J O U R N A L O F U L T R A S T R U C T U R E A N D M O L E C U L A R S T R U C T U R E R E S E A R C H 97, 1-9 (1986)

Ultrastructural and Biochemical Alterations Produced in Rat Adrenal Cortex by 4-Aminopyrazolopyrimidine

HENRIQUE ALMEIDA,* M . M . MAGALH/~ES,* TERESA A. M . SERRA,t

C. HIP6LITO-REIS,~ AND MARIA C. MAGALHAES*

*Institute of Histology and Embryology and ~Laboratory of Biochemistry, Faculty of Medicine of Porto, and Centers of*Experimental Morphology, tExperimental Cytology, and ~Pharmacology and Chemical

Biopathology of the University of Porto (INIC), Porto, Portugal

Received March 4, 1987, and in revised form May 25, 1987

The ultrastrnctural and biochemical changes produced by 4-aminopyrazolo[3,4-d]pyrimidine (4- APP), on zona fasciculata ceils of rat adrenal cortex are described. Male rats weighing ~ 200 g were injected intraperitoneaUy with 50 mg/kg/day of 4-APP for 3 days; the controls were injected with buffer. All animals were sacrificed on the 4th day and the adrenals from some of them were processed for electron microscopy. The adrenals from the remaining rats were used for measure- ments of gland cholesterol and corticosterone; the latter was also measured in the blood. In 4-APP- treated rats the zona fasciculata cells exhibited an increase in the amount of smooth endoplasmic reticulum and in the number of free ribosomes, often arranged in polyribosomes, and a decrease in the number of lipid droplets. The nucleus showed scarce condensed chromatin and nucleolar fragmentation. The quantitative analysis showed a significant increase of the volumetric density of endoplasmic reticulum and a significant decrease of the lipid droplets in treated rats when compared with controls. Concerning the nucleus, the volumetric density of condensed chromatin significantly decreased, while the relative volume of fibrillar centers, and of granular and vacuolar components increased. In treated rats, the adrenal cholesterol and corticosterone concentrations and the blood corticosterone level were significantly decreased. These data show that 4-APP has a remarkable effect on corticosteroidogenesis and depletes the pool of adrenal cholesteryl esters, and these data stress the importance of plasma cholesterol in the steroidogenesis; on the other hand the drug appears to have a direct effect on the nucleus. © 1986 Academic Press, Inc.

In the last two decades numerous studies have demonstrated the role of plasma li- poproteins as cholesterol carriers to be used as substrate for steroidogenesis (Gwynne and Strauss, 1982). In the rat, a major portion of the cholesterol used by the adrenal cells for corticosteroidogenesis is derived from the circulating lipoproteins (Morris and Chaikoff, 1959; Dexter et al., 1970), namely from the high density lipoproteins (HDL) (Mahley and Holcombe, 1977; Andersen and Dietschy, 1978). 4-Aminopyrazo- lo[3,4-d]pyrimidine (4-APP), an adenine analog, has been shown to depress lipopro- rein synthesis and secretion by the liver (Henderson, 1963; Shiffet al., 1971). This led some investigators to use this drug to evaluate adrenal steroid metabolism, under

low cholesterol supply. They observed that plasma corticosterone (Andersen and Diet- schy, 1978) and intraglandular cholesterol levels were decreased, though the adrenal rate of cholesterol synthesis from acetate was enhanced (Andersen and Dietschy, 1976, 1978; Balasubramaniam et al., 1977). On the other hand the morphological effects of 4-APP on adrenal cortex cells have scarcely been studied and only with respect to the cytoplasm (Szab6 et al., 1980; Szab6 et al., 1984; Murakoshi et al., 1984; Maz- zocchi et al., 1986).

Following a preliminary presentation on the nucleolar effects of this drug (Almeida et al., 1987) we decided to evaluate quali- tat ively and quant i ta t ively the changes which occur in the cytoplasm and nucleus

0889-1605/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

ALMEIDA ET AL.

o f t h e z o n a f a s c i c u l a t a ce l l s o f t h e r a t a d r e n a l

g l a n d a f t e r 4 - A P P a d m i n i s t r a t i o n a n d c o r -

r e l a t e t h e m w i t h s o m e b i o c h e m i c a l a s s a y s .

MATERIALS AND METHODS

Twenty-six, 2-month-old male rats weighing - 2 0 0 g, from the colony of the Gulbenkian Institute of Sci- ence, Oeiras, Portugal, were used. They were divided into two groups of 13 rats each and kept in the same room conditions with free access to water and labo- ratory diet.

The animals of the first group received an intraper- itoneal injection of 50 mg/kg/day of 4-APP (Sigma Chemical Co.) in 10 m M sodium phosphate, pH 3.5, at 10:00 AM for 3 days. The rats of the second group (controls) were injected with an appropriate volume of 10 m M sodium phosphate under the same conditions.

Morphological Studies

At 10:00 AM of the 4th day, the animals were ether anesthetized, and the adrenals were removed and cut into small pieces. The fragments were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, at 4°C, for 2 hr, rinsed in buffer with 7% sucrose over- night, postfixed in 1% osmium tetroxide in veronal- acetate buffer, pH 7.3, at 4°C, for 2 hr, dehydrated in ascending concentrations of ethanol, and Epon embed- ded. Semithin sections, 1 or 2 ~*m thick, were stained with methylene blue-azur II (Richardson et aL, 1960) and used for selection of the zona fasciculata. For elec- tron microscopy, golden ultrathin sections were stained with alcoholic uranyl acetate for 15 min and lead citrate for 10 min, and observed in a Jeol 100B electron mi- croscope.

For the regressive staining procedure (Bernhard, 1969), some pieces were fixed in 2.5% glutaraldehyde in Sorensen's phosphate buffer, pH 7.3, for 2 hr at room temperature. After dehydration and Epon embedding, golden ultrathin sections were cut and stained with 5% uranyl acetate for 1 rain, 0.2 M EDTA in distilled water for 10 rain, and lead citrate for 1 rain.

Quantitative Studies

Five animals of each group were used for morpho- metric studies. Golden ultrathin sections of the zona fasciculata from three Epon blocks per animal were cut and 8 micrographs per rat were taken at random at × 6000, so that 40 micrographs were studied for each animal group. A rectangular test area of 22 x 16 cm

with 102 lines of constant length (1.3 cm) arranged in 17 equidistant and parallel rows and in which the dis- tance between the endpoints of the lines in every di- rection was 1.3 cm was placed randomly on photo- graphic prints that had been enlarged to x 18 000. The number of points which fell on the cytoplasmic struc- tures was used for point-counting volumetry. In the special case of the endoplasmic reticulum the volume was calculated from the points which fell within the area occupied by the cytoplasmic space between the other organelles (mitochondria, Golgi, etc.). Nuclear quantitative studies were performed in the same con- ditions but using microphotographs that had been en- larged to x 27 000. The test area was square and con- tained 112 lines of 1 cm of length arranged in 16 parallel and equidistant rows in which the distance between the endpoints of the lines, in every direction, was also 1 cm. The number of points (Pi) for each cell structure was transformed into volumetric density in agreement with the formula Vv~ = Pi/PT (Weibel and Bolender, 1973) expressed as a percentage (PT is the total number of test points). The average values were compared by the Student's t test.

Biochemical Studies

Biochemical analytical procedures were performed using eight 4-APP-treated rats and eight controls. The adrenals were removed, dissected free from surround- ing fat and connective tissue, washed with ice-cold saline, and blotted. The two adrenals from each animal were weighed and homogenized in ice-cold 0.1 Mphos- phate buffer, pH 7.4, using a Potter-Elvejhem homog- enizer with a motor-driven Tefon pestle. For the as- sessment of adrenal and plasma corticosterone the fluorometric method of Mejer and Blanchard (1973) was used; fluorescence was read on a Perkin-Elmer spectrofluorometer at an excitation of 470 nm and an emission of 520 nm. Total and free cholesterol were determined by the method of Sperry and Webb (1950). Protein contents were determined by the method of Lowry et al. (1951); Lab-trol (Dade Diagnostic, Inc.) was used as a standard. All determinations were done in duplicate. The average values were compared by the Student's t test.

RESULTS

M o r p h o l o g i c a l O b s e r v a t i o n s

A l t h o u g h t h e a n i m a l s t r e a t e d w i t h 4 - A P P

a p p e a r e d t o e a t a n d d r i n k n o r m a l l y t h e y h a d

Fig. 1. Zona fasciculata cell of a control animal. Note the usual ultrastructure of the cytoplasm with mito- chondria (m), smooth endoplasmic reticulum (er), and lipid droplets (1). Fixation, glutaraldehyde and osmium tetroxide; staining, uranyl acetate and lead citrate, x 18 000.

FIG. 2. Treated rat. Observe the smooth endoplasmic reticulum (er) with numerous ribosomes often arranged in polyribosomes with helical form (arrows). The mitochondria (m) appear to be smaller and slightly denser. Go, Golgi complex. Fixation and staining are as in Fig. 1. x 18 000.

3

4 ALMEIDA ET AL.

FIG. 3. Treated animal. A detail of helical arrange- ment of the ribosomes (arrows). Fixation and staining are as in Fig. 1. x 36 000.

loss o f weight and activity. The adrenal glands were soft, reddish, and increased in weight when compared with controls. In the light microscope, the morphology of the ad- renal cortex zona fasciculata was similar to that previously described (Deane, 1962). In 4-APP-treated rats, the general architecture o f the zona fasciculata had partially lost the cordonal type, becoming looser, and the blood capillaries appeared dilated and con-

tained numerous erythrocytes. The cell cy- toplasm exhibited few lipid droplets and the nuclei seemed enlarged, paler, and with less condensed chromat in than the control nu- clei. The nucleoli exhibited irregular outline and presented areas o f different staining af- finities.

Electron microscope examinat ion o f the zona fasciculata o f control rats showed the usual ultrastructure (Figs. 1 and 4). In treat- ed rats, the lipid droplets were much less numerous and often had a moderately dense content. The smooth endoplasmic reticu- lure appeared increased and numerous free r ibosomes were scattered in the cytoplasm (Fig. 2); often they were arranged in poly- r ibosomes with curious helical forms (Figs. 2 and 3). The mi tochrondr ia sometimes ap- peared to be smaller and slightly denser. The other organelles presented no alter- ations. The nuclei o f treated rats seemed larger than those o f controls. The condensed chromat in and nucleolus-associated chro- mat in were scarce (Figs. 5 and 6). The re- ticular aspect o f the nucleolus was lost (Figs. 5 and 6); nucleolar components appeared fragmented with separation o f the dense fi- brillar and granular components as was al- ready described (Almeida et aL, 1987).

The quantitative study concerning the cy- toplasm showed a significant increase of the smooth endoplasmic reticulum volumetr ic density and a significant decrease o f the lip- id droplets relative vo lume in treated ani- mals, when compared with normal ones (Table I). The mi tochondr ia /peroxisome numerical ratio increased from 18.2 in con- trois (1878 mi tochondr ia and 103 peroxi- somes were counted on 40 micrographs) to 62.1 in treated rats (1864 mi tochondr ia and

FIG. 4. Control rat. The nucleus (N) exhibits the usual fine morphology with dispersed chromatin (dch), patches of condensed chromatin (cch), and two nucleoli (nu). In the nucleolus observe the dense fibrillar (short arrow) and granular (long arrow) components and the fibrillar centers (arrow head). Fixation and staining are as in Fig. 1. x 18 000.

FIG. 5. Treated animal. In the nucleus (N) the scarcity of condensed chromatin and the nucleolar fragmen- tation with separation of the dense fibrillar (short arrow) and granular (long arrow) components are evident~ The fibrillar centers (arrow head) appear to be surrounded by a small amount of dense fibrillar component, dch, dispersed chromatin. Fixation and staining are as in Fig. 1. x 12 000.

i ! ' ~ .

H ~

6 ALMEIDA ET AL.

FIG. 6. Treated animal. Areas of bleached chromatin (nuch) are scarce, mainly at the nuclear periphery. The nucleolus (nu) exhibits nucleolar fragmentation. N, nucleus. Staining with the EDTA method. × 19 200.

TABLE I

VARIATIONS OF VOLUMETRIC DENSITIES (%) OF THE ORGANELLES OF ZONA FASCICULATA CELLS AFTER 4-APP ADMINISTRATION

Lipid Rats a Mitochondria b Reticulum b Golgi b droplets b Lysosomes b Others b

Treated 34.86 _+ 1.28 54.92 _+ 1.13 1.21 _+ 0.19 4.44 + 0.12 0.45 _+ 0.10 4.12 _+ 0.62 Controls 37.99 _+ 2.12 45.16 _+ 1.82 0.93 _+ 0.23 10.64 _+ 1.78 0.44 + 0.10 4.84 _+ 0.75 P values n.s, < 0.01 n.s. <0.01 n.s. n.s.

a Five animals per group were used; eight photographs per animal were utilized. b Mean values from each animal group expressed as percentage + SE.

TABLE II

VARIATION OF VOLUMETRIC DENSITY (°/0) OF NUCLEAR AND NUCLEOLAR COMPONENTS AFTER 4-APP ADMINISTRATION

Rats a D.C. b C.C. b F.Cp. b G.Cp. b F.Ct. b V.Cp. b

Treated 76.70 _+ 2.62 5.31 _+ 1.64 3.68 _+ 0.17 10.35 +_ 1.28 0.53 + 0.11 3.43 + 0.88 Controls 73.43 + 0.89 19.60 + 0.54 3.12 _+ 0.39 2.52 _+ 0.28 0.20 _+ 0.05 1.23 _+ 0.24 P values n.s. <0.001 n.s. < 0.001 <0.01 < 0.01

a Five animals per group were used; eight photographs per animal were utilized. b Mean values from each animal group expressed as percentage _+ SE. D.C., dispersed chromatin; C.C.,

condensed chromatin; F.Cp., nucleolar dense fibrillar component; G.Cp., nucleolar granular component; F.Ct., fibrillar centers; V.Cp., nucleolar vacuolar component .

ALTERATIONS PRODUCED IN ADRENAL CORTEX BY 4-APP

30 peroxisomes were counted on 40 micro- graphs). The morphometric analysis of the nucleus showed a significant increase of the volumetic density of fibrillar centers and granular and vacuolar components, and a significant decrease of the same parameter for the condensed chromatin (Table II).

B i o c h e m i c a l A n a l y s i s

The biochemical de terminat ions re- vealed that in the plasma of treated rats there was a significant decrease of the cor- ticosterone level in relation to control val- ues (controls, 1.04 + 0.268 ~tg/ml; treated, 0.34 + 0.43 ~tg/ml; P < 0.05). In the treated animals the adrenal contents of esterified cholesterol and corticosterone were signifi- cantly decreased when compared with con- trol values (Table III).

DISCUSSION

At present 4-APP is used extensively to study the adrenal steroid metabolism, since it causes a strong decrease in the cholesterol uptake due to a profound hypocholesterol- emia. Significant drops in the plasmatic li- poproteins (Shift et al., 1971) and choles- terol (Shift et al., 1971; Andersen and Dietschy, 1976; Balasubramaniam et al., 1977; Mikami et al., 1984) after 4-APP administration have been demonstrated.

Our observations show that 4-APP pro- voked marked adrenal biochemical and morphological changes which are probably correlated. The adrenal cholesterol content was significantly decreased, which is in ac- cordance with previous data of other in- vestigators (Balasubramaniam et al., 1977; Kovanen et al., 1980; Mikami et al., 1984) and concomitantly we observed a signifi- cant decrease of the number and volume of lipid droplets, the storage sites ofcholesteryl esters (Fr/ihling et al., 1973; Vahouny et al., 1984-1985). These data suggest that the cholesterol stored in the lipid droplets was used for steroidogenesis since the blood supply of adrenal cholesterol was dimin- ished. This assumption is supported by bio-

chemical studies, which have recently shown enhanced activities 0fthose ester hydrolases which hydrolyze the lipid droplets choles- terol esters and yield free cholesterol for ad- renal metabolic processes (Mikami et al., 1984). Curiously the enzymatic systems re- lated to the adrenal cholesterol esterification such as acyl-CoA synthetase, an enzyme in- volved in the formation of acyl-CoA from fatty acids (Mikami et al., 1984), and acyl- CoA-cholesterol acyltransferase (ACAT), involved in the synthesis of cholesterol es- ters from acyl-CoA and free cholesterol (Balasubramaniam et al., 1977), presented decreased activities in rats treated with 4-APP. The utilization of endogenous stores of cholesterol for corticosteroid synthesis, however, does not appear to be sufficient to maintain the corticosteroidogenesis rate, because both the plasma corticosterone level and the adrenal cholesterol and corticoste- rone concentrations are lower in treated rats than in control animals, as was observed by Andersen and Dietschy (1978), Vahouny et al. (1984-1985), and us.

In our experimental conditions another compensatory pathway, the de novo choles- terol synthesis is presumably working in or- der to produce substrate for steroidogenesis. Andersen and Dietschy (1976) observed that after 4-APP administration the adrenal syn- thetizes cholesterol from acetate and Bala- subramaniam et al. (1977) noted that the activity of the key enzyme of the cholesterol synthesis from acetate, the 3-hydroxy-3- methylglutaryl-CoA reductase, was in- creased. This adrenal compensatory d e novo cholesterol synthesis may be related to the significant increase of the smooth endo- plasmic reticulum observed in our material, which is understandable since it is the sub- cellular site where cholesterol synthesis from acetate takes place.

An adrenal cytoplasmic feature which de- serves some comment is the curious helical arrangement of the polysomes in 4-APP- treated rats. This kind of arrangement ap- pears sometimes in normal cells (Kisilevsky et al., 1973) but it is much more frequent

ALMEIDA ET AL.

TABLE II I

EFFECTS OF 4-APP ON ADRENAL WEIGHT AND BIOCHEMICAL COMPONENTS a

Adrenal weight Protein Total cholesterol Free cholesterol Animals b (rag) (~g/rng of adrenal) 0zg/mg of protein) 0zg/mg of protein)

Treated 57.5 + 3.6 122.7 + 5.0 328.41 __+ 34.90 79.29 ± 11.74 Controls 35.0 ± 2.9 117.4 + 5.3 476.29 _+ 37.81 64.29 + 10.45 P values <0.002 n.s. <0.02 n.s.

Means + SE. b Four animals per group were used.

during cell ma tura t ion (Behnke, 1963; Waddington and Perry, 1963; Echlin, 1965; Jensen, 1968) and after administration of some drugs. 4-APP is known to have an inhibitor effect on protein synthesis (Booth and Sartorelli, 1961); however, that effect was observed within 6 hr after 4-APP administration and was followed by a re- covery to an almost normal level of protein synthesis at 12 hr. Since our material was studied 24 hr after the last 4-APP admin- istration, it is likely that protein synthesis was still recovering and that the peculiar ribosomic arrangement expresses this con- dition. It is interesting to note that other protein synthesis inhibitors, such as ethio- nine (Baglio and Farber, 1965; Shinozuka et al., 1970) lasiocarpine, and aflatoxine (Monneron, 1969), produce striking nucleo- lar changes, but when those changes are dis- appearing and the nucleolus is being re- formed toward normal morphology, an arrangement ofpolysomes similar to the one we observed after 4-APP administration may be seen. In spite of those nucleolar changes the synthesis ofextranucleolar RNA persisted (Lafarge et aL, 1966) which al- lowed Monneron (1969) to suggest the pro- duction of a transient messenger RNA.

Profound nuclear alterations are also caused by 4-APP which may be due to a direct drug effect on the nuclear metabo- lism. The alterations which concern the nu- cleolus are the nucleolar fragmentation with significant increase of fibrillar centers and granular and vacuolar component volu- metric densities. The nucleolar fragmenta- tion may be related to the incorporation of

the 4-APP nucleotide derivative in the RNA, affecting the normal nucleolar RNA syn- thesis and processing (Almeida et al., 1987). The increases of granular component and fibrillar centers are not well understood al- though a relation to an enhanced metabolic activity cannot be excluded (Smetana and Busch, 1974; Medina et al., 1983; Vagner- Capodano et aL, 1984). The vacuolar in- crease seems to be more apparent than real. Indeed, the vacuolar component areas may only be portions of the dispersed chromatin penetrating into the nucleolar fragments.

The significant decrease of condensed chromatin is highly suggestive of an in- creased metabolic activity due to the gene derepression. In some cellular processes, such as organ regeneration and hypertro- phy, steroid hormone action on target ceils, and lymphocyte immune activation, there is a gene derepression with the nuclei ex- hibiting reversible transformation of het- erochromatin to euchromatin; sometimes this modification appears to be associated with nucelolar hypertrophy and conversion ofmonoribosomes into polyribosomes, and is correlated with new synthesis of RNAs (Frenster, 1974). In our material the con- version of condensed chromatin to dis- persed chromatin, the nucleolar fragmen- tat ion, and the special polysomic arrangement, taken on the whole, would be related to the production of a special tran- sient messenger RNA, which is in agree- ment with Monneron's suggestion (1969).

In short, 4-APP produces indirect cyto- plasmic and direct nuclear effects on the ad- renal. Cytoplasmic effects are probably due

ALTERATIONS PRODUCED IN ADRENAL CORTEX BY 4-APP

TABLE III

EXTENDED

Esterified cholesterol Corticosterone (#g/mg of protein) (#g/mg of adrenal)

249.12 _+ 33.26 0.018 +_ 0.005 412.00 +__ 30.26 0.044 _+ 0.004

<0.01 <0.01

to the decrease o f p l a s m a c i rcu la t ing cho-

les te ro l a n d e m p h a s i z e the i m p o r t a n c e o f

e x o g e n o u s cho le s t e ro l a n d o f s to red choles -

t e ro l as subs t ra te sources for co r t i cos te -

ro idogenes i s . T h e effects on the nuc l eus ap-

pea r to be r e l a t ed to the s t i m u l a t i o n o f s o m e

specia l m e t a b o l i c pa thways .

We thank Professor A. Coimbra for critical reading of the manuscript.

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