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[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.
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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.
2369
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TOXICITY OF QUINONEIMINES AND QUINONEDHMINES
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