Molecular mechanisms of quinone cytotoxicity

Quinones are probably found in all respiring animal and plant cells. They are widely used as anticancer, antibacterial or antimalarial drugs and as fungicides. Toxicity can arise as a result of their use as well as by the metabolism of other drugs and various environmental toxins or dietary constituents. In rapidly dividing cells such as tumor cells, cytotoxicity has been attributed to DNA modification. However the molecular basis for the initiation of quinone cytotoxicity in resting or non-dividing cells has been attributed to the alkylation of essential protein thiol or amine groups and/or the oxidation of essential protein thiols by activated oxygen species and/or GSSG. Oxidative stress arises when the quinone is reduced by reductases to a semiquinone radical which reduces oxygen to superoxide radicals and reforms the quinone. This futile redox cycling and oxygen activation forms cytotoxic levels of hydrogen peroxide and GSSG is retained by the cell and causes cytotoxic mixed protein disulfide formation. Most quinones form GSH conjugates which also undergo futile redox cycling and oxygen activation. Prior depletion of cell GSH markedly increases the cell's susceptibility to alkylating quinones but can protect the cell against certain redox cycling quinones. Cytotoxicity induced by hydroquinones in isolated hepatocytes can be attributed to quinones formed by autoxidation. The higher redox potential benzoquinones and naphthoquinones are the most cytotoxic presumably because of their higher electrophilicty and thiol reactivity and/or because the quinones or GSH conjugates are more readily reduced to semiquinones which activate oxygen.     

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

Glutathione – linking cell proliferation to oxidative stress

SignificanceThe multifaceted functions of reduced glutathione (gamma-glutamyl–cysteinyl–glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions.Recent advancesThe intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about −300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment.Critical issuesThe concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined.Future directionsThe nuclear GSH pool provides an appropriate redox environment for essential nuclear functions. Future work will focus on how this essential thiol interacts with the nuclear thioredoxin system and nitric oxide to regulate genetic and epigenetic mechanisms. The characterization of redox-regulated cell cycle proteins in plants, and the elucidation of mechanisms that facilitate GSH accumulation in the nucleus are keep steps to unravelling the complexities of nuclear redox controls.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Alkaline-earth cations enhance ortho-quinone-catalyzed ascorbate oxidation

Ortho-quinones 1,10-phenanthroquinone and beta-lapachone but not para-quinones naphthazarin (NZQ) and 1,4-naphthoquinone enhance ascorbate oxidation in the presence of MgCl(2) and CaCl(2) at constant ionic strength. Alkaline-earth cation chelation is observed for the ortho-semiquinones but not for the para-semiquinones, while no interaction between these quinones (with the exception of NZQ) or ascorbate and these salts was detected, suggesting that semiquinone-metal complexes are responsible for the catalytic action on ascorbate oxidation of these metal salts in the presence of these ortho-quinones. Thus, redox cycling efficiency of the quinones under study here, in the presence of ascorbate, depends not only on the quinone redox potential but also on the semiquinone ability to chelate alkaline-earth cations.     

Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells

Antioxidants in plant cells mainly include glutathione, ascorbate, tocopherol, proline, betaine and others, which are also information-rich redox buffers and important redox signaling components that interact with cellular compartments. As an unfortunate consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plant cells can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from miotosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment. This interaction regulates the appropriate induction of acclimation processes or execution of cell death programs, which are the two essential directions for higher plant cells.     

Effect Of Glutathione On The Redox Transitions Of Naphthohydroquinone Derivatives Formed During Dt-Diaphorase Catalysis

The oxidation of GSH coupled to the redox transitions of 1, Cnaphthoquinone derivatives during DT-diaphorase catalysis was examined. The quinones studied included 1,4-naphthoquinone and its dimethoxy-and hydroxy derivatives and were selected according to their different ability to undergo nucleophilic addition with GSH and the dual effect of superoxide dismutase on hydroquinone autoxidation

GSH was oxidized to GSSG during the redox transitions of the above quinones, regardless of their substitution pattern. This effect was accompanied by an increase of total O₂ consumption, indicating the ability of GSH to support quinone redox cycling. The values for the relationship [O₂]_consumed[GSSG]_formde were, with every quinone examined, above unity. thus pointing to the occurrence of autoxidation reactions other than those involved during GSSG formation

These results are discussed in terms of the functional group chemistry of the quinones and the ther-modynamic properties of the reactions involved in the reduction of the semi- to the hydro-quinone by GSH     

Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed.     

Redox potential of quinones in both electron transfer branches of photosystem I

The redox potentials of the two electron transfer (ET) active quinones in the central part of photosystem I (PSI) were determined by evaluating the electrostatic energies from the solution of the Poisson-Boltzmann equation based on the crystal structure. The calculated redox potentials are -531 mV for A1A and -686 mV for A1B. From these results we conclude the following. (i) Both branches are active with a much faster ET in the B-branch than in the A-branch. (ii) The measured lifetime of 200-290 ns of reduced quinones agrees with the estimate for the A-branch and corroborates with an uphill ET from this quinone to the iron-sulfur cluster as observed in recent kinetic measurements. (iii) The electron paramagnetic resonance spectroscopic data refer to the A-branch quinone where the corresponding ET is uphill in energy. The negative redox potential of A1 in PSI is primarily because of the influence from the negatively charged FX, in contrast to the positive shift on the quinone redox potential in bacterial reaction center and PSII that is attributed to the positively charged non-heme iron atom. The conserved residue Asp-B575 changes its protonation state after quinone reduction. The difference of 155 mV in the quinone redox potentials of the two branches were attributed to the conformation of the backbone with a large contribution from Ser-A692 and Ser-B672 and to the side chain of Asp-B575, whose protonation state couples differently with the formation of the quinone radicals.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO₂. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components—ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate–glutathione (Asc–GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.     

Interaction of antioxidants and their implication in genetic anemia

The generation of reactive oxygen species (ROS) is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, zinc, and the carotenoids). These antioxidant nutrients occupy distinct cellular compartments and among them, there are active recycling. For example, oxidized vitamin E (tocopheroxy radical) has been shown to be regenerated by ascorbate, GSH, lipoic acid, or ubiquinols. GSH disulfides (GSSG) can be regenerated by GSSG reductase (a riboflavin-dependent protein), and enzymic pathways have been identified for the recycling of ascorbate radical and dehydroascorbate. The electrons that are used to fuel these recycling reactions (NADH and NADPH) are ultimately derived from the oxidation of foods. Sickle cell anemia, thalassemia, and glucose-6-phosphate-dehydrogenase deficiency are all hereditary disorders with higher potential for oxidative damage due to chronic redox imbalance in red cells that often results in clinical manifestation of mild to serve hemolysis in patients with these disorders. The release of hemoglobin during hemolysis and the subsequent therapeutic transfusion in some cases lead to systemic iron overloading that further potentiates the generation of ROS. Antioxidant status in anemia will be examined, and the potential application of antioxidant treatment as an adjunct therapy under these conditions will be discussed.     

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP⁺/reduced NADP and NAD⁺/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD⁺/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples. cell proliferation; contact inhibition; glutathione     

Supplemental ascorbate or alpha-tocopherol induces cell death in Cu-deficient HL-60 cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Glutaredoxin 2a,a mitochondrial isoform,plays a protective role in a human cell line under serum deprivation

The roles of mitochondrial glutaredoxin (Grx2a) under serum deprivation were assessed using the human stable HepG2 cell lines overexpressing or down-regulating Grx2a. The Grx2a-overexpressing stable cells displayed enhanced proliferation, decreased reactive oxygen species (ROS) and caspase-3 activity levels, and increased total GSH level, compared to the vector control cells. These characteristics of the overexpressing stable cells were reversed by down-regulating Grx2a in the same cell line. In the limited serum conditions, the Grx2a-overexpressing stable pcDNA3.0/HA-Grx2a cells exhibited higher cellular viabilities and total GSH level, and showed much lower enhancement in ROS and caspase-3 activity levels than the vector control pcDNA3.0/HA cells. However, the Grx2a-down-regulating stable cells gave rise to diminished cellular viabilities and further decreased total GSH level, and contained significantly higher ROS and caspase-3 activity levels, under serum deprivation than the vector control cells. These results suggest that Grx2a plays proliferative and anti-apoptotic roles under serum deprivation.