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J. Biol. Chem., Vol. 277, Issue 1, 104-113, January 4, 2002

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Role of Calcium and Calcium-activated Proteases in CYP2E1-dependent Toxicity in HEPG2 Cells^*

Andres A. Caro and Arthur I. Cederbaum

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, August 15, 2001, and in revised form, October 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca²⁺ and activation of Ca²⁺-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 µM) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 µM) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca²⁺ were inhibited by omission of Ca²⁺ from the extracellular medium, and toxicity was restored by reincorporation of Ca²⁺. An inhibitor of Ca²⁺ release from intracellular stores did not prevent the toxicity or the increase in Ca²⁺, reflecting a role for the influx of extracellular Ca²⁺ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca²⁺ in E47 cells exposed to iron-AA were inhibited by -tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca²⁺ and activation of Ca²⁺-dependent proteases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

According to the calcium hypothesis of cell injury, a perturbation of intracellular Ca²⁺ homeostasis leading to a sustained increase in cytosolic Ca²⁺ is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress, causing the ultimate loss of cell viability through activation of various Ca²⁺-dependent processes (1, 2). Increases of [Ca²⁺]_i can result from an influx of Ca²⁺ from the extracellular medium, redistribution of Ca²⁺ from intracellular stores, or both influx and redistribution. Increases in [Ca²⁺]_i from any source often lead to changes in other fluxes because of activation of phospholipases and proteases (3). Calcium influx in nonexcitable cells occurs through Ca²⁺-selective channels activated secondarily to store depletion and/or through receptor- or second messenger- operated channels, along a decreasing electrochemical gradient (4). Other Ca²⁺ transport systems that can be activated by oxidative stress, include nonselective cation channels and the reverse mode of operation of the Na⁺/Ca²⁺ exchanger (5, 6).

Although intracellular calcium has been suggested to play a role in the oxidative damage of hepatocytes, the effects and source of the oxidative stress-induced intracellular Ca²⁺ increase are currently debatable (6). Treatment of liver cells with several oxidative stress inducer drugs increases [Ca²⁺]_i and produces cell toxicity through necrosis and/or apoptosis, depending on the degree of exposure (dose and duration) (2). The initial source of Ca²⁺ depends on the specific oxidant and cell type employed, since certain toxins cause calcium release from intracellular stores (5, 7), whereas others cause Ca²⁺ influx from the extracellular space (6, 8). Elevated calcium may initiate a cascade of signaling leading to activation of phospholipases A2 and C, endonucleases, or proteases (7, 9-11) and the expression of several immediate early genes including c-fos, c-jun, c-myc, and egr-1 (3). Inhibitors of calcium-dependent proteases and phospholipases can preserve cell viability in anoxic and hypoxic injury in hepatocytes (12).

In contrast to the above, increases of [Ca²⁺]_i may not be essential in all forms of hepatocyte injury (e.g. toxicity by the redox cycling drug naphthazarin was mediated through lysosomal damage and prevented by inhibition of lipid peroxidation and chelation of intralysosomal iron (13)). Hydrogen peroxide-induced cell death in rat hepatocytes was not dependent upon an increase in calcium but was blocked by deferoxamine (14). A Ca²⁺-independent, iron-mediated mechanism of prooxidant-induced cytotoxicity has been proposed (14). Some reports suggest that both calcium-dependent and calcium-independent events contribute to the cell toxicity (15); an influx of extracellular Ca²⁺ ions may aggravate the mechanism of iron-mediated cell injury (16). H₂O₂ toxicity in L929 cells could be switched from a calcium-mediated process (in the absence of glucose) to an iron-mediated process (in the presence of glucose) (17).

Reactive oxygen species (ROS)¹ are involved in mechanisms of early alcohol-induced liver injury. CYP2E1 is induced in hepatocytes by ethanol and is one source of ROS leading to liver injury (18). The role of [Ca²⁺]_i in ethanol-induced liver cell injury remains to be defined. Ethanol administration in vivo increases the calcium content in the liver of rats and mice, and this effect was enhanced by glutathione depletion or co-administration of iron, suggesting a synergism between ethanol and these agents (19, 20). The rise in liver cell calcium content may contribute to the cell injury process associated with toxic agents, or it may be a relatively late consequence of cell injury (21).

To evaluate biochemical and toxicological properties of CYP2E1, HepG2 cell lines overexpressing human CYP2E1 were previously established (22, 23). Ethanol, iron, or polyunsaturated fatty acids such as arachidonic acid were toxic to the cells expressing CYP2E1 but not to control HepG2 cells (24-26). We recently described a synergistic toxicity in CYP2E1-expressing cells caused by the combined addition of iron plus arachidonic acid (27). The potential role, if any, of calcium and/or Ca²⁺-activated hydrolases in these models of CYP2E1-dependent toxicity has not been determined. The objectives of this work were (i) to evaluate if CYP2E1-dependent toxicity in HepG2 cells is dependent on the alteration of Ca²⁺ homeostasis and (ii) to study the activation of calcium-dependent processes during CYP2E1-dependent toxicity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- Ionomycin, TMB-8, and calpeptin were from Calbiochem. PBS was from Roche Molecular Biochemicals. G418 was from Invitrogen (Carlsbad, CA). Ethanol (95%) was from Pharmaco Products (Brookfield, CT). Fluo3-AM and pluronic acid were from Molecular Probes, Inc. (Eugene, OR). Protein concentration was measured using the Bio-Rad DC protein assay. Most other chemicals used were from Sigma. The iron-nitrilotriacetic acid complex (Fe-NTA; 1:3 iron/NTA) was prepared as previously described (26).

Culture and Treatment of Cells-- Two human hepatoma HepG2 cell lines described by Chen and Cederbaum (23) were used as models in this study: E47 cells, which constitutively express human CYP2E1, and C34 cells, which are HepG2 cells transfected with the empty pCI vector. All cell lines were grown in MEM containing 10% fetal bovine serum (FBS) and 0.5 mg/ml G418 supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere in 5% CO₂ at 37 °C. Cells were subcultured at a 1:5 ratio once a week. For the experiments, cells were plated at a density of 30,000 cells/ml and incubated for 12 h in MEM supplemented with 5% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin (MEM_exps). After this period, the medium was replaced with MEM_exps supplemented with arachidonic acid (from 0 to 10 µM). After 12 h of incubation at 37 °C, the medium was removed, and the cells were washed once with PBS to remove unincorporated arachidonic acid. The cells were incubated for an additional 12-h period with MEM_exps. Then the cells were washed once with PBS, and the medium was replaced with MEM_exps without FBS or with S-MEM supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin (S-MEM_exps). S-MEM medium is identical to MEM medium except for the omission of calcium chloride. The medium was supplemented with any additions (e.g. antioxidants, channel blockers, inhibitors) for 1 h, prior to the addition of Fe-NTA (from 0 to 100 µM), which was considered as the initiation of the cellular toxicity phase (time = 0 h). The cells were incubated for variable periods (up to 12 h) before the biochemical analyses. This basic protocol (i.e. preloading with arachidonic acid, washing, adding MEM_exps without FBS or S-MEM_exps, and initiating the toxicity phase by the addition of Fe-NTA) was used for all experiments.

Cytotoxicity Measurements-- 3 × 10⁴ cells were plated onto 24-well plates, and after the corresponding treatment, the medium was removed, and cell viability was evaluated by the MTT test as previously described (27). The absorbance of the formazan produced was measured at a wavelength of 570 nm with background substraction at 630 nm (28). Viability was expressed as follows: 100 × (A_570-630 sample/A_570-630 control). Control refers to incubations in the absence of arachidonic acid and Fe-NTA and was considered as the 100% viability value. Another index of cytotoxicity used was the leakage of lactate dehydrogenase. At the end of the treatment, 1 ml of the medium was collected to measure LDH activity released into the medium (LDH out) using the Sigma LDH-20 diagnostic kit. Cells were washed with PBS, harvested by scraping in 1.0 ml of PBS, and sonicated (20 s, duty cycle 40%, output control 50%). The cellular suspension was centrifuged, and the supernatant was assayed for LDH activity (LDH in). Cytotoxicity was expressed as percentage of LDH release: 100 × (LDH out/LDH out + LDH in). Cell morphology was also assessed under the light microscope (×20) as a third index of viability.

Measurement of Intracellular Calcium-- The intracellular calcium levels were determined with the fluorescent calcium indicator fluo3-AM by flow cytometry. 3 × 10⁵ cells were plated in 10-mm Petri dishes, and at the end of the various treatments the medium was replaced with 3 ml of MEM_exps without FBS plus 2.5 µM fluo3-AM and 0.02% pluronic acid (stock solution × 1000 in Me₂SO). Cells were incubated for 30 min at 37 °C. After loading, the cells were washed in PBS (to remove any dye nonspecifically associated with the cell surface), trypsinized, and resuspended in 1 ml of MEM_exps without FBS plus 5 µg of propidium iodide (PI). PI was used to assay for the viable cell population, since these cells exclude PI, whereas nonviable cells take up this dye. The measurement of [Ca²⁺]_i was performed by flow cytometry analysis of 10,000 cells using Cell Quest software. The increase in calcium level was expressed using the percentage of fluo3 fluorescence intensity in Fe-NTA plus AA-treated cells (F) over that of AA-loaded cells not exposed to Fe-NTA (F₀) (100 × F/F₀) (29). 10 µM ionomycin was applied to one sample before each experiment to check for correct loading of the cells and thus served as a positive control. The inhibitors tested did not interfere with the quantification of the fluorescence (488/525 nm excitation/emission) of a standard fluo3-Ca²⁺ solution.

Lipid Peroxidation Assay-- The production of thiobarbituric acid-reactive substances (TBARS) was assayed as previously described (27). The protein concentration of the cell suspension was determined using a protein assay kit based on the Lowry assay (Bio-Rad DC kit). The concentration of malondialdehyde was calculated from a standard curve prepared using malonaldehyde bisdimethylacetal (30).

Intracellular ROS Measurement-- Intracellular ROS were monitored with 2',7'-dichlorofluorescein diacetate (DCFH-DA) as the probe. 3 × 10⁵ cells were plated in 10-mm Petri dishes, and after the corresponding treatment, the medium was replaced with FBS-free MEM_exps supplemented with 5 µM DCFH-DA (31). The cells were incubated for 30 min at 37 °C, and after this incubation, they were washed with PBS, trypsinized, and resuspended in 1 ml of FBS-free MEM_exps plus 5 µg of PI. 10,000 cells were analyzed by flow cytometry using Cell Quest software.

Antioxidant Activity of Inhibitors-- The possible antioxidant activity of flufenamic acid or calpeptin was evaluated following the protocol described in Ref. 32. Microsomal membranes from control rat liver (250 µg of protein/0.10 ml) were mixed with 0.5 µl of each drug (stock solutions × 200 in ethanol) and preincubated for 10 min at 37 °C in a reaction buffer consisting of 120 mM KCl, 50 mM sucrose, and 10 mM potassium phosphate, pH 7.2. Lipid peroxidation reactions were initiated by the addition of 0.025 mM FeCl₃ chelated by 0.25 mM ADP and 0.83 mM dihydroxyfumaric acid. At various times of incubation, the levels of TBARS were determined as described above. The compounds tested did not interfere with the quantification of malondialdehyde in the standard curve at the concentrations employed.

CYP2E1 Activity-- CYP2E1 activity was measured in microsomes derived from E47 cells and in liver microsomes from acetone-treated rats (1% acetone (v/v) in the drinking water for 7-10 days) by the spectrophotometric analysis of p-nitrophenol hydroxylation as described in Ref. 22. Microsomes from rat liver were employed in an in vitro test for the possible inhibitory activity of flufenamic acid or calpeptin on CYP2E1 catalytic activity. In this case, the drugs were preincubated for 10 min at 37 °C with the microsomal suspension, prior to initiating the reaction by the addition of 0.4 mM p-nitrophenol and 1 mM NADPH. The compounds tested did not interfere with the quantification of 4-nitrocatechol in the standard curve at the concentrations employed.

Measurement of Calpain Activity in Situ-- The activity of calpain in intact cells was assessed with the use of the cell-permeable fluorogenic substrate SUCC-LLVY-AMC (33). Specific proteolysis of the substrate by calpain liberates the fluorescent AMC group, leading to an increase of its fluorescence that is proportional to the proteolytic activity of calpain. 7.5 × 10⁴ cells were plated in 6-well plates, exposed to AA (0-5 µM) for 12 h, washed with PBS, and further incubated for 12 h in MEM_exps as described above. After this, cells were washed with serum-free MEM_exps without phenol red, and 3 ml of this medium plus 25 µM SUCC-LLVY-AMC was added to the cells in each well. After an equilibration period of 5 min, Fe-NTA (0-25 µM) was added, and the cells were incubated at 37 °C for up to 6 h. Fluorescence readings at 360/430-nm excitation/emission were performed at intervals. Controls with calpeptin, a specific calpain inhibitor, were performed by preincubating the cells with the inhibitor for 1 h prior to the addition of the fluorogenic substrate. Blank fluorescence readings (incubation medium without cells, incubated in the same conditions) were subtracted from each data point. -Tocopherol, calpeptin, and TMB-8 at the concentrations used did not interfere with the quantification of AMC fluorescence. 100 µM flufenamic acid slightly quenched the fluorescence of AMC in the medium of E47 cells treated with iron/AA/SUCC-LLVY-AMC (22% of inhibition of fluorescence); this factor was considered in the quantification of calpain activity in the presence of flufenamic acid.

Statistics-- Data are expressed as means ± S.E. of the mean from 3-5 independent experiments. One-way analysis of variance with subsequent post hoc comparisons by Scheffe was performed. p < 0.05 was considered as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CYP2E1-dependent Toxicity in HepG2 Cells Exposed to Iron plus Arachidonic Acid-- Fig. 1A shows that the combination of 25 µM Fe-NTA and 5 µM arachidonic acid was highly toxic in HepG2 cells overexpressing CYP2E1 (E47 cells, 31% viability at 12 h) with respect to cells not expressing detectable activity of CYP2E1 (C34 cells, 75% viability at 12 h). The toxicity in E47 cells was evident starting from early incubation times (i.e. 3 h, 62% viability), while no significant toxicity could be detected in C34 cells exposed to iron plus arachidonic acid at early times (Fig. 1A). Arachidonic acid (up to 10 µM) or Fe-NTA (up to 100 µM) by itself (at 6 h) was not significantly toxic in C34 or E47 cells (Fig. 1, B and C), but the combination of iron and arachidonic acid showed enhanced toxicity in both cell types. The toxicity was greater in cells overexpressing CYP2E1 at all combinations tested. This enhanced toxicity of arachidonic acid and iron was evident at relatively low concentrations of arachidonic acid (1 µM, with 12.5 µM Fe-NTA; Fig. 1B), and Fe-NTA (5 µM, with 5 µM arachidonic acid; Fig. 1C).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   CYP2E1-dependent toxicity of iron and arachidonic acid. A, time curve. C34 (circles) and E47 (squares) cells were incubated for 12 h in MEMexps medium (empty symbols) or in MEMexps medium supplemented with 5 µM arachidonic acid (filled symbols). After washing, cells were cultured for variable periods (up to 12 h) in the absence (empty symbols) or presence of 25 µM Fe-NTA (filled symbols). Viability was measured by MTT reduction activity as described under "Experimental Procedures." B, arachidonic acid dose dependence curve. C34 (circles) and E47 (squares) cells were incubated for 12 h in MEMexps medium supplemented with increasing concentrations of arachidonic acid (from 0 to 10 µM). After washing, cells were cultured for 6 h in the absence (open symbols) or presence of 12.5 µM Fe-NTA (filled symbols). Viability was measured by MTT reduction activity. C, Fe-NTA dose dependence curve. C34 (circles) and E47 (squares) cells were incubated for 12 h in MEMexps medium (empty symbols) or in MEMexps medium supplemented with 5 µM arachidonic acid (filled symbols). After washing, cells were cultured for 6 h in MEMexps medium without FBS, with increasing concentrations of Fe-NTA (from 0 to 100 µM). Viability was measured by MTT reduction activity. At all time points (A) or concentrations of AA (B) or of iron (C), toxicity was significantly greater (p < 0.05) in the E47 cells () than in the C34 cells ().

Intracellular Calcium Concentration in HepG2-derived Cells Exposed to Iron and Arachidonic Acid-- E47 cells exposed to 5 µM arachidonic acid and subsequently to 25 µM Fe-NTA for 3 h showed an increased intracellular calcium content (Fig. 2A, curve ii) with respect to control cells incubated only with arachidonic acid (Fig. 2A, curve i). When 10 µM ionomycin was added to the iron plus arachidonic acid-treated cellular suspension, an additional increase in the fluo3 fluorescence was detected (Fig. 2A, curve iii), suggesting that the intracellular indicator was not saturated under these loading conditions. No significant differences in fluo3 fluorescence were detected between control (not treated with AA plus Fe-NTA) C34 and E47 cells (not shown). Intracellular calcium increased both in arachidonic acid-loaded C34 and E47 cells after exposure to Fe-NTA, starting from early incubation times (i.e. 30 min), although the increase was greater in the cells that overexpressed CYP2E1 at all time points evaluated (Fig. 2B). Permeabilization of the cell membrane with digitonin led to an increase in PI staining but to a loss of the fluo3 fluorescence, so that the quantification of fluo3 fluorescence in our experiments was restricted to PI-negative (i.e. viable) cells. Therefore, the increase in intracellular calcium by exposure to iron plus arachidonic acid occurred prior to the loss of membrane integrity, since only PI negative cells were considered for the quantification of fluo3 fluorescence. This suggests that elevation in calcium levels is an early event in the developing toxicity.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of iron plus arachidonic acid on the level of intracellular calcium. A, typical histogram showing the increase of intracellular calcium in iron plus arachidonic acid-treated cells. E47 cells were exposed to 5 µM arachidonic acid and either 0 µM Fe-NTA (i) or 25 µM Fe-NTA (ii) for 3 h in MEMexps without FBS. The attached cells were then loaded with 2.5 µM fluo3-AM plus 0.02% pluronic acid for 30 min at 37 °C. The cells were washed, trypsinized, and resuspended in PI-supplemented medium for flow cytometry analysis. In a third experiment, the cellular suspension obtained in ii was further supplemented with 10 µM ionomycin and immediately analyzed by flow cytometry (iii). FL1-H stands for fluo3. B, time course study. C34 (circles) and E47 (squares) cells were supplemented with 5 µM arachidonic acid, washed, and exposed to Fe-NTA for variable periods (from 0 to 3 h). Cells were then loaded with fluo3-AM plus pluronic acid, washed, trypsinized, and resuspended in PI-supplemented medium for flow cytometry analysis. Data are presented as 100 × F/F0, where F represents the fluorescence of the PI-negative cells at each particular time, and F0 is the fluorescence of the PI-negative zero time control (cells only loaded with arachidonic acid). At all time points evaluated, fluo3 fluorescence was significantly greater (p < 0.05) in E47 cells than in C34 cells.

Effect of Extracellular Calcium Omission on the Toxicity of Iron and Arachidonic Acid-- The omission of extracellular calcium in the last incubation step was accomplished by working with S-MEM medium lacking fetal bovine serum. The omission of extracellular calcium was not toxic by itself in nontreated E47 cells (Fig. 3A, MTT assay; Fig. 3B, LDH release, open circles). The omission of extracellular calcium, however, significantly decreased the toxicity of iron plus arachidonic acid in E47 cells (Fig. 3, A and B, open squares versus closed squares). For example, AA plus Fe-NTA produced strong toxicity after 6 h of incubation in MEM_exps medium but not in Ca²⁺-free medium for up to 6 h of incubation with Fe-NTA. Toxicity in Ca²⁺-free medium was found after 12 h of incubation with iron, although this remained considerably lower than the toxicity in MEM at that same time point (Fig. 3). E47 cells exposed for 6 h to 5 µM arachidonic acid and 25 µM Fe-NTA in MEM_exps showed substantial morphological changes with respect to control cells; cells were swollen and dispersed with accumulation of intracellular vesicles, and many cells were detached and floated to the top of the culture dish (data not shown). However, morphology in E47 cells exposed to iron plus arachidonic acid for 6 h remained normal in S-MEM_exps in contrast to the changes observed in MEM_exps (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of extracellular calcium omission on the CYP2E1-dependent toxicity of iron plus arachidonic acid. E47 cells were incubated for 12 h in MEMexps medium (circles) or in MEMexps medium supplemented with 5 µM arachidonic acid (squares). After washing, cells were cultured for variable periods (up to 12 h) either in FBS-free MEMexps (filled symbols) or S-MEMexps (empty symbols) in the absence (, ), or presence of 25 µM Fe-NTA (, ). Viability was measured by MTT reduction activity (A) or by LDH leakage (B), as described under "Experimental Procedures." The loss of viability (A) or increase in LDH leakage (B) was significantly greater (p < 0.05) after Fe/AA treatment in MEM () compared with S-MEM () at all time points.

The greater toxicity by AA plus Fe-NTA in MEM than S-MEM media presumably reflects a requirement for Ca²⁺ in the overall toxicity pathway. To validate that these differences were indeed due to the absence of Ca²⁺ in S-MEM, Ca²⁺ was added back to S-MEM, and the toxicity of AA plus Fe-NTA was determined. The addition of up to 0.4 mM Ca²⁺ had no effect on the viability of E47 cells in S-MEM in the absence of AA plus Fe-NTA. However, Ca²⁺ in a concentration-dependent manner restored the toxicity of AA plus Fe-NTA (Fig. 4). The toxicity when 0.4 mM Ca²⁺ was added to the S-MEM began to approach the toxicity found in MEM medium, which contains 1.8 mM Ca²⁺ (Fig. 4). Adding Ca²⁺ to S-MEM at concentrations greater than 0.4 mM could not be evaluated because of precipitation of the medium.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Ca2+ supplementation of S-MEM medium increases the toxicity of iron and arachidonic acid. E47 cells were incubated in the absence of iron and arachidonic acid () or with 1 µM arachidonic acid plus 10 µM Fe-NTA (), 5 µM arachidonic acid plus 10 µM Fe-NTA (), or 10 µM arachidonic acid plus 10 µM Fe-NTA (), following the basic protocol described under "Experimental Procedures," with a last incubation step of 6 h in S-MEMexps. The S-MEMexps was either supplemented with increasing concentrations of Ca2+ (from 0 to 0.4 mM) or replaced with MEMexps medium, which contains 1.8 mM Ca2+. Viability was assessed measuring the MTT reduction activity. Significant loss of viability (p < 0.05) was found at all concentrations of arachidonic acid when supplementing with calcium concentrations greater than 0.2 mM.

Reactive Oxygen Production-- The toxicity of iron and arachidonic acid in E47 cells was previously related to increased ROS generation and lipid peroxidation (27). Experiments were designed to test whether the omission of Ca²⁺ in the extracellular medium inhibited the generation of ROS and lipid peroxidation caused by AA plus Fe-NTA in the E47 cells. ROS generation was tested by measuring the fluorescence of DCFH by flow cytometry. Control E47 cells (Fig. 5i) showed a relatively low fluorescence of DCFH in viable (i.e. PI-negative) cells (lower right quadrant), reflecting the basal generation of ROS. About 31% of the cells lost membrane integrity and accumulated PI (Fig. 5i, upper left quadrant). Permeabilization of the cell membrane with digitonin triggered the loss of DCFH from the intracellular space; thus, only PI-negative (i.e. viable) cells were considered for the quantification of DCFH fluorescence. Arachidonic acid-loaded cells exposed to Fe-NTA for 3 h in MEM had increased PI staining, indicating loss of plasma membrane integrity (58% of cells with high PI staining) (Fig. 5ii). In contrast, cells exposed to iron plus arachidonic acid in S-MEM showed a smaller percentage of cells in the PI-positive quadrant (31% of cells with high PI staining) (Fig. 5iv). However, despite differences in viability in MEM compared with S-MEM, the mean DCFH fluorescence values were the same in MEM and S-MEM in the absence of AA plus Fe-NTA (572 ± 123 and 615 ± 116 AU, Fig. 5, i and iii, respectively) as well as in the presence of AA plus Fe-NTA (1786 ± 217 and 1592 ± 175 AU; Fig. 5, ii and iv, respectively). The addition of AA plus Fe-NTA increased the mean DCFH fluorescence about 3-fold in both MEM and S-MEM.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Effect of calcium omission on membrane permeability (PI staining) and ROS generation (DCFH oxidation) in E47 cells exposed to iron and arachidonic acid. E47 cells were incubated in the absence of Fe-NTA plus arachidonic acid, in MEMexps (i) or S-MEMexps (iii), or exposed to 5 µM arachidonic acid and 25 µM Fe-NTA for 3 h in MEMexps (ii) or S-MEMexps (iv), following the basic culture protocol described under "Experimental Procedures." After the treatment, cells were loaded with DCFH-DA for 30 min at 37 °C, washed, trypsinized, and resuspended in PI-supplemented medium for flow cytometry analysis. Shown are typical dot plots with two-parameter display of data (FL1-H or DCFH and FL2-H or PI). The percentage of PI-positive cells in the upper left quadrant is given for each plot.

Lipid peroxidation in E47 cells exposed to Fe plus arachidonic acid for 3 h in MEM increased about 3-fold with respect to control cells; this increase was similar to that in E47 cells exposed to iron plus arachidonic acid in S-MEM (Table I). Preincubation of arachidonic acid-loaded E47 cells with -tocopherol prior to the 3-h incubation with Fe-NTA decreased the TBARS content of the cells to basal levels (Table I). Taken as a whole, these results indicate that the lower toxicity of Fe plus arachidonic acid in E47 cells in S-MEM medium was not related to an inhibition of ROS generation and lipid peroxidation (i.e. the requirement for Ca²⁺ in the toxicity process was not related to altered ROS production but to some other Ca²⁺-dependent effect).

View this table: [in this window] [in a new window]   Table I TBARS generation in E47 cells E47 cells were incubated either in MEM or S-MEM in the absence or presence of 5 µM AA (preloaded) plus 25 µM Fe-NTA as described under "Experimental Procedures." The last 3h of preincubation in MEM or S-MEM was carried out in the absence of FBS. Following the treatment, production of TBARS was determined in the cellular suspension. A control experiment was done in E47 cells treated with iron plus arachidonic acid in serum-free MEMexps, preincubated with 100 µM -tocopherol (+Fe/AA, MEM + -tocopherol).

Effect of -Tocopherol and TMB-8-- Lipid peroxidation processes mediated by iron can increase calcium influx from the extracellular space through plasma membrane calcium channels (8). If such an influx is related to the CYP2E1-dependent toxicity, then an antioxidant (-tocopherol) should prevent the toxicity and the increase in intracellular calcium mediated by iron plus arachidonic acid in E47 cells. On the other hand, TMB-8, an inhibitor of the release of calcium from intracellular stores (6), should not prevent the toxicity and the increase in intracellular calcium if extracellular calcium is required. -Tocopherol prevented (Fig. 6A) and TMB-8 did not prevent (Fig. 6B) the toxicity of iron plus arachidonic acid in E47 cells. In fact, there was a small increase in toxicity by AA plus Fe-NTA in the presence of TMB-8. The effect of these additions on intracellular calcium levels was determined. -Tocopherol prevented (Fig. 7) the increase in intracellular calcium in E47 cells exposed to arachidonic acid plus Fe-NTA for 3 h, consistent with the prevention of toxicity. This indicates that -tocopherol-sensitive processes such as lipid peroxidation (which was inhibited by -tocopherol; Table I) play a key role in the increase in intracellular calcium induced by Fe/AA in the E47 cells. TMB-8 caused a further elevation in the level of calcium (Fig. 7), consistent with the potentiation of AA plus Fe-NTA toxicity. The omission of extracellular calcium (i.e. S-MEM replacing MEM) prevented the increase of intracellular calcium elicited by iron plus arachidonic acid in E47 cells (Fig. 7), further indicating that extracellular calcium is important for the increase in intracellular calcium induced by Fe/AA in the E47 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of -tocopherol (A) or TMB-8 (B) on the viability of E47 cells. E47 cells were incubated with the indicated concentrations of -tocopherol (A) or TMB-8 (B) in the absence () or presence () of 5 µM AA plus 25 µM Fe-NTA, with the last incubation step of 3 h. Viability was assessed by measuring the MTT reduction activity. The increase in viability was significant (p < 0.05) at all concentrations of -tocopherol (A), whereas the decrease in viability was significant (p < 0.05) at all concentrations of TMB-8 (B).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of calcium omission and several inhibitors on the iron plus arachidonic acid-induced elevation of intracellular calcium levels in E47 cells. Cells were exposed to 5 µM arachidonic acid and 25 µM Fe-NTA (3 h) in MEMexps without FBS in the absence of further addition (B), in S-MEMexps replacing the MEMexps (C), or in FBS-free MEMexps with a preincubation step of 1 h with 100 µM -tocopherol (T) (D), 50 µM TMB-8 (E), 100 µM flufenamic acid (F), or 50 µM calpeptin (G). A, AA-loaded E47 cells in MEMexps not exposed to Fe-NTA, considered as the 100% Ca2+ intracellular level. Calcium levels were analyzed with fluo3 as described under "Experimental Procedures."

Effect of Calcium Channel Blockers on the Toxicity of Iron and Arachidonic Acid in HepG2 Cells Overexpressing CYP2E1-- Experiments were designed to test whether inhibitors of plasma membrane calcium channels were protective against the AA plus Fe-NTA toxicity in E47 cells (Table II). Nifedipine, verapamil, and diltiazem (inhibitors of L-type calcium channels in excitable cells and store-operated channels at higher concentration in liver cells) (34) did not prevent the toxicity. SKF 96365 (an inhibitor of receptor-operated calcium channels) and benzamil (an inhibitor of Na⁺/Ca²⁺ exchange) (34) at 1 and 10 µM were also not protective. Higher concentrations of some of these agents (100 µM) showed increased toxicity by themselves. Flufenamic acid (an inhibitor of nonspecific cation channels) (5) showed significant inhibition of iron plus arachidonic acid toxicity at 100 µM (Table II). The protective effect against toxicity by flufenamic acid was verified at different incubation times (up to 12 h) by the MTT assay (Fig. 8A) and was confirmed by significant inhibition of the AA plus Fe-NTA-catalyzed increase in LDH release (Fig. 8B). Flufenamic acid did not inhibit CYP2E1 catalytic activity (Fig. 9A); nor did it show antioxidant activity against microsomal lipid peroxidation (Fig. 9B), as compared with a known inhibitor of CYP2E1 activity such as 4-methylpyrazole (Fig. 9A) or an antioxidant such as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Fig. 9B, Trolox). Thus, the partial protection against AA plus Fe-NTA toxicity in E47 cells by flufenamic acid was not due to inhibition of CYP2E1 or prevention of ROS/lipid peroxidation. However, flufenamic acid did not inhibit the increase of intracellular calcium triggered by iron plus arachidonic acid (Fig. 7).

View this table: [in this window] [in a new window]   Table II Effect of calcium channel blockers on the toxicity of iron plus arachidonic acid in E47 cells E47 cells were incubated in the absence (Fe/AA) or presence (+Fe/AA) of 5 µM AA (preloaded) plus 25 µM Fe-NTA for 6 h, in the presence of the indicated calcium channel blockers. Viability was assessed as MTT reduction activity, with the no addition control assigned as 100% viability.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Effect of flufenamic acid on the CYP2E1-dependent toxicity of iron plus arachidonic acid. A, E47 cells were incubated in the absence (, ) or presence of 5 µM AA plus 25 µM Fe-NTA (, ) for the indicated time periods, either without (, ) or with 100 µM flufenamic acid (, ). Viability was assessed as MTT reduction activity. The decrease in viability by Fe/AA treatment was significantly (p < 0.05) less in the presence () than in the absence () of flufenamic acid. B, E47 cells were incubated in the absence or presence of 5 µM arachidonic acid plus 25 µM Fe-NTA after a preincubation with the indicated concentrations of flufenamic acid (from 0 to 100 µM). Viability was assessed as LDH leakage. The increase in LDH leakage by Fe/AA was significantly lower (p < 0.05) in the presence of 50 or 100 µM flufenamic acid.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Effect of flufenamic acid on CYP2E1 catalytic activity and microsomal lipid peroxidation. A, CYP2E1 catalytic activity was assessed by PNP hydroxylation in rat hepatic microsomal membranes in the presence () or absence () of 100 µM flufenamic acid or in the presence of 4 mM 4-methylpyrazole (). B, generation of TBARS was assessed in a microsomal system incubated with Fe-ADP and dihydroxyfumaric acid as reductant, in the presence () or absence () of 100 µM flufenamic acid or in the presence of 100 µM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) ().

Role of Calcium-activated Proteases in CYP2E1-dependent Toxicity-- Activation of calcium-dependent proteases (calpain) is thought to play an important role in certain models of oxidative stress-induced toxicity in hepatocytes (1, 9, 10). Fig. 10 shows that the toxicity of iron plus arachidonic acid in E47 cells was significantly prevented by incubation with calpeptin (Fig. 10A) or E64 (Fig. 10B), two specific cell-permeable calpain inhibitors. 50 µM calpeptin did not inhibit the increase of intracellular calcium in E47 cells triggered by iron plus arachidonic acid (Fig. 7); thus, calpeptin was blocking the downstream actions of calcium. The activation of calpain activity was tested using SUCC-LLVY-AMC as a specific fluorogenic substrate. Control experiments showed that a calcium ionophore (ionomycin) increased the fluorescence due to release of AMC as a consequence of the proteolysis of the SUCC-LLVY-AMC substrate, and this was blocked (61%) by preincubation with calpeptin (Fig. 11A). Calpain activation was detected in E47 cells loaded with arachidonic acid beginning after 1 h of exposure to Fe-NTA (Fig. 11B). This activation was significantly inhibited by calpeptin (Fig. 11B). Importantly, calpain activation in E47 cells was also blocked by -tocopherol and flufenamic acid, agents that decrease the Fe/AA toxicity, but not by TMB-8, which was not protective (Table III). Calpain activation (as calpeptin-inhibitable fluorescence of AMC) in C34 cells after exposure to iron plus arachidonic acid was statistically nonsignificant, while in E47 cells it increased 40-fold with respect to nontreated control cells; this may reflect the smaller increase produced by Fe/AA in intracellular calcium in C34 cells compared with E47 cells (Table III). Analogous to experiments with flufenamic acid (Fig. 9), calpeptin had no effect on CYP2E1 activity; nor did it show antioxidant activity at the concentrations used (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Effect of calpain inhibitors on the toxicity of Fe and arachidonic acid. E47 cells were incubated in the absence (, ) or presence of 5 µM arachidonic acid plus 25 µM Fe-NTA (, ) for the indicated time periods, with (, ) or without (, ) 50 µM calpeptin (A) or 100 µM E64 (B). Viability was assessed as MTT reduction activity. The loss in viability by Fe/AA treatment was significantly lower (p < 0.05) in the presence of calpeptin (A, ) or E64 (B, ) than in their absence (A, B, ) at all time periods.

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Calpain proteolytic activity in E47 cells. Activity was determined with the use of the membrane-permeant fluorogenic calpain-specific substrate SUCC-LLVY-AMC. A, fluorescence (360/430-nm excitation/emission) as a function of incubation time with SUCC-LLVY-AMC of E47 cells (), E47 cells plus 0.2 µM ionomycin (), E47 cells plus 50 µM calpeptin (), or E47 cells plus ionomycin plus calpeptin (). B, fluorescence (360/430-nm excitation/emission) as a function of incubation time of E47 cells plus SUCC-LLVY-AMC with the following: no AA or Fe (); 5 µM AA and 25 µM Fe-NTA (); 50 µM calpeptin (); or Fe/AA and calpeptin (). The increase in calpain activity by Fe/AA treatment was significant (p < 0.05) at time periods greater than 2 h, as was the inhibition by calpeptin of this increase.

View this table: [in this window] [in a new window]   Table III Calpain activity in situ C34 and E47 cells were preloaded with 5 µM arachidonic acid, followed by the addition of the indicated compounds (50 µM calpeptin, 100 µM -tocopherol, 50 µM TMB-8, or 100 µM flufenamic acid). After a 1-h incubation, calpain-specific fluorogenic substrate SUCC-LLVY-AMC (25 µM) and 0 or 25 µM Fe-NTA were added, and the cells were incubated for an additional 3 h. Fluorescence of the medium (360/430 nm) was determined after this incubation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The toxicity of iron plus arachidonic acid in E47 cells was causally linked to oxidative stress, as evidenced by elevated lipid peroxidation and the inhibition of TBARS generation and toxicity by -tocopherol. Iron-mediated lipid peroxidation may be initiated through ^·OH-dependent or ^·OH-independent pathways. CYP2E1 may activate lipid peroxidation in the presence of AA plus Fe through the generation of ROS such as O and H₂O₂, and the reduction of iron to its catalytic ferrous form. An alteration of Ca²⁺ homeostasis with elevated intracellular calcium levels was detected at early times (e.g. 30-60 min after exposure of E47 cells to iron plus arachidonic acid). This increase in intracellular calcium is a critical and early event in CYP2E1 and oxidative stress-dependent toxicity because (i) like the toxicity, it was higher in E47 cells than in C34 cells; (ii) it depended on the addition of iron to AA-loaded cells; (iii) toxicity was higher in MEM than in S-MEM; (iv) it developed at early times prior to the loss of plasma membrane integrity; and (v) inhibition of downstream calcium-dependent reactions (e.g. activation of calpain) lowered the toxicity significantly. Although fluo3 is in general a satisfactory Ca²⁺ indicator (36), the increases of intracellular calcium as detected with this agent are likely to be underestimated because of the oxidation of the probe by hydroxyl radicals and peroxidase/H₂O₂ (37). Thus, precaution was taken in expressing the data obtained in relative terms and not in absolute calcium concentration terms.

Influx of calcium from the extracellular medium is a critical and early event in the CYP2E1 plus AA/Fe-NTA toxicity, because the omission of calcium from the extracellular medium lowered this toxicity, and the reincorporation of extracellular calcium restored toxicity. The omission of calcium from the extracellular medium prevented the early increase in intracellular calcium produced by iron plus arachidonic acid in E47 cells, which probably explains the lower toxicity. An inhibitor of the release of calcium from intracellular stores, TMB-8, did not inhibit the toxicity, but it also did not prevent the increase in intracellular calcium. The additional increase in [Ca²⁺]_i produced by TMB-8 plus Fe/AA may reflect a compensatory flux of calcium from other stores or from the extracellular media. In fact, TMB-8 slightly enhanced the Fe/AA toxicity, perhaps as a result of the additional increase in [Ca²⁺]_i. This suggests that extracellular calcium is responsible for the increase in intracellular calcium produced under these conditions. The influx of calcium and the subsequent toxicity depends on the activation of lipid peroxidation processes, since both of these events are inhibited by -tocopherol (for further discussion, see below). Lipid peroxidation in C34 cells exposed to Fe plus AA was lower than in E47 cells (27), which explains the lower increase in intracellular calcium and toxicity in C34 versus E47 cells. The omission of calcium was not toxic to C34 or E47 cells at the time points studied.

Inhibitors of several calcium channels present in liver cells (store-operated, receptor-operated, and Na⁺/Ca²⁺ exchanger) were not protective against the AA plus Fe-NTA toxicity at the concentrations utilized, suggesting that these systems do not participate in the increased influx of calcium. Flufenamic acid, although it inhibited the toxicity, did not inhibit the rise in intracellular calcium, suggesting that this compound acts in later steps after the increase of intracellular calcium already has taken place. Indeed, flufenamic acid partially inhibited the activation of calpain in E47 cells exposed to iron plus AA. Flufenamic acid can inhibit other cellular cysteine proteases such as lysosomal cathepsins B and L (38, 39). Since we could not specifically identify a plasma membrane calcium channel that promotes increased entry of calcium into the E47 cells, it is possible that the net increases in the influx of Ca²⁺ may result from a direct but nonspecific effect of lipid peroxidation on the plasma membrane or on an impairment of the Ca²⁺-ATPase activity, which pumps calcium either out of the cell or into intracellular storage depots. Further studies are required to evaluate these possibilities.

Calpain activation as a result of the Ca²⁺ increase probably plays a major role in the mechanism of necrotic cell death in the AA plus Fe-NTA-treated E47 cells; the time course of calpain activity mirrored the decrease of cell viability, the activation of calpain in iron plus arachidonic acid-treated C34 cells was low compared with E47 cells, and a specific calpain inhibitor blocked calpain activity and the toxicity of iron plus AA in E47 cells. Calpain activity did not increase at very early times (i.e. 1 h) of exposure to Fe-NTA in arachidonic acid-loaded E47 cells, while Ca²⁺ concentration did increase at that early time point, suggesting a possible sequence of events: first calcium overload and then activation of calpain. Oxidative stress inhibits calpain activity stimulated by ionomycin in situ (40), suggesting that the net increase in calpain activity observed in Fe/AA-treated cells may reflect a balance between calpain activation by calcium overload and calpain inhibition by oxidation of the active site cysteine. Importantly, the activation of calpain did mirror the time course of the developing toxicity. Calpeptin did not block CYP2E1 activity or function as an antioxidant; nor did it inhibit the increase in Ca²⁺ in E47 cells treated with iron plus AA, but calpeptin prevented the toxicity and the activation of calpain in these cells. This suggests that calpain was activated before the actual onset of cell death and plays a causal role in cell necrosis. The exact mechanism(s) through which calpain activation leads to toxicity remains to be established. Calpain substrates include cytoskeletal, plasma membrane-associated proteins and signal transduction- and calmodulin-dependent proteins and transcription factors (41). Activation of a mitochondrial calpain-like protease activity in rat liver mitochon-dria can induce the mitochondrial permeability transition, a trigger for cell necrosis (42). Interestingly, cyclosporin A, an inhibitor of the mitochondrial permeability transition, decreased the Fe/AA toxicity in E47 cells (27). Cleavage of Bax was mediated by calpain in apoptotic and necrotic neuronal cells (43). Other calcium-activated enzymes such as phospholipases or endonucleases may also contribute to the developing toxicity, and activation of these enzymes, analogous to the increase in calpain activity, is under study.

The fact that calcium omission completely prevented the toxicity of iron plus arachidonic acid in cells overexpressing CYP2E1 at early times (up to 6 h) but did not prevent TBARS generation or ROS generation suggests that this toxicity is mainly calcium-dependent but not directly due to ROS and lipid peroxidation (although ROS and lipid peroxidation are necessary for the increase in [Ca²⁺]_i; witness the protection by -tocopherol). Toxicity did develop when calcium was omitted, but at later time periods (e.g. 12 h). This suggests that a calcium-independent process (probably iron-dependent lipid peroxidation, since -tocopherol is protective) may be involved at later stages, or else calcium from intracellular stores may be related to this later toxicity. The model shown in Scheme 1 is proposed to account for these results. Thus, lipid peroxidation is central to the toxicity, either promoting calcium entry (a), followed by activation of proteases such as calpain, or acting directly (b) on cellular membranes and macromolecules.

View larger version (9K): [in this window] [in a new window]   Scheme 1.

These results suggest that CYP2E1-dependent necrosis triggered by oxidative stress in HepG2 cells is mediated by an increase in the influx of Ca²⁺ from the extracellular space and activation of Ca²⁺-dependent enzymes such as calpain. CYP2E1 expression may represent an important factor to consider in the alteration of calcium homeostasis in hepatocytes exposed to ethanol. An ethanol-induced increase in hepatocyte Ca²⁺ levels has been observed in some studies but not in others (44). In pathological conditions where CYP2E1 is elevated and is a factor involved in the injury, such as alcoholic liver disease, control of calcium mobilization or inhibition of calcium-activated enzymes may prove to be helpful in controlling the damage. The role of calcium and Ca²⁺-dependent enzymes in various models of alcohol liver damage or other tissue damage and other systems or models of CYP2E1-dependent toxicity warrants further study.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant AA06610 from The National Institute on Alcohol Abuse and Alcoholism.

To whom correspondence should be addressed: Dept. of Pharmacology and Biological Chemistry, Box 1603, Mount Sinai School of Medicine, One Gustave L. Levy Pl., New York, NY 10029. Tel.: 212-241-7285; Fax: 212-996-7214; E-mail: arthur.cederbaum@mssm.edu.

Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M107864200

	ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; PI, propidium iodide; Fe-NTA, iron-nitrilotriacetic acid complex; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; TBARS, thiobarbituric acid reactive substances; AA, arachidonic acid; Fe/AA, iron plus AA; TMB-8, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester; FBS, fetal bovine serum; DCFH-DA, 2',7'-dichlorofluorescein diacetate; SUCC-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr 7-amido-4-methylcoumarin; PBS, phosphate-buffered saline; MEM, minimal essential medium; AMC, 7-amido-4-methylcoumarin; AU, arbitrary units.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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