Metabolism of Testosterone-'4C by Cultured Human Cells

DONNAD. SHANmES,KURTHIRSCHHORN,and MARIA I. NEW

From the Department of Pediatrics, Division of Pediatric Endocrinology,Cornell University Medical College, NewYork 10021

A B S T R A C T The metabolism of "C-labeled testosteroneby cultured human fibroblasts and amniotic fluid cellswas investigated. Radiolabeled testosterone was incu-bated with the cultured cells for 48 hr, and the labeledmetabolites present in the medium were subsequentlyidentified. The major metabolic products of testosteroneformed by cultured fibroblasts were A'-androstenedione,dihydrotestosterone, androsterone, and androstanediol.The amount of testosterone metabolized through each oftwo pathways was calculated and used to form a ratiodesignated the 17f-hydroxyl/17-ketonic ratio. Fibro-blasts from normal male and female children and adultfemales had high 17P-hydroxyl/17-ketonic ratios indi-cating testosterone metabolism occurred primarilythrough the 17P-hydroxyl pathway. There was a changein the pattern of testosterone metabolism with age inmales, i.e., adult males had much lower 17#-hydroxyl/17-ketonic ratios than did male children.

The testosterone metabolism of fibroblast cultures de-rived from three children with testicular feminizationand their mothers was compared to normal age and sex-matched controls. Fibroblasts of children with testicularfeminization metabolized testosterone predominantlythrough the 17-ketonic pathway and manifested a pat-tern of testosterone metabolism distinctly different fromtheir sex and age matched controls. The mothers ofchildren with testicular feminization could be distin-guished from normal females by their much lower 17#-hydroxyl/17-ketonic ratios. The much lower amountsof dihydrotestosterone and androstanediol produced byfibroblasts from patients with testicular feminization as

This work was presented in part at the Annual Meetingof the Endocrine Society, June 1971.

Doctors New and Hirschhorn are recipients of the HealthResearch Council of the City of New York Career ScientistAward under contracts I-481 and I-513, respectively. Dr.Hirschhorn's present address is the Department of Pedi-atrics, Division of Medical Genetics, Mount Sinai Schoolof Medicine, NewYork 10028.

Received for publication 7 October 1971 and in revisedform 3 December 1971.

compared with normals suggests there is a decrease intestosterone 5a-reductase activity in these patients.

Cultured amniotic fluid cells metabolized testosteroneto the same four major metabolites found in fibroblastcultures, but their activity was much lower than that offibroblasts. Most of the amniotic fluid cell cultures mne-tabolized testosterone largely through the 173-hydroxylpathway as did fibroblasts from normal children.

INTRODUCTIONThe technique of cell culture has been widely used inrecent years to investigate the biochemical basis of manyhereditary diseases. Cultured skin fibroblasts and cul-tured amniotic fluid cells have been used to detect geneticdisorders of lipid, amino acid, mucopolysaccharide, andcarbohydrate metabolism (1, 2). By use of the cell cul-ture technique, homozygous and heterozygous stateshave been detected and distinguished for some geneticdisorders.

Sweat et al. in 1958 and Berliner, Swim, andDougherty in 1960 studied the metabolism of proges-terone, cortisol, and corticosterone by cultured humanfibroblasts (3, 4), thus demonstrating the feasibility ofusing the cell culture technique for the study of steroidmetabolism. We wished to apply the technique of cellculture to the diagnosis of genetic disorders of steroidmetabolism utilizing fibroblasts and amniotic fluid cellsin culture. Recently a number of investigators have stud-ied the metabolism of testosterone by human skin takenfrom normal subjects and patients with testicular femini-zation. Gomez and Hsia (5) studied the metabolism oftestosterone-"4C by skin slices obtained from normalsubjects. They identified androstenedione, dihydrotes-tosterone, androstanedione, androsterone, and epiandros-terone as metabolites of testosterone. Wilson and Walker(6) demonstrated a decreased conversion of testosterone

to dihydrotestosterone in skin slices from the labiamajora of patients with testicular feminization. North-cutt, Island, and Liddle (7) found that the amount of

The Journal of Clinical Investigation Volume 51 1972 1459

dihydrotestosterone formed by slices of suprapubic skinwas lower in testicular feminization patients than innormal males. The mother of one of their testicularfeminization patients could not be distinguished from anormal female when the reduction of testosterone todihydrotestosterone by suprapubic skin slices was com-pared. The metabolism of testosterone by skin sliceswas studied by Jenkins and Ash (8) who reported thatsuprapubic skin slices from patients with testicular fem-inization metabolized testosterone in the same manner asdid skin slices from normal males and females. In a studyby Mauvais-Jarvis, Bercovici, and Gauthier (9), tes-tosterone-'4C was administered i.v. while testosterone-'H was applied percutaneously to normal subjects andpatients with testicular feminization. They found thattestosterone 5a-reductase was greatly decreased in theskin of patients with testicular feminization.

Since the metabolism of testosterone by human skinslices in vitro had been amply demonstrated, a studywas undertaken of the metabolism of testosterone bycultured skin fibroblasts obtained from normal individu-als, both male and female of different ages, as well asfrom patients with testicular feminization and theirmothers. Wehoped to be able to demonstrate a differencein the metabolism of testosterone by fibroblasts fromtesticular feminization patients as compared to normalsfor use in diagnosis and as a possible system by whichto study this disease. Metabolism of testosterone by nor-.mal cultured amniotic fluid cells was also investigated inorder to determine to what extent these fetal cells couldbe used to study steroid metabolism.

METHODSCell culturesTo establish skin fibroblast cultures, biopsies were taken

without anesthesia from the extensor surface of the upperarm from all individuals studied. The method of Danes andBearn (10) was used for the establishment of the fibroblastcultures. Cell lines were grown in the tissue culture labora-tories of Dr. Hirschhorn and Dr. Danes. The medium usedwas Eagle's minimum essential medium (MEM) supple-mented with 15% or 30% fetal calf serum, 1%7o penicillin(10,000 U/ml), 1% streptomycin (10,000 ,ug/ml), and 1%L-glutamine (200 mM). Studies of representative fibroblastcultures showed that there were no differences in testos-terone metabolism whether 15% or 30% fetal calf serumwas used. Fibroblast cultures were fed twice weekly andwhen confluent, were subcultured by treatment with 0.25%trypsin.

TABLE IChromatography Systems Used

I:M:W Isooctane: methanol: water (10: 10: 1)M: M: W Mesitylene: methanol: water (10: 10: 1)C: B: M:W Cyclohexane:benzene: methanol:water (10:2.5:10:1.5)D: N: M Decalin: nitromethane: methanol (10: 5: 5)C: M Chloroform: methanol (98:2)

Amniotic fluid obtained by amniocentesis was centrifugedat 250 g for 15 min, and the cells were resuspended inMcCoy's 5A medium (Grand Island Biological Co., GrandIsland, N. Y.) and distributed to 5 ml sterile plastic flasks(Falcon Plastics, Div. of BioQuest, Oxnard, Calif.). Thecell cultures were gassed with 10%o C02 in air and incu-bated at 37° C. Cell cultures were fed twice a week andgassed after each feeding. The complete medium (5 ml)consisted of McCoy's 5A medium (containing 30% fetalcalf serum) supplemented with 1% penicillin (10,000 U/ml), 1%o streptomycin (10,000 jug/ml), and 1% L-glutamine(200 mM). When confluent, the amniotic fluid cells weresubcultured by treatment with 0.25% trypsin. Sex of theamniotic fluid cells was confirmed by karyotypic analysis.

All cells used in these experiments had been subculturedone to six times. The average number of subcultures under-gone by the fibroblasts was three and by the amniotic fluidcells, two.

ChemicalsTestosterone-4-"C ( SA approximately 58.8 mCi/mmole)

was purchased from New England Nuclear Corp. (Boston,Mass.) and purified by chromatography on paper in thesolvent system C: B: M: Wfor 16 hr. (See Table I).Specific activity of this testosterone-'4C was determined byacetylation with acetic anhydride-'H whose specific activityhad previously been determined (11). Tritium-labeled an-drogens obtained from New England Nuclear were checkedfor purity by paper chromatography in several differentsolvent systems and were used without further purification.Tritium-labeled 5a-androstanediols (3a + 313) were a giftfrom Dr. Jean D. Wilson. Reference steroids were pur-chased from Mann Research Laboratories, Inc., N. Y. Allsolvents and reagents used were purified as previouslydescribed (11).

Incubation with testosterone-4-l"CPurified testosterone-4-14C (SA = 49.5 mCi/mmole) was

dissolved in absolute ethanol for addition to the cell cultures.The final concentration of ethanol in the cell culture mediumwas usually 0.5% and never exceeded 1%o. Previous controlstudies on alcohol toxicity had shown that these concentra-tions of ethanol in the media did not affect the growth ormorphology of cultured fibroblasts or amniotic fluid cells.The volume of medium in each cell culture flask was 5 ml.Incubation of the culture with radioactive testosterone wascarried out for 48 hr without cofactors. The medium wasthen removed for extraction of the radioactive steroids (Fig.1). Preliminary experiments had shown that no significantamount of radioactivity remained in the cells after 48 hrincubation, therefore only the medium was subjected tosteroidal analyses. The remaining cells were harvested bytreatment with trypsin and disodium versenate, and theirtotal DNA content was determined by the diphenylaminereaction (12). The medium and cells were stored at -20'Cbefore determination of DNA content and extraction ofsteroids. Tritium-labeled testosterone, androsterone, A4-androstenedione, dihydrotestosterone, and androstanediolwere added to the medium before extraction to estimatelosses during purification. Unlabeled androstanedione wasadded quantitatively to correct for losses of this metabolite.Extraction of steroids from the medium was carried outwith 9 vol of dichloromethane-ethyl acetate (1:1, v: v in aseparatory funnel). The upper aqueous layer was removed,

1460 D. D. Shanies, K. Hirschhorn, and M. I. New

Testosterohe +androstanediol

TLC silicaCHCI3: methanol98: 2 1

Testosterone Androstanediol

C:B:M:W I:M:W10:2.5:10:1.5 10:10:1

Mediu m

Add testosterone-3H and 3H - metabolites

Extract (CH2CI2:EtAc 1:1)

Chromatography (I:M:W 10:10:11

A + DHT Androsterone Androstanedione

M:M:W M: M:W I:M:Wreversed phase reversed phase 10:10:110:10:1 10:10:1

DHT

1:M:W l:M:W10:10:1 10:10:1

FIGURE 1 Purification of testosterone and its metabolites.

and the organic extract was washed once with a i'r vol of0.1 N NaOHand twice with a 'i vol of water. The extractwas evaporated to dryness and the residue was subjected topaper chromatography in the I: M: Wsystem (Table I) for16 hr. The radioactive steroids were located by scanningwith a Packard Radiochromatogram Scanner (Model 7201;Packard Instrument Co., Inc., Downers Grove, Ill.) andcomparison with reference steroids. Radioactive peaks wereeluted with methanol and resubmitted to paper or thin-layerchromatography for further purification. Thin-layer chroma-

tography which was used to separate labeled testosteronefrom the 5a-androstanediols (3a+3,8) was carried out on10 X 40 cm precoated silica gel plates (MN Polygram SilN-HR) obtained from Brinkmann Instruments, Inc., West-bury, N. Y. The solvent system used was chloroform:methanol (98:2), and the running time was approximately3i hr. Testosterone was detected by viewing the plate undera UV-scanner, and androstanediol was detected by stainingreference steroids with 5% phosphomolybdic acid in ethanoland heating at 100°C.

TABLE II3H/14C Ratios on Sequential Purification Steps (A-G)*

A4-Andros- Dihydrotestos-Testosterone Androsterone tenedione terone Androstanediol

A 1.78 A 54.82 B 58.50 B 1.96 G 1.92C 1.90 B 48.43 D 55.80 D 2.07 H 1.84E 1.81 E 53.86 F 56.17 E 1.98

A 26.87 A 29.30 D 10.27 B 36.0 G 1.21C 28.81 B 30.80 F 10.35 D 35.8 H 1.28E 27.85 E 29.62 E 10.58

A 2.58 A 44.80 B 19.64 B 21.2 G 1.99C 2.37 B 45.07 D 20.38 D 20.9 H 1.81E 2.35 F 19.69

A 9.65 A 79.50 B 60.16 B 91.8 G 13.30C 9.27 B 78.94 D 62.50 D 91.9 H 14.52E 9.42 F 61.28

* A, after 1st paper chromatography (system I :M :W); B, after 2nd paperchromatography (system M:M :W); C, after 2nd paper chromatography(system C :B :M :W); D, after 3rd paper chromatography (system I :M :W);E, after acetylation and rechromatography (system D :N :M); F, after reduc-tion with sodium borohydride and rechromatography (system C:B:M:W);G, after one paper chromatography (I :M :W) and one thin-layer chroma-tography (system C :M); H, after two thin-layer chromatographies(system C :M).

Metabolism of Testosterone-'4C by Cultured Human Cells 1461

Each metabolite was chromatographed at least twice, andits 3H/'C ratio was determined by counting in a liquidscintillation counter (Packard Tri-Carb Model 3375). Aminimum of 5,000 events was counted, resulting in a standarderror of less than 1.4%. The efficiency for tritium was 41%,and for 14C, 69%.

Evaluation of the methodSpecificity. Purity of the labeled metabolites was demon-

strated by constancy of the 'H/14C ratio during successivechromatographies and after derivative formation (acetylationor borohydride reduction) (Table II). Acetates of testos-terone, dihydrotestosterone, androsterone, and etiocholanolonewere formed by reacting dried eluates with 100% aceticanhydride (0.1 ml) and anhydrous pyridine (0.1 ml) over-night at room temperature. The borohydride reductionproduct of A'-androstenedione was formed by the methodof Gandy and Peterson (13). The acetylated derivativeswere chromatographed in system D: N: M (Table I) for16 hr. The A'-androstenedione which was reduced to testos-terone was chromatographed in the C: B: M: W system(Table I) for 16 hr.

After preliminary experiments revealed that no furtherpurification was achieved by this step, derivative formationwas eliminated from the method. In early experiments sepa-ration of 3a from 3f8, 5a-androstanediol, indicated that allthe radioactivity was in the 3a-fraction. No attempt wasmade subsequently to separate the 3ca from the 3,-andro-stanediol. Epiandrosterone acetate was separated from dihy-drotesterone (DHT) acetate in chromatography systemD: N: M.

Precision. Results utilizing the same cell line but carry-ing out the incubation at separate times indicate good repro-ducibility between experiments (Table III).

Controls. Addition of testosterone at the end-time per-mitted us to estimate recovery of added unmetabolizedsubstrate. These experiments showed 97-100%o recovery ofadded testosterone.

Methodological losses. Labeled testosterone was incubatedfor 48 hr with 5 ml of complete medium but without cells.Recovery of approximately 94% of the added testosteronewas achieved with no formation of metabolites detected.

TABLE I I IPrecision of Results in Two Separate Experiments Utilizing

the Same Cell Strain from a 7 yr old Girl WithTesticular Feminization

Experiment I Experiment II

Substrate (T-14C), cpm 139,155 55,662Amount of DNA, p4g 18 21Metabolities*

Testosterone 38 33Androsterone 0.61 0.65A4-Androstenedione 2.6 3.2Dihydrotestosterone 1.6 1.5Androstanediols 1.4 3.1

Ratio, 173-hydroxyl/17-ketonic pathway 0.94 1.2

* Expressed as pmoles metabolite/,pg DNA per nmolesubstrate.

After extraction and purification the following percentagesof the tritiated metabolites were recovered: testosterone,22%o; -androsterone, 32%; A'-androstenedione, 33%; dihy-drotesterone, 32%; and androstanediol, 13%. The precedingfigures are the averages for recovery of these metabolitesin 10 representative experiments.

RESULTSAmount of testosterone metabolized by cultured cells.

The amount of testosterone metabolized by the culturedcells was estimated from the amount of metabolic prod-ucts formed. The amounts of testosterone metabolized ineach cell culture experiment are listed in Table IV. Thetotal amount of 14C-radioactivity recovered consisted ofthe amounts of purified 14C-metabolites plus the amountof unreacted testosterone-YC recovered. All values werecorrected for procedural losses. Table IV shows therecoveries of added radioactivity for each experiment.The average recovery of radioactivity for the fibroblastexperiments was 86% and for the amniotic fluid cellexperiments, 87%. It is clear, therefore, that the ma-jority of the metabolic products of testosterone havebeen accounted for by our method and no significantquantity of testosterone or metabolites remained in thecell pellet. In preliminary experiments isolated dihydro-testosterone was acetylated and rechromatographed insystem D: N: M (Table I). No change in the 'H/14Cratio of dihydrotestosterone acetate was observed afterderivative formation. Since epiandrosterone acetateseparates from dihydrotestosterone acetate in this system,it was concluded that no significant amount of epian-drosterone was formed by the cultured cells.

In order to compare the amounts of testosterone me-tabolized by each cell line, taking into account the vari-able cell number, the per cent testosterone metabolizedper /Agram of DNA was computed for each cell line(Table IV). There was no consistent correlation betweenthe amount of testosterone metabolized and the age, sex,or clinical status of the person from whom the cells werederived.

Metabolites of testosterone formed by cultured cells.Preliminary experiments showed that the major metabo-lites of testosterone formed by fibroblast cell cultureswere A4-androstenedione, dihydrotestosterone, andros-terone, and androstanediol (Table V). Androstanedionewas sometimes found to be a minor metabolite of testos-terone representing 1-3% of the added radioactivity. Anattempt was made to identify etiocholanolone as ametabolite of testosterone, but no 14C-radioactivity wasassociated with the tritium-labeled etiocholanolone iso-lated. In order to control variable cell number, DNAvalues were obtained for each flask of cells. Experimentalresults were thus expressed as pmoles of metabolite perigram DNA per nmole of substrate used, thus takinginto account the variability in cell number and in amount

1462 D. D. Shanies, K. Hirschhorn, and M. I. New

TABLE I VAmount of Testosterone-14C Metabolized and Per Cent

Added Radioactivity Recovered

Per cent Per centtestos- added

Per cent terone 14c-radio-testos- metabo- activityterone lized recovered

metabo- per pg (correctedSubjects* lizedt DNA for losses)

Fibroblast experiments2 yr old male (RL)2 yr old male (ES)2 yr old male (RS)2 yr old TF (BS)2 yr old TF (MP)2 yr old TF (BS)2 yr old TF (BS)7 yr old TF (AC)7 yr old TF (AC)2 yr old female (BG)2 yr old female (AR)2 yr old female (ND)Mother, TF (Mrs. C)Mother, TF (Mrs. S)Mother, TF (Mrs. P)Mother, TF (Mrs. C)Adult female (MD)Adult female (JC)Adult female GM)Adult female (MR)Adult male (IL)Adult male (LC)Adult male (JG)Adult male (DC)

81.392.939.980.249.641.657.111.017.651.020.924.761.031.123.740.874.7

104.3419242.311.211.112.5

Amniotic fluid cell experimentsMale fetuses

9 wk (BN) 22.49 wk (BN) 32.714 wk (BA) 8.515 wk (RP) 12.016 wk (MM) 7.416 wk (AB) 7.716 wk (FB) 17.716-19 wk (BM) 5.617 wk (FA) 10.122 wk (BC) 13.528.5 wk (BD) 10.5

Female fetuses14 wk (RC)15 wk (MG)15 wk (MG)15 wk (DB)16 wk (EL)16 wk (RS)16.5 wk (HN)

13.611.614.0

9.110.128.815.9

4.1519.35

3.532.081.271.351.850.610.855.932.521.521.951.881.872.205.453.245.062.452.601.870.502.16

0.500.670.331.200.240.370.980.250.351.070.46

0.590.430.440.230.340.620.71

84.299.651.587.6

107.690.996.679.186.493.994.259.779.291.192.881.891.0

106.041.0

104.497.589.999.556.5

95.269.463.398.882.075.495.096.195.775.485.1

70.2113.88986.474.191.7

103.9

TABLE IV (continued)

Per cent Per centtestos- added

Per cent terone 14C-radio-testos- metabo- activityterone lized recovered

metabo- per pg (correctedSubjects* lizedt DNA for losses)

Female fetuses17 wk (PP) 11.3 0.52 81.717 wk (PP) 11.5 0.52 95.718 wk (BG) 4.7 0.29 95.720 wk (EV) 8.1 0.37 89.4

* TF, testicular feminization; letters in parentheses are theidentifying initials of the subject from whomthe sample wasderived.$ Per cent testosterone metabolized as estimated from meta-bolic products formed.

of substrate used. In fibroblast cultures derived fromnormal male and female children dihydrotestosterone andandrostanediol were the main metabolites of testosterone.Fibroblasts from normal adult females metabolized tes-tosterone mainly to androstanediol and dihydrotestos-terone, while those from normal adult males formedmainly A'-androstenedione and dihydrotestosterone. Theprimary metabolite of testosterone in cultures from pa-tients with testicular feminization and their mothers wasA4-androstenedione.

Metabolites formed from testosterone by cultures ofamniotic fluid cells derived from 19 different male andfemale fetuses are shown in Table VI. The major me-tabolites of testosterone formed by cultured amnioticfluid cells are the same as those formed by fibroblasts,i.e., androsterone, A'-androstenedione, dihydrotestoster-one, and androstanediol. In seven amniotic fluid cell cul-tures derived from males, dihydrotestosterone was themajor metabolic product of testosterone. Cultures fromtwo slightly younger male fetuses and one older malefetus (9, 14, and 28i wk of gestation) formed relativelymore A'-androstenedione. Amniotic fluid cell cultures de-rived from females formed mainly dihydrotestosterone,androstanediol, and A4-androstenedione as metabolic prod-ucts of testosterone. No one metabolite was consistentlyformed in greater amounts than any other. Androsteronewas a relatively minor metabolic product in culturesfrom both male and female fetuses.

In an attempt to elucidate the relationship betweensubstrate concentration and the formation of metabolites,the concentration of testosterone was varied in experi-ments using three different flasks of the same fibroblastcell line (derived from a 2 yr old testicular feminizationpatient). The DNA values for the three flasks weresimilar (38.5 ug, 30.9 ttg, 30.9 Ig). The fibroblasts wereincubated for 48 hr with testosterone-4-14C. The results

Metabolism of Testosterone-"4C by Cultured Human Cells 1463

TABLE VTestosterone Metabolism by Cultured Fibroblasts

Per centconversion

Ratio, of testos-Metabolic products recovered 17,6-hydroxyl/ terone

17-ketonic to DHTSubjects* Substrate DNA T A A4 DHT Adiol Adione pathway + adiol

nmoles Ag pmoles metabolitel;&g DNA/nmole substrate added

2 yr old male (RL) 3.8 20 1.5 0.92 0 13 28 0 45 822 yr old male (ES) 1.5 4.8 14 2.7 0 96 95 0 71 922 yr old male (RS) 2.6 11 10 2.8 1.5 16 15 1.3 7.2 342 yr old TF (BS) 3.9 39 1.9 4.1 15 0.91 0.73 2.2 0.086 6.22 yr old TF (MP) 3.9 39 15 0.46 12 0.46 0.21 0 0.056 2.62 yr old TF (BS) 0.50 31 16 2.6 9.6 0.71 0.55 1.0 0.11 4.02 yr old TF (BS) 1.2 31 13 3.1 13 0.91 1.5 1.0 0.15 7.47 yr old TF (AC) 1.9 18 38 0.61 2.6 1.6 1.4 0.94 5.47 yr old TF (AC) 0.74 21 33 0.65 3.2 1.5 3.1 - 1.2 9.62 yr old female (BG) 1.5 8.6 50 0.70 1.3 39 19 0 29 502 yr old female (AR) 3.8 8.3 88 0.24 1.6 16 7.2 0 13 192 yr old female (ND) 0.74 16 22 0.31 0.80 0.62 14 0 14 24Mother, TF (Mrs. C) 1.9 31 5.8 7.2 4.6 2.9 4.8 1.2 0.64 24Mother, TF (Mrs. S) 3.9 17 36 1.0 14 2.1 1.4 2.2 0.23 5.9Mother, TF (Mrs. P) 3.9 13 54 2.5 11 1.7 3.4 0 0.36 6.5Mother, TF (Mrs. C) 0.74 19 22 3.8 15 1.3 2.1 0.18 6.5Adult female (MD) 1.5 14 12 1.2 0.29 19 34 0.22 35 74Adult female (JM) 2.6 8.1 0.37 3.1 0.12 2.6 45 0 15 39Adult female (JC) 1.2 32 0.52 3.1 0.10 0.87 28 0 9.1 93Adult female (MR) 1.2 38 3.0 1.8 0.21 5.8 17 0 12 87Adult male (IL) 3.9 16 34 2.2 22 1.2 0.86 1.0 0.087 3.4Adult male (LC) 1.5 6.0 131 0.50 6.5 8.8 2.8 0 1.7 7.2Adult male (JG) 1.2 22 40 0.63 2.0 1.1 1.3 0 0.92 5.3Adult male (DC) 2.6 5.8 75 1.0 5.5 9.3 5.7 0 2.3 8.7

T, testosterone; A, androsterone; A4, A4-androstenedione;androstanedione.* See corresponding footnote to Table IV.

are shown in Fig. 2. As the concentration of testosteronein the incubation media was raised from 0.99 to 7.82 X10" M, formation of the four major metabolites (an-drosterone, dihydrotestosterone, A'-androstenedione, andandrostanediol) increased in a linear fashion. More A'-androstenedione was formed with increasing substrateconcentration than any of the other three metabolites.These studies showed that the enzymes metabolizingtestosterone were not saturated at the substrate concen-trations utilized. There was no decline or plateau in theproduction of metabolic products with increasing sub-strate concentrations up to 7.82 X 10-7 M. All experi-ments with fibroblasts utilized substrate concentrationsbetween 0.99 X 10' and 7.8 X 10' M.

To compare the relative importance of the two majorpathways of testosterone catabolism (Fig. 3), the an-drosterone and the A'-androstenedione values were addedtogether as were the dihydrotestosterone and androstane-diol values. The former sum thus represented metabo-

DHT, dihydrotestosterone; adiol, androstanediol; adione,

lites of the 17-ketonic pathway, and the latter, the 17,8-hydroxyl pathway (14, 15). The ratio of these two path-ways (1713-hydroxyl/17-ketonic) was then calculated forall experiments. The results for the fibroblast experi-ments are shown in Table V. The fibroblasts from thetwo 2 yr old patients with testicular feminization metab-olized testosterone primarily through the 17-ketonic path-way. In two cultures derived from a 7 yr old patientwith testicular feminization the 17-ketonic and the 17k-hydroxyl pathways were almost equally utilized. In con-trast, cultures from normal 2 yr old male children me-tabolized testosterone almost exclusively through the 17p-hydroxyl pathway. Normal 2 yr old females also used the17,-hydroxyl pathway primarily. Fibroblast cultures es-tablished from three mothers of children with testicularfeminization showed predominance of the 17-ketonicpathway of testosterone metabolism. These mothers thusshowed a significant reduction in the importance of the17fi-hydroxyl pathway when compared to normal adult

1464 D. D. Shanies, K. Hirschhorn, and M. I. New

TABLE VITestosterone Metabolism by Cultured Amniotic Fluid Cells

Per centconversion

Ratio, of testos-Metabolic products recovered 17,5-hydroxyl/ terone

17-ketonic to DHTSubjects* Substrate DNA T$ A A4 DHT Adiol Adione pathway + adiol

umoles jsg pmoles metabolite/pg DNA/nmole substrate addedMale fetuses

9 wk (BN) 3.6 45 16 0.21 4.0 0.30 0.68 0 0.23 4.29 wk (BN) 3.6 49 7.5 0.35 5.1 0.65 0.61 0.23 6.814 wk (BA) 5.0 26 21 0.13 1.4 0.78 0.93 1.1 4.515 wk (RP) 3.9 10 87 0.31 1.8 5.9 4.0 0 4.7 9.916 wk (MM) 3.6 31 24 0.05 0.53 0.76 1.0 0 3.1 5.616 wk (AB) 2.9 21 32 0.09 0.99 1.0 1.6 0 2.4 15.116 wk (FB) 1.1 18 44 0.26 1.2 6.6 2.0 0 5.7 5.516-19 wk (BM) 1.2 23 40 0 0.64 1.2 0.56 0 2.8 4.117 wk (FA) 3.6 29 30 0.24 0.80 1.5 0.98 0 2.5 7.122 wk (BC) 5.0 13 49 0.34 2.0 5.1 3.2 3.6 10.628.5 wk (BD) 0.74 23 32 0.12 3.2 0.60 0.64 0 0.36 2.9

Female fetuses14 wk (RC) 5.0 23 25 0.19 0.81 2.6 1.7 0.63 4.3 9.815 wk (MG) 2.9 27 38 0.15 1.1 0.96 2.1 0 2.6 8.215 wk (MG) 2.9 32 23 0.16 1.0 2.4 0.74 0 2.6 10.215 wk (DB) 1.9 41 19 0.08 0.83 0.88 0.41 0 1.4 5.316 wk (EL) 3.9 46 14 0.46 4.2 0.54 1.0 0 0.32 7.216 wk (RS) 5.0 30 21 0.17 0.48 2.0 1.1 - 4.8 8.116.5 wk (HN) 1.1 22 39 0.22 0.59 3.6 2.8 7.9 14.117 wk (PP) 3.6 22 32 0.17 1.2 2.3 1.5 2.7 8.217 wk (PP) 2.2 22 38 0.23 2.0 0.81 2.2 0 1.4 6.618 wk (BG) 1.2 16 56 0 1.6 0.88 0.40 0 0.81 2.120 wk (EV) 1.9 22 36 0.10 1.0 1.7 0.77 0 2.3 5.6

* See corresponding footnote to Table IV.See Table V for abbreviations.

females. In the case of normal adult males, the resultsare not consistent. Two experiments had ratios favoringthe 17-ketonic pathway while two others utilized mainlythe 17f-hydroxyl pathway. It is clear, however, thatadult males can be distinguished from adult females;that children with testicular feminization can be distin-guished from normal male children and that mothers ofpatients with testicular feminization can also be distin-guished from normal adult females by virtue of theirdifferent 17P-hydroxyl/17-ketonic pathway ratios.

Amniotic fluid cell cultures from male fetuses, withthree exceptions, metabolized testosterone mainly via the179-hydroxyl pathway (Table VI). One culture froma very young male fetus (BN, 9 wk of gestation) metab-olized testosterone almost exclusively by the 17-ketonicpathway. Although these two experiments are hardlyconclusive, the predominance of the 17,8-hydroxyl path-way in the older fetuses may reflect maturation of theappropriate steroid enzymes. Amniotic fluid cells from

seven female fetuses showed a 17P-hydroxyV17-ketonicratio greater than one, whereas the remaining two fe-male fetuses showed a predominance of the 17-ketonicpathway.

DISCUSSION

It has been shown in this study that cultured skinfibroblasts metabolize testosterone actively. The metabo-lites of testosterone formed by cultured fibroblasts arethe same as those reported for skin slices in vitro (5,6, 8). Previous investigators who studied testosteronemetabolism by skin slices in vitro did not, however,identify androstanediol as a metabolic product of tes-tosterone (5, 8). No 5P-metabolites of testosteronewere identified in the cell culture experiments whichis in agreement with investigations (5, 6, 8) usingtissue slices.

The metabolism of testosterone by fibroblasts derivedfrom patients with testicular feminization was clearly

Metabolism of Testosterone-"C by Cultured Human Cells 1465

040

E/30

E 0.30-

10

0

s 20

O~ ~ 0

0 2 4 6 8Testosterone concentration (moles x 10-7/I liter)

FIGuRE 2 Relationship of substrate concentration to meta-bolic product formation in fibroblast cell cultures.

distinguished from that of fibroblasts derived from age-matched normal males. While normal male childrenutilized the 17j#-hydroxyl pathway almost exclusively,the children with testicular feminization either favoredthe 17-ketonic pathway greatly or metabolized testos-

17-Ketonic pathway

0W4-Androstenedione

10

0H

50-Androstanedione

1>0

HO HOH

59-Androsterone

170lHydroxyl pathway

Testosterone

OH

5g-Androstanolone

1

5a-Androstanediol

FIGuRE 3 Different pathways of testosterone metabolism.

terone equally through both pathways. Cultured cellsfrom children with testicular feminization producedless 5a-reduced metabolites (dihydrotestosterone andandrostanediol) than did cells from normal children.These results suggest that there is a deficiency of tes-tosterone 5a-reductase in fibroblasts of children withtesticular feminization. It is, however, entirely pos-sible that factors other than impaired enzymatic ac-tivity, e.g. decreased nuclear binding of testosterone,were responsible for the lower production of 5a-reducedmetabolites by cultured cells from patients with tes-ticular feminization. Wilson and Walker (6) andNorthcutt, Island, and Liddle (7) have also found de-creased activity of testosterone 5a-reductase in sexskin slices from patients with testicular feminization.

Cultured cells from adult females and adult malescould be distinguished by the metabolic products oftestosterone (17fi-hydroxyl/17-ketonic ratios) but maleand female children could not be clearly distinguishedin this way. There was a change in the pattern oftestosterone metabolism with age in fibroblasts frommales but not in fibroblasts from females. Culturedfibroblasts from male children had much greater 17,8-hydroxyl/17-ketonic ratios than did those from adultmales suggesting a maturational change in testosteronemetabolism. These in vitro results using cultured cellsdo not parallel the physiological in vivo metabolism oftestosterone wherein the adult male would produce moreof the biologically active 5a-reduced metabolites. How-ever, the highly artificial experimental conditions ofcultured cells may not permit extrapolation to in vivometabolism.

It is apparent however, that the use of cultured skinfibroblasts in the study of testosterone metabolismprovides a new diagnostic tool to distinguish childrenwith testicular feminization from normal children. Themetabolism of steroids other than testosterone by cul-tured skin fibroblasts may prove useful in the studyof other steroid hormone disorders. Even organ-specificdisorders such as the adrenogenital syndrome may beamenable to study by the cell culture technique sinceother organ-specific diseases such as Tay Sachs dis-ease (16) and erythropoetic porphyria (17) haveshown expression of their biochemical defects in cul-tured skin fibroblasts.

While the experiments presented here aid in thediagnosis of testicular feminization they do not shedlight on the mechanism causing the difference in themetabolism of testosterone between normal culturedcells and cells from patients with testicular feminiza-tion nor do these observations elucidate the cause ofthe syndrome of testicular feminization. However cul-tured skin fibroblasts may provide a useful model sys-tem in which to study the target cell separate from

1466 D. D. Shanies, Hirschhorn, and M. 1. New

/

the host in a target cell disorder such as testicularfeminization. At present, it is unclear whether the de-ficiency of testosterone 5a-reductase demonstrated invivo is the basic defect in this condition. Stricklandand French (18) provided evidence to the contrary inthat patients with testicular feminization did not re-spond to dihydrotestosterone administration.

An important outcome of this study is the capacityto detect the carrier of the gene for testicular feminiza-tion. The mothers of patients with testicular feminiza-tion could be distinguished from normal women byreference to the pattern of testosterone metabolism oftheir skin fibroblasts. The testicular feminization syn-drome in humans may be an X-linked disease since apresumably homologous mutation in rodents has beenshown to be X-linked (19, 20). Results indicate thatfibroblasts from the mothers of children with testicularfeminization appear to show an abnormal testosteronemetabolism. Therefore, it may be possible to clone thecells of a heterozygous mother and demonstrate themode of inheritance of this disorder. According to theLyon hypothesis (21), if the gene were X-linked inhumans, two clonal populations would result; one nor-mal and the other abnormal with respect to testosteronemetabolism. This cloning technique has been used todemonstrate the mode of inheritance of Lesch-Nyhansyndrome (22).

Amniotic cells in culture have demonstrated a ca-pacity to metabolize testosterone. Thus, a technique isnow available to study fetal metabolism of testosteronewithout interruption of pregnancy. It may be possibleto demonstrate a difference in the production of 5a-reduced metabolites of testosterone in amniotic cellsfrom a fetus with testicular feminization thereby mak-ing the prenatal diagnosis of testicular feminizationpossible. Such studies could be extended to other ster-oidal hormones and would have broad scope in the pre-natal diagnosis of other hormonal disorders and in theinvestigation of the effect of drugs and hormones onprenatal steroid metabolism. In conclusion, the studyof the metabolism of steroids in cell culture has pro-vided a new and powerful tool to study target cell dis-ease and may yield a new approach to prenatal diag-nosis and genetic counseling in hereditary diseases ofsteroid metabolism.

ACKNOWLEDGMENTSWe acknowledge the technical assistance of Mrs. Seu-lainChen, and Misses Joan Martin, Enid Kutinsky, and EnidStern. We are grateful to Dr. Jean Wilson, University ofTexas, Southwestern Medical School, Dallas, Texas, for thelabeled androstanediol and to Dr. Betty Shannon Danes ofthe Department of Medicine, Division of Human Genetics,Cornell University Medical College, for her provision ofcultured fibroblasts for metabolic studies.

This work was supported by U. S. Public Health Service,National Institutes of Health Grant Awards HD 72 andHD02552, and Training Grant Award HD00210; and U. S.Public Health Service, Division of Research Facilities andResources, Clinical Research Center Grant Award RR 47.

REFERENCES1. Hsia, D. Y. Y. 1970. Study of hereditary metabolic dis-

eases using in vitro techniques. Metabolism (Clin.Exp.). 19: 309.

2. Milunsky, A., J. W. Littlefield, V. E. Shih, and L.Atkins. 1970. Prenatal genetic diagnosis (first of threeparts). N. Engl. J. Med. 283: 1370.

3. Sweat, M. L., B. I. Grosser, D. L. Berliner, H. E.Swim, C. J. Nabors, and T. F. Dougherty. 1958. Themetabolism of cortisol and progesterone by cultureduterine fibroblasts, strain U 12-705. Biochim. Biophys.Acta. 28: 591.

4. Berliner, D. L., H. E. Swim, and T. F. Dougherty.1960. Metabolism of [4-'4C] corticosterone by fibro-blasts, strain U 12-79. Biochim. Biophys. Acta. 38: 184.

5. Gomez, E. C., and S. L. Hsia. 1968. In vitro metabolismof testosterone-4-14C and A'-androstene-3,17-dione-4-`4Cin human skin. Biochemistry. 7: 24.

6. Wilson, J. D., and J. D. Walker. 1969. The conversionof testosterone to 5a-androstan-17fi-ol-3-one (dihydro-testosterone) by skin slices of man. J. Clin. Invest. 48:371.

7. Northcutt, R. C., D. P. Island, and G. W. Liddle. 1969.An explanation for the target organ unresponsivenessto testosterone in the testicular feminization syndrome.J. Clin. Endocrinol. Metab. 29: 422.

8. Jenkins, J. S., and S. Ash. 1971. The metabolism oftestosterone by skin in normal subjects and in testicularfeminization. J. Endocrinol. 49: 515.

9. Mauvais-Jarvis, P., J. P. Bercovici, and F. Gauthier.1969. In vivo studies of testosterone metabolism by skinof normal males and patients with the syndrome oftesticular feminization. J. Clin. Endocrinol. Metab. 29:417.

10. Danes, B. S., and A. Bearn. 1969. Cystic fibrosis of thepancreas. A study in cell culture. J. Exp. Med. 129: 775.

11. New, M. I., J. M. Gross, and R. E. Peterson. 1968.Double isotope dilution derivative technique for testos-terone glucuronoside in urine. Acta Endocrinol. 58: 77.

12. Tedesco, T. A., and W. J. Mellman. 1967. Desoxyribo-nucleic acid assay as a measure of cell number in prepa-rations from monolayer cell cultures and blood leuco-cytes. Exp. Cell Res. 45: 230.

13. Gandy, H. M., and R. E. Peterson. 1968. Measurementof testosterone and 17-ketosteroids in plasma by thedouble isotope dilution derivative technique. J. Clin.Endocrinol. Metab. 28: 949.

14. Baulieu, E. E., and P. Mauvais-Jarvis. 1964. Studies ontestosterone metabolism. I. Conversion of testosterone-17a-'H to 5a-androstane-3a, 17fi-diol-17a-3H: a new"17j8-hydroxyl pathway." J. Biol. Chem. 239: 1569.

15. Mauvais-Jarvis, P., J. P. Bercovici, 0. Crepy, and F.Gauthier. 1970. Studies on testosterone metabolism insubjects with testicular feminization syndrome. J. Clin.Invest. 49: 31.

16. Kolodny, E. G., B. S. Uhlendorf, J. M. Quirk, et al.1969. Gangliosides in cultured skin fibroblasts: accumu-lation in Tay-Sachs disease. 12th Int. Conf. Biochem.Probl. Lipids. 39.

Metabolism of Testosterone-"4C by Cultured Human Cells 1467

17. Romeo, G., M. M. Kaback, and E. Y. Levin. 1970.Uroporphyrinogen III cosynthetase activity in fibro-blasts from patients with congenital erythropoieticporphyria. Biochem. Genet. 4: 659.

18. Strickland, A. L., and F. S. French. 1969. Absence ofresponse to dihydrotestosterone in the syndrome of tes-ticular feminization. J. Clin. Endocrinol. Metab. 29:1284.

19. Ohno, S., R. Dofuku, and U. TettenbQrn. 1971. Moreabout X-linked testicular feminization of the mouse asa noninducible (is) mutation of a regulatory locus: 5a-androstan-3a-17,B-diol as the truce inducer of kidney

alcohol dehydrogenase and fi-glucuronidase. Clin. Genet.2:128.

20. Bardin, C. W., J. E. Allison, A. J. Stanley, and L. G.Gumbreck. 1969. Secretion of testosterone by the pseudo-hermaphrodite rat. Endocrinology. 84: 435.

21. Lyon, M. F. 1968. Chromosomal and subchromosomal in-activation. Ann. Rev. Genet. 2: 31.

22. Migeon, B. R., V. M. Der Kaloustian, W. L. Nyhan,W. J. Young, and B. Childs. 1968. X-linked hypoxan-thine-guanine phosphoribosyl transferase deficiency:heterozygote has two clonal populations. Science (Wash-ington). 160: 425.

1468 D. D. Shanies, K. Hirschhorn, and M. I. New