Nagoya ]. med. Sci. 34: 183-190, 1971
ELECTRON MICROSCOPIC STUDIES ON THE ADRENAL
OF RAT TREATED WITH AMINOGLUTETHIMIDE
-WITH SPECIAL REFERENCE TO ACCUMULATION OF
CHOLESTEROL IN INTRAMITOCHONDRIAL VACUOLESGEN
lToH
1st Department of Pathology, Nagoya University School of Medicine
(Director: Prof. Shooichi Sugiyama)
ABSTRACT
Aminoglutethimide ( Elipten, Ciba,) interferes with the biosynthesis of adrenocortical hormones by blocking the conversion of cholesterol to pregnenolone.
Male albino rats were fed with the diet containing aminoglutethimide and were
killed after various time intervals ranging from 24 hours to 7 days. The adrenal
cortices of these animals were studied both by light and electron microscopy.
Special effort was made to determine the intracellular localization of free
cholesterol by an electron microscopic histochemical method using the digitonin
reaction.
The animals killed 24 hours after the initiation of the feeding were found
to have small myelin figures in contact with the mitochondrial membrane in
fasciculata cells which might reflect compensatory rise of ACTH. The animals
fed with the diet for 4 to 7 days were shown to have enlarged adrenal cortex.
Light microscopic examination revealed an increase of the lipid globules both in
size and number in the zona fasciculata. Ultrastructural studies showed swollen,
hypertrophied, bizarre prophiles of the mitochondria with the formation of the
vacuoles. The electron microscopic histochemical method demonstrated digitonincholesterol crystals localized in the vacuoles of the mitochondria, in the perimitochondrial areas and in the lipid globules.
These findings correlate well with the biochemical data which indicate the
intracellular site of steroid bicsynthesis and lend morphological support to the
view that the biosynthesis of pregnenolone from cholesterol takes place within
the mitochondria. These results indicate that aminoglutethimide causes accumulation of cholesterol in the vacuoles of the mitochondria.
INTRODUCTION
Aminoglutethimide which was introduced as an anticonvulsant in 1958 was
later proved to interfere with the biosynthesis of the adrenocortical hormones
by blocking the conversion of cholesterol to pregnenolone 1l 2l. Clinical trials 3l- 7l,
were performed on the patients suffering from Cushing's syndrome 3l due to
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Received for publication February, 25, 1971.
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183
184
G. ITOH
malignant adrenal cortical tumors 4l or hyperfunctioning cortical adenomas 5l .
Morphological studies 3>6l on the adrenal glands of these patients treated with
aminoglutethimide revealed hypertrophy and vacuole formation of the adrenal
cortical cells 6> by light microscopy, and also accumulation of lipid vacuoles
bounded by a single limiting membrane in the cytoplasm of the fasciculata
cells by electron microscopy 8l. In the rat, aminoglutethimide caused accumulation of lipids 6l 9> in the cytoplasm of the faciculata cells which resulted in
destruction of this zone 6>. Electron microscopic studies9l 10l demonstrated vacuole
formation in the mitochondria of the fasciculata cells. Accumulation of lipids3>11>
and cholesterol 2l was also demonstrated biochemically.
Since the work of Hayano et a/. 12 ) in 1956 which indicated that the mitochondrial fraction was necessary for the biosynthesis of pregnenolone from
chlesterol, biochemical works 13>14>15> concerning the intracellular localization of
steroid biosynthesis followed and confirmed that aminoglutethimide inhibited
20a-hydroxylation of the cholesterol side chain 1l and desmolase activity 3l .
These results suggest that aminoglutethimide might cause cholesterol accumulation in the intramitochondrial vacuoles. This speculation has not been proved
in any publication in the past. This investigation was designed, therefore, to
confirm the morphological changes of the adrenal gland of the rat given aminoglutethimide. Special attention was paid to the ultrastructural histochemical
demonstration of cholesterol in the vacuoles of the mitochondria.
MATERIALS AND METHODS
Fourteen male albino rats were fasted for 24 hours, although water was
allowed ad. libitum. The animals were fed with the standard laboratory diet
with 0.6% of aminoglutethimide added. They were killed by decapitation at
the same time each afternoon by the following schedule : 4 rats at 24 hours
after the initiation of the aminoglutethimide diet, 2 rats at the end of the
second day, 4 rats on the fourth day, 4 rats on the seventh day, respectively.
A total of four rats were served for the control study.
One of the rat adrenal glands was fixed in 3% solution of formol for light
microscopic examination. One half of the other side of the adrenal was cut
into blocks, fixed in buffered solution of 3% glutaraldehyde for 2 hours, washed
for 24 hours, post-fixed with buffered solution of 1% osmic acid, dehydrated
in graded alcohol, embedded in epoxy resin and was examined electron microscopically. The other half of the adrenal was fixed, according to Flickinger's
method 16>17>, for 20 hours in buffered solution of 0.1 N caccodylate containing
2% formaldehyde, 2.5% glutaraldehyde and 0.05% calcium chloride, incubated
for 24 hours in buffered solution of caccodylate containing 2% formaldehyde,
2.5% glutaraldehyde, 0.05% calcium chloride and 0.2% digitonin, washed for 24
hours in buffered solution of caccodylate, post-fixed with buffered solution of
CHOLESTEROL IN INTRAMITOCHONDRIAL VACUOLES
185
1% osmic acid, dehydrated in graded acetone, embedded in epoxy resin and
examined electron microscopically.
For orientation semi-thin sections were observed under light microscopy,
trying to recognize the different regions of the gland by their structure.
RESULTS
Hypertrophy of the adrenal cortex was recognized in the rats sacrificed
after 4 and 7 days of aminoglutethimide feeding. The animals belonging to
these groups were found to have increased amounts of lipid globules through
the semi-thin sections of the epoxy resin embedded blocks.
Electron microscopically, small myelin figures (Fig. 1) having contact
with the mitochondrial membrane were frequently found in the fasciculata
cells of the rats sacrificed 24 hours after the initiation of the feeding, while
these structures were rarely seen in the rats sacrificed 4 and 7 days after the
initiation of the aminoglutethimide diet. Dilatation of the vesicular cristae of
the mitochondria (Fig. 2) occurred sporadically 2 days after the onset of the
diet and increased in degree and frequency as time elapsed, resulting in accumulation of many and large vacuoles in the mitochondria of the fasciculata
cells of the rats sacrificed after 4 and 7 days of the feeding (Fig. 3). Lipid
globules were of small size at the earlier stage, while they increased in size
and number at the later stage. Electron-transparent areas at the periphery
of one pole of the lipid globules were frequently observed in the rats fed on
the diet for 4 or 7 days.
Electron microscopic examination of the digitonin treated adrenal tissue
revealed needle-shaped crystals in the intramitochondrial vacuoles (Figs. 4, 5,
6), in the perimitochondrial areas (endoplasmic reticulum) and in the lipid
globules at the periphery of one pole of the lipid globules. The needle-shaped
crystals in the intramitochondrial vacuoles were abundant in the cytoplasm
of the fasciculata cells of the adrenals in rats 4 and 7 days after the initiation
of the aminoglutethimide diet. On the other hand, the needle-shaped crystals
were rarely seen in the fasciculata cells of rats 1 and 2 days after the onset
of the diet.
The crystals in the lipid globules seemed to be located at the electrontransparent areas of the adrenal specimen not treated with digitonin. The
crystals were much more abundant in rats 4 and 7 days after the initiation
of the diet than in those 1 and 2 days after the initiation of the diet.
The needle-shaped crystals were assumed to be tubular in structure and
were composed of 3 to 7 coaxial lamellae. The innermost and outermost layers
of the 3 coaxial lamellae were electron dense while the lamellae inbetween was
electron transparent (Fig. 7). They were 400 to 1000 A in width. The exact
length was not calculated. The electron dense and electron transparent lamella
186
G. ITOH
was 30 A in thickness.
These needle-shaped crystals were entirely lacking in the tissue samples
incubated without digitonin in the medium.
DISCUSSION
Aminoglutethimide inhibits conversion of cholesterol to pregnenolone 1>2>3>
which occurs in the mitochondria 12> and causes compensatory rise of ACTH
production 2>. ACTH produces ultrastructural changes 18>- 24> of mitochondria,
smooth surfaced endoplasmic reticulum and liposome in the faciculata cells of
the adrenal cortex.
The myelin figures noted in this experiment might reflect a compensatory
rise of ACTH. Schwarz et a/. 22 > observed intramit6chondrial vacuoles in fasciculata cells of the rat treated with ACTH. Yoshimura 25 > described large
intramitochondrial vesicles which were produced by stimulation of ACTH. The
intramitochondrial vacuoles observed in this experiment, however, were much
more abundant than those produced by ACTH and also considerably different
in appearance. Racela et a/. 10> confirmed development of intramitochondrial
vacuoles in hypophysectomised rats treated with aminoglutethimide. Taking
into consideration these results, it seems reasonable to interpret that the intra·
mitochondrial vacuoles in this experiment arose as a result of the specific
action of aminoglutethimide.
The digitonin reaction which was initiated by Windaus in 1910 26> for estimation of free cholesterol, was applied for histochemistry by Brunswick in
1922 27> and was further employed for the determination of 3 8-hydroxysteroids
by Clarenburg et al. in 1966 28>. Okros 29> and Levy et a/.30>, independently in
1968 and in 1967, were the first who applied this method to electron microscopic
histochemistry. Okros demonstrated the needleshaped crystals of digitonincholesterol complex in the normal rat adrenal tissue treated with digitonin.
According to his description, the digitonincholesterol crystals were made up
of electron transparent and electron dense coaxial lamellae; the crystals varied
between 6000 and 7000 A in length and 400 to 1000 A in width; the electron
dense and electron transparent lamellae of the crystals were 20 to 30 A in
thickness; the number of the lamellae was 1 to 9. Scallen et a/. 17> recently
demonstrated the needle-shaped crystals of digitonin-cholesterol complex in
the murine liver treated with digitonin and proved that as much as 99% of
the unesterified cholesterol was preserved by this procedure. The needle-shaped
crystals observed in this experiment had entirely the same appearance as those
described by Okros and Scallen et al. Digitonin reacts not only with free
cholestorol but also with 3 8-hydroxysteroids 28>. Whether or not the crystals
of the digitonin-cholesterol complex are different in structure from those of
digitonin-3 8-hydroxysteroids is not known. The latter, however, is scanty in
CHOLESTEROL IN INTRAMITOCHONDRIAL VACUOLES
187
amount. Consequently it is concluded that the needle-shaped cryatals represent
the digitonin-cholesterol complex.
It is generally accepted that cholesterol, the precursor of steroid hormones,
comes mainly from serum free cholesterol in the rat adrenals. Moses et at.s 1>
recently demonstrated by electron microscopic autoradiographs and biochemical
analysis that tritiated cholesterol injected intravenously, though only 9% of
the tritiated cholesterol given, accumulated in the mitochondria. The needleshaped crystals at the perimitochondrial areas observed in this experiment
might, therefore, indicate accumulation of cholesterol of extra-adrenal origin.
Okros 29> observed in 1967 digitonin-cholesterol crystals localized in lipid
globules in normal rat adrenal tissue treated with digitonin by electron
microscopy. Moses et a/. 31 > confirmed that 70 to 80% of tritiated cholesterol
injected intravenously are distributed within lipid globules in the adrenal of
normal rats. In this experiment digitonin-cholesterol crystals were observed
in lipid globules. This means that aminoglutethimide causes accumulation of
cholesterol in lipid globules.
Moses et a/. 31> postulate that cellular mechanisms exist which cause
mobilization of cholesterol from the lipid globules to the mitochondria. Further
studies are needed as to this point.
ACKNOWLEDGEMENT
I express my sincere gratitude to former Prof. M. Miyakawa for his cordial
consideration and to Prof. S. Sugiyama and Prof. H. Tauchi for their king
advice in this experiment. I am indebted to Dr. T. Hirano for his careful
review of the manuscript and to my colleagues for their friendly encouragement
and critisism throughout this work. Thanks are also due to my technical
officials for their kind technical helps.
This is a doctoral thesis judged by Nagoya University inquiry council of medical
degree.
REFERENCES
1) Kahnt, F. W., and R. Neher, Uber die adrenale Steroid-Blosynthese in vitro. III Selektive
Hemmung der Nebennierenrinden-Funktion, Helv. chim. aCta, 49, 725, 1966.
2) Dexter, R. N., Fishman, L. M., Ney, R. L., and Liddle, G. W., Inhibition of adrenal
corticosteroid synthesis byaminoglutethimide. Studies of tne mechanism of action, ].
din. Endocr. and Metabol., 27, 473, 1967.
3) Cash, R., Brough, A. J., Cohen, M. N. P., and Satoh, P. S., Aminoglutethimide (Elipten)
as an inhibitor of Adrenal steroidgenesis; Mechanism of action and therapeutic trial,
]. elin. Endocr. and Metabol., 27, 1239, 1967.
4) Schteingart, D. E., Cash, R., and Conn, J. W., Aminoglutethimide and matastatic adrenal
cancer, ].A.M.A., 198, 1007, 1966.
5) Fishman, L. M., Liddle, G. W., Island, D. P., Fleischer, N., and Kiichel, 0., Effects of
aminoglutethimide on adrenal function in man, J. clin. Endocri. and Metabol., 27, 481,
188
G. ITOH
1967.
6) Camacho, A. M., Cash, R., Brough, A. ]., and W ilroy, R. S., Inhibition of adrenal
steroidgenesis by aminoglutethimide and the mechanism of action, ].A.M.A., 202, 20,
1967.
7) Schteingart, D. E., and Conn,]. W., Effects of aminoglutethimide upon adrenal function
and cortisol metabolism in Cushing's syndrome, ]. clin. Endocri. and Metabol., 27, 1657,
1967.
8) Symington, T, Functional pathology of the human adrenal gland, 406, 1969, E and S
Livingstone Ltd.
9) Marek, ]., Thoenes, M., und Motlik, K., Lipoide Transformation der Mitochondrien in
Nebennierenrindenzellen nach Aminoglutethimid (Elipten, Ciba ), Virchows Arch. Abt.
B. Ze/lpatho., 6, 116, 1970.
10) Racela, A., Azarnoff, D., and Svoboda, D., Mitchondrial cavitation and hypertrophy in
rat adrenal cortex due to aminoglutethimide, Labo. Invest., 21, 52, 1969.
11) Cammcho, A.M., Brough, A.]., Cash, R., and Wilroy, R. S., Adrenal toxicity associated
with the administration of an anticonvulsant drug. ( abstr. ), ]. Pediat., 68, 852, 1966.
12) Hayano, M., Saba, N., Dorfman, R. L., Hechter, 0., Some aspects of the biogenesis of
adrenal steroid hormones, Recent. Progr. Hormone res., 12, 79, 1956.
13) Peron, F. G., and Robidoux, W. F., The biosynthesis of corticoids by rat adrenal
fraction and inhibition of endogenous cholesterol utilization by cobra venom, Biochim.
Biophys. Acta., 82, 125, 1964.
14) lchii, S., Kobayashi, S., and Matsuba, M., Effect of ACTH in vivo on the cholesterol
side chain cleaving enzyme and on steroid hormone precursor cholesterol in r a t
adrenal gland, Steroids, 5, 663, 1965.
15) Ichii, S. and Kobayashi, S., Studies on the biosynthesis of sterol and corticosterone
in rat adrenal gland, Endocrino/o. japan ., 13, 39. 1966.
16) Flickinger, C. ]., The post natal development of the Sertoli cells of the mouse, Z.
Ze/lforsch. Mikroskop. Anat., 78, 92, 1967.
17) Scallen, T. ]., and Dietert, S. E., The quantitative retention of cholesterol in mouse
liver prepared for electron microscopy by fixation in a digitonin-containing aldehy de
solution, 1- Cell Bio/og., 40, 802, 1969.
18) Idelman, S., In International r eview of cytology. Edited by Bourne, G. H., Danielli,
]. F., and ]eon, K. W., Academic Press, New York, 181, 1970.
19) Symington, T., In Functional pathology of the human adrenal gland, Edited by
symington, T. E and S Livingstone Ltd.
20) Ashworth, C. T., Race, ]. G., and Mollenhauer, H. H., Study of functional activity of
adrenocortical cells with electron microscopy, Am. ]. Patho., 35, 425, 1959.
21) Carr, I., The ultrastructure of the human adrenal cortex before and after stimulation
with ACTH, ]. pathol. bact., 81, 101, 1961.
22) Schwarz, W., Merker, H. ]. und Suchowsky, G., Elektronenmikroskopische Unters uchungen tiber die Wirkungen von ACTH und Stress a uf die Nebennierenrinde d er
Ratte, Virchows Arch. path. Anat., 335, 165, 1962.
23) Yamori, T., Matsuura, S., and Sakamoto, S., An electron microscopic study of the
normal and s timulated adrenal cortex in the rat, Z . Ze/lforsch., 55, 179, 1961.
24) Nishikawa, M ., Mur one, I., and Sato, T ., Electron microscopic investigation of t he
adrenal cortex, Endocrinology, 73, 167, 1963.
25) Yoshimura, F., Watanabe, M., Omoto, T ., and Sekiguchi, T ., Functional s ignifcance of
the tubular a g ranular endoplasmic reticulum in the adrenocor tical cells of albin o
rats, Endocri. j apan, 15, 145, 1968.
26) Windaus, A., Uber die quantitative Bestimmung des Cholesterins und der Cholesteripester in eini¥en normalen und patholo~ischep. ;Nieren1 Hoppe-Sellers z. ph;;sio/. Chem ..
CHOLESTEROL IN INTRAMITOCHONDRIAL VACUOLES
189
65, 110, 1910.
27) Brunswick, H., Der mikroskopische Nachweis der Phytosterine uud von Cholesterin
als Digitonin-Steride, Z. wiss. Mikr., 39, 316, 1922.
28) Clarenburg, R. and Chaikoff, I. L., Origin of milk cho!Psterol in the rat: dietar y versus
endogenous sources, f. lipid res., 7, 27, 1966.
29) Okras, I., Digitonin reaction in electron microscopy, Histochemic, 13, 91, 1968.
30) Levy, M., Toury, R., et Ardre, ]., Separation d es membranes mitochondriales. purification et caracterisation enzymatique de Ia membrane externe, Biochim. Biophys.
Acta., 135, 599, 1967.
31) Moses, H. L., Dayis, W. W., Rosenthal, A. S., and Garren, L. D., Adrenal cholesterol:
Localization by electron microscope autoradiography. Science, 163, 203, 1969.
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G. ITOH
EXPLANATION OF FIGURES
MT.
LG.
V AC.
CRYS.;
mitochondria
lipid globule
vacuole
crystal of digitonin-cholesterol complex
FIG. 1. The fasciculata cell of the rat sacrificed 24 hours after the aminoglutethimide
feeding. Note the myelin figures having contact with the mitochondria l membrane.
FIG. 2. The fasciculata cell of the rat 2 days after the initiation of the aminoglutethimide
diet. Note sporadic dilatation of vesicular cristae of the mitochondria.
FIG. 3. The fasciculata cell of the rat 4 days after the initiation of the diet. Note the
enlargement of the mitochondria and accumulation of large vacuoles in the
mitochondria. Those vacuoles contain fragments of membrane like materials.
FIG. 4. The fasciculata cell of the rat 4 days after the initiation of the diet.
Needleshaped crystals of digitonin-cholesterol complex are found in t he vacuoles of the
enlarged mitochondria and in the perimitochondrial endoplasmic r eticulum. (the
adrenal tissue with digitonin treatment)
FIG. 5. The fasciculata cell of the rat 7 days after the initiation of the diet. The needleshaped crystals are also found in the intramitochondral vacuoles and in the
perimitochondrial endoplasmic reticulum. (the adrenal tissue treated with digitonin)
FIG. 6. The fasciculata cell of the r at 7 days after the initiation of the diet. Note the
degenerated mitochondria containing large vacuoles. The needle-sh aped crystals
are also found in the vacuoles. The lipid globule is found to contain the crystals
at its periphery. (the adrenal tissue treated with digitonin)
FIG. 7. Digitonin-cholesterol crystal is assumed to be tubular in structure composed of
a total of three coaxial lamellae. The innermost and outermost layers are
electron dense while the lamella in-between is electron transparent. (the adrenal
tissue treated with digitonin)