Metabolism of steroids by mouse teratocarcinoma cells

Journal of Steroid Biochemistry, Vol. 13, pp. 431 to 437 Pergamon Press Ltd 1980. Printed in Great Britain

METABOLISM OF STEROIDS BY MOUSE TERATOCARCINOMA CELLS ERKKI ANTILA a n d JORMA WARTIOVAARA

Departments of Medical Biology and Pathology, University of Helsinki, 00170 Helsinki 17. Finland

{Received 10 July 1979) SUMMARY

Steroid metabolism was studied in cells of a pluripotent mouse teratocarcinoma (TC) line OC15S1. One to five day old monolayer cultures were incubated for 3 to 20h in the presence of radioactive pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione or estradiol-17//. Extracted radioactivity was analyzed by thin layer chromatography, gas liquid chromatography and recrystallizations to constant specific activity and constant isotope ratio. The substrates were metabolized to different extents and most of the metabolites were excreted into the medium. The age of the culture did not affect the results. Similar types of metabolic conversion of steroids was obtained by cultured mouse embryonic fibroblasts which were used as a reference. The results indicated that the cultured TC cells are not capable of 1, efficient conversion of pregnenolone to progesterone nor of 2. significant modification of the major exogenous estrogen, estradiol-17fl, but are capable of 3. efficient modification of progesterone and of 4. converting pregnenolone to a more polar form, as well as of 5. metabolizing both DHA and androstenedione.

Incubation

INTRODUCTION

Teratocarcinomas (TCI are germ line tumours from which a number of cell lines have been established [1]. Embryonal carcinoma cells form the stem cells of the tumours which in addition usually contain a wide variety of cell types deriving from the three primary embryonic germ layers [2]. The OC15S1 cell line used in the present study is a p!uripotent mouse embryonal carcinoma line which can differentiate in vitro [2] into endoderm like cells also. Enzyme activities for steroid production and metabolism have been demonstrated in early embryos of a number of species [3-7]. Disagreement exists on the exact stage of onset of steroid metabolism in the mouse [6, 8, 9-[. The aim of the present study was to reinvestigate how cultivated TC cells, corresponding to the inner cell mass cells of the blastocyst stage embryo[10], metabolize steroids as preliminarily reported [11].

Two to 4 day old cell cultures were incubated in 10ml culture medium with [4-14C]-pregnenolone (7.9 x 10-7 M, 0.42 #Ci), [4-14C]-progesterone (6.3 x 10-TM, 0.38#Ci), [4-t4C]-DHA (7.4 x 10 " M , 0.38 #Ci), [l,2,6,7(n)-3H]-androstenedione (4 x 10-1° M, 0.31 #Ci) or [2,4,6,7(n)-3H]-estradiol-17~ ( l . 4 x 10-9M, 1.5#Ci). The incubation time was chosen on the basis of pilot experiments for maximal yield of metabolites. During kinetic studies with labelled pregnenolone and progesterone four or five 1 ml samples were taken from the incubation medium after 30 rain, l h, 3 h, 10 h and at the end after 20 h. At term of incubation the medium was collected, The cells were washed three times with phosphate-buffered saline and scraped off from the dish. Both the medium and the cells were acetone treated and processed separately as described below. Controls were made by incubating culture medium without cells with the substrates.

Extraction, purification and characterization of steroids

EXPERIMENTAL

Cell culture Cells used were mouse teratocarcinoma line OC15S1 derived from a single cell clone [12] and secondary fibroblast cultures from strain CBA mice embryos (a gift from Dr. Antti Vaheri, Dept. of Virology, Univ. of Helsinki). Cells were grown in Eagles minimal essential medium supplemented with 10% fetal calf serum. 1 x 106 cells were plated in 90 mm plastic Petri dishes containing 10ml of growth medium. The cells were replated into new dishes every second day. In longer experiments the medium was changed at similar intervals.

Extraction was done by diethylether including partitioning against 1 N NaOH [13]. Alternatively the diethylether extract was evaporated, dissolved into toluene and extracted with 0.5N NaOH (×3, 0.5 vol.). The free neutral steroid fraction was recovered from the toluene phase. The free phenolic steroid fraction was obtained by diethylether extraction from the neutralized NaOH phase. The recoveries of pregnenolone, progesterone, dehydroepiandrosterone, testosterone, androstenedione and estradiol under identical conditions have been 78.0 _+ 2.6, 83.8 + 0.2, 87.8 + 0.4, 83.7_+ 0.8, 82.4 + 0.9 and 82.5 +_ 1.5 percent respectively[14]. Recovery of

431

432

ERKKIANTILAand JORMAWARTIOVAARA

radioactivity in the free steroid fractions extracted from the cells and from the medium was measured from aliquots by liquid scintillation counting. Both extracts were submitted to thin layer chromatography (t.l.c.) for the separation of metabolites [15]. Carrier steroids were not used as fractions from 6-8 incubations were pooled before gas chromatography to obtain detectable amounts of steroids. Solvent systems used were system A for pregnanes and system B for androstanes and estrogens (phenolic steroid fraction). The radioactivity was detected and quantified on t.l.c.-plates by a t.l.c.-scanner (LB2721, Berthold Co., Wildbad, GFR~ Reference steroids were visualized by their U.V.-absorption (240nm) and/or as fluorescent spots after spraying with p-toluene sulfonic acid (20~ in ethanol). The metabolites on t.l.c.plates were divided into six fractions which were numbered in the order of decreasing polarity. The radioactive metabolites were further characterized by rechromatography of pooled fractions in different solvent systems and identified following addition of carrier by chromatographing to constant specific activity [15] or to constant isotope ratio [16] followed by recrystallization [17]. Radioactivity was measured in both crystals and motherliquid. Identity was confirmed if after three crystallizations the isotope ratio (3H/14C) was constant within 5~o limits. Identification of other fractions was done by gasliquid chromatography (g.l.c.) after prepurification in Sephadex LH-20 columns according to Setchell and Shackleton [18]. G.l.c. analysis was performed with a Perkin-Elmer F-30 chromatograph (Beaconsfield, Bucks, England), with a hydrogen flame ionization detector using a packed 3~o OV-1 (2.5 x 1980mm) column. Retention times relative to cholestane of the studied compound and its trimethylsilyl ether [19] were compared to those of similarly treated standards. Protein determination

Amount of cellular protein was determined [20] after extraction by taking samples from the water phase of cell extracts. Corrections for losses were not done. Steroids

The radioactive steroids with following specific activities were purchased from The Radiochemical Centre (Amersham, England): [4-1~]-androstene dione, 60mCi/mmol; [1,2,6,7(n)-3H]-androstenedione 87 Ci/mmol; [4-t4C]-DHA, 55 mCi/mmol; [2,4,6,7(n)3H]-estradiol-17fl, 109 Ci/mmol; [4-14C]-pregnenolone, 53mCi/mmol; [4-~4C]-progesterone, 61mCi/ mmol; [l,2,6,7(n)-3H]-progesterone, 81 Ci/mmol. Reference steroids were mainly obtained from The Steroid Reference Collection (London, England). Progesterone and androstenedione were supplied by Sigma Chemical Co (St. Louis, Mo.). Solvent systems in t.l.c.

A acetone--chloroform (15:85, v/v), B chloroformether (3:1), C benzene--ethanol (9:1), D benzene-

ethanol (4:1), E chloroform--ethanol (9:1), F chloroform-ethanol (19:1), G benzene-ethylacetate (1 : 1), H dichlormethane-acetone (5:1), I cyclohexaneethylacetate-ethanol (45:45:10), K cyclohexaneethylacetate (1:1), L ethylacetate-n-hexane--ethanolacetic acid (144:27:9:20). RESULTS Metabolism of substrates

Recoveries of radioactivity in the ether phase from incubations of teratocarcinoma and fibroblast cultures with different substrates are demonstrated in Table 1. Radioactivity remaining in the water phase was unsignificant (< 5Yo,data not shown). Most of the radioactivity was found in the free neutral steroid fraction extracted from the media. All substrates except estradiol-17fl were extensively metabolized by both cell types (Fig. 1). In the incubation lasting 20 h these substrates were metabolized to over 50~. The estradiol-17fl metabolism was relatively weak even with long incubation times. The metabolites were extensively excreted into the media and only low levels of radioactivity were retained in the cells. The t.l.c, profiles were very similar in both the medium and the cells (Fig. 2). The distribution on t.l.c.-plates of radioactivity in neutral steroid fraction is demonstrated in the case of media from 20 h incubations Fig. 1A-D. With all substrates, except estradiol-17fl (Fig. 1E), accumulation of one main metabolite was detected. In control incubations without cells (data not shown) no conversion of substrates was found. Mouse embryo fibroblasts displayed a similar kind and even more efficient metabolism as teratocarcinoma cells (Fig. 1). Kinetics of pregnenolone and progesterone conversion were studied in two and four day old cultures of teratocarcmoma cells (Fig. 3). With both substrates 80--90~o conversion was obtained in 20 h. Characterization of metabolites

When pregnenolone was used as substrate six fractions were obtained in t.l.c, with both teratocarcinoma cells and mouse embryo fibroblasts (Figs 1A, 2). Fraction V contained progesterone as identified by chromatography and recrystallizations to constant isotope ratio (c.i.r.) and to constant specific activity (c.s.a.) (Table 2). Part of this fraction and part of fraction VI probably contained 5or-reduced progesterone (see progesterone incubation). Fraction IV contained the substrate having a retention time in g.l.c, similar to authentic pregnenoione. The highly polar metabolites in fractions I, II and III remained unidentified. Use of progesterone as substrate yielded six fractions (Figs 1B, 2). Fraction VI cochromatographed in two-dimensional t.l.c. (solvent systems: A, C) with 5~t-pregnane-3,20-dione as carrier. Fraction V was the substrate (see Table 2). Fractions I, II, III and IV remained unidentified. In DHA incubations (Fig. 1C) only the substrate in

8

10 6 2 2

pregnenolone

progesterone

OHA

androstenedione

estradiol-176

4.0~0

.72.1~0.9

1.4~0.5

3.9~0.6

21.2~6,3

69.2~6.5

5.6~1.4

5.1~1.7

3.0~0

nd

82.5~6.6

87.g~1.1

8.2~2.g

82.1~6.3

n = number of incubations, incubation time 20 b nd = not determined

n

Substrata

Teratocarcinoma cells Medium Cells neutral phenolic combined fraction fraction fraction

2

2

2

2

n

42.8~6.6

86.I~0.2

7g.4~4.6

78.2~0

37.6~3.6

nd

11.3~0,I

1.0~0

0.3~0.I

2.3~0.3

1.1~0.4

Fibroblasts Cells Medium neutral phenolic combined fraction fraction fraction

Table 1. Per cent recovery of added radioactive steroids in the ether extract of cells and m e d i u m

g~

O

g

o

434

ERKKI ANTILAand JORMAWARTIOVAARA TE R A T O C A R C I N O M A C E L L S

FIBROBLASTS

n8 ,till 168 i 5 2

It

n 2 ~J9 1 2 3 i 5

L

TERATOCARCINOMA CELLS n2 ~Jg nd

II III IV V Vl

III IV V %#1

"~

n2 ~g

102"2"/

50

n10 Jug t ? 3 t ~ 50

I

It III IV V VI n6 )Jg 2 2 0 i g e

50 ¸

nj..o I II III IV V

50

FIBROBLASTS

II III IV V

lll.,_

n2

Vl

50

50

[-I

II

III i V V

I

II

III IV V VI

| II III IV V V|

I

II II| IV V VI

Fig. 1. Distribution of radioactivity on t.l.c.-plates expressed as per cent of total recovery. Radioactivity on t.l.c, plates is divided into six fractions (I-VI) numbered in the order of decreasiong polarity. Radioactivity extracted from media of 20 h incubations of teratocarcinoma cells and mouse embryo fibroblasts. (The Rr values of fractions with similar numbers differ from each other with different substrates. See Fig. 2). Shaded columns = substrate fraction, n = number of incubations (when n = 2, SD is substituted with range)/~g = amount of cellular protein, nd = not determined. Row A: pregnenolone incubations (neutral steroid fraction). Row B: progesterone incubations (neutral steroid fraction). Row C: DHA incubations (neutral steroid fraction). Row D: androstenedione incubations (neutral steroid fraction). Row E: estradiol-17/3 incubations. fraction IV was identified by g.l.c. In androstenedione incubations (Fig. 1D) in addition to the substrate found in fraction V, testosterone was found in fraction II. Determination of c.s.a, by chromatography and measurement of c.i.r, and c.s.a, by recrystaUizations confirmed this finding (Table 2). Radioactivity l;ound in the phenolic fractions of the above neutral steroid incubations was also studied by t.l.c. Chromatographic mobilities were not similar to the common estrogens, estrone, estradiol-17fl or estriol (solvent systems: B, E, D): The compounds may instead of estrogens be very polar neutral steroids. In estradioi-17fl incubations (Fig. 1E) the substrate was found in fraction IV. Low radioactivity in the other fractions prohibited identification. DISCUSSION

The present results demonstrate that monolayer cultures of the pluripotent mouse teratocarcinoma (TC) cell line OC15S1 and mouse embryo fibroblasts are able to metabolize particularly pregnanes and androstanes. All substrates, specially progesterone but

estradiol-17fl to a lesser extent, were converted to metabolites excreted into the medium. Almost all metabolites were more polar than their substrates. Conversion to biologically active forms were observed in a minor quantity: pregnenolone was converted to progesterone and androstenedione to testosterone. The low yield of progesterone was not, however, due to further metabolism of progesterone as the main metabolite peak found in pregnenolone incubation was not found when progesterone was used as substrate. Previously Sherman and coworkers have reported that cells derived from mouse blastocysts are able to convert exogenous pregnenolone to progesterone [8, 21]. Such conversion has not been demonstrable in younger, preimplantation mouse embryos [22,9] although A5-3fl-hydroxysteroid dehydrogenase (3/~-HSD) activity, involved in the production of progesterone, has been histochemically shown in both trophectoderm and inner cell mass (ICM) cells of mouse and rat embryos [6, 23]. Our results with TC cells are in line with the idea that ICM cells have 3/~-HSD activity. In metabolic studies it is important to take in consideration the needed

Fibroblasts

TC

TC

TC

Fibroblasts

TC

TO

III

IV

V

VI

VII

VIII

IX

TC

180

V

TC

.830

IV

II

1.79

III

I

1.28

II

Cells

2.45

I

before

196

.820

1.73

1.69

androstenedione

progesteFone

pregnenolone

pr'egnenolone

androstenedione

progesterone

pregnenolone

pregnenolone

pregnenolone

Substrate

186

.805

1.74

1.82

2.67

2nd

or s p e c i f i c

2.89

Ist

ratio

Acetone-pentane

V,prezesterone V,progesterone V,progesterone

fr fr fr

ll,testostebone

F,

fr I I , t e s t o s t e r o n e

fr

C,

fr V,progesterone

)

|

} >

K,

V,progesterone

fr

I

H

E

methanol-water

acetone-water

G,

I,

C,

E

I, E

C,

I,

C,

fr V , p r o g e s t e r o n e

~

2517

2451

IX

Systems

5256

4992

VIII

Product

137

589

148

604

Vll

VI

3rd

dpm/m Z

after

or 3H

R e c r y s t a

before

14C

B.

fr V , p r o g e s t e r o n e

162

.799

1.77

1.82

2.68

3rd

activity

C h r o m a t o g r a p h y

Isotope

A.

Table 2. Identification of progesterone and testosterone

ratio

1.02

.710

1.85

1,23

Ist

(cpm/pg)

3H/14C

3H/14C

3H/14C

31!/14C

1.02

.894

1.77

1.19

@

Isotope

1.01

.690

1.60

1 20

2nd

1.06

.757

1.85

1.29

3rd

in c r y s t a l s

1 i i z a t i o n s

0

Q0

m'

436

ERKKI ANTILA and

JORMA WARTIOVAARA

O I

PREGNENOLONE

% 100'

22

A

1 e• ee

50-

eo

I e I

3

20h

t ;. ,•

; SF

B

Vl

e •

V

IV

ee•• III

o% _

• i

II

1°°1~

B

(-,

PROGESTERONE

1 1

J

L' VI

V

I0

20~

Fig. 3. Kinetics of pregnenolone (A) and progesterone (B)

q

SF

3

e•eeo e

IV

III

II

e.

e ~ e = o f

I

conversion to metabolites. Triangles represent 2-day and squares 4-day cultivated teratocarcinoma cells. Number above thesymbols indicates number of samples analyzed. Amount of substrate left is indicated as per cent of radioactivity found in samples taken at different time points of incubation. In A range indicated when not covered by symbol.

0

Fig. 2. The t.l.c,-profiles on the radioactivity extracted from culture medium (continuous line) and from teratocarcinoma cells (dotted line) after 20 h incubation with pregnenolone (A) and progesterone (B). Arrow indicates the substrate fraction. See text for characterization of fractions I to VI. O = origin, SF = solvent system A. threshold concentrations of substrates as Egert et al.[24] have pointed out in studies with progesterone metabolism by rat uterus. The finding that ICM outgrowths in culture, at the time equivalent to midgestation, metabolize progesterone very poorly [cf. 25] as compared to our results on the prominent metabolism of progesterone by corresponding TC cells may result from the difference in initial progesterone concentrations used (ours 6 x 10-7 M, theirs 6 x 10 - s M). Similarly we could not notice metabolites of estradiol-17fl in any larger amounts with the initial concentration of 1.4 x 10-9 M. Nor could we demonstrate formation of estradiol-17/~ from exogenous precursors used. It was somewhat unexpected that the mouse embryo fibroblasts used as reference cells to the pluripotent TC cells gave very similar results with all substrates used. Also, similar chromatographic profiles were found in TC cultures independent of length of cultivation used. With the OC15Sl line the presence of 3fl-HSD activity has been found in both embryonal carcinoma cells and in endodermal cells derived from

the former (our unpublished histochemical observations). This suggests that the capability of embryonal carcinoma cells to metabolize steroids is not sensitive to differentiation. The idea might not be far-fetched that the capacity to metabolize steroids could be a more general characteristic of cells from early embryogenesis on by which means cells could eg. reduce harmful levels of steroids. In addition to TC cells this may apply also to other transformed cells like mouse L-cells in which steroid receptors have been found [26]. Acknowledgements--We wish to thank Prof. Herman

Adlercreutz and Dr. Arto Saute for their valuable comments and critical review of the manuscript. Thanks are also due to Ms. Leea Kauppi and Elina Waris for their skilful technical assistance. This study was supported by grants from the Sigrid Jus61ius Foundation and the National Research Council for Medical Sciences. REFERENCES

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Steroid metabolism in teratocarcinoma cells 4. Perry J. S., Heap R. B. and Amoroso E. C.: Steroid hormone production by pig blastocysts. Nature 245 (1973) 45-47. 5. Dickmann Z. and Dey S. K.: Steroidogenesis in the preimplantation rat embryo and its possible influence on morula-blastocyst transformation and implantation. J. reprod. Fert. 37 (1974) 91-93. 6. Dey S. K. and Dickmann Z.: AS-3fl-hydroxysteroid dehydrogenase activity in mouse morulae and blastocysts. 7th Ann. Meeting Soc. Study Reproduction, Abstract No 150 (1974). 7. Niimura S. and Ishida K.: Histochemical studies of AS-3fl-,20a- and 20fl-hydroxysteroid dehydrogenases and possible progestagen production in hamster eggs. J. Reprod. Fert. 48 (1976) 275-278. 8. Chew N. J. and Sherman M. 1.: Biochemistry of differentiation of mouse trophoblast. AS,3fl-hydroxysteroid dehydrogenase. Biol. Reprod. 12 (1975) 351-359. 9. Antila E., Koskinen J., Niemel~i P. and Saure A.: Steroid metabolism by mouse preimplantation embryos in vitro. Experientia 33 (1977) 1374-1375. 10. Martin G. R. and Evans M. J.: The formation of embryoid bodies in vitro by homogeneous embryonal carcinoma cell cultures derived from isolated single cells. In Teratomas and Differentiation (Edited by M. I. Sherman and D. Solter). Academic Press, New York (1975) pp. 169-187. 11. Antila E. and Wartiovaara J.: Mouse teratocarcinoma cells metabolize exogenous steroids. J. cell Biol. 79 (1978) 202a. 12. McBurney M. W.: Clonal lines of teratocarcinoma cells in vitro: differentiation and cytogenetic characteristics. J. Cell Physiol. 89 (1976) 441-455. 13. Kahri A. I., Pesonen S. and Saure A.: Ultrastructural differentiation and progesterone-X4C metabolism in cultured Cells of fetal rat adrenals under influence of ACTH. Steroidolooia I (1970) 25-64. 14. Saure A.: The biogenesis and metabolism of steroids in the human placenta of 9-14 weeks in vitro. Ann. Acad. Sci. Fenn. A5, 159 (1973) 1-131. 15. Kaartinen E., Laukkanen M. and Saure A.: Metabolism of dehydroepiandrosterone by rat testicular homogenates; Kinetic study at different temperatures; Direct effect of 17fl-oestradiol. Acta Endocr., Copenh. 66 (1971) 50-64.

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16. Antila E.: Early steroid metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris embryos. Differentiation 8 (1977) 71-77. 17. Axelrod L. R., Matthijssen C., Goldzieher J. W. and Pulliam J. F.: Definitive identification of microquantities of radioactive steroids by recrystallization to constant specific activity. Acta Endocr., Copenh. suppl. 99 (1965) 7-65. 18. Setchell K. D. R. and Schackleton C. H. L.: The group separation of plasma and urinary steroids by column cromatography on Sephadex LH-20. Clin. chim. Acta 47 (1973) 381 388. 19. Brooks C. J. W., Chambaz E. and Horning E. C.: Thinlayer and column chromatographic group separations of steroids as trimethylsilyl ethers. Isolation for gasliquid chromatographic analysis of pregnanediol and estriol in pregnancy urine. Analyt. Biochem. 19 (1967) 234-242. 20. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (1951) 265-275. 21. Salomon D. S. and Sherman M. I.: The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells. Develop. Biol. 47 (1975) 394-406. 22. Sherman M. I. and Atienza S. B.: Production and metabolism of progesterone and androstenedione by cultured mouse blastocysts. Biol. Reprod. 16 (1977) 190-199. 23. Dey S. K. and Dickmann Z.: As-3fl-hydroxysteroid dehydrogenase activity in rat embryos on days 1 through 7 of pregnancy. Endocrinology 95 (1974) 321-332. 24. Egert D., Jonat W. and Maass H.: Progesterone in the uterus. V. Correlation of the in vitro progesterone metabolism in the rat uterus on the progesterone concentration. Steroids 26 (1975) 193-214. 25. Sherman M.I., Atienza S. B.,.Satomon D. S. and Wudl L. R.: Progesterone formation and metabolism by blastocysts and trophoblast cells in vitro. In Development in Mammals. (Edited by M. H. Johnson). NorthHolland, Amsterdam, (1977) pp. 209-233. 26. Jung-Testas I., Bayard F. and Baulieu E. E.: Two sex steroid receptors in mouse fibroblasts in culture. Nature 259 (1976) 136 138.