UPTAKE AND LOSS OF TRITIATED OESTRADIOL BY THE ADRENAL GLAND AND BY OESTROGEN-INDUCED ADRENOCORTICAL CARCINOMA IN THE RAT
BETTY G. MOBBS*
Cancer Research Centre, University of British Columbia, Vancouver 168, Canada
(Received 19 March 1970)
SUMMARY
The loss of injected tritiated oestradiol from the adrenals of non-tumour- bearing rats of different endocrine status and from the adrenals and tumours of animals bearing two lines of transplantable oestrone-induced adrenocorti- cal carcinoma has been studied. There was no evidence that adrenals from non-tumour-bearing rats behaved as ‘target organs’ for oestrogen as regards retention of the hormone. As compared with skeletal muscle of the same animals, one tumour line and the host adrenals behaved in a similar way to normal adrenals and did not retain oestradiol, whereas the other tumour line and host adrenals lost the hormone at a somewhat slower rate than muscle.
It is suggested that the behaviour of the host adrenals and of the trans- planted tumours regarding the injected oestrogen was determined more by the hormonal environment of the tissues including the contribution made by the tumour itself, than by specific oestrogen receptors in adrenal tissue.
INTRODUCTION
The induction by oestrone pellets of transplantable oestrogen-responsive carcinoma of the adrenal cortex in rats has been reported by Noble (1967), and at present several lines of these tumours with varying dependence on oestrogen are maintained in this laboratory. Considerable evidence, much of which has been recently summarized by Kitay (1968), suggests that oestrogen has a direct, as well as an indirect, effect via the pituitary on the adrenal, and it was hoped that the investigation described here would indicate whether the adrenal tumour should be regarded as a ‘target organ’ for oestrogens. Classical ‘target organs’, such as the uterus and vagina, have the ability to take up and retain the injected hormone (Jensen & Jacobson, 1960), and this is also a characteristic of some mammary tumours (Folca, Glascock & Irvine, 1961; Jungblut, DeSombre & Jensen, 1967; King, Cowan & Inman, 1965; Mobbs, 1966; Terenius, 1968). Since the retention of injected oestradiol has been shown to be related to the
* Present address: Department of Urology, Queen’s University, Kingston, Ontario, Canada.
responsiveness of breast tumours to oestrogen (Folca et al. 1961; Mobbs, 1966), it was of interest to investigate this relationship in two lines of oestrogen-induced adreno- cortical carcinoma with differing responsiveness to oestrogen.
Additional experiments were carried out to investigate the pattern of uptake of oestrogen by the adrenals in non-tumour-bearing animals of different endocrine status.
MATERIALS AND METHODS
Female inbred hooded rats bearing tumour lines 3A and 4A, each in the tenth transplant generation, were used. The 3A line tumour shows oestrogen dependence in that it grows only in female rats with oestrone pellets. At 45 days of age each animal was implanted subcutaneously in the flank with an oestrone: cholesterol (9:1, w/w) pellet weighing approximately 10 mg, and with approximately 1 mg tumour tissue introduced by trocar subcutaneously in the dorsal neck region. The 4A tumour line shows a lesser degree of oestrogen dependence in that it grows in male or ovariecto- mized female rats, but grows more quickly if oestrogen is present. The tumour was transplanted into 58-day-old female rats which had been ovariectomized the previous day and did not bear oestrone pellets. All tumours were used when the mean diameter was approximately 1.5 cm, 3-4 months after transplantation. At this time the approxi- mate body weight of rats with the 3A tumour line was 200 g, and that of animals bearing the 4A line was 260 g. Animals bearing the 3A line were ovariectomized 4 days before use, and the pellets were removed, dried and weighed. The mean amount of oestrone absorbed was 30 ug/animal/day. All rats were injected s.c. with 16-7 uCi (approx. 0.10 µg) [6,7-3H]oestradiol-178 in 0.17 ml saline: ethanol (15:1, v/v)/100 g body weight. The tumour, adrenals and a sample of thigh muscle (used as control ‘non-target’ tissue) were excised under ether anaesthesia 20-185 min after injection and stored in air-tight vials at - 17 ℃.
Owing to the limited availability of hooded rats, the investigation on the adrenals of non-tumour-bearing animals was carried out mainly with female Sprague-Dawley rats in three groups: (1) immature rats (24 days old, approximate weight 50 g); (2) mature rats (75 days old) ovariectomized 20 days previously, and (3) intact rats, 75 days old. The same dose of [3H]oestradiol was injected as in the tumour-bearing animals, and the adrenals and a sample of thigh muscle were excised from four or five animals from each group 15, 30, 60 and 120 min after injection. Three mature ovariectomized hooded rats were also used for comparison with the Sprague-Dawley animals.
Radioactivity was estimated in four or five replicate samples of each piece of tumour and muscle tissue; the radioactivity in each adrenal was estimated separately. Each sample was digested overnight at 60 ℃ with 2 ml pronase solution (0-1 mg pronase/ml 0.04 M-borate buffer, pH 8-0-8-2, containing 2.19 mg CaCl2.6H2O/1). Free steroids were extracted from the tissue suspension with 15 ml methylene chloride. After centrifuging to separate completely the aqueous and organic phases, 10 ml aliquots of each extract were evaporated to dryness in counting vials. Radioactivity remain- ing in the aqueous phase after methylene chloride extraction was extracted after acidification to pH 1 with 2.5 M-H2SO4 and the addition of ammonium sulphate (1 g/2 ml pronase digest). Extraction was carried out with 4 x 2 ml ether : ethanol
(3:1, v/v). After evaporation, the residue was extracted with 3 x 2 ml ethanol. This was evaporated to dryness in counting vials and redissolved in 2 ml ethanol before addition of phosphor. Previous experiments (Mobbs, 1968) have shown that this method gives 95.0± 2.1 (s.D.) % recovery of free [6,7-3H]oestradiol-176, with 2.3% remaining in the aqueous phase after methylene chloride extraction. The mean recovery for water-soluble metabolites was 65-6 %.
In order to determine if significant amounts of oestrone were present in adrenal tumour after the injection of oestradiol, the remainder of the methylene chloride extracts from the adrenal and tumour tissue from animals bearing the 3A tumour, which had been injected at least 1 h before samples were taken, were pooled. Aliquots were evaporated to dryness, redissolved in ethanol and run on paper chromatograms in the solvent system toluene : heptane or petroleum ether (b.p. 80-100 ℃) : methanol : water (5:5:7:3). Oestradiol and oestrone standards were run simultaneously, and the appropriate zones were cut out, eluted and counted.
All scintillation counting was carried out using a toluene-phosphor scintillation fluid; 4.5 g 2,5-diphenyl-oxazole (PPO) and 0·1 g 1,4-bis-(2-(5-phenyloxozolyl)) benzene (POPOP) per litre of toluene. Ten ml was added to the vials containing free steroid extract and 8 ml to the vials containing extracts of water-soluble metabolites. Counting was carried out in a Packard Tri-Carb scintillation counter (series 3000) at 36 % efficiency, and quenching was corrected for by the internal standard method.
RESULTS
Owing to the small amount of material available for paper chromatography, only two determinations were made of the relative concentration of oestradiol and oestrone in adrenal and tumour tissue. The ratios of oestradiol: oestrone for the tissue of rats bearing the 3A tumour were 2.5 and 2-4 for the adrenals, and 3.4 and 2.6 for the tumour tissue. Thus a considerable amount of the injected oestradiol was metabolized to oestrone; at least part of this metabolism may have taken place in the adrenal tissue itself, as preliminary work in this laboratory (A. F. Burton, personal communi- cation) indicates that the 3A tumour is capable of carrying out the conversion of oestradiol to oestrone in vitro. It has also been shown (Ryan & Engel, 1953) that hyperplastic human adrenal can convert oestradiol to oestrone to some extent.
From 53 determinations of the radioactivity present in the free steroid and water- soluble fractions of adrenal and tumour tissue, the mean percentage of radioactivity present in the former was 94.6+ 2-4 (s.D.) %. The results shown in Tables 1 and 2 and Fig. 1 are therefore based on radioactivity present in the free steroid fractions only.
In the adrenals of all three groups of Sprague-Dawley non-tumour-bearing rats both uptake and loss of radioactivity was rapid. The concentration of tritium in the adrenal was considerably higher than that in the muscle at all times after injection, but the relative rate of loss from both tissues was approximately constant throughout. Thus there was no indication of specific retention of oestradiol by the adrenal. Up to 30 min after injection the concentrations of radioactivity in adrenals from immature and ovarietomized rats were higher than those in the intact animals, but after this there were no significant differences in concentration between the groups, although owing to the small size of the adrenals in the immature animals, the amount of
radioactivity present per adrenal was much less than in the other groups. One hour after injection the concentration of tritiated oestrogen in the adrenals was approxi- mately 2.6 x 10-9 mol/kg tissue and by 150 min after injection it had dropped to about 7.8 x 10-10 mol/kg. There was no evidence that adrenals of the non-tumour- bearing hooded rats behaved differently regarding oestradiol uptake and loss from those of the Sprague-Dawley rats.
Table 1. Tritium in adrenal and muscle of non-tumour-bearing rats after injection of 0.1 µg [6,7-3H]oestradiol-17 8/100 g
Each value was calculated from four or five replicate estimations for muscle (except for 15 min, mature intact) and eight or ten replicate estimations for adrenal (means ±s.D.).
| Time after injection (min) | (c.p.m./mg wet wt) | A:M | (Mean c.p.m./whole adrenal): (Mean c.p.m./mg muscle) | |
|---|---|---|---|---|
| Adrenal (A) | Muscle (M) | |||
| Immature | ||||
| 15 | 483.5+ 154-4* | 54.4± 13.1 | 8.9 | 97-3 |
| 30 | 166·9± 21·7 ** | 24.1± 2.7 | 6-9 | 75-2 |
| 60 | 96.0± 19.3 | 15.2± 3.1 | 6.3 | 76-8 |
| 150 | 30.0± 8·8 | 5.2+ 1-4 | 5-8 | 59.0 |
| Mature, ovariectomized | ||||
| 15 | 386.2± 45.2 ** | 34.8± 11.3 | 11-1 | 470-5 |
| 30 | 205-1± 40.5 | 23.5± 4.8 | 8.7 | 329-4 |
| 60 | 100·7± 7.7 | 13.2± 0-9 | 7-6 | 288.4 |
| 150 | 35.7± 8.4 | 4.6+ 1.1 | 7.8 | 287.5 |
| Mature, intact | ||||
| 15 | 262.7+ 30.3 | 34.9 (33-1, 36-6) | 7.5 | 297-9 |
| 30 | 231.7± 44.4 | 27-3+ 5.1 | 8.5 | 300-9 |
| 60 | 110.3± 31.3 | 12.0± 3.4 | 9.2 | 344-6 |
| 150 | 29.1 ± 8.9 | 3.6± 2.2 | 8.1 | 312.0 |
* P < 0.05; ** P < 0.01 compared with mature intact animals.
A similar pattern of loss was observed for both the host adrenal tissue and the transplanted tumour in animals bearing the 3A tumour (Table 2). In order to mini- mize variations between animals due to possible differences in absorption of the injected hormone from the injection site, the ratio of the concentration of radio- activity present in tumour compared with that in muscle (T:M) and adrenal com- pared with muscle (A:M) was calculated and plotted against time after injection (Fig. 1). Regression analysis showed that there was no significant difference in the behaviour of these three tissues regarding the retention of radioactivity. The con- centration of oestrogen in the tumour was approximately 1 x 10-9 mol/kg tissue 150 min after injection.
In the animals bearing the 4A tumour, the results were somewhat different. Although there was no marked concentration of radioactivity during the time investigated, regression analysis of T : M and A : M plotted against time showed that loss from both the host adrenal and the transplanted tumour was significantly slower than that from muscle (Fig. 1). At 150 min after injection the concentration of tritiated oestrogen in the 4A tumour was approximately 1.8 x 10-9 mol/kg and in the host adrenal 2.4 × 10-9 mol/kg.
The adrenals of rats bearing the 4A line were considerably smaller (mean wt/gland
Table 2. Tritium in transplanted adrenocortical tumour, adrenals and muscle of tumour-bearing rats after injection of 0.1 µg [6,7-3H]oestradiol-178|100 g
(The mean values for tumour and muscle were calculated from four or five replicate estimations on each tissue from a single rat at each time.)
| Time after injection (min) | Tumour | Adrenal | Muscle | (Mean c.p.m./whole adrenal): (Mean c.p.m./mg muscle)* |
|---|---|---|---|---|
| Tumour line 3A | ||||
| 23 | 146.7 (95-7-202.6) | 147.9 (146.3, 149.4) | 23.3 (21.0-24.6) | 156.5 |
| 36 | 118.7 (59-1-162.7) | 157.7 (156-4, 158.9) | 18-6 (16-0-20-5) | 160.1 |
| 45 | 135.4 (114.4-159.1) | 65.8 (65-5, 66.1) | 14.5 (12.8-17.4) | 125.1 |
| 62 | 192.2 (144.5-312.7) | 92.7 (88.7, 96-7) | 15.5 (12.3-18-6) | 186-8 |
| 63 | 56-7 (25-7- 70-5) | 77.2 | 7.4 (5.6-10.2) | 183.8 |
| 82 | 112.7 (83.9-130-8) | 88.5 | 13.8 (9.8-17.0) | 212.4 |
| 90 | 28.0 (17.3- 34.5) | 18.7 (11-3, 26.0) | 2.9 (2.4- 4.8) | 175-8 |
| 102 | 51.2 (50.3- 52.9) | 34.3 (29-7, 38.8) | 5.1 (3.7- 7.2) | 159.8 |
| 122 | 58-5 (51-9- 63-9) | 35.2 (34-4, 36-0) | 5.5 (4-7- 6-6) | 215.4 |
| 145 | 48.8 (37.2- 63.3) | 28.6 (26-3, 30.8) | 7.4 (5.3-11.8) | 116.1 |
| 183 | 27-3 (21-1- 40-0) | 15.7 | 3-9 (2-0- 5.3) | 154.2 |
| Tumour line 4A | ||||
| 36 | 188.1 (158-4-230.6) | 175-3 (147-7, 202.8) | 34.6 (26.8-40.8) | 67.4 |
| 47 | 198.7 (166-4-218.9) | 187-4 (175-7, 199.0) | 33-9 (33-0-36-5) | 55.5 |
| 57 | 137.6 (131.1-167.9) | 176-7 (173.9, 179.5) | 21-1 (19.6-22.4) | 101.3 |
| 58 | 136.5 (43.6-199.6) | 255.7 (254-4, 257.0) | 36-6 (32.3-44.0) | 61.8 |
| 69 | 138.4 (74-3-183.4) | 207-6 (186-9, 228.2) | 23.0 (18.6-26.2) | 117.1 |
| 83 | 151.2 (148.3-158.4) | 203.5 (203-4, 203.6) | 19.8 (18.9-21.3) | 135.7 |
| 94 | 121.7 (80-9-172.1) | 176.1 | 17.2 (13.5-21.7) | 96.2 |
| 102 | 91.8 (73-2-109-4) | 136-7 (116.1, 157.2) | 8.3 (6.1- 9.6) | 156.4 |
| 111 | 61.5 (28-6- 73.5) | 68.0 (65.8, 70.2) | 8.3 (6.4- 9.5) | 183.8 |
| 120 | 88.5 (43-6-110.0) | 88.6 (84-1, 93-1) | 14.0 (13-5-17-6) | 132-6 |
| 153 | 68.9 (51.8- 87-8) | 92.1 (85.1, 99-1) | 4.8 (1.2- 9.8) | 235.2 |
| 179 | 68.2 (56-1- 79.9) | 84-9 (76.1, 93.7) | 6-9 (5.3- 8.0) | 96.6 |
* The ratio mean c.p.m./whole adrenal: mean c.p.m./mg muscle does not alter significantly with time after injection in the rats bearing the 3A tumour line, whereas in rats bearing the 4A line there was a significant relationship with time after injection; r=0.6047, P < 0.05.
13.0 mg) than those of rats bearing the 3A line (mean wt 28.1 mg). The normal adrenal weight for mature female hooded rats is 25-30 mg/gland. The ratios of total c.p.m./gland to c.p.m./mg wet weight of muscle were therefore calculated (Table 2). The total amount of radioactivity in the smaller adrenals in relation to muscle concentration was less than in the larger adrenals early after injection, but after about 70 min, the total amount of radioactivity present in relation to muscle was equal to that in the larger adrenals; i.e. the slower loss of radioactivity from the host adrenals in the rats with the 4A line as compared with those from the 3A line was confirmed. In the non-tumour-bearing rats (Table 1), on the other hand, the small adrenals of
20
T: M
A: M
10
Ratios A : M and T : M
T: M
5
A: M
2
Tumour line 3 A
Tumour line 4A
1
20
60
100
140
180
20
60
100
140
180
Minutes after injection
immature animals took up a smaller total amount of radioactivity than the adrenals of the mature animals, and in all three groups the amount of radioactivity present in the adrenals compared with that in muscle did not alter greatly throughout the experiment. The adrenals in the mature Sprague-Dawley rats were somewhat larger than those in the hooded strain (mean adrenal wt in immature animals, 11.2 mg; ovariectomized mature animals, 38.9 mg; intact mature animals, 34.7 mg), and the total amount of radioactivity taken up was correspondingly larger.
DISCUSSION
There was little evidence from these experiments that normal adrenal tissue or the oestrone-induced adrenocortical carcinomas behave as ‘target organs’ for oestrogen, as regards ability to concentrate injected oestradiol. Previous studies on the uptake
of tritiated oestradiol by the adrenals of non-tumour-bearing rats have been carried out (Chobanian, Brecher, Lille & Wotiz, 1968; Eisenfeld & Axelrod, 1965, 1966; Jensen & Jacobson, 1960). As in the present experiments, no retention of oestradiol by the adrenals was found, and no significant differences in the concentration of oestradiol in the adrenals of immature (100 g), intact and ovariectomized mature animals 1 h after injection (Eisenfeld & Axelrod, 1966). The concentration of oestra- diol at this time was 1.9 x 10-9 mol/kg, of the same order as that found in the experi- ments described above.
Autoradiographic evidence for retention of oestrogens by the adrenal is conflicting : a combined biochemical and autoradiographic study (Michael, 1965) showed no retention of tritiated hexoestrol by the cat adrenal as a whole, but autoradiography showed that the concentration of tritium in the zona fasciculata was considerably greater than that in other regions of the adrenal. This is of interest with respect to the present experiments, as the adrenal tumours studied here are thought to arise from the zona fasciculata. In the mouse, on the other hand, autoradiography demonstrated the most marked concentration of radioactivity in the zona glomerulosa after the injection of large doses of [3H ]oestradiol or [14C]oestrone (Ullberg & Bengtsson, 1963). A recent study of the cellular localization of tritiated oestradiol in the rat adrenal by high resolution autoradiography indicated that all three zones of the adrenal cortex accumulate radioactivity to some extent, but that the radioactivity is not in the nuclei, as in ‘target organs’ such as the uterus (Stumpf, 1969). The discrepancies in the results of different autoradiographic studies may arise from differences in the methods and doses used, and may be at least partly due to differences between species.
A further indication that the adrenal and adrenal tumours are not behaving as ‘target’ tissue is that a considerable amount of oestrone was present in the free steroid extracts. It has been shown that in tissues such as uterus and vagina (Jensen & Jacobson, 1962) and in oestrogen-responsive mammary tumours (King et al. 1965) almost all the radioactivity present after the injection of [3H]oestradiol is in the form of oestradiol.
The adrenals of the host animals showed the same pattern of loss of tritium as the respective tumour lines, although these differed slightly. This suggests that the transplanted tumour is influencing the behaviour of the host adrenal as regards the injected oestradiol; another indication of the differential influence of the two tumour lines on the host adrenal is the small size of the latter in rats bearing the 4A line, although this may be partly due to the ovariectomized state of the host rats (Kitay, 1968). It may be that the different tumour lines have different metabolic activities, thus altering the hormonal environment of the host animals in different ways. Differences in the behaviour with respect to oestradiol uptake of the two tumour lines due to the long-term absence of oestrogen in the 4A line cannot be ruled out, although it was shown that ovariectomy of normal rats 20 days before injection did not affect oestradiol uptake by the adrenals. There may also be long-term effects of oestrone pelleting in the 3A group, but short-term effects are unlikely, since the pellets were removed 4 days before injection and the behaviour of the adrenal tissue was similar to that in normal rats.
One may conclude from these experiments that the interaction between oestradiol
and the adrenal glands and tumours arising from it is determined not so much by the possession of a specific receptor molecule for oestrogens, as in ‘target’ tissues, but by the interplay of many factors in a complex hormonal environment.
This study was supported by the National Cancer Institute of Canada.
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