Experimental brain tumors by transplacental ENU

A ct a Neuropathologica

Acta Neuropathol. (Berl.) 49, 117-122 (1980)

9 Springer-Verlag 1980

Experimental Brain Tumors by Transplacental ENU* Multifactorial Study of the Latency Period D. Schiffer 1, M. T. G i o r d a n a 1, A. M a u r o 1, G. R a c a g n i 2, F. B r u n o 2, S. P e z z o t t a 3, a n d P. Paoletti 3 1 II. Neurological Clinic, University of Turin, v. Cherasco 15, 1-10126, Turin, Italy 2 Institute of Pharmacology and Pharmacognosy, University of Milan, Milan, Italy 3 Neurosurgical Clinic, University of Pavia, Pavia, Italy

Summary. E x p e r i m e n t a l cerebral t u m o r s have been i n d u c e d b y t r a n s p l a c e n t a l E N U . The m o r p h o l o g i c study o f the b r a i n s o f t r e a t e d rats revealed t h a t cellular h y p e r p l a s i a s a p p e a r at the 30th d a y o f e x t r a u t e r i n e life in the p a r a v e n t r i c u l a r white matter, i.e., before the already known "early neoplastic proliferations". C y t o f l u o r i m e t r i c investigations failed to d e m o n s t r a t e differences between t r e a t e d a n d c o n t r o l rats d u r i n g the 1st m o n t h . O n the c o n t r a r y , a d e n y l a t e cyclase activity is very high in t h a t period. The d u r a t i o n o f the latency p e r i o d is discussed.

arrest ( G o e r t t l e r et al., 1970; Bosch, 1977a, b), the earliest t u m o r a l lesions have been m o r p h o l o g i c a l l y o b s e r v e d only after the 2nd m o n t h o f e x t r a u t e r i n e life ( K o e s t n e r et al., 1971; L a n t o s a n d Cox, 1976; Schiffer et al., 1978; L a n t o s a n d Pilkington, 1979). O n the m o r p h o l o g i c a l a n d m o l e c u l a r level we w a n t e d to reinvestigate the tissual events in this 2 - m o n t h interval in those areas, or their precursors, where the earliest t u m o r a l lesions appear.

Material and Methods Key words: E N U t u m o r s - L a t e n c y p e r i o d

The time lapse between the effect o f a c a r c i n o g e n on cells a n d the first a p p e a r a n c e o f t u m o r s has n o t yet been fully clarified. In the case o f E N U it is very difficult to identify the p h e n o t y p i c a l t e r a t i o n s in vivo o f pres u m p t i v e t u m o r cells ( L a e r u m and Rajewsky, 1975). This is due to the p o l y m o r p h i c cellular c o m p o s i t i o n o f the n e r v o u s tissue a n d to the fact t h a t only a m i n o r f r a c t i o n o f its c o n s t i t u e n t cells u n d e r g o e s changes leading to the " m a l i g n a n t " p h e n o t y p e . T h e chemical m e c h a n i s m o f t u m o r i n d u c t i o n by t r a n s p l a c e n t a l E N U is sufficiently k n o w n ( R a j e w s k y a n d G o t h , 1976) a n d the m o d e l is very well established as far as t u m o r c o m p o s i t i o n a n d l o c a t i o n are concerned. W h i l e s h o r t - t e r m effects have been o b s e r v e d within 6 - 48 h after E N U a d m i n i s t r a t i o n , consisting o f necroses, n u c l e a r p y k n o s i s a n d t e m p o r a r y cell cycle * This research was supported by Grants No. 79.00678.96 and No. 79.00664.96 of Progetto Finalizzato "Control of Neoplastic Growth", Consiglio Nazionale delle Ricerche (C.N.R.), Rome Offprint requests to." D. Schiffer, M.D. (address see above)

Pregnant Fisher 344 rats, on the 17th day of gestation, were treated with a single dose of ENU 20 mg/kg i.v. On the newborn rat brains the following investigations were carried out:

Morphologie Study Groups of five rats have been killed every 5 days, starting from birth until the 60th day of extrauterine life. The brains have been fixed in Carnoy at 0~ dehydrated, paraffin-embedded, and cut into 4 ~tm thick serial sections. The staining methods employed were: hematoxylin-eosin (H.-E.), PTAH, luxol fast blue B for myelin. With the progressing development the following zones have been examined: germinal zone, cortex, mantle zone, basal ganglia, medullary center, paraventricular white matter, and subependymal plate. Cell counts have been performed in microscopic fields of 160 x 110/am2. Cytofluorimetric Study On 4 gm thick sections of the brain Feulgen reaction has been carried out with 0.05 % pararosanilin hydrochloride (G. T. Gurr), according to the method described by Prenna et al. (1971). Cytofluorimetric measurements of DNA were performed, according to Prenna et al. (1974 a, b) with Leitz MPV2 microscope photometer. For fluorescence excitation a stabilized high pressure xenon lamp (Osram XBO 75 W/I) was used in combination with the following filters: heat protection KGI (2 ram); SS 592 (10rim half peak) interference filter. Dichromatic mirror TK 580 and Kodak Wratten 92 as barrier filter (620 rim) were employed. Cell DNA was measured in the medullary center and paraventricular white matter of treated and control rats killed on the 11th, 19th, 30th, 45th, and 60th day of extrauterine life.

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Acta Neuropathol. (Bed.) 49 (1980)

Fig. 1, a Germinal zone with many mitoses in 1-day-old rat. Em. Eos. x 278; b Mitoses in the cortex in 1l-day-old rat. Era. Eos. • 928; c Mitoses in the paraventricular white matter of 11-day-old rat. Era. Eos. x 928; d Mitoses in the subependymal plate in 30-day-old rat. Era. Eos. x 928

Adenylate CyclaseActivity Assay Samples of paraventricular white matter of control and treated rats have been examined. Tissue was gently homogenized at 0~ in 50 volumes of 80 mM tris-mateate buffer (pH 7.5). Adenylate cyclase activity was measured using adenosine triphosphate-8-1~C as substrate, as described by Kebabian et al. (1972). The incubation mixture of 100 gl contained 80 mM tris-maleate buffer, 1 mM 14C-ATP (specific activity 7--106cpm/gmol), 2 mM MgSO4, 15 mM phosphocreatine, 10 mM theophylline, 0.5 mM ethyleneglycol-bis-(beta-amino-ethylether)N,N'-tetra acetic acid (EGTA), 50 gg of creatinephosphokinase and tissue homogenate containing 100 gg of protein. After incubation for 2.5' the reaction was stopped by boiling the tubes for 3'. Radioactive cAMP was separated from radioactive ATP using alumina and Dowex columns (Mao and Guidotti, 1974).

the contrary, occasional in the other structures in b o t h animal groups. After the 30th day, they continue to be present in the subependymal layer in considerable amount. In the other structures they disappear with the exception of paraventricular white matter, where they are still present until the 60th day in b o t h animal groups (Fig. 1).

Morphology

F r o m the 30th day on, circumscribed or diffused cell hyperplasias can be observed in the paraventricular white matter (Fig. 2a). Morphologically, they only consist in an increased n u m b e r o f nuclei, which do n o t differ f r o m those o f the n o r m a l paraventricular white matter, i.e., mostly small dark nuclei. In m a n y animals hyperplasias have been recognized only by evaluating the cell density per microscopic field (Fig. 3). Hyperplasias contain mitoses, but in an a m o u n t n o t exceeding that o f the normal white matter.

F r o m birth until the 30th day o f extrauterine life there is no difference between treated and control rats in the ventricular germinal zone, cortex, periventricular white matter, subependymal layer and basal ganglia: mitoses are frequent in the ventricular germinal zone and, on

D u r i n g the 2nd month, reactive astrocytes appear (Fig. 2b) and the lesion becomes the already k n o w n "early neoplastic proliferation" (ENP) described by Koestner et al. (1971), Lantos and Cox (1976), and Schiffer et al. (1978).

Results

D. Schifferet al. : LatencyPeriod of ExperimentalBrain Tumors

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Fig. 2. a Hyperplasiain the hemisphericwhite matter. Em. Eos. x 285; b Appearance of reactive astrocytes. Em. Eos. x 950

Cytofluorimetry The histograms of cellular DNA in the medullary centre and paraventricular white matter are shown in Fig. 4. Most cells are diploid and only few are up to tetraploid. There is no cell with DNA more than tetraploid and there is no difference between treated and control rats until the 60th day.

Adenylate Cyclase Activity Figure 5 shows that on the 15th day of extrauterine life the adenylate cyclase of treated rats almost doubles that of controls. Thereafter, it decreases until the 60th day. In controls the decrease begins only on the 45th day. After the 75th day, in agreement with previous reports (Racagni et al., 1979), the enzyme activity is constantly lower in treated rats than in controls.

Discussion

The present investigation demonstrates that the very early tumoral transformation takes place on the 30th day of extrauterine life as simple cell hyperplasia, i.e., I month earlier than already known (Lantos and Cox, 1976; Schiffer et al., 1978). The longitudinal scheme of tumor development after transplacental ENU, already proposed by Schiffer et al. (1978), may be thus corn-

pleted with the addition of hyperplastic phases on the 30th day of extrauterine life (Fig. 6). The cell composition of hyperplasias is similar to that described by Lantos and Pilkington (1979) in abnormal cell clusters of subependymal plate in 8-week-old rats. It is to be remarked that in the 1st month of life there are few mitoses in the paraventricular white matter, both in treated and control rats, and this is consistent with the finding of few cells up to tetraploid by cytofluorimetry. The dividing cells probably represent glioblasts or the so-called free subependymal cells coming from the subependymal layer and migrating and dividing into the corpus callosum (Lewis, 1968a; Paterson et al., 1973) as light or medium dark oligodendrocytes. The number of mitoses does not increase in hyperplasias. The latency period of tumor development results to be halved, from 2 to 1 month in our observation. However, what happens in this period remains still to be clarified and only speculative considerations can be made. By autoradiography, the cell cycle time in early neoplastic proliferations, i.e., the tumor stage immediately following hyperplasias, results to be about 20 h (Giordana et al., 1979). This figure is not far from that calculated for the subependymal plate in adult rats by Lewis (1968b). On the contrary, the labeling index of ENPs is very low, about 3 ~ (Giordana et al., 1979), in

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