Percutaneous lumbar nucleotomy

The nature of the events whereby the reactive intermediates resulting from the bioactivation of bromobenzene and furosemide induce hepatotoxicity is unknown. To examine a role for disturbances in intracellular calcium homeostasis, secondary to a depletion in cellular reduced glutathione (GSH) and reduced protein thiols (PSHs), isolated mouse hepatocytes were exposed to cytotoxic concentrations of bromobenzene or furosemide. Cytosolic calcium concentration, as well as thiol status, was determined. The incubation of hepatocytes with 3.0 mM bromobenzene, and subsequent additions (1.2 mM) of the agent every hour, resulted in significant GSH depletion. The loss of plasma membrane integrity at 1.5 h preceded both a rise in the cytosolic Ca2+ concentration and depletion of total PSH content. Furosemide (1.0 mM) produced a 70% depletion in cellular GSH content in isolated hepatocytes. The initiation of cell damage occurred concurrently with both a rise in the cytosolic Ca2+ concentration and a depletion of total PSH content 4 h following furosemide addition. Since the increase in cytosolic Ca2+ did not precede cytotoxicity, these results do not support an initiating role for Ca2+ deregulation in bromobenzene and furosemide hepatotoxicities. In addition, depletion of PSH content did not correlate with bromobenzene- or furosemide-induced cytotoxicity.     

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.     

Peroxidase-catalyzed oxidation of 3,5-dimethyl acetaminophen causes cell death by selective protein thiol modification in isolated rat hepatocytes

In this study we used a peroxidase model system (glucose/glucose oxidase and horseradish peroxidase) to investigate the effect of extracellularly generated reactive metabolites of 3,5-Me2-acetaminophen on cell viability and on cellular thiol levels. Incubation of hepatocytes with 3,5-Me2-acetaminophen in the presence of glucose/glucose oxidase and horseradish peroxidase caused a concentration-dependent loss of cell viability. Loss of viability was associated with decreased protein thiol levels. Addition of the reducing agent DTT, but not catalase, during the incubation restored cellular protein thiol levels and arrested the cell killing. Protein thiol depletion occurred selectively to the mitochondrial and microsomal fractions and was specific for a very limited number of protein bands. The data suggest that the oxidative modification of individual protein cysteine residues within the latter two organelle fractions is critically involved in the mechanism of toxicity.     

Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death

Oxidants are important human toxicants. Increased intracellular free Ca2+ may be critical for oxidant toxicity, but this mechanism remains controversial. Furthermore, oxidants damage the endoplasmic reticulum (ER) and release ER Ca2+, but the role of the ER in oxidant toxicity and Ca2+ regulation during toxicity is also unclear. tert-Butylhydroperoxide (TBHP), a prototypical organic oxidant, causes oxidative stress and an increase in intracellular free Ca2+. Therefore, we addressed the mechanism of oxidant-induced cell death and investigated the role of ER stress proteins in Ca2+ regulation and cytoprotection after treating renal epithelial cells with TBHP. Prior ER stress induces expression of the ER stress proteins Grp78, Grp94, and calreticulin and rendered cells resistant to cell death caused by a subsequent TBHP challenge. Expressing antisense RNA targeted to grp78 prevents grp78 induction sensitized cells to TBHP and disrupted their ability to develop cellular tolerance. In addition, overexpressing calreticulin, another ER chaperone and Ca2+-binding protein, also protected cells against TBHP. Interestingly, neither prior ER stress nor calreticulin expression prevented lipid peroxidation, but both blocked the rise in intracellular free Ca2+ after TBHP treatment. Loading cells with EGTA, even after peroxidation had already occurred, also prevented TBHP-induced cell death, indicating that buffering intracellular Ca2+ prevents cell killing. Thus, Ca2+ plays an important role in TBHP-induced cell death in these cells, and the ER is an important regulator of cellular Ca2+ homeostasis during oxidative stress. Given the importance of oxidants in human disease, it would appear that the role of ER stress proteins in protection from oxidant damage warrants further consideration.     

The use of cultured hepatocytes to investigate the mechanisms of drug hepatotoxicity

In the course of biotransformation reactions catalyzed both by cytochrome P450 and by conjugating enzymes, drug-derived reactive metabolites and active oxygen species can appear that may escape the detoxification process, initiating radical chain reactions (e.g., lipid peroxidation), covalently binding to macromolecules (proteins, DNA), or impairing the energetic balance of cells. This is usually followed by alterations of ion homeostasis that precede irreversible biochemical changes and cell death. There are, however, cellular mechanisms of defense that prevent, or repair, the damage caused by these reactive intermediates. Ultimately it is the balance between bioactivation, detoxification, and defense mechanisms that determines whether a compound will or will not elicit a toxic effect. Cultures of hepatocytes, including those of human origin, can be used to elucidate the mechanisms of drug toxicity. This is illustrated in the study of the mechanism of hepatotoxicity by diclofenac. Much less cytotoxicity is observed in nonmetabolizing hepatomas than in hepatocytes. The observed cell dysfunction parallels the biotransformation of the drug, and particularly the formation of the minor metabolite N,5-dihydroxydiclofenac by hepatocytes. This compound is able to inhibit mitochondrial ATP synthesis in hepatocytes.     

Coordinated intercellular calcium waves induced by noradrenaline in rat hepatocytes: dual control by gap junction permeability and agonist

Calcium-mobilizing agonists induce intracellular Ca2+ concentration ([Ca2+]i) changes thought to trigger cellular responses. In connected cells, rises in [Ca2+]i can propagate from cell to cell as intercellular Ca2+ waves, the mechanisms of which are not elucidated. Using fura2-loaded rat hepatocytes, we studied the mechanisms controlling coordination and intercellular propagation of noradrenaline-induced Ca2+ signals. Gap junction blockade with 18 alpha-glycyrrhetinic acid resulted in a loss of coordination between connected cells. We found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of intercellular Ca2+ waves. In addition, our experiments revealed functional differences between noradrenaline-induced Ca2+ signals in connected hepatocytes. These results demonstrate that intercellular Ca2+ signals in multicellular systems of rat hepatocytes are propagated and highly organized through complex mechanisms involving at least three factors. First, gap junction coupling ensures coordination of [Ca2+]i oscillations between the different cells; second, the presence of hormone at each hepatocyte is required for cell-cell Ca2+ signal propagation; and third, functional differences between adjacent connected hepatocytes could allow a 'pacemaker-like' intercellular spread of Ca2+ waves.     

The regulation of reactive oxygen species production during programmed cell death

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.     

Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.     

Rorschach cognitive developmental indices of mentally retarded persons: a comparison with scores on Wechsler Intelligence Scale for children-revised

The complete mechanism by which pathogenic mtDNA mutations cause cellular pathophysiology and in some cases cell death is unclear. Oxidant stress is especially toxic to excitable nerve and muscle cells, cells that are often affected in mitochondrial disease. The sensitivity of cells bearing the LHON, MELAS, and MERRF mutations to oxidant stress was determined. All were significantly more sensitive to H2O2 exposure than their nonmutant cybrid controls, the order of sensitivity was MELAS > LHON > MERRF > controls. Depletion of Ca2+ from the medium protected all cell lines from oxidant stress, consistent with the hypothesis that death induced by oxidant stress is Ca(2+)-dependent. A potential downstream target of Ca2+ is the mitochondrial permeability transition, MPT, which is inhibited by cyclosporin A. Treatment of MELAS, LHON, and MERRF cells with cyclosporin A caused significant rescue from oxidant exposure, and in each case significantly greater rescue of mutant than control cells. The pronounced oxidant-sensitivity of mutant cells, and their protection by Ca2+ depletion and CsA, has potential implications for both the pathophysiological mechanism and therapy of these mitochondrial genetic diseases.     

Mechanism of p-hydroxybenzoate ester-induced mitochondrial dysfunction and cytotoxicity in isolated rat hepatocytes

The relationship between the metabolism and the cytotoxic effects of the alkyl esters of p-hydroxybenzoic acid (parabens) has been studied in freshly isolated rat hepatocytes. Incubation of hepatocytes with propyl-paraben (0.5 to 2.0 mM) elicited a concentration- and time-dependent cell death that was enhanced when enzymatic hydrolysis of propyl-paraben to p-hydroxybenzoic acid was inhibited by a carboxylesterase inhibitor, diazinon. The cytotoxicity was accompanied by losses of cellular ATP, total adenine nucleotide pools, and reduced glutathione, independently of lipid peroxidation and protein thiol oxidation. In the comparative toxic effects based on cell viability, ATP level, and rhodamine 123 retention, butyl- and isobutyl-parabens were more toxic than propyl- and isopropyl-parabens, and ethyl- and methyl-parabens and p-hydroxybenzoic acid were less toxic than propyl-paraben. The addition of propyl-paraben to isolated hepatic mitochondria reduced state 3 respiration with NAD+-linked substrates (pyruvate plus malate) and/or with an FAD-linked substrate (succinate plus rotenone), whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not affected significantly by propyl-paraben. Further, the addition of these parabens caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by propyl-paraben, butyl-paraben, and their chain isomers. These results indicate that a) propyl-paraben-induced cytotoxicity is mediated by the parent compound rather than by its metabolite p-hydroxybenzoic acid; b) the toxicity is associated with ATP depletion via impairment of mitochondrial function related to membrane potential and/or oxidative phosphorylation; and c) the toxic potency of parabens to hepatocytes or mitochondria depends on the relative elongation of alkyl side-chains esterified to the carboxyl group of p-hydroxybenzoic acid.     

Care tracker: a new approach to nursing care in ambulatory settings

It is becoming increasingly clear that mitochondrial Ca2+ uptake from and release into the cytosol has important consequences for neuronal and glial activity. Ca2+ regulates mitochondrial metabolism, and mitochondrial Ca2+ uptake and release modulate physiological and pathophysiological cytosolic responses. In glial cells, inositol 1,4,5-trisphosphate-dependent Ca2+ responses are faithfully translated into elevations in mitochondrial Ca2+ levels, which modifies cytosolic Ca2+ wave propagation and may activate mitochondrial enzymes. The location of mitochondria within neurones may partially determine their role in Ca2+ signalling. Neuronal death due to NMDA-evoked Ca2+ entry can be delayed by an inhibitor of the mitochondrial permeability transition pore, and mitochondrial dysfunction is being increasingly implicated in a number of neurodegenerative conditions. These findings are illustrative of an emerging realization by neuroscientists of the importance of mitochondrial Ca2+ regulation as a modulator of cellular energetics, endoplasmic reticulum Ca2+ release and neurotoxicity.     

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons

To establish direct linkage between the ethanol-inducible cytochrome P450, CYP2E1, ethanol hepatotoxicity, and lipid peroxidation, a HepG2 cell line which expresses human CYP2E1 was established by retroviral infection. Ethanol produced a time-and concentration-dependent cytotoxicity to HepG2 cells expressing the CYP2E1 but not to control cells. The ethanol toxicity was prevented by inhibitors of CYP2E1 and antioxidants. In a similar manner, addition of a polyunsaturated fatty acid such as arachidonic acid produced toxicity to the cells expressing CYP2E1 but not the control cells. Toxicity was associated with enhanced lipid peroxidation and was prevented by antioxidants. The ethanol and arachidonic acid toxicity was apoptotic in nature and was associated with activation of Caspases I and III. The toxicity and apoptosis could be prevented by peptide inhibitors of ICE and by transfection with a plasmid containing the cDNA for human Bcl-2. These results show that this HepG2 cell model can be used to establish a CYP2E1-dependent ethanol hepatotoxicity system, and that induction of a state oxidative stress appears to play a central role in the CYP2E1-dependent apoptosis and cytotoxicity.     

From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state

The permeability transition pore (PTP) is a channel of the inner mitochondrial membrane that appears to operate at the crossroads of two distinct physiological pathways, i.e., the Ca2+ signaling network during the life of the cell, and the effector phase of the apoptotic cascade during Ca2+-dependent cell death. Correspondingly, two open conformations of the PTP can also be observed in isolated organelles. A low-conductance state, that allows the diffusion of small ions like Ca2+, is pH-operated, promoting spontaneous closure of the channel. A high-conductance state, that allows the unselective diffusion of big molecules, stabilizes the channel in the open conformation, disrupting in turn the mitochondrial structure and causing the release of proapoptotic factors. Our current results indicate that switching from low- to high-conductance state is an irreversible process that is strictly dependent on the saturation of the internal Ca2+-binding sites of the PTP. Thus, the high-conductance state of the PTP, which was shown to play a pivotal role in the course of excitotoxic and thapsigargin-induced cell death, might result from a Ca2+-dependent conformational shift of the low-conductance state, normally participating in the regulation of cellular Ca2+ homeostasis as a pH-operated channel. These observations lead us to propose a simple biophysical model of the transition between Ca2+ signaling and Ca2+-dependent apoptosis.     

Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes

BACKGROUND & AIMS: Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A-mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death. METHODS: Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL). RESULTS: Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids. CONCLUSIONS: The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.     

Calcium vs. iron-mediated processes in hydrogen peroxide toxicity to L929 cells: effects of glucose

H2O2 toxicity was studied in L929 cells in the presence and absence of glucose. The data obtained in the absence of glucose suggest a Ca2+-dependent mechanism of cell injury. No evidence was found for any involvement of iron in the process. In particular, cell injury was unaffected by the intracellular iron chelators 2,2'-dipyridyl and deferoxamine or by the hydroxyl radical scavengers DMSO and DMPO. On the other hand, the intracellular Ca2+ chelator BAPTA/AM provided significant protection. The cytosolic Ca2+ level rapidly and consistently increased after H2O2 addition, prior to visible bleb formation and loss of cell viability. Additionally, GSH not only prevented cell death but also significantly decreased cytosolic calcium accumulation. In the presence of glucose, however, Ca2+ does not seem to play any role in H2O2 toxicity. Cell death is now mainly mediated by iron: the iron chelators and hydroxyl radical scavengers prevented cell injury, the increase in cytosolic Ca2+ was significantly less pronounced, and BAPTA/AM did not exert any protection under these conditions. Hence, the metabolic state of the L929 cells, as given by the availability of glucose, decisively determines the biochemical mechanism of H2O2 cell injury.     

The thiol-specific antioxidant enzyme prevents mitochondrial permeability transition. Evidence for the participation of reactive oxygen species in this mechanism

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H2O2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization and membrane protein thiol oxidation, in a concentration-dependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca2+ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.     

Changes in cytosol Ca(2+)-, Mg(2+)-, H(+)-, N(+)-, and K(+)-concentrations in cultivated hepatocytes in hypoxic conditions

AIM: It is assumed that disturbances of cellular ion homeostasis, especially an increase in the cytosolic Ca2+ concentration, are of decisive importance for hypoxic cell injury. The aim of this study is the determination of alterations in the cytosolic Ca2+, Mg2+, H+, Na+ and K+ concentration in cultured hepatocytes during hypoxia. METHODS: The alterations of ion homeostasis under hypoxic conditions were studied in primary cultures of isolated rat hepatocytes by using fluorescence microscopy. RESULTS: The measurements of cytosolic Ca2+ concentration showed no alterations during the first 3-4 h of hypoxia. About 1-2 h before cell injury became evident Ca2+ increased from 147 +/- 28 to 385 +/- 31 nM. Similarly the cytosolic Mg2+ concentration increased from 0.63 +/- 0.05 to 1.42 +/- 0.11 mM in a late stage of hypoxia. In contrast, the cytosolic Na+ concentration increased continuously from 16 +/- 2 mM at start to 76 +/- 9 mM after 5 h of hypoxic conditions. The cytosolic K+ concentration remained constant for 2 h (129 +/- 7 mM) but then decreased down to 31 +/- 18 mM. The intracellular H+ concentration increased slightly under hypoxic conditions, the pH decreased from 7.35 +/- 0.02 to 7.19 +/- 0.04. CONCLUSION: The results indicate that cytosolic Ca2+ plays only a minor role in the pathomechanism of hypoxic hepatocellular injury but suggest an important role of the cytosolic Na+ concentration in this process.     

Evidence of increased excitability in GEPR hippocampus preceding development of seizure susceptibility

3-Benzoylpyridine (3BP) is a major metabolite of HGG-12, and oxime that has been synthesized as a potential antidote to the toxic effects of soman and other anticholinesterases. Structural similarities exist between 3BP, the cytochrome P450 (CYP)-inducer metyrapone (MET) and other 3-substituted pyridines that interact with CYPs. The present study evaluated the regulatory effects of 3BP on CYP expression in rat liver. Both 3BP and MET (100 mg/kg) increased total hepatic microsomal holo-CYP content significantly 24 h after administration to male rats. Pronounced increases in activities mediated by CYP2B (androstenedione 16 beta-hydroxylation and 7-pentylresorufin O-depentylation) were produced by 3BP and MET, which correlated with respective 9- and 14-fold increases in CYP2B immunoreactive protein. In addition, both agents slightly increased rates of microsomal CYP3A-dependent steroid 6 beta-hydroxylation, troleandomycin metabolite complex formation and total CYP3A immunoreactive protein. Induction of the dexamethasone-inducible CYP3A23 mRNA to 4.5- and 2.5-fold of control was detected in liver of MET- and 3BP-induced rats; CYP3A2 mRNA levels were unchanged. Analogous in vitro studies revealed that MET was a preferential inhibitor of CYP3A-mediated steroid 6 beta-hydroxylation activity, but 3BP was inactive against constitutive steroid hydroxylase CYPs. These findings indicate that the structurally related 3BP and MET elicit similar induction effects on CYPs 2B and 3A23 in rat liver after in vivo administration, but differential inhibitory effects of the chemicals on CYP activity in vitro. Recent reports have implicated a microsomal binding site in the induction of CYP3A1/3A23 in rat liver. In light of the present findings, substituted pyridines like 3BP may be useful tools in structure-activity studies to evaluate the physicochemical requirements for binding to this protein.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 microM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 microM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na+/K+-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway.     

Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells

Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812) and grp78 in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine the role of these genes in tolerance to toxic chemicals. Prior heat shock did not protect cells from IDAM but pretreatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or tunicamycin enhanced expression of the endoplasmic reticulum (ER) chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78 and grp94 expression after ER stress. Protection against IDAM due to prior ER stress was also attenuated in pkASgrp78 cells suggesting that ER chaperones of the GRP family are critical for tolerance. Covalent binding of IDAM to cellular macromolecules and depletion of cellular thiols was similar in tolerant and na?ve cells. However, DTTox pretreatment blocked the increases in cellular Ca2+ and lipid peroxidation observed after IDAM treatment. Overexpressing the ER Ca2+-binding protein calreticulin prevented IDAM-induced cell death, the rise in cytosolic Ca2+, and oxidative stress. Although activation of the ER stress response did not prevent toxicity due to Ca2+ influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the mitochondria may be important in toxicity. The data support a model in which induction of ER stress proteins prevents disturbances of intracellular Ca2+ homeostasis, thus uncoupling toxicant exposure from oxidative stress and cell death. Multiple ER stress proteins are likely to be involved in this tolerance response.