of Nrtr,.ocbi,iiii\tr!. Vol 32. pp 187-190 Pergamon Press Ltd. 1979 Printed In Great Britain 0 International Society for Neurochemistry Ltd
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THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES G. KONAT,H. OFFNERand J. CLAUSEN The Neurochemical Institute, 58, Ridmandsgade, DK-2200 Copenhagen N, Denmark (Received 20 June 1978. Recised 7 August 1978. Accepted 28 August 1978) Abstract-Rats (20-day-old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [14C]leucine into the acid-insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt, was added in citro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.
THEBIOSYNTHESIS of myelin membrane in the developing rat brain can be restrained by treating the animals with triethyllead (PbEtJ (KONAT& CLAUSEN,1974). Among different myelin constituents, proteins are the most severely affected and seem to be the limiting factor in the membrane assembly. Furthermore, the deposition of myelin proteins is more hampered by PbEt,-induced intoxication that the deposition of total tissue proteins (KONAT & CLAUSEN, 1976; KONATet al., 1976). Studies in uitro (KONATrt al., 1978) demonstrated that the inhibition of protein synthesis is secondary t o the suppression of cellular energy-generating processes brought about by PbEt,. In uiuo, however, more complicated conditions may arise due to the presence of possible PbEt, metabolites. The aim of the present communication was to study the specificity of PbEt, toward the myelin protein synthesis in the forebrain slices derived from the intoxicated animals as well as in the slices treated with the toxin in uitro. The investigation also included studies on the metabolism of PbEt, in the young rat forebrain during the course of intoxication. MATERIALS A N D METHODS Materials. ~-[U-'~C]Leucine(280 Ci/mol) and [ I4C] toluene standard source (4.15 x lo5 d.p.m./ml) were obtained from New England Nuclear Chemicals GmbH, Dreieichenhain, West Germany, and NCS tissue solubilizer was from Amersham/Searle. Arlington Heights, IL. Triethyllead chloride was agift from The Associated Octel Company Ltd., London. All other chemicals were of analytical grade from Merck, Darmstadt, West Germany. A n i m d s . White female Wistar rats of specific pathogen free type were used throughout the studies. PbEt, (saline solution, 2 mg/ml) was administered intraperitoneally to 20-day-old animals at a dose of 8 mg/kg body weight and successively 4 days later at a dose of 5mg/kg (KONATet al., 1976). All animals were killed by decapitation under
light ether anaesthesia and the forebrains dissected (KONAT & CLAUSEN, 1976). Incubation methods. The tissue was sliced by means of a mechanical tissue chopper t o yield slices 0.36 mm thick. The slices were transferred into 50 ml conical flasks, suspended in 20 vol of Krebs-Ringer bicarbonate buffer fortified with 10 mwglucose (incubation medium) and placed in a water bath at 38°C. The reaction was initiated by adding 1.5 pCi of [14C]leucine and the incubation was carried out with constant shaking under an atmosphere of 95"4 02-59(, COz. At the end of incubation the flasks were placed in ice and the medium decanted. The slices were washed with 20ml of ice-cold saline, suspended again in 20 ml saline and centrifuged at 500 y for 5 min. Preparation of ntplin. Myelin was isolated from the tissue slices by the flotation method (KONAT& CLAUSEN, 1974; 1976) with some modifications. Briefly, the tissue was homogenized in 10 vol of 0.25 M-sucrose, diluted with an additional 20vol of the same sucrose solution and then centrifuged at 27,000 y for 20 min. The pellet was resuspended in 30 ml of 0.88 M-sucrose, overlayed with 20 ml of 0.25~-sucrose and centrifuged for 60min at 106,900 gmar The interfacial material was collected and subjected to two osmotic shocks lasting overnight and 20 min respectively. The osmotically shocked myelin was recycled through the discontinuous gradient step as above, freed of sucrose and lyophilized. The purity of the myelin fraction was confirmed by its protein pattern on polyacrylamide gel electrophoresis and the absence of other subcellular marker enzyme activities (KONAT& CLAUSEN, 1978). Electron microscopy (by courtesy of Prof. 0. BEHNKE) showed typical multilamellar myelin figures and no other recognizable cellular elements. Ratlioactiiity counting. The incorporation of ['4C]leucine into the acid-insoluble protein was determined in the tissue homogenates essentially as described previously (KONATrt a/., 1978). Samples of lyophilized myelin (5 mg) were extracted twice with 8 ml of diethylether-ethanol (3:2. v/v) prior to the solubilization in NaOH and were further processed as the homogenate material. Radioactivity determination was carried out in a Beckman LS-230 liquid scintillation counter with Carbon-14 Above Tritium Iso-Set module. Counting efficiency determined by the internal
187
G. KONAT,H. OFFNER and J. CLAWEN
188
TABLE1. FOREBRAIN WEIGHT A N D RECOVERY OF MYELIN FROM THE TISSUE SLICES AFTER 2 h INCUBATION IK KREBSRINGERBICARBONATE BUFFER AT 38°C
all the lead present in the organ was in the form of PbEt,. There was no detectable PbEt, in the control forebrains at any age, and the total lead (in this case the inorganic lead) content was below I nmol Forebrain Age weight Myelin yield per g wet tissue. The maximal PbEt, level was Group (days) (g) (mg/forebrain) detected 1 day after each intraperitoneal injection. The toxin was constantly removed from the tissue 20 1.04 f 0.03 6.9 f 0.6 Control during the following days. The maximal rate of PbEt, 1.10 k 0.03 10.6 & 0.5 23 Control removal was about 3.5 nmol per day from 1 g of the 23 1.03 & 0.03 9.5 f 0.8 PbEt,-treated 27 1.14 f 0.03 14.2 f 1.0 Control forebrain. Thus, during the 4 days following the first 1.04 0.02* 11.1 f. 1.1* 27 PbEt ,-treated injection, the toxin concentration dropped by 25% as Control 30 1.16 f 0.03 17.2 f 0.9 compared to 21st day level. The maximal PbEt, level 30 1.07 k 0.03* 12.9 k ].I* PbEt,-treated was restored by the second injection and then steadily Control 21.1 1.2 34 1.18 f 0.03 decreased from the 25th day onward. By the 34th 1.10 f 0.03* 34 14.9 1.3* PbEt,-treated day the PbEt, level was less than 10% of the maximal Each value represents the mean ~ s . D for . four to twelve value. animals. The rate of incorporation of ['4C]leucine into the * Values significantly different from corresponding contotal protein in the forebrain slices plotted as a functrol values. tion of age is depicted in Fig. 1. In the case of control standard method using ['4C]toluenc standard was about animals the protein synthetic activity decreased linearly with age. There was about 38% loss in the 6 1 x for the present samples. Lead determination. PbEt, was estimated in the benzene activity over the period of 14 days of the experiment. (1968). For The protein synthesis in the forebrains from PbEt,extracts prepared according to BOLANOWSKA total lead determination the tissue was solubilized with treated animals was initially restrained by about 17% tetramethylammonium hydroxide essentially as described as comparcd to the controls. However, on 30th day by RAsroGI & CLAUSEN (1976). The concentration of t h e the activity began to increase and on 34th day of element was measured in the samples by a Perkin-Elmer life exceeded that of the controls by about 17%. 460 flameless atomic absorption spectrophotometer at Regarding the myelin proteins the synthetic activity 283.3 nm. Statistical eoaluution. Data were analysed by the differ- of the tissue was also age-dependent and had the ences between means and the level of significance was highest value in 20- to about 27-day-old forebrains 1953). (Fig. 2). In animals older than 27 days the activity based upon the Student-t distribution (CROXTON, Differences with P values less than 0.005 were considered declined steadily with age. The intoxication caused to be significant. a significant (4G50%) inhibition of the incorporation during the first 7 days of the experiment. From about 27th day onward the rate of the myelin protein synRESULTS thesis increased in the intoxicated forebrains and The maturation process in the forebrains of intoxicated animals was significantly hampered as seen from both organ weight and myelin deposition (Table 1). The inhibition values were virtually identical to those reported previously (KONATet al., 1976). Table 2 shows the forebrain lead level in the course of intoxication, The values for total and organic lead were very close to each other at a given age. The differences between the corresponding values were all statistically nonsignificant and probably attributable to analytical error. Thus, it can be concluded that
*
350h
TABLE2. CONCENTRATION
OF TOTAL LEAD
A N D I R I E T H Y L L E A D IN THE FOREBRAINS OF IN-
TOXICATEU RATS
Age (days) 21 24 25 27 34
Lead (nmol/g wet weight) Total PbEt, 33.0 f 3.5 22.8 3.4 39.0 f 3.8 24.5 i 3.5 2.0 4.7
*
36.1 26.8 35.1 27.5
f 5.0 k 3.3 f 5.6 f 2.7 3.0 f 1.8
Each value represents the mean from four animals.
+s.D.
Day5
FIG. I. Incorporation of ['4C]leucine into total protein in the forebrain slices prepared from control (open circles)
and PbEt,-treated (solid circles) rats. All incubations were carried out for 2 h. Values are expressed as the radioactivity per forebrain. Each point represents the mean value from four to six animals. Vertical bars indicate S.D.
Triethyllead and myelin protein synthesis
20
I 30
25
I
Days
FIG.2. Incorporation of ['4C]leucine into myelin proteins in the forebrain slices prepared from control (open circles) and PbEt,-treated (solid circles) rats. All incubations were carried out for 2 h. Values are expressed as the radioactivity per forebrain. Each point represents the mean value
from four to six animals. Vertical bars indicate S.D. amounted to about 75% of the control value on 34th day post partum. The objectives of the following experiments were to investigate the uptake of PbEt, by the forebrain slices as well as the effect of the toxin added in v i m on the protein synthesis. The kinetics of thesc processes in the slices prepared from 27-day-old control forebrains is presented in Fig. 3. Under the cxperimental conditions employed in these studies, the level of PbEt, in the tissue was positively correlated with the initial concentration of the toxin in the incubation medium, however, the relationship was not linear. The protein synthesis in the forebrain slices was sensitive to PbEt, added in vitvo into the incubation medium. At every PbEt, concentration studied, the incorporation of the labelled amino-acid into the myelin proteins was inhibited more than the incorporation into the total protein. The difference was pronounced and statistically significant up to an . 1.5 pM-PbEt, alkyllead concentration of 5 p ~ Thus, (initial concentration in the incubation medium) caused 27 and 10% inhibition of the myelin and total protein synthesis respectively. DISCUSSION
The pattern of the forebrain growth and the deposition of myelin in the course of PbEt,-induced intoxication was virtually the same as observed previously (KONATet al., 1976; KONAT& CLAUSEN, 1978) providing evidence for the reproducibility of this hypomyelination model. However, the amount of myelin recovered from the incubated forebrain slices was about 10% lower than the amount recovered from the freshly dissected tissue (KONAT& CLAUSEN,1976; KONATet a/., 1976). The loss of myelin was probably due to the washing of slices at the end of incubation, Y.r j 2 ' I
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189
as this process removed some fine tissue particles leaving only big tissue flakes. Furthermore, SMITH& SEDGEWICK (1975) demonstrated that during the incubation of brain slices in Krebs-Ringer bicarbonate buffer some myelin fragments decrease their intrinsic density. Formation of myelin fraction isopycnic with 0.25 M-sucrose might also contribute to thc lower yield of myelin from the incubated forebrain slices. The present studies made it possible to correlate the observed pathological events with the concentration of PbEt, in the forebrain. All the lead in the tissue was found in the form of PbEt,. PbEt, has been demonstrated to be a stable metabolite of tetraethyllead (CREMER, 1959; STEVENS et a[.. 1960: BOLANOWSKA, 1968). Thus, the present results are in (1968) agreement with data reported by BOLANOWSKA on the cerebral lead content of rats injected with tetraethyllead. Furthermore, human brains from fatal tetraethyllead poisoning also contained PbEt, as a el nl., 1967). The sole form of lead (BOLANOWSKA maximal cerebral PbEt, content was about 35 nmol/g, and this concentration seems to be critical. Administration of the toxin in doses higher than 8 and 5mg/kg for the first and the second injection respectively, resulted in very unequal development of
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3. PbEt, content and protein synthesis in the forebrain slices incubated with different concentrations of the alkyllead. Forebrains of 27-day-old rats were used. To study the PbEt, binding, 1 g of the tissue slices was incubated for 10 min in the incubation medium containing PbEt,. After that time the tissue slices were pelleted by centrifugation. homogenized in redistilled water and PbEt, content was determined. In the protein synthesis experiments the tissue slices prepared from one forebrain were incubated for 2 h as described in Materials and Methods. In the case of the forebrain slices incubated without PbEt, the amount of radioactivity incorporated into the total and the myelin proteins was 262 k 17 x 103c.p.m. and 7.9 k 0.6 x 10' c.p.m. respectively. A+oncentration of PhEt, in the forebrain slices, .-total protein synthesis, 0 -myelin protein synthesis. Each point represents the mean calculated from four to six experiments. S.D. values were below 10% of the corresponding mean values. FIG.
190
G. KONAT, H. OFFNER and J . CLAUSEN
animals within the experimental group. This mani- the incubation medium. It has been postulated that fested by persistent paralysis, severe growth retarda- the synthesis of integral membrane proteins, as comtion and finally death of some of the most affected pared to soluble proteins, requires an elaborate metaanimals (KONAT,unpublished observations). The in- bolic processing in the cell [ROTHMAN& LEKARD, jection schedule used in this laboratory produced pro- 1977). Thus, the difference in the inhibition values for nounced retardation of the forebrain development, the total and for the myelin protein synthesis suggests but there was no mortality and the variation of differ- a disturbance of some processes involved in the postent parameters among PbEt,-treated animals was translational furnishing of the myelin membrane procomparable to the normal individual variation within teins brought about by PbEt,. the control group. The protein synthesis was significantly inhibited in Acknowledgements--Gratitude is expressed to Prof. 0. the slices prepared from the intoxicated animals as BEHNKE,Medicinsk-Anatomisk Institut, University of well as in the slices, to which PbEt, was added in Copenhagen for electron microscopic evaluation of the vitro. At a similar concentration of toxin in the tissue, myelin samples. The determination of lead o n atomic the inhibition values were very close to each other absorption spectrophotometer was performed at the in these two systems. Thus, as can be computed from School of Pharmacy, Copenhagen with kind advice from Dr. BETTYMOGENSEN. the absorption curve in Fig. 3, under the experimental conditions, the PbEt, content of the forebrain slices REFERENCES prepared from 27-day-old intoxicated rats was reduced from 27.5 to 18.5nmol/g due to diffusion of BOLANOWSKA W. (1968) Distribution and excretion of the toxin into the incubation medium. This PbEt, triethyllead in rats. Br. J . ind. M e d . 25, 203-208. concentration produced 17 and 48% inhibition of the BOLANOWSKA W., PIOTROWSKI J. & GARCZYNSKI H. (1967) Triethyllead in the biological material in cases of acute total and myelin protein synthesis respectively. tetraethyllead poisoning. Arch. Toxikol. 22, 278-282. Almost identical (19 nmol/g) PbEt, tissue concenCREMER J. E. (1959) Biochemical studies on the toxicity tration was obtained by suspending 27-day-old conof tetraethyllead and other organo-lead compounds. Br. trol forebrain slices in the incubation medium conJ . ind. Med. 16, 191-199. taining 3 p - P b E t , . In this case, the total and myelin CROXTON F. E. (1953) in Elenlentary Statistics with Appliprotein synthesis were inhibited by 19 and 39% recation in Medicine and the Biological Sciences. Dover, spectively. The results indicate that PbEt, present in New York. the tissue is itself toxic to the CNS cells and is respon- KONATG. &,CLAUSEN J. (1974) The effect of long-term sible for the depressed metabolic activity as exempliadministration of triethyllead on the developing rat brain. Environ. Physiol. Biochein. 4, 236-242. fied by the protein synthesis. J. (1976) Triethyllead-induced hypoG. & CLAUSEN With the decreasing cerebral PbEt, concentration, KONAT myelination in the developing rat forebrain. Expl Neurol. the protein synthetic activity of the forebrain slices 50, 124133. improved. In the case of the total protein, the activity J. (1978) Protein composition of even exceeded the control values on 34th day. It can KONATG. & CLAUSEN forebrain myelin isolated from triethyllead-intoxicated be assumed that in the course of intoxication the foreyoung rats. J . Neurocheni. 30, 907-909. brain cells developed some mechanisms to overcome KONAT G . , OFFNER H. & CLAUSEN J. (1976) Triethylleadthe metabolic suppression caused by PbEt,. When restrained myelin deposition and protein synthesis in the the toxin concentration decreased, the tissue revealed developing rat forebrain. Expl Neurol. 52, 58-65. an activation of the metabolism as compared to the KONAT G., OFFNER H. & CLAUSEN J. (1978) Effect of triethyllead on protein synthesis in rat forebrain. Expl control. Also in the case of the myelin protein synNeurol. 59, 162-167. thesis, the tendency to catch up with the control was J. (1976) Absorption of lead evident during the second half of the intoxication RASTOGIS. C. & CLAUSEN through the skin. Toxicology 6 , 371-376. period. J . (1977) Membrane asymmetry. The incorporation of radiolabelled leiicine into the ROTHMANJ. E. & LENARD Science, N . K 195, 743-753. myelin proteins was more vulnerable than the synSMITHM. E. & SEDGEWICK L. M. (1975) Studies of the thesis of the total cerebral protein with respect to mechanism of demyelination. Regional differences in the inhibitory action of PbEt,. This feature was myelin stability in uitro. J . Neurochern. 24, 763-770. observed in the forebrain slices regardless of whether STEVENS C. D., FELDHAKE C. J. & KEHOE R. A. (1960) Isolathe slices were prepared from the PbEt,-intoxicated tion of triethyl-lead ion from liver after inhalation of animals or whether the toxin was added in vitro into tetraethyl-lead. J . Pharmac. exp. Ther. 128, 90-101.
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THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES G. KONAT,H. OFFNERand J. CLAUSEN The Neurochemical Institute, 58, Ridmandsgade, DK-2200 Copenhagen N, Denmark (Received 20 June 1978. Recised 7 August 1978. Accepted 28 August 1978) Abstract-Rats (20-day-old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [14C]leucine into the acid-insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt, was added in citro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.
THEBIOSYNTHESIS of myelin membrane in the developing rat brain can be restrained by treating the animals with triethyllead (PbEtJ (KONAT& CLAUSEN,1974). Among different myelin constituents, proteins are the most severely affected and seem to be the limiting factor in the membrane assembly. Furthermore, the deposition of myelin proteins is more hampered by PbEt,-induced intoxication that the deposition of total tissue proteins (KONAT & CLAUSEN, 1976; KONATet al., 1976). Studies in uitro (KONATrt al., 1978) demonstrated that the inhibition of protein synthesis is secondary t o the suppression of cellular energy-generating processes brought about by PbEt,. In uiuo, however, more complicated conditions may arise due to the presence of possible PbEt, metabolites. The aim of the present communication was to study the specificity of PbEt, toward the myelin protein synthesis in the forebrain slices derived from the intoxicated animals as well as in the slices treated with the toxin in uitro. The investigation also included studies on the metabolism of PbEt, in the young rat forebrain during the course of intoxication. MATERIALS A N D METHODS Materials. ~-[U-'~C]Leucine(280 Ci/mol) and [ I4C] toluene standard source (4.15 x lo5 d.p.m./ml) were obtained from New England Nuclear Chemicals GmbH, Dreieichenhain, West Germany, and NCS tissue solubilizer was from Amersham/Searle. Arlington Heights, IL. Triethyllead chloride was agift from The Associated Octel Company Ltd., London. All other chemicals were of analytical grade from Merck, Darmstadt, West Germany. A n i m d s . White female Wistar rats of specific pathogen free type were used throughout the studies. PbEt, (saline solution, 2 mg/ml) was administered intraperitoneally to 20-day-old animals at a dose of 8 mg/kg body weight and successively 4 days later at a dose of 5mg/kg (KONATet al., 1976). All animals were killed by decapitation under
light ether anaesthesia and the forebrains dissected (KONAT & CLAUSEN, 1976). Incubation methods. The tissue was sliced by means of a mechanical tissue chopper t o yield slices 0.36 mm thick. The slices were transferred into 50 ml conical flasks, suspended in 20 vol of Krebs-Ringer bicarbonate buffer fortified with 10 mwglucose (incubation medium) and placed in a water bath at 38°C. The reaction was initiated by adding 1.5 pCi of [14C]leucine and the incubation was carried out with constant shaking under an atmosphere of 95"4 02-59(, COz. At the end of incubation the flasks were placed in ice and the medium decanted. The slices were washed with 20ml of ice-cold saline, suspended again in 20 ml saline and centrifuged at 500 y for 5 min. Preparation of ntplin. Myelin was isolated from the tissue slices by the flotation method (KONAT& CLAUSEN, 1974; 1976) with some modifications. Briefly, the tissue was homogenized in 10 vol of 0.25 M-sucrose, diluted with an additional 20vol of the same sucrose solution and then centrifuged at 27,000 y for 20 min. The pellet was resuspended in 30 ml of 0.88 M-sucrose, overlayed with 20 ml of 0.25~-sucrose and centrifuged for 60min at 106,900 gmar The interfacial material was collected and subjected to two osmotic shocks lasting overnight and 20 min respectively. The osmotically shocked myelin was recycled through the discontinuous gradient step as above, freed of sucrose and lyophilized. The purity of the myelin fraction was confirmed by its protein pattern on polyacrylamide gel electrophoresis and the absence of other subcellular marker enzyme activities (KONAT& CLAUSEN, 1978). Electron microscopy (by courtesy of Prof. 0. BEHNKE) showed typical multilamellar myelin figures and no other recognizable cellular elements. Ratlioactiiity counting. The incorporation of ['4C]leucine into the acid-insoluble protein was determined in the tissue homogenates essentially as described previously (KONATrt a/., 1978). Samples of lyophilized myelin (5 mg) were extracted twice with 8 ml of diethylether-ethanol (3:2. v/v) prior to the solubilization in NaOH and were further processed as the homogenate material. Radioactivity determination was carried out in a Beckman LS-230 liquid scintillation counter with Carbon-14 Above Tritium Iso-Set module. Counting efficiency determined by the internal
187
G. KONAT,H. OFFNER and J. CLAWEN
188
TABLE1. FOREBRAIN WEIGHT A N D RECOVERY OF MYELIN FROM THE TISSUE SLICES AFTER 2 h INCUBATION IK KREBSRINGERBICARBONATE BUFFER AT 38°C
all the lead present in the organ was in the form of PbEt,. There was no detectable PbEt, in the control forebrains at any age, and the total lead (in this case the inorganic lead) content was below I nmol Forebrain Age weight Myelin yield per g wet tissue. The maximal PbEt, level was Group (days) (g) (mg/forebrain) detected 1 day after each intraperitoneal injection. The toxin was constantly removed from the tissue 20 1.04 f 0.03 6.9 f 0.6 Control during the following days. The maximal rate of PbEt, 1.10 k 0.03 10.6 & 0.5 23 Control removal was about 3.5 nmol per day from 1 g of the 23 1.03 & 0.03 9.5 f 0.8 PbEt,-treated 27 1.14 f 0.03 14.2 f 1.0 Control forebrain. Thus, during the 4 days following the first 1.04 0.02* 11.1 f. 1.1* 27 PbEt ,-treated injection, the toxin concentration dropped by 25% as Control 30 1.16 f 0.03 17.2 f 0.9 compared to 21st day level. The maximal PbEt, level 30 1.07 k 0.03* 12.9 k ].I* PbEt,-treated was restored by the second injection and then steadily Control 21.1 1.2 34 1.18 f 0.03 decreased from the 25th day onward. By the 34th 1.10 f 0.03* 34 14.9 1.3* PbEt,-treated day the PbEt, level was less than 10% of the maximal Each value represents the mean ~ s . D for . four to twelve value. animals. The rate of incorporation of ['4C]leucine into the * Values significantly different from corresponding contotal protein in the forebrain slices plotted as a functrol values. tion of age is depicted in Fig. 1. In the case of control standard method using ['4C]toluenc standard was about animals the protein synthetic activity decreased linearly with age. There was about 38% loss in the 6 1 x for the present samples. Lead determination. PbEt, was estimated in the benzene activity over the period of 14 days of the experiment. (1968). For The protein synthesis in the forebrains from PbEt,extracts prepared according to BOLANOWSKA total lead determination the tissue was solubilized with treated animals was initially restrained by about 17% tetramethylammonium hydroxide essentially as described as comparcd to the controls. However, on 30th day by RAsroGI & CLAUSEN (1976). The concentration of t h e the activity began to increase and on 34th day of element was measured in the samples by a Perkin-Elmer life exceeded that of the controls by about 17%. 460 flameless atomic absorption spectrophotometer at Regarding the myelin proteins the synthetic activity 283.3 nm. Statistical eoaluution. Data were analysed by the differ- of the tissue was also age-dependent and had the ences between means and the level of significance was highest value in 20- to about 27-day-old forebrains 1953). (Fig. 2). In animals older than 27 days the activity based upon the Student-t distribution (CROXTON, Differences with P values less than 0.005 were considered declined steadily with age. The intoxication caused to be significant. a significant (4G50%) inhibition of the incorporation during the first 7 days of the experiment. From about 27th day onward the rate of the myelin protein synRESULTS thesis increased in the intoxicated forebrains and The maturation process in the forebrains of intoxicated animals was significantly hampered as seen from both organ weight and myelin deposition (Table 1). The inhibition values were virtually identical to those reported previously (KONATet al., 1976). Table 2 shows the forebrain lead level in the course of intoxication, The values for total and organic lead were very close to each other at a given age. The differences between the corresponding values were all statistically nonsignificant and probably attributable to analytical error. Thus, it can be concluded that
*
350h
TABLE2. CONCENTRATION
OF TOTAL LEAD
A N D I R I E T H Y L L E A D IN THE FOREBRAINS OF IN-
TOXICATEU RATS
Age (days) 21 24 25 27 34
Lead (nmol/g wet weight) Total PbEt, 33.0 f 3.5 22.8 3.4 39.0 f 3.8 24.5 i 3.5 2.0 4.7
*
36.1 26.8 35.1 27.5
f 5.0 k 3.3 f 5.6 f 2.7 3.0 f 1.8
Each value represents the mean from four animals.
+s.D.
Day5
FIG. I. Incorporation of ['4C]leucine into total protein in the forebrain slices prepared from control (open circles)
and PbEt,-treated (solid circles) rats. All incubations were carried out for 2 h. Values are expressed as the radioactivity per forebrain. Each point represents the mean value from four to six animals. Vertical bars indicate S.D.
Triethyllead and myelin protein synthesis
20
I 30
25
I
Days
FIG.2. Incorporation of ['4C]leucine into myelin proteins in the forebrain slices prepared from control (open circles) and PbEt,-treated (solid circles) rats. All incubations were carried out for 2 h. Values are expressed as the radioactivity per forebrain. Each point represents the mean value
from four to six animals. Vertical bars indicate S.D. amounted to about 75% of the control value on 34th day post partum. The objectives of the following experiments were to investigate the uptake of PbEt, by the forebrain slices as well as the effect of the toxin added in v i m on the protein synthesis. The kinetics of thesc processes in the slices prepared from 27-day-old control forebrains is presented in Fig. 3. Under the cxperimental conditions employed in these studies, the level of PbEt, in the tissue was positively correlated with the initial concentration of the toxin in the incubation medium, however, the relationship was not linear. The protein synthesis in the forebrain slices was sensitive to PbEt, added in vitvo into the incubation medium. At every PbEt, concentration studied, the incorporation of the labelled amino-acid into the myelin proteins was inhibited more than the incorporation into the total protein. The difference was pronounced and statistically significant up to an . 1.5 pM-PbEt, alkyllead concentration of 5 p ~ Thus, (initial concentration in the incubation medium) caused 27 and 10% inhibition of the myelin and total protein synthesis respectively. DISCUSSION
The pattern of the forebrain growth and the deposition of myelin in the course of PbEt,-induced intoxication was virtually the same as observed previously (KONATet al., 1976; KONAT& CLAUSEN, 1978) providing evidence for the reproducibility of this hypomyelination model. However, the amount of myelin recovered from the incubated forebrain slices was about 10% lower than the amount recovered from the freshly dissected tissue (KONAT& CLAUSEN,1976; KONATet a/., 1976). The loss of myelin was probably due to the washing of slices at the end of incubation, Y.r j 2 ' I
L
189
as this process removed some fine tissue particles leaving only big tissue flakes. Furthermore, SMITH& SEDGEWICK (1975) demonstrated that during the incubation of brain slices in Krebs-Ringer bicarbonate buffer some myelin fragments decrease their intrinsic density. Formation of myelin fraction isopycnic with 0.25 M-sucrose might also contribute to thc lower yield of myelin from the incubated forebrain slices. The present studies made it possible to correlate the observed pathological events with the concentration of PbEt, in the forebrain. All the lead in the tissue was found in the form of PbEt,. PbEt, has been demonstrated to be a stable metabolite of tetraethyllead (CREMER, 1959; STEVENS et a[.. 1960: BOLANOWSKA, 1968). Thus, the present results are in (1968) agreement with data reported by BOLANOWSKA on the cerebral lead content of rats injected with tetraethyllead. Furthermore, human brains from fatal tetraethyllead poisoning also contained PbEt, as a el nl., 1967). The sole form of lead (BOLANOWSKA maximal cerebral PbEt, content was about 35 nmol/g, and this concentration seems to be critical. Administration of the toxin in doses higher than 8 and 5mg/kg for the first and the second injection respectively, resulted in very unequal development of
80 -
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3. PbEt, content and protein synthesis in the forebrain slices incubated with different concentrations of the alkyllead. Forebrains of 27-day-old rats were used. To study the PbEt, binding, 1 g of the tissue slices was incubated for 10 min in the incubation medium containing PbEt,. After that time the tissue slices were pelleted by centrifugation. homogenized in redistilled water and PbEt, content was determined. In the protein synthesis experiments the tissue slices prepared from one forebrain were incubated for 2 h as described in Materials and Methods. In the case of the forebrain slices incubated without PbEt, the amount of radioactivity incorporated into the total and the myelin proteins was 262 k 17 x 103c.p.m. and 7.9 k 0.6 x 10' c.p.m. respectively. A+oncentration of PhEt, in the forebrain slices, .-total protein synthesis, 0 -myelin protein synthesis. Each point represents the mean calculated from four to six experiments. S.D. values were below 10% of the corresponding mean values. FIG.
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G. KONAT, H. OFFNER and J . CLAUSEN
animals within the experimental group. This mani- the incubation medium. It has been postulated that fested by persistent paralysis, severe growth retarda- the synthesis of integral membrane proteins, as comtion and finally death of some of the most affected pared to soluble proteins, requires an elaborate metaanimals (KONAT,unpublished observations). The in- bolic processing in the cell [ROTHMAN& LEKARD, jection schedule used in this laboratory produced pro- 1977). Thus, the difference in the inhibition values for nounced retardation of the forebrain development, the total and for the myelin protein synthesis suggests but there was no mortality and the variation of differ- a disturbance of some processes involved in the postent parameters among PbEt,-treated animals was translational furnishing of the myelin membrane procomparable to the normal individual variation within teins brought about by PbEt,. the control group. The protein synthesis was significantly inhibited in Acknowledgements--Gratitude is expressed to Prof. 0. the slices prepared from the intoxicated animals as BEHNKE,Medicinsk-Anatomisk Institut, University of well as in the slices, to which PbEt, was added in Copenhagen for electron microscopic evaluation of the vitro. At a similar concentration of toxin in the tissue, myelin samples. The determination of lead o n atomic the inhibition values were very close to each other absorption spectrophotometer was performed at the in these two systems. Thus, as can be computed from School of Pharmacy, Copenhagen with kind advice from Dr. BETTYMOGENSEN. the absorption curve in Fig. 3, under the experimental conditions, the PbEt, content of the forebrain slices REFERENCES prepared from 27-day-old intoxicated rats was reduced from 27.5 to 18.5nmol/g due to diffusion of BOLANOWSKA W. (1968) Distribution and excretion of the toxin into the incubation medium. This PbEt, triethyllead in rats. Br. J . ind. M e d . 25, 203-208. concentration produced 17 and 48% inhibition of the BOLANOWSKA W., PIOTROWSKI J. & GARCZYNSKI H. (1967) Triethyllead in the biological material in cases of acute total and myelin protein synthesis respectively. tetraethyllead poisoning. Arch. Toxikol. 22, 278-282. Almost identical (19 nmol/g) PbEt, tissue concenCREMER J. E. (1959) Biochemical studies on the toxicity tration was obtained by suspending 27-day-old conof tetraethyllead and other organo-lead compounds. Br. trol forebrain slices in the incubation medium conJ . ind. Med. 16, 191-199. taining 3 p - P b E t , . In this case, the total and myelin CROXTON F. E. (1953) in Elenlentary Statistics with Appliprotein synthesis were inhibited by 19 and 39% recation in Medicine and the Biological Sciences. Dover, spectively. The results indicate that PbEt, present in New York. the tissue is itself toxic to the CNS cells and is respon- KONATG. &,CLAUSEN J. (1974) The effect of long-term sible for the depressed metabolic activity as exempliadministration of triethyllead on the developing rat brain. Environ. Physiol. Biochein. 4, 236-242. fied by the protein synthesis. J. (1976) Triethyllead-induced hypoG. & CLAUSEN With the decreasing cerebral PbEt, concentration, KONAT myelination in the developing rat forebrain. Expl Neurol. the protein synthetic activity of the forebrain slices 50, 124133. improved. In the case of the total protein, the activity J. (1978) Protein composition of even exceeded the control values on 34th day. It can KONATG. & CLAUSEN forebrain myelin isolated from triethyllead-intoxicated be assumed that in the course of intoxication the foreyoung rats. J . Neurocheni. 30, 907-909. brain cells developed some mechanisms to overcome KONAT G . , OFFNER H. & CLAUSEN J. (1976) Triethylleadthe metabolic suppression caused by PbEt,. When restrained myelin deposition and protein synthesis in the the toxin concentration decreased, the tissue revealed developing rat forebrain. Expl Neurol. 52, 58-65. an activation of the metabolism as compared to the KONAT G., OFFNER H. & CLAUSEN J. (1978) Effect of triethyllead on protein synthesis in rat forebrain. Expl control. Also in the case of the myelin protein synNeurol. 59, 162-167. thesis, the tendency to catch up with the control was J. (1976) Absorption of lead evident during the second half of the intoxication RASTOGIS. C. & CLAUSEN through the skin. Toxicology 6 , 371-376. period. J . (1977) Membrane asymmetry. The incorporation of radiolabelled leiicine into the ROTHMANJ. E. & LENARD Science, N . K 195, 743-753. myelin proteins was more vulnerable than the synSMITHM. E. & SEDGEWICK L. M. (1975) Studies of the thesis of the total cerebral protein with respect to mechanism of demyelination. Regional differences in the inhibitory action of PbEt,. This feature was myelin stability in uitro. J . Neurochern. 24, 763-770. observed in the forebrain slices regardless of whether STEVENS C. D., FELDHAKE C. J. & KEHOE R. A. (1960) Isolathe slices were prepared from the PbEt,-intoxicated tion of triethyl-lead ion from liver after inhalation of animals or whether the toxin was added in vitro into tetraethyl-lead. J . Pharmac. exp. Ther. 128, 90-101.
