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
Journal of Agricultural and Food Chemistry
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
2 ACS Paragon Plus Environment
Page 2 of 31
Page 3 of 31
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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-
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
6 ACS Paragon Plus Environment
Page 6 of 31
Page 7 of 31
Journal of Agricultural and Food Chemistry
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 1013 M to
131
104 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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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 1013 M to 104 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
Journal of Agricultural and Food Chemistry
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 1011 M to 105 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,
10 ACS Paragon Plus Environment
E2 was tested at
Page 11 of 31
Journal of Agricultural and Food Chemistry
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 × 109 M 1.0 × 10-7 M), and “--” indicates undetectable EA at the test
224
concentrations. “NA” means unavailable data.
11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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 × 107 M, 3.6 × 108 M, and 7.9 × 109 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 109 M to 106 M. The
289
EC50 of BPA obtained from the two in vitro assays in this study was 2.5 × 108 M and
290
2.6 × 107 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
14 ACS Paragon Plus Environment
Page 14 of 31
Page 15 of 31
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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.
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
18 ACS Paragon Plus Environment
Page 18 of 31
Page 19 of 31
Journal of Agricultural and Food Chemistry
381
Kaleigh H. Nicastro: 0000-0003-4587-0053
382
Thomas H. Epps, III: 0000-0002-2513-0966
383
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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.
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
24 ACS Paragon Plus Environment
Page 24 of 31
Page 25 of 31
Journal of Agricultural and Food Chemistry
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
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 1
26 ACS Paragon Plus Environment
Page 26 of 31
Page 27 of 31
Journal of Agricultural and Food Chemistry
Figure 2
27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 3
28 ACS Paragon Plus Environment
Page 28 of 31
Page 29 of 31
Journal of Agricultural and Food Chemistry
Figure 4
29 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 5
30 ACS Paragon Plus Environment
Page 30 of 31
Page 31 of 31
Journal of Agricultural and Food Chemistry
Table of Contents Graphic
31 ACS Paragon Plus Environment