ARCHIVES

OF BIOCHEMISTRY

Vol. 282, No. 1, October,

AND

BIOPHYSICS

pp. 26-33,

1990

Modulation of Benzoquinone-Induced Cytotoxicity Diethyldithiocarbamate in isolated Hepatocytes’ Vbronique

V. Lauriault,

Larry

Faculty of Pharmacy,

University

Received

and in revised

March

2,1990,

G. McGirr,

of Toronto,

form

April

Wilson

Toronto,

W. C. Wong,

Ontario,

20,199O

Inc.

Quinones are widely distributed in nature and play essential biological roles in mitochondrial respiration, 1 Supported by the Medical Research Council of Canada. ’ To whom correspondence should be addressed at Faculty macy, 19 Russell Street, University of Toronto, Toronto, Canada M5S 1Al.

26

and Peter J. O’Brien2

Canada

The copper-chelating thiol drug diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced alkylation cytotoxicity by reacting with benzoquinone and forming a conjugate which was identified by fast atom bombardment mass spectrometry as 2-(diethyldithiocarbamate-S-yl) hydroquinone. In contrast to benzoquinone, the conjugate was not cytotoxic to isolated hepatocytes. The thiol reductant dithiothreito1 had no effect on benzoquinone-induced alkylation cytotoxicity. However, inactivation of catalase in the hepatocytes with azide and addition of the reducing agent ascorbate markedly enhanced the cytotoxicity of the conjugate but did not affect benzoquinone-induced cytotoxicity. Furthermore, inactivation of glutathione reductase and catalase in hepatocytes greatly enhanced the cytotoxicity of the conjugate and caused oxidation of GSH to GSSG. The conjugate also stimulated cyanide-resistant respiration, which suggests that the conjugate undergoes futile redox cycling resulting in the formation of hydrogen peroxide which causes cytotoxicity in isolated hepatocytes only if the peroxide detoxifying enzymes are inactivated. Diethyldithiocarbamate does, however, protect uncompromised isolated hepatocytes from benzoquinone cytotoxicity by conjugating benzoquinone, thereby preventing the electrophile from alkylating essential macromolecules. Diethyldithiocarbamate therefore changed the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress, which was less toxic. o isgo Academic Press.

by

of PharOntario,

photosynthesis, and prothrombin synthesis (1). On the other hand, quinones also occur as compounds ofpotential toxicological significance in foodstuffs and environmental pollutants (1) and have been used as anticancer drugs such as diaziquone (an aziridinylbenzoquinone), doxorubicin, and daunorubicin. (2). Quinones may exert their toxicity by direct interaction with cellular nucleophiles such as glutathione and other sulfhydryl-containing proteins (2-4), which results in the formation of conjugates (5-7). Quinones may also exert their toxicity by their ability to undergo oneelectron reduction at the expense of cellular reducing equivalents, a process known as redox cycling, leading to the formation of semiquinone radicals which may activate oxygen (6,8,9). The hydrogen peroxide formed oxidizes intracellular GSH via glutathione peroxidase and may cause oxidative stress by oxidizing essential macromolecules (9). Acetaminophen and bromobenzene may cause liver necrosis as a result of covalent binding to essential macromolecules (10,ll). The parent molecule, 1,4-benzoquinone (BQ),3 is thought to be the ultimate carcinogen of the industrial solvent and gasoline component, benzene, a leukemogen and myelotoxin which is thought to be metabolized to phenol and hydroquinone by the liver. The hydroquinone may then undergo activation by oxidation to benzoquinone by prostaglandin synthetase and/or myeloperoxidase in the bone marrow, the primary target organ (12). Benzoquinone redox cycles, albeit poorly, with either purified NAD(P)H cytochrome P450 reductase, NADH cytochrome b5 reductase (8), or isolated hepato-

3 Abbreviations used: DEDC, diethyldithiocarbamate; DS, disulfiram; DTT, dithiothreitol; BQ, benzoquinone; DETAPAC, diethylenetriamine pentaacetic acid; Asc, ascorbate; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; AZ, azide; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; FAB, fast atom bombardment; GSH, reduced glutathione; GSSG, oxidised glutathione; ESR, electron spin resonance.

0003.9861/90 $3.00 CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

MODTJLATION

OF

BENZOQUINONE

CYTOTOXICITY

cytes (13). However, Rossi et al. (14) demonstrated that the mechanism of benzoquinone-induced toxicity in isolated hepatocytes results from the alkylation of cellular macromolecules and not oxidative stress. Diethyldithiocarbamate (DEDC) is a copper-chelating thiol drug which has been found to protect mice in uiuo against acetaminophen-induced hepatotoxicity (15). DEDC was also shown to protect isolated hepatocytes against N-acetyl-p-benzoquinoneimine, the highly reactive electrophilic compound (16). The protective mechanism of DEDC against N-acetyl-p-benzoquinoneimine was attributed to the formation of a nontoxic conjugate with DEDC (16). Diethyldithiocarbamate has also been reported to protect mice in uiuo against bromobenzene (15), whose cytotoxic metabolites include a bromobenzoquinone (17-19). In the present study, DEDC was found to protect isolated hepatooytes from benzoquinone by forming a nontoxic BQ:DEDC conjugate, thus preventing irreversible alkylation of GSH and of protein thiols by the benzoquinone. However, if catalase or GSH reductase was inactivated, the isolated hepatocytes became susceptible to the conjugate although their susceptibility to benzoquinone was unaffected. Diethyldithiocarbamate therefore changed the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress, which was less toxic. MATERIALS

AND

METHODS

Diethyldithiocarbamate, disulfiram, Trypan blue, fluoro-2,4-dinitrobenzene, iodoacetic acid, GSH, GSSG, sodium azide, and DETAPAC (diethylenetriamine pentaacetic acid) were obtained from Sigma Chemical Co. (St Louis, MO). 1,4-Benzoquinone (BQ) was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WS). BCNU (1,3-(bis(2chloroethyl)-1-nitrosourea) was a gift from Dr Rauth from the Ontario Cancer Institute. Collagenase (from Clostridium histoliticum), Hepes, and bovine serum albumin (BSA) were obtained from BoeringerMannheim (Montreal, Canada). All other chemicals used were of the highest purity that was commercially available. Adult male SpragueeDawley rats (180-200 g) fed ad libitum were used to prepare hepatocytes. Isolated hepatocytes were prepared by collagenase perfusion of the liver as previously described (20). Cells were suspended in round-bottomed flasks continuously rotating in a water bath at 37°C in Krebs-Henseleit bufIer, pH 7.4, supplemented with 12.5 mM Hepes under a gas stream of 95% 0, and 5% CO,. The final incubation volume was 10 ml at a concentration of 10fi cells/ml. Cell viability was determined by measuring the uptake of Trypan blue (final concentration: 0.16%, w/v). DEDC (100 mM) was dissolved in the incubation buffer (pH 7.4) just before use whereas BQ was dissolved at a concentration of 10 mM in DMSO. A stock of BQ:DEDC conjugate was prepared by eluting the conjugate by HPLC as described later. A stock of BQ:DEDC conjugate (10 mM) was dissolved in DMSO and was added to isolated hepatocytes after 30 min of preincubation. DMSO (up to 1%) was not toxic to isolated hepatocytes. The addition of 4 m&f azide to isolated hepatocytes inactivated >98% of the oxygen release observed on addit,ion of 50 pM hydrogen peroxide to hepatocytes plus azide. The total amount of GSH and GSSG in isolated hepatocytes was measured in deproteinized samples (5% metaphosphoric acid) after

BY

27

DIETHYLDITHIOCARBAMATE

derivatization with iodoacetic acid and Auoro-2,4-dinitrobenzene (21). Analysis was carried out by HPLC using a PBondapak NH, column and a Water 6000A solvent delivery system, equipped with a Model 660 solvent programmer, a Wisp 710A automatic injector and a Data Module (Water Associates, Milford, MA). GSH and GSSG were used as external standards. The formation of the BQ:DEDC conjugate was monitored spectrophotometrically using a Beckman DU-7 spectrophotometer. Incubations were carried out in 1.5 ml 0.1 M phosphate bufferpH 6.0 containing 100 pM DEDC and 100 pM BQ in 1% DMSO. A quantitative analysis of the BQ:DEDC conjugate was carried out by HPLC (Beckman 421 A controller, 1lOB solvent delivery module). DEDC (100 PM) and BQ (100 FM) were combined in phosphate buffer (0.1 M at pH 6.0) and the reaction mixture was analyzed by HPLC using a C 18 PBondapak reverse phase column (0.3 mm X 30 cm) eluted at a flow rate of 1 ml/min with a linear gradient system of methanol/ water (lo:90 to 1OO:O) over 20 min (uv, 300 nm). Large scale isolation of the BQ:DEDC conjugate was carried out using HPLC (Beckman 421 A controller, 1lOB solvent delivery module) with a Whatman Partisil M9 lo/50 ODS reverse phase column (9.4 mm X 50 cm). BQ (10 mM) and DEDC (10 mM) were reacted in 0.1 M phosphate buffer, pH 6.0. Samples were injected onto the column and eluted at a flow rate of 3 ml/min with an isocratic solvent system of 60% methanol and 40% water. The conjugate was eluted with a retention time of 14.1 min. Methanol was removed from pooled samples with nitrogen and the sample lyophilized and stored at ~20°C under nitrogen for mass spectrometry analysis. Mass spectra were recorded using a VG Analytical ZAB-IF instrument equipped for fast atom bombardment (FAB). For FAB analysis, compounds were dissolved in glycerol and placed directly on the probe. Oxygen consumption was measured with a Clark oxygen electrode (Yellow Springs Instrument Co., Model 53) with and without isolated hepatocytes in a 2.0.ml chamber at 37°C. Before use, hepatocytes (lOfi cells/ml) were maintained under preincubation conditions, in the Hepes-supplemented Krebs-Henseleit buffer, pH 7.4. KCN (1 mM) was added to inhibit mitochondrial respiration. Other additions are as indicated in the figure legends.

RESULTS Effect of Diethyldithiocarbamate Induced Cytotoxicity

on Benzoquinone-

The permeable thiol drug diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced cytotoxicity. As shown in Fig. lA, hepatocytes treated with 100 yM benzoquinone resulted in 50% hepatocyte cytotoxicity after 30 min of incubation as determined by Trypan blue uptake. However, in the presence of 1 mM DEDC, isolated hepatocytes were protected from BQ (100 PM)-induced cytotoxicity. DEDC (5 mM) was not toxic to isolated hepatocytes (Fig. 1A). Inactivation of hepatocyte catalase with azide did not cause any hepatocyte cytotoxicity and had no effect on alkylation-induced cytotoxicity of BQ. However, in the presence of increasing concentrations of DEDC, BQ-induced cytotoxicity was markedly enhanced as shown in Fig. 1B. Similarly, when hepatocyte catalase was inactivated by obtaining the hepatocytes from cyanamide-treated rats (22) susceptibility to BQ was unaffected but was enhanced if DEDC was present (results not shown).

28

LAURIAULT

ET

AL.

100 1

100 g

s ij

so-

i

80

g

60-

23

60

sd

40-

d

8

20-

g

0

!3 it

5 i

.,.,',.,.,. 0

30

I', 60

90

TIME

120

150

180

40

*O 0

210

(min)

0

30

60

90

TIME

120

150

180

(min)

FIG. 1. (A) Protection against benzoquinone-induced cytotoxicity by diethyldithiocarbamate in isolated rat hepatocytes (10” cells/ml). Cytotoxicity was monitored by Trypan blue uptake at various time points. Hepatocytes were incubated with the following: BQ (100 PM) (m), DEDC (5 mM) (A), DEDC (1 mM) + BQ (100 PM) (0) and no additions (X). (B) Enhancement of benzoquinone-induced cytotoxicity by diethyldithiocarbamate in catalase-inactivated rat hepatocytes. Cytotoxicity was monitored by Trypan blue uptake at various time points. Hepatocytes (10” cells/ml) were incubated with the following: AZ (4 mM) + BQ (75 wM) (O), AZ (4 mM) + BQ (75 pM) + DEDC (1 mM) (m), AZ (4 mM) + BQ (75 FM) + DEDC (250 pM) (0), AZ (4 mM) + BQ (75 pM) + DEDC (150 FM) (0) and no additions (X). Values represent averages of three separate experiments with error bars representing the standard deviations.

Isolation and Identification Conjugate

of the Benzoquinone:DEDC

In order to understand the modulation of benzoquinone cytotoxicity by DEDC, the reaction of DEDC with benzoquinone was studied. As shown in Fig. 2A. benzoquinone (100 PM) reacted with diethyldithiocarbamate (100 PM) in

3; 0.0 L 0 200

, 500

300

Wavelength

(nm)

III1lll’r’ 2421

-4-i 18 15 12 Time (mid

9

6

3

'niect

FIG. 2. (A) Spectra of benzoquinone:diethyldithiocarbamate conjugate versus benzoquinone and DEDC. (- -) BQ (100 fiM); (- -) DEDC (100 PM); (-) time 1 min, BQ:DEDC conjugate (100 PM). (B) HPLC elution profile of the products of the reaction of 1,4-benzoquinone with DEDC. Chromatographic conditions consisted of a linear gradient of methanol/water (lo:90 to 1OO:O) at a flow rate of 1 ml/min over 24 min. Peak 1, hydroquinone, 6.0 min; peak 2, 1,4-benzoquinone, 8.4 min; peak 3,2-(diethyldithiocarbamate-S-yl) hydroquinone, 14.1 min; peak 4, disulfiram, 20.4 min; peak 5, diethyldithiocarbamate, 23.4 min.

0.1 M phosphate buffer, pH 6.0, resulting in a decrease in DEDC absorbance (at 280 nm) and benzoquinone absorbance (at 245 nm). The product was formed with absorbance maxima at wavelengths of 290 and 320 nm. Analysis of the pH 6.0 reaction mixture (BQ( 100 PM) with DEDC(lOO PM) in 0.1 M phosphate buffer) using HPLC indicated the formation of a single compound with a retention time of 14.1 min (uv, 300~rim) (Fig 2B). Under the described conditions, the yield of the BQ: DEDC conjugate formed by this reaction was 99 k 1%. Under these conditions 1 eq of DEDC was needed to completely form the BQ:DEDC conjugate. However, further addition of BQ (100 PM) 1 min after the reaction of BQ (100 PM) with DEDC (100 PM) caused the conjugate peak to decrease in size and hydroquinone was formed and it was suggested that the conjugate reduces BQ to hydroquinone, which had a retention time of 6.0 min. As shown in Fig. 3, FAB mass spectral analysis of the isolated product yielded a base peak with a m/e of 258 corresponding to the (M + 1) ion; a m/e at 280 (M + Nat) and a fragment at a m/e of 224 corresponding to a (M + l-H&S) ion. This was consistent with the structure of 2-(diethyldithiocarbamate-S-yl) hydroquinone. Thus the product formed with absorbance maxima at wavelengths of 290 and 320 nm was the reduced form of the BQ:DEDC conjugate. 2-(diethyldithiocarbamate-S-yl)hydroquiFurthermore, none was also isolated from hepatocytes treated with benzoquinone and DEDC (data not shown). Comparison of the Effects of Benzoquinone and BQ: DEDC Conjugate on Hepatocyte Viability and GSH The comparison of the cytotoxicity of BQ:DEDC conjugate versus BQ was studied in order to determine

MODULATION dy+l 93

lOOI 5oj 1 : 57 0: I’

?!?a .I

OF gly+Na+ ‘15/1

I A.1

BENZOQUINONE

CYTOTOXICITY

2gly + 1 155 2gly+Na+

18

.I

,...,,

BY

29

DIETHYLDITHIOCARBAMATE

M+l 258

207

224

,I

.,.

..,I

M+Na+ 280 I. ..!I%.

.I.

M+Na++ 372 I!.

gly

Mw257

FIG.

3.

FAB

mass spectra

of isolated

benzoquinone:diethyldithiocarbamate

whether the conjugation of DEDC to BQ was the reason for the protection exhibited by DEDC in Fig. 1A. In comparison to BQ-induced cytotoxicity, the BQ:DEDC conjugate (100 PM) was not toxic to isolated hepatocytes. The thiol reductant dithiothreitol (10 mM) did not prevent subsequent benzoquinone-induced alkylation cytotoxicity when added 3 or 10 min after benzoquinone (Fig. 4). Furthermore, as shown in Fig. 4, the cytotoxicity induced by benzoquinone markedly increased with benzoquinone concentration. On the other hand, the BQ: DEDC conjugate, which was approximately fourfold less toxic, was less affected by increasing the concentration.

00 0

30

60

90

120

150

180

210

Time (min) FIG. 4. Comparison of cytotoxicity between BQ (100 FM) and the BQ:DEDC conjugate (100 KM) in isolated hepatocytes. Cell viability was measured by determining Trypan blue uptake at various time points. Hepatocytes ( lo6 cells/ml) were incubated with the following: BQ (50 pM) (W), BQ (100 pM) (O), BQ (100 pM) + DTT (10 mM) added at 10 mins (O), BQ:DEDC conjugate (100 PM) (A), BQ:DEDC conjugate (200 PM) (O), and no addition (X). Values represent averages of three separate experiments with error bars representing the standard deviations.

conjugate.

In order to determine the molecular mechanism of protection by DEDC against BQ cytotoxicity, the effect of the conjugate on intracellular glutathione levels was investigated. BQ (50 pM) rapidly depleted GSH in isolated hepatocytes (Figs. 5A and 5B), which did not recover throughout the course of the experiment, indicating that the depletion of cellular GSH in isolated hepatocytes was likely due to GSH conjugation. The BQ: DEDC conjugate (50 PM), on the other hand, had no effect on GSH or GSSG levels. Oxidative Stress Cytotoxicity Induced by the BQ:DEDC Conjugate in Hepatocytes Deficient in Hydrogen Peroxide Detoxifying Enzymes Since menadione was found to form a conjugate with GSH which could cause reductive oxygen activation as a result of redox cycling (6), it was of interest to determine whether the BQ:DEDC conjugate was cytotoxic to hepatocytes deficient in hydrogen peroxide detoxifying enzymes, thereby explaining the potentiation of benzoquinone cytotoxicity by DEDC in such hepatocytes. Preincubating hepatocytes with azide (4 mM) caused almost complete inhibition of catalase activity, which had no effect on BQ-induced cytotoxicity (14,22) but markedly enhanced the cytotoxicity of the conjugate as shown in Fig. 6A. This suggests that the BQ:DEDC conjugate permeates the hepatocyte and catalyzes cytotoxic hydrogen peroxide formation in isolated hepatocytes. Furthermore, addition of the reducing agent ascorbate (2.5 mM) markedly enhanced the cytotoxicity induced by the conjugate in catalase-inactivated hepatocytes to such an extent that the conjugate was now cytotoxic at 5 pM, i.e., one-tenth of the concentration in the absence of ascorbate (Fig. 6A). By contrast, ascorbate did not affect the cytotoxicity of benzoquinone in catalase-inactivated hepatocytes. Ascorbate alone was also not cytotoxic in these hepatocytes. Inactivation of glutathione

30

LAURIAULT

-d 8o“i

i5 (mill)

1 TIME

io

ET

AL.

~-60

conjugate on hepatocyte GSH (A) and GSSG (B) levels. Hepatocytes (lo6 cells/ml) were incubated with FIG. 5. Effect of BQ and BQ:DEDC the following: BQ (50 PM) (m), BQ:DEDC conjugate (50 pM) (0) and BCNU (50 FM) + azide (4 mM) + BQ:DEDC conjugate (50 fiM) (A). GSH and GSSG levels were determined by the Reed method as described under Materials and Methods. Values represent averages of three separate experiments with error bars representing the standard deviations.

reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (50 for 30 min prior to the addition of the BQ:DEDC conjugate (50 PM) also markedly increased the cytotoxicity without causing cytotoxicity in the absence of the conjugate (Fig. 6B). Furthermore, inactivation of both peroxide detoxification enzymes, catalase and glutathione reductase, further enhanced the cytotoxicity of the BQ:DEDC conjugate as shown in Fig. 6B without causing cytotoxicity in the absence of the conjugate. Since intracellular GSH plays an important role in reducing hydrogen peroxide (20), the extent to which oxidative stress occurs in the cell by the conjugate was investigated by measuring GSSG levels formed. When the PM)

conjugate was added to hepatocytes, in which catalase was inactivated with azide, GSH was oxidized to GSSG by the BQ:DEDC conjugate (50 PM) as shown in Figs. 5A and 5B, thereby showing that the conjugate enters the hepatocyte. Furthermore, when hepatocyte glutathione reductase was inactivated with BCNU (50 PM) the conjugate also caused oxidation of hepatocyte GSH to GSSG. In the absence of conjugate, azide had no effect on hepatocyte GSH levels, whereas BCNU depleted hepatocyte GSH by 47 + 4%. Further evidence showing that oxygen was not required for benzoquinone cytotoxicity was obtained when the 95% 02/5% CO, atmosphere was replaced with 95%

B

o! 30

60

90

120

150

TIME

(mid

160

210

240

270

. , . , . , . , . , . , . , . , . , 0

30

60

90

120

150

160

210

240

270

TIME (min)

FIG. 6. (A) Effect of the inactivation of catalase by azide and the catalysis of redox cycling by ascorbate on the cytotoxicity of the BQ:DEDC conjugate. Cytotoxicity was monitored by Trypan blue uptake at various time points. Hepatocytes (lo6 cells/ml) were incubated with the conjugate (50 pM) (A), azide (4 mM) + BQ:DEDC conjugate (5 FM) following: BQ:DEDC conjugate (50 pM) (O), aside (4 mM) + BQ:DEDC + ascorbate (2.5 mM) at 10 min (w), and no addition (X). (B) Effect of the inactivation of glutathione reductase and catalase on the cytotoxicity of the BQ:DEDC conjugate. Cytotoxicity was monitored by Trypan blue uptake at various time points. Hepatocytes (lo6 cells/ml) were incubated with the following: BQ:DEDC conjugate (50 pM) (O), preincubation of hepatocytes with BCNU (50 pM) for 30 min + BQ:DEDC conjugate (w) and preincubation of hepatocytes with BCNU (50 pM) for 30 min + aside (4 mM) + BQ:DEDC conjugate (A) and no addition (X). Values represent averages of three experiments with error bars representing the standard deviations.

MODIJLATION

OF

BENZOQUINONE

CYTOTOXICITY TABLE

BQ:DEDC

Conjugate-Mediated

BY I

Oxygen Activation

with Hepatocytes

Oxygen

Additions

None

BQ (100 PM) (BQ(lOO

@M)

+ DEDC(lOO

PM))

+ DEDC(200

PM))

BQWO PM) (BQ(200

PM)

Uptake +Hepatocytes’ (nmol. mini’ 10s cells)

~Hepatocytes” ~Asc. 0.03 1.2 2.1 1.4 6.0

+ 0.01 fO.l f 0.3* + 0.1 t 0.8*

31

DIETHYLDITHIOCARBAMATE

+Asc. 0.03 1.3 8.4 1.6 73.6

i * -+ f f

+CN/+Asc.

+CN 0.01 0.1 0.8** 0.2 7.2**

1.4 1.9 2.9 1.4 5.4

f k * * k

0.1 0.3 0.5* 0.2 0.7*

1.4 1.9 9.6 1.8 78.6

f -+ + + k

0.2 0.3 l.l** 0.2 8.2**

Note. Oxygen uptake and cyanide-resistant respiration was measured using a Clark oxygen electrode. ’ Oxygen uptake was measured for BQ (100 and 200 PM) and BQ:DEDC conjugate (100 and 200 pM) with or without ascorhate (Asc.) (1 mM) using 2.ml of KrebssHenseleit buffer, pH 7.4, as medium in electrode at 37°C. * Isolated hepatocytes (10s cells/ml) were incubated (37°C) in Krebs-Henseleit buffer, pH 7.4, (2 ml). Cyanide (1 mM) was added in order to inhibit mitochondrial respiration. Oxygen uptake was measured for benzoquinone (100 PM) and for the BQ:DEDC conjugate (100 and 200 pM) with and without ascorbate (Asc.) (1 mM). The values are expressed as the means of three separate experiments (*SE). * Significantly different from controls (None), P < 0.05. ** Significantly different from controls, P < 0.01.

N2/5% COZ. Hepatocytes were preincubated for 30 min prior to the addition of benzoquinone to decrease the oxygen to approximately 0.5% as measured with a Clark oxygen electrode. The nitrogen atmosphere was maintained for the duration of the experiment. Control hepatocytes maintained their viability for at least 3 h. The susceptibility of these hypoxic hepatocytes to benzoquinone (100 PM) was similar to that found with oxygenated hepatocytes (data not shown).

oxygen uptake with the conjugate in the absence of hepatocytes. Furthermore, the metal chelator, DETAPAC, had no effect on the oxygen consumption of the conjugate (data not shown). Addition of catalase when the oxygen uptake was nearly complete resulted in the release of oxygen. Catalase present at the beginning of the reaction inhibited the rate of oxygen uptake by 43% (data not shown), indicating that 86% of the oxygen uptake was due to hydrogen peroxide formation.

Evidence of Oxygen Activation by the BQ:DEDC Conjugate but not by Benzoquinone

DISCUSSION

In order to determine whether the conjugate underwent futile redox cycling and oxygen activation the effectiveness of the conjugate at inducing cyanide-resistant respiration was investigated. As shown in Table I, in isolated hepatocytes, the conjugate stimulated cyanide-resistant respiration while BQ induced little cyanide-resistant respiration. Although cyanide is a strong nucleophile little reaction between the cyanide and benzoquinone occurred during the course of the respiration experiment. Indeed, 100 PM benzoquinone reacted only slowly with 1 mM cyanide at 37°C (only 46% reacted in 2 h). The formation of BQ:GSH conjugate by reacting benzoquinone with GSH (100 PM) was also not affected even by 10 mM cyanide. The addition of ascorbate markedly enhanced oxygen uptake by the conjugate, confirming that ascorbate catalyzed a futile redox cycling of the conjugate which resulted in oxygen activation. Ascorbate by itself did not stimulate cyanide-resistant respiration in isolated hepatocytes. Ascorbate also catalyzed a marked increase in

The present study investigated the mechanism of how the thiol drug, diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced cytotoxicity and yet enhanced cytotoxicity in hepatocytes whose catalase had been inactivated with azide or whose glutathione reductase had been inactivated with BCNU. The following evidence suggests that DEDC acted as a cell permeant thiol which formed a conjugate with benzoquinone. Furthermore, the conjugate underwent redox cycling and oxygen activation which was only cytotoxic in hepatocytes whose peroxide detoxifying enzymes had been inactivated. The addition and substitution reactions of quinones, which are electrophiles, with nucleophiles, particularly thiols and amino groups, have been extensively documented. The majority of quinones react with thiols by a nucleophilic Michael addition to form a hydroquinone conjugate (23-26). FAB mass spectrometry carried out on the BQ:DEDC conjugate isolated by HPLC shows that benzoquinone also undergoes a nonenzymatic conjugation with DEDC at the C-2-position to form the cor-

32

LAURIAULT

ET

AL. OXIDATIVE

0

Benzoquinone

FIG.

isolated

7.

Hypothetical hepatocytes.

scheme

describing

CYTOTOXICITY

Ascorbate

OH

2.(DEDC-Syl)

STRESS

hydroquinone

the reactive

pathway

and subsequent

responding hydroquinone; 2-(diethyldithiocarbamateS-yl) hydroquinone. Furthermore, in contrast to benzoquinone, the conjugate was not toxic to normal isolated hepatocytes. It was previously shown that alkylating quinones such as benzoquinone are highly toxic but that more substituted quinones such as tetramethylbenzoquinone (14) or 2,3dimethoxy-1,4-naphthoquinone (3) are not cytotoxic even though they readily caused cyanide-resistant respiration as a result of futile redox cycling and oxygen activation. The redox cycling quinones however are cytotoxic if the hepatocyte is compromised by the inactivation enzymes involved in peroxide metabolism such as catalase or GSH reductase (14). Since menadione forms a GSH conjugate, which can undergo futile redox cycling and oxygen activation (6), the ability of the BQ:DEDC conjugate to cause oxidative stress in hepatocytes was investigated. Inactivation of catalase by azide had no effect on benzoquinone-induced cytotoxicity as shown previously (14). However, inactivation of hepatocyte catalase enhanced the susceptibility of hepatocytes to BQ:DEDC conjugate-induced cytotoxicity, suggesting that the conjugate catalyses hydrogen peroxide formation in isolated hepatocytes. No cyanide-resistant oxygen uptake was observed after the addition of benzoquinone to isolated hepatocytes, indicating a lack of significant oxygen activation as a result of redox cycling. By contrast, the BQ:

oxidative

stress

cytotoxicity

of the BQ:DEDC

conjugate

in

DEDC conjugate stimulated cyanide-resistant oxygen uptake, indicating that the conjugate undergoes futile redox cycling and oxygen activation. Upon addition of the reductant ascorbate at 10 min, there was a marked increase in hepatocyte cytotoxicity and cyanide-resistant oxygen uptake by the BQ:DEDC conjugate. In the absence of hepatocytes, ascorbate also catalyzed the futile redox cycling of the conjugate and consequent oxygen activation but not that of benzoquinone. The GSSG formation in hepatocytes by the BQ:DEDC conjugate suggests that GSH oxidation by hydrogen peroxide was faster than GSSG reduction by GSH reductase. Inactivation of hepatocyte glutathione reductase with BCNU also enhanced GSH oxidation as well as BQ:DEDC conjugate cytotoxicity. Thus inactivation of either peroxide detoxication system, i.e., catalase or glutathione reductase, dramatically enhances the cytotoxicity of the BQ: DEDC conjugate. Benzoquinone rapidly depleted hepatocyte glutathione without oxidation and in agreement with previous findings (14), GSH levels did not recover. The thiol reductant dithiothreitol had no effect on benzoquinoneinduced cytotoxicity when added 3 min after benzoquinone. However, hepatocyte glutathione levels were only affected by the BQ:DEDC conjugate if catalase or glutathione reductase was inactivated. The conjugate then caused oxidation of hepatocyte GSH.

MODULATION

OF

BENZOQUINONE

CYTOTOXICITY

These results suggest that the hydroquinone conjugate reacts with molecular oxygen, forming superoxide anions which then dismutate enzymatically or spontaneously to hydrogen peroxide and oxygen (27). The first reaction step probably leads to an autoxidizable semiquinone molecule which then reacts with dioxygen to form another molecule of superoxide anion. Both reactions would lead to a fully oxidized conjugate (13,28,29). Takahashi et al. (7) found by ESR that the GSH conjugates of naphthoquinone form semiquinone radicals. The one-electron reduction of the oxidized conjugate would form the autoxidizable semiquinone radical conjugate and result in futile redox cycling. This reduction may be catalyzed by NAD(P)H-cytochrome P450 reductase, NADH-cytochrome b5 reductase and NADH-ubiquinone oxidoreductase (9,13). The two-electron reduction of certain quinones to their hydroquinone form may also be catalyzed by the flavoenzyme NAD(P)H:oxidoreductase (DT-diaphorase) (29). These mechanisms for futile redox cycling leading to hydrogen peroxide-mediated oxidative stress and toxicity are outlined in Fig. 7. The results presented here together with those presented previously with methyl-substituted benzoquinone derivatives (14) suggest that in the case of benzoquinone, cytotoxicity occurs as a result of extensive alkylation of essential proteins and not oxidative stress. Thus, benzoquinone or its GSH conjugate do not redox cycle enough in the isolated hepatocytes to cause oxidative stress cytotoxicity. In the presence of DEDC however, alkylation of essential macromolecules by benzoquinone is prevented and the cytotoxicity in hepatocytes is averted. The BQ:DEDC conjugate formed participates in redox cycling and oxygen activation leading to hydrogen peroxide formation and can cause cytotoxicity when hepatocyte peroxide detoxifying enzymes such as catalase and glutathione reductase are inactivated. Diethyldithiocarbamate, therefore, changes the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress which was less toxic.

Pang of the Carbohydrate for recording the FAB mass Rokeah Ladies Auxilliary for

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