CELL-FREE PROTEIN SYNTHESIS BY RAT BRAIN IN TRIETHYL TIN INTOXICATION

Arta Ncurol. Scandinav. 50, 103-108, 1974 Department of Neurology, Medical Academy, Poznan, Poland. CELLFREE PROTEIN SYNTHESIS BY RAT BRAIN IN TRIETHYL TIN INTOXICATION M. WENDER, B. ZGORZALEWICZ and A. PIECHOWSKI ABSTRACT An acute cerebral oedcma was induced in rats by administration of tricthyl tin a t a dose of 4 mg per kg body weight. Studies involved tracing of L-[*4C] lcucinc and I,-[14C] proline incorporation to the postmitochondrial fraction of the cerebral white and grey matter. Morphological studies disclosed spongious changes in the white matter (typical for TET intoxication). Biochemical studies demonstrated a pronounced reduction of labelled lcucin incorporation, particularly in the white matter. A reduction of incorporation of the other amino acid studied (proline) was not^ so marked, although significant for the white matter. The results seem to indicate that a diminished protein bio- synthesis is an essential although certainly not the only metabolic cause of the white matter lesion, particularly of the myelin, in TET intoxication. Triethyl tin intoxication is an experimental model which enables us to study basic phenomena occurring in nervous tissue during cytotoxic cerebral oedema. hlorphologically, the oedema precipitated by alkyl tin compounds may be distinguished by a selective lesion of the white matter with formation of vacuoles between myelin sheaths (Aleu et al. 1963, Hirano et al. 1968). This results in permanent spongious changes within the white matter. Therefore, triethyl tin intoxication lends itself well also as a model for studies on the degenerative processes of myelin sheaths. Disturbances in protein metabolism play an important role in the patomechanism of cerebral oedema. In many variations of the process, particularly in those associated with an increased filtration of plasma proteins to nervous tissue, high albumin levels are found in the oedematous fluid (Kaps 1954, Kalsbeck & Cumings 1962, Lowenthal 1964). An increase in the total protein content, described in different models of experimental oedema, is also of interest. Hauser e f al. (1963) The work was performed on behalf of the Committee of Science and Technics (No. 09.4.1.3.2.2.). 104 observed it in cerebral oedema induced by Psyllium seed implantation, while Klatzo et al. (1958)-in oedema provoked by freezing the brain. The same was found in our studies (Wender et al . 1972) on cytotoxic oedema induced by triethyl tin intoxication, in spite of the fact that no parallel increase in the permeability of the blood-brain barrier was detected. The question arises whether the phenomenon results from changes in protein bonds structure of the pre-injury insoluble proteins with subsequent release of soluble products or whether it is caused by an increased biosynthesis of proteins. Experimental studies of Nakazawa (1968) proved the latter to be possible. The author demonstrated an increase in the specific activity of proteins evaluated by the incorporation of glycine, in cerebral oedema provoked by cryoinjury. 'To clarify the problem, which is of importance in understanding the role of protein metabolism disturb- ances in a cytotoxic cerebral oedema, we have undertaken studies of protein synthesis in vitro in thiethyl tin intoxication. MATERIAL AND METHODS Experimental. The experiments were conducted on Wistar rats of either sex, weighing 200 to 300 g. Experimental animals were injected intraperitoneally with 4 mg triethyl tin sulphate (TET) per kg body weight. Three experimental groups comprised animals sacrificed 5 hours, 24 hours and 28 days following TET administration, respectively. ZZistolog(j. For routine histological studies brain sections were fixed in formalin, embedded in parafin, and following proccssing, stained with the Nissl, H + E and Kliiver-Barrera methods. The celloidin-embedded material was stained by the Woelcke method. Biochemistru. Protein biosynthesis was studied by tracing the in vitro L-[14C] leucine and L-[14C] proline incorporation to the postmitochondrial fraction (sepa- rately for the grey and white matter) prepared according to Johnson (1968). A portion of the postmitochondrial preparation (1.0-1.5 mg protein) was in- cubated at 30" C for 30 min in a reaction mixture (0.5 ml) which contained 25 pmoles of tris-HC1 (pH 7.4), 25 pmoles of KCl, 5 pmoles of MgCI,, 5 pmoles of 2-mercaptoethanol, 1.0 pmoles of ATPNa,, 0.1 pmole of GTPNa, and 0.25 pCi of Ir[14C] leucine o r L-[l4C] proline. Labelled amino acids were purchased from the Radiochemical Centre, Amersham. At the same time the control sample was in- cubated for 30 min at 0" C. The acid insoluble protein was extracted by the method of Rubin et al. (1967) and the amount of radioactivity was determined with a USB-2 liquid scintillation counter. The counts per min obtained were computed per mg protein of the postmitochondrial fraction (specific activity). The latter was quanti- fied by the method of Kings ley (1942). RESULTS In experimental animals a clinical syndrome developed, corresponding to that described by Magee et al. (1957) as typical for TET-induced cerebral oedema. 105 Figure I. Spongious changes in the corpus callosum of the TET-intoxicated rat. Kliiuer-Barrera staining. (x 52) . Histological examination in the experimental animals disclosed dif- fuse spongious changes of the white matter in the form of numerous irregular and sharply delineated empty spaces (Figure 1 ) . The changes were seen both in the early and in the late period following intoxication. No glial or mesenchymal reaction could be observed in the oedematous white matter. In the grey matter and particularly in the cerebral cortex, an injury of neurocytes was observed in the form of acute vacuolar degeneration. The Virchow-Robin spaces were broadened everywhere in the brain. The results of L-leucine incorporation into the postmitochondrial fraction in TET intoxication are presented in Table 1. A very marked decrease of incorporation in all the studied periods of experimental disease was found, most pronounced in the white matter of the brain. For the grey matter the decrease is also scen although it is significant only in some experimental groups. The decrease in proline incorpora- 106 tion into the proteins of the postmitochondrial fraction of the brain was less pronounced although statistically significant in all stages for the cerebral white matter and 28 days following intoxication for the grey matter. Details of the results are presented in Table in Table 2. 7-able I . Incorporation o f labelled L-leucine into postmitochondrial fraction in triethyl t in intoxication ( in total count s /min /mg protein). Cerebral white matter Cerebral grey matter Normal values 1208k7G 1566 2 67 5 hours after intoxication 24 hours after intoxication 28 days after intoxication 468 f 31 P < 0.05 440 f 46 P < 0.05 3 0 1 f 1 7 P < 0.05 1041 2 72 P < 0.05 1358 f 141 P < 0.05 755f113 P < 0.05 Number of animals in each group : 10 Mean f S.E. Table 2. Incorporation of labelled L-proline into postmitochondrial fraction in triethgl t in intoxication ( in total count s /min /mg protein). Cerebral white matter Cerebral grey matter Normal values 3 6 5 f 3 7 5 hours after intoxication 24 hours after intoxication 167221 P < 0.05 135214 P < 0.05 28 days after 132211 intoxication P < 0.05 Numbcr of animals in each group: 10 Mean k S.E. 568 2 49 G14f34 P < 0.05 740 f 3 2 P < 0.05 327235 P < 0.05 DISCUSSIOS Studies in the in vitro condition provide an insight into the protein biosynthetic processes in nervous tissue with elimination of effects of the cell membrane permeability. The latter factor is crucial in de- termining of labelled amino acid incorporation into cerebral proteins, when the amino acids are administered intravenously or intraperi- toneally. Hence, particularly valuahle in experimental pathology are the studies performed on the postrnitochondrial fraction, free of mito- 107 chondria, myelin, synaptosomes and cell nuclei (Johnson 1968) . The fraction contains an energy-generating system ; the incorporation is however higher if the system is supplemented by exogenous com- ponents. The effect of TET intoxication on cell-free protein synthesis was studied in the postmitochondrial fraction, isolated from cerebral grey or white matter removed at different times following an acute experi- mental intoxication. The results obtained indicate that alkyl tin com- pounds strongly influence the aniount of radioactive amino acids incorporated into protein. The sudden reduction of protein biosyn- thesis is much more evident in the white matter than in the grey matter, which is in line with the morphological changes being also more evident in the white matter. This may suggest that changes in biosynthesis of proteins--one of the main macromolecular components of nervous tissue--are an important although certainly not the oldy metabolic cause of injury of the white matter, particularly of myelin, in this disease. In our studies we have discovered also that, even after a prolonged period ( 4 weeks after intoxication), the protein bio- synthesis does not reach its starting level. Again, this is in parallel with morphological studies which indicate on the one hand a per- manent type of intramyelinic change and, on the other hand, their exclusively degenerative character with no glial or mesenchymal reaction. Experimental studies performed up till now have been detected a spectrum of deviations in the content and composition of proteins in TET-induced oedema, with no increase in the permeability of cell membranes forming the blood-brain barrier (Bakay 1965). Kalsbeck & Cuminys (1962) detected an additional albumin fraction (fraction 6) in starch gel electrophoresis of TET-intoxicated brain proteins. We were unable to confirm the finding using polyacrylamide gel electro- phoresis (Wender e t al. 1972). The difference may however be accounted for by the divergence in the methods employed. In our studies, protein extraction was preceded by perfusion, aimed at wash- ing out the blood from nervous tissue. In the studies we found an evident increase in the content of soluble proteins in the TET-induced cerebral oedema. We suggested that this may result from changes in physicochemical properties of structural proteins. A further suggestion stated that such changes might result in an increased water-binding ability by the proteins. This could contribute in the development of cerebral oedema in TET-intoxicated animals. Results of the present studies, indicating a markedly reduced incorporation of labelled leucine and proline into cerebral proteins of the postmitochondrial fraction, 108 support the contention. They clearly indicate, that increase in a soluble protein level in nervous tissue cannot be interpreted in terms of in- creased protein biosynthesis, as found for another type of cerebral oedema (following cryoinjury) by Naliazawa (1968). REFERENCES Aleu, F., H. 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Goldstein (1967): The determination of transfer ribo- nucleic acid by aminoacylation. I. Leucine and phenylalanine transfer ribo- nucleic acid from E. coli Bi. Analyt. Biochem. 20, 533-544. Wender, M., A. Piechowski & A. Wajgt (1972): Soluble cerebral proteins in experimental demyelinating processes. Exp. Path. 7 , 290-297. 840-846. Received August 21, 1973. hi. Wender, M.D. Department of Neurology Medical Academy Poznan Poland 49 Przybyszewskiego Str.