* Children's Research Institute, Center for Developmental Pharmacology and Toxicology, and Department of Pediatrics, Children's Hospital, The Ohio State University, Columbus, Ohio 43205; and
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
Received March 8, 2002; accepted June 3, 2002
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ABSTRACT |
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Key Words: glutathione; nuclei; nucleoli; glutathione reductase; glutathione peroxidase; glutathione S-transferases; diquat; Fischer-344 rats; oxidant stress; reactive oxygen species.
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INTRODUCTION |
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Administration of diquat to Fischer-344 and Sprague-Dawley rats causes marked increases in intrahepatic generation of reactive oxygen species (Lauterburg et al., 1984; Lawrence et al., 1976; Nakagawa et al., 1992
; Rikans et al., 1993), and readily produces acute hepatic necrosis in Fischer-344 rats, while Sprague-Dawley rats are remarkably resistant to liver damage (Smith, 1987
, Smith et al., 1985
). More recently, we observed that female Fischer-344 rats were more resistant than were males to oxidant-stress responses and acute hepatic necrosis from diquat treatment (Gupta et al., 2000
). The strain- and sex-dependent differences in susceptibility to diquat-induced hepatic necrosis have provided useful experimental models for studies of oxidant stress responses, particularly in distinguishing oxidative alterations that are observed in the absence of acute lethal cell injury, thereby enabling us to focus our efforts to identify the molecular events that are more tightly coupled with cytotoxicity, and are therefore more likely to contribute to the mechanisms responsible for initiation of cell death by reactive oxygen species in vivo.
Cellular mechanisms for defense against the potentially damaging effects of reactive oxygen species rely heavily upon glutathione (GSH), which also participates in many other functions essential to normal cell physiology (Meister, 1989; Smith and Mitchell, 1989
). The presence of GSH is necessary, but not sufficient, for effective functioning of GSH-dependent antioxidant defense mechanisms, which require the contributions of enzymes that include glutathione peroxidases (GPX), glutathione S-transferases (GSTs), and glutathione reductase (GR). GSH-dependent antioxidant defense mechanisms exhibit significant compartmentalization (Smith et al., 1996
), and mitochondrial expressions have received particular attention, in part due to the relevance of mitochondrial thiol status to apoptotic mechanisms of cell death (Cai et al., 1998
, 2000
; Petronilli et al., 1994
). Proteins that are encoded by the nuclear genome are directed to mitochondria by mitochondrial targeting signals that typically are N-terminal sequences of 20 to 60 amino acids with numerous positively charged and few, if any, negatively charged amino acid residues (Neupert, 1997
; Tamura et al., 1996
, 1997
).
In principle, protection of nuclear contents from damage by reactive oxygen species should be of comparable importance, but less has been published on nuclear compartmentalization of GSH-dependent antioxidant defense mechanisms. Soboll et al.(1995) reported the presence of GSH, GPX, and GSTs in nuclear fractions in the livers of male Wistar rats, but little has been published on possible regulatory mechanisms or responses to stress by these enzymes. The nuclear membrane pore complex shows an exclusion size limit for diffusion of globular proteins of about 60 kDa, and transport from the cytoplasm to the nucleus of proteins larger than about 20 kDa, involves highly organized and regulated mechanisms (Agutter et al., 1994; Garcia-Bustos et al., 1991; Schmidt-Zachmann et al., 1993). Nuclear and nucleolar localizations of proteins are directed by one or two positively charged peptide sequences that are internal in the primary sequence of amino acids, but presumably are localized on the surface of the respective proteins, at least in the conformers that are localized to the nucleus and/or nucleolus.
The objective of the present study was to test the hypotheses that GR, GPX, and GSTs are expressed in rat liver nuclei and that differences in one or more of these antioxidant defense enzymes, either basally or in response to administration of comparably hepatotoxic doses of diquat, would parallel the differences in susceptibility of male and female Fischer-344 rats in this model of acute hepatic necrosis.
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MATERIALS AND METHODS |
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Animals.
Adult male and female Fischer-344 rats were purchased from Harlan Sprague-Dawley (Houston, TX) and were adapted for at least 3 days before study. The animals were kept in an air-conditioned room on a 12-h light/dark cycle, with food and tap water available ad libitum. Diquat was administered, ip, in normal saline, and control animals received equal volumes of saline alone. Six h later, the animals were heavily anesthetized with 200 mg/kg of pentobarbital, ip, and the thoracic and abdominal contents were exposed. Blood samples were obtained by cardiac punctures into heparinized syringes for subsequent determinations of plasma alanine amino transferase (ALT) activities, and livers were removed for isolation of hepatic nuclei.
Isolation of nuclei and nucleoli.
Nuclei and nucleoli were isolated according to the procedure of Higashinakagawa et al.(1972), and the procedure was carried out at 24°C. Briefly, the livers were weighed, minced, and homogenized in 0.25 M sucrose containing 50 mM Tris, 25 mM KCl, and 5 mM MgCl2, pH 7.4. The homogenates were centrifuged at 700 x g for 20 min, the pellets washed once with the sucrose-Tris buffer, and the washed pellets suspended in 10 volumes of 2.3 M sucrose-10 mM MgCl2 with 3 strokes in a Dounce homogenizer. The resulting homogenates were centrifuged at 40,000 x g for 1 h. The nuclear pellets were resuspended in 0.34 M sucrose containing 0.05 mM MgCl2, homogenized gently, and the suspensions visualized under a light microscope. The nuclei were sonicated in a sonicator (10 kC/s) at an output of 200 W for 4560 s. We examined sonicates with a light microscope for the extent of disruption of nuclei. When virtually all the nuclei were disrupted, the sonicates were underlayered with 20 ml of 0.88 M sucrose containing 0.05 M MgCl2 and centrifuged at 2000 x g for 20 min at 4°C. The supernatants were discarded, and the inside surfaces of the centrifuge tubes were wiped well with clean tissue papers. The resulting pellets contained purified nucleoli, which were checked under light microscope by staining them with azure C.
Biochemical assays.
Nuclei were assayed for DNA contents by fluorimetric measurements of dye binding (Hoechst 33258) using a TKO 100 Fluorometer (Hoefer Scientific Instruments, San Francisco, CA). RNA concentrations were determined by the method of Kerr and Seraidarian (1945). Plasma ALT activities were determined using Sigma assay kit (Procedure No.59-UV), and protein concentrations were measured as we have described previously (Gupta et al., 2000).
Western analyses.
Equal amounts of proteins (100 µg) were loaded in each lane and were separated by SDS/2-mercaptoethanol/polyacrylamide (SDSPAGE) slab gel electrophoresis; the stacking and the resolving gels contained 5% (w/v) and 12.5% (w/v) polyacrylamide, respectively, followed by western blotting performed as we have described previously (Gupta et al., 2000). Transblotting to PVDF membranes was performed for 4 h at 25°C, using a Bio-Rad transblot cell (Bio-Rad Laboratories), and the membranes were soaked in a solution of 5% (w/v) non-fat dry milk in 20 mM Tris buffer, pH 7.6, containing 150 mM NaCl and 0.1% Tween-20 (TBS-T) for 30 min at 25°C. The PVDF membranes were incubated overnight with monoclonal antinuclear or antinucleolar antibody (MABs 1277 and 1281, Chemicon Int., Temecula, CA), diluted 1:2500. These antibodies, prepared as purified hybridoma supernatants, were used essentially as recommended by the supplier (www.chemicon.com), and were not characterized further. After rinsing the PVDF membranes with TBS-T for 30 min (6 changes of 50 ml, 510 min each), the membranes were incubated with 50 ml of TBS-T containing horseradish peroxidase-conjugated antiserum (diluted 1:1500) for 4 h at 25°C. Finally, the membranes were rinsed 6 times with TBS (6 changes of 50 ml, 510 min each), and the bands were visualized on the Hyperfilm by enhanced chemiluminescence.
GR activities.
GR activities were assayed as we have described previously (Gupta et al., 2000). Assay mixtures consisted of 83 µmol Tris (pH 8.0), 0.8 µmol of EDTA, 5.70 µmol GSSG in 0.1 M Tris (pH 7.0), and 0.2 µmol NADPH in total volumes of 0.8 ml. To the above mixtures, 0.2 ml of homogenate fractions (generally 1.0 to 1.5 mg of proteins) were added, mixed rapidly, and rates of oxidation of NADPH followed at 340 nm.
Glutathione peroxidase (GPX) activities.
GPX activities with cumene hydroperoxide and hydrogen peroxide (H2O2) were determined according to the method of Lawrence and Burk (1976). Briefly, the reaction mixtures consisted of 50 mM KPO4 (pH 7), 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 U/ml GR, 1 mM GSH, and 1.5 mM cumene hydroperoxide or 0.25 mM H2O2. All ingredients except homogenate fraction and peroxide were combined at the beginning of each day. Homogenate fractions (0.1 ml) were added to 0.8 ml of the reaction mixtures and allowed to incubate for 5 min at room temperature before initiation of the reaction by the addition of 0.1 ml of peroxide solution. Absorbances at 340 nm were recorded for 5 min, and the activities were expressed as mU (µmol NADPH oxidized per min). Blank reactions with homogenate fractions replaced by distilled water were subtracted from each assay.
Glutathione S-transferase (GST) activities.
GST activities with 1-chloro-2,4-dinitrobenzene (CDNB) were determined by the method of Habig et al. (1974). GST- activities were calculated as the differences of GPX activities measured by subtracting the activities measured with H2O2 from the activities measured with cumene hydroperoxide (Reddy et al., 1981
).
Statistics.
Data are expressed as means ± SEM and were analyzed with a 2-way ANOVA and post hoc testing with modified t-tests, using SPSS Version 9.0. Values of p < 0.05 were considered significant.
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RESULTS |
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DISCUSSION |
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Burk observed that selenium-deficient rats showed greatly enhanced susceptibility to diquat-induced liver damage, implicating a critical role for Se-dependent glutathione peroxidase, but not precluding contributions from other selenoproteins or secondary effects of dietary selenium deficiency (Burk et al., 1980). Subsequently, Fu demonstrated greater diquat-induced liver damage in knockout mice lacking expression of GPX-1 (Fu et al., 1999
). These reports thus strongly support the importance of GPX- and GSH-linked functions in antioxidant defense mechanisms relevant to diquat toxicity. However, the magnitudes of differences in GPX and GR activities created in the experimental models reported previously (Burk et al., 1980
; Fu et al., 1999
) are substantially greater than are the gender or treatment differences observed in the nuclear and nucleolar fractions in the present studies (Fig. 3
) or in hepatic homogenates, as we have reported previously (Gupta et al., 2000
).
Bellomo et al.(1992) observed greater fluorescence intensities in nuclei than in cytoplasm of monochlorobimane-treated hepatocytes and interpreted these results as evidence for a distinguishable nuclear compartment of GSH. Monochlorobimane reacts with GSH in a GST-catalyzed reaction to form the same fluorescent thioether (GS-bimane) as is formed by reaction of GSH with monobromobimane, which reacts readily with thiols other than GSH and does not require GST catalysis. Monochlorobimane-derived fluorescence is reasonably attributed to the GSH-derived thioether, but Briviba et al.(1993) observed that preformed GS-bimane thioether microinjected into rat hepatocytes was concentrated rapidly into the nuclei. The nuclear fluorescence reported by Bellomo et al., although probably reflecting nuclear concentrations of GSH-derived bimane thioether, may not have been proportional to nuclear GSH concentrations. In other studies, Voehringer et al.(1998) used rapid centrifugation through silicone oil of nuclei from partially solubilized cells to estimate nuclear GSH contents in cultured cells. These investigators observed nuclear concentrations of GSH that were modulated by cellular expression of Bcl-2 differently than were total cell GSH contents, which suggests nuclear compartmentalization of GSH.
An equally intriguing and potentially important question raised by our data relates to how the cell regulates or otherwise directs expression of GR, GPX, and GST to nuclear, mitochondrial, or cytoplasmic compartments. GR is a product of a single nuclear gene that is expressed in cytosolic, mitochondrial, nuclear, and nucleolar compartments. Examples of nuclear and mitochondrial expression of products of a single nuclear gene have been reported for other proteins (Lakshmipathy et al., 1999; Otterlei et al., 1998; Slupphaug et al., 1993
), and potential mechanisms for differential expression have been described (Danpure, 1995
). In general, the effects of an active N-terminal mitochondrial targeting sequence dominate the effects of nuclear localization sequences, which usually are internal within the protein sequence and are not removed following entry to the nucleus, while many N-terminal mitochondrial targeting sequences are removed following import into mitochondria (Danpure, 1995
; Garcia-Bustos et al., 1991
; Mattaj et al., 1998; Neupert, 1997
; Slupphaug et al., 1993
).
Some proteins involved in cell cycle control move between the nucleus and the cytoplasm in the course of cell cycle progression (Danpure, 1995). Consistent with this possibility, we have observed greater nuclear GR activities of CHO cells during S phase than in cells in other stages of the cell cycle (Rogers et al., 2002
). In addition to the possible contributions of intranuclear thiol redox changes to cell function, DNA uncoiled from histones, as for replication during S phase, would be at increased risk for potential harm from reactive oxygen species, and survival advantages of enhanced antioxidant protection of nuclei could be especially acute in this phase of the cell cycle.
The physical association of GR and GPX with nucleoli suggests a functional relationship or purpose that is likely to be important. Nucleolar associations usually are determined by nucleolar localization sequences. Disruption or deletion of nucleolar localization sequences does not prevent proteins that are normally nucleolar from accumulating in the nucleus, but such modifications result in uniform distribution of the modified protein throughout the nucleus (Rose et al., 1992; Schmidt-Zachmann and Nigg, 1993
). In contrast, nucleolar localization sequences are not sufficient to direct proteins not normally found in the nucleus to nuclear or nucleolar compartments. Although the potential functional purposes of GR and GPX in the nucleus are easy to rationalize, the survival advantages afforded by nucleolar association of GR and GPX are not as obvious. Visintin and Amon (2000) recently suggested that sequestration in the nucleolus may serve to prevent proteins from reaching their normal locations in the cell, in effect inactivating the proteins until their functions are required. These authors suggested that such sequestration and release would be most attractive for proteins active in regulation of cell cycle functions. The associations of GR and GPX with nucleoli are clear, but the functions served by nucleolar sequestration of these enzymes are not evident from the data presently available.
Whatever the relevance of nuclear GR, GPX, and the GSTs in resistance to diquat-induced hepatic necrosis in vivo and cytotoxicity in vitro, these enzymes are likely to contribute in some fashion to cell defenses against adverse effects of reactive oxygen species. The activities of GR and GPX in cell nuclei and nucleoli suggest significant roles for these enzymes, but the specific functions of these proteins may not be limited to antioxidant defense functions. The numerous redox- and thiol/disulfide-dependent mechanisms in regulation of gene expression, signal transduction, and cell cycle progression that have been reported (Arrigo, 1999; Shackelford et al., 2000
; Sun et al., 1996) suggest functions that might be served by the GSH-dependent antioxidant system and related enzymes and systems (Mustacich and Powis, 2000
; Powis and Montfort, 2001
). Alternative hypotheses include those in which nuclear and/or nucleolar GR, GPX and GST function in capacities not directly related to their antioxidant functions, such as have been demonstrated with GPX-4 serving as a structural protein in sperm flagella (Ursini et al., 1999
), GSTpi regulation of JNK activities in cell proliferation (Ruscoe et al., 2001
; Yin et al., 2000
), and GAPDH contributing to several cellular activities from transcriptional/translational regulation to apoptosis in neuronal cells (Sawa et al., 1997
; Shashidharan et al., 1999
). Obviously, further studies will be required to determine whether oxidant-inducible changes in the relative distributions of antioxidant enzymes within nuclear or other subcellular pools could contribute to differences in susceptibility to oxidant-induced injury.
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ACKNOWLEDGMENTS |
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NOTES |
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