THE JOURNAL OF BIOLOGICAL CHEMISTRY @ 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 32, Issue of November 15, pp. 2149621503,1991 Printed in V.S.A.

Effect of Hydroxy Substituent Position on 1,4-Naphthoquinone Toxicity to Rat Hepatocytes*

(Received for publication, May 1, 1991)

Karin Ollinger and Anders Brunmark From the Department of Pathology, University of Linkoping, S-581 85 Linkoping, Sweden

The effect of hydroxy substitution on 1,4-naphtho- quinone toxicity to cultured rat hepatocytes was stud- ied. Toxicity of the quinones decreased in the series 6,8-dihydroxy-1,4-naphthoquinone > S-hydroxy-1,4- naphthoquinone > 1,4-naphthoquinone > Z-hydroxy- 1,4-naphthoquinone, and intracellular GSSG forma- tion decreased in the order 6,8-dihydroxy-1,4-naph- thoquinone > S-hydroxy-l,4-naphthoquinone >> 1,4- naphthoquinone >> Z-hydroxy- 1,4-naphthoquinone. The electrophilicity of the quinones decreased in the order 1,4-naphthoquinone >> S-hydroxy-1,4-naph- thoquinone > 5,8-dihydroxy-1,4-naphthoquinone >> Z-hydroxy-l,4-naphthoquinone. Treatment of the he- patocytes with BSO (buthionine sulfoximine) or BCNU (1,3-bis-Z-chloroethyl-l-nitrosourea) increased S-hy- droxy-1,4-naphthoquinone and 6,8-dihydroxy-1,4- naphthoquinone toxicity, whereas neither BSO nor BCNU largely affected 1,4-naphthoquinone and 2-hy- droxy- 1,4-naphthoquinone toxicity. Dicumarol in- creased the toxicity of 1,4-naphthoquinone dramati- cally and somewhat the toxicity of Z-hydroxy-l,4- naphthoquinone, whereas S-hydroxy- 1,4-naphthoqui- none and 6,8-dihydroxy- 1,4-naphthoquinone toxicity increased only slightly. The toxicity of S,8-dihydroxy- 1 ,4-naphthoquinone decreased dramatically in re- duced O2 concentration, whereas 1,4-naphthoquinone, S-hydroxy-1,4-naphthoquinone, and Z-hydroxy-1,4- naphthoquinone toxicity was not largely affected. It was concluded that 6,8-dihydroxy-1,4-naphthoqui- none toxicity is due to free radical formation, whereas the toxicity of 1,4-naphthoquinone and of S-hydroxy- 1,4-naphthoquinone also has an electrophilic addition component. The toxicity of Z-hydroxy-1,4-naphtho- quinone could not be fully explained by either of these phenomena.

The quinoid structure is widespread in nature and the quinones encompass over 1200 naturally occurring com- pounds (1). Naturally occurring quinones have several differ- ent roles in organisms; they are functional constituents of several biochemical systems (e.g. ubiquinone and vitamin Kl), they are dyes, or they act as defensive compounds. Quinones find use as pharmaceutics (e.g. the anthracycline cytostatics) and they are environmental pollutants (2). The exposure of cells to quinoid compounds may lead to several deleterious consequences. Chronic exposure to quinones may be carcino- genic; several quinones are mutagenic in the Ames test (3,4), as well as to cultures of eucaryotic cells (5). Acute exposure

* This study was supported by Grant 2703-B91-01XAB from the Swedish Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to quinones may cause cell death. During the last decades considerable effort has been made

to understand the mechanism underlying the toxicity of qui- noid compounds. Two main features of quinones are now generally accepted as critical for their cytotoxicity: ( a ) the capacity of quinones to produce oxygen free radicals and ( b ) the electrophilicity of quinones, which enables them to form adducts to cellular constituents. The intracellular free radical production by quinones is thought to be a consequence of O2 reduction by semiquinone intermediates. The semiquinones are formed via one-electron reduction of the corresponding quinone catalyzed by, e.g. NADPH-cytochrome P150 reductase. This process, called redox cycling, generates 0; and Hz02 and may thereby cause oxidative stress. Quinones can also be reduced via a two-electron pathway catalyzed by DT-diaphor- ase. This normally results in formation of a more stable hydroquinone and, therefore, it is considered to be a detoxi- cation pathway. All the quinones studied here are substrates for both NADPH-cytochrome P450 reductase and DT-dia- phorase (6, 7).

The capacity of a quinone to produce oxygen free radicals is dependent on several interrelated chemical properties of the molecule, including the following. (a ) The reduction po- tential must be high enough to allow efficient reduction of the quinone by cellular reductases but not so high that it reduces the rate of electron transfer from the hydro- and semiquinone to 02. Good correlations between rate of electron transfer from hydro- or semiquinones and reduction potentials have been observed, in agreement with the Marcus equation (8,9). ( b ) The pKa value, especially that of the hydroquinone, is important since electron transfer is orders of magnitude higher for the anionic form of the reductant (10) and thus a high pK, value precludes the autoxidation of the hydroqui- none. Semiquinones generally have rather low pK, values, and therefore, the pKa value does not have to be considered for the rate of electron transfer at neutral pH. (c) Another parameter of interest is the stability constant of the semiqui- none (K8), which determines the position of disproportiona- tion equilibria (2 Q7 + 2H’ c, Q + QH2, where Q is quinone) and thereby the steady-state level of semiquinones, as well as the stability of hydroquinones. The electrophilicity, on the other hand, is mainly determined by the electron density at the carbons in the quinoid double bond (C2=C3). In studies of cell toxicity it has to be considered that a quinone that forms a stable hydroquinone will be hindered from electrophilic addition by reduction because the hydroquinone is not an electrophile.

The capacity to produce free radicals is dramatically influ- enced by the substituents present on the quinoid molecule. Table I lists some of the chemical properties that are impor- tant for the capacity of the quinones studied here to produce free radicals. The unsubstituted 1,4-naphthoquinone has an El7 value that allows rather efficient one-electron reduction.

21496

21497 Hydroxynaphthoquinones and Hepatocytes

TABLE I Reduction potentials, dissociation constants, and stability constants for unsubstituted and

hydroxy-substituted 1,4-naphthoquinones Index numbers refer to the source the value was obtained from.

Quinone Reduction potentials P K

El7 E, E " 7 SQ QHz PKS

mV

1,4-Naphthoquinone -140" +306" +83" 4.lZ7 9.329 -7.6b 2-Hydroxy-1,4-naphthoquinone -41531 +135" - 1 40" 4. 7" 9.OZ9 -9.3b 5-Hydroxy-1,4-naphthoquinone -9332 +311" +109' 3.632 -6.8b 5,8-Dihydroxy-1,4-naphthoquinone - 1 1 0 ~ 3 -1533 -6233 2.733 7.fY3 -1.6b

Calculated from C7 and El7 as described previously (27). Calculated from E', and 6 7 as described previously (27). Redox potential was measured as described under "Materials and Methods."

It has a rather low stability of the semiquinone form, a high pK, of the hydroquinone, and a high F7 value and, therefore, the hydroquinone form is quite stable. 2-Hydroxy-1,4-naph- thoquinone, on the other hand, has a very low El7 value. This is caused by stabilization of the quinoid form due to depro- tonation of the hydroxy group at position Cz and tautomeri- zation of the quinoid form and by donation of electrons from the deprotonized hydroxy group to the quinone ring. This very low El7 value makes one-electron reduction of the qui- none less efficient. The rather low P7 of this compound probably favors the autoxidation of the hydroquinone. 5- Hydroxy-1,4-naphthoquinone has similar reduction poten- tials as l,4-naphthoquinone but its semiquinone is a little more stable than that of l,4-naphthoquinone, which may lead to a higher semiquinone concentration and thereby a some- what higher rate of autoxidation. 5,8-Dihydroxy-1,4-naph- thoquinone possesses a highly stabilized semiquinone form, which increases the steady state level of semiquinone and thereby probably increases the efficiency of free radical pro- duction after enzymatic reduction. This compound also has a rather low pK, value for the hydroquinone, which further destabilizes the hydroquinone and favors autoxidation reac- tions. The cellular metabolism of this compound is of great interest because it is a structural analogue to the clinically important anthracycline cytostatics doxorubicin and dauno- rubicin (Scheme I).

We report here on the influence of hydroxy substituents on 1,4-naphthoquinone toxicity to cultured rat hepatocytes and the relation between the underlying mechanism of toxicity and the chemical properties of these 1,4-naphthoquinones, determined by the substitution pattern.

In this study we have used primary hepatocyte cultures, which offered some advantages to our study over hepatocyte suspensions, such as long and stable viability, thus limiting the loss of viability in control cells. Pretreatment of the cells with various agents is also facilitated because these agents are

0 0 1.4-Naphthoquinone 2-Hydroxy-1,bnaphthoquinone

0 W O

OH 0 5-Hydroxy-l A-naphthoquinonc 5,8-Dihydroxy-l,4-naphlhoquinone

SCHEME I

easily removed by changing the medium and only a very low cell death is observed after the pretreatment.

MATERIALS AND METHODS

Chemicals-l,4-Naphthoquinone, 2-hydroxy-l,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 5,8-dihydroxy-1,4-naphthoqui- none were from Aldrich-Chemie (Steinheim, Federal Republic of Germany (F. R. G.)). NADPH, GSH, GSSG, and glutathione reduc- tase were from Boehringer Mannheim (Mannheim, F. R. G.). Insulin, dexamethasone, BSO,' and dicumarol were from Sigma. BCNU was from Bristol-Myers. Fetal calf serum and HEPES were from GIBCO Ltd. (Paisley, Scotland, United Kingdom (U.K.)). Glutamine and Williams E Medium were from Flow Laboratories (Isvine, Scotland, UK). Type I collagen was from Biomedical Technologies, Inc. (Stroughton, MA). 1243-200 ATP monitoring reagent was from Phar- macia (Uppsala, Sweden). All other chemicals were from Merck (Darmstadt, F. R. G.). All chemicals were of the highest purity com- mercially available.

Polarography-Redox potential was measured with a dropping mercury electrode using an EG&G Princeton Applied Research po- larographic analyzer ( E G G , Princeton, NJ). 500 pM of quinone, dissolved in ethanol, was added to a supporting electrolyte of 0.1 M potassium phosphate buffer, pH 7.0.

Enzymes-NADPH-cytochrome PdsO reductase (EC 1.6.2.4) was isolated from rat liver as described (11). Specific activity for NADPH- cytochrome Ptso reductase was 12.6 +mol cytochrome Crduced X min" X mg protein" (measured as described in Ref. 11). Rat liver DT- diaphorase (EC 1.6.99.2, NAD(P)H dehydrogenase (quinone)), pre- pared as described previously (12), was a generous gift from Christina Lind (St. Gorans Hospital, Stockholm, Sweden). Specific activity for DT-diaphorase was 950 nmol 2-chlorophenolindopheno~,,duced X min" X pg protein" (13).

Incubation of Quinones with Reductases-NADPH-cytochrome Paso reductase experiments were performed in 0.2 M potassium phosphate buffer, pH 7.4, containing 20 p~ quinone and 150 p~ NADPH. The reaction was initiated by addition of enzyme (0.925 pg/ml). The assay temperatures was 28 "C. DT-diaphorase experiments were performed in 0.1 M potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose, 20 p M quinone, and 200 p~ NADPH. The reaction was initiated by addition of DT-diaphorase (45.2 ng/ml). The assay tem- perature was 28 "C. The rate of enzymatic reduction was followed by the decrease of NADPH absorption at 340 nm on a Varian DMS-100 UV-VIS spectrophotometer (Varian Svenska AB, Solna, Sweden).

Preparation of Rat Liuer Mitochondria-Mitochondria were pre- pared as described previously (14). Rat liver was homogenized in 9 vohmes of buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.4. The homogenate was centrifuged at 400 X g for 10 min, and the resulting supernatant was centrifuged at 4300 X g for 20 min. The mitochondrial pellet was then washed

The abbreviations used are: BSO, buthionine sulfoximine; GSH, glutathione; GSSG, glutathione disulfide; BCNU, 1,3-bis-2-chloroe- thyl-1-nitrosourea; HEPES, N-2-hydroxyethylpiperazine-N"2-eth- anesulfonic acid; E'?, reduction potential for the couple quinone/ semiquinone at pH 7; E',, reduction potential for the couple semiqui- none/hydroquinone at pH 7; Erm7, reduction potential for the couple quinone/hydroquinone at pH 7; HPLC, high performance liquid chromatography; HBSS, Hanks' buffered saline solution.

21498 Hydroxynaphthoquinones and Hepatocytes

with EDTA-free buffer by centrifugation at 4300 X g for 20 min, resuspended in EDTA-free buffer, and kept on ice before use. Protein was determined by the method of Lowry et al. (15).

Animals-Male Wistar rats (weight 160-200 g) were from ALAB (Solna, Sweden). The rats were fed ad libitum and fasted 16 h prior to use.

Isolution and Culture of Hepatocytes-Isolated hepatocytes were prepared by collagenase perfusion as described previously (16). The technique routinely yields around 200 X lo6 cells/liver with over 90% viability (as determined by trypan blue exclusion). Hepatocytes were suspended in Williams E medium containing 5 pg/ml insulin, 0.335 p M dexamethasone, 2 mM glutamine, and 10% fetal calf serum. The cells were plated at a density of 120,000 cells/cm2 on 35-mm Petri dishes (Costar, Cambridge, MA). Petri dishes were coated with 1.7 pg/cmZ type I collagen prior to use. The cells were cultured at 37 "C in an atmosphere of 5% CO, and 95% air. The cell culture medium was changed 2 h after seeding.

Incubation of Hepatocytes with Quinones-1,4-Naphthoquinone, 2- hydroxy-1,4-naphthoquinone, 5-hydroxy-l,4-naphthoquinone, and 5,8-dihydroxy-1,4-naphthoquinone were dissolved in dimethyl sulf- oxide (final concentration of dimethyl sulfoxide never exceeded 0.3%) and diluted in HBSS (Hanks' buffered saline solution) containing 20 mM HEPES, pH 7.0. It is well known that the level of several detoxication enzymes, as well as GSH concentration, changes with time in culture after isolation of hepatocytes. Therefore, all incuba- tions were started 7 h after seeding. The incubation of hepatocytes with quinones were performed in air at 37 "C unless otherwise stated.

The effect of different Oz pressure was assessed by incubation of hepatocytes in airtight chambers (Flow Laboratories, Isvine, Scot- land, UK) in Nz, air, or O2 atmosphere. The incubation mixture was bubbled with the respective gas during the 20 min before use. This procedure resulted in an oxygen concentration in the medium of 16 p~ (0.50 mg/ml) when the incubation mixture was bubbled with Nz, 172 p~ (5.5 mg/ml) in air, and 834 p M (27 mg/ml) when the incuba- tion medium was bubbled with Oz. The Oz concentration was meas- ured according to Winkler (17). A 2.5-h incubation of the hepatocytes in Nz- or Oz-bubbled HBSS without addition of quinones did not affect the viability of the cells (measured by trypan blue exclusion test as described below).

Determination of Cell Viability-Cell viability was measured with the trypan blue exclusion test (trypan blue dissolved in HBSS at 0.4% final concentration). Quinones interact with most enzymes making enzyme leakage tests (e.g. lactate dehydrogenase) for cell viability impossible. However, the trypan blue exclusion test gives results similar to those of the lactate dehydrogenase leakage test (18).

Measurement of Enzyme Activity-Hepatocytes were incubated with quinones as described above. The cells were lysed by addition of 1% Triton X-100 in a 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Glutathione reductase activity (19) and glutathione peroxidase activity (20) were measured as described pre- viously.

Determination of NADPH-NADPH was determined as described previously (21).

Determination of ATP-ATP levels were determined using the luciferase-linked luminescence method. Hepatocyte cultures, incu- bated as described above, were extracted with 500 p1 of 2.5% trichlo- roacetic acid. A 20-pl sample was added to 180 p1 of an aqueous solution containing 1.2 ng/ml firefly luciferase, 64 nM D-luciferin, 1 mg/ml bovine serum albumin, 10 p M magnesium acetate, 2 nM inorganic pyrophosphate, 2 mM EDTA, and 0.1 M Tris-HC1, pH 7.75. The luminescence was measured using a LKB Wallac 1250 biolumi- nometer (Wallac Oy, Turku, Finland). Assay temperature was 25 'C. ATP levels were calculated against a standard curve.

Determination of GSHIGSSG-At the time points indicated the cells were washed with ice-cold PBS and immediately frozen. 300 pl of a solution containing 0.1% EDTA, 5 p~ cysteine, and 40 mM perchloric acid was added to the dishes, and the cells were detached with a rubber policeman. The suspension was centrifuged at 300 X g for 10 min and the supernatant was used for GSH/GSSG analysis. Separation of GSH and GSSG was performed on a Biophase ODS I 5-pm reverse phase column (250 X 4.3 mm) (Bioanalytical Systems Inc., West Lafayette, IN). The mobile phase consisted of 0.1 M monochloroacetic acid dissolved in deionized triple distilled water, pH 3.0, and was supplied by a Jasco 880-PU pump (Japan Spectro- scopic Co. Ltd, Tokyo, Japan). The flow rate was 1.6 ml/min. GSH and GSSG (R, 3.5 and 8.8 min, respectively) were detected by a BAS LC-4B Amperometric Detector (Bioanalytical Systems Inc., West Lafayette, IN, USA) with a dual Au/Hg electrode in a series arrange-

ment. The potential of the upstream electrode was -1.0 V, and the downstream electrode potential was +0.150 V. GSH and GSSG con- centrations were calculated from standard curves.

Treatment with Inhibitors-In order to inhibit glutathione reduc- tase, hepatocytes were incubated for 30 min with 300 p~ BCNU in Williams E medium supplemented as described above. The activity of glutathione reductase was 11% of the control, and the GSH level was 17% of control cells incubated for the same time without BCNU. At the end of the incubation the cells were washed with PBS and the cells were incubated with quinones as described above. GSH was depleted by incubation with 1 mM BSO, an inhibitor of y-glutamyl- cysteine synthetase (22), dissolved in Williams E medium, for 5 h. At this time GSH levels were 31% of control. DT-diaphorase was inhib- ited with 2.5 p~ dicumarol added together with the quinone in HBSS. GSH concentration in hepatocytes was 60% of the control after a 30- min incubation with 2.5 p M dicumarol. The viability of control cells, incubated with the inhibitors during the same time period, was unaffected.

Determination of the Second Order Rate Constants-The second order rate constant for the electrophilic addition between reduced glutathione and l,4-naphthoquinone5 was determined by mixing equi- molar amounts of quinone and GSH in a 0.1 M potassium phosphate buffer, pH 7.0, at 25 "C. For the reaction with 2-hydroxy-1,4-naph- thoquinone a 100-fold higher concentration of GSH was used, and for this quinone the rate constant was determined as a pseudo-first order rate constant. At different time points samples were withdrawn and the rate constant was calculated from the decrease in quinone and/or GSH concentration. Quinone concentration was determined with HPLC/electrochemical detection as described previously (7) and GSH concentration was measured as described above.

Statistical Analysis-The results were evaluated with Student's t test for matched pairs.

RESULTS

Relation between Position of Hydroxy Substituent on 1,4- Naphthoquinone und Cytotoxicity-Fig. 1 shows the cell via- bility after 1.5-h incubation of the hepatocytes with different concentrations of 1,4-naphthoquinone, 2-hydroxy-1,4- naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 5,8- dihydroxy-1,4-naphthoquinone. Unsubstituted 1,4-naphtho- quinone killed half the number of cells at a concentration of

A B

0 20 10 6 0 B O

a i 0 5 10 IS

D

OH0

1 7 OH 0

P Quinone concentration (pM)

FIG. 1. Relation between quinone concentration and toxic- ity on cultured rat hepatocytes. Cultured rat hepatocytes (120,000 cells/cmz) were incubated in HBSS with the quinones at the concen- trations indicated on the abscissa for 1.5 h at 37 "C and cell viability was estimated by trypan blue exclusion test. A, l,4-naphthoquinone; B, P-hydroxy-l,4-naphthoquinone; C, 5-hydroxy-1,4-naphthoqui- none; D, 5,8-dihydroxy-1,4-naphthoquinone. The values shown in the figure represent the mean f S.E. of 6 experiments with separate hepatocyte preparations.

Hydroxynaphthoquinones and Hepatocytes 21499

about 30 PM (Fig. LA), whereas 2-hydroxy-1,4-naphthoqui- none was much less toxic and killed half the number of cells at a concentration of about 134 PM (Fig. 1B). Both the 1,4- naphthoquinones substituted by hydroxy groups in the ben- zene ring were more toxic than 1,4-naphthoquinone; 5-hy- droxy-1,4-naphthoquinone killed half the number of cells at a concentration of about 8.1 wtM (Fig. IC), and 5,8-dihydroxy- 1,4-naphthoquinone killed half the number of the cells at a concentration of about 3.7 WM (Fig. 1D). Thus, substitution by a hydroxy group in the quinoid ring of 1,4-naphthoquinone decreases toxicity, whereas substitution with hydroxy substit- uents in the @ position of the benzene ring increases toxicity.

Enzyme-catalyzed Reduction of 1,4-Naphthoquinones-To compare the influence of quinone hydroxy substitution pat- tern on the efficiency of one- and two-electron reduction, the quinones were incubated with the flavin enzymes DT-dia- phorase and NADPH-cytochrome P45o reductase and the rate of reduction was measured spectrophotometrically as NADPH oxidation at 340 nm. Table I1 shows that all four quinones studied are substrates for both enzymes and can be reduced at the expense of NADPH. Under aerobic conditions the products can be oxidized by O2 in one-electron steps back to the quinone form, which in turn can be re-reduced by the enzyme in a cyclic manner. All the semiquinones have rather low reduction potentials and are rapidly oxidized back to the respective quinone by 02, resulting in a continuous rate of NADPH consumption, which is mainly limited by the rate of reduction of the quinone by NADPH-cytochrome P,so reduc- tase.

l,4-Naphthoquinone is the best substrate for DT-diaphor- ase and produced the highest initial rate of NADPH oxidation. The hydroquinone form is quite stable and a second, much slower rate, appears after equimolar amounts of quinone and NADPH have been consumed. 5-Hydroxy-1,4-naphthoqui- none and 5,8-dihydroxy-1,4-naphthoquinone are also good substrates for DT-diaphorase, while 2-hydroxy-1,4-naphtho- quinone caused the slowest rate of NADPH oxidation. All hydroxy-substituted 1,4-naphthoquinones are oxidized back to the quinone form at a higher rate than the rate of enzymatic reduction, and therefore, no change in NADPH oxidation rate was observed. No reduction of the quinones via DT-diaphor-

TABLE I1 NADPH oxidation during DT-diaphorase and NADPH-cytochrome P4w reductase-catalyzed reduction of unsubstituted and hydroxy-

substituted 1,4-naphthquinones The rate of enzymatic reduction calculated from the timecourse of

NADPH oxidation. Initial rate is calculated within the first minutes of quinone reduction. Enzymatic reduction of 1,4-naphthoquinone by DT-diaphorase shows a second, slower rate after about 2 min, reflect- ing the autoxidation of the hydroquinone product. Assay conditions: NADPH-cytochrome P4so reductase experiments were performed in 0.2 M potassium phosphate buffer, pH 7.4, containing 20 p~ quinone and 150 PM NADPH. The reaction was initiated by addition of 0.925 pg of enzymejml. Assay temperature was 28 "C. DT-diaphorase ex- periments were performed in 0.1 M potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose, 20 p M quinone, and 200 p M NADPH. The reaction was initiated by the addition of 45.2 ng of enzyme/ml. Assay temperature was 28 "C.

NADPH-cytochrome phorase DT dia-

Quinone reductase initial rate' Initial 2nd

rate" rate"

l,4-Naphthoquinone 3.3 853 64 2-Hydroxy-1,4-naphthoquinone 0.5 26 5-Hydroxy-l,4-naphthoquinone 3.5 790 5,8-Dihydroxy-1,4-naphthoquinone 3.8 336

a nmol NADPH/min/pg enzyme.

ase could be seen after supplementation of the incubation mixtures with 30 pM dicumarol.

Because NADPH-cytochrome Pqw reductase reduces all quinones to their semiquinone form, a correlation with El7

may be expected. Accordingly 5,8-dihydroxy-1,4-naphthoqui- none, 5-hydroxy-l,4-naphthoquinone, and 1,4-naphthoqui- none, which have rather similar El7 values (see Table I), are reduced at similar rates, whereas 2-hydroxy-lP-naphthoqui- none, which has a rather low E17 value, is reduced at a slower rate.

Mitochondrial Cyanide-resistant Respiration Produced by Quinones-Quinones may receive electrons from components of the respiratory chain, a process that could result in free radical formation and thereby mitochondrial and cellular damage. The effect on mitochondrial cyanide-resistant res- piration of the 1,4-naphthoquinones studied is shown in Table 111. The efficiency of the quinones to cause cyanide-resistant respiration decreased in the following order: 1,4-naphthoqui- none > 5-hydroxy-l,4-naphthoquinone > 5,8-dihydroxy-1,4- naphthoquinone > 2-hydroxy-l,4-naphthoquinone. The three former quinones have rather similar El7 values and their efficiency in promoting mitochondrial cyanide-resistant res- piration may be related mainly to their polarity. 2-Hydroxy- 1,4-naphthoquinone has a very low E'7 and only a very low cyanide-resistant respiration is observed during incubation of mitochondria with this quinone.

Effect of Hydroxy Substitution on Electrophilic Addition- Because of their electrophilic character quinones may exert toxic effects by destruction of cellular components, e.g. en- zymes. Table IV gives the rate of addition to GSH of the quinones studied here. The electrophilicity decreased in the order 1,4-naphthoquinone > 5-hydroxy-1,4-naphthoquinone

TABLE 111 Cyanide-resistant mitochondrial respiration in the presence of unsubstituted and hydroxy-substituted 1,l-naphthoquinones

02 consumption was measured with a Clark-type electrode in a 900-pl reaction chamber. Values are shown k S.D., n = 3. Reaction mixture contained 0.1 M Tris-HC1 buffer, pH 7.4, 50 mM KCI, 5 mM succinate, 1 mg of mitochondrial protein/ml, 1 mM KCN, and 20 p M quinone.

Quinone 0, uptake"

Control 2.6 * 0.1 1,4-Naphthoquinone 18.9 * 1.7 2-Hydroxy-1,4-naphthoquinone 4.0 * 0.5 5-Hydroxy-1,4-naphthoquinone 13.7 2 2.0 5,8-Dihydroxy-1,4-naphthoquinone 7.9 k 0.9

a nmol O2 X min" X mg mitochondrial protein".

TABLE IV Rate of electrophilic addition to GSH for unsubstituted and hydroxy-

substituted 1,l-naphthoquinones The reaction mixtures contained the quinone and reduced gluta-

thlone in a 0.1 M potassium phosphate buffer, pH 7.0 at 25". Different concentrations of quinone and GSH were used to facilitate measure- ment of reactant consumption: the reaction mixtures contained 50 p M 1,4-naphthoquinone/50 p M GSH, 100 pM 2-hydroxy-l,4-naph- thoquinonejl0 mM GSH, 10 PM 5-hydroxy-1,4-naphthoquinone/lO p M GSH, and 10 PM 5,8-dihydroxy-1,4-naphthoquinone/lO ~ L M GSH. At different time points samples were withdrawn, and the decrease in quinone and/or GSH concentration was determined with HPLC as described under "Material and Methods."

Quinone M G W I Q I " s-'

l,4-Naphthoquinone 2071 2-Hydroxy-1,4-naphthoquinone 1.6 X 10-~ 5-Hydroxy-1,4-naphthoquinone 105 5,8-Dihydroxy-1,4-naphthoquinone 50

21500 Hydroxynaphthoquinones and Hepatocytes

> 5,8-dihydroxy-1,4-naphthoquinone >> 2-hydroxy-1,4- naphthoquinone. 1,4-Naphthoquinone has a rather high rate of formation of conjugates with GSH and may react with intracellular macromolecules possessing thiol groups. There- fore, it is likely that this compound may be toxic through electrophilic addition reactions. Similarly, Rossi et al. (23) found that 2,6-dimethyl-1,4-benzoquinone, which has a rate constant for the addition to GSH ( k = 1.6 X lo3) similar to that of 1,4-naphthoquinone, was toxic because of addition to cellular components. Furthermore, it is evident that 1,4- naphthoquinone, which has by far the highest rate of electro- philic addition to GSH, is much less toxic than both 5,8- dihydroxy-1,4-naphthoquinone and 5-hydroxy-1,4-naphtho- quinone. This indicates that electrophilic addition is not the main mechanism for expression of toxicity for the latter two quinones. 2-Hydroxy-l,4-naphthoquinone is a very poor elec- trophile because of the very high electron density at CB, caused by the hydroxy substituent, and it is unlikely that this com- pound can mediate cell killing through electrophilic addition.

Effect of Quinone Exposure on Hepatocyte Glutathione Sta- tus-The capacity of the different quinones to cause GSH oxidation and formation of GSSG, probably as a consequence of Hz02 formation via redox cycling, was determined by incubation of the hepatocytes with the respective quinones at a concentration of 3.5 p~ for 10 min. As shown in Fig. 2 the GSH/GSSG ratio decreased more rapidly the more toxic the quinone was. 5,8-Dihydroxy-1,4-naphthoquinone caused a high GSH oxidation and loss of total glutathione (GSH + GSSG), whereas 5-hydroxy-1,4-naphthoquinone caused a smaller GSH oxidation and loss of total glutathione. Incuba- tion of the hepatocytes with 1,4-naphthoquinone and 2-hy- droxy-1,4-naphthoquinone only slightly affected total intra- cellular glutathione levels. Because 5,8-dihydroxy-1,4- naphthoquinone caused the highest GSH oxidation when the hepatocytes were incubated with the quinones at the same concentration it appears that 5,8-dihydroxy-1,4-naphthoqui- none is the most efficient intracellular free radical producer of the compounds tested. This conclusion does not rule out the possibility that GSH oxidation, followed by loss of total

A B C D

FIG. 2. Effect of quinone exposure on hepatocyte intracel- lular GSH and GSSG at the same quinone concentration. Cultured hepatocytes (120,000 cells/cmz) were incubated in HBSS at 37 "C with the different quinones at a quinone concentration of 3.5 p~ for 10 min. The cells were then rinsed with ice-cold PBS and rapidly frozen. GSH and GSSG was extracted, and the levels were determined by HPLC as described under "Materials and Methods." The values shown represent the mean f S.E. of 4 experiments with separate cell preparations. A , 1,4-naphthoquinone; E, 2-hydroxy-1,4- naphthoquinone; C, 5-hydroxy-l,4-naphthoquinone; D , 5,a-dihy- droxy-1,4-naphthoquinone. 100% represents the GSH level in control cells, not exposed to quinone (the GSSG level in control cells was always below the detection limit). No GSSG formation and no change in total glutathione were found in control cells incubated in HBSS for the same time.

glutathione, may be an important factor in the cell killing by the quinones that are not as efficient as 5,8-dihydroxy-1,4- naphthoquinone in producing GSH oxidation and loss of total glutathione. To test this possibility the hepatocytes were incubated with quinones at concentrations that produced a similar degree of cell death (about 21% cell killing) after 30 min. Fig. 3 (A-D) shows the time courses for GSH consump- tion, GSSG formation, and the total intracellular glutathione level in hepatocytes under such conditions. Unsubstituted 1,4-naphthoquinone was the quinone that most efficiently produced oxidation of GSH to GSSG under these conditions; after only 2 min, 67% of the initial GSH content was con- verted to GSSG. In the same time 5,8-dihydroxy-1,4-naphth- oquinone produced 37%, 5-hydroxy-1,4-naphthoquinone pro- duced 50%, and 2-hydroxy-1,4-naphthoquinoneproduced 31% GSSG formation. Thus, by comparing Fig. 3 and the time courses of cell death at the same quinone concentration (Fig. 4) it appears that there is a close link between cell death and cellular glutathione status for all the quinones studied.

Fig. 3 shows further that the GSSG formation occurred very rapidly, and in addition, that there was a slow decrease in the total intracellular glutathione content. As can be seen in Fig. 4 (A-D), the cell viability after 30 min of incubation is around 80% for all the quinones at the concentrations used in the GSH/GSSG measurement, whereas the level of total glutathione in the cells after 30 min of exposure is between 18% (1,4-naphthoquinone) and 40% (5-hydroxy-1,4-naphth- oquinone) of initial intracellular concentration left. Further- more, the decrease in intracellular total glutathione content starts immediately after the addition of the quinones. It seems unlikely, therefore, that the decrease in total glutathione is

A

C

0 10 20 30

B

D

n 10 20 30

Time (mi")

FIG. 3. Effect of quinone exposure on intracellular GSH, GSSG, and total glutathione levels at quinone concentrations producing similar levels of cell death. Cultured hepatocytes (120,000 cells/cm2) were incubated in HBSS at 37 "C with the differ- ent quinones at the concentrations shown in the figure. At the times indicated on the abscissa the cells were rinsed with ice-cold PBS and then rapidly frozen. GSH and GSSG was extracted, and the levels were determined by HPLC as described under "Materials and Meth- ods." The values shown represent the mean f S.E. from 5 experiments with separate cell preparations. A , l,4-naphthoquinone (30 pM); B, 2-hydroxy-1,4-naphthoquinone (300 p ~ ) ; C, 5-hydroxy-l,4-naphtho- quinone (5 pM) ; D, 5,8-dihydroxy-1,4-naphthoquinone (3.5 pM). 100% represents the GSH level in control cells, not exposed to quinone (the GSSG level in control cells was always below the detection limit). No GSSG formation and no change in total glutathione were found in control cells incubated in HBSS for the same time.

Hydroxynaphthoquinones and Hepatocytes 21501

0’ I 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5

Time (hours)

FIG. 4. Time course for hepatocyte killing during quinone exposure. Cultured hepatocytes (120,000 cells/cm2) were incubated in HBSS at 37 “C with the quinones at the concentrations shown in the figure, and cell viability was estimated by trypan blue exclusion test at the times indicated on the abscissa. A, 1,4-naphthoquinone (30 PM); B, 2-hydroxy-1,4-naphthoquinone (300 p ~ ) ; C, 5-hydroxy-1,4- naphthoquinone (5 p ~ ) ; D, 5,8-dihydroxy-1,4-naphthoquinone (3.5 PM). The values shown in the figure are the mean * S.E. from 6 experiments with separate hepatocyte preparations.

caused by leakage out from lysed cells. I t should be pointed out that ATP and NADPH levels were not significantly affected by quinone exposure and no inactivation of glutathi- one reductase or glutathione peroxidase was observed (not shown). Therefore, the GSSG accumulation observed in the above described experiments is probably a consequence of the reduction of H202 by GSH peroxidase at a rate that is greater than the rate by which glutathione reductase can reduce GSSG.

Oxygen Dependence of Quinone Cytotoxicity-Information about the importance of free radicals for the toxicity of these quinones may also be obtained from experiments with differ- ent O2 concentrations in the medium in which the cells are incubated with the quinones. Fig. 5 shows that 1,4-naphtho- quinone and 5-hydroxy-1,4-naphthoquinone have similar tox- icity in 16 pM 0, as in 172 pM o,, while a 834 pM 0, concentration increased their toxicity significantly (p 5 0.05 a n d p 5 0.01, respectively) (Fig. 5). When a higher concentra- tion of 5-hydroxy-l,4-naphthoquinone (10 p ~ ) was used, 91% of the cells were killed in 172 pM O2 concentration, whereas 29% were killed in 16 p~ O2 (not shown). Fig. 5 also shows that the toxicity of 2-hydroxy-1,4-naphthoquinone was inde- pendent of O2 concentration. The 1,4-naphthoquinone whose toxicity was most dependent on the 0, concentration was 5,8- dihydroxy-1,4-naphthoquinone; in 172 p~ 0,92% of the cells were killed by 5,8-dihydroxy-1,4-naphthoquinone, but only 22% of the cells were killed in 16 p~ O2 (p 5 0.001). In 834 I.LM O2 all the cells were killed by 5,8-dihydroxy-1,4-naphtho- quinone.

After 1.5 h of incubation with 1,4-naphthoquinone in 16 pM 0, the hepatocytes contained 10.4 * 1.4% (n = 5) of the initial GSH concentration, and 12.4 f 2.6% was detectable as GSSG. The corresponding values for 2-hydroxy-1,4-naphth- oquinone were GSH = 56.5 & 6.3% and GSSG 15.6 k 3.2%, for B-hydroxy-l,4-naphthoquinone the values were GSH = 55.2 k 11.4% and GSSG = 10.7 k 3.2%, and for 5,8-dihydroxy- 1,4-naphthoquinone the values were GSH = 29.2 & 8.4% and GSSG = 21.0 f 5.4%. Although intracellular GSH and GSSG

A B C D

FIG. 5. Influence of O2 concentration on quinone toxicity on hepatocytes. HBSS, containing the quinones at concentrations shown in the figure, was bubbled with Nz, air, and Oz for 20 min (resulting in concentrations of Oz of 16,172, and 834 pM, respectively), and the solutions were then carefully pipetted on to the cells. The cells were then incubated in the different Oz concentrations shown in the figure for 1.5 h as described under “Materials and Methods,” and cell viability was determined by trypan blue exclusion test. 100% represents the viability of control cells not exposed to quinones. The viability of the control cells was not affected by incubation in the different atmospheres. A , 1,4-naphthoquinone (30 p ~ ) ; B, 2-hydroxy- 1,4-naphthoquinone (300 p ~ ) ; C, 5-hydroxy-1,4-naphthoquinone (5 PM); D, 5,8-dihydroxy-1,4-naphthoquinone (5 p ~ ) . The values shown in the figure are the mean * S.E. from 6 experiments with separate hepatocyte preparations.

content probably is affected by leakage of GSH and GSSG out of the cells the results above still indicate that a lower GSH consumption occurs in 16 p~ 02, probably reflecting a lower rate of free radical production (cf. Fig. 3). Furthermore, it is likely that the relatively low GSH content in the hepa- tocytes exposed to 1,4-naphthoquinone in 16 pM O2 is partially due to electrophilic addition of 1,4-naphthoquinone to GSH.

To check if 16 p~ O2 concentration affected the cellular energy production, the hepatocytes were incubated in HBSS with 16 pM O2 and 172 pM O2 concentration for 1.5 h. We found that 16 p~ O2 concentration of the medium decreased intracellular ATP levels by only 10% as compared with 172 PM O2 (not shown). Thus, it seems most likely that the effect of a lower 0, concentration on quinone toxicity is not related to cellular energy production.

Effect of Enzyme Inhibitors on Quinone Toxicity-By treat- ing the hepatocytes with dicumarol, BSO, and BCNU we wanted to investigate the importance of DT-diaphorase, re- duced glutathione, and glutathione reductase, respectively, in the detoxication processes related to cellular exposure to the quinones. Table V lists the effects of these compounds on quinone toxicity.

BSO treatment prior to incubation of the hepatocytes with quinones resulted in GSH levels that were 31% of control cells without largely affecting viability. This pretreatment increased the toxicity of 5-hydroxy-1,4-naphthoquinone by 59% and that of 5,8-dihydroxy-l,4-naphthoquinone 36%, whereas the toxicity of 1,4-naphthoquinone and of 2-hydroxy- 1,4-naphthoquinone were almost unaffected. Preincubation of the hepatocytes with BCNU decreased the glutathione reductase activity by 89% and the GSH level by 83% with only small effects on cell viability. BCNU pretreatment had a strong influence on the toxicity of 5-hydroxy-1,4-naphtho- quinone and 5,8-dihydroxy-1,4-naphthoquinone. The toxicity of the former increased 45% and that of the latter 81%.

The incubation of the hepatocytes concomitantly with di- cumarol (2.5 pM) and the quinones increased the toxicity of all the 1,4-naphthoquinones tested, although to different ex- tents (Table V). The effect of dicumarol on 1,4-naphthoqui- none cell killing was largest; it was increased by 96%. The

21502 Hydroxynaphthoquinones and Hepatocytes TABLE V

Effect of inhibitors on the toxicity to hepatocytes incubated with unsubstituted and hydroxy-substituted 1,4-naphthoquinones for 30 m i n

Hepatocytes were incubated with 300 ~ L M BCNU for 30 min or 1 mM BSO for 5 h before the addition of the respective quinone. Dicumarol, 2.5 p ~ , was added together with the quinone. Cells were incubated with the quinones, dissolved in HBSS, for 30 min at 37 "C, and the cell viability was measured with trypan blue exclusion test. In order to normalize experiments performed different days the effect of the inhibitors and quinones on cell viability was calculated as percent of the viability of untreated cells (the viability of untreated cells always exceeded 98%). For detailed assay conditions see "Materials and Methods." The values represent the mean t S.E. from five separate cell preparations.

Cell survival

Only quinone BSO BCNU Dicumarol

% of control Control (without quinone) 98.0 f 1.9 95.7 f 2.9 97.4 f 1.6 1,4-Naphthoquinone (30 p ~ ) 73.8 f 3.3 59.2 k 8.4 73.1 f 4.9 2.8 -C 1.4" Z-Hydroxy-1,4-naphthoquinone (100 p ~ ) 84.9 f 1.3 78.8 & 3.3 81.4 k 4.6 35.9 f 4.3b 5-Hydroxy-1,4-naphthoquinone (5 p ~ ) 73.8 f 2.0 30.6 f 5.3" 40.9 f 6.8" 51.1 f 2.5" 5,8-Dihydroxy-l,4-naphthoquinone (2.5 PM) 70.0 f 4.2 45.0 & 9.6' 13.0 f 5.2' 45.4 f 5.4

" p 5 0.01 compared with incubations of hepatocytes with only quinone. p 5 0.001 compared with incubation of hepatocytes with only quinone.

' p 5 0.05 compared with incubation of hepatocytes with only quinone.

cell killing by 2-hydroxy-1,4-naphthoquinone increased 58%, whereas the cell toxicity of the benzene ring /3-hydroxy- substituted 5-hydroxy-1,4-naphthoquinone and 5,8-dihy- droxy-1,4-naphthoquinone increased by 31 and 35%, respec- tively. Dicumarol alone did not affect cell viability, but it decreased the intracellular GSH concentration by 33%. Thus, the effect of dicumarol on the toxicity of 5-hydroxy-1,4- naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone is probably overestimated because decreasing the intracellular GSH content increased the toxicity of the latter two quinones (see above).

DISCUSSION

Quinone cellular toxicity has been ascribed to two basic mechanisms: (a) the formation of quinone adducts to cellular constituents, probably mainly thiol-containing proteins, re- sulting in their inactivation and (b) redox cycling by the quinone resulting in the formation of 0; and H202, followed by GSH oxidation to GSSG. The former process has been demonstrated to be responsible for cell killing by benzoqui- none and several benzoquinone derivatives without the for- mation of GSSG (23). No specific target has been demon- strated for this process, and the explanation for cell killing may very well be an overall inactivation of the cellular meta- bolic machinery. The latter process involves GSH depletion, thereby incapacitating the removal of H202 by glutathione peroxidase, probably followed by generation of hydroxyl rad- ical via a Fenton reaction and, finally, lipid peroxidation. This sequence of events is supported by a study on H2O2 killing of hepatocytes which shows that cell killing requires the forma- tion of 0; as well as iron salts (24).

Although the sequence of events that precedes cell death after quinone exposure is still obscure it is clear that chemical properties of the quinone, determined by its substituents, will determine whether it mediates cell killing via electrophilic addition or redox cycling. Previous studies have shown that mono- or dimethyl substitution of 1,4-naphthoquinone (at position 2 or 2 and 3, respectively) decreases its cytotoxicity (25) either due to lower rate of redox cycling, caused by the lower redox potentials imposed by methyl substitution, or lower rate of electrophilic addition. Below we discuss the evidence obtained that support either mechanism for the quinones studied here.

All the quinones studied cause GSH oxidation followed by

loss of total glutathione suggesting that they mediate Hz02 formation. However, dramatic differences in efficiency in promoting this process between the quinones were observed. 5,8-Dihydroxy-1,4-naphthoquinone was clearly the quinone that most efficiently caused GSH oxidation and loss of total glutathione, suggesting that this compound is the most effi- cient in promoting redox cycling of the compounds studied. This does not rule out the possibility that GSH oxidation and loss of total glutathione may be of importance for the toxicity of the other compounds. This was tested by incubation of the hepatocytes with the different quinones at concentrations where the quinones caused a similar degree of cell death. Under these conditions all quinones caused a similar magni- tude of GSH oxidation and loss of total glutathione, suggest- ing that these phenomena may be important for the toxicity of all the quinones. It seems likely that the decrease in total intracellular glutathione concentration in the experiments shown in Fig. 3 is not mainly due to leakage of GSH out through the ruptured cell membrane. We suggest that the decrease in total glutathione is primarily a consequence of transport of GSSG out of the cell across the cell membrane. A similar mechanism has been observed in perfused liver (26) and is probably a way to increase the intracellular GSH/ GSSG ratio under oxidative conditions where the glutathione reductase activity does not suffice to keep the intracellular GSSG concentration low. Electrophilic addition of the qui- nones to GSH may also contribute to the decrease in total intracellular glutathione concentration, although it could not explain the decrease of total glutathione produced by 2- hydroxy-1,4-naphthoquinone which has an extremely low electrophilicity (see Table IV).

Because GSH depletion is also likely to lead to higher formation of quinone adducts to protein sulfhydryl groups, the explanation for the relationship between GSH depletion/ loss of total glutathione and cell death is not easily distin- guished between effects of free radical formation and inacti- vation of cellular components by adduct formation. However, some conclusions regarding the importance of adduct forma- tion uersus free radical-related oxidative processes may be drawn from the experiments with the glutathione synthesis inhibitor BSO and the glutathione reductase inhibitor BCNU. The effect of BCNU on quinone toxicity decreased in the order 5,8-dihydroxy-1,4-naphthoquinone > 5-hydroxy-1,4- naphthoquinone > 2-hydroxy-1,4-naphthoquinone = 1,4-

Hydroxynaphthoquin

naphthoquinone, whereas the effect of BSO decreased in the order 5-hydroxy-1,4-naphthoquinone > 5,8-dihydroxy-1,4- naphthoquinone > 1,4-Naphthoquinone > 2-hydroxy-1,4- naphthoquinone. The effects of BCNU and BSO are probably not completely distinguishable since a lower initial intracel- lular GSH concentration, caused by BSO pretreatment, would decrease the efficiency of H202 removal by glutathione per- oxidase and a lower activity of glutathione reductase leads to a lower intracellular GSH concentration making electrophilic additions of quinones to protein sulfhydryls more efficient. Despite these limitations it may still be concluded that 5,8- dihydroxy-1,4-naphthoquinone is the compound whose tox- icity is most dependent on oxidative processes, whereas 1,4- naphthoquinone toxicity appears to merely be a function of electrophilic addition and 5-hydroxy-1,4-naphthoquinone takes a position somewhere in between these compounds. 2- Hydroxy-1,4-naphthoquinone toxicity to hepatocytes was only slightly affected by BSO and BCNU treatment.

If one assumes that alterations in the concentration of the final electron acceptor in redox cycling processes, 02, leads to altered rate of formation of free radicals it would be possible to obtain information about the importance of oxidative free radical processes for the toxicity of a quinone. Thus, if a quinone is much less toxic in an atmosphere of reduced O2 pressure than at 21% O2 pressure, this would indicate that its toxicity a t 21% O2 pressure is mainly a consequence of free radical production. Incubation of the hepatocytes with the quinone at increased O2 pressure would indicate whether the quinone is a t all capable of cell killing via free radical forma- tion followed by cellular oxidative processes. 5,8-Dihydroxy- 1,4-naphthoquinone shows the highest dependence on O2 concentration of the compounds tested, strengthening the above conclusions regarding the importance of free radical formation for the toxicity of this compound. The toxicity of 1,4-naphthoquinone appeared not to be largely dependent on free radical formation at atmospheric O2 concentration, but its capacity to participate in redox cycling was demonstrated by the increased toxicity when the cells were exposed to this compound at elevated O2 concentration. 5-Hydroxy-1,4- naphthoquinone again took an intermediate position between the above two compounds whereas the toxicity of 2-hydroxy- 1,4-naphthoquinone was unaffected by O2 pressure.

The increased free radical formation by 1,4-naphthoqui- nones substituted in the 5- and 8-position is, as discussed in the introduction section, likely to be related to the decreased stability of the hydroquinone form of these compounds. This is experimentally confirmed by the dramatic effect by inhi- bition of two-electron reduction via DT-diaphorase on 1,4- naphthoquinone toxicity, a compound that forms a rather stable hydroquinone, as compared with the relatively small effect on the toxicity of 5-hydroxy-1,4-naphthoquinone and 5,8-dihydroxy-l,4-naphthoquinone. This difference may be understood as a consequence of the decreased pK, value of the hydroquinone caused by benzene ring hydroxy substitu- tion, which increases the rate of autoxidation and also favors formation of the easily oxidizable semiquinone (the effect of pK. value on electron transfer from hydroquinones is dis- cussed in Ref. 10, and the formation of semiquinones from hydroquinones and quinones is discussed in Ref. 27).

The results presented above show that hydroxy substitution of 1,4-naphthoquinone at different positions leads to de- creased or increased toxicity to rat liver hepatocytes. Hydroxy substitution in the Cy position renders a quinone that is much less electrophilic and has reduction potentials which make its participation in redox cycling reactions unfavorable, and it is

!ones and Hepatocytes 21503

not clear what the mechanism may be for the toxicity of 2- hydroxy-1,4-naphthoquinone. Monohydroxy substitution at C5 and dihydroxy substitution at C5 and Cs make the quinone less electrophilic, but appear to increase the efficiency of redox cycling.

Acknowledgments-We thank Britt-Marie Gustavsson for help with preparation of hepatocyte cultures and Irene Svensson for per- forming the experiments with mitochondria.

REFERENCES 1. Thomson, R. H. (1987) Naturally Occurring Quinones, Vol. 111,

2. Schuetzle, D. (1983) Enuiron. Health Perspect. 47 , 65-80 3. Tikkanen, L., Matsushima, T., Natori, S., and Yoshihira, K.

(1983) Mutat. Res. 124 , 25-34 4. Chesis, P. L., Levin, D. E., Smith, M. T., Emster, L., and Ames,

B. N. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 1696-1700 5. Price, P. A., Suk, W. A., Skeen, P. C., Chirigos, M. A., and

Huebner, R. J. (1975) Science 187, 1200-1201 6. Ollinger, K., Llopis, J. and, Cadenas, E. (1989) Arch. Biochem.

Biophys. 2 7 5 , 514-530 7. Buffinton, G. D., Ollinger, K., Brunmark, A., and Cadenas, E.

(1989) Biochem. J. 257 , 561-571 8. Rich, P. R., and Bendall, D. S. (1980) Biochim. Biophys. Acta

9. Meisel, D. (1975) Chem. Phys. Lett. 34 , 263-266

Chapman & Hall, London

592,506-518

10. Wardman, P. (1989) J. Phys. Chem. Ref. Data 18 , 1637-1755 11. Ingelman-Sundberg, M., and Glaumann, H. (1980) Biochim. Bio-

12. Hojeberg, B., Blomberg, K., Stenherg, S., and Lind, C. (1981)

13. Emster, L., Danielson, L., and Ljunggren, M. (1962) Biochim.

14. Bellomo, G., Martino, A., Richelmi, P., Moore, G., Jewell, A., and Orrenius, S. (1984) Eur. J . Biochem. 140 , 1-6

15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275

16. Pertoft, H., and Smedsrbd, B. (1986) in Cell Separation (Pretlow, T. G., and Pretlow, T. B., eds) Vol4, pp. 1-24, Academic Press, New York

17. Winkler, L. W. (1888) Ber. Deutsche Chem. Ges. 2 1 , 2843-2854 18. Jauregui, H. O., Hayner, N. T., Driscoll, J. L., Williams-Holland,

R., Lipsky, M. H., and Galletti, P. M. (1981) I n Vitro (Rockuille)

19. Worthington, D. J., and Rosemeyer, M. A. (1974) Eur. J. Biochem. 48, 167-177

20. Gunzler, W. A., and Floh6, L. (1985) in CRC Handbook of Methods for Oxygen Radical Research (Greenwald, R. A., ed) pp. 285- 290, CRC Press, Boca Raton, FL

phys Acta 599,417-435

Arch. Biochem. Biophys. 207, 205-216

Biophys. Acta 58, 171-188

17, 1100-1110

21. Jones, D. P. (1981) J. Chromatogr. 225 , 446-449 22. Dethmers, J. K., and Meister, A. (1981) Proc. Natl. Acad. Sci.

23. Rossi, L., Moore, G . A., Orrenius, S., and O’Brien, P. J. (1986) Arch. Biochem. Biophys. 251 , 25-35

24. Starke, P. E., and Farber, J. L. (1985) J. Bid. Chem. 260,10099- 10104

25. Ross, D., Thor, H., Threadgill, M. D., Sandy, M. S., Smith, M. T., Moldeus, P., and Orrenius, S. (1986) Arch. Biochem. Bio-

26. Akerboom, T., Bilzer, M., and Sies, H. (1982) F E B S Lett. 140,

27. Swallow, A. J. (1982) in Quinones in Energy Conserving Systems,

28. Butler, J., and Hoey, B. M. (1986) J. Free Radicals Biol. & Med.

29. Bailey, S. I., and Ritchie, I. M. (1985) Electrochimica Acta 30, 3-

30. Koppenol, W. H., and Butler, J. (1985) Adu. Free. Radicals Biol.

31. d’Arcy Doherty, M., Rodgers, A., and Cohen, G. M. (1987) J.

32. Mukherjee, T. (1987) Radiat. Phys. Chem. 29, 455-462 33. Land, E. J., Mukherjee, T., and Swallow, A. J. (1983) J. Chem.

U. S. A. 78,7492-9496

phys. 248,460-466

73-76

pp. 59-72, Academic Press, New York

2, 77-81

12

& Med. 1,91-131

Appl. Toricol. 7, 123-129

SOC. Faraday Trans. 179, 391-415