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]. 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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