GLUCOCORTICOID AND MINERALOCORTICOID PATHWAYS IN TWO ADRENOCORTICAL CARCINOMAS: COMPARISON OF THE EFFECTS OF o,p’-DICHLORODIPHENYLDICHLOROETHANE, AMINOGLUTETHIMIDE AND 2-p-AMINOPHENYL-2- PHENYLETHYLAMINE IN VITRO
YVAN TOUITOU, ANDRÉ BOGDAN, ANDRÉ AUZEBY AND JEAN-PAUL DOMMERGUES
Faculty of Medicine Pitié-Salpêtrière, Department of Biochemistry, 91 Boulevard de l’Hôpital, 75634 Paris Cedex 13, and Department of Pediatrics, Hospital Antoine-Béclère, Clamart, France
(Received 5 September 1978)
SUMMARY
The synthesis of glucocorticoids and mineralocorticoids in vitro was studied in an adreno- cortical carcinoma after ablation from an 11.5-year-old boy. This patient had been un- successfully treated with high doses of o,p’-dichlorodiphenyldichloroethane (o,p’-DDD) and aminoglutethimide. These in-vitro results were compared with those obtained with another adrenocortical carcinoma removed from a 26-year-old woman who had received no pre- operative treatment. The sensitivity of these adrenocortical carcinomas to o,p’-DDD, aminoglutethimide and 2-(p-aminophenyl)-2-phenylethylamine (SKF 12185) was investi- gated. Synthesis of cortisol (47%) and corticosterone (45%) in control incubations showed that 113-hydroxylase activity was not affected by the treatment. This explains the raised level of plasma cortisol in the treated child. All three compounds inhibited both 116-hydroxylase and 18-hydroxylase activities up to 95%, depending on the inhibitor. This study shows (a) an inhibitory effect of o,p’-DDD on the steroidogenesis of an adrenocortical carcinoma in vitro, an effect not previously reported in man or laboratory animals, and (b) the in-vitro efficacy of o,p’-DDD and aminoglutethimide on corticosteroidogenesis by a carcinoma un- responsive to treatment in vivo. This discrepancy between data obtained in vivo and in vitro could possibly be explained by either an insufficient ratio of ingested dose : tumour mass, or a malabsorption of the drugs in this patient.
INTRODUCTION
Whilst numerous clinical reports of adrenocortical carcinomas, including their response to inhibitors of adrenal steroid synthesis, have been reported (Bergenstal, Hertz, Lippsett & Moy, 1960; Lippsett, Hertz & Ross, 1963; Hutter & Kayhoe, 1966; Okun, 1970; Lubitz, Freman & Okun, 1973), few studies are available on corticosteroidogenesis of such tumours in vitro.
In the present study, the biosynthesis of both glucocorticoids and mineralocorticoids in vitro by an adrenocortical carcinoma removed from a young boy was studied. This patient did not respond to treatment with 2(o-chlorophenyl)-2(p-chlorophenyl)-1,1-dichloroethane (o,p’-DDD) and aminoglutethimide («(p-aminophenyl)-«-ethylglutarimide). To understand why the treatment had failed, the effects of these two inhibitors, as well as 2(p-aminophenyl)- 2-phenylethylamine (SKF 12185) on adrenocortical steroid biosynthesis in vitro were studied.
The results were compared with those obtained with an adrenocortical carcinoma removed from a 26-year-old woman, who had received no preoperative treatment with inhibitors of steroid synthesis.
MATERIALS AND METHODS Labelled steroids, cofactors and reagents
[1,2-3H]Corticosterone (sp. act. 50 Ci/mmol), [1,2-3H]deoxycorticosterone (sp. act. 46-8 Ci/ mmol), [1,2-3H]deoxycortisol (sp. act. 54.5 Ci/mmol), [4-14C]cortisone (sp. act. 59.8 Ci/ mol), [4-14C]aldosterone (sp. act. 55.0 Ci/mol), [4-14C]corticosterone (sp. act. 59-3 Ci/mol) and [4-14C]cortisol (sp. act. 55.0 Ci/mol) were purchased from New England Nuclear Corporation. The radiochemical purity of these labelled steroids was checked by paper chromatography shortly before use.
[4-14C]18-Hydroxycorticosterone and [4-14C]18-hydroxy-11-deoxycorticosterone were obtained from [4-14C]deoxycorticosterone by incubating rat adrenal homogenates. The radiochemical purity of both steroids was checked as described previously (Touitou, Bogdan, Legrand & Desgrez, 1975).
[4-14C]11-Dehydrocorticosterone was obtained from the chromic acid oxidation of [4-14C]- corticosterone. The protection of the side-chain of corticosterone by acetylation was not necessary (Bush, 1961).
Unlabelled steroids were purchased from Merck (Darmstadt, Germany) and Ikapharm (Ramat-Gan, Israel), NADP+ and potassium hydroxide from Merck, malic acid from Sigma (St Louis, Missouri, U.S.A.) and sterile Earle’s medium from Institut Pasteur (Paris, France). The solvents were of analytical grade as supplied from Merck.
Subjects
Two patients, a boy (11.5 years) and a 26-year-old woman, presented with characteristic symptoms and signs of hypercortisolism. Investigation led in both cases to a diagnosis of probable adrenocortical carcinoma, which was confirmed at subsequent operation. The tumour was removed from the woman without any pretreatment. In contrast, the child was treated with o,p’-DDD (13.5 g/day) for 2 months (total dose 864 g). No improvement in the condition of the child was observed. The levels of steroids in urine and plasma before and after treatment were as follows: urinary 17-oxosteroids, 18.5 mg/24 h before, 16-0 mg/24 h after; dehydroandrosterone, 15-0 mg/24 h before, 12.8 mg/24 h after; plasma cortisol, 36 µg/ 100 ml before, 30 µg/100 ml after; urinary 17-hydroxycorticosteroids, 9-2 mg/24 h before, 8.4 mg/24 h after.
Due to the lack of effect of the treatment with o,p’-DDD, the child received in addition 1 g aminoglutethimide/day on the last 13 days of the treatment period, this treatment produced no improvement. The tumour was removed a week after the end of treatment.
Morphological findings
Histological examination of the tumours showed features of malignancy with cellular pleomorphism, nuclear hyperchromasia and mitotic figures. The carcinoma from the boy was located on the right side, weighed 300 g, was of a greyish appearance with some yellow areas and showed necrotic and haemorrhagic foci. The tumoral lesions were massive and no normal adrenal tissue could be found. The tumour from the woman was located on the left side, weighed 700 g and showed large areas of haemorrhagic and necrotic foci. The carcinoma had crossed the capsule and infiltrated the adjacent fat tissue.
Experimental procedure
Both carcinomas were dissected free of adipose and connective tissues. The experiments were carried out on portions of the two adrenocortical carcinomas which had been weighed,
trimmed and homogenized with a Teflon-glass homogenizer in Earle’s medium buffer; CaCl2 was added to adjust the calcium concentration to 4 mmol/l (Sandor, Fazekas, Lehoux, Leblanc & Lanthier, 1972).
Incubation flasks contained exact amounts of [3H]deoxycorticosterone (8.3 uCi), [3H]corti- costerone (8.9 uCi) or [3H]11-deoxycortisol (3.8 uCi) which were evaporated to dryness, then solubilized with 2-0 ml Earle’s solution 1 h before the incubation started. An NADPH-genera- ting system made up of NADP+ (1 mmol/l) and malic acid (5 mmol/1) neutralized with a 0.1 M- potassium hydroxide solution was added to each incubation flask (Touitou & Legrand, 1970). The final volume of each incubation was 10-0 ml.
To each incubation flask was added either 380 mg homogenized tissue from the boy or 1 g homogenized tissue from the woman. All of the incubations were performed in duplicate under aerobic conditions in a Dubnoff metabolic shaking incubator at 37 ℃ for 2 h.
At the end of the incubation, 15.0 ml acetone were added to stop the reactions. To account for procedural losses during subsequent extraction and identification of synthesized steroids, trace amounts of [4-14C]18-hydroxycorticosterone, [4-14C]aldosterone, [4-14C]corticosterone, [4-14C]11-dehydrocorticosterone, [4-14C]18-hydroxy-11-deoxycorticosterone, [4-14C]corti- sone or [4-14C]cortisol were added to the incubation flasks to be used as internal standards, depending on the tritiated steroid precursor used.
Incubation media were filtered through a glass-cotton layer into a Büchi vessel. The steroids were extracted as previously described (Touitou et al. 1975).
Quantitative measurement of radioactivity was done in a POPOP-PPO toluene scintilla- tion solution with a two-channel liquid scintillation spectrometer (Tri-Carb, model 3255, Packard Instrument Company). Isotope contents were expressed as disintegrations/min. The results were calculated as percentage conversion of the total radioactivity. The data have been corrected for procedural losses.
Isolation and characterization of steroids
The following solvent systems were used for paper chromatography (PC) of labelled steroids and conversion products: PC 1: dichloroethane/ethylene glycol (1 : 1, v/v); PC 2: toluene/ propylene glycol (1 : 1, v/v); PC 3: benzene/formamide (1 : 1, v/v); PC 4: methylcyclohexane/ toluene/formamide (10 : 10 : 1, by vol.); PC 5: benzene/heptane/methanol/water (67 : 33 : 80 : 20, by vol.); PC 6: benzene/methanol/water (10 : 5 : 5, by vol.). Radioactive material was detected on paper chromatograms with a radiochromatogram scanner (Packard, model 7200).
The steroids were separated by descending paper chromatography. Individual isolated metabolites were further run in suitable chromatographic systems in which their isopolarity with carbon-14 internal standards was established. A conversion product was considered pure when a constancy of 3H : 14℃ ratios was established in successive chromatographic systems.
The dried extracts were first chromatographed in system PC 1 for 4 h which allowed the separation of either 18-hydroxycorticosterone and aldosterone (precursors: deoxycortico- sterone or corticosterone), or cortisol and cortisone (precursor: 11-deoxycortisol). Each area was then purified and characterized using the 14C-labelled steroids as standards.
Tritiated 18-hydroxycorticosterone and aldosterone were measured as previously described and evaluated by double isotope dilution (Touitou et al. 1975, 1976).
Corticosterone and 11-dehydrocorticosterone were separated in system PC 2 for 7 h. Corti- costerone was acetylated with unlabelled acetic anhydride into its 21-acetate, the polarity of which was checked in systems PC 4 for 14 h and PC 5 for 4 h. Corticosterone 21-acetate was either oxidized with chromic acid to 11-dehydrocorticosterone 21-acetate (polarity checked in system PC 4 for 7 h), or hydrolysed to corticosterone (polarity checked in system PC 2 for 14 h). 11-Dehydrocorticosterone was characterized by acetylation and the product was checked as described above.
18-Hydroxy-11-deoxycorticosterone was first separated from the other steroids (cortico- sterone, 11-dehydrocorticosterone, deoxycorticosterone) in system PC 2 for 7 h. The steroid was then purified in system PC 3 for 24 h, and oxidized by periodic acid to the lactone of 18- hydroxy-11-deoxycorticosterone (polarity checked in systems PC 3 and PC 5).
Cortisol was purified in system PC 1 for 17 h, then oxidized by chromic acid to 11-oxo- androstenedione (polarity checked in systems PC 5 and PC 6).
Cortisone was purified in system PC 3 for 30 h and oxidized by chromic acid to 11-oxo- androstenedione (polarity checked as above).
RESULTS Synthesis of cortisol and cortisone from 11-deoxycortisol
Of the 11-deoxycortisol substrate, 47% was synthesized to cortisol in the treated carcinoma and 64% in the untreated one (Table 1). The ratios of cortisol : cortisone were respectively 19 and 9. The addition of aminoglutethimide (5 u.mol) inhibited cortisol synthesis by approxi- mately 83% in the treated carcinoma and by 62% in the untreated one. Addition of o,p’-DDD (50 umol) resulted in 28% inhibition of cortisol synthesis in the treated carcinoma but did not alter cortisol synthesis in the untreated one (Table 1).
Table 1. Effect of o,p’-dichlorodiphenyldichloroethane (o,p’-DDD), aminoglutethimide and SKF 12185 on the conversion of [1,2-3H]11-deoxycortisol to cortisol and cortisone by two adrenal cortical carcinomas in vitro
(One carcinoma from a young boy was found to be unresponsive to treatment with o,p’-DDD and amino- glutethimide in vivo; the other from a woman was not treated in vivo. Each flask contained : NADP+ (1 mmol/l), malate (5 mmol/l) and 380 mg homogenized treated carcinoma or 1000 mg homogenized untreated carcinoma in a total volume of 10-0 ml. The o,p’-DDD, aminoglutethimide and SKF 12185 were added directly, as powders, to the incubation flasks. Incubation lasted for 2 h at 37 ℃ in air. All the data are the means of duplicate incubations.)
| Addition to incubation | Cortisol | Cortisone | ||
|---|---|---|---|---|
| Treated (%) | Untreated | Treated | Untreated | |
| (%) | (%) | (%) | ||
| None | 47.3 | 64.4 | 2-50 | 7.33 |
| o,p'-DDD (50 u.mol) | 34-1 | 61.2 | 2.14 | 5.13 |
| Aminoglutethimide (5 pmol) | 8-20 | 25-0 | 5.69 | 6.54 |
| Aminoglutethimide (10 umol) | 8.00 | - | 9-73 | - |
| SKF 12185 (5 umol) | 4.50 | 2.50 | - | 3.30 |
| SKF 12185 (10 umol) | 2.50 | - | 1.42 | - |
With SKF 12185 (5 u.mol) the inhibition was greater than 90% in both carcinomas, the cortisol : cortisone ratio was ten times lower when compared with the control incubations.
Synthesis of corticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone and 11-dehydrocorticosterone from 11-deoxycorticosterone
Conversion rates of deoxycorticosterone to corticosterone were similar (45%) with both carcinomas. However, the addition of aminoglutethimide resulted in greater inhibition of corticosterone synthesis in the treated carcinoma than in the untreated one (Table 2).
18-Hydroxylation of corticosterone and deoxycorticosterone was low in the two carcino- mas and was further lowered by addition of aminoglutethimide. In contrast, 11-dehydro- corticosterone production was increased in the presence of the drug in both carcinomas. Addition of o,p’-DDD (50 p.mol) to the incubations with the untreated carcinoma produced no noticeable changes in the synthesis of these steroids.
Table 2. Biosynthesis of corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone and aldosterone from [1,2-3H]deoxycorticosterone as precursor, without (control) and with addition of aminoglutethimide (AG) or o,p’-dichlorodiphenyldichloroethane (o,p’-DDD)
(The carcinomas were removed at surgery, from a young boy unresponsive to treatment with o,p’-DDD and AG, and from a woman who received no pre-operative treatment. Each flask contained: NADP+ (1 mmol/l), malate (5 mmol/l), and 380 mg homogenized treated carcinoma or 1000 mg homo- genized untreated carcinoma in a total volume of 10-0 ml. The o,p’-DDD and AG were added directly as powders to the incubation flasks. Incubations lasted for 2 h at 37 ℃ in air. All the data are the means of duplicate incubations.)
| Corticosterone | 11-Dehydrocorticosterone | Aldosterone | 18-Hydroxy- corticosterone | 18-Hydroxy-11-deoxy- corticosterone | |||||
|---|---|---|---|---|---|---|---|---|---|
| Addition to incubation | Treated | Untreated | Treated | Untreated | Untreated | Treated | Untreated | Treated | Untreated |
| % | (%) | (%) | % | % | |||||
| None | 45-0 | 45.2 | 12.0 | 3.66 | 0.23 | 0-51 | 0-31 | 4-15 | 2.28 |
| o,p'-DDD (50 µmol) | - | 46-2 | 3-85 | 0-21 | 0-29 | 2.35 | |||
| AG (5 pmol) | 1.46 | 18.3 | 22.8 | 6.78 | 0.13 | 0-17 | |||
| AG (10 mol) | 1.59 | 20-5 | 0.18 | 1.50 | |||||
Synthesis of 18-hydroxycorticosterone and 11-dehydrocorticosterone from corticosterone The synthesis of 18-hydroxycorticosterone from corticosterone in the treated carcinoma was similar to that observed with deoxycorticosterone as substrate. In contrast, the production of 11-dehydrocorticosterone was greater (Table 3).
The addition of the inhibitors resulted in a 32-66% inhibition of 18-hydroxycorticosterone synthesis and a slight increase in 11-dehydrocorticosterone production (Table 3).
Table 3. Effect of o,p’-dichlorodiphenyldichloroethane (o,p’-DDD), aminoglutethimide and SKF 12185 on the conversion of [1,2-3H]corticosterone to 18-hydroxycorticosterone and 11-dehydrocorticosterone by an adrenal cortical carcinoma removed surgically from a young boy
(Experimental conditions were identical to those described in Table 1. The inhibitors were added as powders to the incubation medium. Incubation lasted for 2 h at 37 ℃ in air. All the data are the means of duplicate incubations.)
| 18-Hydroxycorticosterone | 11-Dehydrocorticosterone | |
|---|---|---|
| Addition to incubation | (%) | (%) |
| None | 0.64 | 20-0 |
| o,p'-DDD (50 umol) | 0-23 | 21.4 |
| Aminoglutethimide (5 pzmol) | 0-44 | 23.8 |
| Aminoglutethimide (10 umol) | 0-22 | 25.9 |
| SKF 12185 (5 umol) | 0-40 | 26-4 |
| SKF 12185 (10 u.mol) | 0-30 | 19-7 |
SKF 12185 is 2(p-aminophenyl)-2-phenylethylamine.
DISCUSSION
The data obtained with the two adrenocortical carcinomas in vitro showed active biosynthesis of cortisol and corticosterone from deoxycortisol and deoxycorticosterone respectively. 118-Hydroxylation of these latter steroids was still very high, even in the adrenal tissue obtained from the treated child. These results are consistent with the plasma cortisol level (30 µg/100 ml) observed in this child during treatment. Moreover, the 11-hydroxylation of deoxycortisol and deoxycorticosterone in the treated adrenocortical carcinoma showed clear parallelism.
18-Hydroxylation of corticosterone by the two adrenocortical carcinomas was low in vitro whereas these tumours yielded large amounts of corticosterone. Biosynthesis of aldosterone was found to be low in vitro.
18-Hydroxylation of deoxycorticosterone by the two tumours was found to be higher than that of corticosterone. There was a marked discrepancy in the synthesis of ACTH-dependent steroids by the two tumours. Synthesis of 18-hydroxydeoxycorticosterone, although not negligible, was low when compared with that of cortisol and corticosterone. These results are in excellent agreement with those obtained by Touitou, Bogdan & Luton (1979) and are similar to those of De Nicola, Oliver & Birmingham (1968b), De Nicola, Traikov, Birmingham, Palmer & Ruddick (1970), Melby, Dale, Grekin, Gaunt & Wilson (1972) and Ulick (1976), with human adrenal tissues of various origins.
The difference in 18-hydroxylation of corticosterone and deoxycorticosterone could be due to different biosynthetic loci (zona fasciculata or glomerulosa) or to the existence of two different enzymic processes.
11-Dehydrocorticosteroids (cortisone and 11-dehydrocorticosterone) have been found in all of the incubations. Synthesis of cortisone was greater in the non-treated carcinoma; synthesis of 11-dehydrocorticosterone differed between the two tumours and was higher from corticosterone (20%) than from deoxycorticosterone (12%); however, 11-dehydro- corticosterone synthesis was always low when compared with that by the adrenals of
various animal species (Birmingham, Rochefort & Traikov, 1965; Fazekas & Webb, 1966; De Nicola, Kraulis & Birmingham, 1968a; Fazekas, Sandor & Lanthier, 1970). This syn- thesis of dehydrocorticosterone can be related to the existence of an 110-hydroxycortico- steroid dehydrogenase, active either on corticosterone added as a precursor or corticosterone synthesized from deoxycorticosterone as a precursor.
Addition of o,p’-DDD as a powder to the incubations with the untreated gland did not result in any modification of the synthesis of glucocorticosteroids or mineralocorticosteroids. This lack of effect in vitro could be due to the extreme insolubility of o,p’-DDD itself. The drug may become active through its peripheral metabolism (Touitou et al. 1977), but not by conversion to 2(0-chlorophenyl)-2(p-chlorophenyl)-1,1-dichloroethylene (Touitou & Bogdan, 1978; Touitou, Bogdan & Luton, 1978).
In contrast to its lack of effect on the untreated carcinoma, the addition of the same amount of o,p’-DDD to the incubations with the treated tumour showed a decrease in the synthesis of the steroids, i.e. cortisol from deoxycortisol and 18-hydroxycorticosterone from corticosterone. This is, to our knowledge, the first evidence for an inhibitory effect of o,p’- DDD on human adrenal tissue in vitro.
The dissociation of the effects of o,p’-DDD on the two carcinomas in vitro may indicate a priming action of the presurgical treatment, possibly through a modification of mito- chondrial permeability due to alteration of the mitochondrial structure following in-vivo treatment (Adjovi & Idelman, 1969; Racela, Azarnoff & Svoboda, 1969; Kadioglu & Harrison, 1971).
In contrast to o,p’-DDD, addition of aminoglutethimide to the incubation medium resulted in an inhibition of the synthesis of cortisol (83%), corticosterone (97%) and 18- hydroxycorticosterone (67%). The degree of inhibition obtained by addition of this drug as a powder was very near to that observed previously (Touitou & Legrand, 1971; Touitou et al. 1975) when the drug was added after solubilization in an organic solvent. This work demon- strated the efficacy of o,p’-DDD and aminoglutethimide on adrenocortical carcinomas in vitro whereas no clinical or biological improvement could be seen during treatment with these two drugs in vivo. This discrepancy might be due either to an insufficient ratio of ingested dose : tumour mass or to malabsorption of the orally administered drugs.
The inhibitory effect of SKF 12185 on 18-hydroxycorticosterone and aldosterone synthesis by sheep adrenals in vitro has been demonstrated previously (Touitou et al. 1976). In the present work SKF 12185 was as active as aminoglutethimide on 18-hydroxycorticosterone synthesis, and even more active on 113-hydroxylation of deoxycortisol. The very important inhibitory activity of this drug may allow its use in the treatment of hypercorticism and especially of adrenocortical tumours in order to diminish rapidly the consequences of hypersecretion of the adrenal steroids. Further investigations in vivo will be necessary to show whether the results obtained in vitro indicate that SKF 12185 should be added to the list of adrenal steroidogenesis inhibitors generally used in chemotherapy, i.e. o,p’-DDD and aminoglutethimide.
The authors gratefully acknowledge Roussel Pharmaceutical Corp., CIBA Pharmaceutical Corp. and Smith, Kline and French who provided o,p’-DDD, aminoglutethimide and SKF 12185 respectively. We also wish to thank Professor Thervet for providing adrenocortical tumour tissue and Mrs Micheili for her editorial assistance.
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