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Metabolism of progesterone and testosterone by chick embryo cartilage in vitro
BIOCHIMICA
BB*
ET BIOPHYSICA
ACTA
347
55299
METABOLISM EMBRYO
CARTILAGE
SEI-ITSU Sational
OF PROGESTERONE
MLTROTA** Institute
(Received
IN
September
TESTOSTERONE
BY
CHICK
VITRO”
AND RUN-ICHI
of Radiologicnl
AND
Sciences,
TAMAOKI
Chiba-shi
(Japan)
5th, 1966)
SUMMARY
Epiphyses of femora and tibiae obtained from I3-day-old chick embryos homogenized and incubated with either [4-Xlprogesterone or [4-Xltestosterone
were in
the presence of an NADPH-generating system at 38” for 60 min. The metabolites produced were identified by thin-layer chromatography, chemical transformation and crystallization studies. Progesterone was actively converted to $-pregnane-3cc,zo,%diol via $-pregnane-3,zo-dione and then 3a-hydroxy-$-pregnan-zoone.Testossteronewas metabolized to s/3-androstane-3a,z@diol via IT/?-hydroxy-$-androstan3-one. From these results, it was concluded that embryonic cartilage possesses enzymic activities for the catabolism of the sex steroid hormones in the presence of an NADPH-generating system, and that the configuration of the reduced of progesterone and testosterone was limited to 3a-OH and 58-H.
metabolites
INTRODUCTION
that
Concerning the effect of steroid hormones corticosteroids and sex steroid hormones
in connective tissue, it was reported specifically affected the growth of
cartilage in vitrol. On the other hand, it was demonstrated that chick embryonic cartilage actively converted cortisol to dihydrocortisol, tetrahydrocortisol and I@hydroxyandrostenedione2,8; the effects of cortisol and its metabolites on the growth of cultured bone were also examined3. In this paper, progesterone and testosterone were incubated with homogenate of chick embryo cartilage, and the metabolites thus produced were identified. This is the first report of a demonstration of the metabolism of sex steroid hormones by cartilage. * The fcllowing trivial names have been used in this text: cortisol, rrg,r7cc,21-trihydroxypregn-4I I/J,I 7cc,zI-trihydroxy-$-pregnane-j,zo-dione ; tetrahydrocorene-3,zo-dione ; dihydrocortisol, tisol, 3a.1 I/I, 17a,zr-tetrahydroxy-#-pregnan-zo-one; progesterone, pregn-4-ene-3,zo-dione; testosterone, 17/&hydroxyandrost-+en-3.one. ** Ph. D. Fellow of the University of Tokyo.
B&him.
Biophys.
Acta,
137 (1967) 347-355
S. MUROTA,
348 MATERIALS
B. TAMAOKI
AND METHODS
Radioactive steroids [4-‘4C]Progesterone
(specific activity, 146.5 &/mg) and [4-r4C]testosterone activity, 76.5 &/mg) were purchased from New England Nuclear Corp. Mass., U.S.A.). The radiochemical purity of these steroid preparations was
(specific (Boston, confirmed Tissue
13
by thin-layer
preparation
chromatography
shortly
before use.
ami incubation
Epiphyses of femora and tibiae (0.25 g), obtained from S chick embryos aged days, were homogenized in 4.2 ml of ice-cold Gey’s balanced saline solution4 with
a tight-fitting Teflon-glass homogenizer. After centrifugation of the homogenate at 8ooxg for IO min, 4 ml of the supernatant was mixed with I ml of Gey’s solution containing either [4-*4C]progesterone (0.4. pug, 0.06 ,uC) or i4-14C]testosterone (0.8 pg, 0.06 PC) and incubated at 38” for 60 min with continuous shaking. Examination of an aliquot of the centrifuged homogenate under the microscope showed that it contained practically no unbroken cells. As cofactor-generating system, 3 mg glucose 6-phosphate and 2 Kornberg units of glucose-6-phosphate were added to the flasks. The gas phase was maintained
as a mixture
1.6 mg NADP+, dehydrogenase of 95s/, 0, and
5% CO,. Extraction of the metabolites radioactive metabolites were extracted 3 times Immediately after incubation, with 5 vol. of dichloromethane. The combined extracts were dried over anhydrous Na,SO,, filtered through glass wool and concentrated under reduced pressure at 40’. More than 90% of the radioactivity initially added was recovered at this stage. Separation
of the metabolites
by thin-layer
Thin layers of a 4: I mixture stadt, Germany)
about 025mmin
chromatography
of silica gel G and GF,,,,
thickness,
(E. Merck, A.G., Darm-
werepreparedonglassplates(5
cm x zocm)
using a spreading device. An aliquot of the extract was applied to the thin-layer plate horizontally as a line 3-4 cm in length with a special pipette 2.5 cm above the lower edge of the plate. The plate was placed in a cylindrical glass container with a groundglass cover, containing the developing solvent to a depth of 5-7 mm and developed at 15-zo”, after saturation. The solvent system generally used was benzene-ethyl acetate-acetone (8 : 2 : I, by vol.). Detection of the steroids on the chromatograms Zones of the radioactive metabolites, on the thin-layer chromatograms, were detected with a windowless gas-flow counter equipped with an automatic scanning device (Vanguard 880, Technical Measurement Corp., North Haven, Conn., U.S.A.), and also by taking autoradiograms with X-ray films. The areas of ultraviolet-absorbing steroids were detected under an ultraviolet lamp. Other steroids added as carriers of the radioactive products in chemical transformation procedures were detected using iodine vapor at room temperature and phosphoric acid at 110’ for 5 min.
Biochim.
Biophys.
Ada,
137 (1967)
347-355
METABOLISM
OF SEX STEROIDS
349
IN CARTILAGE
Measurement of the radioactivity The silica gel scraped from the areas of the radioactive
steroids
on the chroma-
tograms detected by radioautography was packed into small glass tubes. The radioactive steroids were eluted exhaustively with a I: I mixture of ethanol and chloroform. Recovery was in the range of 90-95°/0 of the radioactivity applied to the thinlayer plates. The radioactivity of an aliquot of the extracts was measured with a liquid scintillation spectrometer (Nuclear Chicago Corp., Des Plaines, Ill., U.S.A.) in the conventional toluene-PPO-POPOP system. The approximate efficiency of the counting was 70, O’o for radiocarbon. IdentiJicatiola of the rudioactive metabolites An aliquot of the isolated metabolites was mixed with the respective authentic steroids, and thin-layer chromatography was carried out using various solvent systems. A second aliquot was subjected to acetylation with a I :I (by vol.) mixture of pyridine and acetic anhydride, and a third to oxidation with 0.5% CrO, in 90”/; acetic acid, in the usual manner. The chromatographic behavior of the derivatives obtained was compared with that of authentic preparations. The identity of the radioactive products with the carrier steroids was established on the basis of identical mobilities and shapes of the spots detected by radioautograms with those detected by color reactions. Further details of the method of extraction are described in previous papers2+ys.
and identification
of the products
RESULTS
Metabolism of 1@YJ@ogesterone and [$‘“C]testosterone in embryonic cartilage Initial thin-layer chromatography (benzene-ethyl acetate-acetone, 8: 2 :I, by vol.) of the extract from the cartilage homogenate incubated with either 4-l%progesterone or [4-1%]testosterone in the presence of an NADPH-generating system gave a chromatogram showing 3 zones of radioactivity (Fig. I). The RF’s of the radioactive metabolites
of [4-14C]progesterone
were: 0.72 (PI), 0.46 (PJ
and 0.24 (PJ,
while
those of the metabolites of [4-Xltestosterone were: 0.40 (T,), 0.34 (TJ and 0.20 (T3), respectively. Table I shows the dependence of the bioconversion on the cofactor and summarizes the results of quantitative determinations of metabolites. Identification of the metabolite Pl as 5P-preglcane-3,2o-diolze The area designated as P, in Fig. I was extracted and purified by repeated thin-layer chromatography in the benzene-ethyl acetate-acetone (8: 2: I, by vol.) system. P, was neither acetylatable nor oxidizable by the procedures previously mentioned. From its chromatographic behavior, it seemed that P, could be 5a- or $L pregnane-3,zo-dione or a mixture of both. An aliquot of P, was mixed with authentic 5cr- and $Lpregnane-3,2o-dione and chromatographed in a thin layer of silica gel using an pz-heptane-ethyl acetate (5 : 2, by vol.) as solvent system. Clear separation of ga-pregnane-3,zo-dione (RF 0.20) from the $-epimer (RF 0.17) was observed, using the reversible iodine reagent. The radioactivity ot P, was exclusively located in the area of $&pregnane-3,2o-dione. An aliquot of P, was mixed with an authentic preparation of $-pregnane-3,zo-dione and crystallized 4 times from 4 different solBiochim.
Biophys.
4cta,
137 (1967) 347-355
S. MUROTA, B. TAMAOKI
350
Fig. I. Autoradiogram of the thin-layer chromatogram of the radioactive metabolites of [4-14C]progesterone (left) and [4J%jtestosterone (right) obtained from incubated homogenate of chick embryonic cartilage in the presence of an NADPH-generating system at 38” for 60 min. P,, 58. pregnane-3,zo-dione; I’,, 3cr-hydroxy-5B-pregnan-2o-one; P,, 5p-pregnane-3cr,2o/%diol; T,, 17phydroxy-5p-androstan-3-one; T,, unknown; T,, 5/l-androstane-3cr, I 7/Sdiol. (P) and (T) indicate the positions of progesterone and testosterone, respectively.
TABLE
I
QUANTITATIVE
ANALYSIS
OF
METABOLITES
OF
PROGESTERONE
AND
TESTOSTEROXE
IN
CARTILAGE
Homogenate of epiphyses obtained from I3-day-old chick embryo femora and tibiae was incubated with either [4-l%]progesterone or [4-‘Kltestosterone in the presence of an NADPH-generating system at 38” for 60 min. Values for each product are expressed as per cent of the total of metabolites and recovered substrate. (P), recovered progesterone; P,,5,%pregnane-3.20.dione; P,-3w hydroxy-5/Gpregnan-2o-one; P,,5~-pregnane-3cr.zop-diol; (T), recovered testosterone ; T,, I 7/G hydroxy-5P-androstan-3-one; T,, unknown; and T,, 5/?-androstane-3a,I7,%diol. ~~___ Substrate
Metabolite
Progesterone
P, P,
4.4 87.8 7.7 Trace
37.5 Nil Nil 62.5
Testosterone
T, T* T, (T)
23.5 11.3 65.1 Trace
Nil Nil Nil IOO.0
NA DPH-generating system ~___ PYeSe?Ke Absence
-
Biochim.
Biophys.
Acta.
137 (1967)
347-355
METABOLISM OF SEX STEROIDS IN CARTILAGE
vent mixtures. constant,
As shown in Table
within the counting
351
II, the specific activities
error, throughout
of the crystals
remained
the 4 crystallizations.
Identi$cation of the metabolite P, as 3cr-hydroxy-$i-pregnan-zo-one The radioactive substance designated as P, (Fig. I) was acetylatable and oxidizable. The mobility of the oxidized product on thin-layer chromatography showed TABLE
the same
value
as for P, already
identified
as 5/?-pregnan-3,zo-dione.
An
11
IDENTIFICATION
OF
Crystallization
SUBSTANCE
Pl
ANDP,
BY
CRYSTALLIZATIOK
Substance P?
Substance P, Carrier
steroids
5P-Pregnane-3,2o-dione Solvent
___~_
_~_ Specific activity (counts/
-___
_______~ 3a-Hydroxy-5~-pregnan-zo-olze ~ Solvent
(counts/min
mnin per mg)
Dichloromethane-n-heptane Benzene-ethanol Chloroform-methanol Benzene-n-heptane
1st
2nd 3rd 4th Average
f
62.8 = 0.6
S.E.
aliquot
Per m&T)
Dichloromethane-n-heptane Benzene-n-heptane Diethyl ether-n-heptane Chloroform-n-heptane
60.9 63.4 63.5 63.2
_~~____ Specafic activity
98.; 93.5 92.7 93.6 94.6 _+ 1.4 .~
of P, was mixed with 3a-hydroxy-5P-pregnan-zo-one
and its $Lepimer,
and
chromatographed using benzene-ethyl acetate-acetone (8: z : I, by vol.) and n-heptane-ethyl acetate (5 : 2, by vol.) systems; 3cr-hydroxy-5/I-pregnan-zo-one (RF, 0.46 and 0.20) and its j/3-epimer (RF, 0.56 and 0.29) were separated from each other in both systems. The radioactivity of P, was mainly located in the area of SE-hydroxy5,%pregnan-2o-one. After addition of carrier SE-hydroxy-58-pregnan-zo-one, crystallizations were performed from 4 different solvent mixtures and the specific activities of the crystals
were constant
(Table
II).
Identi$cation of the metabolite P, as 5B-Preglaane-3cc,zoB-diol The radioactive substance designated as P, in Fig. I was also acetylatable and oxidizable, and the oxidized product had a chromatographic mobility identical to 5/Spregnane-3,2o-dione. Because of its higher polarity than 3a-hydroxy-5/!-pregnan2o-one (P2), P, appeared to be either 5/Lpregnane-3n, zoa-diol or its ao/Gepimer. An aliquot of P, was mixed with 5/3-pregnane-3a,2oa-diol and its zofi-epimer, and chromatographed in benzene-ethyl acetate-acetone (8 : 2 : I, by vol.) and benzene-chloroform-diethyl ehter-methanol (2 : 2 : I : I, by vol.) systems, where 5/Lpregnane3a,zoudiol (RF, 0.19 and 0.45) was separable from the ao,&epimer (RF, 0.24 and 0.50). The radioactivity of P, was localized exclusively in the spot of carrier 5/Y-pregnane-3a,zo/Sdiol, which was detected by reversible iodine reagent. Crystallization of P, with added carrier 5B-pregnane-3cr,zo#Ldiol from 4 different solvent mixtures gave crystals of constant specific activity (Table III). Identi$cation of the metabolite T, as I7,!3-hydroxy-5/3-androstan-3-one The radioactive substance designated as T, (Fig. I) was acetylatable and oxidizable, and the oxidized product showed similar polarity to 5cr- or $-androstaneRiochim.
Biopltys. Acta, 137 (1967)
347-355
S. MUROTA, B. TAMAOKI
352 TABLE
III
IDENTIFICATIONOF SUBSTANCE
P,
BY
CRYSTALLIZATION
Substance P, ~ ...___. Carviev steroi~P~~ne-30(,2og-diol
1st 2nd 3rd 4th A\-wage
Solvent
Specific activity (countslmin per mg)
Methanol-chloroform Ethyl acetate-n-heptanc Acetone-cyclohexane Ethanol-dichloromethane
248 265 254 255
I S.E.
255.5 m’m 3.5
3,r7-dione (RF, 0.70) on thin-layer chromatography using a benzene-ethyl acetateethanol (8 : 2 : I, by vol.) system. Separation of 5a-androstane-3,r7-dione from its 5/1’epimer by thin-layer chromatography was possible using an n-heptane-ethyl acetate (5 :z, by vol.) system (RF, 0.28 and 0.25, respectively). The radioactivity of the oxidized product of T, was concentrated mainly at the location of carrier 5,%androstane-3,r7-dione. An aliquot of T, was mixed with r7/3-hydroxy-5@-androstan-3-one and chromatographed in the benzene-ethyl acetate-acetone (8 : 2 : I, by vol.) solvent system. Aliquots of the mixture were either acetylated or oxidized, and chromatographed as mentioned above. In each case, carrier steroid could not be obtained free from radioactivity. Crystallisation of a further aliquot of the mixture with an authentic preparation of 17/Ghydroxy-5/&androstan-3-one, gave crystals of constant specific activity (Table TABLE
from 4 different solvent mixtures, IV).
I\
IDENTIFICATION
OF
SUBSTANCE
T,
AND
T, BY
CRYSTALLIZATION
.-__
Crystallization
Substance T,
Substance T,
Carrier sterozd~~-~-___ ..~ r7P-Hvdroxy-5~-androstan-3-one Solvelzt
Specific activity (countslmin
Solve?at
Dichloromethane-n-heptane Benzene-cyclohexane Chloroform-n-heptane Ethyl acetate-cyclohexane
2nd 3rd 4th i\verage + S.E.
_______.-
____
241 237 236 236 237.5 :1 1.1
Specific activi1.v (countslmin
Per msl
Pev w)
_.~ 1st
-
____~______. 5B-Androstane-3a,I7P-diol
Ethanol-dichloromethane Acetone-cyclohexane Methanol-chloroform Ethyl acetate-n-heptanc ~~_
___
249 247 247 247 247.5 _t 0.5
Identijkation of the metabolite T, as 5B-androstane-3cr,I7B_diol The radioactive substance designated T, in Fig. I was also acetylatable and oxidizable. The behavior of the oxidized product of T, on thin-layer chromatography developed with an s-heptane-ethyl acetate (5:z, by vol.) solvent system, indicated that the derivative was 5/$androstane-3,r7-dione. An aliquot of T, was mixed with 5p-androstane-3a,r7@-diol and its 3p-epimer, and chromatographed using the benzene-ethyl acetate-acetone (8: 2 : I, by vol.) system, where 5/3-androstane-3a,r7/Gdiol (RF, 0.20) was separable from its 3/?-epimer (RF, 0.35). The radioactivity of T, was located exclusively in the area of 5/3-androstane-3cr,r7/Ldiol. An aliquot of T, was mixed with 5/Landrostane-3a,r7P-diol, and aliquots of the mixture were subjected Biochim. Biophys.
Acta, 137 (1967) 347-355
METABOLISMOF
SEX STEROIDSIN
353
CARTILAGE
to acetylation or oxidation, and the products were chromatographed in the benzeneethyl acetate-acetone (8 : z : I, by vol.) system. On every thin-layer chromatogram, the mobility as well as the shape of the radioactive spot, detected by radioautography, completely coincided with that of carrier steroid. Crystallization of another aliquot of the mixture from 4 different solvent mixtures gave constant specific activity (Table IV). The metabolite T,, had an almost identical polarity to 3cr-hydroxy-@-androStan-IT-one but repeated crystallizations with an authentic preparation did not give constant specific activity. A scheme for the catabolic pathways of progesterone and testosterone in chick embryo cartilage is shown in Scheme I, together with that of cortisol, established previously2T3. Cortim
0
&/
0
&
H
=0 ---OH
&/ HO
/
0
’
I
i C+b c-o
0
CH*OH
& LIP I 0
H
4
H
H
HO
CH>OH
I
C=O
---OH
LtP
\
0
0
H
CH3
C=O 0
CIIP
HO”
& H
’
H
C-3
OH-C-H
Scheme I. A scheme of the metabolic pathways of from the previous papers2,3) in embryonic cartilage
progesterone,testosterone and cortisol (cited
DISCUSSION
Few reports are available on the metabolism of steroid hormones in peripheral tissue. THOMAS AND DORFMAN' demonstrated 3/3-and I@hydroxysteroid dehydrogenase and A4-gcr-hydrogenase in skeletal-muscle strip of rabbit with androst-d-eneB&him.
Biophys.
Acta,
137
(1967) 347-355
S. MUROTA,B.TAMAOKI
354
3,r7-dione in vitro. BERLINER AND DOUGHERTY~ studied the metabolism of corticosteroids and progesterone in fibroblasts, and reported that metabolism of cortisol was mainly conducted in fibroblasts, except for liversa*O. In this experiment the predominant cells found in the epiphyses used were chondrocytes; adhering connective tissue and periosteum were removed in preparation. The authors demonstrated previously that homogenate tisol to tetrahydrocortiso12
or cultured chick-embryo cartilage actively converted corand now chondrocytes have been found to have an enzyme
system capable of metabolizing sex steroid hormones. It seems that there is a definite pattern to the catabolism of steroid hormones in embryonic cartilage, since, in every case, the functional A4-3-keto group, which is common to progesterone, testosterone and cortisol, uniformly underwent enzymic reduction to the corresponding 3a-hydroxy-$-steroid. The results indicate that, under the conditions used ill m&o, the cartilage tissue had no significant 3P-hydroxysteroid dehydrogenase or A’-ju-hydrogenase activity, but did have 3a-hydroxysteroid dehydrogenase and A”+hydrogenase activity. It has been reported that in liver tissue A4-gcr-hydrogenase is located in the microsomes” and A4-$6hydrogenase in the soluble phase 12, suggesting that these two enzymes show differences in their intracellular distribution and occurrence. It is noteworthy that the embryonic cartilage contained 3a-hydroxysteroid dehydrogenase and A”-5/%hydrogenase but not the 3/G and 5cr-enzymes, indicating the organ specificity of steroid catabolism. U’ith regard to this point, DOUGHERTY, BERLINER ASD BERLINEP have shown the dependence of cortisol metabolism on cell type and organ; for example reticuloendothelial cells of liver show predominantly ‘-2 ring reduction rather than C-zo-keto reduction, whereas those of adrenal show predominantly C-zothat throughout the metabolism of keto reduction. KAPPAS et al. 13$15demonstrated
all C,, and C,, steroids, only those of the 3~OH and 51-H type have specific thermogenie effects. Recently C,,, C,, and C,, steroids with a $-H configuration were also said to disrupt lysosomes and to release 70+80:< of total hydrolase activity from liver granules in vitro, whereas no Sa-isomer solubilized the enzymesl6. These facts suggest that among the metabolites so far regarded as inactive there may be some having new biological activities not present in the original steroid hormone. The authors reported (ref. I) that progesterone inhibited [%]sulfate incorporation into chondroitin sulfate, per unit dry weight of chick embryo femora growing in vitro, whereas the reverse effect was observed with testosterone. The results suggest that sex steroid hormones may have some direct action on cartilage growth. However, whether or not the metabolites of progesterone and testosterone identified in these experiments retained biological activity was not established. In the case of cortisol, the authors have demonstrated that the metabolites of cortisol, are without effect on cartilage growths. ACKNOWLEDGEMENT
The authors
are most
grateful
to Dr. M. SHIKITA
of this laboratory
for his
invaluable advice and discussion.
REFERENCES I H. ENDO AND S. MUROTA, I‘issue Culture, Dr. W. Junk, The Hague, Netherlands, 1965, p. 31. 2 S. MUROTA, M. SHIKITA AND B. TAMAOKI, Biochim. Biophys. Acta. IIT (1966) 421.
3 S.
MUROTA, H. ENDO AND B. TAMAOKI,
Biochim.
Biophys.
,4&a,
137 (1967)
347-355
B&him.
Biophys.
Acta,
136 (1967)
379,
METABOLISM 4 5 6 7 8 g 10 II. 12 ~3 14
OF SEX STEROIDS
IN CARTILAGE
355
ENDO, Eq%l. Cell Res., 21 (1960) 151. SHIKITA, H. KAKIZAKI AND B. TAMAOKI, Steroids, 4 (1964) 521. SHIKITA AND B. TAMAOKI, ~~~c~e~~s~~~, 4 (1965) 1189. Z. THOMAS AND R. I. DORFMAN, J. Biol. Chem., 239 (1964) 762. L. BERLINER AND T. F. DOUGHERTY, Ann. N.Y. Acad. Sci., 88 (1960) 14. F. DOUGHERTY, M. L. BEKLINER AND D. L. BERLINER, ~~~oc~~no~agy, 66 (1960) 550. F. DOUGHERTY AND D. L. BERLINER, in I. PAGE, Connective Tissue, Thrombosis and Athevosclerosis, Academic Press, New York, ‘959. p. 143. J, S. MCGUIRE AND G. M. TOMKINS, J. Biol. Chenz., 235 (1960) 1634. G. M. TOMKINS, J. Biol. Chem., 225 (1957) 13. T. F. DOUGHERTY, D. L. BERLINER AND M. L. BERLINER, ~~etaboZ~~m, IO (1961) 966. _a. KAPPAS, L. HELLMAN, D. K. FUKUSHIMA ANI) T. F. GALLAGHER, J. C&n. Enrlocrinol. Metab.,
H. M. M. P. D. T. T.
16 (1956) 948. 15 A. K~\PP~s AND R. H. PALMER, Pharm. Reviews, 15 (1963) 123. rG G. WEISSMANN AND L. THOMAS, Recent Progr. Hormone Res., 20 (1964) 215. Biochim. Biophys.
Acta, 137 (1967) 347-355
BB*
ET BIOPHYSICA
ACTA
347
55299
METABOLISM EMBRYO
CARTILAGE
SEI-ITSU Sational
OF PROGESTERONE
MLTROTA** Institute
(Received
IN
September
TESTOSTERONE
BY
CHICK
VITRO”
AND RUN-ICHI
of Radiologicnl
AND
Sciences,
TAMAOKI
Chiba-shi
(Japan)
5th, 1966)
SUMMARY
Epiphyses of femora and tibiae obtained from I3-day-old chick embryos homogenized and incubated with either [4-Xlprogesterone or [4-Xltestosterone
were in
the presence of an NADPH-generating system at 38” for 60 min. The metabolites produced were identified by thin-layer chromatography, chemical transformation and crystallization studies. Progesterone was actively converted to $-pregnane-3cc,zo,%diol via $-pregnane-3,zo-dione and then 3a-hydroxy-$-pregnan-zoone.Testossteronewas metabolized to s/3-androstane-3a,z@diol via IT/?-hydroxy-$-androstan3-one. From these results, it was concluded that embryonic cartilage possesses enzymic activities for the catabolism of the sex steroid hormones in the presence of an NADPH-generating system, and that the configuration of the reduced of progesterone and testosterone was limited to 3a-OH and 58-H.
metabolites
INTRODUCTION
that
Concerning the effect of steroid hormones corticosteroids and sex steroid hormones
in connective tissue, it was reported specifically affected the growth of
cartilage in vitrol. On the other hand, it was demonstrated that chick embryonic cartilage actively converted cortisol to dihydrocortisol, tetrahydrocortisol and I@hydroxyandrostenedione2,8; the effects of cortisol and its metabolites on the growth of cultured bone were also examined3. In this paper, progesterone and testosterone were incubated with homogenate of chick embryo cartilage, and the metabolites thus produced were identified. This is the first report of a demonstration of the metabolism of sex steroid hormones by cartilage. * The fcllowing trivial names have been used in this text: cortisol, rrg,r7cc,21-trihydroxypregn-4I I/J,I 7cc,zI-trihydroxy-$-pregnane-j,zo-dione ; tetrahydrocorene-3,zo-dione ; dihydrocortisol, tisol, 3a.1 I/I, 17a,zr-tetrahydroxy-#-pregnan-zo-one; progesterone, pregn-4-ene-3,zo-dione; testosterone, 17/&hydroxyandrost-+en-3.one. ** Ph. D. Fellow of the University of Tokyo.
B&him.
Biophys.
Acta,
137 (1967) 347-355
S. MUROTA,
348 MATERIALS
B. TAMAOKI
AND METHODS
Radioactive steroids [4-‘4C]Progesterone
(specific activity, 146.5 &/mg) and [4-r4C]testosterone activity, 76.5 &/mg) were purchased from New England Nuclear Corp. Mass., U.S.A.). The radiochemical purity of these steroid preparations was
(specific (Boston, confirmed Tissue
13
by thin-layer
preparation
chromatography
shortly
before use.
ami incubation
Epiphyses of femora and tibiae (0.25 g), obtained from S chick embryos aged days, were homogenized in 4.2 ml of ice-cold Gey’s balanced saline solution4 with
a tight-fitting Teflon-glass homogenizer. After centrifugation of the homogenate at 8ooxg for IO min, 4 ml of the supernatant was mixed with I ml of Gey’s solution containing either [4-*4C]progesterone (0.4. pug, 0.06 ,uC) or i4-14C]testosterone (0.8 pg, 0.06 PC) and incubated at 38” for 60 min with continuous shaking. Examination of an aliquot of the centrifuged homogenate under the microscope showed that it contained practically no unbroken cells. As cofactor-generating system, 3 mg glucose 6-phosphate and 2 Kornberg units of glucose-6-phosphate were added to the flasks. The gas phase was maintained
as a mixture
1.6 mg NADP+, dehydrogenase of 95s/, 0, and
5% CO,. Extraction of the metabolites radioactive metabolites were extracted 3 times Immediately after incubation, with 5 vol. of dichloromethane. The combined extracts were dried over anhydrous Na,SO,, filtered through glass wool and concentrated under reduced pressure at 40’. More than 90% of the radioactivity initially added was recovered at this stage. Separation
of the metabolites
by thin-layer
Thin layers of a 4: I mixture stadt, Germany)
about 025mmin
chromatography
of silica gel G and GF,,,,
thickness,
(E. Merck, A.G., Darm-
werepreparedonglassplates(5
cm x zocm)
using a spreading device. An aliquot of the extract was applied to the thin-layer plate horizontally as a line 3-4 cm in length with a special pipette 2.5 cm above the lower edge of the plate. The plate was placed in a cylindrical glass container with a groundglass cover, containing the developing solvent to a depth of 5-7 mm and developed at 15-zo”, after saturation. The solvent system generally used was benzene-ethyl acetate-acetone (8 : 2 : I, by vol.). Detection of the steroids on the chromatograms Zones of the radioactive metabolites, on the thin-layer chromatograms, were detected with a windowless gas-flow counter equipped with an automatic scanning device (Vanguard 880, Technical Measurement Corp., North Haven, Conn., U.S.A.), and also by taking autoradiograms with X-ray films. The areas of ultraviolet-absorbing steroids were detected under an ultraviolet lamp. Other steroids added as carriers of the radioactive products in chemical transformation procedures were detected using iodine vapor at room temperature and phosphoric acid at 110’ for 5 min.
Biochim.
Biophys.
Ada,
137 (1967)
347-355
METABOLISM
OF SEX STEROIDS
349
IN CARTILAGE
Measurement of the radioactivity The silica gel scraped from the areas of the radioactive
steroids
on the chroma-
tograms detected by radioautography was packed into small glass tubes. The radioactive steroids were eluted exhaustively with a I: I mixture of ethanol and chloroform. Recovery was in the range of 90-95°/0 of the radioactivity applied to the thinlayer plates. The radioactivity of an aliquot of the extracts was measured with a liquid scintillation spectrometer (Nuclear Chicago Corp., Des Plaines, Ill., U.S.A.) in the conventional toluene-PPO-POPOP system. The approximate efficiency of the counting was 70, O’o for radiocarbon. IdentiJicatiola of the rudioactive metabolites An aliquot of the isolated metabolites was mixed with the respective authentic steroids, and thin-layer chromatography was carried out using various solvent systems. A second aliquot was subjected to acetylation with a I :I (by vol.) mixture of pyridine and acetic anhydride, and a third to oxidation with 0.5% CrO, in 90”/; acetic acid, in the usual manner. The chromatographic behavior of the derivatives obtained was compared with that of authentic preparations. The identity of the radioactive products with the carrier steroids was established on the basis of identical mobilities and shapes of the spots detected by radioautograms with those detected by color reactions. Further details of the method of extraction are described in previous papers2+ys.
and identification
of the products
RESULTS
Metabolism of 1@YJ@ogesterone and [$‘“C]testosterone in embryonic cartilage Initial thin-layer chromatography (benzene-ethyl acetate-acetone, 8: 2 :I, by vol.) of the extract from the cartilage homogenate incubated with either 4-l%progesterone or [4-1%]testosterone in the presence of an NADPH-generating system gave a chromatogram showing 3 zones of radioactivity (Fig. I). The RF’s of the radioactive metabolites
of [4-14C]progesterone
were: 0.72 (PI), 0.46 (PJ
and 0.24 (PJ,
while
those of the metabolites of [4-Xltestosterone were: 0.40 (T,), 0.34 (TJ and 0.20 (T3), respectively. Table I shows the dependence of the bioconversion on the cofactor and summarizes the results of quantitative determinations of metabolites. Identification of the metabolite Pl as 5P-preglcane-3,2o-diolze The area designated as P, in Fig. I was extracted and purified by repeated thin-layer chromatography in the benzene-ethyl acetate-acetone (8: 2: I, by vol.) system. P, was neither acetylatable nor oxidizable by the procedures previously mentioned. From its chromatographic behavior, it seemed that P, could be 5a- or $L pregnane-3,zo-dione or a mixture of both. An aliquot of P, was mixed with authentic 5cr- and $Lpregnane-3,2o-dione and chromatographed in a thin layer of silica gel using an pz-heptane-ethyl acetate (5 : 2, by vol.) as solvent system. Clear separation of ga-pregnane-3,zo-dione (RF 0.20) from the $-epimer (RF 0.17) was observed, using the reversible iodine reagent. The radioactivity ot P, was exclusively located in the area of $&pregnane-3,2o-dione. An aliquot of P, was mixed with an authentic preparation of $-pregnane-3,zo-dione and crystallized 4 times from 4 different solBiochim.
Biophys.
4cta,
137 (1967) 347-355
S. MUROTA, B. TAMAOKI
350
Fig. I. Autoradiogram of the thin-layer chromatogram of the radioactive metabolites of [4-14C]progesterone (left) and [4J%jtestosterone (right) obtained from incubated homogenate of chick embryonic cartilage in the presence of an NADPH-generating system at 38” for 60 min. P,, 58. pregnane-3,zo-dione; I’,, 3cr-hydroxy-5B-pregnan-2o-one; P,, 5p-pregnane-3cr,2o/%diol; T,, 17phydroxy-5p-androstan-3-one; T,, unknown; T,, 5/l-androstane-3cr, I 7/Sdiol. (P) and (T) indicate the positions of progesterone and testosterone, respectively.
TABLE
I
QUANTITATIVE
ANALYSIS
OF
METABOLITES
OF
PROGESTERONE
AND
TESTOSTEROXE
IN
CARTILAGE
Homogenate of epiphyses obtained from I3-day-old chick embryo femora and tibiae was incubated with either [4-l%]progesterone or [4-‘Kltestosterone in the presence of an NADPH-generating system at 38” for 60 min. Values for each product are expressed as per cent of the total of metabolites and recovered substrate. (P), recovered progesterone; P,,5,%pregnane-3.20.dione; P,-3w hydroxy-5/Gpregnan-2o-one; P,,5~-pregnane-3cr.zop-diol; (T), recovered testosterone ; T,, I 7/G hydroxy-5P-androstan-3-one; T,, unknown; and T,, 5/?-androstane-3a,I7,%diol. ~~___ Substrate
Metabolite
Progesterone
P, P,
4.4 87.8 7.7 Trace
37.5 Nil Nil 62.5
Testosterone
T, T* T, (T)
23.5 11.3 65.1 Trace
Nil Nil Nil IOO.0
NA DPH-generating system ~___ PYeSe?Ke Absence
-
Biochim.
Biophys.
Acta.
137 (1967)
347-355
METABOLISM OF SEX STEROIDS IN CARTILAGE
vent mixtures. constant,
As shown in Table
within the counting
351
II, the specific activities
error, throughout
of the crystals
remained
the 4 crystallizations.
Identi$cation of the metabolite P, as 3cr-hydroxy-$i-pregnan-zo-one The radioactive substance designated as P, (Fig. I) was acetylatable and oxidizable. The mobility of the oxidized product on thin-layer chromatography showed TABLE
the same
value
as for P, already
identified
as 5/?-pregnan-3,zo-dione.
An
11
IDENTIFICATION
OF
Crystallization
SUBSTANCE
Pl
ANDP,
BY
CRYSTALLIZATIOK
Substance P?
Substance P, Carrier
steroids
5P-Pregnane-3,2o-dione Solvent
___~_
_~_ Specific activity (counts/
-___
_______~ 3a-Hydroxy-5~-pregnan-zo-olze ~ Solvent
(counts/min
mnin per mg)
Dichloromethane-n-heptane Benzene-ethanol Chloroform-methanol Benzene-n-heptane
1st
2nd 3rd 4th Average
f
62.8 = 0.6
S.E.
aliquot
Per m&T)
Dichloromethane-n-heptane Benzene-n-heptane Diethyl ether-n-heptane Chloroform-n-heptane
60.9 63.4 63.5 63.2
_~~____ Specafic activity
98.; 93.5 92.7 93.6 94.6 _+ 1.4 .~
of P, was mixed with 3a-hydroxy-5P-pregnan-zo-one
and its $Lepimer,
and
chromatographed using benzene-ethyl acetate-acetone (8: z : I, by vol.) and n-heptane-ethyl acetate (5 : 2, by vol.) systems; 3cr-hydroxy-5/I-pregnan-zo-one (RF, 0.46 and 0.20) and its j/3-epimer (RF, 0.56 and 0.29) were separated from each other in both systems. The radioactivity of P, was mainly located in the area of SE-hydroxy5,%pregnan-2o-one. After addition of carrier SE-hydroxy-58-pregnan-zo-one, crystallizations were performed from 4 different solvent mixtures and the specific activities of the crystals
were constant
(Table
II).
Identi$cation of the metabolite P, as 5B-Preglaane-3cc,zoB-diol The radioactive substance designated as P, in Fig. I was also acetylatable and oxidizable, and the oxidized product had a chromatographic mobility identical to 5/Spregnane-3,2o-dione. Because of its higher polarity than 3a-hydroxy-5/!-pregnan2o-one (P2), P, appeared to be either 5/Lpregnane-3n, zoa-diol or its ao/Gepimer. An aliquot of P, was mixed with 5/3-pregnane-3a,2oa-diol and its zofi-epimer, and chromatographed in benzene-ethyl acetate-acetone (8 : 2 : I, by vol.) and benzene-chloroform-diethyl ehter-methanol (2 : 2 : I : I, by vol.) systems, where 5/Lpregnane3a,zoudiol (RF, 0.19 and 0.45) was separable from the ao,&epimer (RF, 0.24 and 0.50). The radioactivity of P, was localized exclusively in the spot of carrier 5/Y-pregnane-3a,zo/Sdiol, which was detected by reversible iodine reagent. Crystallization of P, with added carrier 5B-pregnane-3cr,zo#Ldiol from 4 different solvent mixtures gave crystals of constant specific activity (Table III). Identi$cation of the metabolite T, as I7,!3-hydroxy-5/3-androstan-3-one The radioactive substance designated as T, (Fig. I) was acetylatable and oxidizable, and the oxidized product showed similar polarity to 5cr- or $-androstaneRiochim.
Biopltys. Acta, 137 (1967)
347-355
S. MUROTA, B. TAMAOKI
352 TABLE
III
IDENTIFICATIONOF SUBSTANCE
P,
BY
CRYSTALLIZATION
Substance P, ~ ...___. Carviev steroi~P~~ne-30(,2og-diol
1st 2nd 3rd 4th A\-wage
Solvent
Specific activity (countslmin per mg)
Methanol-chloroform Ethyl acetate-n-heptanc Acetone-cyclohexane Ethanol-dichloromethane
248 265 254 255
I S.E.
255.5 m’m 3.5
3,r7-dione (RF, 0.70) on thin-layer chromatography using a benzene-ethyl acetateethanol (8 : 2 : I, by vol.) system. Separation of 5a-androstane-3,r7-dione from its 5/1’epimer by thin-layer chromatography was possible using an n-heptane-ethyl acetate (5 :z, by vol.) system (RF, 0.28 and 0.25, respectively). The radioactivity of the oxidized product of T, was concentrated mainly at the location of carrier 5,%androstane-3,r7-dione. An aliquot of T, was mixed with r7/3-hydroxy-5@-androstan-3-one and chromatographed in the benzene-ethyl acetate-acetone (8 : 2 : I, by vol.) solvent system. Aliquots of the mixture were either acetylated or oxidized, and chromatographed as mentioned above. In each case, carrier steroid could not be obtained free from radioactivity. Crystallisation of a further aliquot of the mixture with an authentic preparation of 17/Ghydroxy-5/&androstan-3-one, gave crystals of constant specific activity (Table TABLE
from 4 different solvent mixtures, IV).
I\
IDENTIFICATION
OF
SUBSTANCE
T,
AND
T, BY
CRYSTALLIZATION
.-__
Crystallization
Substance T,
Substance T,
Carrier sterozd~~-~-___ ..~ r7P-Hvdroxy-5~-androstan-3-one Solvelzt
Specific activity (countslmin
Solve?at
Dichloromethane-n-heptane Benzene-cyclohexane Chloroform-n-heptane Ethyl acetate-cyclohexane
2nd 3rd 4th i\verage + S.E.
_______.-
____
241 237 236 236 237.5 :1 1.1
Specific activi1.v (countslmin
Per msl
Pev w)
_.~ 1st
-
____~______. 5B-Androstane-3a,I7P-diol
Ethanol-dichloromethane Acetone-cyclohexane Methanol-chloroform Ethyl acetate-n-heptanc ~~_
___
249 247 247 247 247.5 _t 0.5
Identijkation of the metabolite T, as 5B-androstane-3cr,I7B_diol The radioactive substance designated T, in Fig. I was also acetylatable and oxidizable. The behavior of the oxidized product of T, on thin-layer chromatography developed with an s-heptane-ethyl acetate (5:z, by vol.) solvent system, indicated that the derivative was 5/$androstane-3,r7-dione. An aliquot of T, was mixed with 5p-androstane-3a,r7@-diol and its 3p-epimer, and chromatographed using the benzene-ethyl acetate-acetone (8: 2 : I, by vol.) system, where 5/3-androstane-3a,r7/Gdiol (RF, 0.20) was separable from its 3/?-epimer (RF, 0.35). The radioactivity of T, was located exclusively in the area of 5/3-androstane-3cr,r7/Ldiol. An aliquot of T, was mixed with 5/Landrostane-3a,r7P-diol, and aliquots of the mixture were subjected Biochim. Biophys.
Acta, 137 (1967) 347-355
METABOLISMOF
SEX STEROIDSIN
353
CARTILAGE
to acetylation or oxidation, and the products were chromatographed in the benzeneethyl acetate-acetone (8 : z : I, by vol.) system. On every thin-layer chromatogram, the mobility as well as the shape of the radioactive spot, detected by radioautography, completely coincided with that of carrier steroid. Crystallization of another aliquot of the mixture from 4 different solvent mixtures gave constant specific activity (Table IV). The metabolite T,, had an almost identical polarity to 3cr-hydroxy-@-androStan-IT-one but repeated crystallizations with an authentic preparation did not give constant specific activity. A scheme for the catabolic pathways of progesterone and testosterone in chick embryo cartilage is shown in Scheme I, together with that of cortisol, established previously2T3. Cortim
0
&/
0
&
H
=0 ---OH
&/ HO
/
0
’
I
i C+b c-o
0
CH*OH
& LIP I 0
H
4
H
H
HO
CH>OH
I
C=O
---OH
LtP
\
0
0
H
CH3
C=O 0
CIIP
HO”
& H
’
H
C-3
OH-C-H
Scheme I. A scheme of the metabolic pathways of from the previous papers2,3) in embryonic cartilage
progesterone,testosterone and cortisol (cited
DISCUSSION
Few reports are available on the metabolism of steroid hormones in peripheral tissue. THOMAS AND DORFMAN' demonstrated 3/3-and I@hydroxysteroid dehydrogenase and A4-gcr-hydrogenase in skeletal-muscle strip of rabbit with androst-d-eneB&him.
Biophys.
Acta,
137
(1967) 347-355
S. MUROTA,B.TAMAOKI
354
3,r7-dione in vitro. BERLINER AND DOUGHERTY~ studied the metabolism of corticosteroids and progesterone in fibroblasts, and reported that metabolism of cortisol was mainly conducted in fibroblasts, except for liversa*O. In this experiment the predominant cells found in the epiphyses used were chondrocytes; adhering connective tissue and periosteum were removed in preparation. The authors demonstrated previously that homogenate tisol to tetrahydrocortiso12
or cultured chick-embryo cartilage actively converted corand now chondrocytes have been found to have an enzyme
system capable of metabolizing sex steroid hormones. It seems that there is a definite pattern to the catabolism of steroid hormones in embryonic cartilage, since, in every case, the functional A4-3-keto group, which is common to progesterone, testosterone and cortisol, uniformly underwent enzymic reduction to the corresponding 3a-hydroxy-$-steroid. The results indicate that, under the conditions used ill m&o, the cartilage tissue had no significant 3P-hydroxysteroid dehydrogenase or A’-ju-hydrogenase activity, but did have 3a-hydroxysteroid dehydrogenase and A”+hydrogenase activity. It has been reported that in liver tissue A4-gcr-hydrogenase is located in the microsomes” and A4-$6hydrogenase in the soluble phase 12, suggesting that these two enzymes show differences in their intracellular distribution and occurrence. It is noteworthy that the embryonic cartilage contained 3a-hydroxysteroid dehydrogenase and A”-5/%hydrogenase but not the 3/G and 5cr-enzymes, indicating the organ specificity of steroid catabolism. U’ith regard to this point, DOUGHERTY, BERLINER ASD BERLINEP have shown the dependence of cortisol metabolism on cell type and organ; for example reticuloendothelial cells of liver show predominantly ‘-2 ring reduction rather than C-zo-keto reduction, whereas those of adrenal show predominantly C-zothat throughout the metabolism of keto reduction. KAPPAS et al. 13$15demonstrated
all C,, and C,, steroids, only those of the 3~OH and 51-H type have specific thermogenie effects. Recently C,,, C,, and C,, steroids with a $-H configuration were also said to disrupt lysosomes and to release 70+80:< of total hydrolase activity from liver granules in vitro, whereas no Sa-isomer solubilized the enzymesl6. These facts suggest that among the metabolites so far regarded as inactive there may be some having new biological activities not present in the original steroid hormone. The authors reported (ref. I) that progesterone inhibited [%]sulfate incorporation into chondroitin sulfate, per unit dry weight of chick embryo femora growing in vitro, whereas the reverse effect was observed with testosterone. The results suggest that sex steroid hormones may have some direct action on cartilage growth. However, whether or not the metabolites of progesterone and testosterone identified in these experiments retained biological activity was not established. In the case of cortisol, the authors have demonstrated that the metabolites of cortisol, are without effect on cartilage growths. ACKNOWLEDGEMENT
The authors
are most
grateful
to Dr. M. SHIKITA
of this laboratory
for his
invaluable advice and discussion.
REFERENCES I H. ENDO AND S. MUROTA, I‘issue Culture, Dr. W. Junk, The Hague, Netherlands, 1965, p. 31. 2 S. MUROTA, M. SHIKITA AND B. TAMAOKI, Biochim. Biophys. Acta. IIT (1966) 421.
3 S.
MUROTA, H. ENDO AND B. TAMAOKI,
Biochim.
Biophys.
,4&a,
137 (1967)
347-355
B&him.
Biophys.
Acta,
136 (1967)
379,
METABOLISM 4 5 6 7 8 g 10 II. 12 ~3 14
OF SEX STEROIDS
IN CARTILAGE
355
ENDO, Eq%l. Cell Res., 21 (1960) 151. SHIKITA, H. KAKIZAKI AND B. TAMAOKI, Steroids, 4 (1964) 521. SHIKITA AND B. TAMAOKI, ~~~c~e~~s~~~, 4 (1965) 1189. Z. THOMAS AND R. I. DORFMAN, J. Biol. Chem., 239 (1964) 762. L. BERLINER AND T. F. DOUGHERTY, Ann. N.Y. Acad. Sci., 88 (1960) 14. F. DOUGHERTY, M. L. BEKLINER AND D. L. BERLINER, ~~~oc~~no~agy, 66 (1960) 550. F. DOUGHERTY AND D. L. BERLINER, in I. PAGE, Connective Tissue, Thrombosis and Athevosclerosis, Academic Press, New York, ‘959. p. 143. J, S. MCGUIRE AND G. M. TOMKINS, J. Biol. Chenz., 235 (1960) 1634. G. M. TOMKINS, J. Biol. Chem., 225 (1957) 13. T. F. DOUGHERTY, D. L. BERLINER AND M. L. BERLINER, ~~etaboZ~~m, IO (1961) 966. _a. KAPPAS, L. HELLMAN, D. K. FUKUSHIMA ANI) T. F. GALLAGHER, J. C&n. Enrlocrinol. Metab.,
H. M. M. P. D. T. T.
16 (1956) 948. 15 A. K~\PP~s AND R. H. PALMER, Pharm. Reviews, 15 (1963) 123. rG G. WEISSMANN AND L. THOMAS, Recent Progr. Hormone Res., 20 (1964) 215. Biochim. Biophys.
Acta, 137 (1967) 347-355