, B.; Palmese, G.; Stanzione, J.; Ng, H.; Sakkiah, S.; Tong, W.; Sadler, J. Experimental Data Extraction and in Silico Prediction of the Estrogenic Activity of Renewable Replacements for Bisphenol A. Int. J. Environ. Res. Public Health 2016, 13 (12), 705.

469 470 471

(29)

Mauck, J. R.; Bassett, A. W.; Sadler, J. M.; La Scala, J. J.; Napadensky, E.; Reno, K. H.; Stanzione III, J. F. Synthesis and Characterization of a Lignin-Derived Aromatic Polycarbonate. J. Biobased Mater. Bioenergy 2018, 12 (5), 471–476.

472 473 474

(30)

EPA. Endocrine Disruptor Screening Program (EDSP) https://www.epa.gov/sites/production/files/2015-08/documents/081198frnotice.pdf (accessed Mar 1, 2018).

475 476 477

(31)

Soto, A. M.; Maffini, M. V.; Schaeberle, C. M.; Sonnenschein, C. Strengths and Weaknesses of in Vitro Assays for Estrogenic and Androgenic Activity. Best Pract. Res. Clin. Endocrinol. Metab. 2006, 20 (1), 15–33.

478 479 480 481

(32)

Leusch, F. D. L.; De Jager, C.; Levi, Y.; Lim, R.; Puijker, L.; Sacher, F.; Tremblay, L. A.; Wilson, V. S.; Chapman, H. F. Comparison of Five in Vitro Bioassays to Measure Estrogenic Activity in Environmental Waters. Environ. Sci. Technol. 2010, 44 (10), 3853– 3860.

482 483 484 485

(33)

Soto, A. M.; Sonnenschein, C.; Chung, K. L.; Fernandez, M. F. Brogan & Partners The ESCREEN Assay as a Tool to Identify Estrogens : An Update on Estrogenic Environmental Pollutants Olea , Fatima Olea Serrano Source : Environmental Health Perspectives , Vol . 103 , Supplement 7 : Estrogens in The. Environ. Heal. 1995, No. 8.

486 487 488

(34)

IMPORTANT NOTICE BG1Luc4E2 cells are being renamed VM7Luc4E2 cells https://ntp.niehs.nih.gov/iccvam/methods/endocrine/bg1luc/bg1luc-vm7luc-june2016508.pdf (accessed Jan 5, 2018).

489 490 491

(35)

Rogers, J. M.; Denison, M. S. Recombinant Cell Bioassays for Endocrine Disruptors: Development of a Stably Transfected Human Ovarian Cell Line for the Detection of Estrogenic and Anti-Estrogenic Chemicals. In Vitr. Mol. Toxicol. 2000, 13 (1), 67–82.

492 493

(36)

OECD. Test No. 457: BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists; OECD, 2012.

494 495 496

(37)

U.S. Food & Drug Administration. Estrogenic Activity Database (EADB) https://www.fda.gov/ScienceResearch/BioinformaticsTools/EstrogenicActivityDatabaseE ADB/default.htm (accessed Dec 12, 2017).

497 498 499

(38)

Yang, C. Z.; Casey, W.; Stoner, M. A.; Kollessery, G. J.; Wong, A. W.; Bittner, G. D. A Robotic MCF-7:WS8 Cell Proliferation Assay to Detect Agonist and Antagonist Estrogenic Activity. Toxicol. Sci. 2014, 137 (2), 335–349.

500

(39)

Zhang, W.; Fang, Y.; Shi, X.; Zhang, M.; Wang, X.; Tan, Y. Effect of Bisphenol A on the 22 ACS Paragon Plus Environment

Page 22 of 31

Page 23 of 31


EGFR-STAT3 Pathway in MCF-7 Breast Cancer Cells. Mol. Med. Rep. 2012, 5 (1), 41– 47.

501 502 503 504 505

(40)

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


Page 24 of 31

Page 25 of 31


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


Page 26 of 31

Page 27 of 31


Figure 2



Figure 3


Page 28 of 31

Page 29 of 31


Figure 4



Figure 5


Page 30 of 31

Page 31 of 31


Table of Contents Graphic


Evaluation of Estrogenic Activity of Novel Bisphenol A Alternatives

Recommend Documents