Mechanism of Abnormal Production of Adrenal Androgens in Patients with Adrenocortical Adenomas and Carcinomas
YOSHIYUKI SAKAI, TOSHIHIKO YANASE, TAKAYUKI HARA,
RYOICHI TAKAYANAGI, MASAFUMI HAJI, AND HAJIME NAWATA
The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University (Y.S., T.Y., R.T., M.H., H.N.), Fukuoka 812, Japan; and Department of Food and Nutrition, Nakamura Gakuen College (T.H.), Jonan-ku, Fukuoka 814, Japan
ABSTRACT
The production of adrenal androgens can be modulated by the activities of steroidogenic enzymes and by the electron transfer system, NADPH-cytochrome P450 reductase (Red) and cytochrome b5 (b5), both of which have been shown to increase 17,20-lyase activity in vitro. To clarify the mechanism of diminished secretion of adrenal androgens in patients with adrenocortical adenoma and Cushing’s syndrome and of excess secretion in patients with adrenocortical carcinoma, we in- vestigated the enzymatic activities of cytochrome P45017a, 38-hy- droxysteroid dehydrogenase (36-HSD), and Red as well as the content of b5 in five adenomas, three carcinomas, and two normal adrenal glands. An in vitro enzyme assay using a microsomal fraction of the tissues indicated that all the tumors had almost the same degree of 17a-hydroxylase activities as the normal adrenals. However, the rela-
tive activity ratio of 17,20-lyase to 17a-hydroxylase of the three ade- nomas was markedly diminished, and 36-HSD activity was apparently lower in the three carcinomas. The messenger RNA concentrations of P45017a were similar in all tumors, whereas those of 38-HSD were markedly lower in the carcinomas than in other tissues. Both the content of b5 and the activity of Red were significantly lower in the adenomas. These results suggest that low concentrations of adrenal androgens in patients with adrenocortical adenomas are mainly due to low 17,20-lyase activity, which may be explained in part by a lower content of b5 and Red. In addition, high concentrations of adrenal androgens in patients with adrenocortical carcinoma are mainly due to the diminished activity of 36-HSD. (J Clin Endocrinol Metab 78: 36- 40, 1994)
T HE INITIAL step of the steroidogenic pathway in the adrenal cortex is the side-chain cleavage of cholesterol catalyzed by cytochrome P450scc to form pregnenolone, which is converted to progesterone by 30-hydroxysteroid dehydrogenase (36-HSD) activity. Subsequent 17a-hydrox- ylation of pregnenolone or progesterone is a branch point for the formation of cortisol and adrenal androgens in the adrenal gland. The side chain of 17@-hydroxypregnenolone can be cleaved further by a 17,20-lyase reaction to yield an adrenal androgen, dehydroepiandrosterone (DHEA).
It is well established that cytochrome P45017a, a product of a single-copy gene CYP17, can catalyze both the 17a- hydroxylation required for the production of cortisol and the 17,20-lyase reaction leading to the production of adrenal androgens (1, 2). However, secretion of cortisol and androgen dissociates in many clinical situations (3). Although little is known about the mechanism of the dissociated secretion, the electron transfer system might be a clue. Relative activity of 17,20-lyase to 17a-hydroxylase is increased severalfold by elevating the ratio of either cytochrome b5 (b5) (4, 5) or NADPH-cytochrome P450 reductase (Red) (6) to P45017a in vitro.
Cushing’s syndrome secondary to adrenocortical adenoma
provides a good model for studying the dissociation between 17a-hydroxylation and 17,20-lyase activities. Most patients have adenomas that produce low concentrations of androgen (7, 8), and some have adenomas that produce exceptionally high concentrations of androgens. We previously reported higher expression of b5 messenger RNA (mRNA) as well as greater 17,20-lyase activity in adenomas that secreted high concentrations of androgens than in adenomas secreting low concentrations (9). However, the values between the ade- nomas and normal glands were not compared.
In the present study, we reevaluated the tissue content or expression of b5 and Red in adrenocortical adenomas pro- ducing low concentrations of androgens and compared them with those in normal adrenal glands. To clarify the mecha- nism of the high concentrations of adrenal androgens relative to normal concentrations of cortisol in patients with adre- nocortical carcinoma, we also examined the activities and expression of P45017a and 33-HSD and the expression of b5 and Red in these tissues.
Subjects and Methods
Experimental subjects
Five patients with Cushing’s syndrome and benign adrenocortical adenoma and three patients with adrenal carcinoma were studied. Among the patients with adrenal carcinoma, only case 7 showed Cush- ing’s syndrome. The age, sex, and relevant baseline laboratory data are summarized in Table 1. The main difference between these two groups was their serum and/or urine concentrations of adrenal androgens. The
| Patientª | Age | Sex | Tumor histology | Urine | Serum | |||
|---|---|---|---|---|---|---|---|---|
| 17-OHCS (umol/day) | 17KS | Cortisol (nmol/L) | DHEA (nmol/L) | DHEA-S (umol/L) | ||||
| 1 | 38 | F | adenoma | 46.3 | 21.1 | 772 | 3.1 | 1.093 |
| 2 | 44 | F | adenoma | 19.8 | 15.9 | 598 | - | 0.135 |
| 3 | 39 | F | adenoma | 30.9 | 17.3 | 546 | 1.3 | 0.504 |
| 4 | 27 | F | adenoma | 38.6 | 20.8 | 755 | 2.1 | 0.594 |
| 5 | 50 | F | adenoma | 47.4 | 17.6 | 405 | 0.7 | 0.542 |
| 6 | 3 | F | carcinoma | 28.4 | 284.9 | 450 | - | - |
| 7 | 6 | F | carcinoma | 42.2 | 40.2 | 1021 | - | 24.3 |
| 8 | 1 | F | carcinoma | 25.4 | 305.1 | - | - | - |
-, Not determined; 17-OHCS, 17-hydroxy corticosteroids; 17KS, 17ketosteroids.
ª Patients 1-5 in the present study are patients 4, 5, 7, 2, and 3 in our previous study (Ref. 9), respectively.
excretion of 17-ketosteroids was significantly higher in the patients with carcinoma than in the patients with adenomas (P < 0.01). Serum levels of dehydroepiandrosterone-sulfate (DHEA-S) were higher in one cancer patient in whom this was measured.
Tissue samples were obtained from these eight patients at surgery. The histological diagnosis was adrenocortical adenoma in patients 1, 2, 3, 4, and 5 and adrenocortical carcinoma in patients 6, 7, and 8. In addition, two normal adrenal glands were obtained from two patients with renal carcinoma who underwent hemilateral nephrectomy. Imme- diately following surgery, the tissues were stored at -80 C for protein and mRNA analysis.
Preparation of microsomes
Microsomal fractions of human adrenal tumors and normal glands were obtained by the method previously described (9, 10). The micro- somal pellet was suspended in 5 mmol/L HEPES buffer, pH 7.4, con- taining 0.15 mol/L KCI (HEPES-KCI buffer), at a concentration of 20 mg protein/mL; it was stored at -80 C until used. The protein concen- tration was measured by the method of Lowry et al. (11).
In vitro enzyme assay
Microsomal enzyme activities of 17a-hydroxylase, 17,20-lyase, and 38-HSD were measured by in vitro enzyme assay using thin layer chromatography (9, 10). The amount of product formed by each enzyme was linear with time and protein concentration.
Quantitation of mRNA
Total RNA was isolated from tissue by a single-step extraction using acid guanidium thiocyanate, phenol, and chloroform (12). Plasmids containing complementary DNAs (cDNAs) encoding human cytochrome P45017a (13), human Red, and 36-HSD (14) were used, as was human B-actin cDNA (Nippon Gene Co., Toyama, Japan). For Northern blot analysis, samples containing 10 ug total RNA were fractionated on a formaldehyde-1.5% agarose gel and transferred to a nitrocellulose filter by capillary blotting (15). Probes for hybridization included the BamHI insert of the human P45017a cDNA clone pCD17a-H (13), the EcoRI insert of the human Red cDNA clone, and the BamHI-EcoRI insert of the 38-HSD cDNA clone pCMV5H 36-HSD (14). These probes were radiolabeled with [@-32P]deoxycitidine triphosphate using a nick trans- lation kit (Amersham International, Buckinghamshire, England). Pre- hybridization, hybridization, and washing conditions have been de- scribed previously (15, 16). The membrane was washed in 0.1 x SSC (0.15 mol/L NaCI, 0.015 mol/L Na citrate) at 100 C for 5 min to strip off the hybridized probe and was then rehybridized with the next probe. Signal intensities of RNA were quantified by densitometric scanning (FUSIX BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan). P45017a, 38-HSD, and Red expressions were normalized to that of B-actin.
Red activity
Red activity in microsomes was determined by measuring NADPH- dependent cytochrome c reductase activity (17). The reaction mixture (1 mL) contained 100 mmol/L potassium-phosphate buffer (pH 7.4), 20-40 µg microsome, 25 umol/L cytochrome c, 0.5 mmol/L KCN, and 0.1 mmol/L NADPH. The reaction was started by the addition of NADPH. Changes in absorbance at 550 nm were followed at 25 C using the Hitachi 228 A Spectrophotometer (Hitachi Co., Ltd., Tokyo, Japan). The enzyme activity was calculated using an extinction coefficient of 21.1 cm-1 mmol/L-1 at 550 nm (17).
Contents of cytochrome b5
Cytochrome b5 was determined from the difference in spectrum between dithionite-reduced and air-saturated (oxidized) microsomes. The value used for the increase of molar extinction coefficient between 424 nm and 409 nm was 185 cm-1 mmol/L-1 (18).
Statistics
Data are reported as mean + SD. Statistical analysis was performed using Student’s t test. A level of P < 0.05 was accepted as statistically significant.
Results
The enzymatic activities of 17a-hydroxylase, 17,20-lyase, 36-HSD, and Red, and the content of b5 in microsomes of the five adrenal adenomas, three carcinomas, and two nor- mal adrenal glands are shown in Table 2. All tumors and normal glands showed essentially the same degree of 17a- hydroxylase activity. However, 17,20-lyase activity and the relative activity ratio of 17,20-lyase to 17a-hydroxylase in microsomes of the adenomas were markedly lower than in carcinomas and normal glands. The 36-HSD activity was lower in the carcinomas than in the adenomas and normal glands.
Because of RNA degradation, mRNA expressions of P45017a, 36-HSD, and Red were not examined in two adenomas. The amount of P45017a mRNA, when normal- ized to ß-actin mRNA, was the same in the carcinomas as in the other adenomas (Fig. 1). However, the amount of 38- HSD mRNA, normalized to that of -actin, was significantly lower in the adrenal carcinomas (Fig. 1).
Because no difference was observed in the concentration of P45017a mRNA, despite a specific decrease in 17,20-lyase activity and the relative activity ratio of 17,20-lyase to 17a-
| Patient | Tissue | 17,20-Lyase (pmol/mg·min) | 17a-OHase (nmol/mg·min) | 17,20-Lyase 17a-OHase (×10-3) | 38-HSD (nmol/mg· min) | NADPH cyt. c Red. (nmol/mg·min) | Cyt.b5 (nmol/mg) |
|---|---|---|---|---|---|---|---|
| 1 | AA | 27 | 6.25 | 4.3 | 10.3 | 19.8 | 0.29 |
| 2 | AA | 11 | 2.58 | 4.3 | 8.5 | 16.4 | 0.25 |
| 3 | AA | 14 | 2.45 | 5.6 | 10.6 | 41.4 | 0.30 |
| 4 | AA | 16 | 1.73 | 9.1 | 20.5 | 70.0 | 0.32 |
| 5 | AA | 21 | 4.12 | 5.0 | 17.4 | 50.0 | - |
| Mean (5 AAs): ± SD | 17.8 ±6.3 | 3.4 ± 1.8 | 5.7 ± 2.0 | 13.5 ± 5.2 | 39.5 ± 22.1 | 0.29 ± 0.02 | |
| 6 | AC | 149 | 3.10 | 48.0 | 1.5 | 208 | 0.56 |
| 7 | AC | 72 | 2.38 | 30.3 | 2.2 | 138 | - |
| 8 | AC | 270 | 3.32 | 81.3 | 2.6 | 112 | 0.43 |
| Mean (3 ACs): ± SD | 163.7 ± 99.8 | 2.9 ± 0.5 | 53.2 ± 25.9 | 2.1 ±0.5 | 152.2 ± 28.6 | 0.49 | |
| 9 | N | 412 | 4.31 | 95.6 | 7.4 | 108 | 0.54 |
| 10 | N | 132 | 3.15 | 41.9 | 9.0 | 66 | 0.49 |
| Mean (2 Ns): | 272 | 3.7 | 68.8 | 8.2 | 87 | 0.52 |
-, Not determined; AA, adrenocortical adenoma; AC, adrenocortical carcinoma; N, normal adrenal glands; 17a-OHase, 17a-hydroxylase; 38- HSD, 38-hydroxysteroid dehydrogenase; NADPH cyt. c Red, NADPH-cytochrome c reductase; Cyt.b5, cytochrome b5. Values are mean or mean ± SD.
Carcinoma
Adenoma
Normal
Patient
6
7
8
1
2
3
9
10
P45017a
38-HSD
Red.
B-actin
Relative mRNA expression
2
1
-
0
P45017a
3 ₿
-HSD
Red
hydroxylase in adrenal adenomas, the tissue contents of b5 and Red activities in microsomes were examined. As shown in Fig. 2, both the b5 content and the Red activity in micro- somes of the adenomas were significantly lower than those in the carcinomas and normal glands. In addition, the expres- sion of Red in adenomas examined by Northern blot analysis was also less than in carcinomas and normal glands (Fig. 1).
17,20-lyase /17a -OHase
Red
b5
**
**
100-
200.
0.6
80
nmol/min/mg
150.
nmol/mg
2
60-
0.4
100
40.
0.2
50-
20
AA
AC
AA
AC
AA
AC
N
N
N
Discussion
Normal or high concentrations of adrenal androgens are observed in patients with bilateral adrenal hyperplasia and Cushing’s syndrome. In contrast, distinctly low values are seen in patients with adrenal adenomas, making the adrenal androgen concentration helpful in differential diagnosis (7). Elevated plasma androgens in bilateral adrenal hyperplasia can be explained by hyperproduction of ACTH, and low values in adenomas would be due mainly to decreased ACTH secretion, suggested by atrophy of the other adrenal gland. However, an alteration in ACTH secretion does not com- pletely explain the dissociated secretion of cortisol and an- drogen in patients with adrenocortical adenomas. A key enzyme, cytochrome P45017a, is responsible for both cortisol
and androgen production and is under the control of ACTH (19). Cytochrome P45017a is a microsomal P450, which receives two electrons from Red and a molecule of O2 for substrate oxygenation. Cytochrome b5 is also capable of transferring a second electron directly to P450 and has been reported to potentiate the monooxygenase reactions of P45017a (4, 5).
We previously described two cases of adrenocortical ade- noma in patients with Cushing’s syndrome that produced relatively high 17,20-lyase activities and expression of b5; activities of 36-HSD and 17a-hydroxylase, as well as the expression of Red, were not significantly different in these adenomas and in other adenomas that produced low levels of androgens (9). Because b5 is known to increase 17,20- lyase activity relative to 17a-hydroxylase activity in vitro (4, 5), the difference in the content of b5 in the adenomas was thought to be a major cause for the marked difference in 17,20-lyase activity (9). In the present study, 17,20-lyase activity or the relative activity ratio of 17,20-lyase to 17a-hydroxylase and concentrations of b5 were low in adre- nocortical adenomas that produced low concentrations of androgens. In addition, a quantitative comparison of the concentration of Red, determined by Northern blot hybridi- zation, showed that it was also low in these adenomas compared with other tissues. These results suggest that the decrease in the concentrations of b5 as well as of Red may be important in the marked decrease of 17,20-lyase activity observed in adenomas.
The mechanism of high concentration of the adrenal an- drogens relative to the normal concentration of cortisol in patients with adrenocortical carcinoma has long been an intriguing question and is not well elucidated. Although a few reports suggest a decrease in 36-HSD (20, 21), and/or 118-hydroxylase (20) activities in adrenocortical carcinomas, the expression of steroidogenic enzymes and electron transfer systems in the tissues has not been studied until now. In the present study, 36-HSD activity in three carcinomas was significantly decreased compared with the activity in normal adrenal glands and adenomas. The result was confirmed further at the transcriptional level by a specific decrease of 36-HSD mRNA in the carcinomas. However, the ratio of 17,20-lyase to 17«-hydroxylase activities and mRNA concen- tration of P45017a in adrenal carcinomas did not differ significantly from those in normal adrenal glands. The con- tent of Red and b5 in carcinomas was essentially similar to those in normal glands, suggesting little contribution of the electron transfer systems to the modulation of 17,20-lyase activity. A slight increase of Red in adrenocortical carcinomas may reflect the reduced consumption of Red in a subsequent step, such as 21-hydroxylation in microsomes, because of the lower 36-HSD activity. As suggested by previous reports (20, 21), high concentrations of adrenal androgens in patients with adrenocortical carcinomas are caused apparently by the marked reduction in 36-HSD activity, not by an increase in 17,20-lyase activity. The usually large size of such carcino- mas may also contribute to the high concentrations of andro- gens in the patients’ serum.
In summary, different mechanisms are involved in the dissociated secretion of cortisol and adrenal androgens in patients with Cushing’s syndrome secondary to adrenocor- tical adenoma and in patients with adrenocortical carcinoma. The former is brought about by a selective reduction in 17,20- lyase activity, possibly in association with a decrease in the concentrations of electron transport systems whereas, the latter is chiefly caused by the decrease in 36-HSD activity.
Acknowledgments
We greatly appreciate the generosity of Drs. M. R. Waterman and J. I. Mason, University of Texas, Southwestern Medical Center, Dallas, Texas, and C. B. Kasper, University of Wisconsin, Madison, Wisconsin, for providing us with cDNA for P45017a, cDNA for 36-HSD, and cDNA for Red, respectively.
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