Oxidative stress studied in intact mammalian cells

Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.     

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.     

Role of cellular calcium homeostasis in toxic liver injury induced by the pyrrolizidine alkaloid senecionine and the alkenal trans-4-OH-2-hexenal

The pyrrolizidine alkaloid senecionine has been shown to be hepatotoxic, genotoxic, and cytotoxic. However, the biochemical mechanism by which senecionine produces hepatocellular toxicity remains to be elucidated. The role of calcium homeostasis in toxic liver injury was examined in isolated rat hepatocytes treated with senecionine and trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and appropriate cofactors. Hepatocytes treated with senecionine and t-4HH demonstrated greater cytotoxicity (leakage of lactate dehydrogenase) when incubated in the absence of extracellular Ca²⁺ than in its presence. Both compounds elicited an increase in cytosolic Ca²⁺ levels of isolated hepatocytes in the presence of extracellular Ca²⁺ In the following study, senecionine and t-4HH depleted intracellular glutathione levels and induced lipid peroxidation and cytotoxicity in isolated hepatocytes. Pretreatment with the thiolgroup reducing agent dithiothreitol prevented depletion of intracellular glutathione and protected hepatocytes against senecionine and t-4HH-induced lipid peroxidation and cytotoxicity. Both compounds also depleted intracellular ATP and NADPH levels. These results suggest that hepatotoxocity induced by senecionine and t-4HH is not dependent on the influx of extracellular Ca²⁺; however, alterations in intracellular Ca²⁺, possibly associated with depletion of intracellular glutathione, NADPH, and ATP, may play a critical role.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Intracellular Ca2+ Chelators Prevent Dna Damage And Protect Hepatoma 1Clc7 Cells From Quinone-Induced Cell Killing

Exposure of hepatoma lclc7 cells to 2,3-drniethoxy-1.4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca²⁺. DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca²⁺ chelators. Quin 2 or BAPTA, to buffer the increase in cytosolic Ca²⁺ concentration induced by the quinone. DMNQ caused marked NAD⁺ depletion which was prevented when cells were preincubated with 3-aminobenzamide. an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity. or with either of the two Ca²⁺ chelators. However. 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage. NAD⁺ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca²⁺     

Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.     

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′-triphosphate

The incubation of isolated rat hepatocytes with extracellular adenosine 5′-trihosphate (ATP) resulted in an inhibition of Ca²⁺ efflux. The ATP-induced Ca²⁺ accumulation as determined by the increase in phosphorylase a activity and the Ca²⁺ -sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis-[carboxymethyl] aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca²⁺, suggesting that the increased free Ca²⁺ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H-imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca²⁺ -calmodulin process may be involved in the initiation of cytotoxicity.     

The Cytoskeleton As A Target In Quinone Toxicity

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure knoun as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears 10 be due mainly to actin cross-linking and dissociation of α-actinin from the actin network. The major hiochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein dfhydryl groups and the increase cytosolic Ca²⁺ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca²⁺ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma menibrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes. and thus it does not seem to be causatively related to their appeardncc Thiol reductants. such as dithiothreitol. efficiently prevent the oxidation of cytoskeletal protein thiols. the increase in cytosolic free Ca²⁺ concentration and cell blebbing induced by redox cycling. but not alkylating. quinones. These results demonstrate that alkylating and redox cycling quinones cause siinilar structural and biochemical modifications of the cytoskeleton by means of different mechanisms. namely alkylation and oxidation of critical sulfhydryl groups     

Cell calcium, vitamin E, and the thiol redox system in cytotoxicity

The controversial role of extracellular Ca2+ in toxicity to in vitro hepatocyte systems is reviewed. Recent reports demonstrate that extracellular Ca2+-related cytotoxicity is dependent on Ca2+-influenced vitamin E (alpha-tocopherol) content of isolated hepatocytes. Based on a Ca2+-omission model of in vitro oxidative stress, the role of vitamin E in cytotoxicity is further explored. This model demonstrates the interdependence of the GSH redox system and vitamin E as protective agents during oxidative stress. Following chemical oxidant-induced depletion of intracellular GSH, cell morphology and viability are maintained by the continuous presence of cellular alpha-tocopherol above a threshold level of 0.6-1.0 nmol/10(6) cells. alpha-Tocopherol threshold-dependent cell viability is directly correlated with the prevention of the loss of cellular protein thiols in the absence of intracellular GSH. Potential mechanisms for this phenomenon are explored and include a direct reductive action of alpha-tocopherol on protein thiyl radicals, and the prevention of oxidation of protein thiols by scavenging of lipid peroxyl radicals by alpha-tocopherol. It is suggested that in light of the threshold phenomenon of vitamin E prevention of potentially severe oxidative stress-induced cytotoxicity, its use as a protective agent against an oxidative challenge in vivo should be reassessed.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.     

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium,pH and membrane integrity in mammalian cells

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD₅₀ 110 μg/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca²⁺ entry by toxin (0.1–10 μg/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37°C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 μg/ml. No progressive Ca²⁺ entry was observed on toxin addition, instead a rapid rise in intracellular Ca²⁺, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 μg/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.     

Mitochondrial Dysfunction and Reactive Oxygen Species in Excitotoxicity and Apoptosis: Implications for the Pathogenesis of Neurodegenerative Diseases

In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca²⁺ homeostasis and endogenous producers of reactive oxygen species (ROS). Interestingly, the mitochondria have been also implicated as central executioners of cell death. Increased mitochondrial Ca²⁺ overload as a result of excitotoxicity has been associated with the generation of superoxide and may induce the release of proapoptotic mitochondrial proteins, proceeding through DNA fragmentation/condensation and culminating in cell demise by apoptosis and/or necrosis. In addition, these processes have been implicated in the pathogenesis of many neurodegenerative diseases, which share several features of cell death: selective brain areas undergo neurodegeneration, involving mitochondrial dysfunction (mitochondrial complexes are affected), loss of intracellular Ca²⁺ homeostasis, excitotoxicity, and the extracellular or intracellular accumulation of insoluble protein aggregates in the brain.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide. implications for its cytotoxicity.

When isolated hepatocytes were exposed to tert-butyl hydroperoxide (tBOOH) they lost their cellular membrane integrity. Decreased levels of GSH, increased phosphorylase a activity (an indirect index of the amount of free cytosolic Ca²⁺), and increase in the formation of malondialdehyde (MDA)-like products (an index of lipid peroxidation) preceeded the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH), indicating that this later process was the consequence of the former intracellular events. While ATP levels were not modified during the incubation of cells with increasing concentrations of tBOOH, protein synthesis was decreased in a concentration-dependent manner. The glycogen content decreased at the same time as the increase in LDH leakage. The addition of promethazine (PMZ) an antioxidant molecule, prevented the lipid peroxidation, but did not protect cells against the oxidative effects of tBOOH, including loss of membrane integrity. Nevertheless, the addition of GSH to cell suspensions incubated with tBOOH, decreased the formation of MDA-like products, restored the protein synthesis rate, prevented partially the activation of phosphorylase a and preserved cell viability. On the basis of these results, we postulate that both GSH depletion and modification in phosphorylase a activity (Ca²⁺ levels) were the most relevant intracellular events to explain the cytotoxicity of tBOOH.Abbreviations tBOOH tert-butyl hydroperoxides - GSH reduced glutathione - LDH lactate dehydrogenase - MDA malondialdehyde - TBA thiobarbituric acid - PMZ promethazin - BSA bovine serum albumin     

Possible role of ca2+ in heavy metal cytotoxicity

• 1.1. Organic xenobiotic metabolism often results in oxidative stress, involving GSH depletion, alteration of thiol/disulphide balance and peroxidation of membrane lipids. These events can lead to the disruption of Ca²⁺ homeostasis, through impairment of the Ca²⁺ translocases present in cellular membranes. Inhibition of the activity of Ca,Mg-ATPases due to oxidation of their SH groups would lead to uncontrolled rises in cytosolic Ca²⁺ levels resulting in loss of cell viability.
• 2.2. These observations seem to be of interest when interpreting the biochemical mechanisms of heavy metal cytotoxicity. Since these cations (such as Hg²⁺, Cu²⁺, Cd²⁺ and Zn) have an extremely high affinity for SH groups, they may affect the function of SH containing proteins, such as the Ca,Mg-ATPases, as in the case of oxidative stress.
• 3.3. Results are reported indicating that Hg²⁺ may stimulate Ca²⁺ influx through voltage-dependent channels in different experimental systems. Moreover, evidence is presented that heavy metals can inhibit Ca,Mg-ATPase activity and affect mitochondrial functions in the cells of different organisms.
• 4.4. The possibility that heavy metal cytotoxicity is mediated through disruption of Ca²⁺ homeostasis is discussed.

     

Protein kinase C activator PMA reduces the Ca2+ response to antigen stimulation of adherent RBL-2H3 mucosal mast cells by inhibiting depletion of intracellular Ca2+ stores

Activation of protein kinase C has been shown to reduce the Ca²⁺ responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca²⁺ influx, but acceleration of Ca²⁺ efflux and inhibition of Ca²⁺ store depletion by protein kinase C activation have also been described. For adherent RBL-2H3 mucosal mast cells, results from whole-cell patch clamp experiments suggest that protein kinase C activation reduces Ca²⁺ influx, while experiments with intact, fura-2-loaded cells suggest that Ca²⁺ influx is not affected. Here we present single-cell data from Ca²⁺ imaging experiments with adherent RBL-2H3 cells, showing that antigen-stimulated Ca²⁺ responses of phorbol 12-myristate 13-acetate (PMA)-treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca²⁺, indicating that depletion of intracellular Ca²⁺ stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca²⁺]_i was observed, consistent with a slight inhibition of Ca²⁺ influx. However, the major effect of PMA on the antigen-stimulated Ca²⁺ response is to inhibit depletion of intracellular Ca²⁺ stores. We also show that inhibition of protein kinase C did not enhance the Ca²⁺ response to antigen, suggesting that inhibition of the Ca²⁺ response by activation of protein kinase C does not contribute to the physiological response to antigen. J. Cell. Physiol. 181:113–123, 1999. © 1999 Wiley-Liss, Inc.     

Control of the pro-oxidant-dependent calcium release from intact liver mitochondria

Summary

The reduction of molecular oxygen to water provides most of the energy that enables higher organisms to exist. Oxygen reduction is a mixed blessing because incompletely reduced oxygen species are more reactive than molecular oxygen in the ground state and can, when out of control, damage biological molecules. However, incompletely reduced oxygen species may also serve useful functions, as exemplified by their control of mitochondrial Ca²⁺ homeostasis, the understanding of which has improved greatly during the last few years. Hydrogen peroxide can stimulate a specific Ca²⁺ release pathway from intact mitochondria by oxidizing mitochondrial pyridine nucleotides through the activities of glutathione peroxidase, glutathione reductase, and the energy-linked transhydrogenase. Other pro-oxidants such as menadione, alloxan, or divicine also stimulate the specific Ca²⁺ release, because they furnish NAD⁺. The specific Ca²⁺ release requires for its activation the hydrolysis of intramitochondrial NAD⁺ to ADPribose and nicotinamide, and is prevented by inhibitors of NAD⁺ hydrolysis and protein monoADPribosylation. Recent experiments reveal that NAD⁺ hydrolysis and therefore Ca²⁺ release is regulated by vicinal thiols in mitochondria. When reduced or alkylated, the thiols prevent hydrolysis, but when they are cross-linked hydrolysis takes place. Cyclosporine A, which also prevents NAD⁺ hydrolysis, acts distal of these vicinal thiols. Since mitochondrial Ca²⁺ handling is physiologically relevant, its control by pro-oxidants must be added to the growing list of their useful functions.