Estrogenic activity of monomers and initiators used in resin based dental composites
Boonen I1, Vandermarken T1, De Nys S2., Vervliet P3., Covaci A3., Van Landuyt K2., Denison M.S4.,
Elskens, M1.
1

Vrije Universiteit Brussel, Department of Analytical, Environmental and Geo-Chemistry, Brussels,Belgium, 1050, imke.boonen@vub.be; 2 KU Leuven (University of Leuven), Department of Oral HealthSciences, Leuven, Belgium,3000; 3University of Antwerp, Toxicological Centre, Antwerp, Belgium,2000;
4
University of California at Davis, Department of Environmental Toxicology, Davis, USA, 95616
Introduction
Due to the possible negative effects of elementary mercury on human health and the environment, the World HealthOrganization (WHO) has suggested to phase-out using amalgam as a dental restorative1. As an alternative, resin
based dental materials are often used. Even though these dental composites have already been routinely used forseveral decades, their biocompatibility has not yet been fully characterized. The resin matrix of dental composites ismainly based on a mixture of dimethacrylates. Currently, the most used dimethacrylates are Bisphenol A glycidyl
methacrylate (BisGMA), ethoxylated BisGMA (BisEMA), triethyleneglycol dimethacrylate (TEGDMA) andurethane dimethacrylates (UDMA)2. Other than the monomers, the resin matrix also contains an initiator system tostart the polymerization process, as well as stabilizers. Besides the resin, the composite further contains inorganic
filler and a coupling agent, usually an organo-silane, that bonds the reinforcing filler to the resin matrix3.
A problem with using these dental composites is that the polymerization process is not complete, withpolymerization rates ranging from 50-70%, leaving unreacted monomers that can be released into the mouth4. Someof these monomers, such as BisGMA, BisEMA and the less frequently used Bisphenol A Dimethacrylate(BisDMA), are derived from Bisphenol A5. Since BPA is used in the manufacturing process, trace amounts of BPA
may be present in the final composite6.
Bisphenol A is one of the highest volume chemicals produced worldwide, with more than 2 million tons produced
each year7. It’s used in number of different applications, such as epoxy resins, polycarbonate plastics, toys, drinking
containers, medical equipment and thermal receipts8. BPA is also a well-known endocrine disruptor9. Due to thepossible adverse health effects related to the use of BPA, there has been an increasing need for alternatives.
Nowadays, bisphenol analogues such as Bisphenol S, Bisphenol F, Bisphenol C, Bisphenol B etc. are often used as areplacement for BPA10. Since these compounds are structural analogues to BPA, their interactions with the estrogen
receptor might be similar. BPF is interesting in particular, since BPF epoxy resins are used in several consumer
products, including in dental sealants11.
The aim of this study was to determine the estrogenic activity of 9 monomers (BisGMA, TEGDMA, BisEMA (3),BisEMA (6), BisEMA (10), BisDMA, UDMA, BADGE and TCD-DI-HEA) and 3 initiators (CQ, EDMAB,HEMA), as well as BPA and BPF using the Estrogen Responsive Elements Chemical Activated Luciferase GeneExpression (ERE-CALUX) bioassay.
Materials and methods
To start, a range finding experiment was performed for all the compounds to see if there was any estrogenic activity
present, and in which concentration range. A 5 or 10-point consecutive dilution sequence was made (depending onthe potency), starting from a stock solution of 0.1M, with a dilution factor of 10. This provides a broad concentration
range, with treatment concentrations ranging from 10-3M to 10-13M. The process was applied to all compounds,
except for BPA since the active concentration range is already known. Afterwards, for the compounds that show
estrogenic activity, a full dose response curve was made using a 10-point dilution sequence with a dilution factor 2.
All dilutions were made using dimethyl sulfoxide (DMSO) as a solvent and carrier agent.
The ERE-CALUX bioassay was used to determine the estrogenic activity of the compounds. This is a bioanalytical
tool that uses a human breast cancer cell line, which is stably transfected with an ER responsive luciferase reporter
gene (VM7Luc4E2). 17β-Estradiol (E2) was used as a reference standard.

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489

The cells were maintained in α-MEM (alpha Minimum Essential Medium), supplemented with 1% pen-strep, 2% Lglutamine and 10% FBS (Fetal Bovine Serum). The cells are incubated at 37°C, 5% CO2 and 80% humidity. At least
48 hours before dosing, the cells were transferred to DMEM (Dulbecco’s Modification of Eagle’s Medium),supplemented with 4.5% charcoal stripped FBS, 2% L-glutamine, 1% pen-strep and 1% sodium pyruvate. For
harvesting, phenol-red free trypsin was added to collect the cells, which were counted and the cell suspension wasdiluted to 200 000 cells/mL. When seeding 200µL in every well of the 96-well plate, the final amount will be 40 000
cells/well. After incubating for 24h, the medium was removed and each well was dosed with 190µL DMEM,
containing 1% DMSO with the reference standard (E2) or the compound of interest, which are both dosed intriplicate. After incubating the 96-well plate for 19-22 hours, lysis was added and measurements were performed
using a luminometer (Glomax, Promega, USA), which automatically adds luciferine to each well. Data analysis wasperformed in Excel.
Results and discussion
For the analyzed monomers, only BisDMA showed a clear agonistic estrogenic activity, as can be seen in graph 1.
For BisGMA, there was a small agonistic estrogenic activity present, with a response equal to or lower than 20% of
the E2 maximum. At the highest concentrations, cell death occurred which is demonstrated in graph 2 (response dips
below the background value).

100

100

80

80

RLU (%)

120

RLU (%)

120

60

E2BisDMA

60

40

40

20

2001.00E-151.00E-121.00E-091.00E-061.00E-031.00E+00

01.00E-15 1.00E-12 1.00E-09 1.00E-06 1.00E-03 1.00E+00

Concentration (M)Concentration (M)Graph 1: On the left, the results of the range finding experiment for Bisphenol A dimethacrylate (BisDMA). On the right, ERECALUX full dose-response curve for BisDMA. The concentration is depicted in M, the response in Relative Light Units (RLU),
which are scaled to the maximum of the E2 standard curve (100%).

All three BisEMA compounds, TEGDMA, UDMA, BADGE, and TCD-DI-HEA showed no agonistic estrogeniceffects. The lack of estrogenic activity for both TEGDMA and UDMA is to be expected, since both compounds are
not derived from BPA and show no structural relationship to BPA. BisEMA, BADGE and TCD-DI-HEA are alllarge compounds with a high molecular weight, which could explain their inability to bind to the estrogen receptor.
BisDMA on the other hand is rather small, with a very high structural relationship to BPA, which could explain theirsimilar behavior. This indicates that the size of the monomers used has a big influence on their estrogenic activity,
which needs to be taken into account when synthesizing new composites. The 3 initiators that were analyzed, CQ,EDMAB and HEMA, also showed no agonistic estrogenic activity.

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120
100

RLU (%)

80
60E2BisGMA

40
2001.00E-15 1.00E-13 1.00E-11 1.00E-09 1.00E-07 1.00E-05 1.00E-03 1.00E-01

Concentration (M)Graph 2: ERE-CALUX dose response curve for Bisphenol A glycidyl methacrylate (BisGMA). The concentration
is depicted in M, response in Relative Light Units (RLU), scaled to the maximum of the E2 standard curve (100%).

The use of BPA analogues is becoming more popular due to the possible health effects related to BPA. BPF is suchan analogue that is used in the synthesis of epoxy resins which are used in dental sealants. As can be seen fromgraph 3, BPF is a clear estrogenic agonist, which shows a similar behavior to BPA. This shows that caution needs tobe taken when switching to BPA analogues, since most show structural relationships to BPA and they can have
similar effects.

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120.00

RLU (%)

100.0080.00
60.00

E240.00

BPA
BPF

20.00
0.00
1.00E-15 1.00E-13 1.00E-11 1.00E-09 1.00E-07 1.00E-05 1.00E-03
Concentration (M)Graph 3: ERE-CALUX dose response curves for Bisphenol A (BPA) and Bishphenol F (BPF). The concentration isdepicted in M, the response in Relative Light Units (RLU), scaled to the maximum of the E2 standard curve (100%).

Acknowledgements:
This research was supported by the Fund for Scientific Research Belgium (FWO G089016N). The VM7LUC4E2 cellline was developed by the University of California-Davis (USA) with funding from a Superfund Research
Program grant (ES04699) from the National Institute of Environmental Health Sciences.
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