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Roundup Inhibits Steroidogenesis by Disrupting Steroidogenic AcuteRegulatory (StAR) Protein ExpressionLance P. Walsh, 1 Chad McCormick, 1 Clyde Martin,2 and Douglas M. Stocco11Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, USA; 2Department ofMathematics, Texas Tech University, Lubbock, Texas, USA

Recent reports demonstrate that many currently used pesticides have the capacity to disruptreproductive function in animals. Although this reproductive dysfunction is typically characterizedby alterations in serum steroid hormone levels, disruptions in spermatogenesis, and loss of fertility,the mechanisms involved in pesticide-induced infertility remain unclear. Because testicular Leydigcells play a cmcial role in male reproductive function by producing testosterone, we used themouse MA-10 Leydig tumor cell line to study the molecular events involved in pesticide-inducedalterations in steroid hormone biosynthesis. We previously showed that the organochlorine insec-ticide lindane and the organophosphate insecticide Dimethoate directly inhibit steroidogenesis inLeydig cells by disrupting expression of the steroidogenic acute regulatory (StAR) protein. StARprotein mediates the rate-limiting and acutely regulated step in steroidogenesis, the transfer ofcholesterol from the outer to the inner mitochondrial membrane where the cytochrome P450 sidechain cleavage (P450scc) enzyme initiates the synthesis of all steroid hormones. In the presentstudy, we screened eight currendy used pesticide formulations for their ability to inhibit steroido-genesis, concentrating on their effects on StAR expression in MA-10 cells. In addition, we deter-mined the effects of these compounds on the levels and activities of the P450scc enzyme (whichconverts cholesterol to pregnenolone) and the 3p-hydroxysteroid dehydrogenase (3P-HSD)enzyme (which converts pregnenolone to progesterone). Of the pesticides screened, only the pes-ticide Roundup inhibited dibutyryl [(Bu)2]cAMP-stimulated progesterone production in MA-10cells without causing cellular toxicity. Roundup inhibited steroidogenesis by disrupting StAR pro-tein expression, further demonstrating the susceptibility of StAR to environmental pollutants.Key wordsk chemical mixtures, cytochrome P450 side chain deavage, environmental endocrinedisruptor, 313-hydroxysteroid dehydrogenase, Leydig cels, Roundup, steroid hormones, steroido-genesis, steroidogenic acute regulatory protein. Environ Health Perspect 108:769-776 (2000).[Online 12 July2000]ttp://ehpnetl.niehs. nih.govldocsf2000/1 08p769-776wa 7habstract btml

The biosynthesis of all steroid hormonesbegins with the cleavage of the side chain ofcholesterol to form pregnenolone. This reac-tion is catalyzed by the P450scc componentof the cholesterol side chain cleavage enzymesystem (CSCC) located on the matrix side ofthe inner mitochondrial membrane (1).Although this constitutes the rate-limitingenzymatic step in steroidogenesis, the truerate-limiting step is the delivery of cholesterolto the inner mitochondrial membrane andthe P450scc enzyme (2). Because the aqueousdiffision of cholesterol is extremely slow, cho-lesterol cannot readily diffuse to the innermitochondrial membrane at rates capable ofsustaining physiologically relevant levels ofsteroid production (3). To illustrate thispoint, maximal steroid production can beachieved in the absence of stimulation by pro-viding steroidogenic cells with water-solublecholesterol analogs, which can freely diffuse tothe inner mitochondrial membrane (4). Thusthere are mechanisms that mobilize choles-terol from cellular stores to the mitochondriaand which transfer cholesterol from the outerto the inner mitochondrial membrane.

Although the delivery of cholesterol fromcellular stores to the mitochondria is

essential to maintain maximal rates of steroidproduction, the intramitochondrial transferof cholesterol is the key hormonally regulat-ed step. Using protein synthesis inhibitorssuch as cycloheximide and puromycin,investigators have shown that hormone regu-lated steroid production requires rapid denovo protein synthesis (5). Furthermore,cycloheximide treatment, while permittingcholesterol accumulation in the outer mito-chondrial membrane of steroidogenic cells,almost completely blocks cholesterol move-ment to the inner mitochondrial membrane(6). Thus, a hormone-stimulated, rapidlysynthesized, cycloheximide-sensitive proteinis required to mediate the rate-limiting stepin steroidogenesis, the intramitochondrialtransfer of cholesterol.

Numerous studies have been performedto identify and characterize this acute regula-tory factor. Although several proteins havebeen proposed as the acute regulator[reviewed by Stocco and Clark (7?], one ofthese candidate proteins was first describedand characterized by Orme-Johnson et al.(8) as a mitochondrial phosphoprotein thatis rapidly synthesized in response to hor-mone stimulation in rat adrenal cells. Our

laboratory has described a similar protein inmouse MA-10 Leydig tumor cells and hassince purified, cloned, sequenced, andexpressed this protein and named it thesteroidogenic acute regulatory (StAR) pro-tein. The StAR protein fulfills all of the cri-teria of the putative acute regulatory factor(7,9,10). Perhaps the most compelling argu-ment for the role of StAR in steroidogenesiscomes from the finding that, in humans,mutations in the StAR gene cause the diseaselipoid congenital adrenal hyperplasia (lipoidCAH), a condition in which cholesterol andcholesterol esters accumulate and the new-born is unable to synthesize adequate levelsof steroid hormones. Furthermore, StARknockout mice have been generated, andtheir phenotype mirrors that of humanlipoid CAH (11). These observations indi-cate that StAR plays an indispensable role inthe transfer of cholesterol to the P450scc.

Because StAR protein mediates therate-limiting step in steroidogenesis, wehypothesized that, when compared to thesteroidogenic enzymes, StAR protein may beparticularly susceptible to modulation byenvironmental pollutants for a number ofreasons. First, unlike the steroidogenicenzymes that are chronically regulated andhave long half-lives (12), StAR protein is notan enzyme, is acutely regulated, and its activeprecursor form is highly labile. Second, StARprotein expression is critically dependent ontrophic hormone stimulation, making itsusceptible to xenobiotics that disrupt compo-nents of the trophic hormone signaling path-way. In contrast, with the exception ofcytochrome P450 1 7a-hydroxylase/ 17,20-lyase (P450c17), the steroidogenic enzymesretain near-normal steroidogenic enzymecapacity even in the absence of trophic hor-mone stimulation (12). Third, StAR medi-ates the rate-limiting step in steroidogenesis,

Address correspondence to D.M. Stocco,Department of Cell Biology and Biochemistry,Texas Tech University Health Sciences Center,Lubbock, TX 79409 USA. Telephone: (806) 743-2505. Fax: (806) 743-2990. E-mail: [email protected] thank D. Alberts for technical assistance.This work was supported by NIH grant

HD17481 to D. Stucco. L. Walsh was supportedby NIH grant T32-HD07271 and a scholarshipfrom the Lubbock Achievement Awards for CollegeScientists Chapter.Received 3 February 2000; accepted 11 April

2000.

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Articles * Walsh et al.

rendering steroidogenesis extremely sensitiveto disruptions in its expression. Conversely,with the exception of P450scc, which can belimiting, the steroidogenic enzymes are pre-sent in excess amounts (13). Fourth, becauseStAR functions upstream of steroidogenicenzyme activity, the effects of the xenobioticon steroidogenic enzyme activity may be oflittle importance if the xenobiotic also blocksStAR protein expression. Finally, we recentlyshowed that two pesticides, the organochlo-rine insecticide lindane (Sigma, St. Louis,MO), and the organophosphate insecticideDimethoate (BASF Corp., AgriculturalProducts Group, Research Triangle Park,NC), both of which lower serum testos-terone levels in animals, block steroidhormone biosynthesis in Leydig cells byreducing StAR protein expression (14,15).These findings raise the possibility that otherpesticides may also inhibit steroidogenesis bytargeting StAR expression.

Several currently used pesticides disruptsteroid hormone levels and/or reproductivesystem function in animals (16-19). Onebillion pounds of active ingredients and sev-eral times this amount of inert ingredientsare used annually in the United States alone;therefore, the possibility that these com-pounds can affect the reproductive health ofhumans and wildlife in their natural habitatsis of great concern (20). Little information isavailable regarding the effects of pesticides,including Ammo (Zeneca AgriculturalProducts, Wilmington, DE) and Ambush(Zeneca Agricultural Products) and the her-bicides Banvel (Sanex, Inc., Burlington,Ontario, Canada), Cotoran (Ciba-GeigyCorporation, Greensboro, NC), Cyclone(Zeneca Agricultural Products), Fusilade(Zeneca Agricultural Products), Dual (Ciba-Geigy), and Roundup (Monsanto Co., St.Louis, MO) on endocrine system function,despite their widespread use. Therefore, thepresent study was performed to determine ifthese pesticides can disrupt steroid hormonebiosynthesis in the mouse MA-10 Leydigtumor cell line and to determine the site ofsteroidogenic inhibition.

Materials and MethodsChemicals. We purchased Waymouth's MB752/1 medium, horse serum, gentamicinsulfate, lyophilized trypsin-EDTA, phos-phate-buffered saline with Ca2+ and Mg2+(PBS+), and phosphate-buffered saline with-out Ca2+ and Mg2+ (PBS-) from Gibco LifeTechnologies (Gaithersburg, MD). Weobtained [1,2,6,7-N3-H(N)]-progesterone[specific activity (SA) 97 Ci/mmol] and [7-3H(N)]-pregnenolone (SA 21 Ci/mmol)from New England Nuclear (Boston, MA).Antibodies to progesterone were obtainedfrom Holly Hills Biological (Hillsboro, OR).

SU 10603, cyanoketone, and antibodies topregnenolone were generously provided by F.Rommerts, Erasmus University (Rotterdam,The Netherlands). We obtained Percoll andDextran T70 from Pharmacia Fine Chemicals(Uppsala, Sweden). Nunc cell culture dishes,charcoal (Norit), trichloroacetic acid, scinti-verse BD and sodium bicarbonate wereobtained from Fisher Scientific (Houston,TX). Acrylamide, bis-acrylamide, and SDSwere purchased from Bio-Rad (Hercules,CA). Bovine serum albumin (BSA), dibutyryl[(Bu)2]cAMP, 22(R)-hydroxycholesterol(22R-HC), pregnenolone, and progesteronewere purchased from Sigma. We purchasedrabbit antisera to amino acids 88-98 ofmouse StAR protein from Research Genetics(Huntsville, AL) and rabbit antisera to aminoacids 421-441 of rat P450scc enzyme fromChemicon (Temecula, CA). Antisera to puri-fied mouse 3p-hydroxysteroid dehydrogenaseI (3f-HSD) was a generous gift from A.Capponi, University of Geneva (Geneva,Switzerland). We purchased horseradish per-oxidase conjugated goat antimouse IgG fromAmersham (Arlington Heights, IL). StARcDNA was previously cloned in our laborato-ry (21). Bovine P450scc cDNA was a gener-ous gift from M. Waterman, VanderbiltUniversity (Nashville, TN); mouse 3p-HSDI cDNA was a generous gift from A. Payne,Stanford University (Stanford, CA); andmouse 18S rRNA cDNA was a generous giftfrom G. Cornwall, Texas Tech UniversityHealth Sciences Center (Lubbock, TX).

Pesticide formulations selected for studyincluded:* Ammo (300 g/L cypermethrin): (R,S)-o-cyano-3-phenoxybenzyl(1 RXS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclo-propanecarboxylate

* Ambush (240 g/L permethrin): 3-phe-noxybenzyl( 1 R,S)-cis, trans-3-(2,2-dichlorovinyl) -2 ,2-dimethylcyclo-propanecarboxylate

* Fusilade (120 g/L fluazifop-p-butyl): (R)-2-[4-(5-trifluoromethyl-2-pyridyloxy)phe-noxy]propionic acid

* Cyclone (240 g/L paraquat): 1,1'-dimethyl-4,4'-bipyridinium

* Roundup (180 g/L glyphosate): N-(phos-phonomethyl) glycine

* Banvel (480 g/L dicamba): 3,6-dichloro-o-anisic acid

* Cotoran (480 g/L fluometuron): 1,1-dimethyl-3-(a,a,a-trifluoro-m-tolyl) urea

* Dual (958 g/L metolachlor) 2-chloro-6'ethyl-N-(2-methoxy- 1 -methylethyl)acet-o-toluidine.

Glyphosate and X- and y-HCH wereobtained from Sigma.

MA-JO cell culture. The mouse MA-10Leydig tumor cell line was a generous giftfrom M. Ascoli, University of Iowa College

of Medicine (Iowa City, IA). We maintainedcells in Waymouth's MB 752/1 medium +15% horse serum at 37°C, 5% C02, asdescribed previously (22). For dose-response,time-course, steroidogenic enzyme activity,reversibility, and mixture studies, 75,000 cellswere seeded into each well of a 96-well plateand grown overnight. For nuclear run-onanalysis, 50 x 106 cells were seeded onto 25 x25 cm tissue culture dishes and grownovernight. For the remaining studies, 1.5 x106 cells were plated into 100-mm culturedishes and grown until 80% confluent. For allexperiments, medium was removed, cells werewashed twice with PBS+, and serum-freeWaymouth's medium containing the appro-priate treatment was placed on the cells.

Treatment of cells. We stimulated MA-10 cells using a maximal stimulatory dose of(Bu)2cAMP (1 mM). In some studies, opti-mal concentrations of 22R-HC (25 pM) orpregnenolone (10 pM) were provided as asteroidogenic substrate. All treatments wereperformed in serum-free media. Final con-centrations of DMSO and ethanol used aschemical solvents were < 0.4 %.

Dose-response and time-course studies.We stimulated MA-10 cells with (Bu)2cAMPin the presence or absence of the appropriatexenobiotic for 2 hr (in dose-response stud-ies), or 4 hr (in time-course studies), and wemeasured steroid levels and total protein syn-thesis. We calculated the concentration thatinhibits 50% (IC50) values as the slope ofthe linear regression line obtained fromEadie/Hofstee plots of steroidogenesisdose-response data. For steroid determina-tion in Roundup-treated cells, each datapoint is the average ± SE of the means fromat least three separate experiments in whichtreatments were performed in quadruplicate.For progesterone production in cells treatedwith other pesticides, each data point is themean ± SE of four replicates in a singleexperiment that was repeated once.

Radioimmunoassay (RIA). We quanti-fied progesterone by RIA as previouslydescribed (23). Standard curves were preparedin serum-free Waymouth's medium. Analysisof RIA data was performed using a computerprogram specifically designed for this pur-pose. Data are expressed as nanograms permilliliter media.

Determination of total cellular proteinsynthesis. To determine the effects of com-pounds on total protein synthesis, cells weretreated as described previously with theinclusion of 5 pCi/mL Expre35S35S proteinlabeling mix (SA 1,000 Ci/mmol; NewEngland Nuclear). We determined total pro-tein content using a modification of theBradford method (24) on identically platedcells that were not treated with Expre35S35S.After treatment, medium was removed and

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Articles * Roundup disrupts StAR protein expression

cells were solubilized for 2 hr in 0.25 MNaOH at 37°C. Next, we added an equalvolume of cold 20% trichloroacetic acid(TCA) and protein was precipitatedovernight at 40C. TCA-precipitable materialwas transferred onto glass fiber filters using a1225 sampling manifold (Millipore,Bedford, MA), rinsed with 5% TCA, dried,and counted in a liquid scintillation counter.Results were reported as counts per minuteper milligram protein (2 or 4 hr). Each datapoint is the mean ± SE of four replicates in asingle experiment, which was performedthree times.

Determination ofP450scc and 3f3-HSDactivity and reversibility. We determined theeffects of xenobiotics on the combined activ-ities of the P450scc and 3p3-HSD enzymesby adding 22R-HC to MA-10 cells in thepresence or absence of the xenobiotic for 2hr and measuring progesterone production.To determine reversibility, cells were thenrinsed with PBS+, allowed to recover for 24hr in serum-containing medium, and incu-bated again for 2 hr with (Bu)2cAMP and/or22R-HC. Progesterone in the medium wasthen measured. To evaluate P450scc enzymeactivity, 22R-HC was provided as substrateto MA-10 cells in the presence and absenceof the appropriate xenobiotic as well ascyanoketone and SU 10603, inhibitors of30-HSD and P450c17, respectively, for 2hr, and pregnenolone in the medium wasmeasured. To evaluate 3f3-HSD enzymeactivity, pregnenolone was provided as sub-strate, and MA-10 cells were treated in thepresence and absence of the xenobiotic for 2hr, and progesterone in the media was mea-sured. Each data point represents the average± SE of the means from at least three sepa-rate experiments in which treatments wereperformed in quadruplicate.

Isolation of mitochondria and Westernblot analysis. MA-10 cells were stimulatedwith (Bu)2cAMP in the presence or absenceof Roundup for 4 hr, and progesterone inthe media was measured by RIA. We isolat-ed mitochondria by homogenization of thecells followed by differential centrifugation(21). Western blot analysis of mitochondrialprotein was performed as previouslydescribed (25). After detection of StAR,membranes were stripped in 62.5 mM Tris-HCI (pH 6.8), 2% SDS, and 100 mM ,B-mercaptoethanol at 70°C for 30 min,washed in 10 mM Tris-HCI (pH 7.4) and150 mM NaCl twice for 10 min, and thensuccessively probed with P450scc or 3p-HSD antisera. The bands of interest werequantitated using a Biolmage Visage 2000(Biolmage Corp., Ann Arbor, MI) imagingsystem. Values obtained were expressed asintegrated optical density units, as previouslydescribed (26). Each data point represents

the average ± SE of the means from threeseparate experiments in which treatmentswere performed in triplicate.

Isolation ofRNA and Northern blotanalysis. Cells were treated as described forWestern blot analysis. The media wereretained and progesterone was measured byRIA. We isolated total RNA using Trizolreagent (Gibco BRL, Grand Island, NY)according to the manufacturer's protocol.RNA was quantitated and resuspended inRNA sample buffer (0.1 x borate buffer,48% formamide, 6.4% formaldehye, 5.3%glycerol, and 0.27% bromophenol blue). Weperformed Northern blot analysis as previ-ously described (27). We loaded 20 pg totalRNA into each well. Labeling of cDNAprobes for mouse StAR, P450scc, 3,B-HSD,and 18S rRNA was achieved by randompriming (Prime-It II; Stratagene, La Jolla,CA) using [a-32P] dCTP (SA 3,000Ci/mmol; New England Nuclear) accordingto the manufacturer's protocol. Afterhybridization, the blots were washed twice in2 x standard saline citrate (SSC), 1% SDS atroom temperature for 30 min and once in0.1 x SSC, 0.1% SDS at 65°C for 30 min.After Northern blot analysis with StARcDNA, blots were stripped by washing twicein 0.1 x SSC, 1% SDS at 65°C for 30 min,and then successively probed with P450scc,3p-HSD, and 18S rRNA cDNA. We quan-titated the bands of interest, and valuesobtained were expressed as described previ-ously. Each data point represents the average± SE of the means from three separate exper-iments in which treatments were performedin triplicate.

Isolation of nuclei. MA- 10 cells werestimulated with Bu2cAMP in the presence orabsence ofRoundup for 4 hr. After treatment,cells were harvested with a rubber policemanand centrifuged for 5 min at 500 x g, 4°C.The cell pellet was resuspended in ice-coldsucrose I buffer (0.32 M sucrose, 3 mMCaCI2, 2 mM magnesium acetate, 0.1 mMEDTA, 1 mM DTT, 0.5% v/v NonidetP-40, 10 mM Tris-HCI, pH 8.0) andhomogenized with 5 strokes of a Douncehomogenizer. To verify that nuclei were freeof cytoplasmic tags, we inspected them usingan Olympus IMT-2 inverted microscope(Dexter Instrument Co., San Antonio, TX).Then the homogenate was layered onto asucrose cushion consisting of sucrose buffer II(2 M sucrose, 5 mM magnesium acetate, 0.1mM EDTA, 1 mM DTT, 10 mM Tris-HCl,pH 8.0) and centrifuged for 45 min at 30,000x g, 40C. The supernatant was discarded andthe pellet containing nuclei was resuspendedin ice-cold glycerol storage buffer (40% v/vglycerol, 5 mM MgCl2, 0.1 mM EDTA, 50mM Tris-HCl, pH 8.3), frozen on dry ice,and stored in liquid nitrogen.

Nuclear run-on analysis. Nuclei werethawed at room temperature. An equal vol-ume of 2 x reaction buffer (5 mM MgCl2,0.3 M KCl, 5 mM DTT, 10 mM Tris-HCl,pH 8.0) containing 50 jCi/mL [a-32P] uri-dine triphosphate (SA 3,000 Ci/mmol; NewEngland Nuclear), and 0.5 mM cold ATP,guanosine triphosphate, and cytidine triphos-phate (Clontech, Palo Alto, CA) was added.Transcription complexes were elongated byincubating samples in a shaking water bathfor 30 min at 30°C. After incubation, thereaction mixture was digested successivelywith 40 pg/mL DNAse I for 5 min at 30°Cand 160 pg/mL proteinase K for 30 min at42°C. DNAse I and proteinase K wereobtained from Sigma. RNA was extractedwith 5:1 phenol-chloroform, pH 4.3 (FisherScientific), precipitated by the addition of10% TCA, collected onto 0.45 pm MilliporeHA nitrocellulose filters using a vacuummanifold, and rinsed free of unincorporatednucleotides with 5% TCA. RNA capturedonto filters was treated with 25 pg/mLDNAse I for 30 min at 370C, eluted fromfilters with elution buffer (1% SDS, 5 mMEDTA, 10 mM Tris-HCl) for 10 min at65°C, and treated with 30 pg/mL proteinaseK for 30 min at 37°C. The resultant mixturewas extracted with 5:1 phenol-chloroform,pH 4.3, and subjected to a 10-min digestionon ice with 0.200 M NaOH before quench-ing the reaction with 0.290 M Hepes. Weprecipitated RNA by adding 1/10 (v/v) 3 Msodium acetate and 2.5 vol 100% ethanolthen centrifuged it for 30 min at 10,000 x g40C. The resultant RNA pellet was resus-pended in water and an aliqout was countedusing a liquid scintillation counter. We usedequal numbers of nuclei in the in vitro tran-scription assay. Equal counts of RNA werehybridized to target StAR, P450scc, and 18ScDNA inserts and linearized, empty pCMV-5 vector previously immobilized to nylonmembranes (Hybond N+) using a Bio-DotSF microfiltration apparatus (Bio-Rad,Hercules, CA) according to the manufactur-er's protocol. Prehybridization andhybridization were performed under thesame conditions described for Northernblots. We detected radioactivity usinga Phosphorimager 445 SI (MolecularDynamics, Sunnyvale, CA). Signals werequantitated using ImageQuant version 4.1software (Molecular Dynamics) in volumemode, which integrates the intensity ofeach pixel within the defined area. Valueswere obtained as arbitrary units. Each datapoint represents the average ± SE of fiveseparate experiments.

Protein kinaseA (PK4) activity determi-nation. Cells were treated and homogenizedas described in "Isolation of Mitochondriaand Western Blot Analysis." We measured

Environmental Health Perspectives * VOLUME 108 1 NUMBER 8 1 August 2000 771


PKA activity with the SignaTECT cAMP-dependent protein kinase assay system(Promega, Madison, WI), as described in themanufacturer's protocol. Three separateexperiments were performed in which treat-ments were performed in triplicate.

Mixture studies. We stimulated cellswith (Bu)2cAMP in the presence or absenceof the indicated pesticides for 2 hr and wemeasured progesterone. Each data point rep-resents the average ± SE of the means fromthree separate experiments in which treat-ments were performed in triplicate.

Statistical analysis. Statistically significantdifferences were determined by one-way analy-sis of variance and Fisher-protected least-square difference multiple comparison usingthe software program Statview SE + Graphics(Abacus Concepts, Inc., Berkeley, CA).

ResultsProgesterone production and total cellularprotein synthesis. Initial studies were per-formed to determine the effects of severalpesticide formulations on steroidogenesisand total protein synthesis (Figure 1).Unlike the other pesticides, Roundupdecreased progesterone production in adosage-dependent manner (IC50 = 24.4 ±0.67 pg/mL) without inducing a paralleldecrease in total protein synthesis, indicat-ing that this herbicide did not cause acutecellular toxicity or a general disruption intranslation. Because 25 pg/mL Roundupsignificantly (p < 0.01) reduced steroidogen-esis by 65% without affecting total proteinsynthesis, we chose to use this dose for theremaining studies. As Table 1 shows,Roundup also significantly (p < 0.001) dis-rupted steroidogenesis over time withoutinducing a parallel decrease in total proteinsynthesis. Interestingly, the active ingredientin Roundup, glyphosate, did not altersteroidogenesis or total protein synthesis atany dose tested (0-100 gg/mL; datanot shown). These studies indicate thatRoundup may have targeted specific com-ponents of the steroidogenic pathway toinhibit steroid production.

P450scc and -30-HSD enzyme activity,expression, and steroidogenesis. To determineif the inhibitory effect of Roundup on(Bu)2cAMP-stimulated progesterone pro-duction might be due to an inhibition of theactivities of the steroidogenic enzymes,P450scc and/or 3p-HSD, 22R-HC was pro-vided as a substrate and cells were treated for2 hr with Roundup. We used 22R-HC,which can readily diffuse to the P450sccenzyme, bypassing the need for StAR-medi-ated cholesterol transfer, to determinesteroidogenic capacity. Although Roundupsignificantly (p < 0.01) reduced (Bu)2cAMP-stimulated steroidogenesis by 84%,

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To determine if Roundup specificallydisrupted 3i-HSD, P450scc, or bothsteroidogenic enzyme activities, both preg-nenolone-driven progesterone production (ameasure of 33-HSD activity) and 22R-HC-driven pregnenolone production (a measureof P450scc activity) were measured after her-bicide treatment for 2 hr (Figure 3). AlthoughRoundup did not alter 30-HSD enzymeactivity, indicating that the herbicide was notacutely toxic to cells or mitochondria, it

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Table 1. Time-course study of the effects of Roundup on progesterone production and total cellular pro-tein synthesis in MA-10 cells.

2hr 4hrProgesterone Protein synthesis Progesterone Protein synthesis

(ng/mL)* (cpm/mg x 104) (ng/mL)* (cpm/mg x 104)Control 1.92±0.21 56±5.9 2.8±0.42 102±6.5Bu2cAMP (1 mM) 126 ± 20.3 65 ± 4.9 253 ± 13.1 94± 1.5Roundup (25 pg/mL) 21 ± 5.0 40 ± 2.3 47 ± 7.8 75 ± 2.2*For progesterone production at 2 and 4 hr, the difference between (Bu)2cAMP and Roundup ± (Bu)2cAMP was statisti-cally significant (p < 0.001).

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significantly (p < 0.001) reduced P450sccactivity by 61%.

To determine if the decrease in P450sccenzyme activity might have been due to areduction in the levels of this enzyme, and toconfirm that 3p-HSD enzyme levels werenot affected, we determined the effects ofRoundup on the expression of these enzymes.Although this herbicide significantly (p <0.001) blocked steroidogenesis by 94%(Figure 4), Western blot analysis of mito-chondrial protein revealed that it did notalter P450scc or 3p-HSD enzyme proteinlevels (Figures 5 and 6). Moreover, Northernblot analysis also revealed that it did notaffect P450scc mRNA levels (Figure 5).Interestingly, Roundup significantly (p <0.05) reduced 3p-HSD mRNA levels by33% (Figure 6). Because a reduction inP450scc activity alone cannot account forthe observed level of steroidogenic inhibi-tion, the data suggest that this herbicide alsoblocked steroidogenesis before the P450sccenzyme, potentially by reducing cholesterolavailability.

StAR protein and mRNA levels. BecauseStAR protein mediates the transfer of choles-terol to the inner mitochondrial membrane,we determined the effects of Roundup onthe expression of this protein. Western blotanalysis revealed that Roundup significantly(p < 0.01) reduced StAR protein levels by90% (Figure 7). Because StAR levels were

reduced in proportion to steroid levels, andStAR functions upstream of the steroido-genic enzymes, a reduction in StAR proteinlevels alone could account for the observedlevel of steroidogenic inhibition.

To determine if Roundup reduced StARprotein levels by decreasing StAR mRNAlevels, we performed Northern blot analysis(Figure 7). StAR mRNA consists of the 1.6,2.7, and 3.4 kb transcripts, which make up18, 10, and 72%, respectively, of total StARmRNA. Northern blot analysis revealed thatRoundup did not alter StAR mRNA levels,indicating that Roundup disrupted StARprotein expression post-transcriptionally.

Although the importance of the threeStAR transcripts is unknown at this time,Roundup preferentially increased levels ofthe 1.6 and 2.7 StAR transcripts (Figure 7).In fact, this herbicide significantly (p < 0.05)increased levels of the 1.6 and 2.7 kb tran-scripts by 2- and 2.3-fold, respectively.

Roundup may have increased StAR tran-script levels by increasing the rate of StARgene transcription, altering the post-tran-scriptional expression of StAR (e.g., increas-ing StAR transcript stability), or by causing acombination of the two processes. As Figure8 shows, Bu2cAMP increased the rate ofStAR gene transcription 5-fold. However,when cells were stimulated with Bu2cAMPin the presence of Roundup, StAR transcrip-tion increased 8-fold, indicating that

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Figure 2. Effects of Roundup on P450scc and 3p-HSD enzyme activity and steroidogenesis in MA-10 cells.(A) Effects of 2-hr treatment with Roundup on progesterone production. The difference between(Bu)2cAMP and Roundup + (Bu)2cAMP was statistically significant (p < 0.01). (8) Effects of 2-hr treamentwith Roundup on progesterone production after a 24-hr recovery.

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pregnenolone

Figure 3. Effects of Roundup on P450scc and 3,B-HSD enzyme activity. (A) Effects of Roundup on 3p-HSDenzyme activity. (B) Effects of Roundup on P450scc enzyme activity.*Statistically significant differences (p < 0.001).

Roundup may have increased StAR tran-script levels by increasing their synthesis.

PKA activity. A reduction in PKA activi-ty might partly explain the observed reduc-tion in StAR expression and steroidogenesis.To determine if Roundup disrupts PKAactivity, cells were treated for 4 hr. Roundupdid not affect the ability of PKA present incell lysates to phosphorylate the PKA-specif-ic substrate (data not shown), demonstratingthat Roundup inhibited StAR proteinexpression distal to PKA activation.

Effects of a mixture ofpesticides onsteroidogenesis. We previously showed thatDimethoate and cx-, 6-, and y-HCH (lin-dane) inhibit steroidogenesis primarily bydisrupting StAR protein expression (14,15).However, unlike Roundup, these com-pounds reduced StAR expression primarilyby reducing StAR mRNA levels. As shown inFigure 9, when tested individually at concen-trations that were not maximally inhibitory,each pesticide inhibited steroidogenesis by25%. However, when pesticides were testedtogether in a mixture at the same concentra-tions, they inhibited steroidogenesis by only50%, indicating that components in the mix-ture may have interacted antagonistically.

DiscussionWe previously showed that lindane andDimethoate inhibited steroidogenesis by dis-rupting StAR protein expression (14,15).Although these pesticides likely impacteddifferent cellular pathways that regulate theexpression of StAR, they impinged on thesteroidogenic pathway at the level of StARprotein. Therefore, we hypothesized that adisruption in StAR expression might alsoaccount for the reduction in steroidogenesisfollowing treatment with other currentlyused pesticides. In support of this hypothe-sis, the present study showed that Roundupdecreased steroidogenesis by disrupting StARexpression post-transcriptionally. AlthoughStAR has largely been overlooked as a targetfor environmental pollutants in the past, the

400

20

_ 300

11

100 ..... Con,r i

O-

conirol Bu2cAMP Roundup

Figure 4. Effects of Roundup on progesterone pro-duction in MA-10 cells. Cells grown in 100-mmplates were treated as described for Western andNorthern blot analysis in "Materials andMethods."*Statistically significant differences (p < 0.001).

Environmental Health Perspectives * VOLUME 108 1 NUMBER 8 1 August 2000 773


+~~~~~~~~~-K P450sscc00 - - - + P450scc

ch8 + 1 8 S~~~~~~~~~~~~~~~~~~~~~~~~8.! 6 8~~~~~~~~~~~~~~~~~~~~~1.5

r0.5Control Bu2cAMP Roundup Control Bu2cAMP Roundup

Figure 5. Effects of Roundup on P450scc enzyme and mRNA levels. (A) Western blot analysis. (Upperpanel) A representative Western blot; (lower panel) quantitation of immunospecific bands for the P450sccenzyme. (B) Northern blot analysis. (Upper panel) Representative Northern blots for P450scc mRNA and18S rRNA; (lower panel) bands for P450scc mRNA and 18S rRNA were quantitated and data expressed asP450scc mRNA/18S rRNA.

kDa

4742 30HSD

TI T

.-0 3lHSD

.4- 18S

1.5aWVb 1.0

CO 0.5CCL

Control Bu2cAMP Roundup

Figure 6. Effects of Roundup on 3,B-HSD enzyme and mRNA levels. (A) Western blot analysis. (Upperpanel) A representative Western blot; (lower panel) quantitation of immunospecific bands for the 3,B-HSDenzyme. Lighter shades indicate 47-kDa exposure; darker shades indicate 42 kDa exposure. (B) Northernblot analysis. (Upper panel) Representative Northern blots for 3p-HSD mRNA and 18S rRNA; (lower panel)bands for 313-HSD mRNA and 18S rRNA were quantitated and data expressed as 33-HSD mRNA/18SrRNA.*Statistically significant differences (p < 0.05).

present findings suggest that StAR may besusceptible to at least some environmentalpollutants that inhibit steroidogenesis andimpair fertility.

Although Roundup decreased steroido-genesis, the active ingredient of this herbi-cide, glyphosate, did not alter steroidproduction, indicating that at least one othercomponent of the formulation is required to

disrupt steroidogenesis. Because the formula-tion of Roundup is proprietary, further stud-ies are needed to identify the components inRoundup and their ability to disruptsteroidogenesis.

Recently, researchers have focused on theability of xenobiotics to inhibit steroidogenicenzyme activity, overlooking StAR protein as

an important site of steroidogenic inhibition.However, because a reduction in StAR levelsalone is sufficient to explain the effects ofRoundup on steroidogenesis, the fact thatRoundup inhibited P450scc activity is of lit-tle importance. Previous studies have shownthat the accumulation of the 30-kDa form ofStAR protein in mitochondria reflects theamount of cholesterol delivered to the innermitochondrial membrane over time. Thus,for a given decrease in StAR protein levels

there is a proportional decrease in cholesteroldelivery to the P450scc enzyme. In the pre-

sent study, Roundup reduced StAR proteinlevels by 90%, implying that of every 100molecules of cholesterol available for trans-

port to the inner mitochondrial membrane,only 10 molecules actually reached theP450scc enzyme. Roundup inhibited 22R-HC-driven pregnenolone production(P450scc activity) by 71%, indicating that ofthe 10 molecules of cholesterol that reachedthe enzyme, only 3 were converted to preg-

nenolone. Because Roundup did not alter3r-HSD activity, these 3 molecules ofpregnenolone were then converted to prog-

esterone. Thus, based on these calculations,we would expect that this herbicide wouldinhibit steroidogenesis by 97% (90% byinhibiting StAR expression and 7% byinhibiting P450scc activity). In fact,Roundup reduced steroidogenesis by 94%,which is close to this theoretical approxima-tion. Furthermore, although Roundupreduced 30-HSD levels by 30%, it did not

affect its activity, supporting previous studieswhich have shown that the steroidogenicenzymes, with the exception of P450scc, are

present in excess amounts. For example, in

one study, testosterone production wasunaffected, even though levels of P450c17were reduced by 90% (28). In contrast,because StAR mediates the rate-limitingstep in steroid biosynthesis, steroidogenesisis sensitive to small changes in StAR proteinexpression. The present study illustrates theimportance of StAR-mediated cholesteroltransfer in environmental pollutant-inhibit-ed steroidogenesis.

Although the transcriptional regulationof StAR has been the subject of intenseresearch since StAR was cloned in 1994[reviewed by Reinhart et al. (29)], the post-transcriptional regulation of StAR hasreceived considerably less attention and ispoorly understood. StAR protein is regulatedpost-transcriptionally by oxysterols, prosta-glandin F20x, and endotoxin, indicating thatthis may be an important level at whichStAR expression is controlled physiologically(30-32). At least three post-transcriptionalevents are required for StAR to have fullsteroidogenic activity and may have beentargeted by Roundup, including translationof the StAR mRNA into protein, associationof StAR with the outer mitochondrial mem-brane (33), and phosphorylation of the StARprotein. Although the mechanism by whichRoundup disrupted StAR protein expressionpost-transcriptionally remains unclear, inhi-bition of de novo protein synthesis and PKAactivity were ruled out. The precursor 37-kDa form of StAR cannot easily be measured,so whether Roundup specifically reducedStAR synthesis or prevented its associationwith mitochondria remains to be determined.Also, because the phosphorylation of StARwas not directly assessed in the present stud-ies, changes in the phosphorylation status ofStAR protein cannot be excluded.

To date, the majority of studies evaluat-ing the effects of toxicants on steroidogenesishave involved exposing steroidogenic cells tosingle compounds. However, humans andwildlife are typically exposed to large num-bers of chemicals simultaneously. Becauseenhanced steroidogenic inhibition mayresult from interactions between differentchemicals present in chemical mixtures,studies were conducted to determine if amixture of currently used pesticides couldinteract synergistically to inhibit steroidoge-nesis. The present study demonstrated thatthe components interacted antagonistically,inhibiting steroidogenesis less than predicted.Several events may have accounted for thisphenomenon. For example, two or morecompounds may have interacted directlywith one another to generate a less toxic pair,interacted antagonistically at the same site tocancel each other's effects, and/or acted addi-tively at one site, which contributes only50% to overall steroid production. Finally,

VOLUME 108 1 NUMBER 8 1 August 2000 * Environmental Health Perspectives

-.22

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= 0.3__e.m


774


_ _"- *- StAR

cD

10


kb3.42.7 StAR

12

0

1.6

0.5

0Control Bu2cAMP Roundup

Figure 7. Effects of Roundup on StAR protein andmRNA levels. IOD, integrated optical densityunits. (A) Western blot analysis. (Upper panel) Arepresentative Western blot; (lower panel) quanti-tation of immunospecific bands for the StAR pro-tein. (B) Northern blot analysis. (Upper panel)Representative Northern blots for StAR mRNAand 18S rRNA; (middle and lower panels) bandscorresponding to the 3.4, 2.7, and 1.6 kb tran-scripts of StAR mRNA and 18S rRNA were quanti-tated and data expressed as (middle panel) thesum of StAR transcripts lODs/18S rRNA IOD or(lower panel) individual StAR transcript IOD/18SrRNA IOD.*Statistically significant differences (p < 0.05).**Statistically significant differences (p < 0.01).

one compound could have altered themetabolism of another compound to gener-ate a less toxic metabolite.

As a result of the important role thatStAR plays in the steroidogenic pathway, itmay prove to be a useful biomarker to evalu-ate endocrine system function in sentinelwildlife species. In fact, a disruption in StARexpression may represent the first event inthe sequence of time-related changes thatunderlie pesticide-induced toxicity and leadto disturbances at the cellular and whole-organism level. Environmental pollutantshave purportedly caused a wide range ofadverse effects including decreased fertility inshellfish, fish, birds, and mammals anddecreased hatching success in fish, birds, andreptiles (34). In many of these cases, abnor-mal steroid hormone levels have been

-0.~ ~ ~-

lBS-i

200

aloo.

0Control Bu2cAMP Bu2cAMP +

Roundup

Figure 8. Effects of Roundup on StAR gene tran-scription. Upper panel, a representative experi-ment is shown. Lower panel, bands for StAR, L19mRNA and 18S rRNA were quantitated by com-puter assisted image analysis.

_ 120

- 100<|80 * L ICD

6040

* sc d ( 0. 0

U 0 0Scc E

Figure 9. Effects of a mixture of Roundup, ax-, 6-and y-HCH, and Dimethoate on steroid produc-tion.*Statistically significant differences (p < 0.05).

measured, indicating the possibility thatthese toxicants may block steroid hormonesynthesis. Although StAR has not yet beenidentified in submammalian species, severalreports indicate the probability that a StAR-like protein may exist in these species. Forexample, hormone-stimulated steroidogene-sis in fish and insects requires the de novosynthesis of a hormone-regulated, rapidlysynthesized, cycloheximide-sensitive, andhighly labile protein that may mediate cho-lesterol transfer for steroid hormone biosyn-thesis (35,36). A disruption in this StAR-likeprotein might contribute to the developmentof reproductive dysfunction in these species.If this putative StAR-like protein is identi-fied and characterized, it may enhance ourunderstanding of the mechanism of pollu-tant-induced steroidogenic inhibition inthese species.

Not only does StAR play an importantrole in steroid production in the gonads, butit is also indispensable for steroidogenesis inthe adrenal glands. As a result, a disruptionin StAR protein expression may impair more

than just fertility. The adrenal glands synthe-size glucocorticoids and mineralocorticoids,and a reduction in StAR expression in theadrenal gland may affect carbohydratemetabolism, immune system function, andwater balance. Because many toxicants thatreduce StAR expression and steroidogenesisin the ovary and testis also reduce StARexpression and steroidogenesis in the adrenalgland, a disruption in StAR protein expres-sion may underlie many of the toxic effectsof environmental pollutants (29).

In conclusion, Roundup disruptedsteroidogenesis in Leydig cells through apost-transcriptional reduction in StAR pro-tein expression. The use of StAR as an endpoint in studies concerning endocrine dis-ruption merits further consideration.

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2. Simpson ER, McCarthy JL, Peterson JA. Evidence thatthe cycloheximide-sensitive site of adrenocorticotropichormone action is in the mitochondrion. Changes inpregnenolone formation, cholesterol content, and theelectron paramagnetic resonance spectra ofcytochrome P-450. J Biol Chem 253:3135-3139 (1978).

3. Phillips MC, Johnson WJ, Rothblat GH. Mechanisms andconsequences of cellular cholesterol exchange andtransfer. Biochim Biophys Acta 906:223-276 (1987).

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11. Caron KM, Soo SC, Wetsel WC, Stocco DM, Clark BJ,Parker KL. Targeted disruption of the mouse gene encod-ing steroidogenic acute regulatory protein providesinsights into congenital lipoid adrenal hyperplasia. ProcNatI Acad Sci USA 94:11540-11545 (1997).

12. Payne AH, O'Shaughnessy PJ. Structure, function andregulation of steroidogenic enzymes in the Leydig cell.In: The Leydig Cell (Payne AH, Hardy MP, Russel LD,eds). Vienna, IL:Cache River Press, 1996;259-286.

13. Nolan CJ, Payne AH. Genotype at the P450scc locusdetermines differences in the amount of P450scc proteinand maximal testosterone production in mouse Leydigcells. Mol Endocrinol 4:1459-1464 (1990).

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regulatory (StAR) protein in mouse MA-10 Leydig cells[Abstract]. In: Proceedings of the Society for the Study ofReproduction, 8-11 August 1998, College Station, Texas.Madison, Wl:Society for the Study of Reproduction,1998;182.

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18. Abd el-Aziz Ml, Sahlab AM, Abd el-Khalik M. Influence ofdiazinon and deltamethrin on reproductive organs andfertility of male rats. Dtsch Tierarztl Wochenschr101:230-232 (1994).

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22. Ascoli M. Characterization of several clonal lines of cul-tured Leydig tumor cells: gonadotropin receptors andsteroidogenic responses. Endocrinology 108:88-95 (1981).

23. Resko JA, Norman RL, Niswender GD, Spies HG. Therelationship between progestins and gonadotropins dur-ing the late luteal phase of the menstrual cycle in rhesusmonkeys. Endocrinology 94:128-135 (1974).

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27. Sutton HG, Fusco A, Cornwall GA. Cystatin-related epi-didymal spermatogenic protein colocalizes with luteiniz-ing hormone-beta protein in mouse anterior pituitarygonadotropes. Endocrinology 140:2721-2732 (1999).

28. Anakwe 00, Payne AH. Noncoordinate regulation of denovo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxy-lase/C17-20 lyase in mouse Leydig cell cultures: relationto steroid production. Mol Endocrinol 1:595-603 (1987).

29. Reinhart AJ, Williams SC, Stocco DM. Transcriptionalregulation of the StAR gene. Mol Cell Endocrinol151:161-169 (1999).

30. Christenson LK, McAllister JM, Martin KO, Javitt NB,Osborne TF, Strauss JF Ill. Oxysterol regulation of

steroidogenic acute regulatory protein gene expression.Structural specificity and transcriptional and posttran-scriptional actions. J Biol Chem 273:30729-30735 (1998).

31. Bosmann HB, Hales KH, Li X, Liu Z, Stocco DM, HalesDB. Acute in vivo inhibition of testosterone by endotoxinparallels loss of steroidogenic acute regulatory (StAR)protein in Leydig cells. Endocrinology 137:4522-4525(1996).

32. Fiedler EP, Plouffe L, Jr., Hales DB, Hales KH, Khan I.Prostaglandin F(2alpha) induces a rapid decline in prog-esterone production and steroidogenic acute regulatoryprotein expression in isolated rat corpus luteum withoutaltering messenger ribonucleic acid expression. BiolReprod 61:643-650 (1999).

33. Arakane F, Kallen CB, Watari H, Foster JA, Sepuri NB,Pain D, Stayrook SE, Lewis M, Gerton GL, Strauss JF Ill.The mechanism of action of steroidogenic acute regula-tory protein (StAR). StAR acts on the outside of mito-chondria to stimulate steroidogenesis. J Biol Chem273:16339-16345 (1998).

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