National Institutes of Health1, Bethesda, Maryland, U. S. A.
IN VITRO METABOLISM OF PROGESTERONE-4-14C IN AN ADRENOCORTICAL CARCINOMA OF THE RAT By David F. Johnson, Katharine C. Snell, Daniel Francois and Erich Heftmann
ABSTRACT
The in vitro metabolism of progesterone-4-14℃ and 11-deoxycorticosterone- 4-14℃ acetate in a rat adrenocortical carcinoma, 494 and 494-H, has been studied and compared with adrenal tissue from normal rats. An accumula- tion of deoxycorticosterone in the tumour tissue with an accompanying decrease of corticosterone and aldosterone, indicates a blockade in the 11- hydroxylation of deoxycorticosterone. A difference in the distribution of radioactive metabolites is observed between incubation experiments with Tumour 494 and 494-H. These findings indicate a correlation between the metabolic activity and the physiological findings in the host.
The cardinal role of progesterone in the biogenesis of adrenocortical hormones is well established, and details of the metabolic pathways may be found else- where (Heftmann & Mosettig 1960). It has also been established (Giroud et al. 1956; Hofmann 1957; Mialhe-Voloss & Baulieu 1958; Angelico & Quintiliani 1958; Ward & Birmingham 1960; Péron 1960) that in the adrenal cortex of the rat the main end-products of progesterone metabolism are corticosterone and aldosterone, resulting from the successive introduction of oxygen functions at carbon atoms 21, 11 and 18.
Alterations in the pathway of progesterone metabolism in human adrenals (Dorfman 1958) have been observed in the adrenogenital syndrome (Bongio- vanni 1958) and in adrenocortical hyperfunction (Dyrenfurth et al. 1960). Berliner et al. (1958) have observed differences in the per cent conversion of progesterone-4-14C between adrenal tissues from patients with Cushing’s syn-
1. Public Health Service, Department of Health, Education, and Welfare.
drome and with mammary carcinoma. Human adrenal adenomata (Bailey et al. 1960) and a rat adrenal tumour (Iglesias & Mardones 1958) have been reported to produce excessive amounts of adrenocortical hormones in vitro. However, Block & Cohen (1960) have found a decreased total synthesis of corticosteroids, a greater diversity of compounds, and an apparent alteration in metabolic pathway in neoplastic adrenal tissue in mice.
Observations at this Institute (Snell & Stewart 1959) on the functional effects of adrenal cortical carcinoma 494 in rats have indicated an abnormality in the corticosteroid production in the animals. Tumour-bearing rats developed poly- uria, polydipsia, degenerative changes in the distal convoluted tubules of the kidney, and atrophy of the adrenal glands, sex organs, lymphatic tissues, and pituitary gland. A subline tumour, 494-H, produced additional effects indi- cative of progestational activity. In vitro experiments are being presented, which point to a decreased ability of the transplantable tumour to carry out the 11-hydroxylation of radioactive steroid precursors.
EXPERIMENTAL
Adrenocortical tumour transplants (carcinoma 494 and 494-H) were allowed to grow in male and female Osborne-Mendel rats for a period of approximately 3 months. The rats were sacrificed and the tumours were removed and placed immediately in ice-cold physiological saline. Within 1 hour of the operation the tumours were incubated by a modification of the method of Saffran & Schally (1955). Between 1-2 g of tumour tissue, or in control experiments a similar amount of normal adrenal gland tissue, was sliced into segments with a razor blade, and placed in 10 ml of Krebs-Ringer bicarbonate buffer, fortified with 200 mg glucose per 100 ml. Preincubation for 2 hour was carried out in an atmosphere of 5 % CO2 - 95 % O2 at 37º C in a Dubnoff shaker. The tissue was then transferred to another flask, containing fresh buffer and a measured amount of radioactive precursor. An activity of 42 314 c. p. m. was used in experiments with progesterone-4-14℃ and 59 000 c. p. m. in experiments with deoxy- corticosterone-4-14C acetate. Following incubation for 2 hours under conditions described for preincubation, the medium was drawn off and placed in a 50-ml centrifuge tube. It was extracted with three 10-ml portions of redistilled dichloromethane. Emulsions were broken by centrifuging for several minutes at 1200 r. p. m. The combined dichloro- methane extracts were evaporated to dryness under vacuum at 40º C.
Reference steroids (200 ug each) were added as carriers to the residues, which were analyzed by partition chromatography on columns, as previously described (Johnson et al. 1956). The fractions obtained were divided in half, one aliquot was analyzed by ultraviolet (U. V.) absorption at 240 mu and the other by reduction of Blue Tetra- zolium (B. T.). The former aliquots were subsequently concentrated and quantitatively transferred to concentric ring copper planchets, using a multiple plating device designed in this laboratory (François, unpubl.). Each fraction was counted in an automatic thin window Geiger counter (Nuclear of Chicago) at a preset count of 3200. The results of all three assays were then plotted and the total radioactivity in peaks coinciding with the U. V. absorption and B. T. reduction of the added standards were calculated.
RESULTS
Fig. 1 shows the chromatogram resulting from the incubation of progesterone- 4-14C with normal rat adrenal gland tissue. Radioactivity peaks coincide with the peaks of progesterone (P), deoxycorticosterone (Q), corticosterone (B), as well as with other peaks. The large peak of radioactivity just following the cortisone peak was established as being due to aldosterone by comparing the mobility of this fraction with that of authentic aldosterone in both paper and column chromatography. A number of other peaks are evident, which have not been identified and may or may not represent corticosteroids.
Figs. 2 and 3 represent the results from three experiments involving incuba- tion of adrenal tumour tissue with radioactive precursors. The lower part of Fig. 2 indicates the positions of the added carrier steroids, as before. Carriers were added for each experiment, but are not shown for Fig. 3. Fig. 2 shows the distribution of radioactivity following incubation of Tumour 494 with progesterone-4-14C. Comparing it with that of the normal adrenal tissue (Fig. 1), the most obvious difference is in the radioactivity of deoxycorticosterone and aldosterone. Aldosterone occurs in tubes 125-135 and is not identical with the radioactive compound (140-155) just preceding cortisol. Other striking dis- similarities are readily apparent.
The lower curve in Fig. 3 is the chromatogram resulting from the incubation of Tumour 494 with deoxycorticosterone-4-14C acetate. The relative quantities of deoxycorticosterone, corticosterone, and aldosterone are of the same order
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of magnitude as in the foregoing experiment, but some of the other peaks in the curve are now absent.
The top curve in Fig. 3 shows the chromatogram resulting from the incubation of Tumour 494-H with progesterone-4-14C. The most striking difference be- tween this chromatogram and the two previous ones is the large amount of
| Sample | Deoxy- corticosterone | Corticosterone | Aldosterone |
|---|---|---|---|
| Normal Rat Adrenal Tissue and Progesterone-4-14C | 1.27 | 13.77 | 8.42 |
| Necrotic Adrenal Tumour Tissue 494 and Progesterone-4-14℃ | 46.62 | 6.76 | 3.21 |
| Adrenal Tumour Tissue 494 and Progesterone-4-14C | 6.00 | 3.13 | 0.49 |
| Adrenal Tumour Tissue 494-H and Progesterone-4-14C | 7.74 | 1.21 | 0.50 |
| Adrenal Tumour Tissue 494 and Deoxycorticosterone-4-14C acetate | - | 0.62 | 0.30 |
radioactivity in two peaks slightly less polar than cortisol. One of these peaks can be seen in the two preceeding curves but in smaller quantity. Another peak can be seen just following deoxycorticosterone, but because of its proximity to deoxycorticosterone, it is difficult to tell whether this is an addi- tional peak or not. It is quite evident that three are major differences between the metabolic activity of tumour strains 494 and 494-H. On the basis of a report by Eisenstein (1956), the radioactive material located in the peak to the left of corticosterone was chromatographically compared with authentic pregn-4-ene- 11a,21-diol-3,20-dione(11-epicorticosterone), but found not to be identical with that steroid.
Table 1 shows the per cent conversion of added precursors by various tissues to deoxycorticosterone, corticosterone, and aldosterone respectively. It was cal- culated on the basis of the radioactivity of precursor added per gram of tissue. It should be emphasized that the analysis is based upon the activity present in the medium after incubation and that higher radioactivity would undoubtedly have been obtained if the tissues had been macerated prior to extraction.
DISCUSSION
Our results indicate a qualitative difference in the metabolic activity between Adrenal cortical carcinoma 494 and normal rat adrenal tissue. The latter con- verts deoxycorticosterone to corticosterone and aldosterone with such efficiency that only small amounts of deoxycorticosterone can be found in the adrenal
glands of the rat (Ward & Birmingham 1960). While only 1.27 % of the ad- ministered progesterone-4-14C was accounted for as deoxycorticosterone in in- cubation experiments with normal rat adrenal tissue (Table 1), 6.00 % was converted to deoxycorticosterone by Tumour 494 and 46.62 % by a necrotic specimen of this tumour. Deoxycorticosterone is further metabolized by normal rat adrenal glands to give corticosterone (13.77 % of the administered pro- gesterone) and aldosterone (8.42 %), while the tumour tissue has a greatly decreased ability to convert deoxycorticosterone to corticosterone (3.13 %) and aldosterone (0.49 %). The corresponding values for the necrotic specimen are higher, but still below the normal figures. The low rate of conversion of deoxy- corticosterone-4-C14 to corticosterone and aldosterone by the tumour confirms the observation that the blockade occurs in the 11-hydroxylation.
The subline tumour 494-H, which produces progestational effects in the tumour-bearing rat (Snell & Stewart 1959) may have been expected to show an inability to metabolize progesterone. However, it resembles Tumour 494 as far as progesterone metabolism is concerned (Table 1). On the other hand, it shows a different distribution pattern of radioactive metabolites (Fig. 3 - Top).
The effect on water metabolism and endocrine organs of the host are con- sistent with a blockade in 11-hydroxylation of deoxycorticosterone with a re- sulting accumulation of this intermediate in the animal organism. The findings of Mulay (1960), that an adrenocortical carcinoma, produced in the rat by feeding it p-dimethylaminobenzene, protects the animal against an otherwise lethal dose of potassium chloride is of interest in this connection. Excessive production of deoxycorticosterone could very likely afford such protection.
ACKNOWLEDGEMENTS
The authors are indebted to Dr. Maurice M. Pechet of the Research Institute for Medicine and Chemistry, Cambridge, Massachusetts and Dr. W. Klyne, Medical Research Council, London, England, for generous gifts of reference compounds, to Mr. Edward J. Soban for technical assistance, and to Dr. Erich Mosettig for advice and encouragement.
REFERENCES
Angelico R. & Quintiliani M .: Rend. ist. super. sanità 21 (1958) 671.
Bailey R. E., Slade C. I., Lieberman A. H. & Luetscher J. A .: J. clin. Endocr. 20 (1960) 457.
Berliner M. L., Berliner D. L. & Dougherty T. F .: J. clin. Endocr. 18 (1958) 109.
Block E. & Cohen A. I .: J. nat. Cancer Inst. 24 (1960) 97.
Bongiovanni A .: J. clin. Invest. 37 (1958) 1342.
Dorfman R. I .: Ciba Foundation Colloq. Endocr. 12 (1958) 62.
Dyrenfurth I., Lucis O. J., Beck J. C. & Venning H .: J. clin. Endocr. 20 (1960) 765.
Eisenstein A. B .: Proc. Soc. exp. Biol. (N. Y.) 91 (1956) 657.
Giroud C. J. P., Saffran M., Schally A. V., Stachenko J. & Venning E. H .: Proc. Soc. exp. Biol. (N. Y) 92 (1956) 855.
Heftmann E. & Mosettig E .: Biochemistry of Steroids, Reinhold Publ. Corp., New York (1960).
Hofmann F. G .: Endocrinology 60 (1957) 382.
Iglesias R. & Mardones E .: Brit. J. Cancer 12 (1958) 20.
Johnson D. F., Heftmann E. H. & Hayden A. L .: Acta endocr. (Kbh.) 23 (1956) 341.
Mialhe-Voloss C. & Baulieu E .: C. R. Soc. Biol. (Paris) 246 (1958) 639.
Mulay A. S .: Endocrinology 66 (1960) 769.
Péron F. G .: Endocrinology 66 (1960) 458.
Saffran M. & Schally A. V .: Endocrinology 56 (1955) 523.
Snell K. C. & Stewart H. L .: J. nat. Cancer Inst. 22 (1959) 1119.
Ward P. J. & Birmingham M. K .: Biochem. J. 76 (1960) 269.
Received on February 13th, 1961.