The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 247, No. 1, May 15, pp. 183-189,1986

The Role of Mitochondrial Glutathione and Cellular Protein Sulfhydryls in Formaldehyde Toxicity in Glutathione-Depleted Rat Hepatocytes ROBERT Department

of Pharwuzcolcgy, Received

July

H. KU1 University

29,1985,

AND

RUTH

of Texas

and in revised

E. BILLINGS’ Medical form

School, February

Houston,

Texas

77225

3,1986

Depletion of cellular GSH by diethyl maleate (DEM) potentiates CHzO toxicity in isolated rat hepatocytes and it was postulated that this increase in toxicity is due to the further decrease in GSH caused by CHzO in DEM-pretreated hepatocytes (1). The present investigation was conducted to investigate further the effects of CHzO, DEM, and acrolein (a compound which is structurally related to CHzO and DEM) on subcellular GSH pools and on protein sulfhydryl groups (PSH). CHzO caused a decrease in cytosolic GSH but had no effect on mitochondrial GSH either in previously untreated hepatocytes or in DEM-pretreated hepatocytes in which GSH was approximately 25% of control. DEM decreased both cytosolic and mitochondrial GSH but it did not produce toxicity. Neither CHaO (up to 7.5 mM) nor DEM (20 mM) decreased PSH. However, in cells pretreated with 1 mM DEM, CHzO (7.5 mM) decreased PSH and this effect preceded cell death. Acrolein decreased both cytosolic and mitochondrial GSH and it also decreased PSH significantly prior to causing cell death. CHzO and acrolein stimulated phosphorylase a activity, indicative of an increase in cytosolic free Ca’+, by a PSH-independent and PSH-dependent mechanism, respectively. These results suggest that the further depletion of cellular GSH by CHzO in DEM-pretreated cells is not due to the depletion of mitochondrial GSH. CHzO toxicity in DEM-pretreated cells is, however, correlated with depletion of PSH. The critical sulfhydryl protein(s) responsible for cell death remain to be more clearly defined. o 1s~ Academic press. lnc. Glutathione (GSH) plays a protective role in cells (2-4) and is found in two distinct pools, the cytosolic pool, which contains about 85% of the total cellular GSH, and the mitochondrial pool, which contains the remaining 15% (5-10). During normal aerobic metabolism mitochondria utilize large quantities of dioxygen and generate oxygen free radicals and hydrogen peroxide (11). Since the GSH-dependent peroxidases are used in the detoxification of these reactive oxygen species (12), depletion of

mitochondrial GSH could severely compromise the ability of mitochondria to effectively detoxify them and result in peroxidative damage to cell membranes. Meredith and Reed (10,13) have suggested that depletion of the cytosolic GSH pool is not as sensitive an indicator of cellular toxicity as depletion of mitochondrial GSH. They have suggested that the maintenance of the mitochondrial GSH pool is critical in protecting against the cellular toxicity caused by ethacrynic acid (10) and the combination of adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)3 (13). More-

i Present address: Environ Corporation, The Flower Mill, 1000 Potomac St., N.W., Washington, D.C. 20007. a To whom correspondence should be addressed at Department of Pharmacological Sciences, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, Calif. 94080.

3 Abbreviations used: BCNU, 1,3-bis(2-chloroethyl)1-nitrosourea; DEM, diethylmaleate; PSH, protein sulfhydryls; LDH, lactate dehydrogenase; GDH, glutamate dehydrogenase; DTNB, 5,5’-dithiobis(2-chloroethyl)nitrobenzoic acid. 183

0003-9861/86 Copyright All rights

$3.00

8 1986 by Academic Press. Inc. of reproduction in any form reserved.

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AND

over, it was proposed that the reason why diethyl maleate (DEM) is not cytotoxic even though it depletes cytosolic GSH substantially is because it does not affect mitochondrial GSH (10). Romero et al. (14) reported that the toxicity of phorone (2,6dimethyl-2,5-heptadiene-4-one) occurred when the mitochondrial GSH pool was depleted below 40% of control. Cellular GSH has been suggested to act as a buffer against the oxidation of protein sulfhydryls (PSH) which are critical in maintaining normal cellular function (15). Depletion of cellular GSH below a certain level may result in the oxidation of critical PSH and result in cellular toxicity. Enzymes which are highly sensitive to oxidative stress, i.e., the accumulation of oxygen radicals and alteration of cellular redox state, include the Ca2+-dependent ATPases which function to maintain cytosolic Ca2+ within narrow limits (16). Numerous important cellular functions are regulated by cytosolic Ca2+ (17, 18) and perturbation of cytosolic Ca2+ may result in cell death (19,20). In fact, the increase in cellular Ca2+ and cell death have long been associated although the critical steps connecting the two events are still unclear. The existence of a “threshold” level of GSH, below which chemical-induced toxicity is produced, has frequently been suggested. Decreasing the cellular concentration of GSH below a threshold level is associated with CH20 toxicity in isolated rat hepatocytes (1). CH20 decreased cellular GSH levels to a greater extent in DEMpretreated hepatocytes and resulted in a stimulation of lipid peroxidation and cell death. Depletion of cellular GSH (probably by extrusion of the CH,O-GSH adduct, Shydroxymethylglutathione), stimulation of lipid peroxidation, and cell death were inextricably associated in CH20 plus DEMtreated hepatocytes (1). For example, both lipid peroxidation and cell death ensued upon depletion of cellular GSH and these events were prevented by antioxidants such as a-tocopherol. To investigate how depletion of cellular GSH results in cell death the present study was initiated. The focus of these experiments was to determine whether the fur-

BILLINGS

ther decrease in cellular GSH by CH20 in DEM-pretreated hepatocytes and the ensuing toxicity is associated with depletion of mitochondrial GSH or depletion of cellular PSH and perturbation of cytosolic Ca2+. In addition, the effects of CH20 and DEM individually on these parameters were assessed, as well as the effects of acrolein, a compound which is structurally similar to both CH20 (aldehyde) and DEM (a&unsaturated carbonyl). It is also a potent GSH-depleting agent (21,22). Our results suggest that perturbation of protein sulfhydryls by CH20 in DEM-pretreated hepatocytes is associated with the toxicity observed. MATERIALS

AND

METHODS

Chemicals CHaO, 37% solution, acrolein, and dibutyl phthalate were obtained from Aldrich Chemical Company, Inc. (Milwaukee, Wis.). Collagenase (type IV), glutathione reductase, bovine serum albumin (fraction V), diethyl maleate, and other organic chemicals were purchased from Sigma Chemical Company (St. Louis, MO.). All inorganic chemicals were of reagent quality and were purchased from commercial suppliers. Hepatocyte isolation Male Sprague-Dawley rats (Timco Breeding Laboratories, Houston, Tex.) weighing 225-406 g were housed in wire-bottom cages with access to water and food (standard Purina laboratory chow) ud libitum. Hepatocytes were isolated according to the collagenase perfusion technique previously described (23) at approximately the same time of day (lo:30 AM) to minimize the interference which diurnal variation of GSH may cause (24). After isolation, the cells were suspended to produce a concentration of 35 X 106cells/ml in Krebs-Henseleit bicarbonate buffer, pH 7.6, containing 2% bovine serum albumin. Cells were counted using a hemacytometer and light microscope. Initial viability estimates were determined for each cell preparation by the percentage of cells which excluded trypan blue. Only preparations with initial viability greater than 90% were used. Hepatocyte incubation Incubations of 5-ml volumes were carried out in sealed 25-ml Erlenmeyer flasks equilibrated with 95% Oa/5% COB in an orbital shaking water bath at 100 oscillations/min and 37°C. In experiments involving DEM and CH20, 1 mM DEM was added 10 min before CHcO addition. CH,O and acrolein were dissolved in HaO. DEM was dissolved in dimethyl sulfoxide and added in a final volume of 30 ~1. This volume of dimethyl sulfoxide did not have an effect on any of the parameters assessed. Determination of cell viability. Cellular toxicity was assessed by measuring the leakage of the cytosolic

GLUTATHIONE

DEPLETION

AND

enzyme lactate dehydrogenase (LDH) into the incubation medium by the method of Moldeus et al. (26). Percentage viability of the cells was calculated using the total cellular content of LDH as the 100% value. These cells were disrupted with Triton X-100 before LDH assay (26). &fit&on&i& isolutim Mitochondria were isolated from hepatocytes according to the method of Meredith and Reed (10). Essentially, the hepatocyte incubation suspension was treated with digitonin (0.2 mg/ml) to disrupt the cell membrane, and in a 1.9-ml microcentrifuge tube, 600 pl of the disrupted cell suspension was layered on 500 ~1 of dibutyl phthalate which had been placed above 600 ~1 of either 10% perchloric acid or 40% glycerol. After incubation at room temperature for 2 min, centrifugation of the tube at 13,OOOg at room temperature yielded rapid, quantitative sedimentation of intact mitochondria into the bottom layer while cytoplasmic components remained above the dibutyl phthalate. Enzyme assays for glutamate dehydrogenase (GDH) and LDH were performed in the glycerol layer while GSH was determined in the perchloric acid layer. Complete recovery of mitochondria was verified by measuring GDH activity, a mitochondrial enzyme, in the top layer. Cytosolic contamination of the bottom layer was assessed by measuring LDH activity in the bottom layer and the extent of contamination was negligible. Assays. GDH was assayed according to Frieden (25). Glutathione was determined according to the enzymatic cycling method using glutathione reductase and 5,5’-dithiobis(2-chloroethyl)nitrobenzoic acid (DTNB) as described by Akerboom and Sies (27). This method measures both reduced and oxidized glutathione and will be referred to in the text as “GSH” because the hepatocyte concentration of GSSG is negligible (1). PSH were determined according to the method described by Bellomo et al. (16). Briefly, DTNB was added to an aliquot of hepatocyte incubation suspension which had been resuspended in 10 mM Tris-HCl buffer, pH 7.4, containing 0.9% NaCl, precipitated with 6.5% trichloroacetic acid, and finally resuspended in 0.5 M Tris-HCl buffer, pH 7.4. Absorbance at 412 nm was measured as an estimate of PSH and quantitated using a standard curve generated using GSH as the species reacting with DTNB. Cytosolic Ca*+ was determined by measuring the activity of phosphorylase a as described by Hue et al. (28). Briefly, the hepatocyte incubation suspension was disrupted with digitonin (0.2 mg/ml) and an aliquot of cytosol was incubated with phosphorylase a substrates under conditions which favored the liberation of inorganic phosphate. The reaction was stopped at 0,30, and 60 min and the amount of inorganic phosphate released was measured according to LeBel et al. (29). Protein was determined according to the methods of Lowry et al. (30). Statistical analysis. Data were expressed as the means and standard errors of the mean. Statistical

FORMALDEHYDE evaluations between two-tailed Student’s used as the criterion

185

TOXICITY

sample means were made by the t test. In all cases, P < 0.05 was of significance. RESULTS

Mitochmdrial

GSH

Figure 1 shows the effect of CHzO on cytosolic GSH, mitochondrial GSH, and cell viability after 60 min of incubation. CHzO (0.0 to 7.5 mM) depleted cytosolic GSH but had no effect on mitochondrial GSH. At these CHzO concentrations, no cellular toxicity was produced as determined by leakage of the cytosolic enzyme LDH. Figure 2 shows the effect of DEM on cytosolic GSH, mitochondrial GSH, and cell viability after 60 min of incubation. DEM (0.0 to 2.5 mM) depleted cytosolic GSH as well as mitochondrial GSH. At 2.5 mM DEM, cytosolic GSH and mitochondrial GSH were 20 and 25% of control, respectively. No loss in cell viability was detected at these concentrations. At higher concentrations of DEM (up to 20 mM), cellular GSH was undetectable, which indicates

0.0 2.5

IC3-4201,

5.0

7.5

mM

FIG. 1. Effect of CH,O on cytosolic GSH (m), mitochondrial GSH (O), and cell viability (A) after 60 min of incubation. Each point represents the mean + SE of three experiments. The 100% values are as follows: cytosolic GSH: 21 f 2.0 nmol/lO”cells; mitochondrial GSH: 2.9 -t 0.4 nmol/106 cells; cell viability: as determined by leakage of LDH after Triton X-100 treatment.

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BILLINGS

centrations of CH20 and DEM that were slightly below lethal concentrations, neither CHzO (7.5 mM) nor DEM (20 mM) depleted PSH. The combination of CH20 (7.5 mM) and DEM (1 mM) or acrolein (2 mM) after 30 min of incubation depleted PSH significantly without causing toxicity. These treatments caused a further decrease in PSH and significant toxicity after 60 min of incubation. Phosphorylase

0 0.0

0.5

1.0 [DEMI,

1.5 2.0 mM

2.5

FIG. 2. Effect of DEM on cytosolic GSH (m), mitochondrial GSH (a), and cell viability (A) after 60 min of incubation. Each point represents the mean f SE of three experiments. The 100% values are as follows: cytosolic GSH: 22 f 1.6 nmol/106 cells; mitochondrial GSH: 2.8 f 0.3 nmol/lO’ cells; cell viability: as determined by leakage of LDH after Triton X-100 treatment.

that cytosolic as well as mitochondrial GSH was essentially completely depleted. Even under these conditions, no toxicity was observed (data not shown). The effect of CH20 in DEM-pretreated hepatocytes on cytosolic GSH, mitochondrial GSH, and cell viability is shown in Fig. 3. CHaO caused a further depletion of cytosolic GSH beyond the level depleted by DEM alone. There was a progressive loss in cell viability with increasing CHzO concentrations and the extent of cell death was much greater in DEM-pretreated cells than in cells not pretreated with DEM (Fig. 1). Figure 4 shows the effect of acrolein (3 mM) on cytosolic GSH, mitochondrial GSH, and cell viability. Acrolein was found to deplete cytosolic GSH and mitochondrial GSH substantially within the first 30 min of incubation. Cell death was observed after 60 min of incubation; it was almost complete after 90 min of incubation. Protein

SuljTzydryls

Table I shows the effect of various treatments on PSH and cell viability. At con-

a Activity

The effect of various treatments on phosphorylase a activity is shown in Table II. DEM (1 or 5 mM) had no effect on phosphorylase a activity whereas CHBO (7.5 mM) or acrolein (1 mM) significantly stimulated phosphorylase a activity. None of these treatments were cytotoxic. The quantitative relationship between percentage increase in phosphorylase a activity and increase in cytosolic Ca2’ is unknown; therefore, it is not possible to infer

0.0

2.5 104201,

5.0

7.5

mM

FIG. 3. Effect of CHaO on cytosolic GSH (D), mitochondrial GSH (O), and cell viability (A) in DEMpretreated hepatocytes after 60 min of incubation. DEM (1 mM) was added 10 min prior to CHaO addition. Each point represents the mean f SE of three experiments. The 100% values are as follows: cytosolic GSH: 6.9 + 0.6 nmol/106 cells; mitochondrial GSH: 1.9 f 0.3 nmol/lO’ cells; cell viability: as determined by leakage of LDH after Triton X-100 treatment.

GLLJTATHIONE

DEPLETION

AND

FORMALDEHYDE

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TOXICITY TABLE

II

EFFECT OF CHaO, DEM, AND ACROLEIN ON PHOSPHORYLASE a ACTIVITY IN ISOLATED RAT HEPATOCYTES % Phosphorylase a activity

Treatment Untreated CHaO (7.5 mM) DEM (1 mM) DEM (5 mM) Acrolein (1 mM)

0

30

60

90

MINUTES

FIG. 4. Effect of acrolein on cytosolic GSH (m), mitochondrial GSH (O), and cell viability (A). The concentration of acrolein was 3 mM. Each value represents the mean + SE of four experiments. The 100% values are as follows: cytosolic GSH: 33 f 7 nmol/106 cells; mitochondrial GSH: 1.8 f 0.6 nmol/106 cells; cell viability: as determined by leakage of LDH after Triton X-100 treatment.

100 146 + 93 * 107 + 146 +

6* 8 7 4*

Note. Phosphorylase a activity was determined after 30 min of incubation. The 100% value for phosphorylase a activity in untreated cells was 0.062 U/lo6 cells/min. Each point represents the mean + SE of three experiments. * Statistically significant (P < 0.05) from untreated control.

from the data in Table II that CHBO and acrolein have equivalent effects on cytosolic Ca2+ concentrations. DISCUSSION

TABLE

I

EFFECT ON CHaO, DEM, CHaO + DEM, ACROLEIN ON PROTEIN SULFHYDRYLS AND CELL VIABILITY

Treatment Untreated CHaO (7.5 mM) DEM (20 mM) CHaO (7.5 mM) + DEM (1 mM)-30 min CH,O (7.5 mM) + DEM (1 mM) Acrolein (2 mM)-30 min Acrolein (2 mM)

% PSH

AND

% Viability

99 2 3 103 f 4 94 t 3

94f 92f 92f

1 2 1

85 + 4*

94*

1

67 3~ 5* 81 f 4* 73 f 4*

15 * lo* 94f 1 85 + 3*

Note. Unless otherwise indicated, incubations were terminated after 60 min. In experiments involving CHaO and DEM, DEM was added 10 min prior to CHaO. The 100% value for PSH, determined at t = 0 min, was 53 + 2.5 nmol/mg protein. The 100% value for cell viability was determined by leakage of LDH after Triton X-100 treatment. Each value represents the mean + SE of three experiments. * Statistically significant (P < 0.05) from untreated control.

The enhanced toxicity of CH20 in GSHdepleted hepatocytes is apparently not due to an effect of CH20 on mitochondrial GSH. CH20 had no effect on mitochondrial GSH either in cells treated with CH20 alone or in cells pretreated with DEM. Moreover, the results suggest that depletion of mitochondrial GSH is not necessarily associated with cell death. DEM at a concentration of 2.5 mM depleted mitochondrial GSH to 25% of control without causing toxicity. In fact, depletion of cellular GSH per se may not be sufficient to cause cell death since higher concentrations of DEM depleted cellular GSH to undetectable levels and no toxicity was observed. Conversely, the loss of PSH which occurs with acrolein and the combination of CH20 and DEM are correlated with toxicity. A significant loss of PSH preceded the loss of cell viability after the addition of 2 mM acrolein or after the addition of CHzO to cells pretreated with 1 mM DEM. Thus, CH20 toxicity in DEM-pretreated hepatocytes appears to be due to oxidation of critical protein sulfhydryl groups.

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Nicotera et al. (20) have associated the loss of critical PSH with inactivation of Ca2+-dependent plasma membrane ATPase. Inactivation of this enzyme results in the loss of Ca2+ homeostasis and increased cytosolic Ca2+ levels. They have shown that agents such as menadione (2-methyl-1,4-naphthoquinone) and t-butyl hydroperoxide cause substantial depletion of cellular GSH, followed by depletion of PSH and an increase in cytosolic Ca2+. Our results with acrolein and with DEM are consistent with this hypothesis. DEM does not alter PSH and thus does not alter cytosolic Ca2+ and is not cytotoxic whereas both of these events occur with acrolein, which is cytotoxic. The combination of CHzO and DEM also causes substantial GSH depletion, followed by PSH depletion. However, CHzO alone increases cytosolic Ca2+ but it does not alter PSH. This indicates that an increased level of cytosolic Ca2+ without corresponding changes in PSH does not itself lead to cell death. The mechanism by which CH20 increases cytosolic Ca2+ is unknown, but it is apparently unrelated to significant loss of PSH. One possible mechanism is by inhibition of mitochondrial oxidative phosphorylation (31) which interferes with ATP production and impairs the ability of mitochondria to sequester Cazf (18). Mitochondria avidly regulate cytosolic Ca2+. It appears, however, that the increased cytosolic Ca2+ level caused by CH20 (as measured by enhanced phosphorylase a activity) is insufficient to cause cell death. Instead, cell death is more closely associated with changes in PSH. More direct, quantitative measures of cytosolic Ca2+ concentrations are required to more accurately define the relationship between these two effects. The ability of menadione and t-butyl hydroperoxide to exert an oxidative stress, i.e., accumulation of oxygen free radicals, is well documented. Acrolein can be metabolized to highly reactive species such as the epoxide, glycidaldehyde, and perhaps to other reactive species which may stimulate lipid peroxidation (32). The mechanism by which CH20 in DEM-pretreated hepatocytes may cause free radical accumulation and stimulate lipid peroxidation

BILLINGS

(1) is not known. CH20 may stimulate free radical accumulation weakly. In cells with sufficient GSH, these radicals would be adequately sequestered. Thus, CH20 alone does not deplete PSH. In DEM-pretreated hepatocytes, there is insufficient GSH to sequester these free radicals generated in the presence of CH20, PSH is depleted, and lipid peroxidation ensues. In support of this sequence is the temporal relation of these effects. Changes in PSH precede cell death (Table I) and it has also been observed that significant lipid peroxidation requires 60 min of CH20 incubation in DEM-treated cells, which coincides with the time of cell death (1). DEM itself apparently does not stimulate the accumulation of free radicals, which would explain why, although a potent depleter of cellular GSH, it does not alter PSH. Apparent differences in the toxicity of CH20 in DEM-treated cells between experiments (e.g., Table I and Fig. 3) are due to the steepness of both the time course and concentration curve (1). For example, cellular viability decreases from about 50 to 15% in the 60 to 90 min time period. In summary, CH20 toxicity in DEM-pretreated hepatocytes is not associated with mitochondrial GSH depletion but it is associated with depletion of PSH. The effects of acrolein are also consistent with the hypothesis that decreased levels of PSH lead to cell death. The loss of PSH apparently leads to altered function of critical proteins. The critical protein(s) may be Ca2+dependent ATPases which result in increased cytosolic Ca2+ levels. However, increased Ca2+ levels caused with CH20 by a different mechanism do not lead to cell death. These results suggest that other sulfhydryl proteins may also function in maintaining cellular integrity. The nature of these proteins requires further investigation. The combination of CH20 and DEM may be a useful model for identifying these critical proteins. ACKNOWLEDGMENTS This Health

work was supported by National Grants ES2868 and ES7090.

Institutes

of

GLUTATHIONE

DEPLETION

AND

FORMALDEHYDE

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