[CANCER RESEARCH 47, 2363-2370, May 1, 1987]

Role of Metabolism and Oxidation-Reduction Cycling in the Cytotoxicity ofAntitumor Quinoneimines and Quinonediimines1

Garth Powis,2 Ernest M. Hodnett, Kenneth S. Santone,3 Kevin Lee See, and Deborah C. Melder

Department of Pharmacology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 [G. P., K. S. S., K. L. S., D. C. M.] and Department of Chemistry, OklahomaState University, Stillwater, Oklahoma 74078 ¡E.M. HJ

ABSTRACT

Quinone(di)iinines are nitrogen analogues of quiñonesin which one orboth quinone oxygens are replaced by an ¡minogroup. A series ofquinone(di)imines with antitumor activity has been studied for its in vitrochemical reactivity, metabolism, acute toxicity to primary cultured rathepatocytes, and growth-inhibitory activity with Chinese hamster ovary(CHO) cells. The quinone(di)iniines exhibited a wide range of activity assubstrates for metabolism by hepatic microsomal flavoenzymes. Themaximum rate of quinone(di)imine metabolism was more than 7.5-foldgreater than reported for metabolism of quiñones. Some qui-none(di)imines formed free radicals that could be detected by electronspin resonance spectroscopy. 2-Amino-l,4-naphthoquinoneimine gave ashort-lived electron spin resonance signal that could be detected onlyunder aerobic conditions. 2,3',6-Trichloroindophenol gave an electron

spin resonance signal in air that was stable for 24 h. Most qui-none(di)imines underwent oxidation-reduction cycling to form the super-oxide aniónradical, but some quinone(di)imines, although rapidly metabolized, formed little or no Superoxide aniónradical. Quinone(di)imineswere relatively toxic to hepatocytes and CHO cells, and some qui-none(di)imines were more toxic to one cell type than the other. The log1-octanol/water partition coefficient showed an optimal value of 2.61 fortoxicity against both cell types. In hepatocytes the more toxic qui-none(di)imines were the most rapidly metabolized. For a subgroup ofquinone(di)imines toxicity to hepatocytes and CHO cells appeared to berelated to the ability to form a semiquinone(di)imine free radical. Toxicityof quinone(di)imines to hepatocytes and CHO cells was not related toSuperoxide aniónradical formation, and toxicity to CHO cells was notaffected by exclusion of oxygen during exposure of the cells to thecompounds. The rate of chemical addition of quinone(di)imines to reducedglutathione did not correlate with toxicity. An understanding of themechanisms of acute toxicity and growth-inhibitory activity of qui-none(di)imines could lead to the design of more selective quinonoidantitumor agents.

INTRODUCTION

Quiñonesoccur widely in nature and have been extensivelystudied for their cytotoxic and antitumor properties ( 1). Someof the most useful agents for the treatment of human cancerare quiñones(2, 3). Several mechanisms have been proposed toaccount for the cytotoxic properties of quiñones. Because oftheir electrophilic properties quiñonescan react directly withcellular nucleophiles, including soluble and protein thiol groups(4, 5), and may inhibit critical processes in the cell. Somequiñonesintercalate between the base pairs of DNA leading toblockage of DNA, RNA, and protein synthesis (6) while otherquiñonesstabilize binding of nuclear topoisomerase II to DNAresulting in protein-associated DNA strand breaks (7, 8). Critical membrane functions can be altered by quiñones(9, 10). Amechanism receiving extensive study that might explain the

Received 9/2/86; revised 11/13/86, 1/21/87; accepted 1/23/87.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Grant CA33712.2To whom requests for reprint should be addressed, at Department of Phar

macology, Mayo Clinic and Foundation, 200 First Street, S.W., Rochester, MN55905.

' Recipient of NIH Training Grant CA0944I.

cytotoxicity of quiñonesis metabolism to form reactive species(11-14).

Quiñones are metabolized by flavoproteins that catalyzeeither one- or two-electron reduction. Single-electron reductionof quiñones is catalyzed by flavoenzymes such as NADPH-cytochrome P-450 reducÃ-ase(EC 1.6.2.4), NADH-cytochrome¿»sreducÃ-ase (EC 1.6.22), or xanthine oxidase (EC 1.2.3.2),resulting in the formation of a reactive semiquinone free radical.The semiquinone free radical can bind directly to DNA, protein,and lipid (IS, 16) or transfer an electron to a sensitive site inthe cell (17). Under aerobic conditions the semiquinone freeradical is most likely to react with molecular oxygen to formthe Superoxide anión radical (18). In this process the parentquinone is regenerated leading to oxidation-reduction cyclingof the quinone (19). The Superoxide anión radical undergoesspontaneous or enzymatic dismutation to form hydrogen peroxide which, in the presence of trace amounts of iron, reactswith more Superoxide anión radical to form hydroxyl radical(20). Some semiquinone free radicals may react directly withhydrogen peroxide to form hydroxyl radical (21). The hydroxylradical is a powerful oxidizing species that can damage anumber of cellular macromolecules (22) and may lead to celldeath (23). A consequence of oxidation-reduction cycling of aquinone is oxidative stress to the cell with depletion of cellularreduced pyridine nucleotide and/or formation of reactive oxygen species producing depletion of intrat-ellular thiols; this mayaffect Ca2+ homeostasis and lead to eventual cell death (24).Two-electron reduction of quiñones is catalyzed by enzymessuch as NAD(P)H:(quinone-acceptor)oxidoreductase (quinonereducÃ-ase,EC 1.66.9.2) and xanthine oxidase (EC 1.2.3.2) (25,26). Although two-eleclron reduclion of quiñonesmighl leadto the formation of species that bind covalently to criticalmacromolecules (11, 14, 15), it is generally believed to offer acellular protective mechanism against quinone toxicity. Thisoccurs by diverting quiñonesfrom electrophilic attack or oxidation-reduction cycling, and converting them to hydroqui-nones suitable for conjugation with glucuronic acid or sulfatefor export from the cell (24, 27). There remain several unanswered questions relating to the cytotoxicity of quiñones.Thefirst is whether direct chemical reaction or metabolism is moreimportant for cytotoxicity. A second question is whether metabolism, perhaps involving formation of reactive drug intermediates, or oxidation-reduction cycling with formation ofreactive oxygen species is more important for cytotoxicity. Athird question is whether there is a difference in the mechanismfor nonspecific cytotoxicity and antitumor activity of quininoidcompounds and, if so, whether this can be exploited to developmore selective antitumor quininoid compounds.

Quinoneimines and quinonediimines (hereafter referred to asquinone(di)imines) are nitrogen analogues of quiñoneswhereone or both quinone oxygens are replaced by an imino group.Quinone(di)imines have been shown to possess antitumor activity in animal models (28-31). A quinoneimine with activityagainst human cancer is actinomycin D (32), while 9-hydrox-yellipticine can be converted by metabolism to a quinoneimine

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TOXICITY OF QUINONEIMINES AND QUINONEDIIMINES

(33). Quinone(di)imines have many of the same chemical properties as quiñonesincluding the ability to undergo single-electron reduction to a free radical (34). However, little is knownof the biochemistry of quinone(di)imines and whether theyundergo enzymatic reduction and oxidation-reduction cycling.

We have examined the ability of a series of antitumor qui-none(di)imines to undergo enzymatic reduction and to formreactive oxygen species. In the course of this work we found awide range of metabolism among different quinone(di)iminesand identified some quinone(di)imines which, although extensively metabolized, did not form reactive oxygen species. Thesecompounds presented as with the opportunity to study thecontribution of direct electrophilic attack, metabolism, oxidalive stress, and oxygen radical formation to the cytotoxicity ofquinonoid compounds. Primary cultures of rat hepatocytes wereused to measure the nonspecific acute cytotoxicity of qui-none(di)imines and a Chinese hamster ovary cell line to measurethe growth-inhibitory activity.

MATERIALS AND METHODS

Quinoneimines and quinonediimines4 were synthesized as previouslydescribed (29, 31). /VyV'-Dichloro-2-sulfonicacid-l,4-benzoquinonedi-

imine (NSC 34493) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute(Bethesda, MD). NADPH, NADH, ferricytochrome c, Superoxide dis-mutase, crystalline bovine serum albumin, epinephrine, 2,6-dichloroin-dophenol, and scopoletin (7-hydroxy-6-methoxy-coumarin) were purchased from Sigma Chemical Co., St. Louis, MO. Monobromobimane(Thiolyte) was purchased from Calbiochem Biochemicals, San Diego,CA. Acetylated ferricytochrome c was prepared from ferricytochromec by the method of Azzi et al. (35).

Male rats of the Sprague-Dawley strain (Sprague-Dawley, Madison,WI) weighing 150 to 200 g, allowed free access to food and water, wereused for all studies. Hepatic microsomes were prepared from a homog-enate of rat liver in 4 volumes of 0.25 M sucrose by differentialcentrifugation as described by Ernster et al. (36). The microsomes werewashed once in 0.15 M KCI and suspended in 0.15 M KCI at aconcentration of 10 mg protein/ml. Protein was assayed by the dyebinding method of Bradford (37) using a commercial test kit (BioradLaboratories, Richmond, CA) and crystalline bovine serum albumin asa standard. Hepatocytes were prepared by perfusing the liver with lowCa2* medium and collagenase as previously described (19). Hepatocyte

viability immediately after isolation was determined by trypan blueexclusion and was routinely greater than 90%. Chinese hamster (HA-1) cells were obtained from the laboratory of Dr. George Hahn, StanfordMedical Center, Stanford, CA, and maintained as bulk culture mono-layers in multiple 75-cm2 flasks containing EMEM5 (Gibco, Grand

Island, NY). The medium was changed 3 times per wk, and cells in

'The compounds used in the study are: 1, W-bromo-l,4-benzpquinoneimine;2, 2.6-dibromo-/V-chloro-l,4-benzoquinoneimine; 3, 1,4-benzoquinone oxime; 4,2-methoxy-l,4-benzoquinone oxime; 5, 3-bromo-l,4-benzoquinone oxime; 6, 2-amino-l,4-naphthoquinoneimine hydrochloride; 7, jV,/V"-diacetyl-2-amino-l,4-naphthoquinoneimine; 8, /VjV-dimethylindoaniline; 9, 2-acetamido-A'JV-dimeth-ylindoaniline; 10, indophenol. sodium salt; II, 2,3',6-trichloroindophenol, sodium salt; 12, 2,6-dichloroindophenol trifluoroacetate; 13. yvyV'-dichloro-l,4-benzoquinonediimine; 14, /VyV-dichloro-2-methoxy-1,4-benzoquinonediimine;15. yVJV-dichloro-2-chloro-1,4-benzoquinonediimine; 16, AyV'-dichloro-2-nitro-1,4-benzoquinonediimine; 17, AyV'-dichloro-2-sulfonic acid-1,4-benzoquinonediimine; 18, /VyV'-dibromo-I.4-benzoquinonediimine; 19, /V^V'-dibromo-2-methyl-1,4-benzoquinonediimine; 20, A'JV'-dibromo-2-methoxy-1,4-benzoquinonediimine; 21, yVJV'-dibromo-2-chloro-l,4-benzoquinonediimine; 22, /V^V'-dibromo-2-nitro-l,4-benzoquinonediimine. Menadione is 2-methyl-l,4-naphtho-quinone. Diaziquone is 2,5-bis(l-aziridinyl)-3,6-diazo-l,4-cyclohexadiene-l,4-diyl-bis(carbamic acid)diethyl ester.

'The abbreviations used are: EMEM, Eagle's minimal essential medium

supplemented with 10% fetal calf serum, 100 Mgof streptomycin/ml, and 2 mMglutamine; CHO, Chinese hamster ovary: ESR, electron spin resonance; EC»,effective concentration required to release 50% of hepatocyte intracellular láclatedehydrogenase above background release in the absence of compound; 1C»,50%inhibitory concentration; MR, molar refraction with dimensions of volume.

exponential growth were passaged each wk for a maximum of 15 wkusing medium containing 0.05% trypsin and 0.01% EDTA. The CHOcell line was Mycoplasma free by culture (Virology Laboratory, MayoClinic).

Microsomal metabolism of quinone(di)imines was measured by theinitial rate of oxidation of NADPH or NADH at 340 nm in anincubation mixture containing 300 fimol Tris-HCl buffer, pH 7.4, 15Minol MgCh, 0.3 jimol EDTA, and either 0.1 mg or 1 mg microsomalprotein, all in a final volume of 3 ml at 37°C.The quinone(di)imines,

dissolved in 10 M'dimethyl sulfoxide, were added 30 s before additionof 3 Mmol NADPH or NADH dissolved in 10 >/lwater. The dimethylsulfoxide had no effect upon NADPH or NADH oxidation. Metabolismwas corrected for the slow background rate of oxidation of NADPHand NADH by quinone(di)imine in the absence of microsomes. Super-oxide anión radical formation was measured as the difference in theinitial rate of reduction of acetylated ferricytochrome c at 550 nm, inthe presence and absence of Superoxide dismutase, 33 /¿g/ml,using anextinction coefficient of 19.6 mM"' cm"' (35). The incubation mixture

contained, in addition to the components described previously, 60 MMacetylated ferricytochrome c. In a few studies Superoxide aniónradicalformation was measured by following the oxidation of 1 mivi epinephrine to adrenochrome at 480 nm, using an extinction coefficient of 4.02mivr1 cm"1 (38). Superoxide anión radical release by freshly isolatedhepatocytes in suspension at IO6 cells/ml of Dulbecco's phosphate-

buffered saline containing 10 mM glucose was measured by the reduction of acetylated ferricytochrome c, 60 MMat 37'C, in the presence

and absence of Superoxide dismutase, 33 ng/ml (19). Microsomalhydrogen peroxide formation was measured at room temperature bythe decrease in fluorescence of scopoletin as described by Thurman etal. (39), without added azide. Oxygen utilization by microsomal incubations and by freshly isolated hepatocytes was measured at 37°Cusing

a Clark oxygen electrode (Yellow Springs Instrument Company, YellowSprings, OH).

The reaction of quinone(di)imines with reduced glutathione wasdetermined by measuring the decrease in the concentration of reducedglutathione with time. Quinone(di)imine dissolved in 20 ¿ildimethylsulfoxide giving a final concentration of 0.2 mM was added to a solutionof Dulbecco's phosphate-buffered saline, pH 7.4, containing 0.2 mM

reduced glutathione. The mixture was allowed to react at room temperature, and samples were taken at 0, 1, 2, 5, 15, 30, and 60 min.Reduced glutathione was measured by a modification of the method ofFahey et al. (40), in which reduced glutathione was reacted withmonobromobimane to form a stable, fluorescent adduci that could beseparated by reversed-phase high-performance liquid chromatography.Disappearance of reduced glutathione was fitted to a mono- or biex-ponential decay curve using the NONLIN nonlinear least-squaresregression analysis program (41), and rate constants were calculated.

ESR measurements were made with an IBM-ER200 spectrometer

equipped with a TM cylindrical cavity. Instrument settings were: fieldset, 3485 G; microwave frequency, 9.81 GHz; modulation/receiverfrequency, 100 kHz; microwave attenuation, 12 dB; detector current,200 n\. g-Values were calculated against 2,2-diphenyl-l-picrylhydrazylas a standard at g = 2.0036. Incubations were conducted in a quartzflat cell under anaerobic or aerobic conditions at room temperaturewith a system containing 15 ^mol quinone(di)imine, 3 mg microsomalprotein, 3 iimol NADPH, 30 /imol glucose 6-phosphate, 3 units glucose-6-phosphate dehydrogenase, 15 /jmol MgCb, and 150 /¿molpotassiumphosphate, pH 7.4, in a final volume of 3 ml.

Nonspecific toxicity of quinone(di)imines was measured by the leakage of cytosolic lactate dehydrogenase from primary cultures of rathepatocytes over a range of quinone(di)imine concentrations as previously described (42). Results were expressed as the ECso.

Growth inhibitory activity of quinone(di)imines was measured as theinhibition of colony formation by CHO cells. CHO cells in log-phasegrowth were plated in 25-cnr plastic culture flasks at multiple densitiessuch that final counts of between 100 and 200 colonies/flask wereobtained following exposure to compounds. Flasks containing cells and5 ml EMEM were maintained at 37"C in an incubator with 5%

CO2:95% air at 100% relative humidity for 24 h to allow attachmentof cells. Medium was decanted and replaced with 5 ml of fresh warmed

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TOXICITY OF QUINONEIMINES AND QUINONEDIIMINES

medium. The flasks were then gassed for 30 min with either sterilehumidified 5% CO2:95% air or 5% CO2:95% N2 through needlesinserted through a silicone seal in the cap of the flask. After this timethe flasks were placed in the incubator at 37*C for a further 2 h to

allow metabolism by the cells to remove residual dissolved oxygen inmedium. Quinone(di)imine, dissolved in 10 n\ dimethyl sulfoxide, wasinjected anaerobically through the cap seal. After 4-h exposure toquinone(di)imine at 37°C,medium was removed and the flasks were

washed S times with S ml of fresh, warmed growth medium beforeallowing the cells to grow for 10 days at 37'C in incubators with 5%

CO2:95% air at a relative humidity of 100%. Exposure of CHO cells toanaerobic conditions for this time had no effect upon colony formation.After 10 days, medium was removed and flasks were washed with warm0.9% NaCI solution. Colonies were stained with 0.2% crystal violet inmethanol for 10 min, rinsed with tap water, and counted manually.Quadruplicate flasks were used for each quinone(di)imine concentration. Colony formation data were Fitted to a monoexponential survivalcurve using the NONLIN nonlinear least-squares regression analysisprogram (41 ). Variance of the quinone(di)imine concentration requiredto produce 1C,,, was obtained from the variance of the intercept andslope using a Taylor series expansion. Dose-response curves wererepeated at least 3 times. Groups of data were compared using Student's

t test (43).Correlation of data with structural parameters was conducted as

previously described (29) using various procedures of the StatisticalAnalysis Systems (44). The structural variables included the /•'and K of

Swain and Lupton (45), which indicate the ability of substituents towithdraw or donate electrons by a general field effect (/•')or a resonanceeffect (R). Appropriate /•'and H values for substituents attached to the

quinonoid ring were totalled to obtain composite values for eachcompound. MR was used as a measure of size of the substituentsattached to the quinonoid ring. Values of MR, I', and R are those

published by Mansch et al. (46). Log P of the 1-octanol/water partitioncoefficient of each compound was determined as previously described(47) for some of the compounds or calculated for closely relatedcompounds by use of the substituent constant of Mansch et al. (46).Log P values for the compounds used in the toxicity studies were asfollows: Compound 1, 1.12; Compound 3, 1.08; Compound 6, 1.47;Compound 8, 2.02; Compound 9, 0.96; Compound 11, 4.10; Compound 12, 3.14; Compound 13, 0.80; Compound IS, 1.54; and Compound 11,-0.90.

RESULTSMicrosoma] Metabolism. Quinone(di)imines exhibited a wide

range of activity as substrates for metabolism by hepatic micro-somal NADPH-cytochrome P-450 reducÃ-ase and NADH-cy-tochrome bs reducÃ-ase(Fig. 1). In general quinoneimines exhibited a faster rate of metabolism than quinonediimines. The

10-B

810'S IO1" Î03 IO410°"

10' W3

Superoxide formation (nmol/min/mgÃ-

Fig. 1. Metabolism of quinoneimines and quinonediimines, and Superoxideaniónradical formation by the hepatic microsomal fraction, I. with NADH ascofactor, and II. with NADPH as cofactor. Metabolism was measured as theoxidation of reduced pyridine nucleotide. Quinone(di)imines (see Footnote 4)were added to hepatic microsomal suspension at a concentration of Io J \i. Thecontinuous lines are computer-generated regressions. For NADH, r = 0.653 (P< 0.01 ). Compounds 8 and / / which formed no Superoxide with NADH (indicatedby arrows) were omitted from this analysis. For NADPH, r = 0.577 (P < 0.01).Compounds 8, 12, and 15 which formed no Superoxide with NADPH (indicatedby arrows) were omitted from this analysis.

most rapid metabolism was seen with 2-amino-l,4-naphthoqui-noneimine, AVV-diacetyl-2-amino-1,4-naphthoquinoneimine,yvyV-dimethylaniline, indopheno!, and 2,3',6-trichloroindo-

phenol (Compounds 6, 7, 8, 10, and 11, respectively). The Kmof 2-amino-l,4-naphthoquinoneimine with NADPH as cofactorwas 5.2 fi\i and with NADH as cofactor, 19.6 pM. The maximum rate of metabolism of quinone(di)imines with NADPHor NADH as cofactor was more than 7.5-fold greater than seenwith simple quiñonesunder similar conditions (19, 48).

The ability of quinone(di)imines to undergo single-electronreduction by microsomal flavoenzymes and to transfer an electron to oxygen to form Superoxide anión radical was alsostudied. The Km for 2-amino-l,4-naphthoquinoneimine-de-pendent Superoxide anión radical formation with NADPH ascofactor was 1.8 /¿Mand with NADH as cofactor, 30.3 UM.There was a significant positive correlation between metabolismof quinone(di)imines and the formation of Superoxide aniónradical, with both NADPH and NADH as cofactor (Fig. 1).Some quinone(di)imines clearly did not fit this relationship andunderwent rapid metabolism but formed little or no Superoxideanión radical. They were /V,A'-dimethylindolaniline, indo-phenol, 2,3',6-trichloroindophenol, 2,6-dichloroindophenoltrifluoroacetate, and Ar,A^'-dichloro-2-chloro-l,4-benzoquino-

nediimine (Compounds 8, 10, 11, 12, and 15). The compoundswere not being metabolized by microsomal DT-diaphorase asshown by the inability of 30 UM dicumarol, a potent inhibitorof DT-diaphorase (49), to inhibit metabolism (results notshown).

Studies were conducted with selected quinone(di)imines tomeasure Superoxide anión radical formation using an alternative assay, the oxidation of epinephrine, to rule out interferenceby the quinone(di)imines of the reduction of acetylated cyto-chrome c by Superoxide anión radical. Epinephrine oxidationis not an ideal way to measure Superoxide anión radical formation because the product, adrenochrome, may be reducedback to epinephrine by the microsomal system (38). It does,however, provide qualitative confirmation of the presence ofSuperoxide anión radical formation measured by acetylatedcytochrome c reduction. The effect of quinone(di)imines onmicrosomal hydrogen peroxide formation and oxygen utilization was also studied (Table 1). Quinone(di)imines that wererapidly metabolized but produced little or no Superoxide aniónradical as measured by reduction of acetylated ferricytochromec also failed to oxidize epinephrine. Furthermore, the samequinone(di)imines did not stimulate microsomal oxygen utilization or microsomal hydrogen peroxide formation. Qui-none(di)imines that were rapidly metabolized and formed su-peroxide anión radical by both assays also formed hydrogenperoxide, probably by dismutation of the Superoxide aniónradical (22), and stimulated microsomal oxygen utilization.

ESR Studies. The ability of quinone(di)imines to form a freeradical when incubated with hepatic microsomes and NADPHwas studied by ESR spectroscopy. 2-Amino-l,4-naphthoqui-noneimine (Compound 6) gave a strong ESR signal, g = 2.0037,under anaerobic conditions but gave no signal under aerobicconditions (Fig. 2). The signal reached a maximum at 5 minand had disappeared by 1 h. A'-Bromo-1,4-benzoquinoneimine

(Compound 1) also gave a weak ESR signal under anaerobicconditions but not under aerobic conditions. 2,3',6-Trichloroin-

dophenol (Compound 11) gave an ESR signal, g = 2.0060, withhyperfine splitting under both aerobic and anaerobic conditions(Fig. 2). The signal reached a maximum at 5 min and was stilldetectable after 24 h in air. 2,6-Dichlorophenolindophenol trifluoroacetate (Compound 12) also gave a weak ESR signal

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TOXICITY OF QUINONEIMINES AND QUINONEDIIMINES

Table 1 Quinarte(di)imine-dependent microsomal oxygen metabolismThe compounds studied were: 1, A bromo-1.4 bcrmiquinoneimine; 6, 2-amino-l,4-naphthoquinoneimine hydrochloride; 8, A'.,Vdimcihvliniloanilinc: 9, 2-

acetamido-yVJV-dimethylindoaniline; 11, 2,3',6-trichloroindophenol; and 12, 2,6-dichloroindophenol trifluoroacetate. NADPH was the cofactor for all the studies.The quinone(di)iinines were used at a concentration of Id 4 M, except for assay of H2O2 where there was interference with the assay and a quinone(di)imineconcentration of 3 x 10~7 M was used. Superoxide aniónradical (Or) formation was measured using reduction of acetylated ferricytochrome c (acytochrome c) or

epinephrine oxidation.

TOXICITY OF QUINONEIMINES AND QUINONEDHMINES

Table 2 Toxicity of quinone(di)imines to cultured rat hepatocytes and their effects on microsomal metabolism and hepatocyte cellular oxygen metabolismEC»values are for the release of lactate dehydrogenase. NADH was the cofactor used for microsomal metabolism studies. Quinone(di)imines were added at a

concentration of 10 4M to microsomes or to suspensions of freshly isolated hepatocytes. Oxygen utilization was measured in the presence of 25 MMantimycin A. Thecompounds used were: 1, jV-bromo-l,4-benzoquinoneimine; 3, 1,4-benzoquinone oxime; 6, 2-amino-l,4-naphthoquinoneimine hydrochloride; 8, ,V.,Viliniellivi induanitine; 9, 2-acetamido-yVJV-dimethylindoaniline; 11, 2,3',6-trichloroindophenol; 12, 2,6-dichlorophenolindophenol trifluoroacetate; 13, /V,A"-dichloro-l,4-benzoqui-nonediimine; I5, /VJV'-dichloro-l-chloro-1,4-benzoquinonediimine; and 17,,V,\" -dich loro 2 sul ton k: acid-1,4-benzoquinonediimine. Quinone(di)imines were divided

into two groups, toxic (K(\„< 100 Mg/ml) and nontoxic (ECM > 100 fig/ml), and differences were analyzed by the rank sum test to obtain P (43) and by stepwisediscriminant analysis (58).

Microsomal metabolism Hepatocyte metabolism

CompoundNontoxic1

31712EC»(Mg/ml)61

1.7 ±124.3°

306.7 ±46.81000*

202.7 ±10.1NADH

oxidation(nmol/min/mg)245.1

±18.750.4 ±3.9

170.3 ±6.5279.9 ±21.9O2'

formation

(nmol/min/mg)108.2

±2.912.2 ±1.043.6 ±4.142.6 ±7.8Or

formation(nmol/min/106cells)0.69

±0.031.47 ±0.301.05 ±0.120.00 ±0.00O2

utilization(nmol/min/lO*cells)20.1

±1.915.4 ±1.513.1 ±0.0313.8 ±0.5

Toxic689

111315

2.2 ±0.334.9 ±4.524.3 ±7.171.3 ±0.317.7 ±1.917.7 ±1.5

1208.5 + 65.61827.6 ±89.41940.1 ±29.72852.6 ±115.7

811.6 + 43.7211.8 ±8.3

0.50

4.80 ±0.060.00 ±0.000.45 ±0.090.24 ±0.020.45 ±0.030.84 ±0.30

>0.50

106.3 + 5.677.5 + 5.473.1 ±0.722.3 ±0.319.4 ±1.014.7 ±0.6

>0.50" Mean ±SE of 3 determinations.b Highest concentration tested.

Table 3 Quinone(di)imine inhibition of colony formation by Chinese hamsterovary cells in culture

!('

TOXICITY OF QUINONEIMINES AND QUINONEDIIMINES

the fast reaction probably involves a one-electron reduction ofthe quinone(di)imine, since resonance would help to stabilizethe semiquinone(di)imine of these compounds. It should benoted also that the structures of compounds in Group A havearomatic moieties that would stabilize the semiqui-none(di)imine by delocalizing a lone electron. Toxicity of GroupA compounds to hepatocytes and CHO cells showed a weakcorrelation with K¡of r = 0.806 and 0.829 (P < 0.10, > 0.05in both cases), respectively. When K2 was compared with structural parameters the closest correlation was with the field effectF (r = 0.85, P < 0.05). This indicates that the slow reaction isassisted by a general release of electrons in a reaction such as asubstitution reaction. K2 did not correlate with toxicity tohepatocytes or CHO cells. Compounds of Group A react biologically and chemically different to Compounds of Group B.Compounds of Group A gave little or no Superoxide aniónradical when metabolized by hepatic microsomes, whereas compounds of Group B formed relatively large amounts of super-

oxide aniónradical.Correlation of Toxicity with Structural Parameters. The tox

icity data were correlated with the structural variables F, R,MR, and log P. Only log P showed a significant correlationwith toxicity. With the addition of a (log P)2 term, the signifi

cance of the factor was further increased. For hepatocyte toxicity, EC50 = -395-log P + 74.3 (log P)2 + 537 with r = 0.81(P < 0.02). For CHO cell toxicity, IC50 = -490-log P + 91.9(log P)2 + 441 with r = 0.96 (P < 0.01). For both types of

toxicity, there was a maximum toxicity (minimum EC5oor IC50)at log/'=2.61.

DISCUSSION

The toxicity of simple quiñonessuch as menadione to hepatocytes has been attributed to oxidation-reduction cycling ofthe quinone with depletion of mitochondrial reduced pyridinenucleotides and oxidation of soluble and protein thiols (24, 51).There follows altered intracellular Ca2+ homeostasis which may

be an early event in quinone cytotoxicity. Quiñonesalso reactdirectly with soluble thiols, and this could be an additionalfactor in their cytotoxicity (50). So far it has not been possibleto distinguish between a direct reaction of quiñoneswith cellularnucleophiles, metabolism to the semiquinone free radical, depletion of reduced pyridine nucleotide, formation of reactiveoxygen species, or a combination of these processes as mechanisms for quinone toxicity.

Quinone(di)imines have similar chemical properties to quiñones(34) and might be expected to undergo metabolism inthe same way as quiñones.We have previously reported that N-acetyl-/>-benzoquinoneimine, the putative hepatotoxic metabolite formed from acetaminophen, undergoes reduction byNADPH-cytochrome P-450 reducÃ-aseprobably to a semiqui-noneimine free radical (52). However, W-acetyl-p-benzoqui-noneimine does not undergo oxidation-reduction cycling anddoes not stimulate Superoxide aniónradical formation or oxygen utilization. To our knowledge the only other nonhetero-cyclic quinoneimine whose metabolism has been studied is 2,6-dichloroindophenol. Although originally considered to undergotwo-electron reduction by microsomal NADPH-cytochrome P-450 reducÃ-ase(53), 2,6-dichloroindophenol has more recentlybeen shown to form an ESR-deteclable free radical, presumablyihe semiquinoneimine, when reduced by a NADPH-forlifiedmicrosomal preparalion (54). 2,6-Dichloroindophenol does notundergo oxidation-reduclion cycling to form Superoxide aniónradical (55). The results with yV-acelyl-p-benzoquinoneimine

and 2,6-dichloroindophenol suggested thai quinoneimines as agroup mighl noi undergo oxidation-reduclion cycling in ihesame way as quiñones. jV-Acetyl-p-benzoquinoneimine readsrapidly wilh reduced glulalhione (56), and il has been suggesledlhal the hepaloloxicity of Ar-acelyl-/?-benzoquinoneimines is due

lo a direcl chemical reaction with depletion of intracellularreduced glutalhione and oxidalion of prolein thiol groups by./V-acelyl-p-benzoquinoneimine. This raised Ihe possibility thatthe toxicily of other quinone(di)imines mighl be due lo direclchemical reaclion wilh critical cellular nucleophiles. We wereinterested to see, iherefore, whelher quinone(di)imines could bemelabolized by flavoenzymes, whelher ihey formed free radicalsand slimulaled oxygen radical produclion, and how iheir lox-icily relaled lo metabolism and chemical reactiviiy. We choselo use a series of quinone(di)imines lhal has previously beenshown lo have in vivo aniiiumor aclivily againsl Sarcoma 180(28-30).

The quinone(di)imines exhibited a wide range of activily assubslrales for microsomal metabolism with the most activequinone(di)imines being considerably better substrales lhansimple quiñonesunder the same conditions (48). Many of thequinone(di)imines tesled formed Superoxide anión radical inproportion lo their rale of metabolism. The mean ratio ofSuperoxide aniónradical formation lo reduced pyridine nucleo-lide for quinoneimines was 0.92 and for quinonediimines, 0.61,compared to a theoretical ratio of 2. Some of ihe qui-none(di)imines gave ESR speclra when reduced by hepalicmicrosomes and NADPH, indicaling semiquinone(di)iminefree radical formalion. 2-Amino-l,4-naphlhoquinoneimineandA'-bromo-l,4-benzoquinoneimine gave ESR speclra only under

anaerobic condilions, and Ihe speclra disappeared when oxygenwas inlroduced inlo the medium. 2,3',6-Trichloroindophenol

and 2,6-dichloroindophenol trifluoroacelale gave slable signalsthat were not affecled by oxygen. In general, compounds wilha more aromalic structure give more slable free radicals. 2-Amino-l,4-naphthoquinoneimine, which has a naphthylmoiely, gave a slronger signal lhan Ar-bromo-l,4-benzoqui-noneimine, which has a less aromatic struclure. Bolh indophen-ols have a phenyl ring and gave ESR signals, bul Ihe signalwilh 2,6-dichloroindophenol irifluoroacetale was weaker, probably because the trifluoroacetale group holds Ihe eleclrons morelighlly, ihus reslricting their delocalizalion. The resulls of IheESR studies taken together wilh evidence of Superoxide aniónradical formalion suggesl lhal 2-amino-l,4-naphlhoquinoneim-ine and JV-bromo-l,4-benzoquinoneimine are reduced lo a semiquinoneimine free radical lhal, in Ihe presence of oxygen,undergoes oxidalion-reduclion cycling lo form the Superoxideaniónradical and regenerates Ihe pareni quinoneimine. 2,3',6-

Trichloroindophenol and 2,6-dichloroindophenol irifluoroace-lale, on ihe olher hand, are reduced lo a slable free radical lhatdoes not react, or only slowly, with oxygen lo form Ihe super-oxide aniónradical. This may be because ihe eleclronic chargeon ihe nilrogen alom of ihe semiquinoneimine of an indophenolis prolecled from allack by oxygen because of Ihe phenyl grouplhal il bears. The finding of quinone(di)imines lhal were melabolized lo semiquinone(di)imine free radicals lhal did or didnoi undergo oxidation-reduction cycling to form Superoxideaniónradical presented us with the opportunity of sludying Iherelationship belween chemical reaclivity, metabolism, free radical formalion, oxidalion-reduclion cycling, and ihe cyloloxicily

of quinone(di)imines.The quinone(di)imines were very toxic to cultured hepalo-

cyles. 2-Amino-l,4-naphthoquinoneimine was approximately10-fold more toxic to cultured hepatocytes than was menadione

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TOXICITY OF QUINONE1MINES AND QUINONEDHMINES

under similar conditions. Quinone(di)imines that were morerapidly metabolized in vitro exhibited the greatest toxicity tocultured hepatocytes. Our studies only measured metabolismby microsomal flavoenzymes, but it is likely that a similarpattern of quinone(di)imine metabolism will exist for otherflavoenzymes, such as mitochondria! NADH:ubiquinone oxi-doreductase, as has been reported for simple quiñones(19,48).Significantly, there was no association between acute toxicityof quinone(di)imines to hepatocytes and microsomal superox-

ide anión radical formation, hepatocyte Superoxide anión formation, or quinone(di)imine enhancement of oxygen utilizationby intact hepatocytes. This finding would appear to rule outformation of reactive oxygen species as a cause of the acutetoxicity of quinone(di)imines to hepatocytes.

Studies on growth inhibition of CHO cells by qui-none(di)imines showed no significant association between inhibition of colony formation and microsomal quinone(di)iminemetabolism or formation of reactive oxygen species. Exposureof CHO cells to quinone(di)imines under anaerobic conditionsproduced a small decrease in the activity of two quinoneimines(/V-bromo-l,4-benzoquinoneimine and 2-amino-l,4-naphtho-quinoneimine) that formed reactive oxygen species. This mightsuggest a contribution of reactive oxygen species to growthinhibition by these compounds, although several other quinone(di)imines that also formed reactive oxygen species wereunaffected by anaerobic conditions. Overall, the results suggestthat reactive oxygen species are not a major factor in the growthinhibition by quinone(di)imines. It was not possible to studythe toxicity of quinone(di)imines to cultured hepatocytes underanaerobic conditions, since hypoxia itself was acutely toxic tothe hepatocytes.6

Although the metabolism of quinone(di)imines appears to beimportant for their toxicity it is not possible, based on theresults of this study alone, to propose a mechanism for toxicity.Quinone(di)imines are likely to undergo both one- and two-

electron enzymatic reduction by cells. Phenylenediamines,which can be regarded as the fully reduced form of qui-none(di)imines, are considerably less cytotoxic to CHO cellsthan the parent quinone(di)imines. It is unlikely, therefore, thatthe products of two-electron reduction of quinone(di)imines arethe species responsible for cytotoxicity. In fact, analogy toquiñones(27, 57) would suggest that two-electron reduction ofquinone(di)imines by an enzyme such as quinone reducÃ-asemight protect cells against quinone(di)imine cytotoxicity. Qui-none(di)imines can be enzymatically reduced to the semiqui-none(di)imine free radical, but not all quinone(di)iminesundergo oxidation-reduction cycling to form reactive oxygenspecies. Failure to transfer an electron to oxygen to formSuperoxide aniónradical might be because of rapid dismutationof the semiquinone(di)imine free radical to parent quinone(di)imine and phenylene(di)imine (25), because the semi-quinone(di)imine free radical is relatively stable, or because ofother rapid electron transfer reactions. Wurster free radicalsformed by one-electron oxidation of phenylenediamines aregenerally much more stable than free radicals produced byoxidation of diols (54). As previously noted the free radicalformed by microsomal reduction of 2,3',6-trichloroindophenol

was stable for several hours in air and did not form Superoxideaniónradical.

The only significant relationship between a structural parameter and the toxicity of quinone(di)imines to either hepatocytesor CHO cells was with log P, the 1-octanol/water partition

coefficient. There was an optimum log P of 2.61 for both typesof toxicity. This suggests that the ability of quinone(di)imine tocross lipid membranes to reach critical sites within the cellwhile still retaining hydrophilicity is an important feature indetermining relative toxicity.

There was no correlation between the toxicity of qui-none(di)imines to hepatocytes or CHO cells and the rate ofadduct formation between quinone(di)imines and reduced glu-tathione. This appears to rule out direct addition of qui-none(di)imines to reduced glutathione leading to depletion ofcellular thiols as a general mechanism of quinone(di)iminetoxicity. It might, however, be an important mechanism forindividual quinone(di)imines, as has been suggested for N-acetyl-/>-benzoquinoneimine (56). We also cannot rule out that

quinone(di)imines exhibit a different pattern of chemical reactivity with other critical cellular sites that accounts for theirtoxicity. For a subgroup of quinone(di)imines, toxicity to hepatocytes showed a weak association with the ability to undergorapid reaction, probably electron transfer, with reduced glutathione, thus indirectly implicating the semiquinone(di)iminefree radical in the toxicity of these compounds. It may berelevant that this subgroup of quinone(di)imines did notundergo oxidation-reduction cycling and formed little or no

Superoxide anión radical when enzymatically reduced, whichmay explain why toxicity was more closely related to semiqui-none(di)imine free radical formation than the other qui-none(di)imines, where the semiquinone(di)imine free radicalwas destroyed by rapid reaction with oxygen.

A significant correlation was seen between quinone(di)iminehepatotoxicity and CHO cell toxicity which might suggest asimilar mechanism of toxicity in the two cell types. Alternatively, there could be a different mechanism of toxicity withentry of quinone(di)imines into the cell being the rate-controlling step. It should be noted that, despite the overall correlationbetween hepatocyte or CHO cell toxicity, some qui-none(di)imines differed in their toxicities predicted by thisgeneral relationship by up to an order of magnitude. Furtherstudy of these compounds might reveal a mechanism for thedifferential toxicity, which could lead to synthesis of compounds with selective growth-inhibitory activity toward tumorcells and less nonspecific toxicity for other cells.

In summary, we have found that there is a hierarchy of effectsrelating the toxicity of quinone(di)imines to their structure andmetabolism. A balance between lipophilicity and hydrophilicityis important for ensuring that the quinone(di)imines can crosscell membranes and gain access to critical cellular sites. Inhepatocytes, at least, the more toxic quinone(di)imines undergomore rapid metabolism. For a subgroup of quinone(di)imines,toxicity to hepatocytes and CHO cells is associated with theability to form the semiquinone(di)imine free radical. Toxicityof the quinone(di)imines does not correlate with oxidation-reduction cycling and formation of Superoxide aniónradical orwith their direct addition reaction with reduced glutathione.There is an overall correlation between toxicity of qui-none(di)imines to hepatocytes and CHO cells, but some compounds exhibited selective toxicity for one cell type or the other.

ACKNOWLEDGMENTS

6 Unpublished observations.The excellent secretarial work of Wanda Rhodes is gratefully ac

knowledged.

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