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Disruption of steroidogenesis: Cell models for mechanistic

investigations and as screening tools

Alex Odermatt, Petra Strajhar, Roger T. Engeli

Published manuscript

Contribution: Carefully reviewed the section on Leydig cell culture models. Collected and

overviewed references about the most important Leydig cell models in the literature.

Aims: Provided an overview of all available gonadal and adrenal cell lines regarding their

suitability as screening tools for steroidogenic disruption by xenobiotics.

Results: This review demonstrates the species specific differences between rodent and human

cell lines, and highlights that steroidogenesis is altered in most cell lines due to their tumor origins.

Conclusion: The currently available cell lines are limited as screening tools for steroidogenesis.

Review

Disruption

of

steroidogenesis:

Cell

models

for

mechanistic

investigations

and

as

screening

tools

AlexOdermatt*,PetraStrajhar,RogerT.Engeli

SwissCenterforHumanToxicologyandDivisionofMolecularandSystemsToxicology,DepartmentofPharmaceuticalSciences,Pharmacenter,Universityof

Basel,Klingelbergstrasse50,4056Basel,Switzerland

ARTICLE INFO

Articlehistory:

Received8October2015

Receivedinrevisedform31December2015

Accepted20January2016

Availableonline22January2016

Keywords: Adrenal Testis Ovary Leydig Granulosa

Endocrinedisruptingchemical

Invitro

ABSTRACT

In themodernworld, humansare exposedduring theirwholelife toalargenumberofsynthetic chemicals.Someofthesechemicalshavethepotentialtodisruptendocrinefunctionsandcontributeto thedevelopmentand/orprogressionofmajordiseases.Everyyearapproximately1000novelchemicals, usedinindustrialproduction,agriculture,consumerproductsoraspharmaceuticals,arereachingthe market,often withlimited safetyassessmentregardingpotential endocrineactivities. Steroids are essential endocrinehormones,and theimportance of thesteroidogenesis pathwayasatargetfor endocrinedisruptingchemicals(EDCs)hasbeenrecognizedbyleadingscientistsandauthorities.Cell lineshaveaprominentroleintheinitialstagesoftoxicityassessment,i.e.formechanisticinvestigations andforthemediumtohighthroughputanalysisofchemicalsforpotentialsteroidogenesisdisrupting activities.Nevertheless,theusershavetobeawareofthelimitationsoftheexistingcellmodelsinorder toapplythemproperly,and thereis agreatdemandforimprovedcell-based testingsystems and protocols.Thisreviewintendstoprovideanoverviewoftheavailablecelllinesforstudyingeffectsof chemicalsongonadalandadrenalsteroidogenesis,theiruseandlimitations,aswellastheneedforfuture improvementsofcell-basedtestingsystemsandprotocols.

ã2016ElsevierLtd.Allrightsreserved.

Contents

1. Introduction ... 9 2. Steroidogenesis ... 10 3. Leydigcellmodelstoinvestigatesteroidogenesis ... 11 4. Cell-basedsystemstostudyeffectsofEDCsonovariansteroidogenesis ... 14 5. Adrenalcellmodelstoinvestigatedisruptionofsteroidogenesis ... 15 6. Conclusionsandoutlook ... 17 Acknowledgements ... 17 References... 17

1.Introduction

Thereisanincreasinginterestintheidentificationofchemicals thatinterferewiththeendocrinesystem.TheEndocrineSociety definesanendocrinedisruptingchemical(EDC)asan“exogenous chemicalormixtureofchemicalsthatcaninterferewithanyaspect of hormone action” [1]. It is important, in our opinion, to distinguishbetweentransientinfluencesfollowedbyadaptation

and disruptionofendocrinefunctionsleadingtoadversehealth effects.ThisisconsideredbytheEuropeanUnion(EU)thatdefines an EDC asan“exogenous substancethat causes adversehealth effectsinanintactorganism,oritsprogeny,secondarytochanges inendocrinefunction”[2,3].Theprotectionofhumanhealthand theenvironmentis ofhighpriorityformajor organizationsand regulatoryauthorities.Regardingthelargenumberofchemicals thatneedtobetestedforpotentialendocrinedisruptingeffects,in programssuchasREACH(Registration,Evaluation,Authorization andRestrictionofChemicals,http://ec.europa.eu/growth/sectors/ chemicals/reach/index_en.htm), the EPA’s EDSP (Environmental

* Correspondingauthor.

E-mailaddress:[email protected](A.Odermatt).

http://dx.doi.org/10.1016/j.jsbmb.2016.01.009

0960-0760/ã2016ElsevierLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Journal

of

Steroid

Biochemistry

&

Molecular

Biology

Protection Agency’s Endocrine Disruptor Screening Program,

http://www.epa.gov/endo/) or the FDA (U.S. Food and Drug Administration) guidelines for drug development (http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Gui- dances/), it is important to first evaluate the most relevant chemicals,i.e.chemicalswithevidenceofcausingadverseeffects andfor which relevant exposureis knownor canbe expected. Besides chemicals used in industrial production, agriculture, electronics,andconsumerproducts,thesafetyofpharmaceuticals andfoodconstituentsneedtobeassessed.Thus,ahugenumberof chemicalsneedtobetestedforawiderangeofpossibleadverse effects,includingsuchcausedbyadisruptionofsteroidhormone action.

Amongstotherendocrinehormones,steroidsplaycrucialroles in the regulation of nearly all physiological processes. Several reportsprovided evidencefor an associationofdisturbances of steroid hormone action caused by exogenous chemicals with developmentaldefects[4], infertilityand reproductivedysfunc- tions[5,6],testicular,prostateandbreastcancer[7–9],obesityand diabetes [10–12], immune disorders and neurobehavioral and learning dysfunctions [13,14]. Further research is needed to identifyother chemicals disrupting steroid hormone action, to evaluatethemechanismsbywhichsuchchemicalsdisruptsteroid hormoneaction,andtoassessthecriticalexposurewindowsand concentrations that are relevant regarding development and progressionofdiseases.

For the initialendocrinesafety testing ofa largenumber of chemicals,improved in silicoand in vitroassays are needed to facilitatetheprioritizationofchemicalsforfurthertoxicological investigations. Cell-based steroidogenesis assays represent a suitablestarting point toassessdisturbances ofsteroidbiosyn- thesis,inducedbydirectinhibitionofsteroidogenicenzymesorby affectingtheirexpression.Theadvantageofthecell-basedmodels isthatseveralenzymesandreceptorsrequiredforthesynthesisof steroids, as well as the signaling pathways regulating their activities,maybe covered in a single assay. In addition tothe identificationof potentiallyhazardouschemicals,thecell-based steroidogenesisassays allow first mechanistic insights intothe mode-of-actionofEDCs;however,theusersneedtobeawareof thelimitations ofthesystemappliedinordertoavoiddrawing inappropriateconclusionsandover-interpretationofresults.This review focuses on the cell lines that are available to study steroidogenesis,theiradvantagesandlimitations,andtheexisting

gaps for early safety testing of chemicals disrupting steroid homeostasis.

2.Steroidogenesis

Primaryorgansthatareproducingsteroidsfromtheirprecursor cholesterolincludetheadrenalglandsandthegonads,withtestes in males and ovaries in females. Additionally, in females the placentaproduceshighamountsofprogesteroneduringpregnan- cy[15].Otherorgansexpressingsteroidogenic enzymesinclude the brain [16,17], the intestinal tract [18] and the skin [19]. However, the steroids produced in these tissues seem to be restricted to affect local rather than systemic levels, and the relevanceofsteroidogenesisinthesetissueswillnotbediscussed. The major steroidogenic organs synthesize steroids de novo fromcholesterolthatiseitherproduceddirectlybythecellfrom acetyl-CoAor taken upfromdietarycholesterol boundtolow- densitylipoproteins(LDL)inthecirculation(foracomprehensive review see [20]). Cholesterol can be esterified, stored in lipid droplets and be released by the activity of hormone-sensitive lipase.Therate-limitingstepinadrenalandgonadalsteroidogen- esis is the uptake of cholesterol into the mitochondria. The steroidogenic acute regulatory protein (StAR) facilitates the transferofcholesterolfromtheoutertotheinnermitochondrial membrane,anditsconversiontopregnenolonebythecytochrome P450sidechaincleavageenzyme(P450scc,CYP11A1)incoopera- tion with adrenodoxin reductase that functions as an electron transfer protein of CYP11A1 [20]. Dependent on the organ, pregnenoloneisthenfurtherconvertedbytissue-andcelltype- specific enzymes into androgens, estrogens, glucocorticoids or mineralocorticoids.

Thecortexoftheadulthumanadrenalsisresponsibleforthe productionofmineralocorticoidsinthezonaglomerulosa,gluco- corticoids in the zona fasciculata and precursors of active androgens in the zona reticularis (Fig. 1). The zona reticularis expresses high levels of CYP17A1 [21], which possesses 17a- hydroxylaseactivityfortheformationof17a-hydroxypregneno- lone and 17,20-lyase activity for the subsequent formation of dehydroepiandrosterone (DHEA). The high expression of cyto- chromeb5,inthepresenceofcytochromeP450reductase,allows efficient17,20-lyaseactivitythatisneededfortheproductionof DHEA [20,22]. Additionally, the zona reticularis expresses high levelsofthesteroidsulfotransferaseSULT2A1thatisresponsible

for the formation of sulfated DHEA (DHEAS) [23], the most abundantsteroidinhumanblood [24].Importantly,3b-hydrox- ysteroid dehydrogenase 2 (3b-HSD2) is expressed in the zona reticularisatverylowlevels,thusleadingtoonlylowamountsof

D4-androstene-3,17-dione (androstenedione) production [20]. Since 17b-hydroxysteroid dehydrogenasetype 3 (17b-HSD3)is absentand17b-HSD5(AKR1C3)expressedatverylowlevelsinthe zonareticularis [21,25],only verylow levelsof testosteroneare producedbytheadrenals[26,27].CYP21A2isabsentinthezona reticularis, thus no mineralocorticoids and glucocorticoids are formedinthislayer[20].

In the zona fasciculata pregnenolone is converted to 17a- hydroxypregnenolone by CYP17A1, and pregnenolone and 17a-hydroxypregnenolone are converted to progesterone and 17a-hydroxyprogesterone,respectively,by3b-HSD2.Mostofthe progesteroneformedisalso17a-hydroxylated.Furthermetabo- lismbyCYP21A2leadsto11-deoxycortisolandloweramountsof 11-deoxycorticosterone that are further converted by CYP11B1, which is specifically expressed in this zone, into cortisol and corticosterone,respectively[20,28].Cytochromeb5isexpressedat backgroundlevelsinthezonafasciculata[21],resultinginverylow CYP17A1 17,20-lyase activity and thus low amounts of DHEA formation[20].Thezonafasciculataexpressesthemelanocortin-2- receptor and is therefore responsive to adrenocorticotrophic hormone(ACTH)[20,28].

ThezonaglomerulosadoesnotexpressCYP17A1,andpregnen- oloneisconvertedtoprogesteroneby3b-HSD2andfurtherto11- deoxycorticosterone by CYP21A2, and to corticosterone and aldosteronebyCYP11B2.Intheadrenals, CYP11B2expressionis restrictedtothezonaglomerulosaandtheproductionofaldoste- roneisregulatedbyangiotensinIIreceptors[20].

The humanfetal adrenalsproduce high amounts ofDHEAS, which is abolished soon afterbirth where theadrenals mainly consist of a zona glomerulosa and a zona fasciculata and thus produce mineralocorticoids and glucocorticoids [29]. The zona reticularis actively starts producing adrenal androgens at adre- narcheataround6–8yearsofageandreachingpeaklevelsinthe thirddecadeoflife,beforedeclininggradually[30,31].

Inthetestis, steroidogenesisisrestrictedtotheLeydig cells. TheyconvertpregnenolonebyCYP17A1into17a-hydroxyprege- nenolone and further to DHEA (Fig. 2). Because of the high expressionof3b-HSD2and17b-HSD3buttheabsenceofSULT2A1, DHEA is not sulfated and therefore further converted to androstenediol, or by a lower extent to androstenedione, and subsequentlytotestosteroneinLeydigcells[20,32].Furthermore, CYP21A2,CYP11B1and CYP11B2areabsent,thusnogluco-and mineralocorticoids are produced. Testicular steroidogenesis is under thecontrol of humanchorionicgonadotropin (hCG) and luteinizinghormone(LH).

In the ovaries, steroidogenesis is mediated by theca and granulosacells. Thegranulosacells arelocatedin theavascular cellularcompartmentsurroundingtheoocyte,andthethecacells reside in the ovarianstroma; these cellular compartments are separatedbythebasalmembrane.Thethecaandgranulosacells bothexpressStARandCYP11A1[33].Becausegranulosacellsdo notexpressCYP17A1[34],theycansynthesizepregnenolonefrom cholesterol and they convert it further to progesterone in the corpus luteum (Fig. 3) [20]. However, for the production of estrogens,pregnenoloneneedstobesecretedfromthegranulosa cellsandtakenupbythethecacells,oritisproduceddirectlyby thethecacells,toformDHEA.Thethecacellsexpress3b-HSD2and convertDHEAintoandrostenedione[35].Androstenedioneisthen deliveredbacktothegranulosacellsforthearomatase-dependent production of estrogens[34].Granulosa cells alsoexpress17b- HSD1,whichisneededfortheconversionofestroneintoestradiol. Therearecycle-dependentchangesinovariansteroidogenesis:in thelutealphasetheluteinizedgranulosacellsaresuppliedwith sufficient cholesterol, due to enhanced vascularization of the previouslyavascularcompartment,andelevatedLHlevelsenhance theexpressionofCYP11A1and3b-HSD2,resultinginthesynthesis of high amounts of progesterone [33]. In the follicular phase, follicle stimulating hormone (FSH) enhances the expression of aromataseand17b-HSD1fortheproductionofincreasedamounts of estradiol from theca cell-derived androstenedione. LH also activatesLHreceptorsonthecacellstoinduceCYP17A1expression, therebyenhancingandrogenprecursorsforestrogenproductionby granulosa cells. Thus, a tight control of the cooperation of granulosaandthecacellfunctionisessentialfortheappropriate regulationofestradiolsynthesis.

3.Leydigcellmodelstoinvestigatesteroidogenesis

Three independent large epidemiological studies revealed a declineinmaleserumtestosteronelevelsinthegeneralpopulation

[36–38].Obesitywasidentifiedasacontributingfactorforsome but notall observations[39].Increasingevidence suggeststhat exposurestoEDCscontributetomalereproductivediseasesand thatpreventionofEDCexposuresmayreducetheburdenofmale reproductivehealthproblems[40].Asanexample,cryptorchidism is a typical impairment following exposure to antiandrogenic chemicals during male sexual development[41]. Evidence was providedthatlevelsofpolybrominateddiphenylethers(PBDEs)in

humanbreastmilkareassociatedwithcongenitalcryptorchidism, althoughacontributionofotherenvironmentalfactorscannotbe excluded[42].PDBEshavebeenshownininvitrostudiestodirectly antagonizeARactivity(IC50ofapproximately5mMforthemixture DE-71 in an MDA-kb2 cell model expressing an AR-dependent luciferasereporter),and PDBEsadditivelyand/or synergistically actedwithotherARantagonisticcompounds[43].Ininvivostudies PDBEs were shown to cause diminished growth of androgen- dependenttissuesandadelayinpubertyinthemaleratfollowing apubertalexposureto60and120mg/kg/dayoftheDE-71mixture

[44].Althoughsuchhighexposurelevelsareunlikelytobereached inhumans,thefactthathumansareexposed toa multitudeof compounds that may exert additive or synergistic effects emphasizestheneedforthescreeningofchemicalsforpotential antiandrogenic effects. Because of the high public demand to reduceanimaltesting[45],improvedcell-basedassaysareneeded thatallow theidentificationofchemicalsdisruptingthebiosyn- thesisof steroidsand thegaining ofinsightsintothemode-of- actionofsuchchemicals.

There are several immortalized rodent Leydig cell lines available for studying the regulation of steroidogenesis and to assesstheimpactofsubstancesonsteroidhormoneproduction. However, to our knowledge, no human Leydig cell model is currentlyavailablethatcanbeusedforscreeningpurposesandfor toxicologicalstudies.TheavailablerodentLeydigcelllineshave been derived from spontaneous tumors, upon experimental induction,orbyinvitroimmortalization.Allofthesecellsystems havetheirlimitations,assomeofthesteroidogenicenzymesand regulatorypathwaysareexpressedatverylowlevels,ifatall,likely asaresultoftheselectionofcellclonesthatrapidlyproliferateand becauseofdedifferentiationandlossofinitialphenotypeduring prolongedcultivation.

ProbablythemostwidelyusedimmortalizedLeydigcelllineis MA-10[46].MA-10, therelatedMA-12,and thefrequentlyused mLTC-1 are all derived from a C57Bl/6 Leydig cell tumor (designatedM5480)[47].Thesecell linesexpress LH receptors, andincubationwithLH/hCGinducescAMP-dependentsteroido- genesis.MA-10cellsalsoexpressmouseepidermalgrowthfactor receptor(EGFR),whichsuppressesthehCG-inducedsteroidogen- esis[48].Inboth,MA-10andmLTC-1,progesteronewasthemain steroid being produced, in line with the observation that the originaltumorM5480secretedprogesterone butonly verylow amountsoftestosterone,andthetwocelllinesdisplayedsimilar functional characteristics [47]. These observations suggest that 3b-hsd1activityisdominantoverCyp17a1;therefore,pregneno- lone is mainly converted into progesterone, with only minor amounts being further converted into androstenedione and testosterone.Forthesereasons,weproposethat,usingprogester- oneasaread-out,MA-10andmLTC-1cellscanserveassuitable modelstodetectchemicalsthataffecttheinductionofsteroido- genesis,thecAMP-andPKA-dependentsignaling,orthatdirectly inhibit the activities of StAR, Cyp11a1 or 3b-hsd1. Due tothe generationofonlylowamountsoftestosteronebythesecelllines, itisdif_ficulttoquantitativelyassesstheeffectofchemicalsthat disruptCyp17a1or17b-hsd3activities.Nevertheless,themRNA expression of key steroidogenic enzymes, including StAR, Cyp11a1 and 3b-hsd1, and to a lesser extent that of Cyp17a1and17b-hsd3,hasbeendetected inMA-10andmLTC- 1 cells, and has been found to be affected upon exposure to chemicalmodulators[49–51].

MA-10 cells are appliedby many investigatorsto studythe impactofEDCsontheregulationofsteroidogenesis;onlyafewwill bementionedinthisreviewasexamples.Recentstudiesoneffects ofbisphenolA (BPA)and itsanalogsonsteroidogenesisinMA- 10cells suggestedthat tetrabromobisphenolA (TBBPA)concen- tration-dependently increased testosterone production at

concentrationsof3mMandhigher,whilebisphenolS(BPS)had no effect and BPA and bisphenol F (BPF) induced testosterone secretion only at very high concentrations (30 and 100mM, respectively)[49,50].Followingincubationofthecellsfor48hin the presence of 10mM of TBBPA, BPF or BPS an increased production of progesterone, and in the case of TBBPA of 17a- hydroxyprogesterone and androstenedione, was measured. Fur- thermore,incubationofcellswith10mMofBPF,BPSorTBBPAled to an elevated expression of 5a-reductase 1, indicating an increasedproductionof5a-androstanedioneanddihydrotestos- terone. Importantly, the authors provided evidence that the TBBPA-mediated increase in testosterone production may be duetoaninhibitionoftheeffluxofandrogenprecursorsrequired for testosterone synthesis by the multidrugresistanceproteins MRP1andMRP4[50].Theseobservationsemphasizetheneedto includesteroidtransportersintheassessmentofEDCsandprovide afurtherexplanationforthelowamountoftestosteroneproduced byMA-10cellsunderbasalconditions.

MA-10cells werealsoused tostudydirecteffects ofmono- phthalates on testicular steroidogenesis [52]. The LH-induced productionofcAMPandprogesteronewassignificantlyinhibited in MA-10 cells treated with 30mM of mono(2-ethylhexyl) phthalate(MEHP),whereastestosteroneproductionwassignifi- cantlylowereduponincubationofthecellswith1mMMEHP,3mM monobutylphthalate (MBP), 10mM mono-n-oxtylphthalate (MnOP) or 3mM monebenzylphthalate (MBeP) but not in the presenceofmonoethylphthalate(MEP)ormonomethylphthalate (MMP)[52].Atthehighconcentrationof100mMMEHPthemRNA expression levels of StAR, Cyp11A1 and Cyp17A1 were down regulated.Interestingly,inmLTC-1cells(notinducedbyLH)the phthalates di-n-butyl phthalate (DBP), MBP, di(2-ethylhexyl) phthalate (DEHP) and MEHP seemed to increase testosterone productionatlowconcentrationsof0.001to0.1mMbutinhibited at high concentrations of 100mM. Interestingly, the mRNA expressionlevelsofCyp11A1,Cyp17and3b-HSD1weredecreased evenatconcentrationsaslowas0.1mM[53,54].Also,theimpactof themajor metabolitesofMEHPand DEHPontheexpressionof steroidogenic genes has been analyzed, suggesting that the metabolite2-ethylhexanalmightinhibitLeydigcelltestosterone formation, although this effect was only observed at high