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Effect of cadmium on acute-phase protein synthesis in perfused rat liver
TOXICOLOGY
AND
APPLIED
46,803-805
PHARMACOLOGY
SHORT Effect of Cadmium
(1978)
COMMUNICATIONS
on Acute-Phase Protein Perfused Rat Liver’
Synthesis
in
Effect of Cadmium on Acute-Phase Protein Synthesis in Perfused Rat Liver. ZAK, I., AND A. (1978). Toxicot. A&. Pharmacol. 46, 803-805. Injection of cadmium (1 mg/kg, ip) stimulated the synthesis of fibrinogen, seromukoid, and glycoproteins, while the [i4C]leucine incorporation into albumin and total plasma proteins was slightly reduced in the perfused rat liver. DUBIN,
Cadmium, when administered either parenterally or orally, is known to accumulate in the livers and kidneys of experimental animals as a cadmium-metallothionein complex (Cherian and Shaikh, 1975). The influence of cadmium on plasma protein synthesis has only been partially explored (Axelsson and Piscator, 1966; Piscator and Axelsson, 1970). The present study was aimed at showing that cadmium provokes the increase of some plasma protein synthesis not only in vivo (iak and Steibert, 1978) but also in perfused rat livers. METHODS
Male Wistar rats (312 + 27 g) were used. The animals maintained on a standard pellet diet were allowed food and tap water ad libitum. CdCl, was freshly dissolved in pyrogen-free saline and injected ip (1 mg of Cdz+/kg) 48 hr before the experiment. This interval was selected as corresponding to the maximum acute-phase response (iak and Steibert, 1978). Liver perfusion and protein isolation were carried out as described previously (Koj and Dubin, 1974), but the experiment was started by the addition of 5 ,&i of or,-[i4Clleucine. The glycoprotein fraction (B) was separated from 5 ml of dialyzed plasma on a Con A-Sepharose (1.5 x 10 cm) column according to the procedure described by Liener et al., (1973). Electrophoresis in polyacrylamide gel showed that fraction (A) not adsorbed on a Con A-Sepharose column containing mainly albumin. Protein contents in plasma and liver preparations were measured by the method of Lowry et al., (1951), with bovine serum albumin as standard. The 14C radioactivities were determined using a dioxane-based Bray scintillation mixture in a Packard liquid scintillation spectrometer Model 2 111 with the efficiency of 75%. RESULTS
AND
DISCUSSION
The experiments were carried out in two groups: (I) on livers from control rats injected ip with 1.0 ml of pyrogen-free saline; and (II) on livers from cadmium-injected i Department of Animal Biochemistry, Institute of Molecular Grodzka 53,31-001 Krakow, Poland. 803
Biology, Jagiellonian University, ul. ca 1-008x/78/0463-0803 502.00/0 Copyright @ 19‘78 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
804
SHORT COMMUNICATIONS
rats. The mean results of six perfusions for each group are given in Fig. 1, as 14C specific radioactivity incorporated into particular proteins. Injection of cadmium stimulated the synthesis of fibrinogen, seromukoid, and glycoproteins, while the 14C in albumin and total plasma proteins was slightly reduced. Only values for the relative activities of fibrinogen and seromukoid were significantly increased in comparison with the control group at p < 0.05. The values obtained for glycoproteins isolated on a Con A-Sepharose column were also increased, but the variability of the results is responsible for the lack of statistical significance.
FIG. 1. Effect of injected cadmium (1 mg of Cd/kg, ip) on protein synthesis in perfused rat livers. The results are the mean of six experiments.
Axelsson and Piscator (1966) demonstrated a slight reduction in albumin concentration and elevation in cr,-, CQ-, and Pglobulin of Cd-injected rabbits. On the other hand, Piscator and Axelsson (1970) could not demonstrate any alterations in the concentrations of serum albumin or globulin in chronic cadmium poisoning of rabbits. They concluded that liver function in the Cd-intoxicated rabbits was normal in spite of the high concentrations of Cd accumulated in the liver. Fibrinogen and some other plasma glycoproteins known as acute-phase reactants (AP-reactants) are synthesized in the liver in increased amounts after various injuries (Koj, 1974). The mechanism of induced synthesis of AP-reactants is not fully understood, but data indicate that it includes enhanced formation of specific mRNA. Weser and Hiibner (1970) and Hidalgo et al., (1976) observed that cadmium does not inhibit the RNA polymerase activity of rat liver nuclei 10 hr after animals were injected with l-2 mg/kg and that Cd*+ increases RNA polymerase activity after this time. A similar phenomenon in respect to protein synthesis was observed in our experiments, and it was accompanied by a rise in specific activities of fibrinogen and seromukoid fraction indicating the occurrence of typical acute-phase response. However, we did not observe increased specific activities of total plasma protein in contrast to rats with local inflammation evoked by turpentine injection (Koj and Dubin, 1974). This may be caused by a considerable decrease of albumin specific activities in cadmium-injected
SHORT
COMMUNICATIONS
805
rats. Perhaps the inhibition of albumin synthesis in acute intoxication with cadmium is more pronounced than in the animals with local inflammation. In conclusion we suggest that the observed increase of some synthesis of plasma proteins may represent a two-step process. At first, cadmium accumulates in the liver as a cadmium-metallothionein complex, and later this leads to increased protein synthesis. REFERENCES B., AND PISCATOR, M. (1966). Serum proteins in cadmium poisoned rabbits. Arch. Environ. Health 12, 374-38 1. CHERIAN, M. G., AND SHAIKH. Z. A. (1975). Metabolism of intravenously injected cadmiumbinding protein. Biochem. Biophys. Res. Commun. 65, 863-869. HIDALGO, H. A., KOPPA, V., AND BRYAN, S. E. (1976). Effect of cadmium on RNA-polymerase and protein synthesis in rat liver. FEBS Lett. 64, 159-162. KOJ, A. (1974). Acute-phase reactants, In Structure and Function of Plasma Proteins. (A. C. Allison, ed.), Vol. 1, pp. 73-131. Plenum Press, New York and London. KOJ, A., AND DUBIN, A. (1974). On the hormonal modulation of acute-phase plasma protein synthesis in perfused rat liver. Acta Biochim. Polon. 21, 159-167. LIENER, I. E., GARRISON, 0. R., AND PRAVDA, Z. (1973). The purification of human serum a,antitrypsin by affinity chromatography on concanavalin A. Biochem. Biophys. Res. Commun. 5 1,436-443. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. PISCATOR, M., AND AXELSSON, B. (1970). Serum proteins and kidney function after exposure to cadmium. Arch. Environ. Health 21,604-608. WESER, U., AND H~BNER, L. (1970). Cd*+, Mn*+ and Zn*+ induced synthesis of nuclear RNA in the liver of normal and adrenalectomized rats. FEBS Lett. 10, 169-174. YAK, I., AND STEIBERT, E. (1978). The influence of cadmium on the rat serum level of seromukoid. Brom. Chem. Toksykol. (in press). AXELSSON,
IWONA ZAK ADAM DUBIN’
Institute of Occupational Medicine in the Mining and Metallurgical Industry 41-200 Sosnowiec. Poland Received February 16,1978; accepted July 28,1978
AND
APPLIED
46,803-805
PHARMACOLOGY
SHORT Effect of Cadmium
(1978)
COMMUNICATIONS
on Acute-Phase Protein Perfused Rat Liver’
Synthesis
in
Effect of Cadmium on Acute-Phase Protein Synthesis in Perfused Rat Liver. ZAK, I., AND A. (1978). Toxicot. A&. Pharmacol. 46, 803-805. Injection of cadmium (1 mg/kg, ip) stimulated the synthesis of fibrinogen, seromukoid, and glycoproteins, while the [i4C]leucine incorporation into albumin and total plasma proteins was slightly reduced in the perfused rat liver. DUBIN,
Cadmium, when administered either parenterally or orally, is known to accumulate in the livers and kidneys of experimental animals as a cadmium-metallothionein complex (Cherian and Shaikh, 1975). The influence of cadmium on plasma protein synthesis has only been partially explored (Axelsson and Piscator, 1966; Piscator and Axelsson, 1970). The present study was aimed at showing that cadmium provokes the increase of some plasma protein synthesis not only in vivo (iak and Steibert, 1978) but also in perfused rat livers. METHODS
Male Wistar rats (312 + 27 g) were used. The animals maintained on a standard pellet diet were allowed food and tap water ad libitum. CdCl, was freshly dissolved in pyrogen-free saline and injected ip (1 mg of Cdz+/kg) 48 hr before the experiment. This interval was selected as corresponding to the maximum acute-phase response (iak and Steibert, 1978). Liver perfusion and protein isolation were carried out as described previously (Koj and Dubin, 1974), but the experiment was started by the addition of 5 ,&i of or,-[i4Clleucine. The glycoprotein fraction (B) was separated from 5 ml of dialyzed plasma on a Con A-Sepharose (1.5 x 10 cm) column according to the procedure described by Liener et al., (1973). Electrophoresis in polyacrylamide gel showed that fraction (A) not adsorbed on a Con A-Sepharose column containing mainly albumin. Protein contents in plasma and liver preparations were measured by the method of Lowry et al., (1951), with bovine serum albumin as standard. The 14C radioactivities were determined using a dioxane-based Bray scintillation mixture in a Packard liquid scintillation spectrometer Model 2 111 with the efficiency of 75%. RESULTS
AND
DISCUSSION
The experiments were carried out in two groups: (I) on livers from control rats injected ip with 1.0 ml of pyrogen-free saline; and (II) on livers from cadmium-injected i Department of Animal Biochemistry, Institute of Molecular Grodzka 53,31-001 Krakow, Poland. 803
Biology, Jagiellonian University, ul. ca 1-008x/78/0463-0803 502.00/0 Copyright @ 19‘78 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
804
SHORT COMMUNICATIONS
rats. The mean results of six perfusions for each group are given in Fig. 1, as 14C specific radioactivity incorporated into particular proteins. Injection of cadmium stimulated the synthesis of fibrinogen, seromukoid, and glycoproteins, while the 14C in albumin and total plasma proteins was slightly reduced. Only values for the relative activities of fibrinogen and seromukoid were significantly increased in comparison with the control group at p < 0.05. The values obtained for glycoproteins isolated on a Con A-Sepharose column were also increased, but the variability of the results is responsible for the lack of statistical significance.
FIG. 1. Effect of injected cadmium (1 mg of Cd/kg, ip) on protein synthesis in perfused rat livers. The results are the mean of six experiments.
Axelsson and Piscator (1966) demonstrated a slight reduction in albumin concentration and elevation in cr,-, CQ-, and Pglobulin of Cd-injected rabbits. On the other hand, Piscator and Axelsson (1970) could not demonstrate any alterations in the concentrations of serum albumin or globulin in chronic cadmium poisoning of rabbits. They concluded that liver function in the Cd-intoxicated rabbits was normal in spite of the high concentrations of Cd accumulated in the liver. Fibrinogen and some other plasma glycoproteins known as acute-phase reactants (AP-reactants) are synthesized in the liver in increased amounts after various injuries (Koj, 1974). The mechanism of induced synthesis of AP-reactants is not fully understood, but data indicate that it includes enhanced formation of specific mRNA. Weser and Hiibner (1970) and Hidalgo et al., (1976) observed that cadmium does not inhibit the RNA polymerase activity of rat liver nuclei 10 hr after animals were injected with l-2 mg/kg and that Cd*+ increases RNA polymerase activity after this time. A similar phenomenon in respect to protein synthesis was observed in our experiments, and it was accompanied by a rise in specific activities of fibrinogen and seromukoid fraction indicating the occurrence of typical acute-phase response. However, we did not observe increased specific activities of total plasma protein in contrast to rats with local inflammation evoked by turpentine injection (Koj and Dubin, 1974). This may be caused by a considerable decrease of albumin specific activities in cadmium-injected
SHORT
COMMUNICATIONS
805
rats. Perhaps the inhibition of albumin synthesis in acute intoxication with cadmium is more pronounced than in the animals with local inflammation. In conclusion we suggest that the observed increase of some synthesis of plasma proteins may represent a two-step process. At first, cadmium accumulates in the liver as a cadmium-metallothionein complex, and later this leads to increased protein synthesis. REFERENCES B., AND PISCATOR, M. (1966). Serum proteins in cadmium poisoned rabbits. Arch. Environ. Health 12, 374-38 1. CHERIAN, M. G., AND SHAIKH. Z. A. (1975). Metabolism of intravenously injected cadmiumbinding protein. Biochem. Biophys. Res. Commun. 65, 863-869. HIDALGO, H. A., KOPPA, V., AND BRYAN, S. E. (1976). Effect of cadmium on RNA-polymerase and protein synthesis in rat liver. FEBS Lett. 64, 159-162. KOJ, A. (1974). Acute-phase reactants, In Structure and Function of Plasma Proteins. (A. C. Allison, ed.), Vol. 1, pp. 73-131. Plenum Press, New York and London. KOJ, A., AND DUBIN, A. (1974). On the hormonal modulation of acute-phase plasma protein synthesis in perfused rat liver. Acta Biochim. Polon. 21, 159-167. LIENER, I. E., GARRISON, 0. R., AND PRAVDA, Z. (1973). The purification of human serum a,antitrypsin by affinity chromatography on concanavalin A. Biochem. Biophys. Res. Commun. 5 1,436-443. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275. PISCATOR, M., AND AXELSSON, B. (1970). Serum proteins and kidney function after exposure to cadmium. Arch. Environ. Health 21,604-608. WESER, U., AND H~BNER, L. (1970). Cd*+, Mn*+ and Zn*+ induced synthesis of nuclear RNA in the liver of normal and adrenalectomized rats. FEBS Lett. 10, 169-174. YAK, I., AND STEIBERT, E. (1978). The influence of cadmium on the rat serum level of seromukoid. Brom. Chem. Toksykol. (in press). AXELSSON,
IWONA ZAK ADAM DUBIN’
Institute of Occupational Medicine in the Mining and Metallurgical Industry 41-200 Sosnowiec. Poland Received February 16,1978; accepted July 28,1978