Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

Bellomo, Giorgio; Vairetti, Mariapia; Stivala, Lucianna; Mirabelli, Francesca; Richelmi, Plinio; Orrenius, Sten

doi:10.1073/pnas.89.10.4412

Cited by 209 publications

(141 citation statements)

References 37 publications

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“…Although this mitochondrial GSH is sensitive to depletion by many antitumor agents, it is relatively insensitive to BSO depletion. Likewise, many reports have suggested that GSH pools in nuclei are more resistant than those in the cytoplasm to BSO depletion (42)(43)(44)(45)(46). However, since our knowledge is rather limited at this point, it is speculated that these compartmentalized GSH levels may play a dominant role in controlling intracellular ROIs and gene regulation compared with those in the cytoplasmic compartment.…”

Section: Discussionmentioning

confidence: 99%

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Yamane¹,

Furuichi²,

Song³

et al. 1998

Journal of Biological Chemistry

151

109

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Expression of the MRP1 gene encoding the GS-X pump and of the ␥-GCSh gene encoding the heavy (catalytic) subunit of the ␥-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like ␥-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1,4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably ␥-GCSh-transfected cell lines down-regulated endogenous MRP1 and ␥-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and ␥-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and ␥-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and ␥-GCSh expression.Human MRP1 (multidrug resistance protein) encoded by MRP1 was first isolated by molecular cloning from doxorubicinselected multidrug-resistant lung cancer cells (Ref. 1; reviewed in Ref. 2). Studies using plasma membrane vesicles prepared from MRP1-overproducing cell lines demonstrated increased ATP-dependent, high-affinity transport activities of cysteinyl leukotrienes (e.g. LTC 4 ) 1 (3, 4). Deletion of homologous MRP1 alleles in mice results in impaired response to inflammatory stimulus in these animals because LTC 4 is a potent mediator of the inflammation reaction (5). These findings suggest that MRP1 encodes the previously described GS-X (ATP-dependent glutathione S-conjugate export) pump (6). In addition to transporting LTC 4 and its related glutathione S-conjugates, naturally occurring organic conjugates, including 17␤-estradiol (17␤-D-glucuronide), and bile salt conjugates, including 6␣-glucuronosylhydrodeoxychlorate and 3␣-sulfatolithocholytaurine, are also good substrates for the MRP1/GS-X pump (7-9). There are also reports suggesting that GSH may serve as a cofactor in MRP1/GS-X pump-mediated drug transport (8, 11). In addition, the MRP1/GS-X pump is responsible for the release of GSSG from cells. This active export of GSSG is considered to be an important mechanism to maintain the reduced status of intracellular thiols under oxidative stress (12, 13). These observations underscore the importance of GSH for the function of MRP1.Biosynthesis of GSH is controlled by multiple enzyme systems (reviewed in Refs. 14 and 15). The first step of GSH biosynthesis, catalyzed by ␥-...

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Section: Discussionmentioning

confidence: 99%

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Yamane¹,

Furuichi²,

Song³

et al. 1998

Journal of Biological Chemistry

151

109

View full text Add to dashboard Cite

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“…Before the toxic dose was given, the glutathione content in the liver of pretreated rats was reduced to 60% of that of the controls ( fig. 3A), indicating depletion of the cytosolic (extranuclear and -mitochondrial) pool (Bellomo et al 1992;Meredith & Reed 1982). This may be the result of either low synthesis rate or high consumption rate of glutathione.…”

Section: Discussionmentioning

confidence: 99%

Autoprotection in Acetaminophen Intoxication in Rats: The Role of Liver Regeneration

Dalhoff

Laursen

Bangert³

et al. 2001

Pharmacology & Toxicology

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Autoprotection by acetaminophen, i.e. increased resistance to toxic effects caused by pretreatment, is a wellknown phenomenon. The purpose of the present work was to identify mechanisms for increased acetaminophen tolerance induced by pretreatment of rats. One group of female Wistar rats (pretreated rats) received acetaminophen orally in increasing doses (1 to 4.3 g/kg) twice a week for 3 weeks, one group (naïve rats) received the vehicle. At time zero pretreated rats received a toxic dose of 7.5 g/kg (100% lethal in naïve rats), and naïve rats received a toxic dose of 4.3 g/ kg. Blood and liver tissue were collected before and 12, 24, 36, and 48 hr after the toxic dose and were analysed for hepatic glutathione and cysteine contents, hepatic glutathione-S-transferase and blood alanine aminotransferase activity, as well as acetaminophen concentration in plasma. Steady-state mRNA levels of proteins involved in acetaminophen detoxification, cell division and acute phase response were measured, liver tissue was examined for proliferating cell nuclear antigen and degree of hepatocyte necrosis. Six naïve rats not receiving acetaminophen served as controls. The mortality was the same in pre-treated and naïve rats (33 percent). Thus, pretreatment increased the tolerance twice. Before the toxic dose pretreated rats compared to control rats had higher activity of glutathione-S-transferase (liver) and alanine aminotransferase (serum), higher hepatic mRNA level of glutathione-S-transferase and g-glutamylcysteine synthetase heavy and light chain subunits, and lower hepatic concentration of glutathione, cysteine and mRNA of CYP1A2 than control rats. After the toxic dose, the mRNA levels of glutathione-S-transferase, g-glutamylcysteine synthetase heavy and light chain subunits, and CYP1A2 in naïve rats rose, approaching those of pretreated rats. Proliferating cell nuclear antigen labelling was high in pretreated rats, while only slightly increased in a few of the naïve rats. Necrotic hepatocytes were found at all time intervals in pretreated rats, and in naïve rats they appeared after 12 hr, peaking after 36 hr. Pretreatment increased the tolerance to acetaminophen toxicity twice, as estimated by mortality. The data indicate that pretreatment may reduce the relative production of toxic metabolites, but it primarily enhances the protection against these metabolites by regenerating hepatocytes.

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“…Interestingly, nuclear GSH has been shown to be rather resistant to depletion by, e.g. buthionine sulfoximine, N-ethylmaleimide, and maleate, respectively, in comparison to the cytoplasmic GSH, suggesting that nuclear GSH is important against oxidative damage of the nuclear DNA and nuclear proteins (64). Reactions between proteins and GSH or GSSG alone are rather slow and unspecific.…”

Section: Fig 6 Multiple Tissue Northern Blot Analysis Of Human Grx1mentioning

confidence: 99%

Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

Lundberg¹,

Johansson²,

Chandra³

et al. 2001

Journal of Biological Chemistry

304

260

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Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSHdisulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2-31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5 region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

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Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

Cited by 209 publications

References 37 publications

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Autoprotection in Acetaminophen Intoxication in Rats: The Role of Liver Regeneration

Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

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