Exp Toxic PathoI1992; 44: 81-85 Gustav Fischer Verlag lena
Poznan Academy of Medicine, Department of Histology and Embryology, Poznan, Poland
Comparative studies on the effects of amino glutethimide on hamster and rat adrenal cortex By L. K. MALENDOWICZ Received: April 11, 1990; Accepted: August 1, 1990
Address for correspondence: Dr. LUDWIK K. MALENDOWICZ, Poznan Academy of Medicine, Department of Histology and Embryology, 6 Swiecicki Str., PL-60-781 Poznan, Poland Key words: aminoglutethimide; adrenal cortex; stereology; ACTH; cortisol; corticosterone
Summary
Adult female , intact or steroid suppressed hamsters were treated twice daily with 7 mg aminoglutethimide phosphate (AG) for 5 days while intact female rats received 14mg AG per injection. AG resulted in an increase in adrenal gland weight of both hamster and rat. In the hamster AG had no effect on the amount of lipid droplets while in the rat a slightly higher number of fine lipid vacuoles was seen. In the hamster, enlargement of the gland was due to hyperplasia of the zona reticularis cells while in the rat the number of glomerulosa and fasciculata cells increased. AG had no effect on the adrenal cortex of steroid suppressed hamsters. The serum cortisol level was markedly higher in AG-treated hamsters while the corticosterone level was notably lower in AG-administered rats. In both hamsters and rats, AG-treatment did not change the serum ACTH level. Thus the study demonstrated different responses of the hamster and rat adrenal cortex to AG.
Introduction Aminoglutethimide (a:-ethyl-a:-p-aminopheny l-glutarimide ; AG) is a well known drug inhibiting adrenal cholesterol desmolase activity (DEXTER et al. 1967; COHEN 1968). Due to the inhibition of the cole sterol side-chain cleavage activity, AG administration leads to the accumulation of cholesterol and its esters in adrenocortical cells, thus resulting in steatotic degeneration of parenchymal cells (RACELA et al. 1969; MAREK et al. 1970; STARKA and MOTLIK 1971; ITOH 1971, 1978; MALENDOWICZ 1972a, b, c, 1973; YARRINGTON 1983; ZAK 1983). Most of these studies employed rats, similar alterations, however, were also found in AG-treated birds (HOLMES and PHILLIPS 1976). On the other hand, comparative studies of KADIOGLU AND HARRISON 1975) demonstrated that in the Mongolian gerbil (Meriones unguiculatus) the adrenocortical cells showed less marked alterations after AG administration than those of the rat. The adrenal cortex of the hamster differs markedly from the rat adrenal gland. The hamster adrenal gland is almost com-
pletely devoid of lipid droplets and stored cholesterol esters and it secretes cortisol as a principal glucocorticoid. In this species biosynthesis of steroid hormones mainly depends on the intraadrenal synthesis of cholesterol from small molecules of the substrate, and the cortisol secretion rate is lower than in other mammals (LEHOUX and LEFEBVRE 1980); JANSEN and BIRKENHAGEN 1981; MALENDOWICZ and NUSSDORFER 1984; SPADY and DIETSCHY 1985; Iw AKI et al. 1985). Therefore the aim of the study was to compare, by means of stereology, the reactivity of the adrenal cOltex of the hamster and the rat to prolonged administration of small doses of AG.
Materials and Methods Adult female hamsters (Mesocricetus auratus Waterhouse) and rats of the Wi star strain were employed in this study. Animals were maintained under standardized conditions of light (14L: lOD) and temperature (22 ± 2°C) and fed with laboratory pellets with free access to tap water. Aminoglutethimide phosphate (AG, Elipten, Ciba) was dissolved in 0.025 M citrate buffer (pH 4.0; 70mg/ml). Hamsters were administered twice daily with 7 mg AG for 5 days while rats received 14mg AG per injection. Control animals were treated with solvent only. The dose of AG was chosen according to ROBBA et al. (1987). One group of hamsters was treated s.c. daily with 0.25mg cortisone acetate (Upjohn). After weighing, animals were decapitated (12h after the last injection) and trunk blood collected (with I mg NaF/ml). The adrenals were promptly removed, cleaned of adherent adipose tissue and weighed.
Stereology The left adrenal gland was fixed for 24h in Bouin's fluid at 22 ± 1°C, embedded in paraffin wax and serially sectioned at a thickness of 5-6I-lm. Sections were stained with haematoxylin and eosin. The sections were analysed by differential point counting as described by WEIBEL (1979). In the first stage of analysis, using magnification of approximately x 100 and a simple square lattice test system of type A Exp Toxic Pathol 44 (1992) 2
81
variation 4 %. Corticosterone (in the rat) was measured by sulfuric acid fluorescence (GUILLEMIN et al. 1959).
(WEIBEL 1979), the volume fractions of the individual adrenocortical zones and of the medulla and capsula were estimated. The analysis was performed on each 5th section of the gland. In the second stage, the volume fractions of parenchymal cell nuclei, of cytoplasm and of connective tissue together with blood vessels (stroma) were estimated, and the number of nuclear profiles of adrenocortical cells per unit area of section counted on a screen at a final magnification of x 3,000. Detailed description of these methods and of subsequent calculations have been described (NIKICICZ et al. 1984 ; MALENDOWICZ 1987).
Statistical treatment of the results. The data obtained from each animal were averaged per experimental group, and the standard error was calculated. Comparison among the experimental groups was performed by Student's t-test.
Results AG administration resulted in an increase in adrenal gland weight of both hamster and rat (tables 1 and 3). The hamster adrenal cortex only sporadically contained lipid droplets, and AG had no effect on the amount of visible vacuoles. On the other hand, in the zona fasciculata cells of AG treated rats a slightly higher number of fine lipid droplets was seen. In the hamster AG evoked an enlargement of the zona reticularis (ZR) while in the rat adrenal cortex glomerulosa (ZG) and fasciculata (ZF) zones were larger than in controls. In the hamster adrenal cortex AG decreased the average volume of the ZR cells and increased the number of ZR cells and the number of parenchymal cells in the entire gland. On the other hand in the rat, AG had no effect on the average volume of parenchymal cells in the gland while the number of adrenocortical cells increased in the ZG and ZF, and in the entire gland. AG had no effect on the adrenal cortex of steroid suppressed hamsters (table 2).
Hormonal assays Serum was separated and stored at - 20 DC until hormonal assays. ACTH was estimated, in unextracted serum by the means of RIA using the commercial kit "RIA-mat ACTH" (Mallinckrodt Diagnostica). As stated by manufacturer, antibodies against ACTH react with human ACTH (both 1-24 fragment and a whole molecule, cross-reaction 100 %) while there is no cross-reaction with exMSH, l3-endorphin , 13-lipotropin, and other tropic honnones of the adenohypophysis. The intra-assay variance for ACTH was 5 %, while the inter-assay variance was 7 % . Cortisol was quantitated in un extracted hamster serum by RIA with RIA cortisol (1 25 1) kit of Fannos Diagnostica (Finland). With that kit cross-reactions with progesterone and corticosterone were respectively < 0.01 and
Poznan Academy of Medicine, Department of Histology and Embryology, Poznan, Poland
Comparative studies on the effects of amino glutethimide on hamster and rat adrenal cortex By L. K. MALENDOWICZ Received: April 11, 1990; Accepted: August 1, 1990
Address for correspondence: Dr. LUDWIK K. MALENDOWICZ, Poznan Academy of Medicine, Department of Histology and Embryology, 6 Swiecicki Str., PL-60-781 Poznan, Poland Key words: aminoglutethimide; adrenal cortex; stereology; ACTH; cortisol; corticosterone
Summary
Adult female , intact or steroid suppressed hamsters were treated twice daily with 7 mg aminoglutethimide phosphate (AG) for 5 days while intact female rats received 14mg AG per injection. AG resulted in an increase in adrenal gland weight of both hamster and rat. In the hamster AG had no effect on the amount of lipid droplets while in the rat a slightly higher number of fine lipid vacuoles was seen. In the hamster, enlargement of the gland was due to hyperplasia of the zona reticularis cells while in the rat the number of glomerulosa and fasciculata cells increased. AG had no effect on the adrenal cortex of steroid suppressed hamsters. The serum cortisol level was markedly higher in AG-treated hamsters while the corticosterone level was notably lower in AG-administered rats. In both hamsters and rats, AG-treatment did not change the serum ACTH level. Thus the study demonstrated different responses of the hamster and rat adrenal cortex to AG.
Introduction Aminoglutethimide (a:-ethyl-a:-p-aminopheny l-glutarimide ; AG) is a well known drug inhibiting adrenal cholesterol desmolase activity (DEXTER et al. 1967; COHEN 1968). Due to the inhibition of the cole sterol side-chain cleavage activity, AG administration leads to the accumulation of cholesterol and its esters in adrenocortical cells, thus resulting in steatotic degeneration of parenchymal cells (RACELA et al. 1969; MAREK et al. 1970; STARKA and MOTLIK 1971; ITOH 1971, 1978; MALENDOWICZ 1972a, b, c, 1973; YARRINGTON 1983; ZAK 1983). Most of these studies employed rats, similar alterations, however, were also found in AG-treated birds (HOLMES and PHILLIPS 1976). On the other hand, comparative studies of KADIOGLU AND HARRISON 1975) demonstrated that in the Mongolian gerbil (Meriones unguiculatus) the adrenocortical cells showed less marked alterations after AG administration than those of the rat. The adrenal cortex of the hamster differs markedly from the rat adrenal gland. The hamster adrenal gland is almost com-
pletely devoid of lipid droplets and stored cholesterol esters and it secretes cortisol as a principal glucocorticoid. In this species biosynthesis of steroid hormones mainly depends on the intraadrenal synthesis of cholesterol from small molecules of the substrate, and the cortisol secretion rate is lower than in other mammals (LEHOUX and LEFEBVRE 1980); JANSEN and BIRKENHAGEN 1981; MALENDOWICZ and NUSSDORFER 1984; SPADY and DIETSCHY 1985; Iw AKI et al. 1985). Therefore the aim of the study was to compare, by means of stereology, the reactivity of the adrenal cOltex of the hamster and the rat to prolonged administration of small doses of AG.
Materials and Methods Adult female hamsters (Mesocricetus auratus Waterhouse) and rats of the Wi star strain were employed in this study. Animals were maintained under standardized conditions of light (14L: lOD) and temperature (22 ± 2°C) and fed with laboratory pellets with free access to tap water. Aminoglutethimide phosphate (AG, Elipten, Ciba) was dissolved in 0.025 M citrate buffer (pH 4.0; 70mg/ml). Hamsters were administered twice daily with 7 mg AG for 5 days while rats received 14mg AG per injection. Control animals were treated with solvent only. The dose of AG was chosen according to ROBBA et al. (1987). One group of hamsters was treated s.c. daily with 0.25mg cortisone acetate (Upjohn). After weighing, animals were decapitated (12h after the last injection) and trunk blood collected (with I mg NaF/ml). The adrenals were promptly removed, cleaned of adherent adipose tissue and weighed.
Stereology The left adrenal gland was fixed for 24h in Bouin's fluid at 22 ± 1°C, embedded in paraffin wax and serially sectioned at a thickness of 5-6I-lm. Sections were stained with haematoxylin and eosin. The sections were analysed by differential point counting as described by WEIBEL (1979). In the first stage of analysis, using magnification of approximately x 100 and a simple square lattice test system of type A Exp Toxic Pathol 44 (1992) 2
81
variation 4 %. Corticosterone (in the rat) was measured by sulfuric acid fluorescence (GUILLEMIN et al. 1959).
(WEIBEL 1979), the volume fractions of the individual adrenocortical zones and of the medulla and capsula were estimated. The analysis was performed on each 5th section of the gland. In the second stage, the volume fractions of parenchymal cell nuclei, of cytoplasm and of connective tissue together with blood vessels (stroma) were estimated, and the number of nuclear profiles of adrenocortical cells per unit area of section counted on a screen at a final magnification of x 3,000. Detailed description of these methods and of subsequent calculations have been described (NIKICICZ et al. 1984 ; MALENDOWICZ 1987).
Statistical treatment of the results. The data obtained from each animal were averaged per experimental group, and the standard error was calculated. Comparison among the experimental groups was performed by Student's t-test.
Results AG administration resulted in an increase in adrenal gland weight of both hamster and rat (tables 1 and 3). The hamster adrenal cortex only sporadically contained lipid droplets, and AG had no effect on the amount of visible vacuoles. On the other hand, in the zona fasciculata cells of AG treated rats a slightly higher number of fine lipid droplets was seen. In the hamster AG evoked an enlargement of the zona reticularis (ZR) while in the rat adrenal cortex glomerulosa (ZG) and fasciculata (ZF) zones were larger than in controls. In the hamster adrenal cortex AG decreased the average volume of the ZR cells and increased the number of ZR cells and the number of parenchymal cells in the entire gland. On the other hand in the rat, AG had no effect on the average volume of parenchymal cells in the gland while the number of adrenocortical cells increased in the ZG and ZF, and in the entire gland. AG had no effect on the adrenal cortex of steroid suppressed hamsters (table 2).
Hormonal assays Serum was separated and stored at - 20 DC until hormonal assays. ACTH was estimated, in unextracted serum by the means of RIA using the commercial kit "RIA-mat ACTH" (Mallinckrodt Diagnostica). As stated by manufacturer, antibodies against ACTH react with human ACTH (both 1-24 fragment and a whole molecule, cross-reaction 100 %) while there is no cross-reaction with exMSH, l3-endorphin , 13-lipotropin, and other tropic honnones of the adenohypophysis. The intra-assay variance for ACTH was 5 %, while the inter-assay variance was 7 % . Cortisol was quantitated in un extracted hamster serum by RIA with RIA cortisol (1 25 1) kit of Fannos Diagnostica (Finland). With that kit cross-reactions with progesterone and corticosterone were respectively < 0.01 and
