1

Photolysis of estrone generates estrogenic photoproducts with higher

activity than the parent compound

Yasmine Souissia*

, Said Kinania, Stéphane Bouchonnet

a, Sophie Bourcier

a, Christian

Malosseb, Michel Sablier

a, Nicolas Creusot

c, Enrico Mombelli

d, Selim Aït-Aïssa

c*

a École Polytechnique, Laboratoire des Mécanismes Réactionnels, CNRS, route de Saclay, F-

91128 Palaiseau cedex, France

b Unité de Spectrométrie de Masse Structurale et Protéomique - Institut Pasteur, UMR 3528

CNRS, 25-28, rue du Dr Roux, F-75015 Paris, France

c Unité Écotoxicologie in vitro et in vivo, Institut National de l’Environnement Industriel et

des Risques (INERIS), BP2, F-60550 Verneuil en Halatte, France

d Unité Modélisation en Toxicologie et Écotoxicologie, Institut National de l’Environnement

Industriel et des Risques (INERIS), BP2, F-60550 Verneuil en Halatte, France

* Corresponding authors : yasmine@dcmr.polytechnique.fr, selim.ait-aissa@ineris.fr

Souissi et al, Environmental Science and Pollution Research, 2014,

21(13) 7818-7827

2

Abstract

In the present study, we aimed to evaluate the effect of UV-visible irradiation on the

estrogenicity of an estrone aqueous solution by using chemical analysis associated with an in

vitro bioassay and in silico analysis. An estrone aqueous solution was irradiated with an UV-

visible high pressure mercury lamp. By using the MELN in vitro cellular bioassay, based on

the induction of a luciferase reporter gene upon the activation of the estrogen receptor by

chemicals, we showed that the estrogenic potency of the solution increased after irradiation.

High performance liquid chromatography fractionation of the photolyzed solution followed

by in vitro testing of fractions allowed the quantitation of the estrogenic potency of each

fraction. Nine photoproducts were detected and characterized by liquid chromatography-mass

spectrometry coupling. The observed estrogenic activity is mediated by mono and multi-

hydroxylated photoproducts; it is influenced by the position of hydroxyl groups on the

steroidal skeleton. In addition, a structure-activity analysis of the hydroxylated photoproducts

confirmed their ability to act as estrogen receptor ligands.

Keywords: estrone, photodegradation products, LC-MS analysis, in vitro bioassay, QSAR

3

Introduction

Endocrine disrupting compounds (EDCs) can interfere with the hormonal activities of aquatic

lives, animals, and probably also humans. EDCs are a cause of major environmental concern

due to their dissemination in aquatic environmental compartments and their ability to induce

developmental and/or reproductive dysfunctions by interfering with the endocrine system of

exposed organisms (Harris et al. 2011; Jobling et al. 1998). The identification of the main

contaminants responsible of disrupting effects in aquatic media while ascribing significance to

each contaminant is challenging. The evidence collected to date from Europe, the United

States, and other parts of the world have clearly identified steroid estrogens as one of the most

potent chemical classes present in domestic effluents and surface waters (Benotti et al. 2009;

Creusot et al. 2013; Duong et al. 2010; Jobling and Tyler 2006; Lopes et al. 2010). These

steroid hormones are recognized to make their way into the aquatic environment through

sewage discharge and human and animal waste disposal. In addition, it is now well-

established that sewage treatment plant effluents, defined as a complex mixture of both

natural and manmade chemicals, can contain estrogenic contaminants at concentrations high

enough to provoke noticeable endocrine disruption in fish (Brion et al. 2004; Harries et al.

1997; Hinfray et al. 2010; Jobling et al. 1998; Labadie and Budzinski 2005). In the effluent of

a conventional wastewater treatment plant, the concentrations in estrogen hormones may

range from a few nanograms per litre to micrograms per litre, estrone (E1) and 17-estradiol

being widely described as major contributors of the estrogenic potency of effluents (Khanal et

al. 2006). Analytical determinations of natural and synthetic estrogen concentrations were

recently carried out using liquid chromatography coupled with tandem mass spectrometry in

various water samples collected in the area of Paris. E1 concentrations ranging from 0.1 to

15.7 ng/L were measured in the collected samples (Miège et al. 2009). The broad and regular

distribution of E1 in the aquatic environment leads to consider it as a likely potential reservoir

of estrogenic activity, and a source for the generation of degradation products of likely

adverse effects (Miège et al. 2009). E1 is frequently reported as one of the major contributors

of estrogenic activity observed in environmental waters and accounting for a significant part

of the total estrogenic potency of natural steroidal hormone excretion by human beings and

livestock (Khanal et al. 2006). Removal efficiencies of E1 from sewage treatment plant

effluents may greatly differ according to the treatment processes employed in these plants and

results in non-negligible E1 release in the aquatic media. Activated sludge treatments have

been reported to be quite inefficient for the elimination of E1 leading even sometimes to E1

outlet levels larger than the E1 inlet levels (Baronti et al. 2000). The frequent detection of E1

in the environment makes it necessary to find advanced treatment processes with high

removal efficiency, such as photolysis, but also to consider its possible degradation under

solar irradiation and to investigate the possible formation of estrogenic photoproducts. There

is obviously a need for the identification of the chemical transformation pathways taking

place in environmental samples (Escher and Fenner 2011). Although the concentration of the

4

parent steroid hormones in natural aquatic environments is in the very low nanogram per liter

range, it is important to understand their environmental fate due to their high biological

potency.

The identification of photoproducts generated upon photolysis of E1 under UV-visible

irradiation conditions has been previously investigated in our laboratory (Souissi et al. 2012).

Nine major photoproducts were characterized through liquid chromatography coupled with

tandem mass spectrometry (LC-MS/MS) experiments. All the photoproducts were

characterized by the presence of the phenolic ring of E1, and were therefore considered as

potential EDCs. In the present study, by using the in vitro reporter cell line MELN (Balaguer

et al. 2001), we show that UV-visible irradiated E1 aqueous solution is significantly more

estrogenic than the non treated E1 solution. In an attempt to further characterize generated

active products, they were isolated by fractionation using high performance liquid

chromatography (HPLC) and the estrogenic activity of each fraction has been determined.

The biological activities measured are discussed in the light of the identified chemical

structures and on the basis of a structure-activity analysis related to ER binding.

Material and methods

Chemicals

Estrone (E1, 98% purity) was purchased from Sigma (St Quentin Fallavier - France), as well

as HPLC grade methanol, acetonitrile, dimethylsulfoxide and formic acid.

Photolysis experiments

A home-made photo reactor was used for photolysis experiments. This reactor consisted of

six quartz tubes placed in a circle around a high-pressure mercury lamp (HPL-N 125W/542

E27 SG, Philips - Ivry sur Seine, France) in an ultrasonic bath (Bioblock Scientific - Illkirch,

France). The luminous flux emitted from the lamp was reported by the manufacturer to be

6200 lm. The tubes were filled with 30 mL of the stock solution of estrone at 0.01 mg/mL.

The reactor was cooled by water circulation to avoid uncontrolled heating of the irradiated

solution. The temperature was held constant at 25 ± 2 °C. The solution was homogenized by

sonication during irradiation because of the low solubility of estrone in water (Souissi et al.

2012). We checked that sonication did not induce noticeable effects on estrone degradation.

Each photolysis experiment was performed in triplicate for HPLC fractionation, LC-MS/MS

analysis and bioassays estrogenic activity assessment.

5

Photoproducts extraction using Solid Phase Extraction

Solid-phase extractions (SPE) were carried out prior to all analysis. Each sample was

extracted using a SPE cartridge (Oasis®

HLB, Waters, France). The cartridge was conditioned

with 3 mL of methanol and 3 mL of distilled water. 4 mL of a 5:95 methanol/water mixture

were used for washing the SPE cartridge. After elution of 10 mL of sample at a rate of

approximately 60 drops min−1

, the cartridge was vacuum-dried and eluted with 8 mL of

methanol and 4 mL of acetonitrile with 0.1% formic acid, successively. The extracts were

concentrated at ambient temperature under a gentle nitrogen stream until dryness. The same

procedure was systematically applied to the dried sample: dissolution in 200 µL of an

acidified LC-MS mobile phase of H2O/CH3CN (60:40) with 0.1% formic acid for LC/MS

analysis, dissolution in 400 µL of DMSO for bioassay analysis (in this latter case, the

effective quantity of sample was equivalent to 4 mL of estrone solution).

HPLC fractionation

In order to investigate the biological activity of estrone photoproducts, a fractionation

experiment was set up. Photoproducts of estrone were separated on a C18 column (Pursuit-

XRs Ultra 2.8 C18 50 x 2.0 mm, Varian, France) by HPLC (Acquity, Waters) using a UV

detector with a wavelength scanning range from 275 to 285 nm. Elution was performed with a

flow of 0.2 mL/min using the gradient described in Table 1. Fractions were collected with a

solvent front delay of 30 sec and a sampling rate of 20 points/sec. The collected fractions

were merged into twelve main fractions on the basis of the chromatographic separation. Each

main fraction was divided into two parts which were blown to dryness under a nitrogen flow.

One part was dissolved in the mobile phase (at t = 0) for LC-MS/MS analysis while the other

part was reconstituted in 200 µL DMSO for the assessment of the estrogenic activity.

Table 1

Chemical identification by liquid chromatography-mass spectrometry coupling (LC-

MS/MS)

LC-MS/MS analysis was used to assess estrone degradation and identify photoproducts in the

photolyzed solution. The chemical structures of these photoproducts were elucidated based on

(i) collision induced dissociation experiments, (ii) high resolution measurements (iii) the

analysis of isotope labelled molecules and model compounds. Analytical conditions,

characterization strategy and photoproducts identification have been described in details

previously (Souissi et al. 2012). In the present study, the same conditions were used to check

for photoproducts occurrence in both the photolyzed estrone solution and HPLC fractions.

6

Bioassay description

Estrogenic activity in reporter gene assay

The MELN reporter cell line, kindly provided by Dr Balaguer (INSERM, France), has been

used in order to assess the estrogenic activity of fractions containing estrone and its

photoproducts. It was obtained by stable transfection of MCF-7 human breast cancer cells by

an ERE-bGlob-Luc-SVNeo plasmid (Balaguer et al. 2001). Cells were routinely cultured in

phenol red containing Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with

5% foetal calf serum (FCS), 1% nonessential amino acids and penicillin/streptomycin (50

U/mL each) in a 5% CO2 humidified atmosphere at 37 °C. For experiments, cells were left to

incubate for 2 days in phenol red free DMEM supplemented with 3% dextran charcoal

coated-FCS (DCC medium) before seeding in white opaque 96-wells culture plates at a

density of 50,000 cells per well. Serial dilutions of reference chemicals, DMSO extracts of

photolyzed solutions or HPLC fractions were added in triplicates 24 h later and then left to

incubate for 16 h. DMSO concentration in the culture medium was always 0.1% v/v,

including in cell controls. At this concentration, no effect on cell viability or luciferase

activity was observed. At the end of the incubation period, the medium was removed and

replaced by 50 µL of DCC medium containing 0.3 mM of D-luciferin and the luminescence

signal was measured in living cells for 2 s per well with a microtiter plate luminometer

(µBeta, Wallac). Relative luminescence units (RLU) were converted to relative response units

expressed as percent of maximal luciferase induced by the positive control (17β-estradiol (E2)

at 10 nM).

Data analysis and determination of bioassay-derived estrone-equivalents

Modelling of dose-response curves and determination of EC50 (i.e. concentration giving 50%

of the maximum luciferase induction) were done with the Regtox 7.5 macro using the Hill

equation model (freely available at http://www.normalesup.org/~vindimian/), as described

previously (Kinani et al. 2010). Estrogenic activity of the HPLC fractions was expressed as

E1-equivalents, which was determined as the ratio of the EC50 of estrone (E1), expressed as

g/L, to that of the fraction expressed as equivalent-litre of initial aqueous estrone solution per

litre (L-eq/L).

Structure-Activity analysis

The OECD (Q)SAR Toolbox v3.1.0.21 was used in order to have an insight into the possible

relationship that can exist between the structure of the photoproducts and their estrogenic

activity. The OECD (Q)SAR Toolbox can be freely downloaded from the OECD website; it

contains two modules (ER binding profiler and rtER Expert System) that can categorize

7

chemicals according to their binding potency (OECD 2013). The ER binding profiler was

evaluated by Mombelli (Mombelli 2012) and it showed an acceptable performance in

categorizing chemicals with respect to their biding affinities towards estrogen receptors.

Results and discussion

Photo-treatment of estrone solution and isolation of degradation products

During photolysis experiments, estrone concentration dropped from the initial value (0.01

mg/mL) to reach about 75% of degradation after 90 min of irradiation. Before starting with

bioassays, we first confirmed the occurrence of the nine photoproducts in the photolyzed

estrone solution, in line with our previous study (Souissi et al. 2012). These photoproducts

were isolated by preparative HPLC (Fig. 1), yielding 12 fractions that were all collected. Sub-

samples of the fractions were analysed by LC-MS, in order to confirm the presence of the

targeted compound in each fraction. The structures of photoproducts and their assignment to

fractions 1 to 12 are reported in Table 2. The fractions 2 and 5 were collected though no

photoproducts were identified at the corresponding retention times. The fraction 9

corresponds to the remaining estrone in the photolyzed solution. Considering that C7 and C9

include three polar groups (3 hydroxy groups for C7, 1 hydroxy and 2 carbonyl functions for

C9), both compounds would be expected to elute before estrone in reverse-phase HPLC.

Steric effects or molecular dipole moments are useless to rationalize this elution order.

Nevertheless, mass spectrometric data (high resolution measurements and collision induces

dissociation pathways) leave no doubt about the number of hydroxy and carbonyl groups

carried by the structures (Souissi et al. 2012).The retention time of compound C8 is the most

puzzling. The HR measurement gives m/z 541.3323 (Souissi et al. 2012) for the main ion

(MH+), which corresponds to a C36H45O4 raw formula and thus to a dimer of estrone. A non-

covalent dimer formed in the electrospray source has been first considered but discarded

given that the collision energy required to dissociate the pseudomolecular ion was as high are

for other photoproducts. Results obtained with deuterated estrone confirmed this could not be

an impurity. Its potentially high polarity might favor its elution and lead to a retention time

much shorter than expected for a big size compound.

Recent studies reported that the UV-B-mediated degradation of estrone leads to the

photoproduction of lumiestrone, a little known 13α-epimer form of estrone (Trudeau et al.

2011). Both epimers can be easily separated in HPLC using a C18-type column (Whidbey et

al. 2012). Lumiestrone, which exhibits moderate estrogenic activity, has not been detected in

the present work. A perfect coelution of estrone and lumiestrone seems very unlikely

considering previous works. It is more likely that lumiestrone was not produced under our

irradiating conditions.

Fig. 1, Table 2

8

Effect of UV-visible irradiation on the estrogenic activity of estrone in aqueous solution

Estrogenic activity of photolyzed and non photolyzed estrone solutions

The estrogenic potency of irradiated and non-irradiated estrone solutions has been assessed by

using the MELN assay. For this purpose, two samples of equal volume were taken: the first

one from the solution photolyzed for 90 minutes and the second one from the reference (non

photolyzed) solution. Both samples underwent SPE extraction before being diluted at

different levels. The concentration at each level was estimated based on that of estrone in the

non-irradiated solution. Estimating concentrations in such a way introduces a non-negligible

uncertainty due to the assumption that SPE extraction yields of photoproducts are not so

different than that of estrone. As shown in Fig. 2, significant dose-dependent induction of

luciferase activity was obtained for both irradiated and non-irradiated solutions. However, at

an equivalent concentration (concentration in E1 for the reference solution, in E1 +

photoproducts in the irradiated solution), a higher estrogenic activity was observed with the

photolyzed solution (Fig. 2) as a shift of the dose-response curve to the left side of the graph

was observed after treatment. Such increased overall estrogenic activity suggests the photo-

induced formation of compounds more active than E1 itself. It is to be noticed that a control

solution has also been tested. This sample consisted in ultra-pure water submitted to the same

experimental steps than the estrone reference photolyzed solution. This procedural blank,

which has been demonstrated to be non-active in the MELN assay (data not shown),

confirmed that the estrogenic potency of our samples was inherent to estrone and/or its

photoproducts and not to reagents used for the assay or to any other estrogenic contaminant.

Fig. 2

The main observed chemical transformations that affected the original compound were mono-

and multi-hydroxylation (Table 2). The photolytic process resulted in the introduction of

hydroxyl groups into different rings (cycles A, B and C) of the parent compound depending

on the photoproducts. The enhancing effect that hydroxyl groups can have on the estrogenic

potency is well known for hydroxylated estradiol metabolites. For instance, the affinity of the

3-hydroxy metabolite of estradiol has been proven to be higher than that of the 4-hydroxy

metabolite using a receptor binding assay with the human estrogen receptor (Kuiper et al.

1997). In our study, such structural modifications may reflect a higher estrogenic potential of

hydroxylated estrone as compared to the parent compound.

Estrogenic activity of RP-HPLC fractions

In order to investigate estrogenic activity of isolated estrone photoproducts, the different

fractions collected by preparative HPLC were tested for estrogenic activity. Fig. 3 shows that

9

all individual fractions were able to induce luciferase activity in MELN cells but with various

potencies in terms of EC50. This result confirms the finding that estrone photoproducts that

exert estrogenic activity were formed after photolysis.

On the basis of dose-response curves modeling (Fig. 3), activities could be expressed in terms

of estrone-equivalents (E1-eq) for individual fractions and significant differences were

observed among fractions (Fig. 4). Those variations may be explained by two factors: the

structural differences between the photodegradation compounds (Table 2) and their

concentration within the fractions. Fractions 7, 8, 9 and 10 displayed the highest estrogenic

activity followed by fractions 4, 11 and 12. In the fraction 9, which contains estrone

remaining in the photolyzed solution, around 1 mg E1-eq/L was measured, which corresponds

to 10% of initial E1 concentration. This range value is, to some extent, in line with the 75%

loss of estrone measured by LC-MS in the photolyzed solution, while a further loss of

material may have occurred during the fractionation process. Regarding the other fractions, a

quantitative relationship between E1-eq and photoproducts concentration in fraction cannot be

established in the absence of analytical standards necessary to measure concentrations.

Nevertheless, on the basis of chromatographic peak areas, some trends can be highlighted. For

instance, fractions 9 and 10 have almost similar E1-eq while E1 peak (in fraction 9) is much

more intense than C7 peak (in fraction 10), which may reflect that C7 is less abundant but

more potent than E1. Similar hypothesis can be drawn when comparing C6a and C6b in

fraction 8 and E1 in fraction 9. However, obtaining purified standards will be necessary to

confirm experimentally these hypotheses.

Fig. 3 and Fig. 4

The fractions 2 and 5 were found as weakly estrogenic (0.004 and 0.012 mg E1-eq/L,

respectively) although no photoproducts were identified in those fractions due to the poor

separation of the corresponding chromatographic peaks during the fraction collection process.

This observation highlights the great sensitivity of biological assays and their ability to screen

the potential presence of a contaminant not detected by physico-chemical approaches.

These parallel observations indicate that relationships between the structures of the

photoproducts and their estrogenic activities can be analyzed in a systematic way. Such an

analysis can help in prioritizing chemicals for experimental testing by identifying structural

features that are critical in determining efficient interactions with estrogen receptors. For these

reasons, structure-activity relationships of the photoproducts were further analyzed by means

of the OECD (Q)SAR Toolbox and other existing knowledge.

Structure-Activity analysis

10

All the photoproducts but C8 contained a structural alert for ER binding (i.e. a chemical

fragment which is known to mediate binding to the receptor) when analyzed with the ER

binding profiler (Table 3). Conversely, all the chemicals did not belong to chemical classes

that are known to rtER Expert System. All the photoproducts can potentially bind to the

estrogen receptor since they contain within their structure an OH group and other structural

requirements (e.g. molecular weight cut-offs) that are needed for a specific interaction with

the ER (Mombelli 2012). The only exception to this general trend is represented by the

photoproduct C8 that is characterized by a molecular weight greater than 500 Da. Chemicals

characterized by large volumes (approximated by molecular weights) are regarded as non-

binders by the profiler since the dataset that enabled its implementation did not include any

ER-binder with a molecular weight greater than 500 Da as indicated in the documentation of

the OECD Toolbox. The results presented in table 3 conform to what is reasonable to expect

from a general purpose prioritization tool which is not specifically trained to recognize the

effect of minor structural modifications within a specific class of chemicals (Mombelli 2012).

In other words, from a prioritization point of view, the presence within a sample of estrone

derivatives bearing an OH group is in itself a structural characteristic that should trigger

further investigations since their structures are very similar to endogenous hormones.

It is worth observing that existing structural knowledge allows for the formulation of

arguments that go beyond the results reported in table 3 since the binding pocket of the

estrogen receptor has been extensively characterized. For instance, the reduction in activity of

compound C9 could be explained by fact that the immediate hydrophobic surroundings of

position 7 are not favorable for polar interactions between the ER and the keto-group of the

chemical (Anstead et al. 1997). The attenuation of estrogenic activity of compounds C3a and

C3b is also not surprising since these chemicals are characterized by substitutions at position

6 and this position is intolerant to any kind of modification. Moreover, these chemicals are

also characterized by a keto group at position 15 that is intolerant to a replacement with

oxygen or a keto-group at position 12 that can only accommodate small nonpolar groups

(Anstead et al. 1997).

Similarly, compound C4 presents OH groups at positions that are known to be intolerant to

polar groups (position 12) or any kind of substitution (position 6). The low estrogenic activity

of compounds C1 and C2 also seems to have a structural basis since intramolecular hydrogen

bonds between the three OH groups of the A-ring could reduce binding affinities (Anstead et

al. 1997). A supporting argument for a weaker affinity in the presence of adjacent hydroxyl

groups is also provided by Bentz and coauthors since 2-hydroxyestrone is reported to be less

estrogenic than estrone (Bentz et al. 2005). Moreover, compound C1 also has an OH group at

position 6 which, as already discussed, cannot tolerate any substitution.

11

On the other hand, it is more difficult to find a rationale for the most active compounds. For

instance, compound C6a and C6b displayed a high activity while bearing a mono-

hydroxylation at position 11 or 12. This is somewhat inconsistent with existing knowledge

since position 11 is known to be rather intolerant to polar groups and a decrease in binding

affinity should therefore be expected. Similarly, the only available information about position

12 indicates that it should only be able to accommodate small nonpolar groups (Anstead et al.

1997). Therefore the results presented in this paper suggest a peculiar binding mode of these

estrone derivatives. This binding mode could, for instance, make the carbonyl at position 17

more accessible to a receptor H-bond donor site and increase binding affinities (Anstead et al.

1997). The increased activity of compound C7 could be caused by the OH group at position

14 adopting a configuration which is known to enhance the activity of estradiol (Bohl et al.

1987).

As far as compound C5 is concerned, current knowledge allows for limited insight into what

can possibly happen since position 12 has been scarcely investigated and positions 6 and 14

can have an opposite effect on binding affinities: a 14 configuration increases binding

affinities (Bohl et al. 1987) whereas position 6 of the steroidal backbone is intolerant to both

polar and nonpolar groups (Anstead et al. 1997).

Among the structural observations that can be made for the identified photoproducts, the case

of compound 8 is perhaps the most interesting. Indeed, this chemical still maintains an

appreciable activity even if its molecular weight is greater than 500 Da, a threshold beyond

which no binding is generally expected (OECD 2013). It appears therefore that despite its

size, compound 8 can still form thermodynamically convenient interactions with the ER by

adopting a suitable configuration. This atypical behavior could display some similarities with

the binding mode of raloxifene (MW = 473.6, not far from the critical threshold) that can

effectively interact with the ER even if it cannot be entirely accommodated by its binding

pocket. This special binding mode is achieved thanks to a structural adaptation of the ER

prompted by the bulky side chain of raloxifene that displaces helix-12 of the ER while

protruding from the pocket between helix-3 and helix-11 (Brzozowski et al. 1997).

A major rearrangement of the binding pocket is therefore likely to take place also in the case

of compound 9 since it is difficult to imagine an effective interaction of this chemical that

does not reflect a significant induced fit of the receptor. More importantly, this finding

suggests that expanding existing databases by including more chemicals with a molecular

weight greater than 500 Da could unravel unknown binding modes that have been overlooked

thus far despite their potential impact on endocrine disruption.

Table 3

12

Conclusion

Most of the identified photoproducts of estrone exhibit an estrogenic potency, very likely

higher than that of estrone. Estrogenicity measurements remain estimations given that

fractionation did not allow accurate isolation of all the photoproducts due to the strong

resemblance in the chemical structures of some of them. Further investigations using purified

compounds would be necessary to firmly confirm the estrogenic activity of identified

photoproducts. Nevertheless, these results suggest that, in spite of its increasing use for water

disinfection, UV-visible irradiation may not constitute a suitable degradation process for

estrone elimination from aqueous media.

Acknowledgements

This work was partly supported by the French Ministry of Ecology, Sustainable Development

and Energy (P190) via INERIS. We wish to thank two anonymous reviewers for helping to

improve the quality of the manuscript.

References

Anstead, G.M., Carlson, K.E. and Katzenellenbogen, J.A. (1997) The estradiol pharmacophore:

Ligand structure-estrogen receptor binding affinity relationships and a model for the receptor

binding site. Steroids 62, 268-303.

Balaguer, P., Boussioux, A.M., Demirpence, E. and Nicolas, J.C. (2001) Reporter cell lines are useful

tools for monitoring biological activity of nuclear receptor ligands. Luminescence 16, 153-158.

Baronti, C., Curini, R., D'Ascenzo, G., Di Corcia, A., Gentili, A. and Samperi, R. (2000) Monitoring

natural and synthetic estrogens at activated sludge sewage treatment plants and in a receiving river

water. Environ Sci Technol 34, 5059-5066.

Benotti, M.J., Trenholm, R.A., Vanderford, B.J., Holady, J.C., Stanford, B.D. and Snyder, S.A. (2009)

Pharmaceuticals and Endocrine Disrupting Compounds in US Drinking Water. En Environ Sci

Technol 43, 597-603.

Bentz, A.T., Schneider, C.M. and Westerlind, K.C. (2005) The relationship between physical activity

and 2-hydroxyestrone, 16 alpha-hydroxyestrone, and the 2/16 ratio in premenopausal women

(United States). Cancer Causes & Control 16, 455-461.

Bohl, M., Schubert, G., Koch, M., Reck, G., Strecke, J., Wunderwald, M., Prousa, R. and Ponsold, K.

(1987) Quantitative Structure-Activity-Relationships of Estrogenic Steroids Substituted at C-14,

C15. J Steroid Biochem Mol Biol 26, 589-597.

Brion, F., Tyler, C.R., Palazzi, X., Laillet, B., Porcher, J.M., Garric, J. and Flammarion, P. (2004)

Impacts of 17 beta-estradiol, including environmentally relevant concentrations, on reproduction

after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

Aquat Toxicol 68, 193-217.

13

Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E., Bonn, T., Engstrom, O., Ohman, L.,

Greene, G.L., Gustafsson, J.A. and Carlquist, M. (1997) Molecular basis of agonism and

antagonism in the oestrogen receptor. Nature 389, 753-758.

Creusot, N., Tapie, N., Piccini, B., Balaguer, P., Porcher, J.M., Budzinski, H. and Aït-Aïssa, S. (2013)

Distribution of steroid- and dioxin-like activities between sediments, POCIS and SPMD in a

French river subject to mixed pressures. Environ Sci Pollut Res 20, 2784-2794.

Duong, C.N., Ra, J.S., Cho, J., Kim, S.D., Choi, H.K., Park, J.H., Kim, K.W., Inam, E. and Kim, S.D.

(2010) Estrogenic chemicals and estrogenicity in river waters of South Korea and seven Asian

countries. Chemosphere 78, 286-293.

Escher, B.I. and Fenner, K. (2011) Recent Advances in Environmental Risk Assessment of

Transformation Products. Environ Sci Technol 45, 3835-3847.

Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, M., Sumpter, J.P., Taylor, T. and

Zaman, N. (1997) Estrogenic activity in five United Kingdom rivers detected by measurement of

vitellogenesis in caged male trout. Environ Toxicol Chem 16, 534-542.

Harris, C.A., Hamilton, P.B., Runnalls, T.J., Vinciotti, V., Henshaw, A., Hodgson, D., Coe, T.S.,

Jobling, S., Tyler, C.R. and Sumpter, J.P. (2011) The Consequences of Feminization in Breeding

Groups of Wild Fish. Environ Health Perspect 119, 306-311.

Hinfray, N., Palluel, O., Piccini, B., Sanchez, W., Aït-Aïssa, S., Noury, P., Gomez, E., Geraudie, P.,

Minier, C., Brion, F. and Porcher, J.M. (2010) Endocrine disruption in wild populations of chub

(Leuciscus cephalus) in contaminated French streams. Sci Total Environ 408, 2146-2154.

Jobling, S., Nolan, M., Tyler, C.R., Brighty, G. and Sumpter, J.P. (1998) Widespread sexual disruption

in wild fish. Environ Sci Technol 32, 2498-2506.

Jobling, S. and Tyler, C.R. (2006) Introduction: The ecological relevance of chemically induced

endocrine disruption in wildlife. Environ Health Perspect 114, 7-8.

Khanal, S.K., Xie, B., Thompson, M.L., Sung, S.W., Ong, S.K. and Van Leeuwen, J. (2006) Fate,

transport, and biodegradation of natural estrogens in the environment and engineered systems.

Environ Sci Technol 40, 6537-6546.

Kinani, S., Bouchonnet, S., Creusot, N., Bourcier, S., Balaguer, P., Porcher, J.M. and Aït-Aïssa, S.

(2010) Bioanalytical characterisation of multiple endocrine- and dioxin-like activities in sediments

from reference and impacted small rivers. Environ Pollut 158, 74-83.

Kuiper, G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S. and Gustafsson, J.A.

(1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen

receptors alpha and beta. Endocrinology 138, 863-870.

Labadie, P. and Budzinski, H. (2005) Determination of steroidal hormone profiles along the Jalle

d'Eysines River (near Bordeaux, France). Environ Sci Technol 39, 5113-5120.

Lopes, L.G., Marchi, M.R.R., Souza, J.B.G., Moura, J.A., Lorenzon, C.S., Cruz, C. and Amaral, L.A.

(2010) Estrogens in Natural and Treated Waters in the Region of Jaboticabal - Sao Paulo, Brazil.

Quimica Nova 33, 639-643.

Miège, C., Gabet, V., Coquery, M., Karolak, S., Jugan, M.L., Oziol, L., Levi, Y. and Chevreuil, M.

(2009) Evaluation of estrogenic disrupting potency in aquatic environments and urban wastewaters

by combining chemical and biological analysis. TrAC Trends in Analytical Chemistry 28, 186-195.

Mombelli, E. (2012) Evaluation of the OECD (Q)SAR Application Toolbox for the profiling of

estrogen receptor binding affinities. SAR QSAR Environ Res 23, 37-57.

OECD. (2013) Supporting Information for the ER binding profiler http://www.oecd.org/env/ehs/risk-

assessment/theoecdqsartoolbox.htm.

14

Souissi, Y., Bourcier, S., Bouchonnet, S., Genty, C. and Sablier, M. (2012) Estrone direct photolysis:

By-product identification using LC-Q-TOF. Chemosphere 87, 185-193.

Trudeau, V.L., Heyne, B., Blais, J.M., Temussi, F., Atkinson, S.K., Pakdel, F., Popesku, J.T., Marlatt,

V.L., Scaiano, J.C., Previtera, L. and Lean, D.R.S. (2011) Lumiestrone is photochemically derived

from estrone and may be released to the environment without detection. Front Endocrinol 2,

doi.10.3389/fendo.2011.00083.

Whidbey, C.M., Daumit K.E., Nguyen T.-H., Ashworth D.D., Davis J.C.C., Latch D.E. (2012)

Photochemical induced changes of in vitro estrogenic activity of steroid hormones. Water Res 46,

5287-5296.

15

Figures

Fig. 1. HPLC-UV chromatogram of the irradiated solution of estrone and fractionation.

Fig. 2. Estrogenic activity of non-treated and photolyzed estrone solutions as reflected by

luciferase induction in MELN cells. Dilution values on the x-axis represent the fraction of the

initial 10 mg/L sample concentration in the reference solution remaining in each measured

point. This dilution factor accounts for the concentrating effects of SPE, fractionation

processes, and dilution into the cell culture medium. For example, a dilution factor of 1E-06

represents a 106-fold dilution of the initial 10 mg/L reference solution. Results are expressed

as relative light units. Values are means of triplicates ± standard deviation."

0

200

400

600

800

1000

1200

1400

1600

1800

1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04

Re

lati

ve li

ght

un

its

Dilution factor

No treatment

After photolysis

16

Fig. 3. Dose-response curves of luciferase induction in MELN cells by F1–F12 HPLC

fractions of the photolyzed estrone solution. Results are expressed as a percentage of maximal

luciferase activity induced by E2 at 10 nM. Values are means of triplicates (standard

deviations were always below 8%). Concentrations of fractions are given as final dilution

factor of the initial 10 mg/L estrone solution, i.e. considering both concentration factor

through SPE and fractionation processes and dilution factor of DMSO extract in cell culture

medium. Values are means of triplicates ± standard deviation.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1.E-7 1.E-5 1.E-3 1.E-1

Luci

fera

se a

ctiv

ty(r

ela

tive

to

E2

10

nM

)

Dilution factor

F1

F2

F3

F4

F5

F6

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1.E-7 1.E-5 1.E-3 1.E-1

Luci

fera

se a

ctiv

ity

(re

lati

ve t

o E

2 1

0 n

M)

Dilution factor

F7

F8

F9

F10

F11

F12

17

Fig. 4. Estrogenic activity of HPLC fractions (F1 to F12), expressed as g of E1-equivalents

per L-equivalent of initial photolyzed estrone solution (± confidence intervals at 95%).

Table 1. Chromatographic gradient for fractionation experiments

Time (min) H2O/0.1% AF ACN/0.1% AF

0.0 60 40

11.0 60 40

11.1 0 100

18.0 0 100

18.1 60 40

25.0 60 40

1.E-07

1.E-06

1.E-05

1.E-04

1.E-03

1.E-02

F1 (C1)

F2 F3 (C2)

F4 (C3a, C3b)

F5 F6 (C4)

F7 (C5)

F8 (C6a, C6b)

F9 (E1)

F10 (C7)

F11 (C8)

F12 (C9)

Estr

on

e-E

q (

g/L)

18

Table 2. See attached file.

Table 3. Structure-Activity results according to the ER Binding Profiler of the OECD

(Q)SAR Toolbox. The rtER Expert System did not recognize any chemical class and for this

reason it was omitted from the table.

ID Fractions ER Binding Profiler

C1 F1 Very strong binder, OH group

C2 F3 Strong binder, OH group

C3a F4 Strong binder, OH group

C3b F4 Strong binder, OH group

C4 F6 Strong binder, OH group

C5 F7 Strong binder, OH group

C6a F8 Strong binder, OH group

C6b F8 Strong binder, OH group

C7 F10 Very strong binder, OH group

C8 F11 Non binder, MW>500

C9 F12 Strong binder, OH group