Fine Structural and Biochemical Effects of Aminoglutethimide and o,p’-DDD on Rat Adrenocortical Carcinoma 494 and Adrenals
ROBERT N. MOORE, DAVID P. PENNEY, AND KATHY T. AVERILL Departments of Anatomy and Dental Research, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT Rats bearing adrenocortical carcinoma 494 were injected daily for 7, 14, or 21 days with aminoglutethimide (AG) or o,p’-DDD. Reversibility of these steroidogenic inhibitors was determined by injecting other animals for either 14 or 21 days and sacrificing them 14 days later. While the drugs had little effect on body or tumor growth, plasma corticosterone levels were reduced a maximum of 88% in normal and 95% in tumor-bearing rats during AG chemotherapy. These levels were unaltered in normal rats by o,p’-DDD and reduced a maximum of 64% in tumor-bearing animals. Relative adrenal weights generally increased during chemotherapy and then returned to control levels. These changes were mainly due to alterations in the lipid and mitochondrial volume fractions. Lipid increased with both drugs while mitochondria increased with o,p’-DDD and decreased with AG. Cholesterol ester levels paralleled the lipid stereology more closely with AG than o,p’-DDD. With both drugs the most notable changes in tumor fine structure was a decrease in mitochondrial internal membranous vesicles and matrical density. Adrenal mitochondria had the irreg- ular, elongated forms characteristic of tumor-bearing animals and were vacuo- lated (AG) or had internal rings (o,p’-DDD). The large lipid droplets observed during chemotherapy with both drugs were replaced by numerous small droplets in recovery periods.
In humans aminoglutethimide (AG; Elipten- Ciba) and o,p’-DDD have been reported to be effective in the chemotherapy of adrenocorti- cal carcinoma (Schteingart et al., ‘66; Talley, ‘70; Touitou et al., ‘78). Patients treated with AG initially develop symptoms characteristic of adrenal insufficiency and exhibit decreased secretions of cortisol and aldosterone (Ca- macho et al., ‘66; Fishman et al., ‘67). This steroidogenic block may be overcome by a compensatory increase in endogenous ACTH, and cortisol secretion eventually approaches pretreatment levels (Cash et al., ‘67).
The mechanism of action of AG is unknown, but the primary biochemical site is believed to be between cholesterol and A 5-pregneno- lone (Dexter et al., ‘67; Cash et al., ‘67), where it competitively inhibits the mitochondrial desmolase side chain-spliting enzyme, choles- terol (Cohen, ‘68; Kahnt and Neher, ‘66). Sec- ondarily, AG has been reported to cause a decrease in 113-hydroxylation (Wilroy et al., ‘68).
In addition to these biochemical studies, numerous morphological reports on the effects of AG on the rat adrenal cortex have appeared in the literature (Racela et al., ‘69; Marek et al., ‘70, ‘71; Itoh, ‘71; Magalhaes and Magal- haes, ‘72). The drug causes loss of normal adrenocortical zonation, with slight or no change in the zona glomerulosa and marked hypertrophy of cells in the zonae fasciculata and reticularis. On the fine structural level, cytoplasmic lipid and smooth endoplasmic re- ticulum (SER) were markedly increased, and mitochondria were characterized by rarefac- tions of the matrix, segmental loss of the outer membrane, and focal, progressive dilation of
Robert N. Moore’s present address is: Department of Orthodontics, School of Dentistry and Department of Anatomy, School of Medicine, West Virginia University, Morgantown, West Virginia 26506.
Reprint requests should be addressed to David P. Penney.
Received November 11, 1979: Accepted January 21, 1980.
the tubulovesicular cristae and formation of large membrane-bound cavities.
Clinical observations have shown o,p’-DDD to be capable of producing regressing of adre- nal cortical carcinoma and its metastases (Bergenstal et al., ‘59; Gallagher et al., ‘62). The drug has been shown to cause similar decreases in dogs (Vilar and Tullner, ‘59) and to interfere with the mechanism of action of ACTH which stimulates the conversion of cho- lesterol to pregnenolone. A secondary site of action is the partial inhibition of the intram- itochondrial conversion of 11-deoxycortisol to cortisol (Hart and Straw, ‘71 a,b).
More recently, Touitou et al. (‘78) have dem- onstrated the inhibitory effects of o,p’-DDD on 11-hydroxylase and 18-hydroxylase activi- ties in the adrenals of humans with Cushing’s syndrome who were treated with o,p’-DDD. Hart et al. (‘73) reported on the acute mor- phological effects of o,p’-DDD in the dog. Cells of the zonae fasciculata and reticularis were characterized by irregularly-shaped nuclei with coarsely-clumped, abnormal chromatin, loss of regular cellular outline, shrinking, loss of lipid, and eventual rupture. Fine structur- ally, the first acute changes were swelling, dissolution, and rupture of mitochondria in the zonae fasciculata and reticularis. Later, degenerative changes in the endoplasmic re- ticulum and nucleus were observed along with progressive dissolution of the internal con- tents of the cells. The zona glomerulosa was not affected to such an extent. No histological or fine structural investigations of the effects of o,p’-DDD on the rat adrenal cortex have been published.
In contrast to the numerous investigations of the effects of AG and o,p’-DDD on normal adrenocortices and human carcinomas, the use of a laboratory animal model to more precisely elucidate the biological effects of these drugs on an adrenal carcinoma has been limited. Ney et al. (‘69) used rat adrenocortical carci- noma 494 and demonstrated that AG inhibited tumor steroidogenesis in vitro and reduced plasma corticosterone levels in vivo. o,p’-DDD has been briefly reported to have no effect on a transplantable mouse adrenocortical tumor or on the adrenal-castration tumor of the ce X dba mouse (Nichols, ‘61).
In a previous communication (Moore et al., “78), an integrated electron microscopic, ster- eological, and biochemical analysis of rat ad- renocortical carcinoma 494 has been pre- sented. This will serve as the model system for the studies of the effects of aminogluteth- imide and o,p’-DDD which will be presented here.
MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 80-100 grams were implanted with adrenocortical carcinoma 494 as previously described (Moore et al., ‘78). When tumors had become palpable, usually 7-10 days following transplantation, host animals were divided randomly into groups of at least ten rats per group. Either AG (40 mg/kg body weight) or o,p’-DDD (900 mg/kg body weight) was administered daily for 7, 14, or 21 days. The AG was dissolved in citrate buffer (pH 4.0) and injected intraperi- toneally, while o, p’-DDD was dissolved in corn oil and injected orally. Two “recovery” groups were injected for 14 or 21 days and then sacrificed 14 days following the last in- jection. The same chemotherapeutic regime was also administered to similar numbers of non-tumor rats. As controls, citrate buffer (AG sham) or corn oil (o,p’-DDD sham) was inject- ed on the same volume/weight basis to normal and tumor-bearing rats using the time se- quence previously described. The methodology for the physiologic, electron microscopic, ster- ologic, and biochemical analyses has been pre- viously presented (Moore et al., ‘78).
RESULTS
Tumor growth, body and adrenal weights
As the physiological data were consistent for all groups, only the results of the long- term studies of tumor growth and body weight will be presented in detail. Injection of ami- noglutethimide for 21 days followed by a 14- day recovery period (Fig. 1) had little effect on the mean diameter of the tumor or on body weight. While the growth rates of the animals and tumors were different because of a differ- ence in their initial size, there was no discern- able change in the growth pattern after dis- continuation of therapy. During the first few days of o,p’-DDD administration (Fig. 2), an- imals became nervous, had diarrhea, and body weights decreased. After about a week, body weights returned to control levels. Mean tu- mor diameter was not markedly influenced by the drug or its withdrawal.
In contrast, relative adrenal weights (Fig. 3) in both normal and tumor-bearing animals were affected by the drugs. In normal animals treated with AG, relative adrenal weights were increased over control values, and the difference became more significant with the duration of treatment. With cessation of ther- apy, the return to control levels was rapid. Administration of o,p’-DDD in normal ani- mals resulted in larger increases in relative adrenal weight, which slowly began to return
BODY WEIGHT AND MEAN TUMOR DIAMETER AG AND CITRATE 21/14 DAYS
420
400
AG
380
BODY WEIGHT
360
TUMOR DIAMETER
BODY WEIGHT (gm)
340
320
300
280
CITRATE
260
BODY WEIGHT
MEAN TUMOR DIAMETER (mm)
240
TUMOR DIAMETER
45
220
40
200
35
180
30
160
25
140
20
LAST DAY OF THERAPY
15
10
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33 3
5
EXPERIMENTAL PERIOD IN DAYS
420
400
O,P’-DDD
BODY WEIGHT (gm)
380
BODY WEIGHT
360
TUMOR DIAMETER
340
320
CORN OIL
300
O BODY WEIGHT
280
TUMOR DIAMETER
260
MEAN TUMOR DIAMETER (mm)
240
45
40
35
30
25
20
LAST DAY OF THERAPY
15
10
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
5
EXPERIMENTAL PERIOD IN DAYS
SIGNIFICANCE OF DIFFERENCE
I
p< 0.05
2 p< 0.025
DRUG
3 p < 0.01
mg / 100gm BODY WEIGHT
4
p < 0.005
SHAM
28
· NOT SIGNIFICANT
24
AMINOGLUTETHIMIDE
O,P’- DDD
20
16
29
1
12
8
4
0
7* 14| 2|2 |4* 21*
7* 144 21* 14* 211
74 144 214 144 2
213
72 144 211
14 14
14* 214
14 14
14 14
14 14
NORMAL
TUMOR BEARING
NORMAL
TUMOR BEARING
EXPERIMENTAL PERIOD IN DAYS
to control levels following discontinuation of the drug. In tumor-bearing animals, the drugs had a similar influence on relative adrenal weights, with o,p’-DDD exhibiting the most pronounced and prolonged effects.
Tumor morphology
Adrenocortical tumors which had been treated with AG for 7 days showed very slight fine structural alterations when compared to typical non-treated tumor cells (Moore et al., ‘78 and Fig. 4). The most pronounced differ- ence was that tumor cells from AG-treated animals frequently contained large (approxi- mately 5u in diameter), membrane-bound vac- uoles which were completely electron lucent and were considered to be degenerative mito- chondria (Fig. 5). In addition, at 14 days many lysomsomal dense bodies and phagosomes of somewhat smaller diameter were frequently observed, suggesting removal of the function- ally-impaired mitochondria by autophagocy- tosis. After 14 and especially after 21 days of continuous chemotherapy, mitochondria of the tumor cells were vacuolated and had de- creased numbers of internal membranous ves- icles (Fig. 6). In recovery groups, mitochondria with varying internal morphology and matr- ical electron density were present. Some con- tained an electron-lucent matrix and had a few tubular cristae transversing almost the entire diameter of the organelle, while the majority possessed a matrix of normal electron
density and had many tortuous tubular cistae. Tumor fine structure appeared to be unaffected by citrate buffer (AG sham).
Like AG, o,p’-DDD did not markedly alter the fine structure of the tumor. The most notable changes were again in the mitochon- dria which, with continued therapy, showed a progressive decrease in the numbers of inter- nal vesicles and in matrical electron density (Fig. 7). In some mitochondria from the 21- day group, the matrix was very electron lucent and was traversed by a single internal mem- brane. While such a configuration is sugges- tive of mitochondrial division, the event per se was not observed. In recovery groups the matrical density was similar to that of the untreated tumor, but the number of mitochon- drial tubules appeared to be diminished, sug- gesting incomplete recovery. No fine structur- al changes attributable to corn oil (o,p’-DDD sham) were observed.
Adrenocortical morphology
Following 7 consecutive days of AG admin- istration, adrenocortical fine structure differed only slightly from that of tumor-bearing con- trols (Fig. 8). While no changes in cells of the zona glomerulosa were observed, in cells of the zonae fasciculata and reticularis, mito- chondria were irregular in form, and elongat- ed and large lipid droplets were observed. Some mitochondria exhibited a focal loss of internal membranous vesicles, but generally
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these aberrations were not as severe as those seen in adrenocortices of normal rats treated with AG for 7 days (Fig. 9). After 14 and 21 days of continuous AG therapy in tumor-bear- ing rats, mitochondrial matrices became more electron lucent and membranous internae de- creased. In addition, the mitochondria in the 21-day AG treatment group also had irregular and elongated forms characteristic of adreno- cortical mitochondria of tumor-bearing ani- mals (Fig. 8). In recovery groups, lipid droplets decreased in size, while mitochondria re- mained elongated with bizarre internal struc- ture.
Since they have not previously been report- ed and are necessary for the understanding of the influences of the tumor, the fine structural effects of o,p’-DDD on the normal rat adrenal cortex will be briefly described. Following 7 consecutive days of o,p’-DDD administration, adrenocortical cells of the zonae fasciculata and reticularis exhibited no marked fine struc- tural changes of any organelle other than the mitochondria (Fig. 10), and some cells ap- peared to be entirely normal morphologically.
Mitochondrial profiles were elongated and irregular with loss of matrical electron density and decreased numbers of intramitochondrial vesicles. In most mitochondria the vesicles were retained only around the periphery of the organelle, while in others there were long tubular internae which traversed the matrix. Many abnormal mitochondria had double membrane rings at their centers. There were not fine structural changes of zona glomeru- losa cells of this or any other time period in animals which had received o,p’-DDD.
After 14 and 21 days of continuous admin- istration, these morphological changes per- sisted but appeared to be less severe. At this time, there were also one or two large (10 u diameter) lipid droplets present in many cells of the inner zones. Following the recovery periods, mitochondria were more regular in profile and contained a more tubulovesicular internal organization as compared to the pre- vious groups. However, internal membranes were much less numerous than would be seen in the normal mitochondrion. While some large lipid droplets were still present, the majority of the cells had numerous small drop- lets.
The adrenocortices of tumor-bearing ani- mals treated with o,p’-DDD for 7 days had the fine structure characteristic of both influences. Some mitochondria were of the elongated type seen in the zona fasciculata and reticularis of tumor-bearing animals (Fig. 8), while others
were round in profile with a matrix of normal electron density and diminished numbers of internal membranes. Some of these mitochon- dria had the internal rings characteristic of o,p’-DDD, and there was evidence of fusion of atypical mitochondria through disruption of external membranes. No fine structural changes of cells of the zona glomerulosa of this or subsequent experimental periods were observed.
After 14 and 21 days of chemotherapy, there was a progressive decline in the integrity of the mitochondria, with loss of internal mem- branes and the appearance of one to three electron-dense homogeneous bodies which were similar to lipid in appearance. These were found either free in the matrix or within one of the ring structures. Following 21 days of o,p’-DDD treatment, large lipid droplets were also frequently present in the adrenals of tumor-bearing animals. In the recovery groups lipid droplets became elongated in form, and mitochondria displayed increased numbers of tubulovesicular cisternae while still possessing the internal ring structure.
Tumor stereology
In keeping with the fine structural obser- vations, neither AG or o,p’-DDD evoked sta- tistically significant quantitative alterations of tumor nuclear or cytoplasmic volume frac- tions (Table 1). The mitochondrial volume fraction remained at control levels during in- jection of AG, and was only elevated in the 7- day o,p’-DDD group. The lipid volume fraction showed no significant change during AG chemotherapy but significantly decreased in recovery periods, suggesting increased utili- zation for corticosteroidogenesis. A similar, but less pronounced trend was seen with o,p’- DDD.
Adrenocortical stereology
Compared to tumor-bearing control values, the adrenocortical cellular volume fraction (Table 1) was elevated after 7 days of AG treatment. The change was accompanied by a decrease in the nuclear and an increase in the cytoplasmic volume fractions, the latter being due to the significantly increased amount of lipid. After 14 days of chemotherapy, a similar pattern was observed. However, the cytoplas- mic volume fraction was only slightly greater than control due to a decreased mitochondrial volume fraction and a significant increase in lipid. In subsequent groups, the nuclear vol- ume fraction did not differ significantly from control values, but variation in the mitochon-
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| Experimental Groupª | Extra- cellular space and non- parenchymal cells | Necrotic tissue | Cellb | Cytoplasmc | Nucleus | Mito- chondria | Lipid | Lysosomal dense bodies | |
|---|---|---|---|---|---|---|---|---|---|
| 7 Days | |||||||||
| Adrenal | Control | 23.3 ± 1.4 | 0.3 ± 0.3 | 76.4 ± 1.5 | 61.9 ± 1.3 | 14.5 ± 1.4 | 33.8 ± 1.2 | 10.1 ± 1.3 | 0.4 ± 0.1 |
| AG | 11.0 ± 1.4 | 0 | 89.0 ± 1.5 | 82.1 ± 1.6 | 6.9 ± 1.8 | 32.9 ± 1.3NS | 28.3 ± 2.44 | 0.1 ± 0.04 | |
| o,p'-DDD | 10.7 ± 1.8 | 0.02 ± 0.02 | 89.3 ± 1.8 | 81.3 ± 1.4 | 8.0 ± 1.2 | 46.7 ± 1.54 | 12.8 ± 1.0NS | 0.3 ± 0.1 | |
| Tumor | Control | 11.3 ± 1.5 | 8.3 ± 1.8 | 80.4 ± 1.7 | 55.4 + 1.6 | 25.0 ± 2.1 | 10.5 ± 0.6 | 3.6 ± 1.0 | 0.6 ± 0.1 |
| AG | 13.0 ± 1.2 | 4.1 ± 1.5 | 82.9 ± 1.2 | 58.5 ± 1.8 | 24.4 ± 2.5 | 10.6 ± 0.7NS | 1.9 ± 0.4NS | 1.4 ± 0.3 | |
| o,p'-DDD | 14.6 ± 1.1 | 5.6 ± 1.9 | 79.8 ± 1.3 | 60.5 ± 1.8 | 19.3 ± 2.0 | 13.7 ± 0.83 | 3.4 ± 0.6NS | 0.7 ± 0.1 | |
| 14 Days | |||||||||
| Adrenal | Control | 21.5 ± 3.3 | 0.2 ± 0.2 | 78.3 ± 3.1 | 68.3 ± 2.6 | 10.0 ± 2.4 | 30.5 ± 3.4 | 6.5 ± 1.3 | 0.7 ± 0.2 |
| AG | 20.6 ± 3.3 | 0.5 ± 0.4 | 78.9 ± 2.1 | 72.8 ± 1.8 | 6.1 ± 1.4 | 20.2 ± 1.43 | 32.1 ± 2.6+ | 0.4 ± 0.1 | |
| o,p'-DDD | 6.6 ± 1.5 | 0 | 93.4 ± 1.5 | 85.5 ± 1.4 | 7.9 ± 1.4 | 47.2 ± 1.74 | 21.7 ± 1.84 | 0.3 ± 0.1 | |
| Tumor | Control | 8.6 ± 0.7 | 1.3 ± 0.6 | 90.1 ± 1.3 | 65.7 ± 1.2 | 22.4 ± 1.4 | 13.5 ± 0.6 | 2.5 ± 0.5 | 0.6 ± 0.1 |
| AG | 9.0 ± 0.8 | 0.6 ± 0.4 | 90.4 ± 1.1 | 62.6 ± 2.3 | 27.8 ± 2.4 | 14.5 ± 0.8NS | 2.0 ± 0.5NS | 1.4 ± 0.2 | |
| o,p'-DDD | 15.3 ± 1.2 | 5.0 ± 1.3 | 79.7 ± 1.1 | 59.8 ± 1.6 | 19.9 ± 1.7 | 12.5 ±0.7NS | 3.1 ± 0.6NS | 1.1 ± 0.1 | |
| 21 Days | |||||||||
| Adrenal | Control | 15.0 ± 2.0 | 0.1 ± 0.1 | 84.9 ± 2.0 | 72.8 ± 1.8 | 12.1 ± 2.0 | 39.0 ± 1.7 | 10.5 ± 1.4 | 0.6 ± 0.1 |
| AG | 17.2 ± 2.6 | 0.7 ± 0.5 | 82.1 ± 2.5 | 69.1 ± 2.3 | 13.0 ± 1.9 | 30.9 ± 1.84 | 9.1 ± 1.5NS | 0.5 ± 0.1 | |
| o,p'-DDD | 5.6 ± 1.1 | 0.3 ± 0.3 | 94.1 ± 1.2 | 87.8 ± 1.3 | 6.3 ± 1.4 | 56.8 ± 1.84 | 18.9 ± 1.84 | 0.7 ± 0.2 | |
| Tumor | Control | 7.5 ± 1.0 | 8.7 ± 2.3 | 83.8 ± 1.6 | 57.6 ± 1.8 | 26.2 ± 2.3 | 12.6 ± 0.6 | 3.6 ± 1.0 | 0.7 ± 0.1 |
| AG | 9.6 ± 1.3 | 5.3 ± 1.1 | 85.1 ± 1.3 | 59.2 ± 1.2 | 25.9 ± 1.2 | 12.1 ± 0.9NS | 5.6 ± 1.1NS | 1.2 ± 0.3 | |
| o,p'-DDD | 13.7 ± 1.5 | 4.1 ± 1.2 | 82.2 ± 1.6 | 61.2 ± 1.8 | 21.0 ± 2.0 | 13.8 ± 0.7NS | 7.2 ± 1.22 | 1.1 ± 0.2 | |
| 14/14 Days | |||||||||
| Adrenal | Control | 18.5 ± 3.0 | 1.3 ± 0.9 | 80.2 ± 1.9 | 70.0 ± 2.8 | 10.2 ± 1.8 | 30.6 ± 2.0 | 9.2 ± 1.5 | 1.3 ± 0.2 |
| AG | 8.4 ± 1.9 | 0 | 91.6 ± 2.0 | 83.9 ± 1.6 | 7.7 ± 1.5 | 31.9 ± 1.6NS | 21.1 ± 2.54 | 0.5 ± 0.2 | |
| o,p'-DDD | 24.4 ± 1.6 | 0 | 75.6 ± 1.4 | 63.3 ± 1.6 | 12.4 ± 1.6 | 29.5 ± 1.6NS | 17.7 ± 1.83 | 0.6 ± 0.1 | |
| Tumor | Control | 17.2 ± 1.1 | 1.2 ± 0.7 | 81.6 ± 1.6 | 57.8 ± 1.5 | 23.8 ± 1.7 | 13.7 ± 0.6 | 4.9 ± 1.0 | 0.3 ± 0.1 |
| AG | 17.1 ± 1.8 | 2.1 ± 1.0 | 82.2 ± 1.1 | 57.0 ± 2.0 | 23.8 ± 2.0 | 14.3 ± 0.8NS | 1.7 ± 0.63 | 0.3 ± 0.1 | |
| o,p'-DDD | 13.1 ± 1.0 | 2.0 ± 0.8 | 84.9 ± 1.0 | 60.0 ± 1.6 | 24.3 ± 1.6 | 15.1 ± 0.6NS | 1.9 ± 0.33 | 0.6 ± 0.1 | |
| 21/14 Days | |||||||||
| Adrenal | Control | 15.0 ± 2.0 | 0.5 ± 0.4 | 76.0 ± 1.7 | 84.5 ± 1.9 | 8.5 ± 1.6 | 40.2 ± 1.4 | 11.0 ± 1.2 | 0.7 ± 0.1 |
| AG | 20.3 ± 2.8 | 0 | 79.7 ± 2.5 | 69.7 ± 2.2 | 10.0 ± 2.0 | 29.7 ± 1.44 | 10.6 ± 0.9NS | 0.3 ± 0.1 | |
| o,p'-DDD | 15.8 ± 2.0 | 0.1 ± 0.1 | 84.1 ± 1.8 | 77.3 ± 1.5 | 6.8 ± 1.1 | 45.8 ± 1.82 | 14.4 ± 1.4NS | 0.2 ± 0.5 | |
| Tumor | Control | 13.5 ± 0.8 | 0.2 ± 0.2 | 86.3 ± 1.8 | 63.9 ± 1.2 | 22.4 ± 2.3 | 16.1 ± 0.9 | 5.3 ± 0.8 | 0.7 ± 0.8 |
| AG | 17.1 ± 1.8 | 2.1 ± 1.0 | 80.1 ± 1.4 | 55.7 ± 1.6 | 24.4 ± 2.0 | 13.4 ± 0.62 | 1.8 ± 0.44 | 0.9 ± 0.2 | |
| o,p'-DDD | 17.6 ± 1.5 | 7.2 ± 1.4 | 75.2 ± 1.5 | 54.5 ± 1.6 | 20.7 ± 1.7 | 11.0 ± 0.84 | 4.6 ± 0.9NS | 0.8 ± 0.1 |
a Days following initial tumor palpation or equivalent in normal.
” Cell = total volume-(extracellular + necrotic).
” Cytoplasm = cell-nucleus. Significance of difference from control: ’ p<0.05; 2 p<0.025; 3 p<0.01; + p<0.005; NS Not significant.
drial and lipid fractions caused fluctuations in the cytoplasmic volume. The mitochondrial volume fraction continued to be less than control after 21 and 21/14 days. In the 14/14 day group, however, the value returned to control levels. In contrast, the volume fraction of lipid was increased at 14/14 days and re- mained at control levels in the 21 and 21/14 day groups.
With o,p’-DDD the cell and cytoplasmic vol- ume fractions increased with treatment and decreased during the recovery periods. The majority of this change was accounted for by parallel changes in the mitochondrial volume fraction. The lipid volume fraction also in- creased during o,p’-DDD chemotherapy. The values remained significantly elevated in the 14/14 day group and returned to control levels in the 21/14 day group.
Tumor biochemistry
Cholesterol levels in tumors of animals treated with AG did not differ from controls in the 7 and 14 day groups but were signifi- cantly elevated after 21 days (Table 2). In recovery periods, cholesterol values were sig- nificantly less than controls in the 14/14 day group. Cholesterol esters showed the same trend during chemotherapy, with values re- maining elevated in the 21/14 day group. With o,p’-DDD treatment, cholesterol increased af- ter 7 days but remained near or below control values in the periods of longer duration. Cho- lesterol esters showed no significant changes during chemotherapy but were less than con- trols in the recovery groups. Tumor lipid phos- phate levels were not significantly altered by either drug.
Adrenocortical biochemistry
For the most part, AG had little effect on adrenal cholesterol. Cholesterol esters were significantly elevated at 14 days. o,p’-DDD also did not significantly change the choles- terol content of the adrenal glands and, in contrast to AG, did not markedly alter choles- terol ester levels. Lipid phosphate levels were slightly depressed in the 7- and 14-day AG- treated animals, but did not differ from control values in the other groups or in any of the o,p’-DDD-treated animals.
Plasma corticosterone
In normal animals AG significantly reduced plasma corticosterone values in the 14-day group (Table 3). This reduction was even greater in tumor-bearing animals and, when
coupled with the elevated plasma corticoster- one levels generally seen in these animals, made the effects of AG even more pronounced. In contrast, the only significant influence of o,p’-DDD treatment was in the 7-day tumor- bearing group, where the plasma corticoster- one value was significantly less than in the control.
DISCUSSION
While AG was relatively well tolerated by the animals, o, p’DDD was not, and for the first week animals were cachexic. From the growth data (Figs. 1, 2) it appears that neither AG nor o,p’-DDD had much affect on the size of the tumor. AG significantly reduced plasma corticosterone levels throughout treatment in tumor-bearing animals and at 14 days in nor- mal animals (Table 3), suggesting the drug reduced corticosteroidogenesis in both the tu- mor and adrenal. In contrast, o,p’-DDD did not significantly alter plasma corticosterone levels in the normal animals and only slightly reduced them in tumor-bearing animals.
Relative adrenal weights varied more di- rectly with administration of the two drugs, generally increasing during chemotherapy and then diminishing during recovery. The effects of AG were more rapidly overcome than were those of o,p’-DDD, quite possibly reflect- ing the fact that o,p’-DDD is stored in adipose tissue (Cueto and Brown, ‘58) and may be released over a longer period of time than is the water-soluble AG. It is important to note that while both drugs increased relative ad- renal weight, only AG significantly reduced plasma corticosterone levels, suggesting that the mechanism of action of these two drugs is different.
Adrenal
Fine structurally, cells of the adrenal cor- tices of AG- and o,p’-DDD-treated tumor-bear- ing rats reflected both the effects of the tumor and chemotherapy, such as tumor-induced mi- tochondrial elongation (Nickerson, “73), AG- induced vacuolation (Marek et al., ‘70), and o,p’-DDD-induced rings. When compared to normal animals, adrenal cortical fine struc- tural changes induced by AG appeared 7 days later in tumor-bearing animals, whereas no temporal differential appeared between o,p’- DDD-treated groups.
The changes in morphologically-defined ad- renal lipid in the AG groups were paralleled by the biochemical analyses, which showed alterations in cholesterol ester levels with
| Experimental Group | Cholesterol (C) | Cholesterol esters (CE) | Ratio C/CE | Lipid phosphate | |
|---|---|---|---|---|---|
| 7 Days Adrenal | |||||
| Control | 0.66 ± 0.09 | 12.42 ± 0.68 | 0.05 ± 0.01 | 0.22 ± 0.01 | |
| AG | 0.88 ± 0.21NS | 26.50 ± 4.18NS | 0.03 ± 0.01NS | 0.17 ± 0.0044 | |
| o,p'-DDD | 0.76 ± 0.08NS | 14.53 ± 2.49NS | 0.06 ± 0.01NS | 0.18 ± 0.02NS | |
| Tumor | Control | 0.23 ± 0.02 | 0.09 ± 0.04 | 4.5 ± 3.4 | 0.08 ± 0.01 |
| AG | 0.17 ± 0.02NS | 0.07 ± 0.02NS | 4.2 ± 1.5NS | 0.09 ± 0.01NS | |
| o,p'-DDD | 0.45 ± 0.021 | 0.10 ± 0.01NS | 4.7 ± 0.6NS | 0.07 ± 0.01N$ | |
| 14 Days | |||||
| Adrenal | Control | 0.86 ± 0.04 | 8.15 ± 0.38 | 0.11 ± 0.01 | 0.23 ± 0.01 |
| AG | 1.31 ± 0.191 | 48.84 ± 0.354 | 0.03 ± 0.0034 | 0.18 ± 0.0041 | |
| o,p'-DDD | 0.67 ± 0.01NS | 11.14 ± 1.85NS | 0.06 ± 0.01NS | 0.19 ± 0.01NS | |
| Tumor | Control | 0.37 ± 0.04 | 0.08 ± 0.01 | 4.9 ± 0.7 | 0.08 + 0.01 |
| AG | 0.38 ± 0.04NS | 0.09 ± 0.01NS | 4.4 ± 0.6NS | 0.08 ± 0.01NS | |
| o,p'-DDD | 0.24 ± 0.05NS | 0.12 ± 0.03NS | 3.4 ± 1.4NS | 0.05 ± 0.01NS | |
| 21 Days | |||||
| Adrenal | Control | 1.15 ± 0.25 | 12.10 ± 0.22 | 0.07 ± 0.01 | 0.21 ± 0.01 |
| AG | 1.06 ± 0.05NS | 18.46 ± 4.30NS | 0.07 ± 0.02NS | 0.22 ± 0.01NS | |
| o,p'-DDD | 0.82 ± 0.06NS | 9.34 ± 0.99NS | 0.10 ± 0.02NS | 0.15 ± 0.02NS | |
| Tumor | Control | 0.35 ± 0.02 | 0.14 ± 0.02 | 3.3 ± 0.8 | 0.06 ± 0.01 |
| AG | 0.65 ± 0.044 | 0.45 ± 0.044 | 1.6 ± 0.14 | 0.06 ± 0.01NS | |
| o,p'-DDD | 0.30 ± 0.06NS | 0.16 ± 0.02NS | 2.2 ± 0.7NS | 0.07 ± 0.01NS | |
| 14/14 | |||||
| Days | |||||
| Adrenal | Control | 1.04 ± 0.18 | 11.19 ± 0.07 | 0.10 ± 0.02 | 0.19 ± 0.01 |
| AG | 1.28 ± 0.15NS | 17.16 ± 2.35Ns | 0.08 ± 0.01NS | 0.18 ± 0.01NS | |
| o,p'-DDD | 0.53 + 0.18NS | 14.48 ± 1.99Ns | 0.12 ± 0.01NS | 0.21 ± 0.03NS | |
| Tumor | Control | 0.80 ± 0.12 | 0.45 ± 0.01 | 1.8 ± 0.4 | 0.04 ± 0.01 |
| AG | 0.56 ± 0.053 | 0.48 ± 0.05NS | 1.4 ± 0.2NS | 0.04 ± 0.01NS | |
| o,p'-DDD | 0.33 ± 0.064 | 0.22 ± 0.04NS | 1.6 ± 0.2NS | 0.08 ± 0.01NS | |
| 21/14 | |||||
| Days | |||||
| Adrenal | Control | 1.18 ± 0.28 | 6.60 ± 1.38 | 0.15 ± 0.06 | 0.19 ± 0.01 |
| AG | 0.40 ± 0.22NS | 14.47 ± 4.64NS | 0.03 ± 0.01' | 0.17 ± 0.01NS | |
| o,p'-DDD | 1.24 ± 0.23NS | 21.23 ± 2.551 | 0.06 ± 0.01NS | 0.20 ± 0.03NS | |
| Tumor | Control | 0.06 ± 0.04 | 0.52 ± 0.05 | 1.2 ± 0.1 | 0.05 ± 0.01 |
| AG | 0.49 ± 0.04NS | 0.74 ± 0.073 | 0.7 ± 0.12 | 0.04 ± 0.01NS | |
| o,p'-DDD | 0.62 ± 0.06NS | 0.22 ± 0.044 | 3.5 ± 1.31 | 0.08 ± 0.01NS |
Significance of difference from control:
‘p < 0.05; 2 p < 0.025; 3 p < 0.01; 4 p < 0.005; NS Not significant.
| Experimental Group | 7 Days | 14 Days | 21 Days | 14/14 Days | 21/14 Days |
|---|---|---|---|---|---|
| Normal | 9.2 + 2.4 | 4.1 ± 1.8 | 8.3 ± 2.6 | 6.8 ± 2.0 | 4.8 ± 0.8 |
| AG | 5.1 ± 1.0N$ | 0.5 ± 0.54 | 2.3 ± 1.6 NS | 6.4 ± 2.2NS | 2.3 ± 0.52 |
| o,p'-DDD | 10.7 ± 3.4NS | 9.2 ± 2.9NS | 10.3 ± 3.5N$ | 5.0 ± 0.7NS | 3.3 ± 0.9N$ |
| Tumor-bearing | 15.2 ± 4.4 | 12.4 ± 3.1 | 16.2 ± 6.0 | 6.6 ± 1.2 | 7.5 ± 0.5 |
| AG | 2.8 ± 0.82 | 1.7 ± 1.43 | 0.8 ± 0.43 | 1.1 ± 0.63 | 3.6 ± 1.2' |
| o,p'-DDD | 5.5 ± 0.93 | 6.0 ± 1.9NS | 10.1 ± 0.7N$ | 11.5 ± 0.6N8 | 9.0 ± 1.4N$ |
relatively little change in the cholesterol frac- tion, which is low compared to the adrenal gland (Table 2; Mason and Robidoux, ‘78). These results are consistent with the report of Raggatt et al. (‘72), who noted a three-fold cholesterol ester increase in the adrenocortices removed from a patient with Cushing’s syn- drome who was treated with AG.
The primary action of AG inhibits side chain cleavage of cholesterol to A5-pegnenolone (Cohen, ‘68), which could account for the ob- served rapid increase in total lipid volume and cholesterol esters, while the secondary effect on the intramitochondrial enzyme 118-hy- droxylase could contribute to the delayed loss of normal mitochondrial fine structure, both
observations confirmed by stereological data. The increased endogenous ACTH, secondary to reduced steroidogenesis, may be sufficient to overcome the cholesterol-45-pregnenolone inhibition, but not that of the 118-hydroxyla- tion. Such an interpretation would explain the temporal differences observed between mito- chondrial and lipid responses to AG in the adrenal.
Although Hart and Straw (‘71 a,b) have reported similar sites of action of o,p’-DDD to AG, the significantly rapid increase in the mitochondrial volume fraction and lack of significant changes in cholesterol esters and plasma corticosterone values suggest a differ- ent mechanism of action of the two drugs in the adrenal. The large lipid droplets observed in o,p’-DDD-treated adrenals probably con- sisted of free fatty acids or triglycerides, since both of these potential steroid precursors were increased in qualitative thin layer chromatog- raphy. Thus, o,p’-DDD, known to be seques- tered in fat deposits, may alter general lipid metabolism to a greater extent than does AG.
Tumor
There also appears to be a temporal delay in the effects of AG on the tumor as compared to the adrenal. While cholesterol esters in- creased significantly at 14 days in the adre- nals of tumor-bearing animals, cholesterol and cholesterol esters increased at 21 days in the tumor. Although the latter increases are sta- tistically significant, they are small in abso- lute magnitude and, in general, the biochem- ical findings parallel the stereological data which show that the lipid fraction remains essentially unchanged during treatment. The cause of the temporal delay is obscure, but most likely it is due to a diminished capability of the tumor to synthesize cholesterol esters in response to AG therapy. A less efficient cholesterol ester synthetase mechanism would impede the esterification and storage of cho- lesterol and require the tumor to synthesize glucocorticoids directly from plasma cholester- ol and/or cholesterol esters. Such a concept is consistent with the observations (Moore et al., ‘78) that the large tumor “lipid” droplets are not deposits of cholesterol esters, as found in the normal adrenal gland (Moses et al., ‘69).
When compared to the nontreated tumor (Moore et al., ‘78), AG- and o,p’-DDD-induced changes in tumor fine structure were subtle, the most prominent being a loss of mitochon- drial integrity. Since the tumor is relatively insensitive to ACTH (Ney et al., ‘69), and both
the primary and secondary sites of action of these drugs are on reactions catalyzed by intramitochondrial enzymes (Stachenko and Giround, ‘64), it appears that AG and o,p’- DDD may have a direct influence on tumor mitochondria. This is further substantiated by the fact that the fine structural changes in the mitochondria of adrenocortical cells are more extensive and more characteristic of the drug than of diminished amounts of ACTH, as de- scribed for adrenocortices of hypophysectom- ized mice (Fujita, ‘72).
From the various parameters studied, it appears that the effects of AG and o,p’-DDD are more positively manifested in the hosts’ adrenal glands than in the tumor. This is most likely due to the interference by the drug of trophic and steroidogenic regulation by ACTH, which is generally lacking in the tu- mor. However, the drugs evoke some impair- ment of tumor structure and function and appear to have potential usefulness as con- stituents of a balanced chemotherapeutic re- gime, as has been reported by Child et al. (‘76).
ACKNOWLEDGMENTS
The authors are indebted to Dr. Robert L. Ney, University of North Carolina, for supply- ing the adrenocortical carcinoma, to Dr. J. J. Chart, Ciba Pharmaceutical, for the aminog- lutethimide, to Dr. Guido V. Marinetti, Univ- ersty of Rochester, for assistance with the biochemical determinations, and to Mrs. Su- san Walker for sectioning the tissues for elec- tron microscopy.
This work was supported by U.S.P.H.S. Grants CA 11198 and DE-00003, and Ameri- can Cancer Society Grant IN-18N.
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