Nuclear and Nucleolar Glutathione Reductase, Peroxidase, and ...

TOXICOLOGICAL SCIENCES 69, 279 –285 (2002) Copyright © 2002 by the Society of Toxicology

Nuclear and Nucleolar Glutathione Reductase, Peroxidase, and Transferase Activities in Livers of Male and Female Fischer-344 Rats Lynette K. Rogers,* ,1 Sanjiv Gupta,† Stephen E. Welty,* Thomas N. Hansen,* and Charles V. Smith* *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

The present studies were to test the hypotheses that glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities are expressed in nuclei and nucleoli of rat liver cells, and that differences in activities of these enzymes would correlate with the greater resistance of female than of male Fischer-344 rats to hepatic necrosis in vivo, mediated by reactive oxygen species generated by redox-cycling metabolism of diquat. Adult male and female Fischer-344 rats were treated with comparably hepatotoxic doses of diquat (0.1 or 0.2 mmol/kg, respectively), or equal volumes of saline, ip. Six hours later, the livers were harvested, and purified nuclei and nucleoli were isolated by differential centrifugation. Nuclear GR activities in male and female rats were 12 and 15 mU/mg protein, and nucleolar activities were 30 and 51 mU/mg protein, respectively, p < 0.05. Some differences between male and female rats in nuclear and nucleolar activities of GPXs and GSTs were observed, as were some differences in the respective diquat-treated animals, but implications of these differences for susceptibility to diquat-induced oxidant stress effects are not apparent. Nuclear GR, GPX, and GST probably contribute to antioxidant defense mechanisms, but the functions served by localization of GR and GPX in nucleoli are less evident. Key Words: glutathione; nuclei; nucleoli; glutathione reductase; glutathione peroxidase; glutathione S-transferases; diquat; Fischer-344 rats; oxidant stress; reactive oxygen species.

Extensive investigations of a wide variety of diseases and experimental models have led to a general acceptance that reactive oxygen species contribute to many examples of cell and tissue damage. Studies to date have greatly improved our understanding of the effects of reactive oxidants and relationships with cell damage. However, many of the most basic principles and concepts underlying the molecular mechanisms responsible for initiation and progression of oxidant-mediated cell damage remain poorly understood. Efforts to identify critical mechanisms have been hindered by the fact that the alterations caused by exposure of already complex biological systems to the nonspecific effects of most toxicants usually 1 To whom correspondence should be addressed at Children’s Research Institute, 700 Children’s Drive, Columbus, OH 43205. Fax: (614) 722-2774. E-mail: [email protected].

produce a bewildering array of products with even greater ranges of pathophysiological properties. As a result of these molecular and functional complexities, transformations that might initiate or mediate oxidant injury are intermingled with reactions occurring as a result of injury and with transformations that may not be obligatory as either causes or consequences of cell killing (Smith, 1991). 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 strainand 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

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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.

40,000 ⫻ g for 1 h. The nuclear pellets were resuspended in 0.34 M sucrose containing 0.05 mM MgCl 2, 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 45– 60 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 MgCl 2 and centrifuged at 2000 ⫻ 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.

MATERIALS AND METHODS

GR activities. GR activities were assayed as we have described previously (Gupta et al., 2000). Assay mixtures consisted of 83 ␮mol Tris (pH 8.0), 0.8 ␮mol of EDTA, 5.70 ␮mol GSSG in 0.1 M Tris (pH 7.0), and 0.2 ␮mol 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.

All chemicals used were of analytical grade. Reagents for electrophoresis were obtained from Bio-Rad (Hercules, CA). Diquat was generously provided by Dr. Ian Wyatt of Imperial Chemical Industries (Zeneca, Macclesfield, Cheshire, England). 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 2– 4°C. Briefly, the livers were weighed, minced, and homogenized in 0.25 M sucrose containing 50 mM Tris, 25 mM KCl, and 5 mM MgCl 2, pH 7.4. The homogenates were centrifuged at 700 ⫻ 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 MgCl 2 with 3 strokes in a Dounce homogenizer. The resulting homogenates were centrifuged at

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 ␮g) were loaded in each lane and were separated by SDS/2-mercaptoethanol/polyacrylamide (SDS–PAGE) 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, 5–10 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, 5–10 min each), and the bands were visualized on the Hyperfilm by enhanced chemiluminescence.

Glutathione peroxidase (GPX) activities. GPX activities with cumene hydroperoxide and hydrogen peroxide (H 2O 2) were determined according to the method of Lawrence and Burk (1976). Briefly, the reaction mixtures consisted of 50 mM KPO 4 (pH 7), 1 mM EDTA, 1 mM NaN 3, 0.2 mM NADPH, 1 U/ml GR, 1 mM GSH, and 1.5 mM cumene hydroperoxide or 0.25 mM H 2O 2. 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 (␮mol 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-chloro2,4-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 H 2O 2 from the activities measured with cumene hydroperoxide (Reddy et al., 1981).

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served in the nucleolar preparations are unlikely to be attributable to non-nucleolar contamination of these fractions. GR activities were readily measurable in hepatic nuclei of male and female Fischer-344 rats, and specific activities (expressed per mg of protein) were higher in nucleolar fractions than in the nuclear fractions (Fig. 2). The higher specific activities (mU/mg of protein) of GR in the nucleolar fractions than in the corresponding nuclear preparations, in conjunction with the results presented in Figure 1, in which the nucleolar fractions were determined to be essentially free of contamination with nuclear components, indicate that the GR activities in the nucleolar fractions are not artifacts of incomplete separation from other nuclear components. Nuclear GR activities were higher in diquat-treated animals of both sexes than in their respective saline-treated controls. Similarly, GR activities were higher in the nucleolar fractions of diquat-treated male rats than in the respective saline-treated controls, but GR activities were lower in the nucleolar fractions of diquat- than FIG. 1. Western blot analyses of nuclear and nucleolar fractions with antinuclear (A) and antinucleolar (B) antibodies. As indicated, in each series, lane 1 is from a saline-treated male and lane 2 from a saline-treated female. Lanes 3 and 4 are from separate diquat-treated male rats, and lanes 5 and 6 from diquat-treated females. The antinuclear antibody used in (A) was raised against proteins from purified nuclei after separation of nucleoli. In the present analyses, the nuclear preparations we analyzed were of whole purified nuclei and contain nucleolar and extranucleolar proteins, whereas our nucleolar preparations contain no detectable extranucleolar proteins.

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.

RESULTS

Both the isolated nuclear and nucleolar fractions demonstrated strong reactivities with the antinucleolar antibody, consistent with the presence of nucleolar proteins in both preparations (Fig. 1). However, the antinuclear antibody, which was raised against nucleoplasmic proteins using supernatants from sedimentation of nucleoli from purified nuclei, showed essentially no reactivity with our nucleolar fractions, which therefore appear to be substantially free of contamination with extranucleolar proteins. The levels of non-nucleolar proteins in our nucleolar preparations were at or below the limits of detection in both male and female rats, whether the animals had been treated with diquat or with saline alone. No marked differences in proteins were observed between male and female rats in the Western analyses of nuclear and nucleolar proteins, whether the rats had been treated with diquat or saline. The RNA–DNA ratios in the isolated nucleoli were around 1.25 (data not shown), which is in agreement with ratios reported previously by Higashinakagawa (1972) when nucleoli were isolated in the presence of Mg ⫹⫹ ions, and are slightly higher than the ratios of around 1.02 observed with nucleoli isolated in the presence of Ca ⫹⫹. Thus, the enzyme activities we ob-

FIG. 2. Glutathione reductase activities in the nuclear and nucleolar fractions of livers of male and female Fischer-344 rats. Livers were collected from the animals 6 h after administration of saline or diquat (0.1 mmol/kg for males and 0.2 mmol/kg for females), the nuclear and nucleolar fractions were isolated by differential centrifugation, and enzyme activities and protein concentrations measured as described in Materials and Methods. Data are means ⫾ SEM, n ⫽ 4 per group. Two-way ANOVA indicated effects of diquat and gender on GR activities in nuclei, and an effect of gender and an interaction of diquat and gender on GR activities in nucleoli. Common letters indicate groups that are different from each other by modified t-tests, p ⬍ 0.05.

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FIG. 3. Glutathione peroxidase activities with hydrogen peroxide (H 2O 2), cumene hydroperoxide (CumOOH), and glutathione S-transferase-␣ (GST-␣) in the nuclear and nucleolar fractions of the livers of male and female Fischer-344 rats. Livers were collected from the animals 6 h after administration of saline or diquat, as indicated (0.1 mmol/kg for males and 0.2 mmol/kg for females), the nuclear and nucleolar fractions were isolated by differential centrifugation, and enzyme activities and protein concentrations measured as described in Materials and Methods. Data are means ⫾ SEM, n ⫽ 4 per group. Two-way ANOVAs indicated an effect of diquat on GPX activities with cumene hydroperoxide and an effect of gender on GST-␣ activities in nuclei. An effect of diquat and an interaction between diquat and gender on GPX activities with H 2O 2 also were indicated, as were effects of both diquat and gender and an interaction on GPX activities with CumOOH. Common letters indicate groups that are different from each other by modified t-tests, p ⬍ 0.05.

of saline-treated female rats. The doses of diquat administered, 0.1 mmol/kg in the male rats, and 0.2 in the females, were selected to cause comparable oxidant stress responses and comparable, but limited extents of hepatic damage in both sexes (Gupta et al., 2000). In the animals in the present studies, the plasma ALT activities in the diquat-treated male and female rats were 909 ⫾ 279 and 202 ⫾ 10 IU/L (means ⫾ SEM), respectively, indicating slightly greater damage in the male than in the female rats, but appropriate for the goals of the present studies. Analyses of the data within treatment groups against ALT activities also showed no significant correlations between GR and ALT activities (data not shown). GPX activities in nuclear and nucleolar fractions (Fig. 3) of these same rats were comparable and were not affected by diquat in the male rats, but activities were lower in the female rats given diquat than in the respective saline-treated controls. In both the nuclear and nucleolar fractions, GPX activities measured using cumene hydroperoxide in the assays were comparable to the activities measured with H 2O 2, and activities observed in the diquat-treated female rats were lower than in the control animals (Fig. 3). The nuclear activities of GST-␣, which were calculated by subtracting the GPX activities measured with H 2O 2 from the GPX activities measured using cumene hydroperoxide, are relatively low (Fig. 3). In the isolated nucleolar fractions, GST-␣ activities were below the

limits of detection of this method. We therefore measured GST activities with CDNB and observed GST activities in the nucleolar fractions that were measurable, but were much lower than in the nuclear fractions (Fig. 4). In male rats, nuclear fraction GST activities measured with CDNB were not different in diquat-treated animals from controls; however, in female rats, nuclear and nucleolar GST activities were lower in the animals treated with diquat than in those given saline. We calculated the ratios of nuclear to nucleolar activities of the enzymes presented in Figures 2– 4. Diquat administration decreased the nuclear to nucleolar ratios of GR activities in male rats, but the same ratios were greater in diquat-treated female rats than in saline-treated female rats (Fig. 5). Nuclear/ nucleolar GST activities increased similarly in diquat-treated female rats, although no effect in male rats was noted. No effects of diquat on ratios of GPX activities were observed, but

FIG. 4. Glutathione S-transferase activities with 1-chloro-2,4-dinitrobenzene (CDNB) in the nuclear and nucleolar fractions of the liver of male and female Fischer-344 rats. Livers were collected from the animals 6 h after administration of saline or diquat (0.1 mmol/kg for males and 0.2 mmol/kg for females), the nuclear and nucleolar fractions isolated by differential centrifugation, and GST activities measured using CDNB as described in Materials and Methods. Data are means ⫾ SEM, n ⫽ 4 per group. Two-way ANOVAs indicated effects of diquat and gender on GST activities in both nuclei and nucleoli. Common letters indicate groups that are different from each other by modified t-tests, p ⬍ 0.05.

NUCLEAR AND NUCLEOLAR GR, GPX, AND GST IN RAT LIVER

FIG. 5. Ratios of nuclear:nucleolar activities of GSH-dependent antioxidant enzymes. The individual nuclear GR, GPX, and GST activities of each sample were divided by the individual nucleolar activities of the same sample. Data are means ⫾ SEM, n ⫽ 4 per group. Two-way ANOVAs indicated an effect of diquat on GR activities ratios, and an effect of diquat on GST (CDNB) activities ratios as well as an interaction between diquat treatment and sex. Common letters indicate groups that are different from each other by modified t-tests, p ⬍ 0.05.

the modest difference in GPX (H 2O 2) activities in diquattreated female and male rats was statistically significant. DISCUSSION

The data support our first working hypothesis, that GR, GPX, and GST activities are observed in hepatic nuclei and nucleoli of both male and female Fischer-344 rats. The greater GR activities in the hepatic nucleolar fractions from female rats than from the male rats (Fig. 2) correlate with greater resistance to liver injury in the former (Gupta et al., 2000), but this association will require critical examination in greater detail. 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).

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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

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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. ACKNOWLEDGMENTS This work was supported by GM44263 from the National Institutes of Health.

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