in vitro cortisol biosynthesis by a testicular tumor

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in vitro CORTISOL BIOSYNTHESIS BY A TESTICULAR TUMOR I

Paige Ko Besch, David J o Watson, Roger D ° Barry, George J o Hamwi, Jack Mostow and Grant Gwinup Departments of Obstetrics and Gynecology and Medicine Ohio State University College of Medicine Columbus 10, Ohio Received April 5, 1963

ABSTRACT Incubation of a testicular tumor with progesterone-4-14C resulted in the isolation and identification of cortisol and cortisone as well as the tentative identification of seven other compounds° It is concluded that this tumor of the testis represents adrenocortical cell rests and was activated by prolonged elevated levels of ACTH. In vitro steroid biosynthetic studies of a testicular tumor from a 14-year-old white male with third-time recurrent Cushing's Syndrome yielded the information reported here. A detailed clinical report of this case is being reported elsewhere. 2 MATERIALS AND METHODS Tissue: On July 3, 1961, a left orchiectomy was done, yielding a tumor measuring 35 x 25 x 20 mm and weighing approximately 18 grams. Immediately upon its removal it was quick-frozen in liquid nitrogen= The specimen was thawed, and the tissue incubation was begun November 3, 1961o Urines: The urines were collected following various clinical manipulations as described in Table Io They were stored at -20 ° C until utilized. The chromatography was carried out as previously reported. 3 Incubation: Portions of the tumor were minced and 005 gram aliquots were added to beakers containing Krebs-bicarbonate-buffer, adjusted to pH 7.4, and containing 0.49% nicotinamide. Radioactive substrates were previously added to the buffer, which also contained the following: adenosine triphosphate (.0.4 mM), diphosphopyridine nucleotide (.0.4 mM), triphosphopyridine nucleotide (,0ol mM), glucose-6phosphate (..0.5 raM), fumaric acid (,i.0 mM), and glucose-6-phosphate dehydrogenase (.I mg/incubation beaker). The incubation was carried out for three hours at 37 ° C in an atmosphere of 95% 02-5% CO 2.

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Table I. Fractionation of the Urinary 17-oxo-steroids fol Iowing various clini ca l manipulations a Pre-Surgical b Compoundsc Androsterone Etiocholanolone Dehydroepiandrosterone I I-oxygenated compounds

Post-Sur~ i ca I

Stim=d Supp.e

Cont. f

Stim.

Cont.

Supp° Cont.

3.09 3.75 2.13 2.28

2.55 2.01 0.98 1.48

2.63 1.95 1.27 1035

2.01 1.98 I000 1.45

2.05 1.78 1.05 1.35

2.26 2.01 2.08 1.88

2.01 I °60 1.20 1.38

a) Each value represents the average of three determinations, b) Pre-Surgical control specimen destroyed, c) In order below: 5a-androstan-3a-ol-17-one; 513-androstan-3aol-17-one; androst-5-ene-313-ol-17-one; and the II oxo- and 1113-ol- derivatives of androsterone and etiocholanolone, d) ACTH, 40 uts q 12 hrs x 2 days° e) Dexamethasone, 1.5 mg q 8 hrs x 3 days. f) Control values.

Substrates: Progesterone-4-14C (pregn-4-ene-3,20-dione) and androstenedione-4-14C (androst-4-ene-3, 17-dione) were used at 1.125 (25 pC) and 5.375 (25 IJC) IsM levels Iespectivelyl per beaker. 4 Radiochemical purity was established prior to use of the two 4C-labe led compounds. Extraction and Analysis: Each incubation was terminated by the addition of ethyl acetate and subsequently extracted with 6 x I volume of ethyl acetate. The combined extracts were concentrated under nitrogen to an oily residue. The residues were chromatographed as previously described5; all paper chromatograms were run on washed Whatman No. I filter paper. Solvents used in the chromatographic systems were either redistilled or used as reagent grade chemicals° Following the initial chromatography1 radioactive areas were located by scanning with a 4 pi Baird-Atomic Paperchromatogram Scanner or a Packard Radiochromatogram Scanner; areas of interest were eluted and rechromatographed in the appropriate systems for the resolution of the isolated steroids. Oxidation and reduction reactions1 as well as acetylations, were performed as previously described. 5 Radio-quantitation of aliquots of eluted compounds were done in the Packard Tri-Carb Liquid Scintillation Spectrometer. A counting standard error of less than 5% was achieved in the toluene-PPO-POPOP system; arithmetic correction for quenching and self-absorption phenomena was made by use of 14C-toluene additions of a known number. The counter-current distribution was carried out as reported by Carstensen, 6 in a 100 tube E° C. automatic CoC°D. apparatus. Melting points were taken on a NageI-Axelrod apparatus and are uncorrected.

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J

T / P 9 - - "/'2 HRS

,w

~o

to

io

GN

Figure I. A scan of the radioactive compounds on the 72 hr paper chrornatogram run in the toluene/propylene glycol system at 33 ° C.

RESULTS The residues from the two incubations were initially chromatographed in the toluene/propylene glycol (T/PG) system for 72 hrs at 33 ° C. The radioactive scan of the paper chromatogram of the androstenedione-4-14C incubation residue, contained no radioactive areas.

On subsequent paper chromatography of the run-off, compounds

of a less polar nature were found as later described. The initial paper chromatogram, however, of the

progesterone-4-14Cincubation

residue did indeed contain radioactive

areas. These radioactive areas are seen in Figure I. These radioactive areas were eluted and rechromatographed in systems designed to achieve the greatest resolution of the suspected compound from similar compounds. The area of greatest interest, from the progesterone-4-14C incubation, was the peak labeled F. Identification of Cortisol (pre~ln-4-ene-1113,17a, 21-triol-3,20-dione):

Following the

elution of peak F, 50 I~gm of cortisol was added and rechromatographed in the chloroform/formamide (C/F) system; in this system the radioactivity was found to migrate with the ultra-violet absorbing area.

Following elution, this area was re-run in the follow-

ing systems in which, again, the radioactivity and UV area occupied the same position:

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80%

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HI~O-L>O~ E I O H : O . 0 5 % ( W / V ) N o C l 50%

GAMMA

CHCl 3 -50%n-HEXANE

~~

n - :50 o , BTZ e= CPM.

300-

~1=

200-

K=2.52

I00-

6

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I0

14

18

22

26

CPM .10(30

• 800

240mu • 600

• 400

• 200

30

Figure 2. Counter-current distribution of biosynthetic cortisol-4-14C and added inert cortisol. The line is the theoretical distribution curve for a partition coefficient (K) of 2.52.

chloroform (10%)-toluene/ethylene glycol (cT/EG), Bush B5, T/PG, Bush C system. After the radioactive peak and UV absorbing area were found to migrate identically in the above five systems, an acetylation reaction was carried out on an aliquot and rechrornatographed in the above systems; again, the UV area ran with the radioactivity° At this time, 10 mg of inert cortisol was added to the remaining portion and crystallized three times from aqueous methanol giving crystals containing 2958, 2914, and 2983 cpm per mg (MP 216.5 ° C, 217° C, not done third time) and final mother liquor residue having 3001 cpm per mg. A sodium bismuthate oxidation was carried out on the crystallized material; chromatography revealed two UV positive-radioactlve areas. The most polar area failed to migrate and was eluted and identified as unoxidized starting material° A less polar UV positive-radioactive area was identified as 1113-hydroxyandrostenedione (a ndrost-4-ene-1113 -o1-3 , 17-dione). Elution and crystallization with added 1113-hydroxyandrostenedione gave crystals containing 107 and 125 cpm per rag. The mother liquor contained 117 cpm per mg. The crystallized compound was chromato-

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ANALYSIS OF G.G.D. GORTISOL

400

550,

300

2

4

6

8

I0

12

14

Figure 3. C oC.D. of cortisol-4-14C. Relation between log specific activity and the tube number under the peak.

graphed in the T/PG, ligroin/propylene glycol (L/PG), methylcyclohexane/formamide (M/F) systems and failed to separate from authentic 1113-hydroxyandrostenedione. A chromic acid oxidation was carried out on the crystallized cortisol; againt two areas were located. The area remaining on the starting line was identified as unoxidized starting material and the less polar area was identified in T/PG, L/PG and M/F as IIoxo--androstenedione (androst-4-ene-3tll, 17-trione). Attempts to crystallize this area were unsuccessful° The counter-current distribution of the crystallized cortisol (third crystallization) was carried out in the 80% HOH-20O~ EtOH:0.05% (w/v) NaCI/50% CHCI3-50O~ n-Hexane system.6 The tube contents were prepared for analysis by drying in vacuo from the frozen state and dissolving the residue in ethanol. Aliquots were taken for: blue tetrazolium reaction, h max (readings at 225, 240 and 255 mla), and radioactive counting. The results, following calculation and normalization of the curves, are presented in Figure 2. A single peak of material absorbing at 240 mla as

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well as giving a positive blue tetrazolium reaction coincided with the single radioactive peak (K = 2o52)o The points agree well with the plotted theoretical curve, thus indicating no evidence of lack of homogenityo The mean specific activity in the region of the peak was 2950 cpm per rag. Further, an analysis of the CoCoDo was carried out according to Baggett and Engel7; this is presented in Figure 3. it can be seen no regression exists. At this point, the cortisol was considered radiochemically pure° Identification of Cortisone ~regn-4-ene-17a, 21-diol-3, II, 20-trione): Similarly, the peak labeled E was eluted and chromatographed in the above described systems. An acetylation product was formed, as well as the chromic acid derivative. Each radioactive derivative migrated in the appropriate systems along with the inert carrier. Three crystallizations of the biologically synthesized cortisone gave crystals with 2006, 2093, and 2055 cpm per mgo No further identification was carried out. Identification of 6~-OH-Cortisol (pre~ln-4-ene-6~, I1~, 17a,21-tetraol-3,20-dione):

Inas-

much as this compound was isolated in a very small quantity, only a few identification procedures were used° Following acetylation, the radioactive compound was found not to separate from authentic 6~-hydroxycortisol in four chromatographic systems. Also, the sodium bismuthate oxidation product failed to resolve from authentic 6{3, IlJ3-dihydroxyandrostenedione (androst-4-ene-6~, 1113-diol-3, 17-dione). No further identification was carried out° Attempted identification of Peak X. Initially, this peak was thought to be aldosterone. After several preliminary chromatograms, it was obvious that this compound was no._taldosterone. A great amount of chromatographic data of this compound, of the acetate, of the chromic acid oxidation product, as well as the sodium bismuthate oxidation product has been obtained, yet to date the identity remains unknown. Additional work currently is

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underway= Tentative iderttificatJon of remaining compounds from Progesterone-4-14C incubation: The following compounds were isolated: I) pregn-4-ene-17a,21-diol-3,20-dione (I.67 x 106 cpm); 2)pregn-4-ene-IIJ3,21-dioi-3,20-dlone (5=75 x 106.cpm); 3)pregn-4-ene-6j3--ol-3, 20-dione (0.30 x l06 cpm); 4) pregn-4-ene-17a-ol-3,20-dione (2.17 x 106 cpm); 5) pregn4-ene-3, 11,20-trione (0.11 x 106 cpm), as well as unconverted (10.15 x 106 cpm) starting material. These tentative identifications were made by admixed paper chromatograms, acetylation reactions, reductions and oxidations when appropriate. Tentative identification of remaining compounds from Androstenedione-4-14C incubation: The following compounds were isolated: I)androst-4-ene-6~-ol-3, 17-dione (0.15 x 106 cpm); 2)androst-4-ene-1713-oi-3-one (0.50 x 106 cpm); 3)androst-4-ene-3,11,17-trione (0.34 x 106 cpm); 4) and two, yet unidentified, metabolites. The most polar compoundt X', was found to represent the major conversion product (0°88 x 106 cpm) while the least polar compound, X" t was the minor product (0°04 x 106 cpm)o Unconverted androstenedione-4-14C (25.24 x 106) was also recovered. Again, these tentative identifications were made on the basis of admixed paper chromatograms, acetylation reactions, oxidations and reductions wherever appropriate=

DISCUSSION A complete review of the literature relevant to testicular tumors seems unwarranted in light of the excellent review by Savard et al 8 Fairly conclusive evidence was obtained to establish the identity of the in vitro biosynthetically prepared cortisol-4-14C. This was carried out primarily in order to establish radiochemical purity and secondarily to demonstrate the introduction of the II~t

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17a and 21-hydroxyl groups. With the establishment of these enzyme activities in the testicular tissue, then, by inference, less rigid criteria could be used for the tentative identification of other compounds containing one or more of these hydroxyl groups. From Table 2, it can be seen that more cortisone was isolated than cortisol. This implies that the 1113--oidehydrogenase was extremely active in this testicular tumor. Bush, 9 in a recent and fascinating review, has pointed out that the axial Ilia-hydroxyl group is even less stable than the typical axial groups and is far more easily and rapidly oxidized than is any other secondary hydroxyl group in the steroids° The substrate specificity of the i1~-oi dehydrogenase remains somewhat a controversy and is only poorly understood. In this incubation, no dehydrocorticosterone (pregn-4-ene-21-ol-3, l I, 20-trione) was found. [t is unexplained why the IlJ3-ol-dehydrogenase was active in converting cortisol to cortisone and yet failed to convert corticosterone into the li-dehydro-analogue° Perhaps two separate dehydrogenases are required for these two oxidations° |nterestingly, Savard 8 and Dominquez 10 failed to find any II-oxo-compounds, although several 1113-ol steroids were reported by each. In this tumor, androstenedione was converted to II-oxo--androstenedione, yet, no II~-ol intermediate could be identified. The polar compound, as yet unidentified and referred to as X', was conclusively shown to be a compound other than I I[~-hydroxyandrostenedione. Further, this tumor again differs from those studied by Savard e.t a l and Dominquez as the present incubation did indeed produce I I~-hydroxyiated compounds from progesterone. The former authors suggested that two different 11(3-hydroxylases may exist, i . e . , one each for the CI9 and C21 steroids. Dominquez reported, however, when ll-deoxycorticosterone (pregn-4-ene-21-ol-3,20-dione) was utilized as the substrate, small but

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Table 2. Labeled compounds isolated from the progesterone-4-14C incubation Compound Formeda Cortisol Cortisone X 6~-OH-Cortisol Reichstein S Corticosterone 6~-OH-Progesterone 17a-OH-Progesterone I I-oxo-Progesterone Progesterone

Total cpm x 106 0.60 4.27 1.32 0.06 1.67 5.75 0.30 2.17 0. II lO. 15

per cent converted 0.022 15.55 4.80 0.002 6.07 20.90 0.01 7.90 0.004 (41,80)

Production b 10-8 Moles 0.025 17.50 5 °40 0.002 6.83 23.51 0.011 8.89 0.005 unconverted

a) only cortisol and cortisone were extensively identified; the remaining compounds were only tentatively identified as reported in the text. b) product of 0.5 gm of tissue during a 3 hr incubation.

significant amounts of corticosterone were identified. This tumor, in vitro, did not synthesize 17-oxo-steroids from progesterone; it is likely that the urinary 17-oxo-steroids represent both nontumorous testicular, as well as hepatic, metabolism of endogenous substrateso Although this patient had a bilateral subtota I adrena lectomy previous to this testicu iar tumor, it is possible the remaining adrenal tissue accounted for the approximate i.0 mg of urinary dehydroepiandrosterone after surgery. This may represent the autonomous secretion of this tissue, as ACTH failed to elicit a response; the excess 1.0 mg of the 3~-ol-17-oxo--steroid prior to surgery may represent the tumor contribution. That the testicular tumor was contributing to the urinary 17-hydroxycorticoid values was evidenced by the prompt decrease in the values following surgery; this is in keeping with the marked clinical improvement postoperatively. 2 Perhaps further evidence against a direct in vivo production of i7-keto-

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steroids may be obtained from the work of Axelrod and Goldziehero Ii They report evidence to substantiate their theory that reduction of the C-20 ketone must precede the scission of the side chain, after the 17a-hydroxyl group has been introduced. We found neither reduced form Ca or 13). This is in marked contrast to Dominquez who reported both 20a- and 2(~-reductase activity. The former was predominate in competing for C-20 keto-reductions o Histologists have long considered the presence of crystailoids of Reinke as specific identification for Leydig Cells. 12 These crystalloids have been seen in hilus cell tumors of the ovary, but have never been described in adrenocortical tissue. No histological evidence for the presence of these crystalloids could be found in this testicular tumor, thus strongly suggesting that this tumor developed from adrenocortical cell rests= As discussed in the clinical paper, these rests remained dormant until elevated levels of ACTH induced hyperplasia and promoted steroidogenesis resulting in Cushingoid clinical changes. These clinical changes were reversed after surgery° In a previous paper 13 we confirmed the advantages of biochemical means for the identification of a tumor, originally suggested by Sandbergo14 The present paper reports a tumor which was originally described as an undifferentiated tumor of the testis= By biochemical means, however, it was possible to show that this tissue was capable of synthesizing cortisol, and could be considered an adrenocarticai tumor. We consider this evidence further proof of the validity and value of this method of tumor characterization o

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REFERENCES I. This work was supported in part by the following grants: USPHSGrant 2A51184C5)4d) from The National Institute of Arthritis and Metabolic Diseases; The Clinical Research Unit under Grant AM-05078-02, USPHS; and by an Institutional Grant from the American Cancer Society, 1416-E. This is communication number 28 from the Steroid Research Laboratories of the Department of Obstetrics and Gynecology; we wish to thank Dr. J. C. Ullery, Chairman, for his continued support of this project° Portions of this study have been reported in abstract form in: EXCERPTA ME DICA (.international Congress Series)51, 236 41962); ABSTRACT ENDOCRINE SOCIETY, 44th MEETING, NOo 116 41962); and CLINICAL RESEARCH 10, 297 41962)° 2° Hamwi, Go Jo, Gwinup, G., Mostow, J. Ho and Besch, P. K., J. CLIN. ENDOCRINOL. (in press)= 3o Watson, Do J. and Besch, P. Ko, CLINICAL CHEMISTRY 8, 284 (.1962)o 4= A preliminary incubation with 0.2 HC of progesterone-4-14C demonstrated many conversion products. It was felt that by using a very large amount of radiolabeled starting material, these various metabolites would afford, after purification, radiolabeled steroid standards not otherwise available to this laboratory° 5o Besch, Po K., Brownell, Ko A., Hartman, Fo A. and Watson, Do J., ACTA ENDOCRINOL. 39, 355 (1962)o 6° Carstensen, Ho, ACTA SO2. MED. UPSALo 61, 26 (,1956)o 7= Bagger,, Bo and Engel, LoLo, Jo BIOL. CHEM. 229, 443 (1957)o 8= Savard, Ko, Dorfman, Ro Io, Baggett, B., Fielding, Lo Lo, Engel, Lo Lo, McPherson, H. T., Lister, Lo Mo, Johnson, D. So, Hamblen, Eo Co, and Engel, F= L., J. CLIN. INVEST. 39, 534 (1960)o 9. Bush, Io Eo, PHARMACOLo REV. 14, 317 (1962)o I0o Dominquez, Oo V., J. CLIN. ENDOCRINOLo 21, 663 (1961)= I1o AxeJrod, L. Ro and Goldzieher, J o W., J. CLIN. ENDOCRINOL. 22, 431 (1962). 12. Reinke, Fo, ARCH. MIKR. ANATo 47, 34 41896). 13o Besch, P. Ko, Byron, R. C., Barry, Ro D., Teteris, No J o, Hamwi, G° J°, Vorys, No and Ullery, J o C=, AM. J. OBST. AND GYNEC. (in press)° 14o Sandberg, A= Ao, Slaunwhite, W° Ro, Jackson, J= Eo and Frawley, To Fo, J° CLINo ENDOCRINOLo 22, 929 (,1962).