- Email: [email protected]
Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study
Toxicology 161 (2001) 129– 138 www.elsevier.com/locate/toxicol
Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study Stamatios E. Theocharis a,*, Alexandra P. Margeli a,b, Spyridon D. Skaltsas a, Chara A. Spiliopoulou a, Antonios S. Koutselinis a a
Department of Forensic Medicine and Toxicology, Medical School, Uni6ersity of Athens, 11527 Goudi, Athens, Greece b Laboratory of Biochemistry, ‘Aghia Sophia’ Children’s Hospital, 11527 Goudi, Athens, Greece Received 6 November 2000; received in revised form 21 December 2000; accepted 11 January 2001
Abstract Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl4) administration. A single intraperitoneal injection of 1 ml CCl4/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl4 administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl4, in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl4 administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl4, being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carbon tetrachloride; Injury; Liver; Metallothionein; Rats; Regeneration
1. Introduction
* Corresponding author. 21, Thessalias street; Zografou; GR 15772; Athens; Greece. Tel.: +30-1-7780114; fax: +30-17780114. E-mail address: [email protected] (S.E. Theocharis).
Metallothionein (MT) was first discovered by Margoshes and Valle (1957) as a cadmium (Cd) binding protein. Later on Piscator (1964) mentioned a marked increase of MT content in Cd exposed rabbits, as a metal detoxification mecha-
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 1 ) 0 0 3 4 0 - 7
130
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
nism. MTs are a family of heavy metal binding proteins with a large degree of sequence homology. They are single-chain proteins of approximately 6000 Da, characterized by a very high proportion of cysteine residues, which results in several high affinity Cd and/or zinc (Zn) binding sites (Waalkes and Ward, 1989). There are four classes of similar MT proteins, designated MT-1 through -4, defined on the basis of small differences in amino acid sequence and charge characteristics, which can be resolved chromatographically (Cousins, 1983; Hamer, 1986; Kagi et al., 1984; Kagi, 1993). High concentrations of MT are induced in various tissues of the fetus and neonate at the end of term and during the initial postnatal period, respectively (Waalkes and Goering, 1990; Suzuki et al., 1994). During this time period the liver contains the highest concentration of MT (Brady, 1982; Wong and Klaassen, 1979). The elevated levels of MT observed during such situations of high Zn demand are thought to provide Zn within rapidly growing tissues. It has also been mentioned that MT is expressed during hepatocellular proliferation, after partial hepatectomy (PH) in rats (Cain and Griffiths, 1984; Margeli et al., 1994; Ohtake and Koga, 1978; Tsujikawa et al., 1994). Although PH is a wonderful model for triggering physiological mechanisms responsible for regulating hepatocyte proliferation and differentiation, many differences have been identified between regeneration in this model, and in that occurring after chemically induced liver injury. Carbon tetrachloride (CCl4) often serves as an hepatotoxicity inducing agent and its effects have been widely studied and mechanisms have also been proposed for the necrosis produced in the liver (Burr et al., 1993; Diaz-Gomes et al., 1975; Kalf et al., 1987; Slater, 1966; Slater and Sawyer, 1971). In vivo, CCl4 is metabolized to a trichloromethyl free radical (.CCl3), which initiates changes leading eventually to severe cellular damage. Acute CCl4 administration in rats results in hepatic damage, followed by a regenerative process, which peaks later on (Burr et al., 1993). Liver regeneration in vivo has usually been obtained from studies following PH in rats. Induction of hepatocyte proliferation after injury induced by a single intraperitoneal administration of the toxin CCl4 is well docu-
mented, and the necrosis induced by this agent is similar to that of acute drug-induced liver necrosis in humans. In the present study we investigated the expression of hepatic MT over the time course of injury and regeneration induced by acute CCl4 intoxication in rats, by examining alterations in the distribution of this protein, using immunohistochemical techniques.
2. Methods
2.1. Experimental animal model Male Wistar rats (Hellenic Pasteur Institute, Athens, Greece) weighing 190 –230 g each, were used in this study. They had free access to food and water, were kept in an air-conditioned room at 21°C with a 12 h light/dark cycle, and were handled humanely, in accordance with the National Institute of Health guidelines for care and use of laboratory animals. Experiments were started between 07:00 and 09:00 h. The animals were injected intraperitoneally with 1 ml of CCl4/kg body weight, diluted 1:10 in olive oil, under light ether anesthesia (diethyl ether per anesthesia, Codex, Carlo Erba, Milan, Italy). Rats were killed by cardiac puncture at 0, 12, 24, 36, 48, 60 and 72 h post-CCl4 treatment, (seven animals/time point). Immediately after exsanguination, the livers were removed, cleaned and weighed. Small portions of the livers were kept frozen at −80°C to be analyzed for their DNA content and their thymidine kinase (TK, EC 2.7.1.21) activity, while another portion was separated and immersed in buffered formalin solution for histological examination.
2.2. DNA biosynthesis and TK acti6ity One hour before the rats were killed, each animal was injected intraperitoneally with 25 mCi of tritiated thymidine/100 g body weight (Amersham Corp, Buckinghamshire, UK). The livers were homogenized in ice-cold deionized water, and the DNA was extracted according to the method of Munro and Fleck (1966). The determination of DNA in the liver tissue residues was based on the
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
reaction between deoxyribose and diphenylamine as described by Richards (1974). The specific activity of DNA was calculated from the radioactivity measured by a liquid scintillation counter (1211 Rackbeta; LKB-Wallac, Turku, Finland) and the amount of DNA that was determined colorimetrically. Results were expressed as counts per minute (cpm) incorporated/microgram of hepatic DNA (cpm/mgDNA). The enzymatic activity of TK was determined by measuring the in vitro conversion of deoxythymidine to deoxythymidinemonophosphate (Kahn et al. 1988) in the supernatant obtained after homogenization of liver portions and centrifugation at 105 000× g for 1 h at 4°C with an ultracentrifuge (model L5-75; Beckman Instruments, Fullerton, CA). Triplicate aliquots of each sample were spotted onto diethylaminoethyl cellulose discs. The disks were counted for their radioactivity content in a liquid scintillation counter (1211 Rackbeta, LKB-Wallac). The protein was determined by the method of Lowry et al. (1951). The activity of the enzyme TK was expressed as cpm incorporated/min/milligram of protein (cpm/min/ mg protein).
2.3. Enzymatic e6aluation Blood samples were collected from all animals via cardiac puncture. The samples were allowed to clot and the serum was removed by centrifugation at 1000× g for 10 min. All serum samples were sterile, hemolysis-free, and were kept at 4°C before determination of the enzymatic activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (IFCC, 1978), with commercially available kits, using a random access chemistry analyzer (RA-1000; Technicon Instruments Corp., Tarrytown, N.Y.). The activity of the enzymes AST and ALT was expressed as IU/l.
131
number so that the examiner was unaware of the group to which the specimen belonged. The morphologic changes produced in the liver by the intraperitoneal administration of CCl4 were examined during the time course of the whole experiment. The mitotic index was assayed as the number of mitoses observed/ten high power fields (HPF) chosen at random.
2.5. Immunohistochemistry Sections of 4 mm thickness were dewaxed in xylene and brought to water through graded alcohols. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide (BDH Ltd, Poole, England) in methanol for 30 min at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum (Dakopatts, Glostrup, Denmark) diluted 1:5 in phosphate buffered saline (PBS) for 20 min. A mouse (IgG1k) monoclonal antibody that reacts with human and rat MT (Zymed, San Francisco, CA) was used in this study. This antibody reacts with both MT-1 and MT-2 of rat and human origin. The sections were incubated for 1 h at room temperature, with the primary antibody diluted 1:50 in PBS. After washing three times with PBS, sections were incubated for 30 min at room temperature with linking reagent (biotinylated, rabbit, anti-mouse immunoglobulins, diluted 1:200 in PBS) and rinsed three times with PBS and with the peroxidase-conjugated streptavidin label respectively, for 20 min at room temperature. Peroxidase activity was developed in 0.5% (v/v) 3,3%-diaminobenzidine hydrochloride (DAB; Sigma, Saint Louis, MO) in PBS containing 0.03% (v/v) hydrogen peroxide for 5 min. Sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted in gelatin (BDH). The intensity of staining was graded as mild, moderate and intense.
2.4. Histopathology 2.6. Statistical analysis All liver sections were fixed in 4% volume/volume (v/v) buffered formalin solution and embedded in paraffin wax. Sections were cut at 4 mm and stained with hematoxylin and eosin (H&E). All specimens were randomized and given a code
The Wilcoxon test for unpaired measurements was applied to analyze the data. The results were considered as statistically significant when PB 0.05.
132
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
3. Results Animals killed at 0 h constitute the control group for the whole experiment. The administration of CCl4 caused increased activities of serum hepatic enzymes AST and ALT, compared to those observed at the time point of 0 h, (P B 0.001), presenting a peak 36 h after treatment (Fig. 1). The liver sections stained by H&E were examined for necrotic cells, extent of inflammation, and hepatocyte proliferation during the experiment after CCl4 injection. At 12 h after CCl4 administration, the perivenular and midzonal regions exhibited coagulative-type necrosis of hepatocytes. Many hepatocytes appeared having either basophilic (pyknotic) or fragmented (karyorrhectic) nuclei. Macrovesicular fatty change was also evident in some hepatocytes in the vicinity of necrotic areas. A heavy inflammatory infiltrate adjacent to necrotic liver cells was present in centrilobular areas while in the portal area the
hepatocytes remained unaffected. Twenty-four hours after CCl4 injection, hepatocytes in the centrilobular areas presented degenerative changes, while lymphocytic and polymorphonuclear infiltrations were also noted (Fig. 2). Additionally extensive steatosis and foamy degeneration of hepatocytes was observed. Coagulative necrosis was also prominent at 36 h postCCl4 treatment and mitotic activity appeared in the remnant hepatocytes. At 48 h after CCl4 administration, lytic type necrosis, with apparent apoptotic figures in hepatocytes was found in centrilobular areas. The hepatocytes throughout the lobule presented enhanced mitotic activity. The hepatocyte proliferation, as a tissue repair process following hepatic injury due to CCl4 administration, was also evident by the increased indices of regeneration, such as rate of DNA biosynthesis, hepatic TK activity and number of mitoses in hepatocytes. DNA biosynthesis peaked at 48 and 60 h after CCl4 administration (Table 1). TK activity peaked at 36 and 48 h after the
Fig. 1. Serum AST and ALT activities represented by filled and open bars respectively, in CCl4-treated rats examined at different time points post-toxin administration. Results represent the findings in seven rats/time point and are expressed as IU/l. Vertical bars represent the SD from the mean.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
133
Fig. 2. Extensive centrilobular necrosis observed in the liver sections of CCl4-treated rats, at 24 h post-toxin administration. The vessel in the center is a central vein. The large clear cells surrounding the perivenular necrotic zone are hepatocytes ballooned due to macrovesicular steatosis (H&E, magnification X 125). Fig. 3. Intense MT immunoreactivity in the cytoplasm and the nuclei of centrilobular hepatocytes and endothelial cells of the central vein in liver sections of CCl4-treated rats, at 36 h post-toxin administration. The tracer is peroxidase and specific signal appears brown in colour. Counterstained with Mayer’s hematoxylin (magnification X 400).
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
134
injection of the toxic agent (Table 1). Mitotic activity was increased from 36 h post-CCl4 administration, peaked at 48 and 60 h, while later on it progressively decreased (Table 1). MT was detected in isolated cells of the hepatic parenchyma, and in hepatocytes around the central veins, as early as 12 h post-CCl4 administration, presenting an intense pattern of staining in the nuclei and mild staining in the cytoplasm. At 24 h after the administration of CCl4, intense nuclear and cytoplasmic patterns of staining for MT were observed in hepatocytes located in the vicinity of inflammatory infiltrations and necrotic areas. At 36 h the intensity of staining was higher and MT immunoreactivity was prominent in all hepatocytes. Central vein endothelial cells presented nuclear and cytoplasmic patterns of staining (Fig. 3). The maximum expression of MT occurred at the time point of 48 h, in the cytoplasm and nuclei of almost all hepatocytes (Fig. 4 and Fig. 5). At 60 h after CCl4 injection, the hepatocytes presented mild cytoplasmic intensity for MT. At the further time points examined, positivity for both cytoplasmic and nuclear MT staining was noted only in isolated hepatocytes. Kupffer and stellate cells did not show any MT immunoreactivity during the course of the experiment.
4. Discussion CCl4 has previously been shown to exert its acute toxic effects on the liver (Slater and Sawyer,
1971; Diaz-Gomes et al., 1975). The administration of CCl4 in rats caused severe liver injury recognized histopathologically, being also confirmed by the increased activities of serum hepatic enzymes AST and ALT. The development of hepatic necrosis becomes prominent as early as 12 h after the administration of CCl4 and peaks at 36 h. Later on, a regenerative process of hepatocytes occurred, as tissue repair is a biological response that accompanies chemically-induced liver injury. In response to CCl4-induced hepatotoxicity, an increase in the intensity of MT immunoreactivity was noted as early as 12 h following exposure to the toxin, with a maximum at 48 h, the time coinciding with the peak of hepatocyte proliferation. MT is a small cysteine-rich protein, thought to be mainly involved in metal regulation and detoxification, implicated also in cell growth and differentiation (Moffatt et al., 1995; Webb, 1987). It has already been mentioned that, in cases of hepatocellular proliferation as during hepatic regeneration after PH, an induction of liver MT occurs (Cain and Griffiths, 1984; Margeli et al., 1994; Moffatt et al., 1995; Tohyama et al., 1993). It is known that the physiological function of MT is related to Zn homeostasis, so the MT produced supplies Zn to other sites within the cell, as this element is not only essential for various aspects of cell division including DNA, RNA and protein synthesis (Brady, 1982), but is also an important cofactor in various enzymatic systems. It has been reported by Cain and Griffiths (1984) that both MT isoforms, MT-1 and MT-2 are expressed in
Table 1 Indices of liver regeneration in CCl4-treated rats examined at different time points post-toxin administrationa Time (h)
DNA synthesis (cpm/mg DNA)
TK activity (cpm/min/mg protein)
Mitotic index (mitoses/10 HPF)
0 12 24 36 48 60 72
11 9 2 33 93 35 94 44 9 4 187 915 120 918 55 96
13009 110 38799 364 38159 290 72509 650 82959 795 55459 560 41009 380
non detectable 3 90.3 4 90.5 109 0.8 24 9 3 16 9 2 9 90.8
a
Values are expressed as mean 9SD.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
135
Fig. 4. Maximum MT immunoreactivity in all hepatocytes in liver sections of CCl4-treated rats, at 48 h post-toxin administration. The tracer is peroxidase and specific signal appears brown in colour. Counterstained with Mayer’s hematoxylin (magnification X 125).
the liver of partially hepatectomized rats. We have also previously shown the time course of MT expression in the liver after 70% PH (Margeli et al., 1994), using immunohistochemical techniques. It has been shown that either acute phase response induction and infammation (DiSilvestro and Carlson, 1992) or Zn administration (Cagen and Klaassen, 1979; Clarke and Lui, 1986) inhibit CCl4 induced hepatotoxicity in rats. Inflammation and Zn injections, both protectors against CCl4 injury, are not known to produce any other common effects than the induction of MT. The toxic effects of CCl4 on the liver are theorized to involve free radicals (Recknagel and Glende, 1989) and MT can scavenge free radicals in vitro (Thomas et al., 1986; Thornalley and Vasak, 1985). There is also evidence that metabolites of injected CCl4 react with MT in vivo (Cagen and Klaassen, 1979). Alterations of MT expression were observed in the liver during injury and regeneration induced by CCl4 administration. In our study the immunohistochemical expression of MT during the first 24 h following CCl4 administration presented cellular distribution similar to that found for HSP-27 and -70, suggesting a role
for MT protein during stressful states (Theocharis et al., 1999), in order to protect the liver against toxin-induced injury. In the present study MT expression in the liver was correlated with the indices of hepatic proliferation. At the time of maximum hepatocyte proliferation, MT was highly expressed in the liver. The induction of MT during hepatic proliferation after PH caused decreased serum Zn levels in partially hepatectomized rats (Margeli et al., 1994). This shift of Zn, from the blood circulation to liver tissue, reflects the essential role of Zn in liver regeneration. Hepatic Zn, temporarily sequestered by MT, is available to DNA and RNA polymerases, which are Zn requiring metalloenzymes (Hambridge et al., 1986) and are associated with cell replication, differentiation and growth. Hepatic proliferation may also produce an increase demand for regulatory proteins with ‘Zn binding fingers’ (Klug and Rhodes, 1987). The unique structure of MT may facilitate metal ion transfer between MTs and other metal-requiring proteins, including enzymes. Zn bound to MT should be available to cooperate in the regenerative liver process. It has been recently shown that the ad-
136
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
ministration of synthetic oligonucleotides with sequences complementary to the mRNA coding for human MT-2 inhibited MT production causing cells to die from metal toxicity (Takeda et al., 1994). Additionally, Arora et al. (1998) using a 17-mer antisense phosphorothioate oligonucleotide, complimentary to the start site of the MT-1 mRNA sequence, reported a decrease of MT-1 expression and consequently an inhibition of the peak of hepatocyte replication 24 h post-PH. Changes in subcellular localization of MT during cellular differentiation have been studied in the muscle cell lines L6 and H9C2. Initiation of apoptosis by free radical generation resulted in alterations and differential expression of MT in the nucleus and cytoplasm of affected cells. The H9C2 cells contained high levels of MT, differentiated slowly, and had a low incidence of apoptotic bodies compared to the L6 cell line (Apostolova et al., 1999). Apoptotic cell death is a process frequently occurring in toxin-induced liver injury, depending on the dosage and route of toxin administration, on the time of experimenta-
tion and on the assay used for apoptosis detection. Apoptosis in hepatocytes was found as an early feature of CCl4 toxicity (Shi et al., 1998). At 24 h post-CCl4 administration, increased apoptosis was found using the TUNEL assay (Hong and Park, 1997). In our study, we detected apoptotic figures that represent the end stage of the apoptotic process, using routine histological examination by H&E staining, at 48 h post-CCl4 injection. Additionally, apoptosis in hepatocytes was also observed during the fibrotic process occurring in the liver, 1 week post-CCl4 administration (Cabre et al., 1999). Although MT is detected as a cytoplasmic protein in hepatocytes of adult liver (Brady, 1982; Margeli et al., 1994; Webb, 1987), it can be localized in the hepatocyte nuclei in human fetal liver bound to Zn and Cu. Both nuclear and cytoplasmic localizations of MT have been observed in several tumors, mainly in regions of enhanced proliferation. Transient co-localization of Zn and MT has been shown in vitro, in differentiating myoblasts and 3T3-L1 fibroblasts and during the G1 to S phase progression of the cell
Fig. 5. Percentage of hepatocytes positive for MT indicated by dotted bars, in CCl4 treated-rats examined at different time points post-toxin administration. Results represent the findings in seven rats/time point. Vertical bars represent the SD from the mean.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
cycle. Several mechanisms have been proposed for the import and retention of MT in the nucleus, including signal transduction pathways. The high levels of MT in the nucleus of cells under certain conditions may be related to the increased Zn requirement for several metalloenzymes and transcription factors during rapid growth (Cherian and Apostolova, 2000). It has also been reported that cytoplasmic MT expression protects against the cytotoxicity of heavy metals, whereas its nuclear one protects against the cytotoxicity of mutagenic agents (Woo and Lazo, 1997). Our study describes the time course of MT expression in the liver, during toxin (CCl4)-induced injury and regeneration. The induction of MT in rat liver was more prominent during the proliferative process occurring after CCl4 administration. The role of MT induction during this process of injury and regeneration needs to be further investigated by blocking MT expression using antisense oligonucleotides in models of toxin-induced liver injury.
References Apostolova, M.D., Ivanova, I.A., Cherian, M.G., 1999. Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity. Toxicol. Appl. Pharmacol. 159, 175–184. Arora, V., Iversen, P., Ebadi, M., 1998. Manipulation of metallothionein expression in the regenerating rat liver using antisense oligonucleotides. Biochem. Biophys. Res. Commun. 246, 711– 718. Brady, F., 1982. The physiological function of metallothionein. Trends Biochem. Sci. 7, 143–145. Burr, A.W., Carpenter, M.R., Hines, J.E., Gullick, W.J., Burt, A.D., 1993. Intrahepatic distribution of transforming growth factor-alpha (TGF-a) during liver regeneration following carbon tetrachloride-induced necrosis. J. Pathol. 170, 95 – 100. Cabre, M., Ferre, N., Folch, J., Paternain, J.L., Hernandez, M., del Castillo, D., Joven, J., Camps, J., 1999. Inhibition of hepatic cell nuclear DNA fragmentation by zinc in carbon tetrachloride-treated rats. J. Hepatol. 31, 228–234. Cagen, S.Z., Klaassen, C.D., 1979. Protection of carbon tetrachloride-induced hepatotoxicity by zinc, role of metallothionein. Toxicol. Appl. Pharmacol. 51, 107–116. Cain, K., Griffiths, B., 1984. A comparison of isometallothionein synthesis in rats after partial hepatectomy and parenteral zinc injection. Biochem. J. 217, 85–92.
137
Cherian, M.G., Apostolova, M.D., 2000. Nuclear localization of metallothionein during cell proliferation and differentiation. Cell. Mol. Biol. 46, 347– 356. Clarke, I.S., Lui, M.K., 1986. Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol. 64, 1104– 1110. Cousins, R.J., 1983. Metallothionein-aspects related to copper and zinc metabolism. J. Inherit. Metab. Dis. 6, 15 – 21. Diaz-Gomes, M.I., Decastro, C.R., Dacosta, N., Defenos, O.M., Deferreyra, E.C., Castro, J.A., 1975. Species differences in carbon tetrachloride-induced hepatotoxicity: The role of CCl4 activation and of lipid peroxidation. J. Appl. Pharmacol. 34, 102– 114. DiSilvestro, R.A., Carlson, G.P., 1992. Inflammation, an inducer of metallothionein, inhibits carbon-tetrachloride-induced hepatotoxicity in rats. Toxicol. Lett. 60, 175– 181. Hamer, D.H., 1986. Metallothionein. Ann. Rev. Biochem. 55, 913– 951. Hambridge, K.M., Casey, C.E., Krebs, N.F., 1986. Zinc. In: Mertz, W. (Ed.), Trace Elements in Human and Animal Nutrition. Academic Press Inc, New York, pp. 1 – 137. Hong, S.W., Park, C., 1997. The effect of aflatoxin B1 on the expression of early response genes and transforming growth factor-alpha in CCl4 induced rat liver injury. Yonsei. Med. J. 38, 167– 177. IFCC, 1978. International Federation of Clinical Chemistry: Committee on standards, Enzyme Panel. Clin. Chem. 24, 720– 721. Kagi, J.H.R., Vasak, M., Lerch, K., Gilg, D., Hunzika, P., Bernhard, W.R., Good, M., 1984. Structure of metallothionein. Environ. Health. Perspect. 54, 93 – 104. Kagi, J.H.R., 1993. Evolution, structure and chemical activity of class I metallothioneins: an overview. In: Suzuki, K.T., Imura, N., Kimura, M. (Eds.), Metallothionein III. Biological Roles and Medical Implications. Birkhauser, Berlin, pp. 29 – 56. Kahn, D., Svanas, G., Eagopn, P., Makowka, L., Podesta, L., Chapchap, P., Starzl, T.E., VanThiel, D.H., 1988. Effect of an antiandrogenic H2 receptor antagonist on hepatic regeneration in rats. J. Lab. Clin. Med. 112, 232– 239. Kalf, G.F., Post, G.B., Snyder, R., 1987. Solvent toxicology: recent advances in the toxicology of benzene, the glycerol ethers, and carbon tetrachloride. Annu. Rev. Pharmacol. Toxicol. 27, 399– 427. Klug, A., Rhodes, D., 1987. Zinc fingers: a novel protein motif for nucleic acid regulation. Trends Biochem. Sci. 12, 464– 469. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265– 275. Margeli, A.P., Theocharis, S.E., Yannacou, N., Spiliopoulou, C., Koutselinis, A., 1994. Metallothionein expression during liver regeneration after partial hepatectomy in cadmium-pretreated rats. Arch. Toxicol. 68, 637– 642. Margoshes, M., Valle, B.C., 1957. A cadmium protein for equine kidney cortex. J. Am. Chem. Soc. 79, 4813– 4814.
138
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
Moffatt, P., Plaa, G.L., Denizeau, F., 1995. Induction of metallothionein gene expression by epidermal growth factor and its inhibition by transforming growth factor-beta and dexamethasone in rat hepatocytes. Hepatology 21, 1038– 1044. Munro, H.N., Fleck, A., 1966. Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78 – 88. Ohtake, H., Koga, M., 1978. Purification and characterization of zinc-binding protein from the liver of the partially hepatectomized rat. Biochem. J. 183, 683–690. Piscator, M., 1964. On cadmium in normal human kidneys together with a report on the isolation of metallothionein from livers of cadmium exposed rabbits. Nord. Hyg. 45, 76– 82. Recknagel, R.O., Glende, E.A., 1989. The carbon tetrachloride hepatotoxicity model: free radicals and calcium homeostasis. In: Miquel, J., Quintanilha, A.T., Weber, H. (Eds.), Handbook of Free Radicals and Antioxidants in Biomedicine. CRC Press, Boca Raton, Florida, pp. 1–16. Richards, G.M., 1974. Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57, 369–376. Shi, J., Aisaki, K., Ikawa, Y., Wake, K., 1998. Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride. Am. J. Pathol. 153, 515–525. Slater, T.F., 1966. Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature 209, 36 – 40. Slater, T.F., Sawyer, B.C., 1971. The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fraction in vitro. Biochem. J. 123, 815–821. Suzuki, K., Nakajima, K., Otaki, N., Kimura, M., 1994. Metallothionein in developing human brain. Biol. Signals 3, 188– 192. Takeda, A., Norris, J.S., Iversen, P.L., Ebadi, M., 1994. Antisense oligonucleotide of c-myc discriminates between zinc-and dexamethasone-induced synthesis of metallothionein. Pharmacology 48, 119–126.
Theocharis, S.E., Koutselini, H., Spiliopoulou, C., Margeli, A.P., Koutselinis, A., 1999. Intrahepatic distribution of heat shock protein -27 and -70 in carbon tetrachloride intoxicated rats [abstract]. Toxicol. Lett. 109 (Suppl. 1), 29. Thomas, J.P., Bachowski, G.J., Girotti, A.W., 1986. Inhibition of cell membrane lipid peroxidation by cadmium and zinc-metallothionein. Biochim. Biophys. Acta 884, 448– 461. Thornalley, P.J., Vasak, M., 1985. Possible role of metallothionein in the protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36 – 44. Tohyama, C., Suzuki, J.S., Hemelraad, J., Nishimura, N., Nishimura, H., 1993. Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy. Hepatology 18, 1193– 1201. Tsujikawa, K., Suzuki, N., Sagawa, K., Itoh, M., Sugiyama, T., Kohama, Y., Otaki, N., Kimura, M., Mimura, T., 1994. Induction and subcellular localization of metallothionein in regenerating rat liver. Eur. J. Cell. Biol. 63, 240– 246. Waalkes, M., Ward, J., 1989. Induction of hepatic metallothionein in male B6C3F1 mice exposed to hepatic tumor promoters: effects of phenobarbital, acetaminophen, sodium barbital and di(2-ethyl-hexyl)phthalate. Toxicol. Appl. Pharmacol. 100, 217– 226. Waalkes, M., Goering, P., 1990. Metallothionein and other cadmium-binding proteins: recent developments. Chem. Res. Toxicol. 3, 281– 288. Webb, M., 1987. Metallothionein in regeneration, reproduction and development. Experientia 52 (Suppl.), 483– 498. Wong, K., Klaassen, C., 1979. Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J. Biol. Chem. 254, 12399– 12403. Woo, E.S., Lazo, J.S., 1997. Nucleocytoplasmic functionality of metallothionein. Cancer Res. 57, 4236– 4241.
.
Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study Stamatios E. Theocharis a,*, Alexandra P. Margeli a,b, Spyridon D. Skaltsas a, Chara A. Spiliopoulou a, Antonios S. Koutselinis a a
Department of Forensic Medicine and Toxicology, Medical School, Uni6ersity of Athens, 11527 Goudi, Athens, Greece b Laboratory of Biochemistry, ‘Aghia Sophia’ Children’s Hospital, 11527 Goudi, Athens, Greece Received 6 November 2000; received in revised form 21 December 2000; accepted 11 January 2001
Abstract Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl4) administration. A single intraperitoneal injection of 1 ml CCl4/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl4 administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl4, in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl4 administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl4, being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carbon tetrachloride; Injury; Liver; Metallothionein; Rats; Regeneration
1. Introduction
* Corresponding author. 21, Thessalias street; Zografou; GR 15772; Athens; Greece. Tel.: +30-1-7780114; fax: +30-17780114. E-mail address: [email protected] (S.E. Theocharis).
Metallothionein (MT) was first discovered by Margoshes and Valle (1957) as a cadmium (Cd) binding protein. Later on Piscator (1964) mentioned a marked increase of MT content in Cd exposed rabbits, as a metal detoxification mecha-
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 1 ) 0 0 3 4 0 - 7
130
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
nism. MTs are a family of heavy metal binding proteins with a large degree of sequence homology. They are single-chain proteins of approximately 6000 Da, characterized by a very high proportion of cysteine residues, which results in several high affinity Cd and/or zinc (Zn) binding sites (Waalkes and Ward, 1989). There are four classes of similar MT proteins, designated MT-1 through -4, defined on the basis of small differences in amino acid sequence and charge characteristics, which can be resolved chromatographically (Cousins, 1983; Hamer, 1986; Kagi et al., 1984; Kagi, 1993). High concentrations of MT are induced in various tissues of the fetus and neonate at the end of term and during the initial postnatal period, respectively (Waalkes and Goering, 1990; Suzuki et al., 1994). During this time period the liver contains the highest concentration of MT (Brady, 1982; Wong and Klaassen, 1979). The elevated levels of MT observed during such situations of high Zn demand are thought to provide Zn within rapidly growing tissues. It has also been mentioned that MT is expressed during hepatocellular proliferation, after partial hepatectomy (PH) in rats (Cain and Griffiths, 1984; Margeli et al., 1994; Ohtake and Koga, 1978; Tsujikawa et al., 1994). Although PH is a wonderful model for triggering physiological mechanisms responsible for regulating hepatocyte proliferation and differentiation, many differences have been identified between regeneration in this model, and in that occurring after chemically induced liver injury. Carbon tetrachloride (CCl4) often serves as an hepatotoxicity inducing agent and its effects have been widely studied and mechanisms have also been proposed for the necrosis produced in the liver (Burr et al., 1993; Diaz-Gomes et al., 1975; Kalf et al., 1987; Slater, 1966; Slater and Sawyer, 1971). In vivo, CCl4 is metabolized to a trichloromethyl free radical (.CCl3), which initiates changes leading eventually to severe cellular damage. Acute CCl4 administration in rats results in hepatic damage, followed by a regenerative process, which peaks later on (Burr et al., 1993). Liver regeneration in vivo has usually been obtained from studies following PH in rats. Induction of hepatocyte proliferation after injury induced by a single intraperitoneal administration of the toxin CCl4 is well docu-
mented, and the necrosis induced by this agent is similar to that of acute drug-induced liver necrosis in humans. In the present study we investigated the expression of hepatic MT over the time course of injury and regeneration induced by acute CCl4 intoxication in rats, by examining alterations in the distribution of this protein, using immunohistochemical techniques.
2. Methods
2.1. Experimental animal model Male Wistar rats (Hellenic Pasteur Institute, Athens, Greece) weighing 190 –230 g each, were used in this study. They had free access to food and water, were kept in an air-conditioned room at 21°C with a 12 h light/dark cycle, and were handled humanely, in accordance with the National Institute of Health guidelines for care and use of laboratory animals. Experiments were started between 07:00 and 09:00 h. The animals were injected intraperitoneally with 1 ml of CCl4/kg body weight, diluted 1:10 in olive oil, under light ether anesthesia (diethyl ether per anesthesia, Codex, Carlo Erba, Milan, Italy). Rats were killed by cardiac puncture at 0, 12, 24, 36, 48, 60 and 72 h post-CCl4 treatment, (seven animals/time point). Immediately after exsanguination, the livers were removed, cleaned and weighed. Small portions of the livers were kept frozen at −80°C to be analyzed for their DNA content and their thymidine kinase (TK, EC 2.7.1.21) activity, while another portion was separated and immersed in buffered formalin solution for histological examination.
2.2. DNA biosynthesis and TK acti6ity One hour before the rats were killed, each animal was injected intraperitoneally with 25 mCi of tritiated thymidine/100 g body weight (Amersham Corp, Buckinghamshire, UK). The livers were homogenized in ice-cold deionized water, and the DNA was extracted according to the method of Munro and Fleck (1966). The determination of DNA in the liver tissue residues was based on the
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
reaction between deoxyribose and diphenylamine as described by Richards (1974). The specific activity of DNA was calculated from the radioactivity measured by a liquid scintillation counter (1211 Rackbeta; LKB-Wallac, Turku, Finland) and the amount of DNA that was determined colorimetrically. Results were expressed as counts per minute (cpm) incorporated/microgram of hepatic DNA (cpm/mgDNA). The enzymatic activity of TK was determined by measuring the in vitro conversion of deoxythymidine to deoxythymidinemonophosphate (Kahn et al. 1988) in the supernatant obtained after homogenization of liver portions and centrifugation at 105 000× g for 1 h at 4°C with an ultracentrifuge (model L5-75; Beckman Instruments, Fullerton, CA). Triplicate aliquots of each sample were spotted onto diethylaminoethyl cellulose discs. The disks were counted for their radioactivity content in a liquid scintillation counter (1211 Rackbeta, LKB-Wallac). The protein was determined by the method of Lowry et al. (1951). The activity of the enzyme TK was expressed as cpm incorporated/min/milligram of protein (cpm/min/ mg protein).
2.3. Enzymatic e6aluation Blood samples were collected from all animals via cardiac puncture. The samples were allowed to clot and the serum was removed by centrifugation at 1000× g for 10 min. All serum samples were sterile, hemolysis-free, and were kept at 4°C before determination of the enzymatic activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (IFCC, 1978), with commercially available kits, using a random access chemistry analyzer (RA-1000; Technicon Instruments Corp., Tarrytown, N.Y.). The activity of the enzymes AST and ALT was expressed as IU/l.
131
number so that the examiner was unaware of the group to which the specimen belonged. The morphologic changes produced in the liver by the intraperitoneal administration of CCl4 were examined during the time course of the whole experiment. The mitotic index was assayed as the number of mitoses observed/ten high power fields (HPF) chosen at random.
2.5. Immunohistochemistry Sections of 4 mm thickness were dewaxed in xylene and brought to water through graded alcohols. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide (BDH Ltd, Poole, England) in methanol for 30 min at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum (Dakopatts, Glostrup, Denmark) diluted 1:5 in phosphate buffered saline (PBS) for 20 min. A mouse (IgG1k) monoclonal antibody that reacts with human and rat MT (Zymed, San Francisco, CA) was used in this study. This antibody reacts with both MT-1 and MT-2 of rat and human origin. The sections were incubated for 1 h at room temperature, with the primary antibody diluted 1:50 in PBS. After washing three times with PBS, sections were incubated for 30 min at room temperature with linking reagent (biotinylated, rabbit, anti-mouse immunoglobulins, diluted 1:200 in PBS) and rinsed three times with PBS and with the peroxidase-conjugated streptavidin label respectively, for 20 min at room temperature. Peroxidase activity was developed in 0.5% (v/v) 3,3%-diaminobenzidine hydrochloride (DAB; Sigma, Saint Louis, MO) in PBS containing 0.03% (v/v) hydrogen peroxide for 5 min. Sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted in gelatin (BDH). The intensity of staining was graded as mild, moderate and intense.
2.4. Histopathology 2.6. Statistical analysis All liver sections were fixed in 4% volume/volume (v/v) buffered formalin solution and embedded in paraffin wax. Sections were cut at 4 mm and stained with hematoxylin and eosin (H&E). All specimens were randomized and given a code
The Wilcoxon test for unpaired measurements was applied to analyze the data. The results were considered as statistically significant when PB 0.05.
132
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
3. Results Animals killed at 0 h constitute the control group for the whole experiment. The administration of CCl4 caused increased activities of serum hepatic enzymes AST and ALT, compared to those observed at the time point of 0 h, (P B 0.001), presenting a peak 36 h after treatment (Fig. 1). The liver sections stained by H&E were examined for necrotic cells, extent of inflammation, and hepatocyte proliferation during the experiment after CCl4 injection. At 12 h after CCl4 administration, the perivenular and midzonal regions exhibited coagulative-type necrosis of hepatocytes. Many hepatocytes appeared having either basophilic (pyknotic) or fragmented (karyorrhectic) nuclei. Macrovesicular fatty change was also evident in some hepatocytes in the vicinity of necrotic areas. A heavy inflammatory infiltrate adjacent to necrotic liver cells was present in centrilobular areas while in the portal area the
hepatocytes remained unaffected. Twenty-four hours after CCl4 injection, hepatocytes in the centrilobular areas presented degenerative changes, while lymphocytic and polymorphonuclear infiltrations were also noted (Fig. 2). Additionally extensive steatosis and foamy degeneration of hepatocytes was observed. Coagulative necrosis was also prominent at 36 h postCCl4 treatment and mitotic activity appeared in the remnant hepatocytes. At 48 h after CCl4 administration, lytic type necrosis, with apparent apoptotic figures in hepatocytes was found in centrilobular areas. The hepatocytes throughout the lobule presented enhanced mitotic activity. The hepatocyte proliferation, as a tissue repair process following hepatic injury due to CCl4 administration, was also evident by the increased indices of regeneration, such as rate of DNA biosynthesis, hepatic TK activity and number of mitoses in hepatocytes. DNA biosynthesis peaked at 48 and 60 h after CCl4 administration (Table 1). TK activity peaked at 36 and 48 h after the
Fig. 1. Serum AST and ALT activities represented by filled and open bars respectively, in CCl4-treated rats examined at different time points post-toxin administration. Results represent the findings in seven rats/time point and are expressed as IU/l. Vertical bars represent the SD from the mean.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
133
Fig. 2. Extensive centrilobular necrosis observed in the liver sections of CCl4-treated rats, at 24 h post-toxin administration. The vessel in the center is a central vein. The large clear cells surrounding the perivenular necrotic zone are hepatocytes ballooned due to macrovesicular steatosis (H&E, magnification X 125). Fig. 3. Intense MT immunoreactivity in the cytoplasm and the nuclei of centrilobular hepatocytes and endothelial cells of the central vein in liver sections of CCl4-treated rats, at 36 h post-toxin administration. The tracer is peroxidase and specific signal appears brown in colour. Counterstained with Mayer’s hematoxylin (magnification X 400).
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
134
injection of the toxic agent (Table 1). Mitotic activity was increased from 36 h post-CCl4 administration, peaked at 48 and 60 h, while later on it progressively decreased (Table 1). MT was detected in isolated cells of the hepatic parenchyma, and in hepatocytes around the central veins, as early as 12 h post-CCl4 administration, presenting an intense pattern of staining in the nuclei and mild staining in the cytoplasm. At 24 h after the administration of CCl4, intense nuclear and cytoplasmic patterns of staining for MT were observed in hepatocytes located in the vicinity of inflammatory infiltrations and necrotic areas. At 36 h the intensity of staining was higher and MT immunoreactivity was prominent in all hepatocytes. Central vein endothelial cells presented nuclear and cytoplasmic patterns of staining (Fig. 3). The maximum expression of MT occurred at the time point of 48 h, in the cytoplasm and nuclei of almost all hepatocytes (Fig. 4 and Fig. 5). At 60 h after CCl4 injection, the hepatocytes presented mild cytoplasmic intensity for MT. At the further time points examined, positivity for both cytoplasmic and nuclear MT staining was noted only in isolated hepatocytes. Kupffer and stellate cells did not show any MT immunoreactivity during the course of the experiment.
4. Discussion CCl4 has previously been shown to exert its acute toxic effects on the liver (Slater and Sawyer,
1971; Diaz-Gomes et al., 1975). The administration of CCl4 in rats caused severe liver injury recognized histopathologically, being also confirmed by the increased activities of serum hepatic enzymes AST and ALT. The development of hepatic necrosis becomes prominent as early as 12 h after the administration of CCl4 and peaks at 36 h. Later on, a regenerative process of hepatocytes occurred, as tissue repair is a biological response that accompanies chemically-induced liver injury. In response to CCl4-induced hepatotoxicity, an increase in the intensity of MT immunoreactivity was noted as early as 12 h following exposure to the toxin, with a maximum at 48 h, the time coinciding with the peak of hepatocyte proliferation. MT is a small cysteine-rich protein, thought to be mainly involved in metal regulation and detoxification, implicated also in cell growth and differentiation (Moffatt et al., 1995; Webb, 1987). It has already been mentioned that, in cases of hepatocellular proliferation as during hepatic regeneration after PH, an induction of liver MT occurs (Cain and Griffiths, 1984; Margeli et al., 1994; Moffatt et al., 1995; Tohyama et al., 1993). It is known that the physiological function of MT is related to Zn homeostasis, so the MT produced supplies Zn to other sites within the cell, as this element is not only essential for various aspects of cell division including DNA, RNA and protein synthesis (Brady, 1982), but is also an important cofactor in various enzymatic systems. It has been reported by Cain and Griffiths (1984) that both MT isoforms, MT-1 and MT-2 are expressed in
Table 1 Indices of liver regeneration in CCl4-treated rats examined at different time points post-toxin administrationa Time (h)
DNA synthesis (cpm/mg DNA)
TK activity (cpm/min/mg protein)
Mitotic index (mitoses/10 HPF)
0 12 24 36 48 60 72
11 9 2 33 93 35 94 44 9 4 187 915 120 918 55 96
13009 110 38799 364 38159 290 72509 650 82959 795 55459 560 41009 380
non detectable 3 90.3 4 90.5 109 0.8 24 9 3 16 9 2 9 90.8
a
Values are expressed as mean 9SD.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
135
Fig. 4. Maximum MT immunoreactivity in all hepatocytes in liver sections of CCl4-treated rats, at 48 h post-toxin administration. The tracer is peroxidase and specific signal appears brown in colour. Counterstained with Mayer’s hematoxylin (magnification X 125).
the liver of partially hepatectomized rats. We have also previously shown the time course of MT expression in the liver after 70% PH (Margeli et al., 1994), using immunohistochemical techniques. It has been shown that either acute phase response induction and infammation (DiSilvestro and Carlson, 1992) or Zn administration (Cagen and Klaassen, 1979; Clarke and Lui, 1986) inhibit CCl4 induced hepatotoxicity in rats. Inflammation and Zn injections, both protectors against CCl4 injury, are not known to produce any other common effects than the induction of MT. The toxic effects of CCl4 on the liver are theorized to involve free radicals (Recknagel and Glende, 1989) and MT can scavenge free radicals in vitro (Thomas et al., 1986; Thornalley and Vasak, 1985). There is also evidence that metabolites of injected CCl4 react with MT in vivo (Cagen and Klaassen, 1979). Alterations of MT expression were observed in the liver during injury and regeneration induced by CCl4 administration. In our study the immunohistochemical expression of MT during the first 24 h following CCl4 administration presented cellular distribution similar to that found for HSP-27 and -70, suggesting a role
for MT protein during stressful states (Theocharis et al., 1999), in order to protect the liver against toxin-induced injury. In the present study MT expression in the liver was correlated with the indices of hepatic proliferation. At the time of maximum hepatocyte proliferation, MT was highly expressed in the liver. The induction of MT during hepatic proliferation after PH caused decreased serum Zn levels in partially hepatectomized rats (Margeli et al., 1994). This shift of Zn, from the blood circulation to liver tissue, reflects the essential role of Zn in liver regeneration. Hepatic Zn, temporarily sequestered by MT, is available to DNA and RNA polymerases, which are Zn requiring metalloenzymes (Hambridge et al., 1986) and are associated with cell replication, differentiation and growth. Hepatic proliferation may also produce an increase demand for regulatory proteins with ‘Zn binding fingers’ (Klug and Rhodes, 1987). The unique structure of MT may facilitate metal ion transfer between MTs and other metal-requiring proteins, including enzymes. Zn bound to MT should be available to cooperate in the regenerative liver process. It has been recently shown that the ad-
136
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
ministration of synthetic oligonucleotides with sequences complementary to the mRNA coding for human MT-2 inhibited MT production causing cells to die from metal toxicity (Takeda et al., 1994). Additionally, Arora et al. (1998) using a 17-mer antisense phosphorothioate oligonucleotide, complimentary to the start site of the MT-1 mRNA sequence, reported a decrease of MT-1 expression and consequently an inhibition of the peak of hepatocyte replication 24 h post-PH. Changes in subcellular localization of MT during cellular differentiation have been studied in the muscle cell lines L6 and H9C2. Initiation of apoptosis by free radical generation resulted in alterations and differential expression of MT in the nucleus and cytoplasm of affected cells. The H9C2 cells contained high levels of MT, differentiated slowly, and had a low incidence of apoptotic bodies compared to the L6 cell line (Apostolova et al., 1999). Apoptotic cell death is a process frequently occurring in toxin-induced liver injury, depending on the dosage and route of toxin administration, on the time of experimenta-
tion and on the assay used for apoptosis detection. Apoptosis in hepatocytes was found as an early feature of CCl4 toxicity (Shi et al., 1998). At 24 h post-CCl4 administration, increased apoptosis was found using the TUNEL assay (Hong and Park, 1997). In our study, we detected apoptotic figures that represent the end stage of the apoptotic process, using routine histological examination by H&E staining, at 48 h post-CCl4 injection. Additionally, apoptosis in hepatocytes was also observed during the fibrotic process occurring in the liver, 1 week post-CCl4 administration (Cabre et al., 1999). Although MT is detected as a cytoplasmic protein in hepatocytes of adult liver (Brady, 1982; Margeli et al., 1994; Webb, 1987), it can be localized in the hepatocyte nuclei in human fetal liver bound to Zn and Cu. Both nuclear and cytoplasmic localizations of MT have been observed in several tumors, mainly in regions of enhanced proliferation. Transient co-localization of Zn and MT has been shown in vitro, in differentiating myoblasts and 3T3-L1 fibroblasts and during the G1 to S phase progression of the cell
Fig. 5. Percentage of hepatocytes positive for MT indicated by dotted bars, in CCl4 treated-rats examined at different time points post-toxin administration. Results represent the findings in seven rats/time point. Vertical bars represent the SD from the mean.
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
cycle. Several mechanisms have been proposed for the import and retention of MT in the nucleus, including signal transduction pathways. The high levels of MT in the nucleus of cells under certain conditions may be related to the increased Zn requirement for several metalloenzymes and transcription factors during rapid growth (Cherian and Apostolova, 2000). It has also been reported that cytoplasmic MT expression protects against the cytotoxicity of heavy metals, whereas its nuclear one protects against the cytotoxicity of mutagenic agents (Woo and Lazo, 1997). Our study describes the time course of MT expression in the liver, during toxin (CCl4)-induced injury and regeneration. The induction of MT in rat liver was more prominent during the proliferative process occurring after CCl4 administration. The role of MT induction during this process of injury and regeneration needs to be further investigated by blocking MT expression using antisense oligonucleotides in models of toxin-induced liver injury.
References Apostolova, M.D., Ivanova, I.A., Cherian, M.G., 1999. Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity. Toxicol. Appl. Pharmacol. 159, 175–184. Arora, V., Iversen, P., Ebadi, M., 1998. Manipulation of metallothionein expression in the regenerating rat liver using antisense oligonucleotides. Biochem. Biophys. Res. Commun. 246, 711– 718. Brady, F., 1982. The physiological function of metallothionein. Trends Biochem. Sci. 7, 143–145. Burr, A.W., Carpenter, M.R., Hines, J.E., Gullick, W.J., Burt, A.D., 1993. Intrahepatic distribution of transforming growth factor-alpha (TGF-a) during liver regeneration following carbon tetrachloride-induced necrosis. J. Pathol. 170, 95 – 100. Cabre, M., Ferre, N., Folch, J., Paternain, J.L., Hernandez, M., del Castillo, D., Joven, J., Camps, J., 1999. Inhibition of hepatic cell nuclear DNA fragmentation by zinc in carbon tetrachloride-treated rats. J. Hepatol. 31, 228–234. Cagen, S.Z., Klaassen, C.D., 1979. Protection of carbon tetrachloride-induced hepatotoxicity by zinc, role of metallothionein. Toxicol. Appl. Pharmacol. 51, 107–116. Cain, K., Griffiths, B., 1984. A comparison of isometallothionein synthesis in rats after partial hepatectomy and parenteral zinc injection. Biochem. J. 217, 85–92.
137
Cherian, M.G., Apostolova, M.D., 2000. Nuclear localization of metallothionein during cell proliferation and differentiation. Cell. Mol. Biol. 46, 347– 356. Clarke, I.S., Lui, M.K., 1986. Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol. 64, 1104– 1110. Cousins, R.J., 1983. Metallothionein-aspects related to copper and zinc metabolism. J. Inherit. Metab. Dis. 6, 15 – 21. Diaz-Gomes, M.I., Decastro, C.R., Dacosta, N., Defenos, O.M., Deferreyra, E.C., Castro, J.A., 1975. Species differences in carbon tetrachloride-induced hepatotoxicity: The role of CCl4 activation and of lipid peroxidation. J. Appl. Pharmacol. 34, 102– 114. DiSilvestro, R.A., Carlson, G.P., 1992. Inflammation, an inducer of metallothionein, inhibits carbon-tetrachloride-induced hepatotoxicity in rats. Toxicol. Lett. 60, 175– 181. Hamer, D.H., 1986. Metallothionein. Ann. Rev. Biochem. 55, 913– 951. Hambridge, K.M., Casey, C.E., Krebs, N.F., 1986. Zinc. In: Mertz, W. (Ed.), Trace Elements in Human and Animal Nutrition. Academic Press Inc, New York, pp. 1 – 137. Hong, S.W., Park, C., 1997. The effect of aflatoxin B1 on the expression of early response genes and transforming growth factor-alpha in CCl4 induced rat liver injury. Yonsei. Med. J. 38, 167– 177. IFCC, 1978. International Federation of Clinical Chemistry: Committee on standards, Enzyme Panel. Clin. Chem. 24, 720– 721. Kagi, J.H.R., Vasak, M., Lerch, K., Gilg, D., Hunzika, P., Bernhard, W.R., Good, M., 1984. Structure of metallothionein. Environ. Health. Perspect. 54, 93 – 104. Kagi, J.H.R., 1993. Evolution, structure and chemical activity of class I metallothioneins: an overview. In: Suzuki, K.T., Imura, N., Kimura, M. (Eds.), Metallothionein III. Biological Roles and Medical Implications. Birkhauser, Berlin, pp. 29 – 56. Kahn, D., Svanas, G., Eagopn, P., Makowka, L., Podesta, L., Chapchap, P., Starzl, T.E., VanThiel, D.H., 1988. Effect of an antiandrogenic H2 receptor antagonist on hepatic regeneration in rats. J. Lab. Clin. Med. 112, 232– 239. Kalf, G.F., Post, G.B., Snyder, R., 1987. Solvent toxicology: recent advances in the toxicology of benzene, the glycerol ethers, and carbon tetrachloride. Annu. Rev. Pharmacol. Toxicol. 27, 399– 427. Klug, A., Rhodes, D., 1987. Zinc fingers: a novel protein motif for nucleic acid regulation. Trends Biochem. Sci. 12, 464– 469. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265– 275. Margeli, A.P., Theocharis, S.E., Yannacou, N., Spiliopoulou, C., Koutselinis, A., 1994. Metallothionein expression during liver regeneration after partial hepatectomy in cadmium-pretreated rats. Arch. Toxicol. 68, 637– 642. Margoshes, M., Valle, B.C., 1957. A cadmium protein for equine kidney cortex. J. Am. Chem. Soc. 79, 4813– 4814.
138
S.E. Theocharis et al. / Toxicology 161 (2001) 129–138
Moffatt, P., Plaa, G.L., Denizeau, F., 1995. Induction of metallothionein gene expression by epidermal growth factor and its inhibition by transforming growth factor-beta and dexamethasone in rat hepatocytes. Hepatology 21, 1038– 1044. Munro, H.N., Fleck, A., 1966. Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78 – 88. Ohtake, H., Koga, M., 1978. Purification and characterization of zinc-binding protein from the liver of the partially hepatectomized rat. Biochem. J. 183, 683–690. Piscator, M., 1964. On cadmium in normal human kidneys together with a report on the isolation of metallothionein from livers of cadmium exposed rabbits. Nord. Hyg. 45, 76– 82. Recknagel, R.O., Glende, E.A., 1989. The carbon tetrachloride hepatotoxicity model: free radicals and calcium homeostasis. In: Miquel, J., Quintanilha, A.T., Weber, H. (Eds.), Handbook of Free Radicals and Antioxidants in Biomedicine. CRC Press, Boca Raton, Florida, pp. 1–16. Richards, G.M., 1974. Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57, 369–376. Shi, J., Aisaki, K., Ikawa, Y., Wake, K., 1998. Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride. Am. J. Pathol. 153, 515–525. Slater, T.F., 1966. Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature 209, 36 – 40. Slater, T.F., Sawyer, B.C., 1971. The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fraction in vitro. Biochem. J. 123, 815–821. Suzuki, K., Nakajima, K., Otaki, N., Kimura, M., 1994. Metallothionein in developing human brain. Biol. Signals 3, 188– 192. Takeda, A., Norris, J.S., Iversen, P.L., Ebadi, M., 1994. Antisense oligonucleotide of c-myc discriminates between zinc-and dexamethasone-induced synthesis of metallothionein. Pharmacology 48, 119–126.
Theocharis, S.E., Koutselini, H., Spiliopoulou, C., Margeli, A.P., Koutselinis, A., 1999. Intrahepatic distribution of heat shock protein -27 and -70 in carbon tetrachloride intoxicated rats [abstract]. Toxicol. Lett. 109 (Suppl. 1), 29. Thomas, J.P., Bachowski, G.J., Girotti, A.W., 1986. Inhibition of cell membrane lipid peroxidation by cadmium and zinc-metallothionein. Biochim. Biophys. Acta 884, 448– 461. Thornalley, P.J., Vasak, M., 1985. Possible role of metallothionein in the protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36 – 44. Tohyama, C., Suzuki, J.S., Hemelraad, J., Nishimura, N., Nishimura, H., 1993. Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy. Hepatology 18, 1193– 1201. Tsujikawa, K., Suzuki, N., Sagawa, K., Itoh, M., Sugiyama, T., Kohama, Y., Otaki, N., Kimura, M., Mimura, T., 1994. Induction and subcellular localization of metallothionein in regenerating rat liver. Eur. J. Cell. Biol. 63, 240– 246. Waalkes, M., Ward, J., 1989. Induction of hepatic metallothionein in male B6C3F1 mice exposed to hepatic tumor promoters: effects of phenobarbital, acetaminophen, sodium barbital and di(2-ethyl-hexyl)phthalate. Toxicol. Appl. Pharmacol. 100, 217– 226. Waalkes, M., Goering, P., 1990. Metallothionein and other cadmium-binding proteins: recent developments. Chem. Res. Toxicol. 3, 281– 288. Webb, M., 1987. Metallothionein in regeneration, reproduction and development. Experientia 52 (Suppl.), 483– 498. Wong, K., Klaassen, C., 1979. Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J. Biol. Chem. 254, 12399– 12403. Woo, E.S., Lazo, J.S., 1997. Nucleocytoplasmic functionality of metallothionein. Cancer Res. 57, 4236– 4241.
.