Reaction mass of 2,4,6-tris(1-phenylethyl)phenol and bis(1-phenylethyl)phenol and o-(1-phenylethyl)phenol

Additional ecotoxological information

Administrative data

Endpoint:

additional ecotoxicological information

Remarks:

endocrine system modulation in vitro Transcriptional Activation assays on z ebrafish estrogen receptors

Type of information:

other: experimental result obtained on one of the main constituents

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

additional ecotoxicological information

Remarks:

endocrine system modulation in vitro Transcriptional Activation assays on z ebrafish estrogen receptors

Type of information:

other: experimental result obtained on one of the main constituents

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: was to assess the potential endocrine disrupting properties in aquatic vertebrate species of potential impurities or degradation products of tri-styrenated phenol (TSP): mono -MSP- and di-styrenated -DSP- phenol. A previous study conducted on in vitro immortalized luciferase-reporter cell line models revealed a weak but significant agonist effect of MSP on mammalian oestrog en receptors ERα and ERβ. Thus, a similar in vitro strategy was developed using immortalized lucife rase-reporter cell lines expressing the oestrogen nuclear receptors of zebrafish (Danio rerio) which could be putative targets for MSP and DSP.
- Short description of test conditions: 2,4-DSP was tested on the transactivation of zebrafish oest rogen receptors: zfERα, zfERβ1, zfERβ2.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of study / information:

In vitro

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10E-2M for storage at -20°C.

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2,4-DSP presented a non-specific activation of luciferase at 10 μM and general to xicity was observed for concentrations above 33 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was qu antified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.
 Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation i n the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relati ve to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERα cells, 2,4-DSP did not show any significant ERα agonist activity, the observed increase in luminescence starting at 10 μM could not be distinguished from a nonspecific effect (see above). No inhibition of the signal was observed in the presence of 2,4-DSP. An increase in luminescence was detected for 2,4-DSP at 10 and 33 μM and corresponded to a non-specific and/or toxic effect on the cells.

zfERβ1

2,4-DSP was tested on HELN zfERβ1 for agonist or antagonist properties. 2,4-DSP induced an increa se in luminescence at 10 and 33 μM corresponding to a non-specific or general toxic effect.
 In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERβ1 were displayed by 2,4-DSP. The slight increase in luminescence in the presence of 33 μM of 2,4-DSP was due to a non-specific activity or general toxicity as previously demonstrated on the control HELN.

zfERβ2

2,4-DSP was tested on HELN zfERβ2 for agonist or antagonist properties.
 The weak increase in luminescence observed in the presence of 10 and 33 μM 2,4-DSP was due to non-specific activation of luciferase or a general toxic effect
 In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 2,4-DSP showed no i nhibition of the signal induced by 10E-10 E2, thus 2,4-DSP did not act as an antagonist on zfERβ2.

The increase in luminescence observed at 33 μM 2,4-DSP was due to a non-specific activation of luciferase or a general toxic effect.

 

Conclusions:

2,4-DSP was tested on the transactivation of zebrafish oestrogen receptors: zfERα, zfERβ1, zfERβ2. Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.
2,4-DSP did not show any significant zfERα, zfERβ1, zfERβ2 agonist activity. 2,4-DSP did not show any zfERα, zfERβ1, zfERβ2 antagonist activity. The increase in luminescence observed, was due to a non-specific activation of luciferase or a general toxic effect.

Executive summary:

The present study performed in vitro on HELN luciferase reporter cell lines provides new information on the putative interference of styrenated phenol 2,4-DSP with zebrafish oestrogen receptors: zfERα, zfERβ1 and zfERβ2. Prior to evaluating the effects on the above receptors, non-specific effects of 2,4- DSP were tested on non-transfected ER HELN cell lines. Clearly, a general cellular toxic effect was observed at 33 μM for 2,4-DSP.

2,4-DSP was dissolved in DMSO at 10E-2M for storage at -20°C.

All cell lines used in this study were derived from the human HeLa immortal cell line. In 1951, the HeLa cell line was derived from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows studies from different laboratories and across time to be compared (Balaguer et al., 1999).

HeLa cells were stably transfected with luciferase-reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo plasmid, giving the HELN cell line, which served as a negative control. To obtain HELN -zfERα,β1andβ2 cell lines, HELN cells were transfected with oestrogen receptor zfESR1 (zfERα), zfESR2a (zfERβ1) or zfESR2b (zfERβ2) coding sequence under the control of a ubiquitous promoter: pSG5-zfERα-puro, pSG5-zfERβ1 -puro and pSG5 -zfERβ2-puro plasmids respectively (Pinto et al., 2014). Hence, zfERα, zfERβ1 and zfERβ2 were expressed by the HELN cells and could bind putative estrogenic compounds. When oestradiol or xenoestrogens are applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of zfERα, zfERβ1 or zfERβ2. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in theβ-globin promoter. Activation of ERE induces the recruitment of transcription regulators and the onset of downstream luciferase expression.

Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red, 1 g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5 mg/mL puromycin and 1mg/mL G418 and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 37°C.

Before testing, cells were plated for 24h at 20,000 cells/well (200 μL) on 96-well culture plates (Greiner CellStar) in the presence of 200 μL of test medium: DMEM without phenol red, 1 g/L glucose, 5% FCS-DCC, 1% penicillin/streptomycin and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 28°C.

All chemicals to be tested were diluted at their final concentrations in the test medium.

Cells were incubated with the molecules to be tested for 16 hours. After incubation, the tested medium was removed and replaced with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96-well plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2-second integration time /well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary units: 100% was given by the reference ligand. Each sample was tested in four replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6; 10E-6; 3.16 10E-7; 10E-7; 3.16 10E-8; 10E-8). In the case of a positive response and thus an incomplete curve, successive dilutions were performed until a complete dose response curve was obtained. These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For zfERα, zfERβ1 and zfERβ2 antagonist properties were tested in the presence of 0.1 nM 17-β-oestradiol (E2).

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2,4-DSP presented a non-specific activation of luciferase at 10 μM and general toxicity was observed for concentrations above 33 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.

Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

2,4-DSP was tested on HELN zfERα, zfERβ1 and zfERβ2 for agonist properties. The increase in luminescence observed in the presence of 10 and 33 μM 2,4-DSP was due to non-specific activation of luciferase or a general toxic effect.

2,4-DSP was tested on HELN zfERα, zfERβ1 and zfERβ2 for antagonist properties. In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERα, zfERβ1 and zfERβ2 were displayed by 2,4-DSP. An increase in luminescence was detected for 2,4-DSP at 10 μM (zfERα) and 33 μM(zfERα, zfERβ1 and zfERβ2) and corresponded to a non- specific and/or toxic effect on the cells.

Conclusion:

2,4-DSP did not show any significant zfERα, zfERβ1, zfERβ2 agonist activity. 2,4-DSP did not show any zfERα, zfERβ1, zfERβ2 antagonist activity. The increase in luminescence observed, was due to a non- specific activation of luciferase or a general toxic effect.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.c., CaselIas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., Nicolas, J.-C., 2005.In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry 340,295-302.

- Pinto, C., Grimaldi, M., Boulahtouf, A., Pakdel, F., Brion, F., Aït-Aïssa, S., Cavaillès, V., Bourguet, W., Gustafsson, J.-A., Bondesson, M., Balaguer, P., 2014.Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Toxicology and Applied Pharmacology 280, 60-69.

 

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

additional ecotoxicological information

Remarks:

endocrine system modulation in vitro Transcriptional Activation assays on z ebrafish estrogen receptors

Type of information:

other: experimental result obtained on one of the main constituents

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: was to assess the potential endocrine disrupting properties in aquatic vertebrate species of potential impurities or degradation products of tri-styrenated phenol (TSP): mono -MSP and di-styrenated -DSP- phenol. A previous study conducted on in vitro immortalized luciferase-reporter cell line models revealed a weak but significant agonist effect of MSP on mammalian oestroge n receptors ERα and ERβ. Thus, a similar in vitro strategy was developed using immortalized lucifer ase-reporter cell lines expressing the oestrogen nuclear receptors of zebrafish (Danio rerio) which
could be putative targets for MSP and DSP.
- Short description of test conditions: 2,6-DSP was tested on the transactivation of zebrafish oestr ogen receptors: zfERα, zfERβ1, zfERβ2.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of study / information:

In vitro

Specific details on test material used for the study:

Chemicals sent by Solvay were dissolved in DMSO at 10E-2M for storage at -20°C.

Details on results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2,6-DSP presented a non-specific activation of luciferase at 10 μM and general toxicity was observed for concentrations above 33 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.
 Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation i n the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERα cells, 2,6-DSP did not show any significant ERα agonist activity, the observed increase

in luminescence starting at 10 μM could not be distinguished from a nonspecific effect (see above). No inhibition of the signal was observed in the presence of 2,6-DSP.

zfERβ1

2,6-DSP was tested on HELN zfERβ1 for agonist or antagonist properties.
 In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERβ1 were displayed by 2,6-DSP. The slight increase in luminescence in the presence of 33 μM of 2,6-DSP was due to a non-specific activity or general toxicity as previously demonstrated on the control HELN.

zfERβ2

2,6-DSP was tested on HELN zfERβ2 for agonist or antagonist properties. The weak increase in luminescence observed in the presence of 10 and 33 μM 2,6-DSP was due to non-specific activation of luciferase or a general toxic effect.
 In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 2,6-DSP showed no inhibition of the signal induced by 10E-10 E2, thus 2,6-DSP did not as an antagonist on zfERβ2. The increase in luminescence observed at 33 μM 2,6-DSP was due to a non-specific activation of luciferase or a general toxic effect.

 

Conclusions:

2,6-DSP was tested on the transactivation of zebrafish oestrogen receptors: zfERα, zfERβ1, zfERβ2. Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.
2,6-DSP did not show any significant zfERα, zfERβ1, zfERβ2 agonist activity. 2,4-DSP did not show any zfERα, zfERβ1, zfERβ2 antagonist activity. The increase in luminescence observed, was due to a non-specific activation of luciferase or a general toxic effect.

Executive summary:

The present study performed in vitro on HELN luciferase reporter cell lines provides new information on the putative interference of styrenated phenol 2,6-DSP with zebrafish oestrogen receptors: zfERα, zfERβ1 and zfERβ2. Prior to evaluating the effects on the above receptors, non-specific effects of 2,6- DSP were tested on non-transfected ER HELN cell lines. Clearly, a general cellular toxic effect was observed at 33 μM for 2,6-DSP.

2,6-DSP was dissolved in DMSO at 10E-2M for storage at -20°C.

All cell lines used in this study were derived from the human HeLa immortal cell line. In 1951, the HeLa cell line was derived from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows studies from different laboratories and across time to be compared (Balaguer et al., 1999).

HeLa cells were stably transfected with luciferase-reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo plasmid, giving the HELN cell line, which served as a negative control. To obtain HELN -zfERα,β1andβ2 cell lines, HELN cells were transfected with oestrogen receptor zfESR1 (zfERα), zfESR2a (zfERβ1) or zfESR2b (zfERβ2) coding sequence under the control of a ubiquitous promoter: pSG5-zfERα-puro, pSG5-zfERβ1 -puro and pSG5 -zfERβ2-puro plasmids respectively (Pinto et al., 2014). Hence, zfERα, zfERβ1 and zfERβ2 were expressed by the HELN cells and could bind putative estrogenic compounds. When oestradiol or xenoestrogens are applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of zfERα, zfERβ1 or zfERβ2. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in theβ-globin promoter. Activation of ERE induces the recruitment of transcription regulators and the onset of downstream luciferase expression.

Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red, 1 g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5 mg/mL puromycin and 1mg/mL G418 and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 37°C.

Before testing, cells were plated for 24h at 20,000 cells/well (200 μL) on 96-well culture plates (Greiner CellStar) in the presence of 200 μL of test medium: DMEM without phenol red, 1 g/L glucose, 5% FCS-DCC, 1% penicillin/streptomycin and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 28°C.

All chemicals to be tested were diluted at their final concentrations in the test medium.

Cells were incubated with the molecules to be tested for 16 hours. After incubation, the tested medium was removed and replaced with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96-well plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2-second integration time /well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary units: 100% was given by the reference ligand. Each sample was tested in four replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6; 10E-6; 3.16 10E-7; 10E-7; 3.16 10E-8; 10E-8). In the case of a positive response and thus an incomplete curve, successive dilutions were performed until a complete dose response curve was obtained. These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For zfERα, zfERβ1 and zfERβ2 antagonist properties were tested in the presence of 0.1 nM 17-β-oestradiol (E2).

Results:

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2,6-DSP presented a non-specific activation of luciferase at 10 μM and general toxicity was observed for concentrations above 33 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.

Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

2,6-DSP was tested on HELN zfERα, zfERβ1 and zfERβ2 for agonist properties. The increase in luminescence observed in the presence of 10 (zfERα) and 33 μM (zfERα and zfERβ2) 2,6-DSP was due to non-specific activation of luciferase or a general toxic effect.

2,6-DSP was tested on HELN zfERα, zfERβ1 and zfERβ2 for antagonist properties. In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERα, zfERβ1 and zfERβ2 were displayed by 2,6-DSP. An increase in luminescence was detected for 2,6-DSP at 33 μM (zfERβ1 and zfERβ2) and corresponded to a non-specific and/or toxic effect on the cells.

Conclusion:

2,6-DSP did not show any significant zfERα, zfERβ1, zfERβ2 agonist activity. 2,6-DSP did not show any zfERα, zfERβ1, zfERβ2 antagonist activity. The increase in luminescence observed, was due to a non- specific activation of luciferase or a general toxic effect.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.c., CaselIas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., Nicolas, J.-C., 2005.In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry 340,295-302.

- Pinto, C., Grimaldi, M., Boulahtouf, A., Pakdel, F., Brion, F., Aït-Aïssa, S., Cavaillès, V., Bourguet, W., Gustafsson, J.-A., Bondesson, M., Balaguer, P., 2014.Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Toxicology and Applied Pharmacology 280, 60-69.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

additional ecotoxicological information

Remarks:

endocrine system modulation in vitro Transcriptional Activation assays on z ebrafish estrogen receptors

Type of information:

other: experimental result obtained on one of the main constituents

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: was to assess the potential endocrine disrupting properties in aquatic vertebrate species of potential impurities or degradation products of tri-styrenated phenol (TSP): mono -MSP and di-styrenated -DSP- phenol. A previous study conducted on in vitro immortalized luciferase-reporter cell line models revealed a weak but significant agonist effect of MSP on mammalian oestroge n receptors ERα and ERβ. Thus, a similar in vitro strategy was developed using immortalized lucifer ase-reporter cell lines expressing the oestrogen nuclear receptors of zebrafish (Danio rerio) which
could be putative targets for MSP and DSP.
- Short description of test conditions: 2,6-DSP was tested on the transactivation of zebrafish oestr ogen receptors: zfERα, zfERβ1, zfERβ2.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of study / information:

In vitro

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2-MSP did not present any non-specific activity up to 10 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was q uantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.
 Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relat ive to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERα cells, 2-MSP presented a dose-related ERα agonist activity with an EC50 of 118.6 μM ( being less potent than 4-MSP with an EC50 of 20.56 μM).
 No inhibition of the signal was observed in the presence of 2-MSP.

zfERβ1

2-MSP was tested on HELN zfERβ1 for agonist or antagonist properties. 2-MSP displayed no agonist activity.
 In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no ant agonistic properties on zfERβ1 were displayed by 2-MSP.

zfERβ2

2-MSP was tested on HELN zfERβ2 for agonist or antagonist properties. 2-MSP displayed no agonist activity.
 In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 2-MSP showed no inhibition of the signal induced by 10E-10 E2, thus 2-MSP did not act as an antagonist on zfERβ2.

Conclusions:

2-MSP presented a dose-related ERα agonist activity with an EC50 of 118.6 μM (being less potent than 4-MSP with an EC50 of 20.56 μM).
2-MSP did not show any antagonist properties on HELN zfERα.
2-MSP displayed no agonist and no antagonist activity on zebrafish oestrogen receptors zfERβ1 and zfERβ2.

Executive summary:

The present study performed in vitro on HELN luciferase reporter cell lines provides new information on the putative interference of styrenated phenol 2-MSP with zebrafish oestrogen receptors: zfERα, zfERβ1 and zfERβ2. Prior to evaluating the effects on the above receptors, non-specific effects of 2- MSP were tested on non-transfected ER HELN cell lines. On the HELN cell line, 2-MSP did not present any non-specific activity up to 10 μM.

2-MSP was dissolved in DMSO at 10E-2M for storage at -20°C.

All cell lines used in this study were derived from the human HeLa immortal cell line. In 1951, the HeLa cell line was derived from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. The last property is important since it allows studies from different laboratories and across time to be compared (Balaguer et al., 1999).

HeLa cells were stably transfected with luciferase-reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo plasmid, giving the HELN cell line, which served as a negative control. To obtain HELN -zfERα,β1andβ2 cell lines, HELN cells were transfected with oestrogen receptor zfESR1 (zfERα), zfESR2a (zfERβ1) or zfESR2b (zfERβ2) coding sequence under the control of a ubiquitous promoter: pSG5-zfERα-puro, pSG5-zfERβ1 -puro and pSG5 -zfERβ2-puro plasmids respectively (Pinto et al., 2014). Hence, zfERα, zfERβ1 and zfERβ2 were expressed by the HELN cells and could bind putative estrogenic compounds. When oestradiol or xenoestrogens are applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of zfERα, zfERβ1 or zfERβ2. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in theβ-globin promoter. Activation of ERE induces the recruitment of transcription regulators and the onset of downstream luciferase expression.

Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red, 1 g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5 mg/mL puromycin and 1mg/mL G418 and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 37°C.

Before testing, cells were plated for 24h at 20,000 cells/well (200 μL) on 96-well culture plates (Greiner CellStar) in the presence of 200 μL of test medium: DMEM without phenol red, 1 g/L glucose, 5% FCS-DCC, 1% penicillin/streptomycin and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 28°C.

All chemicals to be tested were diluted at their final concentrations in the test medium.

Cells were incubated with the molecules to be tested for 16 hours. After incubation, the tested medium was removed and replaced with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96-well plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2-second integration time /well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary units: 100% was given by the reference ligand. Each sample was tested in four replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6; 10E-6; 3.16 10E-7; 10E-7; 3.16 10E-8; 10E-8). In the case of a positive response and thus an incomplete curve, successive dilutions were performed until a complete dose response curve was obtained. These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For zfERα, zfERβ1 and zfERβ2 antagonist properties were tested in the presence of 0.1 nM 17-β-oestradiol (E2).

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 2-MSP did not present any non-specific activity up to 10 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.

Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERα cells, 2-MSP presented a dose-related ERαagonist activity with an EC50 of 118.6 μM (being less potent than 4-MSP with an EC50 of 20.56 μM).

No inhibition of the signal was observed in the presence of 2-MSP.

zfERβ1

2-MSP was tested on HELN zfERβ1 for agonist or antagonist properties. 2-MSP displayed no agonist activity.

In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERβ1 were displayed by 2-MSP.

zfERβ2

2-MSP was tested on HELN zfERβ2 for agonist or antagonist properties. 2-MSP displayed no agonist activity.

In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 2-MSP showed no inhibition of the signal induced by 10E-10 E2, thus 2-MSP did not act as an antagonist on zfERβ2.

Conclusion:

2-MSP presented a dose-related ERα agonist activity with an EC50 of 118.6 μM (being less potent than 4-MSP with an EC50 of 20.56 μM).

2-MSP did not show any antagonist properties on HELN zfERα.

2-MSP displayed no agonist and no antagonist activity on zebrafish oestrogen receptors zfERβ1, zfERβ2.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.c., CaselIas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., Nicolas, J.-C., 2005.In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry 340,295-302.

- Pinto, C., Grimaldi, M., Boulahtouf, A., Pakdel, F., Brion, F., Aït-Aïssa, S., Cavaillès, V., Bourguet, W., Gustafsson, J.-A., Bondesson, M., Balaguer, P., 2014.Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Toxicology and Applied Pharmacology 280, 60-69.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: was to assess the potential endocrine disrupting properties in aquatic vertebrate species of potential impurities or degradation products of tri-styrenated phenol (TSP): mono -MSP and di-styrenated -DSP- phenol. A previous study conducted on in vitro immortalized luciferase-reporter cell line models revealed a weak but significant agonist effect of MSP on mammalian oestroge n receptors ERα and ERβ. Thus, a similar in vitro strategy was developed using immortalized lucifer ase-reporter cell lines expressing the oestrogen nuclear receptors of zebrafish (Danio rerio) which
could be putative targets for MSP and DSP.
- Short description of test conditions: 2,6-DSP was tested on the transactivation of zebrafish oestr ogen receptors: zfERα, zfERβ1, zfERβ2.
- Parameters analysed / observed: Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

GLP compliance:

not specified

Type of study / information:

In vitro

Test material

Test material information

Constituent 1

Reference substance name:

p-(1-phenylethyl)phenol

EC Number:

217-864-1

EC Name:

p-(1-phenylethyl)phenol

Cas Number:

1988-89-2

Molecular formula:

C14H14O

IUPAC Name:

4-(1-phenylethyl)phenol

Test material form:

solid

Details on test material:

- Density: 1.074 g/cm3

Results and discussion

Any other information on results incl. tables

Details on results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 4-MSP did not present any non-specific activity up to 10 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2. Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERα cells, 4-MSP presented a dose-related ERα agonist activity; 4-MSP with an EC50 of 20. 56 μM being more potent than 2-MSP, whose EC50 was 118.6 μM.
 No inhibition of the signal was observed in the presence of 4-MSP.

zfERβ1

4-MSP was tested on HELN zfERβ1 for agonist or antagonist properties.4-MSP presented a partial ag onist activity detected at 10 and 33 μM, with an apparent EC50 estimated at 25.51 μM.
 In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERβ1 were displayed by 4-MSP.

zfERβ2

4-MSP was tested on HELN zfERβ2 for agonist or antagonist properties. 4-MSP presented a weak partial agonistic activity detected at 10 and 33 μM, its EC50 was estimated at 85.27 μM. In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 4-MSP showed no inhibition of the signal induced by 10E-10 E2, thus 4-MSP did not act as an antagonist on zfERβ2.

Applicant's summary and conclusion

Conclusions:

4-MSP presented a weak agonist activity on the three zebrafish ERs, with estimated EC50 of 20.56, 25.51 and 85.27 μM on zfERα., zfERβ1 and zfERβ2 respectively.
4-MSP did not show any antagonist properties on HELN zfERα, zfERβ1 and zfERβ2.
Although 2-MSP has a weak partial agonist activity on zfERα the overall estrogenic activity is mostly due to 4-MSP which exerts a partial agonist activity on the three zebrafish ERs with a potency order:
zfERα. = zfERβ1 > zfERβ2
The relative potencies of 4-MSP compared to a mixture of nonylphenol were 40 times less potent on zfERα, 6 times less potent on zfERβ1 and 150 times less potent on zfERβ2.

Executive summary:

Many endocrine disruptors exert their harmful effects by interacting with nuclear receptors, which in turn leads to deregulation of gene transcription affecting various cellular processes and having deleterious effects on the organism and/or its progeny in the long term. Among these nuclear receptors are the steroid hormone receptors (glucocorticoid, mineralocorticoid, oestrogen, progestagen and androgen receptors). Some environmental chemicals resemble endogenous hormones and can falsely exert agonist or antagonist effects, leading to undesired activity in the cell. These chemicals are called endocrine disruptors (EDs).

The aim of the present study was to assess the potential endocrine disrupting properties in aquatic vertebrate species of potential impurities or degradation products of tri-styrenated phenol (TSP): mono -MSP- and di-styrenated -DSP- phenol. A previous study conducted on in vitro immortalized luciferase- reporter cell line models revealed a weak but significant agonist effect of MSP on mammalian oestrogen receptors ERαand ERβ. Thus, a similar in vitro strategy was developed using immortalized luciferase- reporter cell lines expressing the oestrogen nuclear receptors of zebrafish (Danio rerio) which could be putative targets for MSP and DSP.

The two isomers of DSP (2,4-DSP and 2,6-DSP), and the two isomers of MSP (2-MSP and 4-MSP), were tested on the transactivation of zebrafish oestrogen receptors: zfERα, zfERβ1, zfERβ2, expressed in immortalized luciferase-reporter cell line models. Dose-response curves were obtained and agonist, antagonist or inverse agonist properties were evaluated.

The main results showed that only 4-MSP presented a significant estrogenic activity on zebrafish oestrogen receptors (zfERα, zfERβ1, zfERβ2) with an observed EC50 of 20.56 μM on zfERα, 25.51 μM on zfERβ1 and 85.27 μM on zfERβ2.

Overall, these in vitro transactivation studies support the hypothesis that 4-MSP displays weak agonist properties on zebrafish oestrogen receptors, and its relative potency was found to be 10 times less than Bisphenol-A and 20 times less than nonylphenol tested under similar experimental conditions.

Material and methods:

The present study performed in vitro on HELN luciferase reporter cell lines provides new information on the putative interference of styrenated phenol 4-MSP with zebrafish oestrogen receptors: zfERα, zfERβ1 and zfERβ2. Prior to evaluating the effects on the above receptors, non-specific effects of 4- MSP were tested on non-transfected ER HELN cell lines. On the HELN cell line, 4-MSP did not present any non-specific activity up to 10 μM.

4-MSP was dissolved in DMSO at 10E-2M for storage at -20°C.

All cell lines used in this study were derived from the human HeLa immortal cell line. In 1951, the HeLa cell line was derived from a cervical cancer and showed remarkably proliferative properties and phenotypic stability. This last property is important since it allows studies from different laboratories and across time to be compared (Balaguer et al., 1999).

HeLa cells were stably transfected with luciferase-reporter oestrogen-responsive gene: ERE-βGlob- Luc-SV-Neo plasmid, giving the HELN cell line, which served as a negative control. To obtain HELN -zfERα,β1andβ2 cell lines, HELN cells were transfected with oestrogen receptor zfESR1 (zfERα), zfESR2a (zfERβ1) or zfESR2b (zfERβ2) coding sequence under the control of a ubiquitous promoter: pSG5-zfERα-puro, pSG5-zfERβ1 -puro and pSG5 -zfERβ2-puro plasmids respectively (Pinto et al., 2014). Hence, zfERα, zfERβ1 and zfERβ2 were expressed by the HELN cells and could bind putative estrogenic compounds. When oestradiol or xenoestrogens are applied in the cell culture medium, the ligand binds to the ligand-binding domain (LBD) of zfERα, zfERβ1 or zfERβ2. Ligand binding induces a change in receptor conformation and the release of chaperones. This change in conformation is associated with ER dimerization and nuclear translocation. Once in the nucleus, receptor dimers bind to ERE located upstream in theβ-globin promoter. Activation of ERE induces the recruitment of transcription regulators and the onset of downstream luciferase expression.

Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red, 1 g/L glucose and supplemented with 5% de-steroided foetal calf serum (FCS-DCC), 1% penicillin/streptomycin, 0.5 mg/mL puromycin and 1mg/mL G418 and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 37°C.

Before testing, cells were plated for 24h at 20,000 cells/well (200 μL) on 96-well culture plates (Greiner CellStar) in the presence of 200 μL of test medium: DMEM without phenol red, 1 g/L glucose, 5%

FCS-DCC, 1% penicillin/streptomycin and kept in a water saturated (95% humidity) 95% air, 5% CO2 atmosphere at 28°C.

All chemicals to be tested were diluted at their final concentrations in the test medium.

Cells were incubated with the molecules to be tested for 16 hours. After incubation, the tested medium was removed and replaced with culture medium supplemented with 0.3mM luciferin for 10 minutes. Then the 96-well plate was placed in a luminometer (Microbeta Wallac Luminometer) and bioluminescence was measured (2-second integration time /well) (Pillon et al., 2005). Results were expressed in luminescence arbitrary units: 100% was given by the reference ligand. Each sample was tested in four replicates and each experiment was performed twice. Tested molecules were used at different concentrations (10E-5; 3.16 10E-6; 10E-6; 3.16 10E-7; 10E-7; 3.16 10E-8; 10E-8). In the case of a positive response and thus an incomplete curve, successive dilutions were performed until a complete dose response curve was obtained. These curves were then analysed and the concentration giving 50% of the maximal effect (EC50) was estimated for each compound. For zfERα, zfERβ1 and zfERβ2 antagonist properties were tested in the presence of 0.1 nM 17-β-oestradiol (E2).

Results

Non-specific effects

Non-specific effects on luciferase expression were assessed on cell lines that were not transfected with receptors (HELN). Non-specific effects were quantified in relation to vehicle activity (DMSO). On the HELN cell line, 4-MSP did not present any non-specific activity up to 10 μM.

Oestrogen receptor activity

Estrogenic agonist activity revealed by luciferase expression mediated by ER transactivation was quantified as a percentage relative to the effect induced by 10 nM (10E-8 M) E2.

Estrogenic antagonist activity was revealed by luciferase expression mediated by ER transactivation in the presence of 0.1 nM (10E-10 M) E2. The antagonist effect was quantified as a percentage relative to the effect of 10nM (10E-8 M) E2.

zfERα

In HELN ERαcells, 4-MSP presented a dose-related ERαagonist activity; 4-MSP with an EC50 of 20.56 μM being more potent than 2-MSP, whose EC50 was 118.6 μM.

No inhibition of the signal was observed in the presence of 4-MSP. zfERβ1

4-MSP was tested on HELN zfERβ1 for agonist or antagonist properties.4-MSP presented a partial agonist activity detected at 10 and 33 μM, with an apparent EC50 estimated at 25.51 μM.

In the presence of 0.1 nM E2, no inhibition of the luminescence signal was observed; therefore, no antagonistic properties on zfERβ1 were displayed by 4-MSP.

zfERβ2

4-MSP was tested on HELN zfERβ2 for agonist or antagonist properties. 4·MSP presented a weak partial agonistic activity detected at 10 and 33 μM, its EC50 was estimated at 85.27 μM.

In the presence of a suboptimal concentration of 0.1 nM (10E-10 M) E2, 4-MSP showed no inhibition of the signal induced by 10E-10 E2, thus 4-MSP did not act as an antagonist on zfERβ2.

Conclusion:

4-MSP presented a weak agonist activity on the three zebrafish ERs, with estimated EC50 of 20.56, 25.51 and 85.27 μM on zfERα., zfERβ1 and zfERβ2 respectively. 4-MSP did not show any antagonist properties on HELN zfERα, zfERβ1 and zfERβ2.

Although 2-MSP has a weak partial agonist activity on zfERα the overall estrogenic activity is mostly due to 4-MSP which exerts a partial agonist activity on the three zebrafish ERs with a potency order: zfERα. = zfERβ1 > zfERβ2.

The relative potencies of 4-MSP compared to a mixture of nonylphenol were 40 times less potent on zfERα, 6 times less potent on zfERβ1 and 150 times less potent on zfERβ2.

References:

- Balaguer, P., François, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., Nicolas, J.c., CaselIas, C., 1999.Reporter cell lines to study the estrogenic effects of xenoestrogens.Sci. Total Environ. 233, 47-56.

- Pillon, A., Servant, N., Vignon, F., Balaguer, P., Nicolas, J.-C., 2005.In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters. Analytical Biochemistry 340,295-302.

- Pinto, C., Grimaldi, M., Boulahtouf, A., Pakdel, F., Brion, F., Aït-Aïssa, S., Cavaillès, V., Bourguet, W., Gustafsson, J.-A., Bondesson, M., Balaguer, P., 2014.Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Toxicology and Applied Pharmacology 280, 60-69.