EXPERIIv~ENTALNEUROLOGY
Effect
59,
of Triethyllead G. KONAT,
The
Neurochemical
Institute,
162-167 (1978)
on Protein
Synthesis
H. OFFNER,
AND J. CLAUSEN
58 Rddmandsgade, Received
Sep tenzber
DK-2200
in Rat Forebrain
Copenhagen
N,
Denmark
6,1977
Triethyllead ( PbEt) inhibited both oxygen utilization and L-[ U-l’C]leucine incorporation into acid-insoluble protein in a cell suspension prepared from 20-day-old rat forebrain. At the same concentrations of PbEtr, protein synthesis was inhibited more than respiration. The cell-free protein synthesis studied in the cytopalsmic fraction was not affected by the neurotoxin. Thus, PbEta does not directly interfere with the protein synthesizing systems and the observed inhibition is secondary to the suppression of the tissue oxidative metabolism.
INTRODUCTION Previous studies from our laboratory demonstrated that poisoning with triethyllead (PbEts) hampers the cerebral myelination processesin young rats (6-S). Unlike in the undernutrition model of experimental hypomyelination (9)) PbEts-induced poisoning resti-ains specifically brain tissue development (6). The suppressionof myelin biosynthesis is probably caused by inhibition of energy-generating processesbrought about by PbEts (1, 3). Furthermore, investigations by Wender et al. (11, 12) on cerebral protein synthesis revealed the inhibitory effect of triethyltin (SnEt,) at the level of amino acid activation. Because PbEt, closely resembles SnEtj in its biochemical activity (l), the present studies were designed to check whether or not there is any specificity of PbEt, toward the protein synthesizing system in the rat brain. MATERIALS
AND
METHODS
Triethyllead chloride was kindly supplied as a gift from The Associated Octel Company Ltd., London. L- [ U-14C]leucine (specific activity 280 Ci/ Abbreviations : TCA-trichloroacetic acid; ATP-adenosine 5’-triphosphate ; GTPguanosine 5’-triphosphate; KRCO&-Krebs-Ringer bicarbonate buffer with 10 g/liter glucose ; PbEk-triethyllead ; SnE&,-triethyltin. 162
0014-4886/78/0591-0162$02.00/O Copyright 0 1578 by Acadenkc P&a, Inc. All rights of reproduction in any form reserved.
TKIETIIYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
163
mol) was obtained from New England Chemicals GmbH, Dreieichenhain, West Germany, and NCS tissue solubilzer was from Amersham/Searle, Arlington Heights, Illinois. All other chemicals were of analytical grade from Merck, Darmstadt, West Germany. Two-day-old white female Wistar rats of specific pathogenic free type were obtained from Veterinary Surgeon Mpllle@rd’s Centre Ltd., Denmark, and maintained as previously ‘described (8) until the age of 20 days postpartum. The system of brain cell suspension (5) was utilized to study cellular protein synthesis and respiration. One forebrain was cut with scissors (50 random cuts) into small pieces, mixed with 5 vol Krebs-Ringer bicarbonate buffer fortified with 10 g/liter glucose (KRCOaG buffer), and gently pressed out of a glass syringe equipped with a 40-mm-long 1Pgauge needle. The pressing out procedure was repeated 10 times and the cell suspension was finally diluted with 25 ml of the same KRCOaG buffer. The incubation mixture for protein synthesis contained 600 ~1 freshly prepared cell suspension (equivalent to 19.5 mg wet weight of the original forebrain), 550 nCi [“Cl leucine, and KRCOSG buffer added to a total volume of 650 ~1. The tubes were flashed with 95% 02-5s COZ, covered with Parafilm, and incubated 30 min with shaking at 38°C unless otherwise indicated. The blanks were incubated at 0°C. At the end of incubation the tubes were placed in an ice bath, 5 ml cold saline was added, and the tubes were centrifuged 5 min at 2709 and 4°C. The pellet was suspended in 3 ml saline and solubilized by the addition of 4 drops 1 N NaOH. After 15 min at room temperature, 4 ml 10% trichloroacetic acid (TCA) was added and the tubes were centrifuged. The pellet was washed three additional times with 5 ml 5% TCA and once with ethanol. The protein precipitate was solublized in 1 ml NCS tissue solublizer. Heating at 50°C for 10 min promoted solubilization. After cooling, 15 ml scintillating fluid [6 g PPO (2,5-diphenyloxazole), 75 ml POPOP (1,4bis-2- (S-phenyloxazolyl) benzene), and toluene to a total volume of 1 liter] was added and the samples were counted for 14C in a Beckman LS-230 liquid scintillation counter for 100 min each. The cell-free protein synthesis was studied in the cytoplasmic fraction (cytosole + microsomes) of the forebrain under conditions similar to those described by Wender et al. (11). The tissue was homogenized in 5 vol medium composed of equal volumes of 0.5 M sucrose and TKM buffer (0.1 M Tris-HCl, 0.1 M KCl, 20 mM MgC12, pH 7.4) and supplemented with 2-mercaptoethanol to a final concentration of 20 mM. The homogenate was centrifuged 15 min at 12,000g to ‘obtain postmitochondrial supernatant (cytoplasmic fraction). A 250-J sample of the cytoplasmic fraction (about 1.5 mg protein) was transferred into a test
164
KONAT,
OFFNER,
AND
CLAUSEN
tube, mixed with 250 ~1 TKM buffer containing 1 -01 adenosine-5’triphosphate ( ATP) , 0.1 ~101 guanosine 5’-triphosphate (GTP) , and 500 nCi [‘*C]leucine. Unless otherwise stated, the preparations were incubated 20 min at 38” and the reaction was terminated by immersing the tubes in an ice bath and immediate addition of 4 ml 10% TCA. After 10 min the tubes were centrifuged and the protein precipitate was washed and counted for 14C radiatcivity as described above. Tissue respiration was followed by polarographic measurement of the decrease in dissolved oxygen using the the Clark oxygen electrode. A 1.2-ml sample of the freshly prepared cell suspension was made to 1.3 ml with the KRCOaG buffer, flashed with 95% 0,-S% COZ, sealed with Parafilm, and incubated 10 min with shaking at 38°C. The tube content was quickly transferred into the thermostated cylindrical Plexiglas vessel equipped with a Teflon-coated magnet bar. The vessel was plugged with a closely fitting electrode (care being taken to remove any air bubbles from the reaction mixture) and the rate of oxygen consumption was determined. The respiration rate in the cell suspension prepared from 20-day-old forebrain as calculated per gram original tissue was 602 f 52 nmol Oz/min. This value was attained after about 5-min incubation and was constant for the following 20 min. Protein was determined by the method of Lowry et al. (10) with crystalized bovine serum albumin as a standard. RESULTS
AND
DISCUSSION
Initial experiments on protein synthesis were carried out to establish the kinetics of the basic systems, namely, cytoplasmic fraction and cell suspension prepared from the rat forebrain. The time course of [‘“Cl leucine incorporation into acid-insoluble protein during a l-h period is depicted in Fig. 1. The kinetics of protein synthesis in the cytoplasmic fraction was the same as that observed by Johnson (4) with the constant decrease in the rate of incorporation. Optionally 20 min, rather than the 30 min used by Wender et al. (11)) was chosen for the standard incubation time. On the other hand, the cell suspension showed a linear rate of incorporation for about 40 min and a considerable decrease thereafter. When computed on the basis of forebarin fresh weight, the initial rate of protein synthesis in the cell suspension was much higher than in the cytoplasmic fraction. This fact, together with the initially constant rate of incorporation, is an indication of relative metabolic integrity of the cells in suspension. In both systems (cellular and cell-free) the protein synthesis was proved to be proportional to the tissue concentration in a range from 50 to 150% of the standard amount.
TRIETEIYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
165
1
FIG. 1. Time course of r.-[U-‘YZ]leucine incorporation into acid-insoluble protein in cellular (solid circles) and cell-free (open circles) systems. The preparations obtained from %&day-old forbrains were incubated for various times and assayed by the standard procedure (see Materials and Methods). Assays were done in duplicate. Each point is an average of three preparations.
The protein synthesis and respiration in the cell suspension were sensitive to PbEt,; however, the former process revealed the greater vulnerability. The inhibition curves are biphasic (Fig. 2). With concentrations of PbEts to about 50 PM the inhibition increases suddenly and reaches about 60 and 35% of control values for protein synthesis and respiration, respectively. Beyond 50 PM the inhibition values increase more slowly.
i1
FIG. 2. Effect of PbEt, on protein synthesis (solid circles) and respiration (open circles) in cell suspension prepared from ZO-day-old rat forebrain. The assays were carried out according to the standard procedure as described in Materials and Methods, and except that the indicated amounts of PbEt, were added to the assay system.
166
KONAT,
OFFNER,
AND
CLAUSEN
Thus, 0.2 mM PbEts caused 80% inhibition of protein synthesis and 65% inhibition of oxygen consumption. The exposure of nervous cells in culture to micromolar concentrations of PbEts leads to progressive degeneration and death of the cells (2). Various cellular elements reveal different susceptibility toward this neurotoxin. Thus, above a certain threshold concentration of PbEts, the observed slowing down of respiration and protein synthesis may be due to increasing mortality among the cells. Actually, a progressive decrease in the respiration rate was observed with cells incubated with PbEts at concentrations higher than 46 pM. Below this concentration the rate was constant for at least 20 min (cf. Materials and Methods). Thus, the inhibition values presented in Fig. 2 were calculated from the respiration rates measured after IO-min incubation. Because the rates of protein synthesis represent averages for a 30-min period, it is not possible to compare the effect of high concentrations of PbEts (second phase or the inhibition curves) on these two processes. To circumvent the problem of endogenous energy supply for protein synthesis the cytoplasmic fraction was used in further experiments. The cell-free system used in this investigation had ATP as the sole energy source (11). Thus, any changes in the [lX]leucine incorporation into acidinsoluble protein should reflect the interference with sensu stricto protein biosynthesis. As seen from Table 1, protein synthesis in the cell-free system was not affected by PbEts. No inhibition was observed even at PbEts concentrations lethal to the cells in suspension (second phase of the inhibition curves). In view of the present results the suppression of protein synthesis by PbEts in the forebrain tissue is secondary to the inhibition of cellular energy-generating systems, namely, glucose oxidation (3) and mitochondrial processes pertaining to oxidative phosphorylation (1). Unlike SnEts (11, TABLE Cell-Free
Protein
Synthesis
Concentration
of PbEtr
1
in the Presence
of Triethyllead
(FM )
[W]Leucine incorporation ($& of control)
23.4 46.8 93.6 187.2
97 102 100 98
a The cytoplasmic fraction as described in Materials and fraction equivalent to 50 mg L-[U-Wlleucine into protein averages f SD obtained from
f f f f
(PbEt,)D
4 5 6 6
was prepared from 20-day-old rat forebrain and incubated Methods. The reaction mixture contained the cytoplasmic wet weight of the original forebrain. The incorporation of in the absence of PbEt, was 2520 f 132 cpm. Values are five preparations.
TRIETHYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
167
12), PbEts shows no specificity toward processes involved in protein biosynthesis. Incorporation of the radioactive amino acid into protein was restrained more than oxygen consumption in the presence of PbEts and this feature probably reflects the utmost sensitivity of protein synthesizing systems to changes in the oxidative metabolism of the cells (5). REFERENCES 1.
2.
3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
W. N., J. E. CREMER, AND C. J. THRELFALL. 1962. Trialkylleads and oxidative phosphorylation. A study of the action of trialkylleads upon rat liver mitochondria and rat brain cortex slices. Biochem. Pharnzacol. 11: 835-846. AMMITZB~LL, T., T. KOBAYASI, I. GRUNDT, AND J. CLAUSEN. 1977. Toxicology of triethyllead, methylmercury and cadminum, determined in chick embryo brain cultures. 19th Meeting of the European Society of Toxicology, Copenhagen, No. 54. CREMER, J. E. 1962. The action of triethyl tin, triethyl lead, ethyl mercury and other inhibitors on the metabolism of brain and kidney slices in vitro using substrates labelled with “C. J. Neurochenz. 9 : 289-298. JOHNSON, T. C. 1968. Cell-free protein synthesis by mouse brain during early development. J. Neuroclze?n. 15 : 1189-1194. JOHNSON, T. C., AND M. W. LUTTGES. 1966. The effects of maturation on in vitro protein synthesis by mouse brain cells. J. N.eurochem. 13: 545-552. KONAT, G., AND J. CLAUSEN. 1974. The effect of long-term administration of triethyllead on the developing rat brain. Emkon. Physiol. Biochem. 4: 236-242. KONAT, G., AND J. CLAUSEN. 1976. Triethyllead-induced hypomyelination in the developing rat forebrain. Exp. Neural. 50 : 124-133. KONAT, G., H. OFFNER, AND J. CLAUSEN. 1976. Triethyllead-restrained myelin deposition and protein synthesis in the developing rat forebrain. Exp. Newel. 52 : 58-65. KRIGMAN, M. R., AND E. L. HOGAN. 1976. Undernutrition in the developing rat: Effect upon myelination. Bruin Res. 107 : 239-255. LOWRY, 0. H., N. J. ROSEBROC’GH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chew. 193: 265-275. WENDER, M., B. ZGORZALEWICZ, AND A. PIECHOWSIX. 1974. Cell-free protein synthesis by rat brain in triethyl tin intoxication. Acta Neural. Scatzd. 50: 103-108. WENDER, M., B. ZGORZALEWICZ, AND A. PIECHOWSE;I. 1975. Activity of sRNAamino acyl synthetases in TET-induced brain oedema. Neuropatol. Pal. 13: 415-421. ALDRIDGE,
Effect
59,
of Triethyllead G. KONAT,
The
Neurochemical
Institute,
162-167 (1978)
on Protein
Synthesis
H. OFFNER,
AND J. CLAUSEN
58 Rddmandsgade, Received
Sep tenzber
DK-2200
in Rat Forebrain
Copenhagen
N,
Denmark
6,1977
Triethyllead ( PbEt) inhibited both oxygen utilization and L-[ U-l’C]leucine incorporation into acid-insoluble protein in a cell suspension prepared from 20-day-old rat forebrain. At the same concentrations of PbEtr, protein synthesis was inhibited more than respiration. The cell-free protein synthesis studied in the cytopalsmic fraction was not affected by the neurotoxin. Thus, PbEta does not directly interfere with the protein synthesizing systems and the observed inhibition is secondary to the suppression of the tissue oxidative metabolism.
INTRODUCTION Previous studies from our laboratory demonstrated that poisoning with triethyllead (PbEts) hampers the cerebral myelination processesin young rats (6-S). Unlike in the undernutrition model of experimental hypomyelination (9)) PbEts-induced poisoning resti-ains specifically brain tissue development (6). The suppressionof myelin biosynthesis is probably caused by inhibition of energy-generating processesbrought about by PbEts (1, 3). Furthermore, investigations by Wender et al. (11, 12) on cerebral protein synthesis revealed the inhibitory effect of triethyltin (SnEt,) at the level of amino acid activation. Because PbEt, closely resembles SnEtj in its biochemical activity (l), the present studies were designed to check whether or not there is any specificity of PbEt, toward the protein synthesizing system in the rat brain. MATERIALS
AND
METHODS
Triethyllead chloride was kindly supplied as a gift from The Associated Octel Company Ltd., London. L- [ U-14C]leucine (specific activity 280 Ci/ Abbreviations : TCA-trichloroacetic acid; ATP-adenosine 5’-triphosphate ; GTPguanosine 5’-triphosphate; KRCO&-Krebs-Ringer bicarbonate buffer with 10 g/liter glucose ; PbEk-triethyllead ; SnE&,-triethyltin. 162
0014-4886/78/0591-0162$02.00/O Copyright 0 1578 by Acadenkc P&a, Inc. All rights of reproduction in any form reserved.
TKIETIIYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
163
mol) was obtained from New England Chemicals GmbH, Dreieichenhain, West Germany, and NCS tissue solubilzer was from Amersham/Searle, Arlington Heights, Illinois. All other chemicals were of analytical grade from Merck, Darmstadt, West Germany. Two-day-old white female Wistar rats of specific pathogenic free type were obtained from Veterinary Surgeon Mpllle@rd’s Centre Ltd., Denmark, and maintained as previously ‘described (8) until the age of 20 days postpartum. The system of brain cell suspension (5) was utilized to study cellular protein synthesis and respiration. One forebrain was cut with scissors (50 random cuts) into small pieces, mixed with 5 vol Krebs-Ringer bicarbonate buffer fortified with 10 g/liter glucose (KRCOaG buffer), and gently pressed out of a glass syringe equipped with a 40-mm-long 1Pgauge needle. The pressing out procedure was repeated 10 times and the cell suspension was finally diluted with 25 ml of the same KRCOaG buffer. The incubation mixture for protein synthesis contained 600 ~1 freshly prepared cell suspension (equivalent to 19.5 mg wet weight of the original forebrain), 550 nCi [“Cl leucine, and KRCOSG buffer added to a total volume of 650 ~1. The tubes were flashed with 95% 02-5s COZ, covered with Parafilm, and incubated 30 min with shaking at 38°C unless otherwise indicated. The blanks were incubated at 0°C. At the end of incubation the tubes were placed in an ice bath, 5 ml cold saline was added, and the tubes were centrifuged 5 min at 2709 and 4°C. The pellet was suspended in 3 ml saline and solubilized by the addition of 4 drops 1 N NaOH. After 15 min at room temperature, 4 ml 10% trichloroacetic acid (TCA) was added and the tubes were centrifuged. The pellet was washed three additional times with 5 ml 5% TCA and once with ethanol. The protein precipitate was solublized in 1 ml NCS tissue solublizer. Heating at 50°C for 10 min promoted solubilization. After cooling, 15 ml scintillating fluid [6 g PPO (2,5-diphenyloxazole), 75 ml POPOP (1,4bis-2- (S-phenyloxazolyl) benzene), and toluene to a total volume of 1 liter] was added and the samples were counted for 14C in a Beckman LS-230 liquid scintillation counter for 100 min each. The cell-free protein synthesis was studied in the cytoplasmic fraction (cytosole + microsomes) of the forebrain under conditions similar to those described by Wender et al. (11). The tissue was homogenized in 5 vol medium composed of equal volumes of 0.5 M sucrose and TKM buffer (0.1 M Tris-HCl, 0.1 M KCl, 20 mM MgC12, pH 7.4) and supplemented with 2-mercaptoethanol to a final concentration of 20 mM. The homogenate was centrifuged 15 min at 12,000g to ‘obtain postmitochondrial supernatant (cytoplasmic fraction). A 250-J sample of the cytoplasmic fraction (about 1.5 mg protein) was transferred into a test
164
KONAT,
OFFNER,
AND
CLAUSEN
tube, mixed with 250 ~1 TKM buffer containing 1 -01 adenosine-5’triphosphate ( ATP) , 0.1 ~101 guanosine 5’-triphosphate (GTP) , and 500 nCi [‘*C]leucine. Unless otherwise stated, the preparations were incubated 20 min at 38” and the reaction was terminated by immersing the tubes in an ice bath and immediate addition of 4 ml 10% TCA. After 10 min the tubes were centrifuged and the protein precipitate was washed and counted for 14C radiatcivity as described above. Tissue respiration was followed by polarographic measurement of the decrease in dissolved oxygen using the the Clark oxygen electrode. A 1.2-ml sample of the freshly prepared cell suspension was made to 1.3 ml with the KRCOaG buffer, flashed with 95% 0,-S% COZ, sealed with Parafilm, and incubated 10 min with shaking at 38°C. The tube content was quickly transferred into the thermostated cylindrical Plexiglas vessel equipped with a Teflon-coated magnet bar. The vessel was plugged with a closely fitting electrode (care being taken to remove any air bubbles from the reaction mixture) and the rate of oxygen consumption was determined. The respiration rate in the cell suspension prepared from 20-day-old forebrain as calculated per gram original tissue was 602 f 52 nmol Oz/min. This value was attained after about 5-min incubation and was constant for the following 20 min. Protein was determined by the method of Lowry et al. (10) with crystalized bovine serum albumin as a standard. RESULTS
AND
DISCUSSION
Initial experiments on protein synthesis were carried out to establish the kinetics of the basic systems, namely, cytoplasmic fraction and cell suspension prepared from the rat forebrain. The time course of [‘“Cl leucine incorporation into acid-insoluble protein during a l-h period is depicted in Fig. 1. The kinetics of protein synthesis in the cytoplasmic fraction was the same as that observed by Johnson (4) with the constant decrease in the rate of incorporation. Optionally 20 min, rather than the 30 min used by Wender et al. (11)) was chosen for the standard incubation time. On the other hand, the cell suspension showed a linear rate of incorporation for about 40 min and a considerable decrease thereafter. When computed on the basis of forebarin fresh weight, the initial rate of protein synthesis in the cell suspension was much higher than in the cytoplasmic fraction. This fact, together with the initially constant rate of incorporation, is an indication of relative metabolic integrity of the cells in suspension. In both systems (cellular and cell-free) the protein synthesis was proved to be proportional to the tissue concentration in a range from 50 to 150% of the standard amount.
TRIETEIYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
165
1
FIG. 1. Time course of r.-[U-‘YZ]leucine incorporation into acid-insoluble protein in cellular (solid circles) and cell-free (open circles) systems. The preparations obtained from %&day-old forbrains were incubated for various times and assayed by the standard procedure (see Materials and Methods). Assays were done in duplicate. Each point is an average of three preparations.
The protein synthesis and respiration in the cell suspension were sensitive to PbEt,; however, the former process revealed the greater vulnerability. The inhibition curves are biphasic (Fig. 2). With concentrations of PbEts to about 50 PM the inhibition increases suddenly and reaches about 60 and 35% of control values for protein synthesis and respiration, respectively. Beyond 50 PM the inhibition values increase more slowly.
i1
FIG. 2. Effect of PbEt, on protein synthesis (solid circles) and respiration (open circles) in cell suspension prepared from ZO-day-old rat forebrain. The assays were carried out according to the standard procedure as described in Materials and Methods, and except that the indicated amounts of PbEt, were added to the assay system.
166
KONAT,
OFFNER,
AND
CLAUSEN
Thus, 0.2 mM PbEts caused 80% inhibition of protein synthesis and 65% inhibition of oxygen consumption. The exposure of nervous cells in culture to micromolar concentrations of PbEts leads to progressive degeneration and death of the cells (2). Various cellular elements reveal different susceptibility toward this neurotoxin. Thus, above a certain threshold concentration of PbEts, the observed slowing down of respiration and protein synthesis may be due to increasing mortality among the cells. Actually, a progressive decrease in the respiration rate was observed with cells incubated with PbEts at concentrations higher than 46 pM. Below this concentration the rate was constant for at least 20 min (cf. Materials and Methods). Thus, the inhibition values presented in Fig. 2 were calculated from the respiration rates measured after IO-min incubation. Because the rates of protein synthesis represent averages for a 30-min period, it is not possible to compare the effect of high concentrations of PbEts (second phase or the inhibition curves) on these two processes. To circumvent the problem of endogenous energy supply for protein synthesis the cytoplasmic fraction was used in further experiments. The cell-free system used in this investigation had ATP as the sole energy source (11). Thus, any changes in the [lX]leucine incorporation into acidinsoluble protein should reflect the interference with sensu stricto protein biosynthesis. As seen from Table 1, protein synthesis in the cell-free system was not affected by PbEts. No inhibition was observed even at PbEts concentrations lethal to the cells in suspension (second phase of the inhibition curves). In view of the present results the suppression of protein synthesis by PbEts in the forebrain tissue is secondary to the inhibition of cellular energy-generating systems, namely, glucose oxidation (3) and mitochondrial processes pertaining to oxidative phosphorylation (1). Unlike SnEts (11, TABLE Cell-Free
Protein
Synthesis
Concentration
of PbEtr
1
in the Presence
of Triethyllead
(FM )
[W]Leucine incorporation ($& of control)
23.4 46.8 93.6 187.2
97 102 100 98
a The cytoplasmic fraction as described in Materials and fraction equivalent to 50 mg L-[U-Wlleucine into protein averages f SD obtained from
f f f f
(PbEt,)D
4 5 6 6
was prepared from 20-day-old rat forebrain and incubated Methods. The reaction mixture contained the cytoplasmic wet weight of the original forebrain. The incorporation of in the absence of PbEt, was 2520 f 132 cpm. Values are five preparations.
TRIETHYLLEAD
AND
BRAIN
PROTEIN
SYNTHESIS
167
12), PbEts shows no specificity toward processes involved in protein biosynthesis. Incorporation of the radioactive amino acid into protein was restrained more than oxygen consumption in the presence of PbEts and this feature probably reflects the utmost sensitivity of protein synthesizing systems to changes in the oxidative metabolism of the cells (5). REFERENCES 1.
2.
3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
W. N., J. E. CREMER, AND C. J. THRELFALL. 1962. Trialkylleads and oxidative phosphorylation. A study of the action of trialkylleads upon rat liver mitochondria and rat brain cortex slices. Biochem. Pharnzacol. 11: 835-846. AMMITZB~LL, T., T. KOBAYASI, I. GRUNDT, AND J. CLAUSEN. 1977. Toxicology of triethyllead, methylmercury and cadminum, determined in chick embryo brain cultures. 19th Meeting of the European Society of Toxicology, Copenhagen, No. 54. CREMER, J. E. 1962. The action of triethyl tin, triethyl lead, ethyl mercury and other inhibitors on the metabolism of brain and kidney slices in vitro using substrates labelled with “C. J. Neurochenz. 9 : 289-298. JOHNSON, T. C. 1968. Cell-free protein synthesis by mouse brain during early development. J. Neuroclze?n. 15 : 1189-1194. JOHNSON, T. C., AND M. W. LUTTGES. 1966. The effects of maturation on in vitro protein synthesis by mouse brain cells. J. N.eurochem. 13: 545-552. KONAT, G., AND J. CLAUSEN. 1974. The effect of long-term administration of triethyllead on the developing rat brain. Emkon. Physiol. Biochem. 4: 236-242. KONAT, G., AND J. CLAUSEN. 1976. Triethyllead-induced hypomyelination in the developing rat forebrain. Exp. Neural. 50 : 124-133. KONAT, G., H. OFFNER, AND J. CLAUSEN. 1976. Triethyllead-restrained myelin deposition and protein synthesis in the developing rat forebrain. Exp. Newel. 52 : 58-65. KRIGMAN, M. R., AND E. L. HOGAN. 1976. Undernutrition in the developing rat: Effect upon myelination. Bruin Res. 107 : 239-255. LOWRY, 0. H., N. J. ROSEBROC’GH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chew. 193: 265-275. WENDER, M., B. ZGORZALEWICZ, AND A. PIECHOWSIX. 1974. Cell-free protein synthesis by rat brain in triethyl tin intoxication. Acta Neural. Scatzd. 50: 103-108. WENDER, M., B. ZGORZALEWICZ, AND A. PIECHOWSE;I. 1975. Activity of sRNAamino acyl synthetases in TET-induced brain oedema. Neuropatol. Pal. 13: 415-421. ALDRIDGE,
