Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17Β-Hydroxysteroid Dehydrogenases

International Journal of Molecular Sciences

Article Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17β-Hydroxysteroid Dehydrogenases

Roger T. Engeli 1, Simona R. Rohrer 1, Anna Vuorinen 1, Sonja Herdlinger 2, Teresa Kaserer 2, Susanne Leugger 1, Daniela Schuster 2,* and Alex Odermatt 1,* ID 1 Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; [email protected] (R.T.E.); [email protected] (S.R.R.); [email protected] (A.V.); [email protected] (S.L.) 2 Computer-Aided Molecular Design Group, Institute of Pharmacy/Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria; [email protected] (S.H.); [email protected] (T.K.) * Correspondence: [email protected] (D.S.); [email protected] (A.O.); Tel.: +43-512-507-58253 (D.S.); +41-61-207-1530 (A.O.)

Received: 20 July 2017; Accepted: 14 September 2017; Published: 19 September 2017

Abstract: Parabens are effective preservatives widely used in cosmetic products and processed food, with high human exposure. Recent evidence suggests that parabens exert estrogenic effects. This work investigated the potential interference of parabens with the estrogen-activating enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) 1 and the estrogen-inactivating 17β-HSD2. A ligand-based 17β-HSD2 pharmacophore model was applied to screen a cosmetic chemicals database, followed by in vitro testing of selected paraben compounds for inhibition of 17β-HSD1 and 17β-HSD2 activities. All tested parabens and paraben-like compounds, except their common metabolite p-hydroxybenzoic acid, inhibited 17β-HSD2. Ethylparaben and ethyl vanillate inhibited 17β-HSD2 with IC50 values of 4.6 ± 0.8 and 1.3 ± 0.3 µM, respectively. Additionally, parabens size-dependently inhibited 17β-HSD1, whereby hexyl- and heptylparaben were most active with IC50 values of 2.6 ± 0.6 and 1.8 ± 0.3 µM. Low micromolar concentrations of hexyl- and heptylparaben decreased 17β-HSD1 activity, and ethylparaben and ethyl vanillate decreased 17β-HSD2 activity. However, regarding the very rapid metabolism of these compounds to the inactive p-hydroxybenzoic acid by esterases, it needs to be determined under which conditions low micromolar concentrations of these parabens or their mixtures can occur in target cells to effectively disturb estrogen effects in vivo.

Keywords: 17β-hydroxysteroid dehydrogenase; estrogen; xenobiotic; endocrine disrupting chemical; in silico; in vitro

1. Introduction Paraben compounds are widely used as additives in cosmetic products and processed food due to their broad spectrum preservative efﬁciency, low cost, and low toxicity [1,2]. Additionally, parabens possess excellent chemical stability and are biodegradable. Chemically, parabens are alkyl esters of p-hydroxybenzoic acid of which propyl- and methylparaben are the most frequently used parabens in cosmetic products. The preservative effects of parabens are at least in part due to disturbances of membrane transport and mitochondrial function in microorganisms [3,4]. European Union (EU) authorities permit a paraben content in cosmetic products of 0.4% for one ester compound and 0.8% for mixtures of esters (EU Council Directive, Cosmetic Products, 76/768/EEC/M11). Generally, mixtures of parabens are used to increase preservative efﬁciency. Lately, the acceptance of the use of parabens

Int. J. Mol. Sci. 2017, 18, 2007; doi:10.3390/ijms18092007 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2007 2 of 13 Int. J. Mol. Sci. 2017, 18, 2007 2 of 13 hastheir decreased favorable among properties, chemical there and is a pharmaceutical tendency to replace companies parabens and, with despite other their compounds favorable because properties, of therepotential is a disturbances tendency to replaceof endocrine parabens effects with [5]. other compounds because of potential disturbances of endocrineParabens effects can [ 5enter]. the systemic circulation via oral intake or by transdermal penetration, which was Parabensconfirmed can by enterthe detection the systemic of systemic circulation pa viaraben oral concentrations intake or by upon transdermal exposure penetration, to these whichcompounds was conﬁrmed [6–8]. However, by the detectionparabens ofare systemic very rapidly paraben metabolized concentrations to p-hydroxybenzoic upon exposure toacid these by compoundsesterases in the [6– 8liver]. However, and in the parabens skin, followed are very by rapidly excretion metabolized via the urine to p -hydroxybenzoic[9]. Parabens are acidmainly by esterasesexcreted as in theglycine, liver su andlfate, in theand skin, glucuronide followed conjugates by excretion [10]. via The the urinetopical [9 application]. Parabens of are paraben mainly excretedcontaining as products glycine, sulfate,more likely and contributes glucuronide to conjugates the systemic [10 ].paraben The topical concentration application than of their paraben oral containingintake due to products the rapid more intestinal likely contributesand hepatic tometa thebolism systemic [11]. paraben This assumption concentration is supported than their by oral the intakefact that due the to main the rapidhuman intestinal paraben and exposure hepatic is metabolismdue to the extensive [11]. This use assumption of personal is care supported products by [12]. the fact thatParabens the main have human been parabenassociated exposure with disturbanc is due to thees extensiveof estrogen use hormone of personal action care and products potential [12]. estrogenicParabens activities have of been parabens associated have withbeen extensively disturbances investigated of estrogen in hormone the past decades actionand [6,9]. potential In 2004, estrogenicDarbre et al. activities reported of the parabens detection have of been unconjugat extensivelyed parabens investigated in breast in the ca pastncer decadestissue, triggering [6,9]. In 2004,furtherDarbre investigations et al. reported into estrogenic the detection activities of unconjugated of parabens parabens [13]. Estrogens in breast cancerare primary tissue, female triggering sex furtherhormones investigations playing a central into estrogenicrole in a variety activities of phys of parabensiological actions [13]. Estrogens in females are and primary males. In female females, sex hormonesestrogens primarily playing a centralregulate role sexual in a varietydevelopment of physiological of the reproductive actions in females tissues and and males. development In females, of estrogenssecondary primarilysexual characteristics regulate sexual at puberty development [14]. Es oftrogens the reproductive trigger target tissues gene expression and development mainly by of secondaryacting through sexual estrogen characteristics receptors at α puberty and β (ER [14α]., EstrogensERβ) [15]. triggerThe 17β target-hydroxysteroid gene expression dehydrogenase mainly by α β α β β actingtypes 1 through and 2 (17 estrogenβ-HSD1 receptorsand 17β-HSD2)and regulate(ER , ERthe local)[15 ].balance The 17 between-hydroxysteroid potent and dehydrogenase weakly active β β typesestrogens. 1 and While 2 (17 17-HSD1β-HSD1 and converts 17 -HSD2) the regulate weakly theactive local estron balancee (E1) between into the potent most and potent weakly estrogen active β estrogens.estradiol (E2), While 17β 17-HSD2-HSD1 catalyzes converts the the opposite weakly reaction active estrone and decreases (E1) into the the local most concentrations potent estrogen of β estradiolactive estrogens (E2), 17 [16]-HSD2 (Figure catalyzes 1). Thus, the compounds opposite reactioninhibiting and 17β decreases-HSD2 result the in local increased concentrations local active of β activeestrogen estrogens concentrations [16] (Figure and can1). increase Thus, compounds the transcription inhibiting of estrogenic 17 -HSD2 target result genes, in thereby increased causing local activeestrogenic estrogen effects. concentrations Conversely, andcompounds can increase inhibiting the transcription 17β-HSD1 ofpossess estrogenic anti-estrogenic target genes, properties thereby β causingby reducing estrogenic local active effects. estrogen Conversely, concentrations. compounds A high inhibiting 17β-HSD2 17 to 17-HSD1β-HSD1 possess ratio in anti-estrogenic ERα-positive β β propertiesbreast cancer by reducingpatients has local been active shown estrogen to po concentrations.sitively correlate A highwith 17the-HSD2 survival to 17of ER-HSD1α-positive ratio inpatients ERα-positive [17]. This breast finding cancer illustrates patients the has importance been shown of proper to positively 17β-HSD2 correlate function. with the survival of ERα-positive patients [17]. This ﬁnding illustrates the importance of proper 17β-HSD2 function.

Figure 1. Interconversion of potent (estradiol) and weakly active estrogens (estrone) by Figure 1. Interconversion of potent (estradiol) and weakly active estrogens (estrone) by 17β-hydroxysteroid 17β-hydroxysteroid dehydrogenase (17β-HSD) enzymes. dehydrogenase (17β-HSD) enzymes.

Most of the studies addressing interferences of parabens with estrogen hormone action focused on the estrogenestrogen receptors.receptors. Possible interferences of parabens with pre-receptor control enzymes modulating endocrine functions, such as hydroxysteroidhydroxysteroid dehydrogenases (HSDs), remain to be investigated. Following Following indications indications from from a virtual a virtual screening screening of a of cosmetic a cosmetic products products database, database, the thepresent present study study addresses addresses potential potential endocrine endocrine disrupti disruptingng effects effects of of parabens parabens on on estrogen estrogen homeostasis homeostasis through inhibition ofof humanhuman estrogen-metabolizingestrogen-metabolizing enzymesenzymes inin vitro.vitro. 2. Results 2. Results Prior to the in vitro testing, the virtual screening of a cosmetic chemical database using 17β-HSD2 Prior to the in vitro testing, the virtual screening of a cosmetic chemical database using 17β- ligand-based pharmacophore models (Figure2A,B) [ 18], which are based on experimentally-veriﬁed HSD2 ligand-based pharmacophore models (Figure 2A,B) [18], which are based on experimentally- inhibitors, revealed parabens as possible 17β-HSD2 inhibitors. The chemical database contained verified inhibitors, revealed parabens as possible 17β-HSD2 inhibitors. The chemical database over 9000 compounds that are approved as bioactive molecules, carrier materials, and preservative contained over 9000 compounds that are approved as bioactive molecules, carrier materials, and preservative additives in cosmetic products. Models 1 and 2 returned 23 and 37 hits, respectively, while model 3 found only one compound. After merging the hit lists and removing duplicates, 49

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Int.Int.additives J.J. Mol.Mol. Sci.Sci.in 2017 cosmetic,, 18,, 20072007 products. Models 1 and 2 returned 23 and 37 hits, respectively,3 of 13 while model 3 found only one compound. After merging the hit lists and removing duplicates, unique hits remained. Among these hits, seven belonged to the chemical class of parabens unique49 unique hits hits remained. remained. Among Among these these hits, hits, seven seven belonged belonged to tothe the chemical chemical class class of of parabens (ethylparaben, phenylparaben, propylparaben, butylparaben, isopropylparaben, isobutylparaben, (ethylparaben, phenylparaben, pr propylparaben,opylparaben, butylparaben, isopropylparaben, isobutylparaben, and 2-phenoxyethyl p-hydroxybenzoate, Figure 2C). and 2-phenoxyethyl p-hydroxybenzoate, Figure2 2C).C). Therefore, this class of compounds was subjected to biological testing. In total, ten commercially- Therefore, this this class class of compounds of compounds was subjected was to subjected biological totesting. biological In total, testing.ten commercially- In total, available parabens, its main metabolite p-hydroxybenzoic acid, and four paraben-like compounds, availableten commercially-available parabens, its main parabens, metabolite its mainp-hydroxybenzoic metabolite p-hydroxybenzoic acid, and four acid,paraben-like and four compounds, paraben-like such as the virtual hit ethyl vanillate, were analyzed in vitro (Figure 3). suchcompounds, as the virtual such ashit the ethyl virtual vanillate, hit ethyl were vanillate, analyzed were in vitro analyzed (Figure in vitro3). (Figure3).

Figure 2. (((AA)) Virtual Virtual screening screening strategy strategy using using pharmacophore models for 1717β-HSD2 inhibitors described earlier [18]. [18]. Virtual Virtual hits hits are are required to map all features with the exception of the optional hydrogen bond acceptor. ( B) Full models with with exclusion exclusion volumes volumes serving serving as as steric steric restrictions restrictions for for the the size ofof hittinghitting compounds.compounds. ( C(C)) The The virtual virtual hits hits phenylparaben phenylparaben and and isopropylparaben isopropylparaben ﬁt to fit model to model 1 and 1 andethylparaben, ethylparaben, phenylparaben, phenylparaben, propylparaben, propylparaben, butylparaben, butylparaben, isopropylparaben, isopropylparaben, and and isobutylparabenisobutylparaben fitﬁt into model 2. For reasons of clarity, exclusion volumes are notnot shown.shown.

FigureFigure 3. 3. ChemicalChemicalChemical structuresstructures ofof thethe testedtested parabensparabens parabens,,, andand vanillicvanillic and and gallic gallic acid acid derivatives. derivatives.

All tested parabens, as well as methyl vanillate, ethyl vanillate, ethylethyl gallate,gallate, andand butylbutyl gallategallate inhibited 17β-HSD2 at a concentration of 20 µM (Figure 4A). The common paraben metabolite p- hydroxybenzoic acid was inactive. For the two most potent compounds, IC50 values were determined. hydroxybenzoic acid was inactive. For the two most potent compounds, IC50 values were determined.

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All tested parabens, as well as methyl vanillate, ethyl vanillate, ethyl gallate, and butyl gallate inhibited 17β-HSD2 at a concentration of 20 µM (Figure4A). The common paraben metabolite Int. J. Mol. Sci. 2017, 18, 2007 4 of 13 p-hydroxybenzoic acid was inactive. For the two most potent compounds, IC50 values were determined. Ethylparaben and ethyl vanillate inhibited 17β-HSD2 with an IC50 of 4.6 ± 0.8 µM and 1.3 ± 0.5 µM[19], Ethylparaben and ethyl vanillate inhibited 17β-HSD2 with an IC50 of 4.6 ± 0.8 µM and 1.3 ± 0.5 µM respectively (Figure4B). A mixture of ethyl- (6 µM) and hexylparaben (12 µM) showed additive effects [19], respectively (Figure 4B). A mixture of ethyl- (6 µM) and hexylparaben (12 µM) showed additive in terms of 17β-HSD2 inhibition compared to each individual compound (Figure5). effects in terms of 17β-HSD2 inhibition compared to each individual compound (Figure 5).

Figure 4. (A) Inhibition of estrone formation by parabens in human embryonic kidney (HEK-293) cell Figure 4. (A) Inhibition of estrone formation by parabens in human embryonic kidney (HEK-293) cell lysates expressing human 17β-HSD2. The conversion of estradiol to estrone was measured in lysates of lysates expressing human 17β-HSD2. The conversion of estradiol to estrone was measured in lysates HEK-293 cells transiently transfected with 17β-HSD2 in the presence of 200 nM radiolabeled estradiol of HEK-293 cells transiently transfected with 17β-HSD2 in the presence of 200 nM radiolabeled and parabens or paraben-like compounds at a final concentration of 20 µM. 17β-HSD2 inhibitor estradiol and parabens or paraben-like compounds at a final concentration of 20 µM. 17β-HSD2 BM13 served as positive control (compound 22 in [18]). Statistical analyses were performed using inhibitor BM13 served as positive control (compound 22 in [18]). Statistical analyses were performed one-way analysis of variance (ANOVA) test followed by a Dunnett post-hoc test. All tested compounds using one-way analysis of variance (ANOVA) test followed by a Dunnett post-hoc test. All tested significantly inhibited 17β-HSD2 activity (p ≤ 0.001) expect for p-hydroxybenzoic acid, which was not compounds significantly inhibited 17β-HSD2 activity (p ≤ 0.001) expect for p-hydroxybenzoic acid, which significant. (B) Concentration-dependent inhibition of 17β-HSD2 by ethylparaben and ethyl vanillate. was not significant. (B) Concentration-dependent inhibition of 17β-HSD2 by ethylparaben and ethyl Results represent the mean ± standard deviation (SD) of three independent measurements. vanillate. Results represent the mean ± standard deviation (SD) of three independent measurements.

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Figure 5. 5. AdditiveAdditive inhibitory inhibitory effects effects of two of two parabens parabens on 17 onβ-HSD2 17β-HSD2 activity. activity. The effect The effectof 6 µM of ethyl- 6 µM ethyl-(2-P) or (2-P) 12 µM or 12hexylparabenµM hexylparaben (6-P) or(6-P) a mixture or a mixture of both compounds of both compounds on 17β-HSD2 on 17 βactivity-HSD2 was activity assessed was assessedin lysates in of lysates transiently-transfected of transiently-transfected HEK-293 HEK-293 cells. Dimethylsulfoxide cells. Dimethylsulfoxide (DMSO) (DMSO) vehicle vehicle served served as a asnegative a negative control control and nordihydroguaiaretic and nordihydroguaiaretic acid acid(NDGA) (NDGA) as a positive as a positive control control [19]. Experiments [19]. Experiments were wereperformed performed three times, three times,independently. independently. Statistica Statisticall analyses analyses were performed were performed using one-way using ANOVA one-way ANOVAfollowed followedby a Dunnett by a post-hoc Dunnett test. post-hoc Both test.tested Both parabens tested parabensand the combination and the combination significantly signiﬁcantly inhibited inhibited17β-HSD2 17 (pβ ≤-HSD2 0.01). (p ≤ 0.01).

Additionally, effects effects of of parabens parabens and and paraben-like paraben-like compounds compounds on the on activity the activity of 17β of-HSD1 17β-HSD1 were wereanalyzed. analyzed. Small Smallparabens parabens such as such the as main the mainmetabolite metabolite p-hydroxybenzoicp-hydroxybenzoic acid, acid,methyl-, methyl-, and andethylparaben ethylparaben did not did inhibit not inhibit 17β-HSD1. 17β-HSD1. In contrast In contrast larger larger parabe parabensns size-dependently size-dependently inhibited inhibited 17β- 17HSD1β-HSD1 activity activity (Figure (Figure 6A).6A). Methyl Methyl vanillate, vanillate, ethy ethyll vanillate, vanillate, ethyl ethyl gallate gallate and and butyl butyl gallate diddid notnot inhibit 1717ββ-HSD1.-HSD1. ICIC5050 valuesvalues were were determined determined for for the the two two most most potent potent parabens parabens inhibiting inhibiting 17β-HSD1 17β- (FigureHSD1 (Figure6B). Hexyl- 6B). Hexyl- and heptylparaben and heptylparaben showed showed IC 50 ICvalues50 values of of2.6 2.6± ±0.6 0.6µ µMM andand 1.8 ± 0.30.3 µM,µM, respectively, inin 1717ββ-HSD1-HSD1 lysatelysate assays.assays. Intact Intact human human granulosa COV434 cells, endogenouslyendogenously expressing 17β-HSD1, were used to determinedetermine ICIC5050 values of hexylparaben (3.5 ±± 1.31.3 µM)µM) and heptylparaben (4.9 ± 1.01.0 µM),µM), indi indicatingcating that that parabens parabens are are able to penetrate cell membranes and inhibit the enzyme (Figure6 6C).C). The size-dependent structure-activity-relationshipstructure-activity-relationship of the tested parabens regarding 17β-HSD1 inhibition was additionally investigated using molecular docking experiments based on an existing crystal structure.structure. TheThe compoundscompounds positions positions in in the the active active site site were were predicted predicted in anin an orientation orientation typical typical for 17forβ -HSD117β-HSD1 inhibitors. inhibitors. The esterThe wasester coordinated was coordinated by a bifurcated by a bifurcated hydrogen bondhydrogen with thebond catalytically with the activecatalytically amino active acids amino Ser142 acids and Tyr155.Ser142 and On bothTyr155. other On sidesboth ofother this sides central of anchor,this central the anchor, ligand wasthe contactingligand was hydrophobiccontacting hydrophobic parts of the pa bindingrts of the site. bindingIn vitro site., the In activityvitro, the of activity the parabens of the increasedparabens withincreased the sizewith of the the size hydrophobic of the hydrophobic chain. Indeed, chain. extended Indeed, hydrophobicextended hydrophobic contacts werecontacts observed were forobserved the more for the active more compounds. active compounds. Additionally, Additionally, the orientation the orientation of the binding of the sitebinding was site variable. was Whilevariable. the While long chainthe long parabens chain parabens preferably preferably occupied occupied the hydrophobic the hydrophobic binding binding site containing site containing Phe259, mostPhe259, smaller most compounds smaller compounds were positioned were withpositioned the side with chain the into side the directionchain into of the cofactordirection NADPH of the (Figurescofactor 7NADPH and A1). (Figures 7 and A1). For comparison, wewe alsoalso calculatedcalculated cLogP cLogP values values and and compared compared them them to to the thein in vitro vitroinhibition inhibition of 17of β17-HSD1β-HSD1 (Table (Table1). 1).

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FigureFigure 6.6. ((AA)) Inhibition Inhibition of of 17 17β-HSD1β-HSD1 activity activity by byparabens. parabens. 17β-HSD1 17β-HSD1 activity activity was measured was measured in lysates in lysatesof transiently-transfected of transiently-transfected HEK-293 HEK-293 cells, cells, using using DMSO DMSO as asa negative a negative,, and and apigenin apigenin as as aa positive,positive, controlcontrol [[20].20]. Statistical Statistical analyses analyses were were performe performedd using using one-way one-way ANOVA ANOVA followed followed by a by Dunnett a Dunnett post- post-hochoc test. test.P-hydroxybenzoicP-hydroxybenzoic acid, acid, methylparaben, methylparaben, and and ethylparaben ethylparaben did didnot notsignificantly signiﬁcantly inhibit inhibit the theactivity activity of of17β 17-HSD1.β-HSD1. All All other other tested tested compou compoundsnds significantly signiﬁcantly inhibited inhibited 17β 17-HSD1β-HSD1 (p (≤p 0.001).≤ 0.001). (B) (BConcentration-dependent) Concentration-dependent inhibition inhibition of of 17 17ββ-HSD1-HSD1 by by hexyl- hexyl- and and heptylparaben. TheThe conversionconversion of of estroneestrone to to estradiol estradiol was was measured measured in in lysates lysates of of HEK-293 HEK-293 cells cells transfected transfected with with 17 17ββ-HSD1-HSD1 and and ( C(C)) in in intactintact human human COV434 COV434 granulosa granulosa cells ce endogenouslylls endogenously expressing expressing 17β-HSD1. 17β-HSD1. Results Results represent represent the mean the ±meanSD of ± threeSD of independentthree independent experiments. experiments.

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FigureFigure 7. 7. ((A,B) The The most most active active 17β 17-HSD1β-HSD1 inhibitor inhibitor heptylparaben heptylparaben docked docked into the into enzymes the enzymesactive activesite. site.(C,D) ( CAmino,D) Amino acids acidsof the ofbinding the binding site are site shown are shownin ball-and-stick in ball-and-stick style (white—carbon, style (white—carbon, red— red—oxygen,oxygen, blue—nitrogen, blue—nitrogen, yellow—sulfur, yellow—sulfur, green—phosp green—phosphor).hor). The cofactor The cofactor NADP NADP (NAP) (NAP) is colored is colored in insalmon pink. pink. The The docked docked parabens parabens are are shown shown in instick stick style. style. Inhibitors Inhibitors with with a smaller a smaller substituent, substituent, likelike benzylparaben, benzylparaben, can can alsoalso adopt adopt a a flipped ﬂipped position. position. Protein-ligand Protein-ligand interactions interactions are color-coded: are color-coded: red redarrow: arrow: hydrogen hydrogen bond bond acceptors; acceptors; yellow yellow sphere: sphere: hydrophobic. hydrophobic. The The ligand ligand binding binding site site surface surface is is coloredcolored by by aggregated aggregated hydrophilicity hydrophilicity (blue)/hydrophobicity (blue)/hydrophobicity (yellow). (yellow).

TableTable 1. 1.Correlation Correlation of of 17 17ββ-HSD-HSD inhibitioninhibition and cLogP cLogP values values of of tested tested parabens. parabens. Molecular 17β-HSD2 % 17β-HSD1 % Compound 17β-HSD2 % 17β-HSD1 % cLogP Compound MolecularWeight Weight (g) (g) Inhibition at 20 µM Inhibition at 20 µM cLogP Inhibition at 20 µM Inhibition at 20 µM p-hydroxybenzoic acid 138.12 0 ± 5 7 ± 11 0.86 p-hydroxybenzoic acid 138.12 0 ± 5 7 ± 11 0.86 methylparaben 152.15 50 ± 6 0 ± 7 0.95 methylparaben 152.15 50 ± 6 0 ± 7 0.95 ethylparabenethylparaben 166.18 166.18 74 74 ±± 4 4 7 7± ±6 6 1.34 1.34 propylparabenpropylparaben 180.20 180.20 37 37 ±± 6 6 37 37 ±± 5 5 1.73 1.73 isopropylparabenisopropylparaben 180.20 180.20 60 60 ±± 1 1 32 32 ±± 9 9 1.73 1.73 ± ± butylparabenbutylparaben 194.23 194.23 30 30 ± 7 7 52 52 ± 1010 2.12 2.12 isobutylparaben 194.23 53 ± 7 65 ± 4 1.97 phenylparabenisobutylparaben 214.22 194.23 53 64 ±± 7 6 65 35 ±± 4 1 1.97 2.38 hexylparabenphenylparaben 222.28 214.22 64 58 ±± 612 35 80 ±± 1 5 2.38 2.90 benzylparabenhexylparaben 228.25 222.28 58 64 ± ±126 80 67 ±± 5 9 2.90 2.23 heptylparabenbenzylparaben 236.31 228.25 64 76 ±± 6 3 67 83 ±± 9 6 2.23 3.29 heptylparaben 236.31 76 ± 3 83 ± 6 3.29 3. Discussion 3. Discussion Parabens have successfully been used as preservative additives for more than 50 years. However, in recentParabens years, following have successfully the detection been of parentused as paraben preservative compounds additives in female for more breast than tumors, 50 years. possible estrogenicHowever, effects in recent were years, extensively following investigated the detection [13]. Theof parent first weakparaben estrogenic compounds effects in of female parabens breast were reportedtumors, by possibleRoutledge estrogenic et al. using effects a yeast-basedwere extensiv estrogenely investigated receptor [13]. (ER) The assay first [21 weak]. Methyl-, estrogenic ethyl-, propyl-,effects andof parabens butylparaben were reported were found by Routledge to have et weak al. using estrogenic a yeast-based effects. estrogen Butylparaben receptor was (ER) the assay most [21]. Methyl-, ethyl-, propyl-, and butylparaben were found to have weak estrogenic effects. potent estrogenic paraben identified, but it was still 10,000 times less potent than estradiol. Furthermore, Butylparaben was the most potent estrogenic paraben identified, but it was still 10,000 times less Routledge et al. reported minor estrogenic effects of parabens in vivo. Subcutaneous application of potent than estradiol. Furthermore, Routledge et al. reported minor estrogenic effects of parabens in high doses of butylparaben (600–1200 mg/kg/day) significantly increased uterotrophic response vivo. Subcutaneous application of high doses of butylparaben (600–1200 mg/kg/day) significantly in rats. However, it was approximately 100,000 times less potent than the positive control estradiol increased uterotrophic response in rats. However, it was approximately 100,000 times less potent (0.4than mg/kg/day the positive). Thecontrol oral estradiol administration (0.4 mg/kg/day). of butylparaben The oral failed administration to increase of uterotrophic butylparaben response failed to [21 ]. Thisincrease observation uterotrophic may beresponse explained [21]. byThis highly observat abundantion may paraben be explained metabolizing by highly enzymes,abundant paraben located in the intestine and liver. Miller et al. showed estrogenic activity of benzyl-, butyl-, propyl-, ethyl-, Int. J. Mol. Sci. 2017, 18, 2007 8 of 13 and methylparaben in a yeast-based estrogen assay. Benzylparaben was reported to be the most active paraben despite being 4000-fold less potent than estradiol [22]. However, it is difficult to extrapolate in vivo data from mice and data from yeast-based assays to humans. Due to a rapid metabolism of parabens it is unlikely that estrogenic effects through direct estrogen receptor activation can cause harmful effects in humans. In the present study, we addressed an alternative mode-of-action for possible estrogenic effects of parabens and identified several compounds potentially interfering with local estrogen metabolism by inhibiting 17β-HSD1 and 17β-HSD2. Benzylparaben inhibited 65% of 17β-HSD2 activity at a concentration of 20 µM (Table1). The benzylparaben concentration used in the activity assays of this study was 100 times in excess to the substrate concentration. Thus, compared to the 4000-fold (EC50: benzylparaben = 80 µM) [22] lower effect of benzylparaben observed on ERα than estradiol (EC50: estradiol = 20 nM) [22], inhibition of 17β-HSD2 and subsequently elevated local estradiol concentrations are more likely to disturb the regulation of estrogenic target genes. This is somewhat more pronounced in the case of ethylparaben, showing very weak effects on ERα activity, but inhibiting 17β-HSD2 with an IC50 of 4.6 µM. Byford et al. reported estrogenic effects of methyl-, ethyl-, propyl-, and butylparaben in estrogen-dependent human breast cancer cell line MCF7 [23]. Parabens were able to competitively displace estradiol from binding to ERα in these cells. To displace estradiol from binding to its receptor a 1,000,000-fold molar excess of parabens was used. Additionally, parabens increased the proliferation of MCF7 cells, while in ERα receptor-negative MDA-MB-231 cells parabens showed no increase in proliferation at equal concentrations. In both cases butylparaben was the most active estrogenic paraben tested. Similar results were published by Okubo et al. showing ER-dependent proliferation of MCF7 cells treated with methyl-, ethyl-, propyl-, butyl-, isopropyl-, or isobutylparaben at concentrations that were 100,000 to 1,000,000 higher than estradiol [24]. However, the ERα-mediated estrogenic effects were not potent and it remains unclear whether these effects were indeed due to the activation of ERα or whether other pathways are involved. Our results suggest that the inhibition of 17β-HSD2 by parabens in cells endogenously expressing 17β-HSD2, and the subsequently elevated estradiol concentrations, should be considered in case of ethylparaben-mediated estrogenic effects. The main metabolite of parabens, p-hydroxybenzoic acid, was reported by Pugazhendhi et al. to be slightly estrogenic [25]. They reported that p-hydroxybenzoic acid competitively displaced estradiol from ERα at a 106 to 107-fold molar excess. Despite the fact that p-hydroxybenzoic acid is the main metabolite of all parabens and its presence in serum, the paraben concentrations needed to observe estrogenic effects are several orders of magnitude higher than the circulating concentrations, questioning the concerns about their potential estrogenic properties in human. Additionally, p-hydroxybenzoic acid is a highly hydrophilic compound with no evidence for bioaccumulation but rapid excretion via the urine [9]. Parabens were shown to inhibit sulfotransferases (SULTs) in human epidermal keratinocytes and skin cytosolic fractions and therefore block local estrogen conjugation and inactivation [26]. It was suggested that a chronic application together with a potential accumulation of parabens in the skin might lead to increasing local estrogen concentrations due to the inhibition of SULTs. Butylparaben was the most potent paraben tested with an IC50 of 37 ± 5 µM. Thus, parabens are thought to be rather weak inhibitors of SULTs; however, subcutaneous accumulation of parabens due to extensive use of dermally applied cosmetic products might actually lead to endocrine disruption due to the interference with estrogen sulfation by parabens. The inhibition of SULTs was the first study that showed estrogenic effects of parabens without direct modulation of ER activity. Parabens inhibiting SULTs and 17β-HSD2 both may exert pro-estrogenic effects by locally increasing estradiol concentrations. The IC50 of 4.6 µM for ethylparaben inhibiting 17β-HSD2 was eight times lower than that for butylparaben inhibiting SULTs, suggesting that estrogenic effects of parabens by inhibiting 17β-HSD2 may be more relevant than the inhibition of SULTs [26]; however, experimental differences and tissue- and cell-specific differences need to be taken into account when comparing effects by these different mechanisms. Int. J. Mol. Sci. 2017, 18, 2007 9 of 13

Since parabens are rapidly metabolized, they may interfere with other estrogen catabolic or conjugating enzymes, thereby leading to elevated estrogen levels. Several studies showed that parabens can easily penetrate rat [27], rabbit [28], and human skin [7]. Daily application of cosmetic products for one month led to an accumulation of methylparaben in the stratum corneum (SC) of the human fore arm. However, two days after stopping daily application, methylparaben was no longer detected [29] in the SC. These results indicate the possibility of local accumulation of parabens in the SC. However, only a daily and extensive use of products containing parabens will eventually lead to accumulation. Despite the rapid metabolism of parabens to p-hydroxybenzoic acid, original parent paraben compounds can be detected in human plasma [30], seminal plasma [30], urine [10], and milk [31]. It is supposed that the detection of systemic paraben concentrations is due to dermal, rather than oral, application of products containing parabens because of highly active intestinal and liver esterase activities [6,11]. This report showed a novel possible mode-of-action for parabens by interfering with local estrogen metabolism via inhibition of 17β-HSDs. Importantly, additive effects of parabens have been observed (Figure5). Often, a mixture of parabens is added to the cosmetic formulation to improve preservative efficiency. Studies showed that various parabens can be detected systemically, substantiating the importance of investigating potential additive effects [7], as we could show for 17β-HSD2 inhibition. Additive effects clearly increase their estrogenic potential and are important to take into account for further evaluation of the estrogenic potential of parabens. However, further investigations on additive and/or synergistic effects of different paraben mixtures are needed. Inhibition of 17β-HSD2 was not correlated to the molecular size or cLogP values as exemplified by the equal activity of ethylparaben and heptylparaben. This study suggests that depending on the tissue expression of 17β-HSD1 and 17β-HSD2, parabens might exert pro-estrogenic or anti-estrogenic effects. Additionally, in tissues expressing both enzymes, the estrogenic effect is likely to be cell-specific depending on the respective enzyme expressed, and the net tissue estrogenic effect might be minimal if a specific paraben or a certain paraben mixture is able to block both enzymes with similar efficiencies. Taking into account that smaller parabens (ethyl-, methyl- and propylparaben) are more commonly used as preservatives than larger parabens, disruption of estrogenic effects by parabens through inhibition of 17β-HSD2 are more likely to occur than inhibition of 17β-HSD1. In comparison, 17β-HSD1 inhibition by parabens was strongly dependent on the size and lipophilicity of the side chain (Table1). As a rule of thumb, the more and extensive hydrophobic contacts the paraben could form with the 17β-HSD1 binding site (Figure7), the more active was the compound. This context will aid in the future construction of an improved pharmacophore model for 17β-HSD1 inhibitors.

4. Materials and Methods

4.1. Cosmetic Products Database The CAS number of compounds approved as cosmetics ingredients in the European Union was collected from the European Commission homepage [32]. The structures of the compounds were retrieved from the Pubchem database [33] using PipelinePilot (Pipeline Pilot 9.1, Biovia Inc., San Diego, CA, USA, 2014). In case multiple structures were obtained for a CAS number, only the entry with the highest number of defined stereo-centers was kept. Salts and counter ions were removed using the wash molecules protocol in MOE, version 2011.10 (Molecular Operating Environment (MOE) 2011.10, Chemical Computing Group ULC, Montreal, QC, Canada). Discovery Studio, version 4.0 (Discovery Studio 4.0; Biovia Inc., San Diego, CA, USA, 2014) was employed to generate tautomers and isomers (in case stereo-centers were undefined) using the default settings. The 3D database was prepared for virtual screening by calculating conformational models for each molecule using LigandScout (LigandScout 3.03b, InteLigand GmbH, Vienna, Austia, 2014) and the build-in Omega [34] FAST option (max. 25 conformers per molecule). The final database consisted of 9045 unique entries represented by 16,939 structures including tautomeric and isomeric forms. Conformational Int. J. Mol. Sci. 2017, 18, 2007 10 of 13 analysis was successful for 16,553 compounds, which was the final number of entries in the virtual screening database.

4.2. Virtual Screening and Property Calculation The cosmetic products database was screened using LigandScout 3.03b with default settings. Standard physicochemical properties such as the cLogP value were calculated within this program, as well.

4.3. In Vitro Testing Human embryonic kidney cells (HEK-293, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modiﬁed Eagle’s medium solution (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Connectorate, Dietikon, Switzerland), 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES buffer (Gibco life technologies, Carlsbad, CA, USA) at pH 7.4, and 1% of non-essential amino acids solution (Sigma-Aldrich). Endogenous 17β-HSD1 activity assays were performed in intact COV434 cells (Sigma-Aldrich). ◦ Cells were cultured under standard condition (5% CO2, 37 C) in DMEM (Sigma-Aldrich) supplemented with 100 µg/mL streptomycin and 100 U/mL penicillin (Gibco Life Technologies), 10% FBS (Connectorate), and 2 mM L-glutamine (Sigma-Aldrich). Lysate activity assays were performed as previously described. Brieﬂy, HEK-293 cells (ATCC) were transiently transfected with expression constructs for human 17β-HSD1 or 17β-HSD2 using the calcium phosphate precipitation method. Lysates of HEK-293 cells expressing 17β-HSD1 or 17β-HSD2 were incubated at 37 ◦C for 10 min in TS2 buffer (100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM MgCl2, 20 mM Tris-HCl, pH 7.4), containing either the inhibitor at the respective concentration or solvent (0.2% DMSO). 17β-HSD1 activity was determined in the presence of 200 nM estrone, including 50 nCi of [2,4,6,7-3H]-estrone and 500 µM NADPH. Whereas, the 17β-HSD2 activity was performed in the presence of 200 nM estrone, including 50 nCi of [2,4,6,7-3H]-estradiol and 500 µM NAD+. To determine 17β-HSD1 activity in COV434 cells, cells were seeded (50,000) into 96-well plates and incubated for 90 min with 200 nM estrone, including 50 nCi of [2,4,6,7-3H]-estrone in serum-free charcoal treated media containing either the inhibitor at the respective concentration or solvent (0.1% DMSO). Both activity essays were stopped by adding a 1:1 ratio of 2 mM unlabeled estrone and estradiol in methanol. Radiolabeled steroids were obtained from Perkin-Elmer (Boston, MA, USA) whereas normal steroids and cofactors were purchased from Sigma-Aldrich. Estrogens were separated by thin layer chromatography and samples were analyzed using scintillation counting. All data were collected from at least three independent measurements.

4.4. Docking Molecular docking studies were performed using GOLD (Gold version 5.2, CCDC, Cambridge, UK) [35]. The protein data bank entry 3hb5 [36] was prepared using the protein preparation wizard. The water molecule 377 was set to “toggle and spin”, which means that it can be replaced by the ligand. ChemScore was used as scoring function. Docking validation was carried out by re-docking of the steroidal X-ray crystal structure ligand. To avoid conformational bias, the 2D structure of the ligand was generated using ChemDraw (ChemBioDraw version 14, PerkinElmer, Waltham, MA, USA, 2016), converted into a structure-data ﬁle (sd ﬁle) with Pipeline Pilot, and built up as a 3D starting structure using OMEGA. The root-mean-square error (RMSD) of the binding pose of the X-ray structure compared to the best-ranked docking structure was 0.920 Å.

5. Conclusions The present study identiﬁed several parabens inhibiting 17β-HSD1 and 17β-HSD2. Parabens can exert estrogenic effects by inhibiting 17β-HSD2. Inhibition of 17β-HSD2 prevents local inactivation of the active estrogen E2. All tested parabens and paraben-like structures, except the main metabolite Int. J. Mol. Sci. 2017, 18, 2007 11 of 13

p-hydroxybenzoicInt. J. Mol. Sci. 2017, 18 acid,, 2007 interfered with the activity of 17β-HSD2 at a concentration of 20 µM. The11 of most13 potent compounds had IC50 values in the low micromolar range and these parabens showed additive inhibitorylower concentrations effects. The than potential those activating estrogenic the effects ER or reportedinhibiting in SULTs this studyas previously were observed reported at[8,22– lower concentrations26]. Whereas most than of those the activatingtested parabens the ER were or found inhibiting to interfere SULTs with as previously 17β-HSD2 reportedactivity, thereby [8,22–26 ]. increasing local E2 concentrations, only larger parabens were found to inhibit 17β-HSD1. The most Whereas most of the tested parabens were found to interfere with 17β-HSD2 activity, thereby increasing frequently used parabens in cosmetic products are short parabens; therefore, inhibition of 17β-HSD1 local E2 concentrations, only larger parabens were found to inhibit 17β-HSD1. The most frequently by parabens used in cosmetic products is of minor relevance regarding health issues. Nevertheless, used parabens in cosmetic products are short parabens; therefore, inhibition of 17β-HSD1 by parabens this study revealed a size-dependent 17β-HSD1 inhibition by parabens, which could be explained by used in cosmetic products is of minor relevance regarding health issues. Nevertheless, this study extensive hydrophobic contacts in the enzyme’s binding site. revealed a size-dependent 17β-HSD1 inhibition by parabens, which could be explained by extensive hydrophobicAcknowledgments: contacts This in study the enzyme’s was supported binding by the site. Swiss Center for Applied Human Toxicology (2017/01/01– 2018/12/31) and the Swiss Federal Office of Public Health (FOPH) (2016/03/01–2016/12/31) (to Alex Odermatt) Acknowledgments:and the Austrian ScienceThis studyFund (P26782, was supported to Daniela by Schu thester). Swiss Daniela Center Schuster for Applied is an Ingeborg Human Hochmair Toxicology (2017/01/01–2018/12/31)Professor of the University and of Innsbruck. the Swiss We Federal thank Thie Officerry ofLanger, Public University Health (FOPH) of Vienna, (2016/03/01–2016/12/31) and InteLigand GmbH (to Alex Odermatt) and the Austrian Science Fund (P26782, to Daniela Schuster). Daniela Schuster is an Ingeborg for providing an academic license for LigandScout. Hochmair Professor of the University of Innsbruck. We thank Thierry Langer, University of Vienna, and InteLigand GmbHAuthor for Contributions: providing an academic Daniela Schuster license and for LigandScout.Alex Odermatt conceived and designed the experiments and wrote Authorthe paper. Contributions: Simona R. DanielaRohrer and Schuster Susanne and Leugger Alex Odermatt performed conceived all the andin vitro designed experiments. the experiments Roger T. and Engeli wrote thesupervised paper. Simona the experiments, R. Rohrer analyzed and Susanne data, provided Leugger Figures, performed and allwrote the thine paper. vitro experiments. Teresa Kaserer Roger compiled T. Engelithe supervisedcosmetic theproducts experiments, database analyzed and established data, provided the docking Figures, workflow. and wrote Anna the Vuorinen paper. Teresaand Sonja Kaserer Herdlinger compiled theperformed cosmetic productsthe virtual database screening and and establisheddocking experiments. the docking workflow. Anna Vuorinen and Sonja Herdlinger performed the virtual screening and docking experiments. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data in the writing of the manuscript; and in the of the study; in the collection, analyses, or interpretation of data in the writing of the manuscript; and in the decisiondecision to to publish publish the the results. results.

AppendixAppendix A A

FigureFigure A1. A1.Predicted Predicted protein-ligand protein-ligand interactionsinteractions of the tested parabens parabens with with the the 17 17β-HSD1β-HSD1 binding binding site (S-docking). site (S-docking).

Int. J. Mol. Sci. 2017, 18, 2007 12 of 13

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