- Email: [email protected]
Steroid metabolism by mouse submaxillary glands. I. in vitro metabolism of testosterone and 4-androstene-3,17-dione
247
STEROID METABOLISM BY MOUSE SUBMAXILLARY GLANDS.
I.
IN VITRO METABOLISM OF TESTOSTERONE _-
AND
4-ANDROSTENE-3,17-DIONE
James C. Coffey
Dental Research Center and the Department of Pediatrics University of North Carolina Chapel Hill, North Carolina 27514
Received:
615173
ABSTRACT
Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17$-hydroxy-5ff-androstan-3-one and 5a-androstane-3o,17g-diol. Testosterone was converted primarily to 5a-androstane-3@,17B_diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17B-hydroxy-5o-androstan-3-one, and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.
INTRODUCTION
Androgen is influential in the structural and biochemical configuration of the rodent submaxillary gland.
Sexual dimorphism of the
STEROIDS
248
22:2
convoluted tubules is influenced by testosterone (1,2). gland ep~thelial-e~ide~al
Submaxillary
growth factor (3,4), nerve growth factor
(5), lethal factor (6,7), lymphoid tissue inhibitory factor (8,9), RNA synthesis (lo), and protease (11,12) are all sensitive to androgen. Some enzymes involved in steroid metabolism have been demonstrated in vitro in rodent submaxillary glands. --
Cholesterol has been synthe-
sized from acetate, 3B-hydroxy-5-pregnen-20"one and 3B-hydroxy-5androsten-17-one from cholesterol, and progesterone from 3$-hydroxy5-pregnen-ZO-one (13,14).
The rodent submaxillary gland has also
been shown to convert progesterone to 5cu-pregnane-3,20-dione and 4-androstene-3,17-dione
(13,15).
The cited studies show that the rodent submaxillary gland is responsive to androgens and that some enzymes required for steroid metabolism are present in the tissue. taken to determine if
the
The present studies were under-
requisite enzymes for converting androgens
to reduced products are found in submaxillary glands, as has been described for other androgen-sensitive tissues.
MATERIALS AND METHODS Materials Testosterone-1,2-3H (57.8 Ci/mM), testosterone-4-14C (53 mCi/mM), 4-androstene-3,17-dione-l,2-3H (42 CifmH), 4-androstene-3 17-dione4-14C (45 mCi/mM), and 17@-hydroxy-So-androstan-3-one-4- 14, (56.1 mCi/mM) were obtained from New England Nuclear. Radiochemical purity of these steroids was checked by thin-layer chromatography prior to use. NADP, glucose-6-phosphate and glucose-6-phosphate dehydrogenase type XV, were obtained from Sigma. Thin-layer chromatography was performed on 5 x 20 cm glass plates coated with either silica gel GF-254 or aluminum oxide GF-254 obtained from E. Merck, AG. All other chemicals and solvents were reagent grade and were used without further purification. Chemical procedures Steroids were acetylated at room temperature overnight in a mixture
Aug. 1973
STEROIDS
249
of equal volumes of acetic anhydride and pyridine. The pyridine-acetic anhydride mixture was removed by evaporation -in vacua after successive additions of ethanol. Steroid acetates were saponified at room temperature overnight in 1 ml of 1 N KOH in methanol. The mixture was diluted with water, and the steroids extracted 5 times with chloroform. The chloroform extract was washed 1 time with N/l0 HCl and then with water until neutral. Preparation and incubation of homogenates Male CD-1 mice, 6 months old, were killed by cervical dislocation. The submaxillary glands were rapidly removed and collected in iced beakers. The submaxillary glands were dissected free of other tissue, minced with scissors, and the mince washed with cold 0.154 M KCl. The washed mince was then homogenized in the medium described by Kase et al. (16). A Dual1 homogenizer, cooled in ice, was used. Each incubation flask contained 250 mg of homogenized submaxillary gland tissue in 3.0 ml of 18.2 mM sodium phosphate buffer (pH 7.4) containing 0.32 mmoles of KC1 and 0.0125 mmole of MgC12. When a NADPH-generating system was utilized 0.005 mmole of NADP, 0.0425 mmole of glucose-&phosphate, and 1.5 units of glucose-&phosphate dehydrogenase were also included in each incubation flask. The testosterone-1,2-3H (Gp,Ci)or 4-androstene3,17-dione-1,2-3H (6pCi), mixed with cold carrier to give a final concentration of O.lpM, were previously added to the incubation flasks and dissolved in 0.1 ml of propylene glycol. Incubations were carried out for either 15 or 180 min at 37OC in a shaking water bath and were stopped by the addition of LO ml of a mixture of chloroform:methanol (2:l). Immediately after the addition of chlorofonn:methanol 2OOpg each of carrier testosterone, 4-androstene-3,17-dione, 17/3-hydroxy-5oandrostan-3-one, androsterone, 5a-androstane-3o,l7$-diol and 5o- and 5p-androstane-3,17-dione were added to each flask. Extraction and purification of metabolites The homogenates were extracted with 25 vol of chloroform:methanol (2:l). The solvents were evaporated in vacua, and the residues were dissolved in chloroform and washed with water. Aliquots of the extra& were chromatographed on 0.5 mm thick silica gel plates in benzene:ethyl acetate (3:l) developed twice. Radioactive peaks were located with a Packard Model 7200 Kadiochromatogram Scanner. Carrier steroids were located under UV light or by spraying with ethanolic iodine solution. With both substrates, four bands of radioactivity corresponding to the following areas were obtained in the initial chromatography step: polar steroids at the origin (area l), testosterone and 5o-androstane3a,17@-diol (area 2), 4-androstene-3,17-dione, androsterone, 17ghydroxy-5a-androstan-3-one (area 3), 5o- and 5$-androstane-3,17-dione (area 4). Area 1, polar material, was scraped off the plates and eluted for counting without further purification. When the area 4 eluate was chromatographed on silica gel in benzene:ethyl acetate 15:1, with four developments, the radioactivity moved near the solvent front and
250
STEROIDS
22:2
was not associated with either To- or 58-androstane-3,17-dione carrier. Radioactivity recovered in this relatively non-polar fraction and the polar fraction remaining at the origin after the initial chromatographic step is included in Tables 2 and 3 to provide a complete account of the distribution of radioactivity. Small aliquots from the eluates of areas 2 and 3 were removed to determine total radioactivity. The remaining portions were acetylated and chromatographed on silica gel plates in benzene:ethyl acetate. Two peaks of radioactivity were obtained on the radioscan of area 2 corresponding to testosterone acetate and So-androstane-3o,178-diol 3,17-diacetate. These two peaks were eluted, aliquots of the eluates taken for counting, and the remaining material saponified. The saponified products were separately chromatographed on aluminum oxide in benzene:ethyl acetate 2:1, developed twice. After scanning, only a single peak of radioactivity corresponding to either testosterone or 5o-androstane-3a,l78_diol was present. The acetylated area 3 eluates also yielded two peaks of radioactivity on the radioscans. One peak was isopolar with &androstene-3,17-dione carrier. The second peak was associated with androsterone and 17$-hydroxy-5o-androstan-3-one carriers. The 4-androstene-3,17-dione peak was eluted and rechromatographed on aluminum oxide in diethyl ether. Again a single radio peak isopolar with 4-androstene-3,17-dione carrier was seen after scanning. The radioactive peak containing a mixture of androsterone and 17@hydroxy-5G-androstan-3-one was eluted, saponified, and the product chromatographed on aluminum oxide in methylene dichloride:diethyl ether (9:l) with two developments. After scanning two peaks were present, one isopolar with androsterone carrier and one isopolar with l78-hydroxy-5o-androstan-3-one carrier. After thin-layer chromatography the total radioactivity was calculated by adding the radioactivity recovered in the final individual peaks and the results for a particular peak were expressed as a percentage of this total. In each case, the total radioactivity recovered was between 85-90% of the incubated amount of steroid. To confirm the identities of metabolites with the chromatographic mobilities of 4-androstene-3,17-dione, androsterone, testosterone, 17@-hydroxy-5a-androstan-17-one and 5cr-androstane-3a,l7B_diol, representative samples were further purified by crystallization from cyclohexane-acetone and ethanol-water (Table I.). Androsterone and ICYandrostane-3o,l7@-diol were crystallized to constant specific activities after addition of 20 mg of carrier steroid. Testosterone, 17S-hydroxy-5a-androstan-3-one and 4-androstene-3,17-dione were crystallized to constant 14C/31i ratios after addition of 20 mg of carrier steroid and the appropriate 14C-steroids. Measurement of radioactivity Radioactivity was measured with a Beckman LS-250 liquid scintillation spectrometer. The material scraped from thin-layer plates was eluted with warm chlorofo~:me~hanol (2:1) and the dried residues of aliquots counted in 1.5ml of toluene containing 0.3% 2,5diphenyloxazole and O.Ol%, 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene. Counting efficiencies of these samples were uniformly 53%56%.
4990t
4950'
4.9
4.9 180
Testosterone **
4.7
4.6
15
4.6
17~-~ydroxy-5~-androstan-3-ona
4.8
4100'
4.6
4.3
4540'
2.1
5.5
4.0
180
180
incubated
4.6 5.0 5.1 5.1
4,6
4130"
4.6
4.7
4000t
4.6
4,6
3900t
5050t
4.4 4.3 4.3 sooot
2.4
5.6 2.0
5.6
3.3
2‘1
5.5
4.0
DPM 3H/DPM14C or DPM/mgt Final ML c3 c4 c2
2.5
5.6
3.8
Cl
17@-Hydroxy-So-androstan-3-one
5wAndrostane-3o,278-diol
180
180
*Q unconverted substrate, Ml, = mother liquor, C = crystallization
Testosterone
Androsterone
4-Androstene~-3,17-dione ;k;k
1.5
180
Testosterone
17~-~ydroxy-5~-androstan-3-on~
15
Incubation time (min)
Testosterone
Metabolite
Crystallization of metabolites of testosterone-l,2-3H and 4-androst~n~-3,17-dione-l,2-3H with male mouse submaxillary gland homogenates.
Substrate
1.
4-Androstene-3,17-dione
Table
252
STEROIDS
22:2
When specific activities were determined, radioactivity data were expressed as dpm using a Cs-137 external standard. When crystallizations to constant 381141: ratios were performed after the addition of the appropriate l4C-labeled steroid, the isotopes were counted simultaneously and calculations based on counting efficiencies obtained with 3H- and 14C-toluene standards. Measurement of steroid weight In the determination of specific activity after crystallization of androsterone and 5a-androstane-3a,l78_dlol carrier weights of androsterone and 5tr-androstane-3@,17B_diol were measured by the optical densities of the sulfuric acid chromogens (17) at 310 mp, and 340 rnl~. respectively.
RESULTS
Incubations with 4-androstene-3,17-dione-1,2-3H
(Table 2).
In the presence of an NADPH-generating system, 4-androstene-3,17dione was actively metabolized by male mouse submaxillary gland tissue. Fifty-four percent substrate conversion occurred in 15 min and 95% conversion occurred in 180 min.
The major metabolite present at
both 15 and 180 min was androsterone, 37% and 58%, respectively. Much small.er amounts of testosterone (3-b%) and 178-hydroxy-5cr-androstan3-one (l-2%) were detected at both times.
Only a trace amount (0.8%) of
5a-androstane-3ru,l78_diol was present at 1.5min but about 7% was present after 180 min of incubation. 4-androstene-3,17-dione was poorly metabolized when the NADPHgenerating system was not included in the incubation medium.
In 15 and
180 min incubations, 93% and 89%, respectively, remained unconverted, At both times only trace amounts of androsterone, 17B-hydroxy-5oandrostan-3-one and 5a-androstane-3o,l7$-diol were present.
Only a
trace of testosterone (0.6%) was present at 15 min but a slight increase in its accumulation (to about 2%) was noted in the longer incubations.
STEROIDS
Aug. 1973
Table 2.
253
Metabolism of 4-androstene-3,17-dione-l,Z-3H submaxillary gland homogenates J;
by male mouse
NADPH generating system Absent Present 180 min 15 min 180 min 15 min -
--
Polar fraction
5.7
14.9
1.7
2.9
Testosterone
3.0
4.1
0.6
2.4
5o-Androstane-3cu,l7@-diol
0.8
6.9
0.2
0.4
4-Androstene-3,17-dione
46.0
5.0
93.5
89.5
Androsterone
37.0
58.0
0.1
0.3
17fi-Hydroxy-Sff-androstan-3-one
1.1
2.0
0.4
0.6
Non-polar fraction
6.3
9.1
3.5
3.9
*Calculated as % of total radioactivity recovered from chromatoplates.
Table 3.
Metabolism of testosterone-1,2-3H by male mouse submaxillary gland homogenates *
NADPH generating system Absent Present 180 min 180 min 15 min 15 min
Polar fraction
3.0
7.2
2.7
2.4
84.0
57.4
94.4
93.1
5o-Androstane-3a,l7g-diof
9.8
31.6
2.0
2.1
4-Androstene-3,X7-dione
0.1
0.1
0.3
0.4
Androsterone
0.1
0.2
0.1
0.1
17$-Hydroxy-Sol-androstan-3-one
0.3
0.6
0.2
0.1
Non-polar fraction
2.8
2.8
Il.2
1.7
Testosterone
Walculated
as % of total radioactivity recovered from chromatoplates.
STEROIDS
254
Incubations with testosterone-l,2-3H
22:2
(Table 3).
Testosterone was not metabolized as actively as 4-androstene-3,17dione by mouse submaxillary gland tissue.
In NADPH-fortified 15 and
180 min incubations, 84% and 57%, respectively of the testosterone remained unconverted.
The major metabolic product at both times was
5~-androstane-3~,17~-diol
(10% and 32%).
Only traces of &androstene-
3,17-dione, 17/3-hydroxy-So-androstan-3-one and androsterone were present. In identical incubations with no co-factors, only about 7% of the testosterone was metabolized in either 15 or 180 min. of So-androstane-3m,l7$-diol
A small amount
(2%) was formed at both times.
Traces of
17/3-hydroxy-5a-androstan-3-one, 4-androstene-3,17-dione and androsterone were present.
DISCUSSION
The -in vitro metabolism of 4-androstene-3,17-dione and testosterone in male mouse submaxillary gland was largely reductive; the products were mainly androsterone and 5c3-androstane-3a,l7B_diol, respectively, when NADPH was available.
The enzymes primarily involved in these
reactions were 5o-reductase and 3o-hydroxysteroid dehydrogenase, those now considered characteristic of other androgen-sensitive tissues such as kidney (18-20), preputial gland (21,22), sebaceous gland (23), scrotal skin (24), and prostate (25-28).
The activity of 17fl-hydroxysteroid dehydrogenase was not marked, especially when testosterone was the substrate.
This result conflicts
with those found in both canine and rat submaxillary gland, where
STEROIDS
Aug. 1973
255
experiments showed that testosterone metabolism was predominately oxidative, the major metabolic product being 4-androstene-3,17-dione (29-31).
The significance of the predominately oxidative androgen
metabolism in canine and rat submaxillary tissue in contrast to the predominately reductive androgen metabolism in mouse submaxillary tissue is unclear. androgen-sensitive
Canine submaxillary gland is not thought to be (32) while rat submaxillary gland, like the mouse
gland, is known to be androgen-sensitive
(12,33).
Even though 5cu-reductase and 3o-hydroxysteroid dehydrogenase activity have been demonstrated in mouse submaxillary gland, it is not conclusive that the androgen-initiated responses of submaxillary tissue are mediated by the same mechanisms proposed for other androgen-sensitive tissues.
Of the acini, intercalated ducts,
convoluted tubules, and intralobular 'striated' ducts of the rodent submaxillary gland (32), the only epithelial element believed responsive to androgen is the convoluted tubule (1,2,4,11).
It is
not possible to say from the available data that androgen-metabolizing capability is confined to convoluted tubules or is a general feature of the whole gland.
ACKNOWLEDGEMENTS
The author greatly acknowledges the technical assistance of Mrs. Dona King with part of this work. This work was supported by PHS Research Grant DE-02668 from the National Institute of Dental Research and in part by General Research Support Grant No. RR 5333 from the General Research Support Branch of the National Institutes of Health.
22:2
STEROIDS
256
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1.
Lacassagne, A., COMPT. REND. 133, 180 (1940).
2.
Rogers, A. W. and Brown-Grant, K., J. ANAT. 109, 51 (1971).
3.
Cohen, S., J.
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Turkington, R. W., Males, J. L., and Cohen, S. 252 (1971).
5.
Levi-Montalcini, R. and Angeletti, P. V. SALIVARY GLANDS AND THEIR SECRETIONS, Editors L. M. Sreebny and J. Meyer, Macmillan, New York, 1964, p. 129.
6.
Hoshino, K. and Lin, C. D., CAN J. PHYSIOL. P~~~O~. (1969).
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8.
Takeda, T. and Grollman, A., AMER. J. PHYSIOL. 215, 1337 (1968).
9.
Kongshaun, P. A. L. and Bliss, J. Q., IMMUNOLOGY l9, 188 (1969).
BIOL. CHEM, 237, 1555 (1962). CANCER RES. 3l,
47, 329
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Charreau, E. H., ACTA PHYSIOL. LAT. AMER. l9, 188 (1969).
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Sreebny, L. M. and Meyer, J., SALIVARY GLANDS AND THEIR SECRETIONS, Editors L. M. Sreebny and J. Meyer, Macmillan, New York, 1964, p. 83.
12.
Riekkinen, P. J. and Niemi, M., ENDOCRINOLOGY 83, 1224 (1968).
13.
Rosner, J. M., Camara, S. A., Cardinali, D. P., and Dieguez, M. E ., ACTA PHYSIOL. LAT. AMER. l5, 221 (1965).
14.
Rosner, J. M., Macome, J. C., and Cardinali, D. P., ENDOCRINOLOGY 85, 1000 (1969).
15.
Charreau, E. H. and Villee, C. A., ENDOCRINOLOGY 82, 630 (1968).
16.
Kase, N., Forchielli, E. and Dorfman, R. I., ACTA ENDOCR. (KOBENHAVN) 37, 19 (1961).
17.
Smith, L. L. and Berstein, S., PHYSICAL PROPERTIES OF STEROID HORMONES, Editor C. L. Engle, Macmillan, New York, 1963, p. 321.
18.
Ritzen, E. M., Nayfeh, S. N., French, F. S. and Aronin, P. A., ENDOCRINOLOGY 9l, 116 (1972).
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19.
Bullock, L. P., Bardin, C. W., and Ohno, S., BIOCHEM. BIOPHYS. RES. COMM. 44, 1537 (1971).
20.
Verhoeven, G. and De Moor, P., EN~CRINOLOGY
21.
Bullock, L., Schneider, G., and Bardin, C. W., LIFE SCI. 9 (Part i), 701 (1970).
22.
Richardson, G. S. and Axelrod, L. R. (1971).
23.
Takayasu, S. and Adachi, K., ENDOCRINOLOGY 90, 73 (1972).
24.
Flamigni, C. A., Collins, W. P., Koullapis, E. N., and Sommerville, I. F., ENDOCRINOLOGY 87, 764 (1970).
25.
Chamberlain, J,, Jagarinec, N., and Ofner, P. 610 (1966).
26.
Wilson, J. D.and Gloyna, R. E., REC. PROG. HOR. RES (1970).
27.
Leav, I., Morfin, R. F., Ofner, P., Cavazos, L. F., and Leeds, E. B., ENDOCR~NOLO~ 89, 465 (1971).
28.
Bruchovsky, N., ENDOCRINOLOGY 89, 1212 (1971).
29.
Mosadomi, H. A. and Ofner, P. 47TH ANN. MEETING INTERNATL, ASSOS. DENT. RESEARCH, March, 1969 (Abstract 504).
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Weiner, A. L., Ofner, P., and Sweeney, E. A., ENDOCRINOLOGY 87, 406 (1970).
31.
Baldi, A. and Charreau, E. H., ENDOCRINOLOGY 90, 1643 (1972).
32.
Huggins, C, and Sommer, J., J. EXP. MED. 97, 663 (1953).
33.
Jacoby, F. and Leeson, C. R., J. ANAT. 93, 201 (1959).
34.
The following trivial names and abbreviations are used in the text of this paper: Cholesterol (5-cholesten-38-01); progesterone (4-pregnene-3,20dione); testosterone (178-hydroxy-4-androsten-3-one); testosterone acetate (17$-acetoxy-4-androsten-3-one); androsterone (3ohydroxy-5a-androstan-17-one); NADP (nicotinamide adenine dinucleotide phosphate).
89, 842 (1971).
ENDOCRINOLOGY 88, 890
BIOCHEM. J. 99,
26, 309
STEROID METABOLISM BY MOUSE SUBMAXILLARY GLANDS.
I.
IN VITRO METABOLISM OF TESTOSTERONE _-
AND
4-ANDROSTENE-3,17-DIONE
James C. Coffey
Dental Research Center and the Department of Pediatrics University of North Carolina Chapel Hill, North Carolina 27514
Received:
615173
ABSTRACT
Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17$-hydroxy-5ff-androstan-3-one and 5a-androstane-3o,17g-diol. Testosterone was converted primarily to 5a-androstane-3@,17B_diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17B-hydroxy-5o-androstan-3-one, and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.
INTRODUCTION
Androgen is influential in the structural and biochemical configuration of the rodent submaxillary gland.
Sexual dimorphism of the
STEROIDS
248
22:2
convoluted tubules is influenced by testosterone (1,2). gland ep~thelial-e~ide~al
Submaxillary
growth factor (3,4), nerve growth factor
(5), lethal factor (6,7), lymphoid tissue inhibitory factor (8,9), RNA synthesis (lo), and protease (11,12) are all sensitive to androgen. Some enzymes involved in steroid metabolism have been demonstrated in vitro in rodent submaxillary glands. --
Cholesterol has been synthe-
sized from acetate, 3B-hydroxy-5-pregnen-20"one and 3B-hydroxy-5androsten-17-one from cholesterol, and progesterone from 3$-hydroxy5-pregnen-ZO-one (13,14).
The rodent submaxillary gland has also
been shown to convert progesterone to 5cu-pregnane-3,20-dione and 4-androstene-3,17-dione
(13,15).
The cited studies show that the rodent submaxillary gland is responsive to androgens and that some enzymes required for steroid metabolism are present in the tissue. taken to determine if
the
The present studies were under-
requisite enzymes for converting androgens
to reduced products are found in submaxillary glands, as has been described for other androgen-sensitive tissues.
MATERIALS AND METHODS Materials Testosterone-1,2-3H (57.8 Ci/mM), testosterone-4-14C (53 mCi/mM), 4-androstene-3,17-dione-l,2-3H (42 CifmH), 4-androstene-3 17-dione4-14C (45 mCi/mM), and 17@-hydroxy-So-androstan-3-one-4- 14, (56.1 mCi/mM) were obtained from New England Nuclear. Radiochemical purity of these steroids was checked by thin-layer chromatography prior to use. NADP, glucose-6-phosphate and glucose-6-phosphate dehydrogenase type XV, were obtained from Sigma. Thin-layer chromatography was performed on 5 x 20 cm glass plates coated with either silica gel GF-254 or aluminum oxide GF-254 obtained from E. Merck, AG. All other chemicals and solvents were reagent grade and were used without further purification. Chemical procedures Steroids were acetylated at room temperature overnight in a mixture
Aug. 1973
STEROIDS
249
of equal volumes of acetic anhydride and pyridine. The pyridine-acetic anhydride mixture was removed by evaporation -in vacua after successive additions of ethanol. Steroid acetates were saponified at room temperature overnight in 1 ml of 1 N KOH in methanol. The mixture was diluted with water, and the steroids extracted 5 times with chloroform. The chloroform extract was washed 1 time with N/l0 HCl and then with water until neutral. Preparation and incubation of homogenates Male CD-1 mice, 6 months old, were killed by cervical dislocation. The submaxillary glands were rapidly removed and collected in iced beakers. The submaxillary glands were dissected free of other tissue, minced with scissors, and the mince washed with cold 0.154 M KCl. The washed mince was then homogenized in the medium described by Kase et al. (16). A Dual1 homogenizer, cooled in ice, was used. Each incubation flask contained 250 mg of homogenized submaxillary gland tissue in 3.0 ml of 18.2 mM sodium phosphate buffer (pH 7.4) containing 0.32 mmoles of KC1 and 0.0125 mmole of MgC12. When a NADPH-generating system was utilized 0.005 mmole of NADP, 0.0425 mmole of glucose-&phosphate, and 1.5 units of glucose-&phosphate dehydrogenase were also included in each incubation flask. The testosterone-1,2-3H (Gp,Ci)or 4-androstene3,17-dione-1,2-3H (6pCi), mixed with cold carrier to give a final concentration of O.lpM, were previously added to the incubation flasks and dissolved in 0.1 ml of propylene glycol. Incubations were carried out for either 15 or 180 min at 37OC in a shaking water bath and were stopped by the addition of LO ml of a mixture of chloroform:methanol (2:l). Immediately after the addition of chlorofonn:methanol 2OOpg each of carrier testosterone, 4-androstene-3,17-dione, 17/3-hydroxy-5oandrostan-3-one, androsterone, 5a-androstane-3o,l7$-diol and 5o- and 5p-androstane-3,17-dione were added to each flask. Extraction and purification of metabolites The homogenates were extracted with 25 vol of chloroform:methanol (2:l). The solvents were evaporated in vacua, and the residues were dissolved in chloroform and washed with water. Aliquots of the extra& were chromatographed on 0.5 mm thick silica gel plates in benzene:ethyl acetate (3:l) developed twice. Radioactive peaks were located with a Packard Model 7200 Kadiochromatogram Scanner. Carrier steroids were located under UV light or by spraying with ethanolic iodine solution. With both substrates, four bands of radioactivity corresponding to the following areas were obtained in the initial chromatography step: polar steroids at the origin (area l), testosterone and 5o-androstane3a,17@-diol (area 2), 4-androstene-3,17-dione, androsterone, 17ghydroxy-5a-androstan-3-one (area 3), 5o- and 5$-androstane-3,17-dione (area 4). Area 1, polar material, was scraped off the plates and eluted for counting without further purification. When the area 4 eluate was chromatographed on silica gel in benzene:ethyl acetate 15:1, with four developments, the radioactivity moved near the solvent front and
250
STEROIDS
22:2
was not associated with either To- or 58-androstane-3,17-dione carrier. Radioactivity recovered in this relatively non-polar fraction and the polar fraction remaining at the origin after the initial chromatographic step is included in Tables 2 and 3 to provide a complete account of the distribution of radioactivity. Small aliquots from the eluates of areas 2 and 3 were removed to determine total radioactivity. The remaining portions were acetylated and chromatographed on silica gel plates in benzene:ethyl acetate. Two peaks of radioactivity were obtained on the radioscan of area 2 corresponding to testosterone acetate and So-androstane-3o,178-diol 3,17-diacetate. These two peaks were eluted, aliquots of the eluates taken for counting, and the remaining material saponified. The saponified products were separately chromatographed on aluminum oxide in benzene:ethyl acetate 2:1, developed twice. After scanning, only a single peak of radioactivity corresponding to either testosterone or 5o-androstane-3a,l78_diol was present. The acetylated area 3 eluates also yielded two peaks of radioactivity on the radioscans. One peak was isopolar with &androstene-3,17-dione carrier. The second peak was associated with androsterone and 17$-hydroxy-5o-androstan-3-one carriers. The 4-androstene-3,17-dione peak was eluted and rechromatographed on aluminum oxide in diethyl ether. Again a single radio peak isopolar with 4-androstene-3,17-dione carrier was seen after scanning. The radioactive peak containing a mixture of androsterone and 17@hydroxy-5G-androstan-3-one was eluted, saponified, and the product chromatographed on aluminum oxide in methylene dichloride:diethyl ether (9:l) with two developments. After scanning two peaks were present, one isopolar with androsterone carrier and one isopolar with l78-hydroxy-5o-androstan-3-one carrier. After thin-layer chromatography the total radioactivity was calculated by adding the radioactivity recovered in the final individual peaks and the results for a particular peak were expressed as a percentage of this total. In each case, the total radioactivity recovered was between 85-90% of the incubated amount of steroid. To confirm the identities of metabolites with the chromatographic mobilities of 4-androstene-3,17-dione, androsterone, testosterone, 17@-hydroxy-5a-androstan-17-one and 5cr-androstane-3a,l7B_diol, representative samples were further purified by crystallization from cyclohexane-acetone and ethanol-water (Table I.). Androsterone and ICYandrostane-3o,l7@-diol were crystallized to constant specific activities after addition of 20 mg of carrier steroid. Testosterone, 17S-hydroxy-5a-androstan-3-one and 4-androstene-3,17-dione were crystallized to constant 14C/31i ratios after addition of 20 mg of carrier steroid and the appropriate 14C-steroids. Measurement of radioactivity Radioactivity was measured with a Beckman LS-250 liquid scintillation spectrometer. The material scraped from thin-layer plates was eluted with warm chlorofo~:me~hanol (2:1) and the dried residues of aliquots counted in 1.5ml of toluene containing 0.3% 2,5diphenyloxazole and O.Ol%, 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene. Counting efficiencies of these samples were uniformly 53%56%.
4990t
4950'
4.9
4.9 180
Testosterone **
4.7
4.6
15
4.6
17~-~ydroxy-5~-androstan-3-ona
4.8
4100'
4.6
4.3
4540'
2.1
5.5
4.0
180
180
incubated
4.6 5.0 5.1 5.1
4,6
4130"
4.6
4.7
4000t
4.6
4,6
3900t
5050t
4.4 4.3 4.3 sooot
2.4
5.6 2.0
5.6
3.3
2‘1
5.5
4.0
DPM 3H/DPM14C or DPM/mgt Final ML c3 c4 c2
2.5
5.6
3.8
Cl
17@-Hydroxy-So-androstan-3-one
5wAndrostane-3o,278-diol
180
180
*Q unconverted substrate, Ml, = mother liquor, C = crystallization
Testosterone
Androsterone
4-Androstene~-3,17-dione ;k;k
1.5
180
Testosterone
17~-~ydroxy-5~-androstan-3-on~
15
Incubation time (min)
Testosterone
Metabolite
Crystallization of metabolites of testosterone-l,2-3H and 4-androst~n~-3,17-dione-l,2-3H with male mouse submaxillary gland homogenates.
Substrate
1.
4-Androstene-3,17-dione
Table
252
STEROIDS
22:2
When specific activities were determined, radioactivity data were expressed as dpm using a Cs-137 external standard. When crystallizations to constant 381141: ratios were performed after the addition of the appropriate l4C-labeled steroid, the isotopes were counted simultaneously and calculations based on counting efficiencies obtained with 3H- and 14C-toluene standards. Measurement of steroid weight In the determination of specific activity after crystallization of androsterone and 5a-androstane-3a,l78_dlol carrier weights of androsterone and 5tr-androstane-3@,17B_diol were measured by the optical densities of the sulfuric acid chromogens (17) at 310 mp, and 340 rnl~. respectively.
RESULTS
Incubations with 4-androstene-3,17-dione-1,2-3H
(Table 2).
In the presence of an NADPH-generating system, 4-androstene-3,17dione was actively metabolized by male mouse submaxillary gland tissue. Fifty-four percent substrate conversion occurred in 15 min and 95% conversion occurred in 180 min.
The major metabolite present at
both 15 and 180 min was androsterone, 37% and 58%, respectively. Much small.er amounts of testosterone (3-b%) and 178-hydroxy-5cr-androstan3-one (l-2%) were detected at both times.
Only a trace amount (0.8%) of
5a-androstane-3ru,l78_diol was present at 1.5min but about 7% was present after 180 min of incubation. 4-androstene-3,17-dione was poorly metabolized when the NADPHgenerating system was not included in the incubation medium.
In 15 and
180 min incubations, 93% and 89%, respectively, remained unconverted, At both times only trace amounts of androsterone, 17B-hydroxy-5oandrostan-3-one and 5a-androstane-3o,l7$-diol were present.
Only a
trace of testosterone (0.6%) was present at 15 min but a slight increase in its accumulation (to about 2%) was noted in the longer incubations.
STEROIDS
Aug. 1973
Table 2.
253
Metabolism of 4-androstene-3,17-dione-l,Z-3H submaxillary gland homogenates J;
by male mouse
NADPH generating system Absent Present 180 min 15 min 180 min 15 min -
--
Polar fraction
5.7
14.9
1.7
2.9
Testosterone
3.0
4.1
0.6
2.4
5o-Androstane-3cu,l7@-diol
0.8
6.9
0.2
0.4
4-Androstene-3,17-dione
46.0
5.0
93.5
89.5
Androsterone
37.0
58.0
0.1
0.3
17fi-Hydroxy-Sff-androstan-3-one
1.1
2.0
0.4
0.6
Non-polar fraction
6.3
9.1
3.5
3.9
*Calculated as % of total radioactivity recovered from chromatoplates.
Table 3.
Metabolism of testosterone-1,2-3H by male mouse submaxillary gland homogenates *
NADPH generating system Absent Present 180 min 180 min 15 min 15 min
Polar fraction
3.0
7.2
2.7
2.4
84.0
57.4
94.4
93.1
5o-Androstane-3a,l7g-diof
9.8
31.6
2.0
2.1
4-Androstene-3,X7-dione
0.1
0.1
0.3
0.4
Androsterone
0.1
0.2
0.1
0.1
17$-Hydroxy-Sol-androstan-3-one
0.3
0.6
0.2
0.1
Non-polar fraction
2.8
2.8
Il.2
1.7
Testosterone
Walculated
as % of total radioactivity recovered from chromatoplates.
STEROIDS
254
Incubations with testosterone-l,2-3H
22:2
(Table 3).
Testosterone was not metabolized as actively as 4-androstene-3,17dione by mouse submaxillary gland tissue.
In NADPH-fortified 15 and
180 min incubations, 84% and 57%, respectively of the testosterone remained unconverted.
The major metabolic product at both times was
5~-androstane-3~,17~-diol
(10% and 32%).
Only traces of &androstene-
3,17-dione, 17/3-hydroxy-So-androstan-3-one and androsterone were present. In identical incubations with no co-factors, only about 7% of the testosterone was metabolized in either 15 or 180 min. of So-androstane-3m,l7$-diol
A small amount
(2%) was formed at both times.
Traces of
17/3-hydroxy-5a-androstan-3-one, 4-androstene-3,17-dione and androsterone were present.
DISCUSSION
The -in vitro metabolism of 4-androstene-3,17-dione and testosterone in male mouse submaxillary gland was largely reductive; the products were mainly androsterone and 5c3-androstane-3a,l7B_diol, respectively, when NADPH was available.
The enzymes primarily involved in these
reactions were 5o-reductase and 3o-hydroxysteroid dehydrogenase, those now considered characteristic of other androgen-sensitive tissues such as kidney (18-20), preputial gland (21,22), sebaceous gland (23), scrotal skin (24), and prostate (25-28).
The activity of 17fl-hydroxysteroid dehydrogenase was not marked, especially when testosterone was the substrate.
This result conflicts
with those found in both canine and rat submaxillary gland, where
STEROIDS
Aug. 1973
255
experiments showed that testosterone metabolism was predominately oxidative, the major metabolic product being 4-androstene-3,17-dione (29-31).
The significance of the predominately oxidative androgen
metabolism in canine and rat submaxillary tissue in contrast to the predominately reductive androgen metabolism in mouse submaxillary tissue is unclear. androgen-sensitive
Canine submaxillary gland is not thought to be (32) while rat submaxillary gland, like the mouse
gland, is known to be androgen-sensitive
(12,33).
Even though 5cu-reductase and 3o-hydroxysteroid dehydrogenase activity have been demonstrated in mouse submaxillary gland, it is not conclusive that the androgen-initiated responses of submaxillary tissue are mediated by the same mechanisms proposed for other androgen-sensitive tissues.
Of the acini, intercalated ducts,
convoluted tubules, and intralobular 'striated' ducts of the rodent submaxillary gland (32), the only epithelial element believed responsive to androgen is the convoluted tubule (1,2,4,11).
It is
not possible to say from the available data that androgen-metabolizing capability is confined to convoluted tubules or is a general feature of the whole gland.
ACKNOWLEDGEMENTS
The author greatly acknowledges the technical assistance of Mrs. Dona King with part of this work. This work was supported by PHS Research Grant DE-02668 from the National Institute of Dental Research and in part by General Research Support Grant No. RR 5333 from the General Research Support Branch of the National Institutes of Health.
22:2
STEROIDS
256
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The following trivial names and abbreviations are used in the text of this paper: Cholesterol (5-cholesten-38-01); progesterone (4-pregnene-3,20dione); testosterone (178-hydroxy-4-androsten-3-one); testosterone acetate (17$-acetoxy-4-androsten-3-one); androsterone (3ohydroxy-5a-androstan-17-one); NADP (nicotinamide adenine dinucleotide phosphate).
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