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18-Hydroxylase activity in the Y1 adrenal cell line
Molecular and Cellular Endocrinology, 66 (1989) 83-91 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
83
02135
1%Hydroxylase activity in the Yl adrenal cell line L.C. Ramirez,
M. Es-souni
and P. Bow-not
Laboratoire de Biochimie des Interactions Cellulaires, Fact&P de Sciences, University de Bourgogne, 21004 Dijon Cedex, France (Received
Key words: Cytochrome
P-450,tp;
Corticosteroid;
9 February
1989; accepted
Gas chromatography-mass
12 June 1989)
spectrometry;
Tumor
cell culture;
(Mouse)
Summary 18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Yl), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20a-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20cY-reduction and 11/3-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. activity of the Yl cells is similar to that of the mouse in vivo which catalyzes The cytochrome P-450,,, the production of an 11/3,18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of lip- to 18-hydroxylated metabolites and of the structure of these metabolites.
Introduction The Yl cell line was originally cloned by Yasumura et al. (1966) from mouse adrenal cortex tumor cultures established by Buonnassisi et al. (1962). Experiments performed with various strains of albino mouse, including the LAFl strain from which the Yl cells originated, led Raman et al. (1964) to conclude that 18-hydroxycorticosterone and aldosterone are the main 18-hydroxylated steroids produced by the species. When adrenal tissue slices from Swiss albino mice were incubated with progesterone, only those two 18-
Address for correspondence: L.C. Ramirez, Laboratoire de Biochimie des Interactions Cellulaires. FacultC de Sciences, Universite de Bourgogne, B.P. 138, 21004 Dijon Cedex. France. 0303-7207/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland.
oxygenated steroids were produced. None of the other possible 18-hydroxylated metabolites, 18-hydroxyprogesterone, 18-hydroxy-ll-deoxycorticosterone or 18-hydroxy-ll-dehydrocorticosterone were detected (Raman et al., 1964; Erickson et al., 1966). The tissue slices also produced 20a-reduced steroids (Ertel and Ungar, 1968). The absence of 17a-hydroxylase activity in the mouse was confirmed both in vivo (Southcott et al., 1957) and in vitro (Hoffman, 1956). Steroid metabolism by the Yl cell line was determined by Pierson et al. (1967) and Kowal and Fiedler (1968). The production of the following seven steroids from pregnenolone was reported: 5-pregnene-3P,20a-diol, progesterone, 11/3-hydroxyprogesterone, 20a-dihydroprogesterone, ll/&hydroxy-20ar-dihydroprogesterone, llketo-20a-dihydroprogesterone, and a derivative of Ltd.
84
1 lp-hydroxy-20a-dihydroprogesterone with an additional hydroxyl group in an undetermined position. Position 18 was excluded after incubation of l&hydroxyprogesterone with the cell line (Kowal and Fiedler, 1968). These results demonstrate the presence of 3P-hydroxysteroid oxidoreductase activity, 1 lp-hydroxylase activity, 11-oxidoreductase activity, and 20a-reductase activity and the loss of 21-hydroxylase activity in the Yl cell line. No 18-hydroxylase activity has ever been reported. New cell cultures established from tumor cells obtained after injection of the Yl cells into the animal, had reestablished 21-hydroxylase activity (Buonassisi et al., 1962). The existence of a single protein expressing both I@-hydroxylase activity and 18-hydroxylase activity was proposed as early as 1976 by Ulick and Rapp and Dahl. This hypothesis has now been vastly documented from data acquired through the study of adrenal pathologies, from experiments in vitro using various species and from experiments using the purified protein. The ratio of lip- to 18-hydroxylation varies according to the species. In the human it is believed to be of the order of 15 : 1 (Ulick, 1976). In purified bovine cytochrome P-450, ,p the ratio has been reported to be 11 : 1 (Watanuki et al., 1977) and 6 : 1 (Sato et al., 1978). Previously the ratio of lip- to 18-hydroxylation had been reported to be between 7 : 1 and 10 : 1 in bovine mitochondria and between 2 : 1 and 3 : 1 in rat mitochondria (Bjorkhem and Kalmar, 1975). The production of 18-hydroxycorticosterone and aldosterone, from ll-deoxycorticosterone, has also been attributed to cytochrome P-450,,, (Ohnishi et al., 1984; Wada et al., 1984; Yanagibashi et al., 1986) although the protein might be somewhat different in the fasciculata and the glomerulosa zones (Lauber et al., 1987). Generally, there is less aldosterone than 18-hydroxycorticosterone produced. In a reconstituted cytochrome P-450,,, prepared from bovine adrenal mitochondria the ratio of 1 &hydroxycorticosterone to aldosterone produced from corticosterone was 50 : 1 (Wada et al., 1984). However, in other preparations from bovine, porcine and rat mitochondria of different zones and with or without treatment the ratio has varied between 2 : 1 and 50 : 1 (Yanagibashi et al., 1986; Lauber et
al., 1987). The ratio also varies depending on the substrate used. Since lip-hydroxylase activity is one of the major enzymatic activities of the Yl cell line, its possible association with 18-hydroxylase and 18dehydrogenase activity was studied using ll-deoxycorticosterone and corticosterone as substrates. Materials and methods Reagents All solvents were pure for analysis quality (Merck) or grade RS HPLC (Carlo Erba). The following reagents were used: sodium borohydride and lithium aluminum hydride from Merck; Omethoxyamine hydrochloride from Pierce Chemical Co.; bis(trimethylsilyl)-trifluoroacetamide and trimethylchlorosilane from Supelco. C,,, C,,, and C,, n-alcanes were purchased from Fluka. Reference steroids Commercially available reference steroids were purchased from Steraloids and Makor Chemicals. Commercially unavailable 20a-reduced steroids containing an 18-methyl group were prepared by chemical reduction of the corresponding 20-0~0 compounds using sodium borohydride in pyridine (Bournot et al., 1989). The reduction in position 20 of 18-hydroxycorticosterone was performed with lithium aluminum hydride in ether as follows. The methoxime derivative in position 3 of the steroid (80 pg) was formed by reaction with Omethoxyamine hydrochloride in pyridine (16 mg/ml) for 15 min at 4°C. After evaporation of the pyridine, 300 pg of lithium aluminum hydride were added in 1.5 ml of anhydrous ether; the reaction was stopped after 15 min at 30” C by addition of water. The reaction products were extracted with ethyl acetate. Cell culture incubations The Yl mouse adrenal cell line from Flow Laboratories was routinely grown in Ham’s FlO medium (Gibco) supplemented with either 2.5% fetal calf serum (Gibco) and 2.5% newborn calf serum (Gibco) or 5% of each serum, 100 IU penicillin/ml and 100 pg streptomycin/ml in a humidified atmosphere of air-CO, (95%-5%) at
85
37” C. The number of dishes necessary for each experiment was prepared by seeding a cell suspension prepared by trypsination of several confluent dishes (50 mg trypsin/lOO ml Ham’s FlO medium without calcium or magnesium salts). These homogenous cell dishes reached confluency in 5-6 days. Two days before the start of the incubation period, cell cultures kept at 5% serum-supplemented medium were changed to 10% serum-supplemented medium. Incubations were performed for 4, 24, 48, and 72 h periods by replacement of the spent medium with 4 ml of fresh medium and addition of the corresponding steroid precursor (100 pg) in 0.01 ml of ethanol. Steroid extraction and analysis Steroids were extracted on Sep-pak C,, cartridges (Waters Associates) and analyzed by high performance liquid chromatography (HPLC) or gas chromatography (GC) as previously described (Ramirez et al., 1982). HPLC analysis was performed on a 10 pm Micropack C,, reversedphase column (Varian) using a 30 : 70% acetonitrile/water solvent gradient programmed linearly in 30 min at a flow rate of 1.2 ml/mm; column temperature was 40” C. GC analysis was performed on a 25 m SE-30 wall-coated fused silica column (Spiral) programmed from 240° C at 1 “/min. The extracted steroids, fractionated or not by HPLC, were analyzed as the methoximetrimethylsilyl (MO-TMS) derivatives by gas chromatography-mass spectrometry (GC-MS). A Ribermag lo-10B quadrupole mass spectrometer (Nermag) coupled to an SE-30 column with helium as the carrier gas, was used. Other conditions were as follows: ion source temperature, 250°C; electron energy, 70 eV; filament current, 0.2 mA. Electron impact spectra were recorded with a Sidar 150-Riber Computer Data System at 3 ms per apparent mass unit (a.m.u.). Methylene unit (M.U.) values (Dagliesh et al., 1966) were calculated during GC-MS analysis using C,, and C,, n-alcanes with an oven temperature program from 230 o C at l”/min. Steroid quantification was done using n-alcane and ll-deoxycorticosterone-21C,, acetate, respectively, as internal standards during GC and HPLC analysis. The response coefficients were calculated from a mixture of reference steroids. The 20a-reduced reference steroids not
available commercially were assumed to have a response coefficient equal to their closest 20-0~0 homologues. Results The metabolites of the exogenous substrates, deoxycorticosterone and corticosterone, were detected together with the endogenous metabolites produced from cholesterol in the serum-supplemented medium. The latter were the same as reported by Pierson et al. (1967) and Kowal and Fiedler (1968). However, under our incubation conditions, the predominant metabolite was liphydroxy-20a-dihydroxy-4-pregnen-3-one, as shown in Fig. 1; minor quantities of progesterone and 20a-dihydroprogesterone were sometimes detected. The qualitative analysis of the metabolites of deoxycorticosterone and corticosterone in this study was made difficult by the large number of metabolites and the very large differences in their quantities. Furthermore, the majority of the MOTMS derivatives of these metabolites form synand anti-isomers which separate during CC analysis (Boumot et al., 1989). Preparative HPLC permitted partial purification of minor metabolites and analysis by GC-MS. In other cases, apparent resolution of compounds eluted under the same peak was obtained during GC-MS analysis by detection of ions specific to each metabolite. A metabolite was considered identified when the M.U. value, and the mass spectrum or the specific ions recorded, were identical to those of the reference compound. The quantitative analysis was simplified by the fact that some of these unresolved compounds were present in negligible amounts, as seen by GC-MS analysis. A complementary quantification by HPLC, where an adequate separation of compounds unresolved by GC is accomplished, allowed confirmation of their quantification. Metabolism
of I I -deoxycorticosterone
(DOC)
Identification of the metabolites of DOC All metabolites produced during the incubation of DOC with the Yl cell line in culture medium
86 TABLE
1
METHYLENE UNIT VALUES OF THE METHOXIME-TRIMETHYLSILYL LITES PRODUCED BY THE Yl ADRENAL CELL LINE
DERIVATIVES
OF DOC AND
Steroids
M.U. values a
21-Hydroxy-4-pregnene-3,20-dione (ll-deoxycorticosterone; DOC)
30.49 (31.02) b
11/3,21-Dihydroxy-4-pregnene-3,20-dione (corticosterone; B)
32.15; 32.20 ’ (32.60; 32.65) h.c
11/3,18,21-Trihydroxy-4-pregnene-3,20-dione (18-hydroxycorticosterone; 18-hydroxyB)
33.25; 33.29 ’
20a,21-Dihydroxy-4-pregnen-3-one (20~dihydro-ll-deoxycorticosterone;
31.42 20wDHDOC)
18,20a,21-Trihydroxy-4-pregnen-3-one (18-hydroxy-20a-dihydro-ll-deoxycorticosterone;
32.68 18-hydroxy-20a-DHDOC
11/3,20a,21-Trihydroxy-4-pregnen-3-one (20wdihydrocorticosterone; 20wDHB)
33.28; 33.33 ’
11/3,18.20a,21-Tetrahydroxy-4-pregnen-3-one (18-hydroxy-20wdihydrocorticosterone;
18-hydroxy-20a-DHB)
20a,21-Dihydroxy-4-pregnene-3,11-dione (20a-dihydro-ll-dehydrocorticosterone;
20~DHA)
34.44; 34.48 ’
32.36
X,20a,21-Trihydroxy-4-pregnen-3-one d (X-hydroxy-20cy-dihydro-ll-deoxycorticosterone; Y,ll/3,20a,21-Tetrahydroxy-4-pregnen-3-one (Y-hydroxy-20wdihydrocorticosterone; ’ b ’ d
32.36 X-hydroxy-20a-DHDOC) d Y-hydroxy-20a-DHB)
33.97
Methylene unit values are calculated with an oven temperature programmed at lo C/mm Minor isomeric peaks. Syn- and anti-isomers of the methoxime derivative in position 3. X and Y, unidentified positions of the additional hydroxyl group, could be identical.
containing
10% serum for 72 h are listed in Table
compound
trum of its MO-TMS
derivative
metabolite
reported
et al., 1985).
(Ramirez
(20~
1 in Fig. 1). The mass spec-
of the rat adrenal
is similar
of DOC
ture,
the most
from 220 ’ C.
prominent
loss of the fragment
1 with their M.U. values. (a) 20ar-Dihydro-ll-deoxycorticosterone DHDGC;
ITS METABO-
to that
previously
(b) 20a-Dihydro-11-dehydrocorticosterone (20a-DHA; compound 3 in Fig. 1). The mass spectrum of the MO-TMS derivative is similar to that of the reference compound. It shows the following molecular and characteristic ions (relative abundance in parentheses): 519 (M)+ (44%); 504 (M-15)+ (8%); 488 (M-31)+ (5%); 416 (M103)+ (100%); 326 (M-103-90)+ (48%); 295 (35%); 276 (19%); 103 (91%). As generally observed for compounds with a 20,21-ditrimethylsiloxy struc-
peaks
result
from
the
of mass 103.
(c) 20a-Dihydrocorticosterone (20a-DHB; compound 6 in Fig. 1). The mass spectrum of its MO-TMS ence
derivative
compound
metabolite
is similar to that of the refer-
and
of DOC
to that
previously
of the rat adrenal reported
(Ramirez
et al., 1985). The most prominent peaks are those arising from the loss of the fragments of mass 103 and 90 a.m.u. corresponding to a CH,OTMS group and a trimethylsilanol, respectively. They are characteristic of steroids with a 20,21-ditrimethylsilyloxy structure (Gustafsson and Sjiivall, (d) 18-Hydroxy-20a-dihydrocorticosterone
1968). (1%
hydroxy-20cy-DHB; compound 9 in Fig. 1). The mass spectrum of the MO-TMS derivative is very similar to that of the corresponding authentic
87 110,20a-dihydroxy4-pregnen-3-one Cholesterol
\
p7 d
C36
4 DO
I
15
20
v-
I
I
25
30
35
40 min
Fig. 1. GC analysis of the MO-TMS derivatives of the metabolites of deoxycorticosterone (DOC) synthesized during its incubation with the Yl adrenal cell line. Chromatogram obtained from the steroid extract before fractionation by HPLC. Analytical conditions were as described in Material and Methods. Proposed structures for steroid metabolites are as follows: 1. 20~DHDOC; 2 and 2’, B and its secondary peak, respectively; 3 and 4, 20~DHA and X-hydroxy-20a-DHDOC; 5, 18-hydroxy-20ol-DHDOC; 6 and 7, 20~DHB and 18-hydroxyB; 8, Y-hydroxy-20(r-DHB; and 9,18-hydroxy-20a-DHB. Other compounds are cholesterol from the incubation medium; 1 lp,20 a-dihydroxy-4-pregnen-3-one, the main endogenous steroid; and C,, n-alcane, the internal standard.
standard (Fig. 2b and 2~). Both spectra show prominent peaks at m/z 578 (M-103)+, 488 (M103-90)+ and 398 (M-103-2 x 90)+. They also contain an ion at m/z 326 which arises from the loss of trimethylsilanol and a fragment of mass 265 which is specific for steroids with an 18,20,21trimethylsilyloxy structure (Boumot et al., 1981). This structure also favors the cleavage between C,, and C,, producing the ion at m/z 205. The
mass spectrum of the same compound with a free llj%hydroxyl group (not derivated) is given for comparison (Fig. 2~). (e) 18-Hydroxy-20ol-dihydro-ll-deoxycorticosterone (18-hydroxy-20ar-DHDOC; compound 5 in Fig. 1). In addition to the ions observed at m/z 490 (M-103)+, 400 (M-103-90)+ and 310 (M-103-2 x 90)’ in the mass spectrum of the MO-TMS derivative of compound 5 (Fig. 3) the base peak is observed at m/z 328 (M-265)+. On the basis of these fragments which characterize the 18,20,21tritritrimethylsilyloxy structure, compound 5 was tentatively identified as 18-hydroxy-20~DHDOC. (f) Corticosterone (B; compound 2 in Fig. 1). (g) 18-Hydroxycorticosterone (18-hydroxyB; compound 7 in Fig. 1). This compound and 20~ dihydrocorticosterone (compound 6) were eluted very close during GC analysis. Compound 7 was identified by GC-MS after previous purification by HPLC. (h) X-Hydroxy-20a-dihydroDOC and Y-hydroxy-20cY-dihydrocorticosterone (compounds 4 and 8 in Fig. 1, respectively). The position of the additional hydroxyl group in these two compounds has not been determined. Their identification is in progress and will be reported separately. Quantification
of the metabolites
of DOC
Analysis by GC and HPLC permitted the quantification of the following metabolites (Table 2): residual DOC, 20a-reduced steroids (2OaDHDOC, 20~DHB, 20~DHA, 18-hydroxy20~DHDOC and 18-hydroxy-20a-DHB), lloxygenated steroids (B, 20~DHB, 18-hydroxy20~DHB, and ~OWDHA), and 18-hydroxylated steroids (18-hydroxy-20a-DHDOC and 18-hydroxy-20a-DHB). The metabolism of DOC, incomplete in 4 h (34.4% residual DOC), is at a maximum after 24 h of incubation (2.1% residual DOC). After 4 h of incubation the predominant metabolites are 20~DHDOC (30.2%) and 20~ DHB (29.5%); corticosterone is a minor metabolite (4.9%). However, after 24 h of incubation there is less 20a-DHDOC (19.0%) and much more 20~DHB (62.7%); there is also a larger accumulation of corticosterone (10.9%). These results imply that the fastest metabolic reaction is the conversion of DOC into 20~DHDOC and that the rate limiting reaction in the formation of ZOWDHB is
M-31-2x90
100
150
200
250
300
350
LOO
L5O
500
550
600
mlz
100
150
200
250
300
350
400
L50
so0
550
600
650
mlz
600
650
mh
100 p_ T 80
.I03
M-90-265 326
(0
I 60
M-K)3-2x90 398 373
M-103-3x90 308
: 5 40. :
205
380
M-03 578
M-X)390
Ii 70 r 100
150
200
250
300
350
450
so0
550
Fig. 2. Mass spectra of the MO-TMS derivatives of the reference compound 18-hydroxy-20~DHB (synthesized chemically from 1%hydroxyB) with a free 11/3-hydroxyl group (a) and with an lip-trimethylsilyloxy group (b). Mass spectrum of the MO-TMS derivative of 18-hydroxy-20a-DHB (compound 9 in Fig. 1) produced from the incubation of DOC with the Yl cell line (c).
20-0~0 group than that of DOC. These results are similar to those reported for the metabolism of exogenous substrates in primary cultures of newborn rat adrenal cells; progesterone was reduced faster than I@-hydroxyprogesterone and DOC faster than corticosterone (Ramirez and Maume, 1984; Raoux and Maume, 1984). The oxidation of the I@-hydroxyl group to 11-0x0 (20a-DHA), a
ll/%hydroxylation. The accumulation of corticosterone also implies a competition for IWhydroxylation between two substrates, DOC and ~OWDHDOC. The accumulation of corticosterone after 24 h of incubation (10.9%) and its diminution during 48 and 72 h incubations (6.7 and 3.1%, respectively), shows a slower rate of reduction of its -
100 M-265 328
‘: GOL
M-103-90
” 60.
400
s .c_ LO0 .103 .t 3 20. E 100
M-103 490
M-103-2x90 310 285.
150
Fig. 3. Mass spectrum
200
250
300
?J4 350
400
450
500
550
of the MO-TMS derivative of compound 5 in Fig. 1 synthesized from DOC by the Yl adrenal identified as 18-hydroxy-20a-DHDOC. Spectrum obtained after purification by HPLC.
600 mh cell line and
89
TABLE
2
PRODUCTION OF DOC METABOLITES (PERCENT OF TOTAL STEROIDS) AS A FUNCTION OF INCUBATION TIME WITH THE Yl ADRENAL CELL LINE Each value is the mean of two determinations of duplicate cell dishes. Range of variation between quantifications is l-68. Metabolites
Incubation
time
4h
24 h
48 h
12 h
DOC B 20a-DHDOC 20(~-DHB 20a-DHA 18-Hydroxy-20 a-DHDOC 18-Hydroxy-20a-DHB
34.4 4.9 30.2 29.5 0.9 _ a _
2.1 10.9 19.0 62.7 3.8 1.4 _
4.2 6.1 2.8 75.1 3.7 3.0 4.5
1.7 3.1 1.2 66.6 10.2 2.2 15.0
20a-Reduced steroids 11-Oxygenated steroids 18-Hydroxylated steroids
60.6 35.2 _
87.0 71.4 1.4
89.1 90.0 7.5
95.2 94.9 17.2
and 18-hydroxy-20&-DHB; there was no 18-hydroxyDOC detected and 18-hydroxycorticosterone was only detected in trace amounts. Incubation of 18-hydroxyDOC and 18-hydroxycorticosterone did not produce the corresponding 20a-reduced metabolites (results not shown), therefore, the formation of 18-hydroxy-20a-DHDOC and 18-hydroxy-20a-DHB implies the 18-hydroxylation of 20a-DHDOC and of 20~DHB. The incubation of 18-hydroxylated steroids in primary cultures of newborn rat adrenal cells also showed little or no formation of the corresponding 20a-reduced compounds (Ramirez and Maume, 1984). The lack of accumulation of 18-hydroxy-20cY-DHDOC indicates the possible 11/3-hydroxylation of this compound in the formation of 18-hydroxy-20a-DHB. Metabolism
of corticosterone
(B)
a Not detected.
minor reaction in 4 h of incubation (0.9%) increases with incubation time (10.2% in 72 h), and not with the availability of substrate (20~DHB). The formation of 18-hydroxylated steroids which occurs only after 24 h of incubation (1.4%) increases after 48 h (7.5%) and again after 72 h (17.2%). The only measurable 18-hydroxylated steroids detected were 18-hydroxy-20a-DHDOC
TABLE
3
PRODUCTION OF CORTICOSTERONE (B) METABOLITES (PERCENT OF TOTAL STEROIDS) AS A FUNCTION OF INCUBATION TIME WITH THE Yl ADRENAL CELL LINE Each value is the mean of two determinations of duplicate cell dishes. Range of variation between quantifications is l-68. Metabolites
Incubation
time
24 h
48 h
12 h
B A 20a-DHB 20a-DHA 18-Hydroxy-ZOa-DHB
36.6 3.9 48.9 1.3 _ a
23.8 1.2 69.4 4.1 1.5
21.4 2.1 55.6 13.8 7.0
20 u-Reduced steroids 11-Dehydrogenated steroids 18-Hydroxylated steroids
56.0 11.2 _
75.0 5.3 1.5
76.4 15.9 7.0
’ Not detected.
Identification of the metabolites of B The metabolites of corticosterone produced by incubation with the Yl cell line under the same conditions as DOC, were identified in the same manner as the metabolites of DOC. The predominant metabolite was 20a-dihydrocorticosterone. Minor metabolites were 20a-dihydro-ll-dehydro18-hydroxy-20a-dihydrocorticorticosterone, costerone (A), 18-hydroxycorticosterone, 1 l-dehydrocorticosterone, and the unidentified Y-hydroxy-20cu-dihydrocorticosterone. Quantification of the metabolites of B Analysis by GC and HPLC permitted the quantification of residual B, 20a-reduced steroids (20~DHB, 20~DHA, and 18-hydroxy-20aDHB), 11-oxygenated steroids (A and 20~DHA), and an 18-hydroxylated steroid (18-hydroxy-20~ DHB). As shown in Table 3, the metabolism of corticosterone was not complete even after 72 h of incubation (21.4% residual corticosterone); the predominant metabolite was 20~DHB (55.6%) with smaller amounts of 20~DHA (13.8%). The slower reduction of corticosterone (36.6% residual corticosterone after 24 h) in contrast to that of DOC (2.1%) is in agreement with the proposition that the main metabolic pathway in the production of 20~DHB from DOC is 20a-reduction followed by ll/Shydroxylation. In the metabolism of corticosterone, as in the metabolism of DOC,
90
oxidation of the lip-hydroxyl group to 11-0x0 (20a-DHA) seems to increase as a function of time (7.3% in 24 h and 13.8% in 72 h) and not as a function of available substrate (20a-DHB). In comparison to the incubation of DOC where 18hydroxylation is detected after 24 h, 18-hydroxy20a-DHB is detected (1.5%) only after 48 h of incubation with corticosterone. There is less 18hydroxy-20a-DHB formed from corticosterone (7.0% in 72 h) than from DOC (15.0%). However, it is not possible to say that DOC (or 20aDHDOC) is a better substrate for 18-hydroxylation than corticosterone (or 20a-DHB) since from the incubation of DOC there is more available substrate (20a-DHB and 18-hydroxy-20aDHDOC). Discussion This study is the first to demonstrate 18-hydroxylase activity in Yl cells. The major 18-hydroxylated metabolite from the incubation of DOC and corticosterone was identified as 18-hydroxy20a-DHB; 18-hydroxy-20a-DHDOC was identified as a minor metabolite from the incubation of DOC. Because of the very strong 20a-oxidoreductase activity in Yl cells, most metabolites, including those 18-hydroxylated, are also 20a-reduced. Although these metabolites represent a small proportion of the total metabolites after 24 h of incubation, the amount of 18-hydroxy-20aDHB produced increases significantly with incubation time. The production of 18-hydroxylated metabolites in Yl cells is similar to that of the mouse adrenal in vivo, that is, very little or no 18-hydroxyDOC and a measurable amount of 18-hydroxycorticosterone (Raman et al., 1964). It is also similar to that of the in vitro system where adrenal tissue slices were reported to produce 18-hydroxycorticosterone but no 18-hydroxyprogesterone or 18hydroxyDOC from progesterone (Raman et al., 1964; Eriksson et al., 1966). In our laboratory, quartered mouse adrenals and mitochondrial preparations produced corticosterone and 18-hydroxycorticosterone from the incubation of DOC; no 18-hydroxyDOC was detected. Therefore, it would seem that the 18-hydroxylated steroids preferentially synthesized by mouse adrenal cells,
in vivo and in vitro, are those which are also hydroxylated in position 1 lp. The cytochrome P-450,,, of Yl cells seems able to express both lip- and 18-hydroxylase activities as do the different cytochromes P-450,,, whose enzymatic activities have been previously characterized (Takemori et al., 1975; Watanuki et al., 1977; Sato et al., 1978; Kim et al., 1983). However, even though much more corticosterone than 18-hydroxyDOC is accumulated, the ratio of lipto 18-hydroxylated compounds cannot be calculated since these products are further metabolized to 1 l/3,1 8-dihydroxylated compounds. The activity of this cytochrome P-450,,,, however, is different from that of newborn rat adrenal cells in primary culture where the predominant metabolites of DOC are corticosterone and 18-hydroxyDOC, and very little 18-hydroxycorticosterone is formed. In these cell cultures the ratio of lip- to 18-hydroxylated compounds is of the order of 2 : 1 (Ramirez et al., 1985). The cytochrome P-450,,, of Yl cells cannot be directly compared to that of bovine cells in culture, since to our knowledge, 18-hydroxylated steroids have not yet been reported in these cell cultures. It is probable that bovine cytochrome P-450,,, activity in cell culture is similar to that of the purified enzyme which catalyzes the formation of 11/3- and 18-hydroxylated metabolites in a ratio of 11 : 1 (Watanuki et al., 1977) or 6 : 1 (Sato et al., 1978). These results indicate that cytochrome P-450,,, activity is dependent on the species, both in terms of the ratio of lip- to 18-hydroxylated metabolites and of the structure of these metabolites. Lately, 18-dehydrogenase activity has also been attributed to cytochrome P-450,,,. In reconstituted systems using purified cytochrome Pfrom different species and from both the 45011, glomerulosa and fasciculata zones, aldosterone was produced from DOC, corticosterone and 18-hydroxycorticosterone (Ohnishi et al., 1984; Wada et al., 1984; Yanagibashi et al., 1986; Lauber et al., 1987). In the Yl cell line, the formation of 18-hydroxy-20a-DHB from DOC and corticosterone led us to look for the formation of aldosterone and its 20a-reduced metabolite. Neither of these products was detected. It is possible that they are produced in amounts undetectable by GC-MS analysis since aldosterone is generally synthesized
91
in much smaller amounts than 18-hydroxylated steroids. The production of aldosterone and/or its 20a-reduced metabolite by Yl cells is being studied using radioactive precursors. This study demonstrates that the Yl cell line is a good cellular model for the study of cytochrome P-450,,, activity, since it retains the 18-hydroxylase activity associated with this enzyme during steroid biosynthesis in the adrenal gland in vivo. Acknowledgements This work was supported by the Direction de la Recherche of the Minis&e de 1’Education Nationale, the Centre National de la Recherche Scientifique (AI 034617) and the Institut National de la Sante et de la Recherche Medicale (U 208). We wish to thank J.P. Berlot for the mitochondrial preparations. References Bjorkhem, I. and Karlmar, K.E. (1975) Eur. J. Biochem. 51, 145-154. Bournot, P., Maume, B.F. and Padieu, P. (1981) J. Steroid Biochem. 14, 1127-1137. Bournot, P., Ramirez, L.C., Pitoizet, N., Maume, B.F. and Padieu, P. (1989) Steroids 53 (in press). Buonassissi, V., Sato, G. and Cohen, A.I. (1962) Proc. Natl. Acad. Sci. U.S.A. 48, 1184-1190. Dagliesch, C.E., Homing, E.C., Homing, M.G., Knox, K.L. and Yarger, Y. (1966) B&hem. J. 101, 792-810. Erickson, R.E., Ertel, R.J. and Ungar, F. (1966) Endocrinology 78, 343-349. Ertel, R.J. and Ungar, F. (1968) Endocrinology 82, 527-534.
Gustafsson, J.A. and Sjovall, J. (1968) Eur. J. Biochem. 6, 236-247. Hoffman, F.G. (1956) Endocrinology 59, 712-715. Kim, C.Y., Sugiyama, T., Okamoto, M. and Yamano, T. (1983) J. Steroid Biochem. 18, 593-599. Kowal, J. and Fiedler, R. (1968) Arch. Biochem. Biophys. 128, 406-421. Lauber, M., Sugano, S., Ohnishi, T., Okamoto, M. and Mtiller, J. (1987) J. Steroid Biochem. 26, 693-698. Ohnishi, T., Wada, A., Nonaka, Y., Okamoto, M. and Yamano, T. (1984) Biochem. Int. 9, 715-723. Pierson, R.W. (1967) Endocrinology 81, 693-707. Raman, P.B., Ertel, R.J. and Ungar, F. (1964) Endocrinology 74, 865-869. Ramirez, L.C. and Maume, B.F. (1984) CR. Sot. Biol. (Paris) 178, 77-83. Ramirez, L.C., Millot, C. and Maume, B.F. (1982) J. Chromatogr. 229, 267-281. Ramirez, L.C., Bournot, P. and Maume, B.F. (1985) J. Steroid B&hem. 22, 249-256. Raoux, R. and Maume, B.F. (1984) CR. Sot. Biol. (Parts) 178, 84-91. Rapp, J.P. and Dahl, L.K. (1976) Biochemistry 15, 1235-1242. Sato, H., Ashida, N., Suhara, K., Itagaki, E., Takemori, S. and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 307-314. Southcott, CM., Bandy, H.E., Newsom, SE. and Darrach, M. (1956) Can. J. Biochem. Physiol. 34, 913-918. Takemori, S., Sato, H., Gomi, T., Suhara, K. and Katagiri, M. (1975) Biochem. Biophys. Res. Commun. 67, 1151-1157. Ulick, S. (1976) Am. J. Cardiol. 38. 814-824. Wada, A., Okamoto, M., Nonaka, Y. and Yamano, T. (<984) Biochem. Biophys. Res. Commun. 119, 365-371. Watanuki, M., Tilley, B.E. and Hall. P.F. (1977) B&him. Biophys. Acta 483, 236-247. Yanagibashi, K., Hanui, M., Shively, J.E., Shen, W.H. and Hall, P. (1986) J. Biol. Chem. 261, 3556-3562. Yasumura, Y., Buonassissi, V. and Sato, G. (1966) Cancer Res. 26. 529-535.
MOLCEL
83
02135
1%Hydroxylase activity in the Yl adrenal cell line L.C. Ramirez,
M. Es-souni
and P. Bow-not
Laboratoire de Biochimie des Interactions Cellulaires, Fact&P de Sciences, University de Bourgogne, 21004 Dijon Cedex, France (Received
Key words: Cytochrome
P-450,tp;
Corticosteroid;
9 February
1989; accepted
Gas chromatography-mass
12 June 1989)
spectrometry;
Tumor
cell culture;
(Mouse)
Summary 18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Yl), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20a-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20cY-reduction and 11/3-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. activity of the Yl cells is similar to that of the mouse in vivo which catalyzes The cytochrome P-450,,, the production of an 11/3,18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of lip- to 18-hydroxylated metabolites and of the structure of these metabolites.
Introduction The Yl cell line was originally cloned by Yasumura et al. (1966) from mouse adrenal cortex tumor cultures established by Buonnassisi et al. (1962). Experiments performed with various strains of albino mouse, including the LAFl strain from which the Yl cells originated, led Raman et al. (1964) to conclude that 18-hydroxycorticosterone and aldosterone are the main 18-hydroxylated steroids produced by the species. When adrenal tissue slices from Swiss albino mice were incubated with progesterone, only those two 18-
Address for correspondence: L.C. Ramirez, Laboratoire de Biochimie des Interactions Cellulaires. FacultC de Sciences, Universite de Bourgogne, B.P. 138, 21004 Dijon Cedex. France. 0303-7207/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland.
oxygenated steroids were produced. None of the other possible 18-hydroxylated metabolites, 18-hydroxyprogesterone, 18-hydroxy-ll-deoxycorticosterone or 18-hydroxy-ll-dehydrocorticosterone were detected (Raman et al., 1964; Erickson et al., 1966). The tissue slices also produced 20a-reduced steroids (Ertel and Ungar, 1968). The absence of 17a-hydroxylase activity in the mouse was confirmed both in vivo (Southcott et al., 1957) and in vitro (Hoffman, 1956). Steroid metabolism by the Yl cell line was determined by Pierson et al. (1967) and Kowal and Fiedler (1968). The production of the following seven steroids from pregnenolone was reported: 5-pregnene-3P,20a-diol, progesterone, 11/3-hydroxyprogesterone, 20a-dihydroprogesterone, ll/&hydroxy-20ar-dihydroprogesterone, llketo-20a-dihydroprogesterone, and a derivative of Ltd.
84
1 lp-hydroxy-20a-dihydroprogesterone with an additional hydroxyl group in an undetermined position. Position 18 was excluded after incubation of l&hydroxyprogesterone with the cell line (Kowal and Fiedler, 1968). These results demonstrate the presence of 3P-hydroxysteroid oxidoreductase activity, 1 lp-hydroxylase activity, 11-oxidoreductase activity, and 20a-reductase activity and the loss of 21-hydroxylase activity in the Yl cell line. No 18-hydroxylase activity has ever been reported. New cell cultures established from tumor cells obtained after injection of the Yl cells into the animal, had reestablished 21-hydroxylase activity (Buonassisi et al., 1962). The existence of a single protein expressing both I@-hydroxylase activity and 18-hydroxylase activity was proposed as early as 1976 by Ulick and Rapp and Dahl. This hypothesis has now been vastly documented from data acquired through the study of adrenal pathologies, from experiments in vitro using various species and from experiments using the purified protein. The ratio of lip- to 18-hydroxylation varies according to the species. In the human it is believed to be of the order of 15 : 1 (Ulick, 1976). In purified bovine cytochrome P-450, ,p the ratio has been reported to be 11 : 1 (Watanuki et al., 1977) and 6 : 1 (Sato et al., 1978). Previously the ratio of lip- to 18-hydroxylation had been reported to be between 7 : 1 and 10 : 1 in bovine mitochondria and between 2 : 1 and 3 : 1 in rat mitochondria (Bjorkhem and Kalmar, 1975). The production of 18-hydroxycorticosterone and aldosterone, from ll-deoxycorticosterone, has also been attributed to cytochrome P-450,,, (Ohnishi et al., 1984; Wada et al., 1984; Yanagibashi et al., 1986) although the protein might be somewhat different in the fasciculata and the glomerulosa zones (Lauber et al., 1987). Generally, there is less aldosterone than 18-hydroxycorticosterone produced. In a reconstituted cytochrome P-450,,, prepared from bovine adrenal mitochondria the ratio of 1 &hydroxycorticosterone to aldosterone produced from corticosterone was 50 : 1 (Wada et al., 1984). However, in other preparations from bovine, porcine and rat mitochondria of different zones and with or without treatment the ratio has varied between 2 : 1 and 50 : 1 (Yanagibashi et al., 1986; Lauber et
al., 1987). The ratio also varies depending on the substrate used. Since lip-hydroxylase activity is one of the major enzymatic activities of the Yl cell line, its possible association with 18-hydroxylase and 18dehydrogenase activity was studied using ll-deoxycorticosterone and corticosterone as substrates. Materials and methods Reagents All solvents were pure for analysis quality (Merck) or grade RS HPLC (Carlo Erba). The following reagents were used: sodium borohydride and lithium aluminum hydride from Merck; Omethoxyamine hydrochloride from Pierce Chemical Co.; bis(trimethylsilyl)-trifluoroacetamide and trimethylchlorosilane from Supelco. C,,, C,,, and C,, n-alcanes were purchased from Fluka. Reference steroids Commercially available reference steroids were purchased from Steraloids and Makor Chemicals. Commercially unavailable 20a-reduced steroids containing an 18-methyl group were prepared by chemical reduction of the corresponding 20-0~0 compounds using sodium borohydride in pyridine (Bournot et al., 1989). The reduction in position 20 of 18-hydroxycorticosterone was performed with lithium aluminum hydride in ether as follows. The methoxime derivative in position 3 of the steroid (80 pg) was formed by reaction with Omethoxyamine hydrochloride in pyridine (16 mg/ml) for 15 min at 4°C. After evaporation of the pyridine, 300 pg of lithium aluminum hydride were added in 1.5 ml of anhydrous ether; the reaction was stopped after 15 min at 30” C by addition of water. The reaction products were extracted with ethyl acetate. Cell culture incubations The Yl mouse adrenal cell line from Flow Laboratories was routinely grown in Ham’s FlO medium (Gibco) supplemented with either 2.5% fetal calf serum (Gibco) and 2.5% newborn calf serum (Gibco) or 5% of each serum, 100 IU penicillin/ml and 100 pg streptomycin/ml in a humidified atmosphere of air-CO, (95%-5%) at
85
37” C. The number of dishes necessary for each experiment was prepared by seeding a cell suspension prepared by trypsination of several confluent dishes (50 mg trypsin/lOO ml Ham’s FlO medium without calcium or magnesium salts). These homogenous cell dishes reached confluency in 5-6 days. Two days before the start of the incubation period, cell cultures kept at 5% serum-supplemented medium were changed to 10% serum-supplemented medium. Incubations were performed for 4, 24, 48, and 72 h periods by replacement of the spent medium with 4 ml of fresh medium and addition of the corresponding steroid precursor (100 pg) in 0.01 ml of ethanol. Steroid extraction and analysis Steroids were extracted on Sep-pak C,, cartridges (Waters Associates) and analyzed by high performance liquid chromatography (HPLC) or gas chromatography (GC) as previously described (Ramirez et al., 1982). HPLC analysis was performed on a 10 pm Micropack C,, reversedphase column (Varian) using a 30 : 70% acetonitrile/water solvent gradient programmed linearly in 30 min at a flow rate of 1.2 ml/mm; column temperature was 40” C. GC analysis was performed on a 25 m SE-30 wall-coated fused silica column (Spiral) programmed from 240° C at 1 “/min. The extracted steroids, fractionated or not by HPLC, were analyzed as the methoximetrimethylsilyl (MO-TMS) derivatives by gas chromatography-mass spectrometry (GC-MS). A Ribermag lo-10B quadrupole mass spectrometer (Nermag) coupled to an SE-30 column with helium as the carrier gas, was used. Other conditions were as follows: ion source temperature, 250°C; electron energy, 70 eV; filament current, 0.2 mA. Electron impact spectra were recorded with a Sidar 150-Riber Computer Data System at 3 ms per apparent mass unit (a.m.u.). Methylene unit (M.U.) values (Dagliesh et al., 1966) were calculated during GC-MS analysis using C,, and C,, n-alcanes with an oven temperature program from 230 o C at l”/min. Steroid quantification was done using n-alcane and ll-deoxycorticosterone-21C,, acetate, respectively, as internal standards during GC and HPLC analysis. The response coefficients were calculated from a mixture of reference steroids. The 20a-reduced reference steroids not
available commercially were assumed to have a response coefficient equal to their closest 20-0~0 homologues. Results The metabolites of the exogenous substrates, deoxycorticosterone and corticosterone, were detected together with the endogenous metabolites produced from cholesterol in the serum-supplemented medium. The latter were the same as reported by Pierson et al. (1967) and Kowal and Fiedler (1968). However, under our incubation conditions, the predominant metabolite was liphydroxy-20a-dihydroxy-4-pregnen-3-one, as shown in Fig. 1; minor quantities of progesterone and 20a-dihydroprogesterone were sometimes detected. The qualitative analysis of the metabolites of deoxycorticosterone and corticosterone in this study was made difficult by the large number of metabolites and the very large differences in their quantities. Furthermore, the majority of the MOTMS derivatives of these metabolites form synand anti-isomers which separate during CC analysis (Boumot et al., 1989). Preparative HPLC permitted partial purification of minor metabolites and analysis by GC-MS. In other cases, apparent resolution of compounds eluted under the same peak was obtained during GC-MS analysis by detection of ions specific to each metabolite. A metabolite was considered identified when the M.U. value, and the mass spectrum or the specific ions recorded, were identical to those of the reference compound. The quantitative analysis was simplified by the fact that some of these unresolved compounds were present in negligible amounts, as seen by GC-MS analysis. A complementary quantification by HPLC, where an adequate separation of compounds unresolved by GC is accomplished, allowed confirmation of their quantification. Metabolism
of I I -deoxycorticosterone
(DOC)
Identification of the metabolites of DOC All metabolites produced during the incubation of DOC with the Yl cell line in culture medium
86 TABLE
1
METHYLENE UNIT VALUES OF THE METHOXIME-TRIMETHYLSILYL LITES PRODUCED BY THE Yl ADRENAL CELL LINE
DERIVATIVES
OF DOC AND
Steroids
M.U. values a
21-Hydroxy-4-pregnene-3,20-dione (ll-deoxycorticosterone; DOC)
30.49 (31.02) b
11/3,21-Dihydroxy-4-pregnene-3,20-dione (corticosterone; B)
32.15; 32.20 ’ (32.60; 32.65) h.c
11/3,18,21-Trihydroxy-4-pregnene-3,20-dione (18-hydroxycorticosterone; 18-hydroxyB)
33.25; 33.29 ’
20a,21-Dihydroxy-4-pregnen-3-one (20~dihydro-ll-deoxycorticosterone;
31.42 20wDHDOC)
18,20a,21-Trihydroxy-4-pregnen-3-one (18-hydroxy-20a-dihydro-ll-deoxycorticosterone;
32.68 18-hydroxy-20a-DHDOC
11/3,20a,21-Trihydroxy-4-pregnen-3-one (20wdihydrocorticosterone; 20wDHB)
33.28; 33.33 ’
11/3,18.20a,21-Tetrahydroxy-4-pregnen-3-one (18-hydroxy-20wdihydrocorticosterone;
18-hydroxy-20a-DHB)
20a,21-Dihydroxy-4-pregnene-3,11-dione (20a-dihydro-ll-dehydrocorticosterone;
20~DHA)
34.44; 34.48 ’
32.36
X,20a,21-Trihydroxy-4-pregnen-3-one d (X-hydroxy-20cy-dihydro-ll-deoxycorticosterone; Y,ll/3,20a,21-Tetrahydroxy-4-pregnen-3-one (Y-hydroxy-20wdihydrocorticosterone; ’ b ’ d
32.36 X-hydroxy-20a-DHDOC) d Y-hydroxy-20a-DHB)
33.97
Methylene unit values are calculated with an oven temperature programmed at lo C/mm Minor isomeric peaks. Syn- and anti-isomers of the methoxime derivative in position 3. X and Y, unidentified positions of the additional hydroxyl group, could be identical.
containing
10% serum for 72 h are listed in Table
compound
trum of its MO-TMS
derivative
metabolite
reported
et al., 1985).
(Ramirez
(20~
1 in Fig. 1). The mass spec-
of the rat adrenal
is similar
of DOC
ture,
the most
from 220 ’ C.
prominent
loss of the fragment
1 with their M.U. values. (a) 20ar-Dihydro-ll-deoxycorticosterone DHDGC;
ITS METABO-
to that
previously
(b) 20a-Dihydro-11-dehydrocorticosterone (20a-DHA; compound 3 in Fig. 1). The mass spectrum of the MO-TMS derivative is similar to that of the reference compound. It shows the following molecular and characteristic ions (relative abundance in parentheses): 519 (M)+ (44%); 504 (M-15)+ (8%); 488 (M-31)+ (5%); 416 (M103)+ (100%); 326 (M-103-90)+ (48%); 295 (35%); 276 (19%); 103 (91%). As generally observed for compounds with a 20,21-ditrimethylsiloxy struc-
peaks
result
from
the
of mass 103.
(c) 20a-Dihydrocorticosterone (20a-DHB; compound 6 in Fig. 1). The mass spectrum of its MO-TMS ence
derivative
compound
metabolite
is similar to that of the refer-
and
of DOC
to that
previously
of the rat adrenal reported
(Ramirez
et al., 1985). The most prominent peaks are those arising from the loss of the fragments of mass 103 and 90 a.m.u. corresponding to a CH,OTMS group and a trimethylsilanol, respectively. They are characteristic of steroids with a 20,21-ditrimethylsilyloxy structure (Gustafsson and Sjiivall, (d) 18-Hydroxy-20a-dihydrocorticosterone
1968). (1%
hydroxy-20cy-DHB; compound 9 in Fig. 1). The mass spectrum of the MO-TMS derivative is very similar to that of the corresponding authentic
87 110,20a-dihydroxy4-pregnen-3-one Cholesterol
\
p7 d
C36
4 DO
I
15
20
v-
I
I
25
30
35
40 min
Fig. 1. GC analysis of the MO-TMS derivatives of the metabolites of deoxycorticosterone (DOC) synthesized during its incubation with the Yl adrenal cell line. Chromatogram obtained from the steroid extract before fractionation by HPLC. Analytical conditions were as described in Material and Methods. Proposed structures for steroid metabolites are as follows: 1. 20~DHDOC; 2 and 2’, B and its secondary peak, respectively; 3 and 4, 20~DHA and X-hydroxy-20a-DHDOC; 5, 18-hydroxy-20ol-DHDOC; 6 and 7, 20~DHB and 18-hydroxyB; 8, Y-hydroxy-20(r-DHB; and 9,18-hydroxy-20a-DHB. Other compounds are cholesterol from the incubation medium; 1 lp,20 a-dihydroxy-4-pregnen-3-one, the main endogenous steroid; and C,, n-alcane, the internal standard.
standard (Fig. 2b and 2~). Both spectra show prominent peaks at m/z 578 (M-103)+, 488 (M103-90)+ and 398 (M-103-2 x 90)+. They also contain an ion at m/z 326 which arises from the loss of trimethylsilanol and a fragment of mass 265 which is specific for steroids with an 18,20,21trimethylsilyloxy structure (Boumot et al., 1981). This structure also favors the cleavage between C,, and C,, producing the ion at m/z 205. The
mass spectrum of the same compound with a free llj%hydroxyl group (not derivated) is given for comparison (Fig. 2~). (e) 18-Hydroxy-20ol-dihydro-ll-deoxycorticosterone (18-hydroxy-20ar-DHDOC; compound 5 in Fig. 1). In addition to the ions observed at m/z 490 (M-103)+, 400 (M-103-90)+ and 310 (M-103-2 x 90)’ in the mass spectrum of the MO-TMS derivative of compound 5 (Fig. 3) the base peak is observed at m/z 328 (M-265)+. On the basis of these fragments which characterize the 18,20,21tritritrimethylsilyloxy structure, compound 5 was tentatively identified as 18-hydroxy-20~DHDOC. (f) Corticosterone (B; compound 2 in Fig. 1). (g) 18-Hydroxycorticosterone (18-hydroxyB; compound 7 in Fig. 1). This compound and 20~ dihydrocorticosterone (compound 6) were eluted very close during GC analysis. Compound 7 was identified by GC-MS after previous purification by HPLC. (h) X-Hydroxy-20a-dihydroDOC and Y-hydroxy-20cY-dihydrocorticosterone (compounds 4 and 8 in Fig. 1, respectively). The position of the additional hydroxyl group in these two compounds has not been determined. Their identification is in progress and will be reported separately. Quantification
of the metabolites
of DOC
Analysis by GC and HPLC permitted the quantification of the following metabolites (Table 2): residual DOC, 20a-reduced steroids (2OaDHDOC, 20~DHB, 20~DHA, 18-hydroxy20~DHDOC and 18-hydroxy-20a-DHB), lloxygenated steroids (B, 20~DHB, 18-hydroxy20~DHB, and ~OWDHA), and 18-hydroxylated steroids (18-hydroxy-20a-DHDOC and 18-hydroxy-20a-DHB). The metabolism of DOC, incomplete in 4 h (34.4% residual DOC), is at a maximum after 24 h of incubation (2.1% residual DOC). After 4 h of incubation the predominant metabolites are 20~DHDOC (30.2%) and 20~ DHB (29.5%); corticosterone is a minor metabolite (4.9%). However, after 24 h of incubation there is less 20a-DHDOC (19.0%) and much more 20~DHB (62.7%); there is also a larger accumulation of corticosterone (10.9%). These results imply that the fastest metabolic reaction is the conversion of DOC into 20~DHDOC and that the rate limiting reaction in the formation of ZOWDHB is
M-31-2x90
100
150
200
250
300
350
LOO
L5O
500
550
600
mlz
100
150
200
250
300
350
400
L50
so0
550
600
650
mlz
600
650
mh
100 p_ T 80
.I03
M-90-265 326
(0
I 60
M-K)3-2x90 398 373
M-103-3x90 308
: 5 40. :
205
380
M-03 578
M-X)390
Ii 70 r 100
150
200
250
300
350
450
so0
550
Fig. 2. Mass spectra of the MO-TMS derivatives of the reference compound 18-hydroxy-20~DHB (synthesized chemically from 1%hydroxyB) with a free 11/3-hydroxyl group (a) and with an lip-trimethylsilyloxy group (b). Mass spectrum of the MO-TMS derivative of 18-hydroxy-20a-DHB (compound 9 in Fig. 1) produced from the incubation of DOC with the Yl cell line (c).
20-0~0 group than that of DOC. These results are similar to those reported for the metabolism of exogenous substrates in primary cultures of newborn rat adrenal cells; progesterone was reduced faster than I@-hydroxyprogesterone and DOC faster than corticosterone (Ramirez and Maume, 1984; Raoux and Maume, 1984). The oxidation of the I@-hydroxyl group to 11-0x0 (20a-DHA), a
ll/%hydroxylation. The accumulation of corticosterone also implies a competition for IWhydroxylation between two substrates, DOC and ~OWDHDOC. The accumulation of corticosterone after 24 h of incubation (10.9%) and its diminution during 48 and 72 h incubations (6.7 and 3.1%, respectively), shows a slower rate of reduction of its -
100 M-265 328
‘: GOL
M-103-90
” 60.
400
s .c_ LO0 .103 .t 3 20. E 100
M-103 490
M-103-2x90 310 285.
150
Fig. 3. Mass spectrum
200
250
300
?J4 350
400
450
500
550
of the MO-TMS derivative of compound 5 in Fig. 1 synthesized from DOC by the Yl adrenal identified as 18-hydroxy-20a-DHDOC. Spectrum obtained after purification by HPLC.
600 mh cell line and
89
TABLE
2
PRODUCTION OF DOC METABOLITES (PERCENT OF TOTAL STEROIDS) AS A FUNCTION OF INCUBATION TIME WITH THE Yl ADRENAL CELL LINE Each value is the mean of two determinations of duplicate cell dishes. Range of variation between quantifications is l-68. Metabolites
Incubation
time
4h
24 h
48 h
12 h
DOC B 20a-DHDOC 20(~-DHB 20a-DHA 18-Hydroxy-20 a-DHDOC 18-Hydroxy-20a-DHB
34.4 4.9 30.2 29.5 0.9 _ a _
2.1 10.9 19.0 62.7 3.8 1.4 _
4.2 6.1 2.8 75.1 3.7 3.0 4.5
1.7 3.1 1.2 66.6 10.2 2.2 15.0
20a-Reduced steroids 11-Oxygenated steroids 18-Hydroxylated steroids
60.6 35.2 _
87.0 71.4 1.4
89.1 90.0 7.5
95.2 94.9 17.2
and 18-hydroxy-20&-DHB; there was no 18-hydroxyDOC detected and 18-hydroxycorticosterone was only detected in trace amounts. Incubation of 18-hydroxyDOC and 18-hydroxycorticosterone did not produce the corresponding 20a-reduced metabolites (results not shown), therefore, the formation of 18-hydroxy-20a-DHDOC and 18-hydroxy-20a-DHB implies the 18-hydroxylation of 20a-DHDOC and of 20~DHB. The incubation of 18-hydroxylated steroids in primary cultures of newborn rat adrenal cells also showed little or no formation of the corresponding 20a-reduced compounds (Ramirez and Maume, 1984). The lack of accumulation of 18-hydroxy-20cY-DHDOC indicates the possible 11/3-hydroxylation of this compound in the formation of 18-hydroxy-20a-DHB. Metabolism
of corticosterone
(B)
a Not detected.
minor reaction in 4 h of incubation (0.9%) increases with incubation time (10.2% in 72 h), and not with the availability of substrate (20~DHB). The formation of 18-hydroxylated steroids which occurs only after 24 h of incubation (1.4%) increases after 48 h (7.5%) and again after 72 h (17.2%). The only measurable 18-hydroxylated steroids detected were 18-hydroxy-20a-DHDOC
TABLE
3
PRODUCTION OF CORTICOSTERONE (B) METABOLITES (PERCENT OF TOTAL STEROIDS) AS A FUNCTION OF INCUBATION TIME WITH THE Yl ADRENAL CELL LINE Each value is the mean of two determinations of duplicate cell dishes. Range of variation between quantifications is l-68. Metabolites
Incubation
time
24 h
48 h
12 h
B A 20a-DHB 20a-DHA 18-Hydroxy-ZOa-DHB
36.6 3.9 48.9 1.3 _ a
23.8 1.2 69.4 4.1 1.5
21.4 2.1 55.6 13.8 7.0
20 u-Reduced steroids 11-Dehydrogenated steroids 18-Hydroxylated steroids
56.0 11.2 _
75.0 5.3 1.5
76.4 15.9 7.0
’ Not detected.
Identification of the metabolites of B The metabolites of corticosterone produced by incubation with the Yl cell line under the same conditions as DOC, were identified in the same manner as the metabolites of DOC. The predominant metabolite was 20a-dihydrocorticosterone. Minor metabolites were 20a-dihydro-ll-dehydro18-hydroxy-20a-dihydrocorticorticosterone, costerone (A), 18-hydroxycorticosterone, 1 l-dehydrocorticosterone, and the unidentified Y-hydroxy-20cu-dihydrocorticosterone. Quantification of the metabolites of B Analysis by GC and HPLC permitted the quantification of residual B, 20a-reduced steroids (20~DHB, 20~DHA, and 18-hydroxy-20aDHB), 11-oxygenated steroids (A and 20~DHA), and an 18-hydroxylated steroid (18-hydroxy-20~ DHB). As shown in Table 3, the metabolism of corticosterone was not complete even after 72 h of incubation (21.4% residual corticosterone); the predominant metabolite was 20~DHB (55.6%) with smaller amounts of 20~DHA (13.8%). The slower reduction of corticosterone (36.6% residual corticosterone after 24 h) in contrast to that of DOC (2.1%) is in agreement with the proposition that the main metabolic pathway in the production of 20~DHB from DOC is 20a-reduction followed by ll/Shydroxylation. In the metabolism of corticosterone, as in the metabolism of DOC,
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oxidation of the lip-hydroxyl group to 11-0x0 (20a-DHA) seems to increase as a function of time (7.3% in 24 h and 13.8% in 72 h) and not as a function of available substrate (20a-DHB). In comparison to the incubation of DOC where 18hydroxylation is detected after 24 h, 18-hydroxy20a-DHB is detected (1.5%) only after 48 h of incubation with corticosterone. There is less 18hydroxy-20a-DHB formed from corticosterone (7.0% in 72 h) than from DOC (15.0%). However, it is not possible to say that DOC (or 20aDHDOC) is a better substrate for 18-hydroxylation than corticosterone (or 20a-DHB) since from the incubation of DOC there is more available substrate (20a-DHB and 18-hydroxy-20aDHDOC). Discussion This study is the first to demonstrate 18-hydroxylase activity in Yl cells. The major 18-hydroxylated metabolite from the incubation of DOC and corticosterone was identified as 18-hydroxy20a-DHB; 18-hydroxy-20a-DHDOC was identified as a minor metabolite from the incubation of DOC. Because of the very strong 20a-oxidoreductase activity in Yl cells, most metabolites, including those 18-hydroxylated, are also 20a-reduced. Although these metabolites represent a small proportion of the total metabolites after 24 h of incubation, the amount of 18-hydroxy-20aDHB produced increases significantly with incubation time. The production of 18-hydroxylated metabolites in Yl cells is similar to that of the mouse adrenal in vivo, that is, very little or no 18-hydroxyDOC and a measurable amount of 18-hydroxycorticosterone (Raman et al., 1964). It is also similar to that of the in vitro system where adrenal tissue slices were reported to produce 18-hydroxycorticosterone but no 18-hydroxyprogesterone or 18hydroxyDOC from progesterone (Raman et al., 1964; Eriksson et al., 1966). In our laboratory, quartered mouse adrenals and mitochondrial preparations produced corticosterone and 18-hydroxycorticosterone from the incubation of DOC; no 18-hydroxyDOC was detected. Therefore, it would seem that the 18-hydroxylated steroids preferentially synthesized by mouse adrenal cells,
in vivo and in vitro, are those which are also hydroxylated in position 1 lp. The cytochrome P-450,,, of Yl cells seems able to express both lip- and 18-hydroxylase activities as do the different cytochromes P-450,,, whose enzymatic activities have been previously characterized (Takemori et al., 1975; Watanuki et al., 1977; Sato et al., 1978; Kim et al., 1983). However, even though much more corticosterone than 18-hydroxyDOC is accumulated, the ratio of lipto 18-hydroxylated compounds cannot be calculated since these products are further metabolized to 1 l/3,1 8-dihydroxylated compounds. The activity of this cytochrome P-450,,,, however, is different from that of newborn rat adrenal cells in primary culture where the predominant metabolites of DOC are corticosterone and 18-hydroxyDOC, and very little 18-hydroxycorticosterone is formed. In these cell cultures the ratio of lip- to 18-hydroxylated compounds is of the order of 2 : 1 (Ramirez et al., 1985). The cytochrome P-450,,, of Yl cells cannot be directly compared to that of bovine cells in culture, since to our knowledge, 18-hydroxylated steroids have not yet been reported in these cell cultures. It is probable that bovine cytochrome P-450,,, activity in cell culture is similar to that of the purified enzyme which catalyzes the formation of 11/3- and 18-hydroxylated metabolites in a ratio of 11 : 1 (Watanuki et al., 1977) or 6 : 1 (Sato et al., 1978). These results indicate that cytochrome P-450,,, activity is dependent on the species, both in terms of the ratio of lip- to 18-hydroxylated metabolites and of the structure of these metabolites. Lately, 18-dehydrogenase activity has also been attributed to cytochrome P-450,,,. In reconstituted systems using purified cytochrome Pfrom different species and from both the 45011, glomerulosa and fasciculata zones, aldosterone was produced from DOC, corticosterone and 18-hydroxycorticosterone (Ohnishi et al., 1984; Wada et al., 1984; Yanagibashi et al., 1986; Lauber et al., 1987). In the Yl cell line, the formation of 18-hydroxy-20a-DHB from DOC and corticosterone led us to look for the formation of aldosterone and its 20a-reduced metabolite. Neither of these products was detected. It is possible that they are produced in amounts undetectable by GC-MS analysis since aldosterone is generally synthesized
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in much smaller amounts than 18-hydroxylated steroids. The production of aldosterone and/or its 20a-reduced metabolite by Yl cells is being studied using radioactive precursors. This study demonstrates that the Yl cell line is a good cellular model for the study of cytochrome P-450,,, activity, since it retains the 18-hydroxylase activity associated with this enzyme during steroid biosynthesis in the adrenal gland in vivo. Acknowledgements This work was supported by the Direction de la Recherche of the Minis&e de 1’Education Nationale, the Centre National de la Recherche Scientifique (AI 034617) and the Institut National de la Sante et de la Recherche Medicale (U 208). We wish to thank J.P. Berlot for the mitochondrial preparations. References Bjorkhem, I. and Karlmar, K.E. (1975) Eur. J. Biochem. 51, 145-154. Bournot, P., Maume, B.F. and Padieu, P. (1981) J. Steroid Biochem. 14, 1127-1137. Bournot, P., Ramirez, L.C., Pitoizet, N., Maume, B.F. and Padieu, P. (1989) Steroids 53 (in press). Buonassissi, V., Sato, G. and Cohen, A.I. (1962) Proc. Natl. Acad. Sci. U.S.A. 48, 1184-1190. Dagliesch, C.E., Homing, E.C., Homing, M.G., Knox, K.L. and Yarger, Y. (1966) B&hem. J. 101, 792-810. Erickson, R.E., Ertel, R.J. and Ungar, F. (1966) Endocrinology 78, 343-349. Ertel, R.J. and Ungar, F. (1968) Endocrinology 82, 527-534.
Gustafsson, J.A. and Sjovall, J. (1968) Eur. J. Biochem. 6, 236-247. Hoffman, F.G. (1956) Endocrinology 59, 712-715. Kim, C.Y., Sugiyama, T., Okamoto, M. and Yamano, T. (1983) J. Steroid Biochem. 18, 593-599. Kowal, J. and Fiedler, R. (1968) Arch. Biochem. Biophys. 128, 406-421. Lauber, M., Sugano, S., Ohnishi, T., Okamoto, M. and Mtiller, J. (1987) J. Steroid Biochem. 26, 693-698. Ohnishi, T., Wada, A., Nonaka, Y., Okamoto, M. and Yamano, T. (1984) Biochem. Int. 9, 715-723. Pierson, R.W. (1967) Endocrinology 81, 693-707. Raman, P.B., Ertel, R.J. and Ungar, F. (1964) Endocrinology 74, 865-869. Ramirez, L.C. and Maume, B.F. (1984) CR. Sot. Biol. (Paris) 178, 77-83. Ramirez, L.C., Millot, C. and Maume, B.F. (1982) J. Chromatogr. 229, 267-281. Ramirez, L.C., Bournot, P. and Maume, B.F. (1985) J. Steroid B&hem. 22, 249-256. Raoux, R. and Maume, B.F. (1984) CR. Sot. Biol. (Parts) 178, 84-91. Rapp, J.P. and Dahl, L.K. (1976) Biochemistry 15, 1235-1242. Sato, H., Ashida, N., Suhara, K., Itagaki, E., Takemori, S. and Katagiri, M. (1978) Arch. Biochem. Biophys. 190, 307-314. Southcott, CM., Bandy, H.E., Newsom, SE. and Darrach, M. (1956) Can. J. Biochem. Physiol. 34, 913-918. Takemori, S., Sato, H., Gomi, T., Suhara, K. and Katagiri, M. (1975) Biochem. Biophys. Res. Commun. 67, 1151-1157. Ulick, S. (1976) Am. J. Cardiol. 38. 814-824. Wada, A., Okamoto, M., Nonaka, Y. and Yamano, T. (<984) Biochem. Biophys. Res. Commun. 119, 365-371. Watanuki, M., Tilley, B.E. and Hall. P.F. (1977) B&him. Biophys. Acta 483, 236-247. Yanagibashi, K., Hanui, M., Shively, J.E., Shen, W.H. and Hall, P. (1986) J. Biol. Chem. 261, 3556-3562. Yasumura, Y., Buonassissi, V. and Sato, G. (1966) Cancer Res. 26. 529-535.