[CANCER RESEARCH 46, 3528-3532, July 1986]
Generation of Reactive Oxygen Radicals through Bioactivation of
Mitomycin Antibiotics1
Chris A. Pritsos and Alan C. Sartorelli2
Department of Pharmacology and Developmental
Connecticut 06510
Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven,
the subsequent reaction of the semiquinone with molecular
oxygen to generate Superoxide and hydroxyl radicals (10-14).
Mitomycin C (MC) is a naturally occurring anticancer agent which
PM, BMY-25282, and BL-6783, are all structural analogues
has been shownto be more cytotoxicto hypoxictumor cells than to their of MC (Fig. 1). PM has greater preferential cytotoxicity to
aerobic counterparts. The mechanism of action of this agent is thought
to involvebiologicalreductiveactivation,to a speciesthat alkylutes DNA. hypoxic EMT6 cells than MC; this phenomenon is due to the
A comparison of the cytotoxicityof MC to EMT6 tumor cells with that fact that although PM is equivalent to MC in producing cyto
of the structural analogues porfiromycin(PM), .•V-(,V',Ai"-diiiielhylaniitoxicity to hypoxic cells, it is significantly less toxic to aerobic
nomethylene)amineanalogueof mitomycinC (BMY-25282),and ;\'-(.!\", cells (15). BMY-25282 and BL-6783, the porfiromycin coun
.V'-ilimeth>laniinoinctliylciie(aminoanalogue of porfiromycin(BL-6783) terpart of BMY-25282, have preferential cytotoxicity to aerobic
has demonstrated that PM is considerably less cytotoxic to aerobic EMT6 cells. BMY-25282 is 10 times more cytotoxic to aerobic
EMT6 cells than MC, whereas BMY-25282 and BL-6783 are signifi EMT6 cells and 4 times more cytotoxic to their hypoxic coun
cantly more toxic. The relative abilities of each of these compounds to terparts than MC.4
generate oxygen free radicals followingbiologicalactivation were mea
Because of the structural similarities of these 4 compounds,
sured. Tumor cell sonicates, reduced nicotinamideadenine dinucleotide we have examined the relative abilities of various biological
phosphate-cytochromec reducÃ-ase,
xanthine oxidase, and mitochondria
systems to reductively activate these antibiotics under aerobic
were used as the biologicalreducing systems. All four mitomycinanti
conditions
to generate oxygen free radicals and have compared
bioticsproducedoxygenradicals followingbiologicalreduction,a process
that may account for the aerobic cytotoxicityof agents of this class. The these activities with their cytotoxicities to oxygenated EMT6
generation of relame amounts of Superoxideand hydroxyl radical were tumor cells. In addition, the relative quantities of Superoxide
also measured in EMT6 cell sonicates. BMY-2S282and BL-6783 pro
and hydroxyl radical generated by each of the mitomycin anti
duced significantly greater quantities of oxygen free radicals with the biotics was measured using EMT6 cell sonicates. DiethyleneEMT6 cell sonicate, reduced nicotinamide adenine dinucleotide phos
triaminepentaacetic acid was included in these reaction mix
phate-cytochromec reducÃ-ase,
and mitochondriathan did MC and PM. tures as an iron chelator to minimize iron-catalyzed HaberIn contrast, BMY-25282and BL-6783 did not generate detectable levels Weiss-induced hydroxyl radical production. The findings dem
of free radicals in the presence of xanthine oxidase, whereas this enzyme
onstrated a correspondence between the toxicity of these agents
was capable of generating free radicals with MC and PM as substrates.
EMT6 tumor cells under aerobic conditions and their capac
MC consistentlyproducedgreater amounts of free radicals than PM with to
ity
to generate oxygen-containing radicals following reduction
all of the reducingsystems. BMY-25282,BL-6783,and MC all generated
by
sonicates
of these cells.
hydroxyl radicals, while PM did not appear to form these radicals. The
findings indicate that a correlation exists between the ability of the
mitomycinantibiotics to generate oxygen radicals and their cytotoxicity
MATERIALS AND METHODS
to aerobic EMT6 tumor cells.
ABSTRACT
INTRODUCTION
MC3 is an antibiotic isolated from Streptomyces caespitosus
which has been shown to be active against a broad spectrum of
animal (1) and human (2) tumors. Furthermore, this agent has
been shown to cause preferential cytotoxicity toward hypoxic
tumor cells relative to their oxygenated counterparts both in
vitro (3-5) and in vivo (6, 7). Studies on the molecular mecha
nism of action of MC have shown that biological activation to
a reactive species is required to produce ¡Ministramicrosslinking of DNA (8). While this appears to be the most plausible
mechanism for the hypoxic cell toxicity of this agent, the futile
oxygen cycling that occurs under aerobic conditions makes it
likely that oxygenated cell toxicity involves reduction to the
semiquinone rather than to the fully reduced species (9), and
Received 9/30/85; revised 3/7/86; accepted 4/2/86.
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.
1This research was supported in part by Grant CH-211 from the American
Cancer Society and USPHS Fellowship CA-07648 from the National Cancer
Institute.
2To whom requests for reprints should be addressed.
3The abbreviations used are: MC, mitomycin C; BL-6783, N-(N',N'-dimethylaminomethylene)amine analogue of porfiromycin; BMY-25282, N-(N',N'-aimethylaminomethylene)amine analogue of mitomycin C; PBS, phosphate-buff
ered saline; PM, porfiromycin; HPLC, high performance liquid chromatography.
MC, PM, BMY-25282, and BL-6783 were provided by Drs. T. W.
Doyle and D. M. Vyas of Bristol Laboratories (Syracuse, NY). Beef
heart mitochondria were generously supplied by Dr. R. S. Pardini
(University of Nevada, Reno, NV). NADPH-cytochrome c reductase
was purified from phenobarbital-treated rabbit liver as previously de
scribed (16). All other materials were purchased from commercial
sources: 5,5-dimethyl-l-pyrroline TV-oxideand diethylenetriaminepentaacetic acid were purchased from Aldrich Chemical Co. (Milwaukee,
WI); acetonitrile (HPLC grade) was from J. T. Baker Chemical Co.
(Phillipsburg, NJ); succinicacid (disodium salt), NADH, NADPH, and
xanthine oxidase (Grade 1 from buttermilk) were from Sigma Chemical
Co. (St. Louis, MO); and fetal bovine serum and Waymouth's medium
were from Grand Island BiologicalCo. (Grand Island, NY).
EMT6 mouse mammary tumor cells were grown at 37°Cin Waymouth's medium supplemented with 15% fetal bovine serum and anti
biotics in a humidified atmosphere of 95% air-5% COj. The character
istics of this cell line have previouslybeen described (17). Cell sonicates
were prepared from exponentially growing monolayer cultures by ex
posing them to 0.05% trypsin in PBS to remove cells from the flasks,
washing with cold PBS, and subsequently resuspending them in the
appropriate buffer system. The cells in an ice bath were disrupted by
three 5-s bursts with a Branson Model 160 sonicator set at 25% of
maximum intensity. Cellular disruption was verified microscopically.
Cytotoxicity studies were performed on EMT6 tumor cells growing
exponentially in 25-cm2Corning plastic culture flasks under the con
ditions described above. Cells were exposed to drug and vehicle or
4 Unpublished results.
3528
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OXYGEN RADICALS GENERATED BY MC ANTIBIOTICS
Compound
Mitomycin
C
-NH2
CH,
Porftromycin
-NH2
BMY-25282
H /CH3
-N=C-N
BL-6783
-N=C-N
"N=C"NXCH
H
/
CH*
Fig. I. Structural formulae of mitomycin antibiotics.
vehicle alone for 60 min at 37'C under 95% air-5% CO2 and then
washed twice with cold PBS before treatment with 0.05% trypsin in
PBS to suspend the cells. The cells were then plated in replicate dishes
in Waymouth's medium plus 15% fetal bovine serum, and the surviving
fraction of cells was measured by colony formation.
Oxygen consumption was used as a measure of oxygen radical
generation (18) and was monitored polarographically with a Gilson
oxygraph equipped with a Clark electrode (19). The enzymatic activa
tion of drug-induced oxygen consumption was assayed by resuspending
EMT6 cells in cold PBS at a concentration of between 3 and 4 x 10'
cells/ml, and subjecting them to sonication as described above. Final
reaction mixtures consisted of 2 ml of EMT6 cell sonicate, 0.5 mM
NADPH, 0.5 mM NADH, and drug or ethanol as a control. All
antibiotics were dissolved in 70% ethanol to a final concentration of
either 1 or 5 mM; dilutions of these stock solutions resulted in no
greater than a 1% final concentration of ethanol in incubation flasks, a
level that did not affect oxygen consumption. Assays were conducted
at 37°C.Cell sonicates and substrates were added first to the respiratory
cell and oxygen consumption was monitored to obtain a baseline rate
for cell sonicate respiration. Once a baseline rate was established, either
drug (in ethanol, 0-1% final concentration) or ethanol (as a vehicle
control) were added and the new rate of oxygen consumption was
ascertained. The drug-induced rate of oxygen consumption was ob
tained by subtracting the rate following drug addition from the rate
prior to its addition. This method of determining drug-induced oxygen
consumption was used for all of the enzyme assays.
Drug-induced oxygen consumption in the presence of xanthine oxi
dase was assayed as described by Pan et al. (20). This was conducted
by incubating 55 ¿igof xanthine oxidase, 2 mM NADH, and 50 mM
phosphate buffer at pH 7.8 in a final volume of 2 ml. A mitomycin
antibiotic in ethanol or ethanol alone was added to initiate the reaction
which was run at 37°C.NADPH-cytochrome c reducÃ-asereaction
mixtures consisted of 2 ml of 50 HIMphosphate buffer (pH 7.4), 0.5
mM NADPH, 0.18 unit of NADPH-cytochrome c reducÃ-ase,and either
drug or an ethanol control; assays were conducted at 30°C.Beef heart
mitochondria, which were isolated by differential centri fugai ion (21)
and aged by repeated freezing and thawing, were uncoupled. Beef heart
mitochondria assay mixtures contained 2 ml of respiratory buffer (0.25
M sucrose, 7.5 mM Mg('b, 15 mM potassium phosphate, and 15 mM
Tris-HCl, pH 7.4), 1 mM KCN, 1 mM NADH, 3.3 HIMsuccinate, 2 mg
of mitochondria! protein, and drug or ethanol as a control. Mitochon
dria, respiratory buffer, and KCN were added initially to the reaction
cell so that cytochrome oxidase could be inhibited prior to the addition
of substrate; assays were run at 37*C. Following a 1-min incubation
useful in identifying the oxygen radical species generated by biological
systems and can be used to compare relative radical production by
various agents in the same system. Prior to sonication, F.MT6 cells
were suspended in 50 mM Tris-HCl buffer (pH 7.8) with 1 mM diethylenetriaminepentaacetic acid and adjusted to a concentration of be
tween 16 and 20 x 10' cells/ml in the same buffer. The reaction mixture
consisted of 200 n\ of cell sonicate, 100 mM x5 dimethyl I-pyrroline
W-oxide, 0.5 mM NADH, 0.5 mM NADPH, and 4.0% ethanol or drug
(in ethanol) in a linai volume of 250 /¿I.The reaction mixture was
incubated for 6 min at 37°C,at which time 25 /./Iof the solution were
injected into the HPLC system which consisted of an Altex ultrasphere
3 firn octadecylsilane 75 x 4.6-mm inside diameter column attached to
a Bioanalytical Systems LC4 A electrochemical detector (Bioanalytical
Systems, Inc., West Lafayette, IN). The detector potential was set at
+0.6 V versus an Ag+-AgCl reference electrode. A Rainin C-18 Microsorb Short-Ones column (Rainin Instrument Co., Woburn, MA) served
as a guard column. The mobile phase consisted of 0.03 M citric acid
(monohydrate), 0.05 M anhydrous sodium acetate, 0.05 M sodium
hydroxide, and 8.5% acetonitrile, brought to pH 5.1 with glacial acetic
acid; the flow rate was 1.0 ml/min. The relative peak areas of the
radicals generated by each drug were determined by cutting the appro
priate peaks from the HPLC tracings and weighing them on an analyt
ical balance.
RESULTS
The data in Table 1 show the concentration of each of the
mitomycin antibiotics that produced 50% kill of EMT6 mouse
tumor cells under aerobic conditions. The order of decreasing
potency of the compounds under these conditions was BMY25282, BL-6783, MC, and PM. To determine whether this
action correlated with the ability of these agents to generate
oxygen radicals, the production of oxygen radicals was mea
sured using different biological reducing systems by monitoring
oxygen consumption, which is a convenient method of measur
ing oxygen radical formation (18).
To ascertain whether the mitomycin antibiotics were capable
of being activated to a state that generates oxygen radicals in a
complete tumor cell system, drug-induced oxygen consumption
was measured in EMT6 cell sonicates supplemented with suf
ficient NADH and NADPH to ensure that these substrates
were not rate limiting. All of the-compounds tested gave a linear
dose dependent rate of oxygen consumption in the presence of
EMT6 cell sonicates and substrate (Fig. 2). BMY-25282 caused
the greatest rate of oxygen consumption followed by BL-6783;
the rate of oxygen consumption produced by MC and PM was
approximately 10-fold lower.
To provide information on systems with the potential to
activate these drugs to forms that generate oxygen radicals,
enzymatic systems which have previously been shown to acti
vate quiñonesto produce oxygen radicals were examined; these
consisted of NADPH-cytochrome c reducÃ-ase(9, 14, 18, 23),
xanthine oxidase (20), and intact mitochondria (14, 24).
The rate of activated drug-induced oxygen consumption pro
duced by NADPH-cytochrome c reductase is shown in Fig. 3.
Table I LDX values for mitomycin antibiotics with aerobic EMT6 cells
EMT6 cells were treated for 60 min with various concentrations of each agent,
removed from flasks by trypsinization, and plated onto replicate plates. Colony
formation was measured after 14 days and the surviving fractions were calculated
from these measurements. LD»values were obtained by extrapolation from the
survival curves, the values that represented the concentration producing 50%
toxicity to the cells. The values represent an average of 3 determinations.
period, mitochondria! respiration was found to be inhibited approxi
mately 95% by the presence of the 1 HIM KCN. The addition of
mitomycin antibiotic and subsequent increase in oxygen consumption
represented drug-induced KCN-insensitive mitochondria! respiration,
or drug-induced oxygen radical generation.
The detection of Superoxide and hydroxyl radical generated by drug
activation by EMT6 cell sonicates was carried out by the HPLC
procedure described earlier (22). This procedure is a qualitative assay
3529
Drug
Mitomycin C
Porfiromycin
BMY-25282
BL-6783
LD»(¡IM)
1.0
2.5
0.02
0.05
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OXYGEN RADICALS GENERATED BY MC ANTIBIOTICS
crated could also account for the observed differences in the
relative cytotoxicities of the mitomycin antibiotics, the oxygen
radical species generated by the 4 compounds were compared
using the EMT6 cell sonicate system, which should best reflect
the intact tumor cell system. The data in Table 2 show the
results of these studies. BMY-25282 and BL-6783 caused sig
nificantly greater production of both Superoxide and hydroxyl
radical than MC and PM. Although the difference observed
between BMY-25282 and BL-6783 for both Superoxide and
hydroxyl radical generation were not statistically significant,
the difference between MC and PM were significant for superoxide production but not for hydroxyl radical production.
DISCUSSION
Quinone-containing compounds are capable of undergoing
one electron reduction and subsequent reaction with molecular
oxygen to generate various oxygen species such as Superoxide,
hydrogen peroxide, and hydroxyl radical (25, 26). The intracellular production of these toxic oxygen species by compounds
of this type have the potential to produce cytotoxicity. Thus,
Adriamycin (27, 28), paraquat (29), menadione (30), and bleomycin (31) are some of the compounds whose cytotoxic actions
have been attributed to the generation of oxygen radicals. The
finding that the reduction of MC leads to the generation of
oxygen radicals (10-14) raises the possibility that these radicals
may be involved in the aerobic cytotoxicity of this agent. The
differences in the cytotoxicities of the 4 mitomycin analogues
shown in Fig. 1, together with their structural similarities,
provided a good series for evaluating the possible role of oxygen
radicals in aerobic toxicity. The substitution of a methyl group
on the aziridine ring portion of the molecule (the R? position
in Fig. 1) resulted in a decreased rate of oxygen radical gener
ation. The substitution of an AH/V',./V'-dimethylaminoniethylene)amine for the amine group on the quinone ring (Ri in
Fig. 1) caused a greatly enhanced rate of oxygen radical gener
ation. Since the substitution at the RI position results in a
change in the redox potential of MC and PM from an £./,of
0.35 V to 0.16 V (32), BMY-25282 and BL-6783 are more
readily reduced, a property that presumably accounts for thenincreased capacity to generate oxygen radicals. BMY-25282
and BL-6783, however, do not appear to be activated by xanthine oxidase, while both PM and MC are activated by this
enzyme to generate oxygen radicals. No information is available
to explain these observations, although one reasonable possi
bility is that steric hindrance caused by the bulkiness of the
substituted amine group at the RI position prevents activation
by xanthine oxidase.
Table 2 Relative quantities of drug-induced oxygen radicals formed in EMT6
cell sonicates containing mitomycin antibiotics
Reaction mixtures consisted of 200 nl of EMT6 cell sonicate (16 to 20 x 10*
cells/ml in 50 min Tris-HCl buffer, pH 7.8), 1 mM diethylenetriaminepentaacetic
acid, 100 HIM 5,5-dimethyl-l-pyrroline
W-oxide, 0.5 mM NADPH, 0.5 mM
NADH, and 4% ethanol. Drugs were added at a final concentration of 40 UM.
and reactions were initiated by the addition of NADPH and NADH. The reaction
mixtures were incubated for 6 min at 37'C and 25 ¿il
of the mixture were used in
the HPLC separation. All values are expressed relative to control samples without
drug which were equated to 1.0. Each value represents an average value ±SD
from at least 5 determinations.
productionCompoundMitomycin
Relative
±0.03°
C
Porfiromycin
0.97 ±0.20
2.12 ±0.20°
BMY-25282
BL-6783Superoxide1.29 1.68 ±0.30°Hydroxyl
* Values with a significant difference of at least P
Student's t test) when compared to the control samples.
radical1.18
±0.10«
1.06 ±0.10
1.35 ±0.16°
1.52 ±0.12°
< 0.05 (by the paired
The findings from these studies demonstrate that the enzy
matic composition of target cell types can affect the reactivity
of various mitomycin antibiotics in that system. Although the
present studies were performed with enzymes from various
mammalian sources, the results are probably representative of
the general interaction of these compounds with the enzyme
systems used. However, direct comparisons to in vivo drug
activation must be made with caution since relative enzyme
concentrations vary between biological systems, and in the
present studies different sources of the enzymes were used and
each assay was optimized for the particular enzyme being
assayed.
BMY-25282 and BL-6783 generated more hydroxyl and
Superoxide radicals than either PM or MC. MC in turn pro
duced more Superoxide and hydroxyl radicals than PM. The
markedly greater production of Superoxide and hydroxyl radi
cals by BMY-25282 and BL-6783 may well contribute to the
greatly enhanced aerobic cytotoxicity of these derivatives, over
that produced by MC and PM, to EMT6 tumor cells.
In summary, the mitomycin antibiotics tested generate var
ious amounts of oxygen free radicals with EMT6 cell sonicates.
The relative quantities of oxygen radicals produced by these
compounds in this system correspond to their relative aerobic
cytotoxicities to EMT6 tumor cells. This relationship suggests
a possible contribution of oxygen radicals to the aerobic cyto
toxicity of these compounds. NADPH-cytochrome c reductase
and xanthine oxidase were the most effective activators of PM
and MC, whereas NADPH-cytochrome c reductase and mitochondrial reductase(s) were the most efficacious systems in
catalyzing the reductive activation of BMY-25282 and BL6783.
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Generation of Reactive Oxygen Radicals through Bioactivation
of Mitomycin Antibiotics
Chris A. Pritsos and Alan C. Sartorelli
Cancer Res 1986;46:3528-3532.
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