Exp Toxic Pathol 1992; 44: 134-137 Gustav Fischer Verlag lena
Department of Histology and Embryology, Poznan Academy of Medicine, Poznan, Poland
Comparative studies on the effect of 4-APP(4-aminopyrazolopyrimidine) on hamster and rat adrenal cortex L. K. MALENDOWICZ
With 3 tables Received: September 10, 1990; Accepted: October 22, 1990
Address for correspondence: Prof. Dr. L. K. MALENDOWICZ, Department of Histology and Embryology, Poznan Academy of Medicine, 6
Swi~cicki
Str., PL- 60- 781 Poznan, Poland
Key words: adrenal cortex; rat; hamster; 4-APP (4-aminopyrazolopyrimidine); stereology
Summary
The study aimed to compare, by means of stereologic methods, the reactivity of the adrenal cortex of the hamster and the rat to prolonged treatment with 4-aminopyrazolo-(3,4d)pyrimidine (4-APP), a drug reducing hepatic secretion of plasma lipoprotein. Adult female hamsters, intact or cortisone suppressed, were administered i.p. daily with 0.5 mg 4-APP for 5 days while intact female rats received 1 mg of the drug per dose. 4-APP resulted in a loss of body weight, with the more profound effect in the hamster. In the rat 4-APP did not change the adrenal gland weight, the volume of the adrenocortical zones, the average volume of adrenocortical cells and the number of parenchymal cells in the gland. Moreover, serum ACTH and corticosterone levels in the rat remained unchanged. In the intact hamster 4-APP decreased the adrenal gland weight, the volume of the zona fasciculata and enhanced the serum cortisol level. In steroid suppressed hamsters 4-APP lowered the adrenal weight, the volume of fasciculata and reticularis zones, the average volume of the fasciculata cells and the number of parenchymal cells in the gland. These findings may suggest the direct inhibitory effect of low 4-APP doses on the hamster adrenal cortex and clearly demonstrate higher susceptibility of the hamster adrenal cortex, if compared with the rat, to 4-aminopyrazolo-pyrimidine.
Introduction Adrenal steroid hormone synthesis depends upon a continuous supply of free cholesterol derived either from plasma lipoprotein or from biosynthesis de novo. Which mechanism of cholesterol utilization dominates is dependent on the species as well as on experimental conditions applied. In most species, among them also in the hamster, there is preference for LDL as the source of extracellular cholesterol while in ACTH treated rat ca 80% of adrenal cholesterol is derived from HDL (GWYNNE et a1. 1976; BALASUBRAMANIAM et a1. 1977; ANDERSEN and DIETSCHY 1978; BROWN et a1. 1979; GWYNNE and HESS 1980; KOVANEN et a1. 1980; LEHOUX and PREISS 1980). In the 134
Exp. Toxic. Pathol. 44 (1992) 3
hamster, the adrenal cortex is almost completely devoid of lipid droplets and stored cholesterol esters and the synthesis of corticosteroids depends mainly on intraadrenal synthesis of cholesterol from small molecules of substrate (LEHOUX and LEFEBVRE 1980; JANSEN and BIRKENHAGEN 1981; MALENDOWICZ and NUSSDORFER 1984; SPADY and DIETSCHY 1985; IWAKI et a!. 1985). The adenine, analogue 4-aminopyrazolo-(3,4d)pyrimidine (4-APP) reduces the hepatic secretion of plasma lipoprotein in rats thus resulting in a marked reduction of plasma and adrenal cholesterol content (SCHIFF et a1. 1971; BALASUBRAMANIAM et a1. 1977; BRECHER and HYUN 1976; MALENDOWICZ et a1. 1987). In 4-APP-induced lipoprotein deficient rats the rate of intraadrenal cholesterol synthesis increases and this effect is connected with impaired steroidogenesis (ANDERSEN and DIETSCHY 1976; BRECHER and HYUN 1978; GWYNN and HESS 1980; SZABO et a1. 1980, 1984; MALENDOWICZ et a1. 1987). As far as structure and steroidogenesis are concerned, the adrenal cOltex of the hamster differs markedly from the rat adrenal gland (for details see MALENDOWICZ and NUSSDORFER 1984; NUSSDORFER 1986). For example, the reactivity of the rat and hamster adrenal cortex to estrogens, which are known to be also a hypocholesterolemic agents, shows marked species differences (GASKIN and KITAY 1970, 1971; MALENDOWICZ et a1. 1982a, b; POPPLEWELL and AZHAR 1987). The aim of the present study was to compare, by means .of stereo logic methods, the reactivity of the adrenal cortex of the hamster and the rat to prolonged treatment with 4-APP, a cholesterol lowering agent.
Materials and Methods , Adult female hamsters (M esocricetus auratus WATERHOUSE) and rats of the Wi star strain were employed in this study. The animals were maintained under standardized conditions of light (14 L: 10 D) and temperature (22 ± 2°C) and fed with laboratory pellets with free access to tap water. 4-APP [4-aminopyrazolo(3,4-d)pyrymidine, Sigma] was
dissolved ex tempore in physiologic solution of saline (adjusted to pH 2.-5) at a concentration I mg/mI. The hamsters were administered i.p. daily with 0.5 mg 4-APP for 5 days while the rats received 1 mg of the drug per dose. The control animals were treated with the solvent only. One group of hamsters was treated s.c. daily with 0.25 mg of cortisone acetate (Upjohn). After weighing, the animals were decapitated (24 h after the last injection) and trunk blood collected (with 1 mg NaF/ml). The adrenals were promptly removed, cleaned of adherent adipose tissue and weighed. Stereology. The left adrenal gland was fixed for 24 h in Bouin's fluid at 22 ± 1°C, embedded in paraffin wax and serially sectioned at a thickness of 5-6 11m. The 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 (WEIBEL 1979), the volume fractions of the individual adrenocortical zones and of the medulla and capsula were estimated. The analysis was pelformed on each fifth 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 (MALENDOWICZ 1987; NIKICICZ et aI. 1984). Hormonal assays. Serum was separated and stored at -20 °C 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, crossreaction 100 %) while there is no cross-reaction with cxMSH , ~ endorphin, ~-lipotropin, and other tropic hormones of the adenohypophysis. The intra-assay variance for ACTH was 5 %, while the inter-assay variance was 7 %. Cortisol was quantitated in unextracted hamster serum by RIA with RIA cortisol ct 25 I) kit of Farmos Diagnostica (Finland) . With that kit cross-reactions with progesterone and corticosterone were respectively < 0.01 and < 2% and interassay variation 4 %. Corticosterone (in the rat) was measured by sulfuric acid fluorescence (GUILLEMIN et aI. 1959). 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 the Student's t-test.
Results 4-APP administration resulted in a loss of body weight in hamsters and rats (tables 1 and 3). In the hamster 4-APP decreased the adrenal gland weight while there was no change in the weight of the rat adrenal gland. 4-APP administration did not change the volume. of adrenocortical zones, the average volume of the adrenocortical cells, and the humber of parenchymal cells in the gland. Only in 4-APP-treated hamsters the volume of the zona fasciculata was lower than in the controls.
Table 1. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female hamster. Results expressed as means ± SE. Control n Body weight (g) Adrenal weight (mg)
7 94 ± 3 16.2 ± 0.8
4-APP 7 64± Id 13 .6±0.4b
Volume of the zones (mrn 3) ZG 2.169±0.173 ZF 6.090 ± 0.231 ZR 4.707 ± 0.336
2.238 ± 0.063 4 .732 ± 0.200° 4 .045 ± 0.165
Volume of cell (11m3) ZG ZF ZR
1,017±42 2,757±173 879 ±25
1,071 ± 27 2,420 ± 143 1,0l8±60
2,115± 197 2,038± 135 4,405 ± 393 8,897 ± 590
2,049± 62 1,866± 123 3,717 ± 227 7,633 ±279
361 ±72 1O.5±1.9
461 ±48 23.5±3.6c
N umber of cells (1 ZG ZF ZR Total Blood level ACTH (pmol/I) cortisol (nmoll!)
X
10 3)
Statistical evaluation of the results by Student's t-test: a - p < 0.05; b - P < 0.02; c - p < 0.01; d - p < 0.001. ZG - zona glomerulosa , ZF - zona fasciculata, ZR - zona reticularis. The serum cortisol level was markedly elevated in 4-APPtreated hamsters while in the rat the level of corticosterone was unchanged. 4-APP treatment did not change the serum ACTH level either in the hamster or in the rat. In steroid suppressed hamsters 4-APP lowered the adrenal weight, the volume of the fasciculata and reticularis zones, the average volume of the fasciculata cells and the number of zona reticularis cells as well as the total number of parenchymal cells in the gland (table 2). On the other hand, the average volume of the zona glomerulasa cells of 4-APP-treated rats was higher than in the controls while the serum ACTH concentration remained unchanged.
Discussion 4-APP, an agent blocking lipoprotein release from the liver and thus reducing the circulating level of lipoprotein cholesterol, has been frequently employed to study the contribution of circulating cholesterol to steroidogenesis in the adrenals. Doses of 4-APP administered into rats varied from 5 mg/lOO g b.wt. in 2 - 3 days experiments (BALASUBRAMANIAM et aI. 1976 ; BROWN et a1. 1979 ; SZABO et aI. 1980; BLANK et a1. 1983; MIKAMI et aI. 1984) to 2 or 1 mg (MALENDOWICZ et aI. 1987 ; NOWAK et aI. 1987; POPPLEWELL and AZHAR 1987). However, daily administration of 0 .5 mg 4-APP per 100 g b.wt. for 3-7 days also resulted in the profound reduction of the serum cholesterol level in the rat and notably disturbed pituitary hormones release (BLANK et al. 1983; MURPHY et al. 1985; MAZZOCCHI et a1. 1986). Regarding the potent toxicity of that Exp. Toxic. Pathol. 44 (1992) 3
135
Table 2. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female, steroid hormone suppressed hamster. Results expressed as means±SE. Cortisone
Cortisone + 4-APP
7 82±4 16.4 ± l.0
7 77±4 13.3 ±0.6a
Volume of the zones (mm 3 ) ZG ZF ZR
2.423 ± 0.190 6.220 ± 0.599 4.813 ± 0.322
2.212 ± 0.086 4.282±0.184c 3.481 ±0.158 c
Volume of cell ([Am 3 ) ZG ZF ZR
896± 38 2,188±165 938±55
1,075 ± 51 b 1,694± 151 a 929±43
2,663 ± 220 2,768±298 4,756±280 1O,187±438
2,612± 143 2,460± 232 3,516± 197 c 8,038±471 c
n Body weight (g) Adrenal weight (mg)
Number of cells (1 ZG ZF ZR Total
X
103)
Blood level of ACTH (pmolll)
444± 63
922± 267
Explanations as in Table I
Table 3. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female rat. Results expressed as means ± SE. Control n
Body weight (g) Adrenal weight (mg)
6 223±8 53.9±1.4
4-APP 6
196 ± 6a 54.0±3.4
Volume of the zones (mm 3 ) 6.520 ± 0.301 7.111 ± 0.431 ZG 25.009 ± 0.846 23.272 ± l.962 ZF 16.767±0.756 17.919± 1.299 ZR Volume of cell ([Am 3 ) ZG ZF ZR Number of cells (1 x 103 ) ZG ZF ZR Total Blood level ACTH (pmolll) corticosterone ([Agll)
929±43 3,562±225 825 ±44
7,058±291 7,208 ± 518 6,738±496 6,874±495 19,524± 1,673 18,644± 1,578 33,456 ± 2,222 32,590 ± 2,207 74.0±23.3 2.30±0.30
Explanations as in Table I
136
971 ± 43 3,332±299 909±49
Exp. Toxic. Pathol. 44 (1992) 3
88.3 ± 20.4 2.20 ± 0.70
adenine analogue, in the present experiments hamsters and rats were administered with relatively low doses of 4-APP. As revealed in the present study 4-APP markedly reduced the body weight in both species with more profound effect in the hamster. This may be a manifestation of the general toxic effect of the drug and it is of interest that cortisone administration prevented 4-APP-induced body weight loss in the hamster. 4-APP administered in higher doses markedly increased the adrenal gland weight in the rat, an effect depending on enlargement of the cortex (SZABO et al. 1980; MALENDOWICZ et al. 1987; NOWAK et al. 1987). This increase was due to hypertrophy and hyperplasia of parenchymal cells. In the present study low doses of 4-APP within 5 days did not change the adrenal gland weight in the rat while in intact or cortisone suppressed hamsters the adrenal weight was notably lower than in the controls. Moreover, zonal and cellular composition of the adrenal cortex was unchanged in 4-APP-treated rats while in the hamster a marked reduction in the volume of the zona fasciculata was found. A more profound effect of 4-APP was observed in cortisone suppressed hamsters in which the drug resulted in the lowering ofthe volume of the fasciculata and reticularis zones, of the size of the fasciculata cells and of the number of parenchymal cells in the gland. These findings may suggest the direct inhibitory effect of 4-APP on the hamster adrenal cortex, however, in the intact hamsters this effect may be partially obliterated by compensatory hepersecretion of ACTH by the nonsuppressed pituitary gland. Such a direct effect of 4-APP on the rat adrenal cortex has been suggested also in our earlier study (Now AK et al. 1987). The results of hormonal estimations do not exclude the possibility of compensatory hypersecretion of ACTH in 4-APP-treated hamsters. In this species basal plasma ACTH levels are very high if compared with other mammals and the values differ markedly among individuals (NOWAK et al. 1990). Therefore it is very difficult to reveal any statistically significant differences in the plasma ACTH level among the groups of experimental hamsters. Moreover, in the present experiment animals were sacrificed 24 h after 4-APP administration and such a one-point sampling time may be insufficient to reveal a possible short-lasting enhanced corticotropin level in the blood. Moreover, increased plasma cortisol levels in 4-APP-treated hamsters strongly suggested an increased stimulation of the hamster adrenal cortex by ACTH, although the impaired inactivation of cortisol by injury, due to 4-APP hepatotoxicity, can not be excluded. If this is the case, the enhanced, due to impaired inactivation, plasma cortisol level may suppress pituitary ACTH secretion and may induce partial atrophy of the adrenal cortex. Contrary to the hamster, in the rat comparable doses of 4-APP had no effect either on the serum ACTH level or on the serum corticosterone concentration. Thus, if compared with the rat, our experiments clearly demonstrate a higher susceptibility of the hamster adrenal cortex to 4-APP.
Acknowledgement The study was supported in part by a grant X-IS from the Poznan Academy of Medicine, Poznan, Poland.
References ANDERSEN JM, DIETSCHY JM: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary and testis of the rat. J Bioi Chern 1978; 253: 9024-9032.
BALASUBRAMANIAM S, GOLDSTEIN JL, FAUST JR, et al.: Lipoproteinmediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J BioI Chern 1977; 252: 1771 1779, BLANK MS, LOUMAYE E, SGOUTAS DS, et aL: Inhibition of pituitary gonadotropin secretion by 4-aminopyrazolo/3,4-d!pyrimidine. Endocrinology 1983; 112: 1973-1979. BRECHER PI, HYUN Y: Effect of 4-amino-pyrazolo-pyrimidine and aminoglutethimide on cholesteryl metabolism and steroidogenesis in the rat adrenal. Endocrinology 1978; 102: 1404-1413. BROWN MS, KOVANEN PT, GOLDSTEIN JL: Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Rec Progr Horm Res 1979; 35: 215-249. GASKIN JH, KlTAY, JI: Adrenocortical function in the hamster: Sex differences and effects of gonadal hormones. Endocrinology 1970; 87: 779-786. - Hypothalamic and pituitary regulation of adrenocortical function in the hamster. Effects of gonadectomy and gonadal hormone replacement. Endocrinology 1971; 89: 1047- 1059. GUILLEMIN R, CLAYTON GW, SMITH JD et al.: Measurement of free corticosteroids in rat plasma: Physiological validation of a method. Endocrinology 1959; 63: 349-358. GWYNNE JT, HESS B: The role of high-density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis. J BioI Chern 1980; 255: 10875-10883. MAHAFEE D, BREWER HB jr. et al.: Adrenal cholesterol uptake from plasma lipoproteins: Regulation by corticotrophin. Proc Natl Acad Sci USA 1976; 72: 4329-4333. IWAKI T, NOGUCHI A, SEKIMOTO T: Sources of extramitochondrial corticoidogenic cholesterol in the adrenal cortex. Jap J Pharmacol 1985; 38: 207-214. JANSEN H, BIRKENHAGEN JC: Liver-lipase-like activity in human and hamster adrenocortical tissues. Metabolism 1981; 30: 428-430. KOVANEN PT, GOLDSTEIN -JL, CHAPPEL DA, et al.: Regulation of lowdensity lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo. J BioI Chern 1980; 255: 5591-5598. LEHOUX JG, LEFEBVRE, A: De novo synthesis of corticosteroids in hamster adrenal glands. J Steroid Biochem 1980; U: 479-485. - PREISS A: Regulation of hamster adrenal 3-hydroxy-3-methylglutaryl coenzyme a reductase activity. Endocrinology 1980; 107: 215-219. MALENDOWICZ, LK: Sex differences in adrenocortical structure and function. XXIV. Comparative morphometric studies on adrenal cortex of intact mature male and female rats of different strains. Cell Tissue Res 1987; 249: 443-449. NUSSDORFER, GG: Sex differences in adrenocortical structure and function. XV . Cellular composition and quantitative ultrastructural study of adrenal cortex of adult quantitative ultrastructural study of adrenal cortex of adult male and female hamster. J Submicrosc Cytol 1984; 16: 715-720. NIKICICZ H. KASPRZAK A: Sex differences in adrenocortical structure and function. VIII. Karyometric and stereo logic studies on adrenals
of intact adult male and female hamsters. Z mikr-anat Forsch 1982a; 96: 81-90. KASPRZAK A, NIKlCICZ H: Sex differences in adrenocortical structure and function. IX. Stereologic studies on the effects of gonadectomy and testosterone or estradiol replacement on adrenal cortex of adult male and female hamsters. Z mikr-anat Forsch 1982 b; 96: 91 - 102. NOWAK M, PALUSZAK J, et al. Adrenocortical function in 4-APP treated rats: A coupled stereological and biochemical study. J Steroid Biochem 1987; 26: 487-492. MAZZOCCHI G, ROBBA C, MENEGHELLI V, et al.: Long term effects of the hypocholesterolaemic drug 4-aminopyrazolopyrimidine on the zona fasciculata of the rat adrenal cortex. J Anat 1986; 149: 1-9. MIKAMI K, NISHIKAWA T, SAITO Y, et al.: Regulation of cholesterol metabolism in rat adrenal glands: Effects of adrenocorticotropin, cholesterol, and corticosteroids on acyl-coenzyme A synthetase and cholesterol ester hydrolases. Endocrinology 1984; 114: 136-140. MURPHY BD, RAJ KUMAR K, McKIBBIN PE, et al.: The effects of hypophysectomy and administration of pituitary hormones on luteal function and uptake of high density lipoproteins by luteinized ovaries and adrenals of the rat. Endocrinology 1985; 116: 1587-1597. NIKICICZ H, KASPRZAK A, MALENDOWICZ LK: Sex differences in adrenocortical structure and function. XIII. Stereologic studies on adrenal cortex of maturing male and female hamsters. Cell Tissue Res 1984; 235: 459-462. NOWAK M, MAJCHRZAK M. MALENDOWlCZ LK: Stereologic studies on the adrenal cortex of 4-APP/4-amino-pyrazolopyrimidine/-induced lipoprotein-deficient rats. Exp Patho11987; 31: 169-173. NUSSDORFER GG, NOWAK KW, et al.: Gestational changes in hamster adrenal cortex: Stereologic and functional studies. Res Exp Med 1990; 190: 163-171. NUSSDORFER GG: Cytophysiology of the adrenal cortex. Int Rev Cytol 1986; 98: 1-405. POPPLEWELL PY, AZHAR S: Effects of aging on cholesterol content and cholesterol-metabolizing enzymes in the rat adrenal gland. Endocrinology 1987; Ul: 64-73. SCHIFF TS, ROHEIM PS, EDER HA: Effects of high sucrose diets and 4amino-pyrazolopyrimidine on serum lipids and lipoproteins in the rat. J Lipid Res 1971; U: 596-603. SPADY DK, DIETSCHY JM:: Rate of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vitro. Biochem Biophys. Acta 1985; 836: 167-175. SZABO D. SOMOGYI J, Acs Z, et aI.: Zonal differences in the distribution and morphology of lipid droplets using 4-amino-pyrazolo-/3,4d! pyrimidine to lower cholesterol level in the rat adrenal. Acta bioI Acad Sci hung 1980; 31: 283-290. SZALAY KSz. TOTH IE et al.: Correlation of lipid droplet content and steroidogenic capacity in zona glomerulosa and fasciculata cells from lipoprotein-deficient rats. Mol Cell Endocrinol 1984; 34: 59-66. WEIBEL ER: Stereologic methods. 1. Practical methods for biological morphometry. Academic Press, New York 1979. pp. 1-415.
Exp. Toxic. Pathol. 44 (1992) 3
137
Department of Histology and Embryology, Poznan Academy of Medicine, Poznan, Poland
Comparative studies on the effect of 4-APP(4-aminopyrazolopyrimidine) on hamster and rat adrenal cortex L. K. MALENDOWICZ
With 3 tables Received: September 10, 1990; Accepted: October 22, 1990
Address for correspondence: Prof. Dr. L. K. MALENDOWICZ, Department of Histology and Embryology, Poznan Academy of Medicine, 6
Swi~cicki
Str., PL- 60- 781 Poznan, Poland
Key words: adrenal cortex; rat; hamster; 4-APP (4-aminopyrazolopyrimidine); stereology
Summary
The study aimed to compare, by means of stereologic methods, the reactivity of the adrenal cortex of the hamster and the rat to prolonged treatment with 4-aminopyrazolo-(3,4d)pyrimidine (4-APP), a drug reducing hepatic secretion of plasma lipoprotein. Adult female hamsters, intact or cortisone suppressed, were administered i.p. daily with 0.5 mg 4-APP for 5 days while intact female rats received 1 mg of the drug per dose. 4-APP resulted in a loss of body weight, with the more profound effect in the hamster. In the rat 4-APP did not change the adrenal gland weight, the volume of the adrenocortical zones, the average volume of adrenocortical cells and the number of parenchymal cells in the gland. Moreover, serum ACTH and corticosterone levels in the rat remained unchanged. In the intact hamster 4-APP decreased the adrenal gland weight, the volume of the zona fasciculata and enhanced the serum cortisol level. In steroid suppressed hamsters 4-APP lowered the adrenal weight, the volume of fasciculata and reticularis zones, the average volume of the fasciculata cells and the number of parenchymal cells in the gland. These findings may suggest the direct inhibitory effect of low 4-APP doses on the hamster adrenal cortex and clearly demonstrate higher susceptibility of the hamster adrenal cortex, if compared with the rat, to 4-aminopyrazolo-pyrimidine.
Introduction Adrenal steroid hormone synthesis depends upon a continuous supply of free cholesterol derived either from plasma lipoprotein or from biosynthesis de novo. Which mechanism of cholesterol utilization dominates is dependent on the species as well as on experimental conditions applied. In most species, among them also in the hamster, there is preference for LDL as the source of extracellular cholesterol while in ACTH treated rat ca 80% of adrenal cholesterol is derived from HDL (GWYNNE et a1. 1976; BALASUBRAMANIAM et a1. 1977; ANDERSEN and DIETSCHY 1978; BROWN et a1. 1979; GWYNNE and HESS 1980; KOVANEN et a1. 1980; LEHOUX and PREISS 1980). In the 134
Exp. Toxic. Pathol. 44 (1992) 3
hamster, the adrenal cortex is almost completely devoid of lipid droplets and stored cholesterol esters and the synthesis of corticosteroids depends mainly on intraadrenal synthesis of cholesterol from small molecules of substrate (LEHOUX and LEFEBVRE 1980; JANSEN and BIRKENHAGEN 1981; MALENDOWICZ and NUSSDORFER 1984; SPADY and DIETSCHY 1985; IWAKI et a!. 1985). The adenine, analogue 4-aminopyrazolo-(3,4d)pyrimidine (4-APP) reduces the hepatic secretion of plasma lipoprotein in rats thus resulting in a marked reduction of plasma and adrenal cholesterol content (SCHIFF et a1. 1971; BALASUBRAMANIAM et a1. 1977; BRECHER and HYUN 1976; MALENDOWICZ et a1. 1987). In 4-APP-induced lipoprotein deficient rats the rate of intraadrenal cholesterol synthesis increases and this effect is connected with impaired steroidogenesis (ANDERSEN and DIETSCHY 1976; BRECHER and HYUN 1978; GWYNN and HESS 1980; SZABO et a1. 1980, 1984; MALENDOWICZ et a1. 1987). As far as structure and steroidogenesis are concerned, the adrenal cOltex of the hamster differs markedly from the rat adrenal gland (for details see MALENDOWICZ and NUSSDORFER 1984; NUSSDORFER 1986). For example, the reactivity of the rat and hamster adrenal cortex to estrogens, which are known to be also a hypocholesterolemic agents, shows marked species differences (GASKIN and KITAY 1970, 1971; MALENDOWICZ et a1. 1982a, b; POPPLEWELL and AZHAR 1987). The aim of the present study was to compare, by means .of stereo logic methods, the reactivity of the adrenal cortex of the hamster and the rat to prolonged treatment with 4-APP, a cholesterol lowering agent.
Materials and Methods , Adult female hamsters (M esocricetus auratus WATERHOUSE) and rats of the Wi star strain were employed in this study. The animals were maintained under standardized conditions of light (14 L: 10 D) and temperature (22 ± 2°C) and fed with laboratory pellets with free access to tap water. 4-APP [4-aminopyrazolo(3,4-d)pyrymidine, Sigma] was
dissolved ex tempore in physiologic solution of saline (adjusted to pH 2.-5) at a concentration I mg/mI. The hamsters were administered i.p. daily with 0.5 mg 4-APP for 5 days while the rats received 1 mg of the drug per dose. The control animals were treated with the solvent only. One group of hamsters was treated s.c. daily with 0.25 mg of cortisone acetate (Upjohn). After weighing, the animals were decapitated (24 h after the last injection) and trunk blood collected (with 1 mg NaF/ml). The adrenals were promptly removed, cleaned of adherent adipose tissue and weighed. Stereology. The left adrenal gland was fixed for 24 h in Bouin's fluid at 22 ± 1°C, embedded in paraffin wax and serially sectioned at a thickness of 5-6 11m. The 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 (WEIBEL 1979), the volume fractions of the individual adrenocortical zones and of the medulla and capsula were estimated. The analysis was pelformed on each fifth 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 (MALENDOWICZ 1987; NIKICICZ et aI. 1984). Hormonal assays. Serum was separated and stored at -20 °C 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, crossreaction 100 %) while there is no cross-reaction with cxMSH , ~ endorphin, ~-lipotropin, and other tropic hormones of the adenohypophysis. The intra-assay variance for ACTH was 5 %, while the inter-assay variance was 7 %. Cortisol was quantitated in unextracted hamster serum by RIA with RIA cortisol ct 25 I) kit of Farmos Diagnostica (Finland) . With that kit cross-reactions with progesterone and corticosterone were respectively < 0.01 and < 2% and interassay variation 4 %. Corticosterone (in the rat) was measured by sulfuric acid fluorescence (GUILLEMIN et aI. 1959). 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 the Student's t-test.
Results 4-APP administration resulted in a loss of body weight in hamsters and rats (tables 1 and 3). In the hamster 4-APP decreased the adrenal gland weight while there was no change in the weight of the rat adrenal gland. 4-APP administration did not change the volume. of adrenocortical zones, the average volume of the adrenocortical cells, and the humber of parenchymal cells in the gland. Only in 4-APP-treated hamsters the volume of the zona fasciculata was lower than in the controls.
Table 1. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female hamster. Results expressed as means ± SE. Control n Body weight (g) Adrenal weight (mg)
7 94 ± 3 16.2 ± 0.8
4-APP 7 64± Id 13 .6±0.4b
Volume of the zones (mrn 3) ZG 2.169±0.173 ZF 6.090 ± 0.231 ZR 4.707 ± 0.336
2.238 ± 0.063 4 .732 ± 0.200° 4 .045 ± 0.165
Volume of cell (11m3) ZG ZF ZR
1,017±42 2,757±173 879 ±25
1,071 ± 27 2,420 ± 143 1,0l8±60
2,115± 197 2,038± 135 4,405 ± 393 8,897 ± 590
2,049± 62 1,866± 123 3,717 ± 227 7,633 ±279
361 ±72 1O.5±1.9
461 ±48 23.5±3.6c
N umber of cells (1 ZG ZF ZR Total Blood level ACTH (pmol/I) cortisol (nmoll!)
X
10 3)
Statistical evaluation of the results by Student's t-test: a - p < 0.05; b - P < 0.02; c - p < 0.01; d - p < 0.001. ZG - zona glomerulosa , ZF - zona fasciculata, ZR - zona reticularis. The serum cortisol level was markedly elevated in 4-APPtreated hamsters while in the rat the level of corticosterone was unchanged. 4-APP treatment did not change the serum ACTH level either in the hamster or in the rat. In steroid suppressed hamsters 4-APP lowered the adrenal weight, the volume of the fasciculata and reticularis zones, the average volume of the fasciculata cells and the number of zona reticularis cells as well as the total number of parenchymal cells in the gland (table 2). On the other hand, the average volume of the zona glomerulasa cells of 4-APP-treated rats was higher than in the controls while the serum ACTH concentration remained unchanged.
Discussion 4-APP, an agent blocking lipoprotein release from the liver and thus reducing the circulating level of lipoprotein cholesterol, has been frequently employed to study the contribution of circulating cholesterol to steroidogenesis in the adrenals. Doses of 4-APP administered into rats varied from 5 mg/lOO g b.wt. in 2 - 3 days experiments (BALASUBRAMANIAM et aI. 1976 ; BROWN et a1. 1979 ; SZABO et aI. 1980; BLANK et a1. 1983; MIKAMI et aI. 1984) to 2 or 1 mg (MALENDOWICZ et aI. 1987 ; NOWAK et aI. 1987; POPPLEWELL and AZHAR 1987). However, daily administration of 0 .5 mg 4-APP per 100 g b.wt. for 3-7 days also resulted in the profound reduction of the serum cholesterol level in the rat and notably disturbed pituitary hormones release (BLANK et al. 1983; MURPHY et al. 1985; MAZZOCCHI et a1. 1986). Regarding the potent toxicity of that Exp. Toxic. Pathol. 44 (1992) 3
135
Table 2. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female, steroid hormone suppressed hamster. Results expressed as means±SE. Cortisone
Cortisone + 4-APP
7 82±4 16.4 ± l.0
7 77±4 13.3 ±0.6a
Volume of the zones (mm 3 ) ZG ZF ZR
2.423 ± 0.190 6.220 ± 0.599 4.813 ± 0.322
2.212 ± 0.086 4.282±0.184c 3.481 ±0.158 c
Volume of cell ([Am 3 ) ZG ZF ZR
896± 38 2,188±165 938±55
1,075 ± 51 b 1,694± 151 a 929±43
2,663 ± 220 2,768±298 4,756±280 1O,187±438
2,612± 143 2,460± 232 3,516± 197 c 8,038±471 c
n Body weight (g) Adrenal weight (mg)
Number of cells (1 ZG ZF ZR Total
X
103)
Blood level of ACTH (pmolll)
444± 63
922± 267
Explanations as in Table I
Table 3. The effect of prolonged 4-APP treatment on some morphometric parameters of the adrenal cortex of the female rat. Results expressed as means ± SE. Control n
Body weight (g) Adrenal weight (mg)
6 223±8 53.9±1.4
4-APP 6
196 ± 6a 54.0±3.4
Volume of the zones (mm 3 ) 6.520 ± 0.301 7.111 ± 0.431 ZG 25.009 ± 0.846 23.272 ± l.962 ZF 16.767±0.756 17.919± 1.299 ZR Volume of cell ([Am 3 ) ZG ZF ZR Number of cells (1 x 103 ) ZG ZF ZR Total Blood level ACTH (pmolll) corticosterone ([Agll)
929±43 3,562±225 825 ±44
7,058±291 7,208 ± 518 6,738±496 6,874±495 19,524± 1,673 18,644± 1,578 33,456 ± 2,222 32,590 ± 2,207 74.0±23.3 2.30±0.30
Explanations as in Table I
136
971 ± 43 3,332±299 909±49
Exp. Toxic. Pathol. 44 (1992) 3
88.3 ± 20.4 2.20 ± 0.70
adenine analogue, in the present experiments hamsters and rats were administered with relatively low doses of 4-APP. As revealed in the present study 4-APP markedly reduced the body weight in both species with more profound effect in the hamster. This may be a manifestation of the general toxic effect of the drug and it is of interest that cortisone administration prevented 4-APP-induced body weight loss in the hamster. 4-APP administered in higher doses markedly increased the adrenal gland weight in the rat, an effect depending on enlargement of the cortex (SZABO et al. 1980; MALENDOWICZ et al. 1987; NOWAK et al. 1987). This increase was due to hypertrophy and hyperplasia of parenchymal cells. In the present study low doses of 4-APP within 5 days did not change the adrenal gland weight in the rat while in intact or cortisone suppressed hamsters the adrenal weight was notably lower than in the controls. Moreover, zonal and cellular composition of the adrenal cortex was unchanged in 4-APP-treated rats while in the hamster a marked reduction in the volume of the zona fasciculata was found. A more profound effect of 4-APP was observed in cortisone suppressed hamsters in which the drug resulted in the lowering ofthe volume of the fasciculata and reticularis zones, of the size of the fasciculata cells and of the number of parenchymal cells in the gland. These findings may suggest the direct inhibitory effect of 4-APP on the hamster adrenal cortex, however, in the intact hamsters this effect may be partially obliterated by compensatory hepersecretion of ACTH by the nonsuppressed pituitary gland. Such a direct effect of 4-APP on the rat adrenal cortex has been suggested also in our earlier study (Now AK et al. 1987). The results of hormonal estimations do not exclude the possibility of compensatory hypersecretion of ACTH in 4-APP-treated hamsters. In this species basal plasma ACTH levels are very high if compared with other mammals and the values differ markedly among individuals (NOWAK et al. 1990). Therefore it is very difficult to reveal any statistically significant differences in the plasma ACTH level among the groups of experimental hamsters. Moreover, in the present experiment animals were sacrificed 24 h after 4-APP administration and such a one-point sampling time may be insufficient to reveal a possible short-lasting enhanced corticotropin level in the blood. Moreover, increased plasma cortisol levels in 4-APP-treated hamsters strongly suggested an increased stimulation of the hamster adrenal cortex by ACTH, although the impaired inactivation of cortisol by injury, due to 4-APP hepatotoxicity, can not be excluded. If this is the case, the enhanced, due to impaired inactivation, plasma cortisol level may suppress pituitary ACTH secretion and may induce partial atrophy of the adrenal cortex. Contrary to the hamster, in the rat comparable doses of 4-APP had no effect either on the serum ACTH level or on the serum corticosterone concentration. Thus, if compared with the rat, our experiments clearly demonstrate a higher susceptibility of the hamster adrenal cortex to 4-APP.
Acknowledgement The study was supported in part by a grant X-IS from the Poznan Academy of Medicine, Poznan, Poland.
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