[CANCER RESEARCH 44, 5638-5643, December 1984]
Role of NADPH:Cytochrome c ReducÃ-aseand DT-Diaphorase in the
Biotransformation of Mitomycin C1
Susan R. Keyes, Paula M. Fracasso, David C. Heimbrook,2 Sara Rockwell, Stephen G. Silgar,3 and
Alan C. Sai-torelli4
Departments of Pharmacology [S. R. K., P. M. F., A. C. S.] and Therapeutic Radiology [S. R.J and Developmental Therapeutics Program, Comprehensive Cancer Center,
Yale University School of Medicine, New Haven 06510, and Department of Molecular Biophysics and Biochemistry, [S. G. S., D. C. H.¡Yale University,
New Haven 06511, Connecticut
ABSTRACT
Hypoxie cells of solid tumors are difficult to eradicate by X-
irradiation or chemotherapy; as an approach to this problem, our
laboratories are investigating the effects of the bioreductive
alkylating agent mitomycin C (MC) on hypoxic cells. This anti
biotic was preferentially toxic to EMT6 mouse mammary tumor
cells and V79 Chinese hamster lung fibroblasts under hypoxic
conditions, but it was equitoxic to Chinese hamster ovary cells
in the presence and absence of oxygen. All cell lines catalyzed
the formation of reactive metabolites under hypoxic conditions
and contained NADPH:cytochrome c reducÃ-aseand DT-diaphor-
ase, two enzymes which may be responsible for the cellular
activation of MC. Although a correlation existed between enzy
matic activities and the formation of reactive metabolites from
MC, there was no correspondence between these parameters
and the degree of cytotoxicity expressed by MC under hypoxic
conditions.
Purified NADPHrcytochrome c reducÃ-ase reduced MC in the
absence of oxygen, with addition of cytochrome P-450 enhanc
ing, but noi participating directly in, the reduction reaction. Ad
dition of NADP+ lo cell sonicates substantially reduced NADPH:-
cylochrome c reducÃ-ase activity, while Ihe formation of reactive
metabolites was affected only slightly; converse results were
observed using mersalyl. Exposure of cell sonicates lo dicumarol
inhibiled DT-diaphorase activity, while the rate of formation of
reactive metabolites of MC was enhanced. The findings suggest
that NADPH:cytochrome c reducÃ-ase and some as yet to be
identified enzyme(s) are important for the reductive activalion of
MC. DT-diaphorase and cytochrome P-450 are not directly in
volved in the activalion of MC, bul they appear lo modulate the
degree of activalion lo reaclive species, which are presumably
responsible for Ihe observed cytotoxicity.
INTRODUCTION
Solid neoplasms contain hypoxic tumor cell populations; since
oxygen-deficient cells are relatively refractory lo convenlional
radiolherapy and mosl chemolherapy, hypoxic cells have Ihe
potential to limit curability (13, 19). As an approach to the
circumvention of this problem, we have been studying the natu-
1This work was supported in part by American Cancer Society Grants CH-211
and PDT 145, USPHS Grant CA-02817, NIH Grants GM-31756 and AM-01160,
and American Heart Association Fellowship 11-104-812.
2 Present address: Department of Pharmacology, Yate University School of
Medicine, New Haven, CT 06510.
3 Present address: Department of Biochemistry, University of Illinois, Urbana, IL
61801.
4 To whom requests for reprints should be addressed.
Received May 21, 1984; accepted August 29, 1984.
CANCER RESEARCH VOL.
5638
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rally occurring antibiolic, MC.5 This drug is aclive against a broad
range of transplantable animal (5) and human (2) tumors, is
preferentially cytotoxic to some hypoxic tumor cell lines in vitro
(11, 23, 27), and is also toxic to hypoxic tumor cells in vivo (25,
26).
Early studies have demonslraled lhal enzymalic bioactivalion
of MC results in cyloloxicily associated wilh cross-linking of
DNA (9). Using liver homogenales, the anaerobic production of
reaclive species from MC has been shown lo require NADPH
and to be catalyzed primarily by enzymes in the microsomal and
nuclear subcellular fractions (12, 28), and some alkylation prod
ucts have been identified (31). Limited studies with purified
enzymes have indicated that both NADPHxytochrome c reduc
Ã-aseand xanthine oxidase actÃ-valeMC under hypoxic conditions,
wilh Ihe concomilanl generalion of a reaclive eleclrophile (14,
21 ). Studies in our laboratories have suggested lhal cytochrome
P-450 might be involved in the anaerobic metabolism of MC (11,
12), and work by others on DT-diaphorase, an enzyme active in
the reduction of quiñones (6, 30), suggests that this enzyme
should also be considered as a possible activator of MC.
Although the mechanism by which MC is metabolically acti
vated has been examined extensively in bacteria and liver, little
is known about the bioaclivation of MC by neoplastic cells.
Previous investigations in our laboratories have demonslraled
that MC is more toxic to EMT6 and Sarcoma 180 tumor cells
under hypoxic conditions than in the presence of air, and lhal
sonicates of these cells are capable of generating a reactive
species from MC (11, 27). However, neilher Ihe enzyme(s) in
lumor cells responsible for activating MC nor the exact biochem
ical basis of Ihe enhanced cyloloxicily in Ihe absence of oxygen
has yet been characterized. The studies described in Ihis paper
were designed to address these issues.
MATERIALS AND METHODS
MC was supplied by the Bristol-Myers Co. (Syracuse, NY). Other
materials were obtained from the following sources: NADP*, NADH,
NADPH, glucose-6-phosphate dehydrogenase (type XII), glucose 6-phos-
phate, dicumarol, mersalyl, 2,6-dichlorophenolindophenol, dilaurylphos-
phatidylcholine, DEAE-cellulose, and 2',5'-ADP agarose (Sigma Chemi
cal Co., St. Louis, MO); fetal bovine serum and Waymouth's medium
(Grand Island Biological Co., Grand Island, NY); DEAE-Trisacryl M and
HA-Utrogel (LKB Instruments, Gaithersburg, MD); gases (Presto Welding
Service Center, North Haven, CT); and 4-<p-nitrobenzyl)pyridine (Aldrich
Chemical Co., Milwaukee, Wl). Chinese hamster V79 cells (subline V79-
8) and CHO cells (subline HA-1) were gifts of Dr. R. Michael Liskay and
Dr. Daniel S. Kapp, Department of Therapeutic Radiology, Yale University
School of Medicine.
5The abbreviations used are: MC, mitomycin C; CHO, Chinese hamster ovary.
44 DECEMBER 1984
 BIOTRANSFORMATION
EMT6 mouse mammary tumor cells, Chinese hamster V79 cells, and
CHO cells were grown at 37° in Waymouth's medium supplemented
with 15% fetal bovine serum and antibiotics in a humidified atmosphere
of 95% air:5% CO2. The characteristics of these 3 cell lines have been
described in detail previously (10,16, 24).
For survival studies, cells were cultured in glass milk dilution bottles
and used in midexponential growth. Cells were exposed to 1.5 UM MC
for various times after pregassing with 95% air:5% CO2 or 95% N2:5%
CO2 for 2 hr (26). The survival of cells was assayed by testing their ability
to form macroscopic colonies (11, 24, 27). Both hypoxic and aerobic
vehicle (50 n\ of 0.9% NaCI solution or 70% ethanol) controls were
included in each experiment; the surviving fractions for hypoxic cultures
were calculated using the hypoxic controls.
Cell sonicates and microsomes were prepared from cells grown as
either suspensions or as monolayers. After being washed with cold
phosphate-buffered saline, the cells were resuspended in approximately
6 volumes of water. After a 10-min incubation on ice, an equal volume
of 1.8% NaCI solution was added, and the cells were disrupted by three
6-sec bursts with a Branson Model 160 sonicator set at 25% of maximum
intensity. Cell sonicates were used for enzyme assays and for preparation
of subcellular fractions. Microsomes were prepared by the method of
Cinti er al. (3) after cell sonicates were adjusted to a concentration of
0.25 M sucrose by addition of 1.25 M sucrose. Microsomes were contam
inated with <5% mitochondria as judged by succinatercytochrome c reduction after the addition of a second aliquot of NADPH (Chart
reductase activity (29) and <1 % cytosol as measured by lactate dehy-
drogenase activity (8), and total protein recovery averaged 92% as
determined by the method of Lowry ef al. (17).
Reactive metabolites of MC were estimated by a modification of the
method of Wheeler ef al. (33) as described previously (11). The absorb-
ance of the adducts of MC and 4-(p-nitrobenzyl)pyridine was measured
at 540 nm. The reaction rate was linear for at least 30 min, and the 5%
final concentration of acetone in which the trapping agent and MC were
dissolved did not affect the enzymatic activation of MC.
Cellular oxidoreductase activities were measured by standard assays.
NADPH: and rotenone-insensitive NADH:cytochrome c reductases were
determined at 30°by monitoring the rate of reduction of cytochrome c
at 550 nm (29, 34), using an extinction coefficient of 27.7 rnw"1 cm"1.
NADHxytochrome bs reductase and DT-diaphorase activities were de
termined at 30°by the reduction of KaFefCNfe (420 nm, «= 1.02 mn/T1
cm~1) and dichlorophenolindophenol (600 nm, < = 21 rriM"1 cm"1), re
spectively (6,29). Cytochrome i>5and cytochrome P-450 were measured
in microsomes by the method of Omura and Sato (20) using an Aminco
DW-2 UV/VIS spectrophotometer in the split-beam mode. The extinction
coefficients used were 185 rtiM~1 cm"1 for cytochrome to5for the wave
length pairs, 425 and 410 nm, and 91 mw"1 cm"1 for the cytochrome P-
450:carbon monoxide complex for the wavelength pairs, 450 and 495
nm.
Cytochrome P-450uiœ and NADPH:cytochrome c reductase were pu
rified from the livers of phenobarbital-induced rabbits by standard pro
cedures (4,7). Both were homogeneous as judged by gel electrophoresis
(15). Enzymatic reduction of MC with purified enzymes was conducted
in a 1-ml solution of 100 mM Tris-HCI (pH 7.0) containing dilaurylphos-
phatidylcholine (30 /¿g/ml),30 MM MC, 0.2 ^M NADPH:cytochrome c
reductase, and varying concentrations of P-45ÛLM2.The reaction was
initiated by the addition of 100 nmol NADPH, and the time course of the
reduction was followed by measuring loss of the absorbance of the
quinone moiety at 363 nm. Anaerobic experiments were performed under
an atmosphere of N2 in a Thunberg-type cuvet, after repeated degassing
and purging with N2. In some experiments, carbon monoxide replaced
the N2 atmosphere. All spectrophotometric observations were performed
in a Gary 219 spectrophotometer at 25°.
RESULTS
Purified NADPH :cytochrome c reductase and cytochrome P-
45ÛLM2were used to reduce MC under hypoxic conditions, since
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OF MITOMYCIN C
previous studies have suggested their importance in the bioac-
tivation of MC (11,12,14, 21). Preliminary experiments showed
that an increase in the reactive reduction product(s) of MC
trapped with 4-(p-nitrobenzyl)pyridine correlated with the disap
pearance of the quinone absorbance at 363 nm (data not shown);
therefore, the latter parameter was monitored in these experi
ments. The anaerobic reduction of MC by NADPH :cytochrome
c reductase or the reductase plus P-450LM2was a typical first-
order reaction between 0 and 50 UM MC, as would be expected
for substrate concentrations significantly below the Km of the
enzymes. Although MC was reduced under hypoxic conditions
by NADPHxytochrome c reductase alone, the rate of reduction
was enhanced by addition of P-450 and reached a maximum at
approximately a 1:1 molar ratio of NADPH:cytochrome c reduc
tase to P-45ÛLM2(Chart 1).
The rate of MC reduction catalyzed by purified NADPH:
cytochrome c reductase and P-450 was not decreased by carbon
monoxide (data not shown). When the reoxidation rate of the
ferrous P-450:carbonyl complex after exhaustion of NADPH was
monitored at 450 nm in the presence of MC, the rate was
approximately 100 times slower than the initial velocity of MC
2). These data support the concept that NADPH:cytochrome c
reductase is the primary reductant of MC, while P-450 acts only
as a modulator of enzymatic activity. Under oxygenated condi-
0006
7 0004 *•
O
0.002
0 0.2 0.4 06
P450 [//M]
Chart 1. Anaerobic reduction of MC by NADPHxytochrome c reductase and
cytochrome P-450. The reaction mixture contained 30 MM MC, 0.2 MM
NADPH:cytcchrome c reductase, dilaurylphosphatidylcholine(30 M9/mt), 100 MM
NADPH, 0 to 0.6 MMcytochrome P-450, and 100 mw Tris-HCI (pH 7.0) in a total
volume of 1 ml. The anaerobic reduction of MC was monitored at 25°by the loss
of the quinone absorbance at 363 nm, and the results show the first-order rate
constant of MC reduction in the absence of oxygen.
I.O IO
S 0.8
o
ç
60.6 6 I
o 1
I.O 2.0 3.0
Time (mm)
Chart 2. Comparison of the rate of cytochrome P-450u«reoxidation to the rate
of MC reduction. The reaction conditions were as described in Chart 1, with 0.6
MMcytochrome P-450 and 30 nmol NADPH. For 0 to 1.5 min, the concentration of
the ferrous:carbonyl complex of P-450 was monitored at 450 nm, and for 1.5 to
3.0 min, the concentration of MC was monitored by the quinone absorbance at
363 nm after a second addition of NADPH.
VOL. 44 DECEMBER 1984
5639
 BIOTRANSFORMATION
lions, the quinone absorbance of MC did not decrease, even
though NADPH was still consumed (data not shown). This finding
is consistent with the scavenging of the MC semiquinone radical
by oxygen, which results in oxygen-induced inhibition of the
activation of MC to a reactive metabolite.
These findings with purified enzymes were compared to the
hypoxic activation of MC in whole cells. As model systems, we
chose EMT6 mouse mammary tumor cells, V79 Chinese hamster
lung fibroblasta, and CHO cells, since these cell lines are com
monly used for investigations on the effects of radiosensitizing
agents and cytotoxic drugs with specificity for hypoxic cells. The
toxicity of MC to these cell lines under hypoxic and oxygenated
conditions is shown in Chart 3. The survival curves for EMT6
and V79 cells were similar, with the survival of the hypoxic cells
being significantly lower than that of aerobic cells and with the
slopes of the hypoxic curves being steeper than those of the
aerobic curves. For CHO cells, there was no significant difference
between aerobic and hypoxic cell survival with short (<2.5 hr)
exposure to MC and only insignificantly greater toxicity to hy
poxic cells at prolonged (>2.5 hr) times of exposure. The data
from 2 and 4 independent experiments are shown for V79 and
CHO cells, respectively; the experimental results for EMT6 cells
were from a single determination, performed during the same
time period with the same lot of MC. The findings with EMT6
cells agreed with those published previously (11), which were
based on independent and more extensive data.
Since most research on the anaerobic metabolism of MC has
been conducted with liver, we analyzed the basic requirements
needed for activation of MC in these cell systems under anaer
obic conditions with an NADPH-regenerating system; all 3 cell
lines activated MC to a reactive species that alkylated the
nucleophile 4-(p-nitrobenzyl)pyridine (Table 1). The greatest
monoalkylating activity was obtained with EMT6 cell sonicates;
substantially lower activities occurred with CHO and V79 cell
sonicates. The generation of reactive metabolites from MC was
inhibited by oxygen and by the absence of exogenous reduced
pyridine nucleotides. Both NADPH and NADH supported the
formation of reactive metabolites from MC, with greater rates of
alkylation occurring in the presence of NADPH; however, neither
cofactor was as efficient as the NADPH-regenerating system.
Hours of
Charts. Survival of aerobic and hypoxic EMT6, V79, and CHO cells as a
function of time after treatment with MC. The cells, grown to midlog phase in glass
bottles, were treated with 1.5 ¡MMC as described in 'Materials and Methods."
Hypoxia was achieved by gassing with 95% N2:5% CO, for 2 hr before treatment
with MC for 1.0 to 3.5 hr, with continued gassing. O, O, A, D, aerobic survival; ». at levels undetectable by our procedures, were involved in the
»,A, •.
hypoxic survival. Each symbol represents a different experiment.
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OF MITOMYCIN C
Table 1
Formation of reactive metabolites from reduction of mitomycin C
The standard reaction mixture contained an NADPH-regenerating system (0.72
m«NADP* :5 my MgCI2:5 mw glucose 6-phosphate: 1.25 unit glucose 6-phosphate
dehydrogenase), 470 MM4-(p-nilrobenzyl)pyridine, and 3 to 5 mg cell sonicate in a
total volume of 1 ml 100 mm Tris-HCI (pH 7.4). The reaction was initiated by the
addition of 300 nmol mitomycin C.
AMO* lO^/min/mg protein
systemAir
cofactorshypoxia*
Cell +
hypoxia*NADH hypoxia"No
lineNADPH-regenerating HypoxiaNADPH
CHO 0.03 ±0.0r0.33 ±0.06 0.18 ±0.03 0.15 ±0.03 0.11 ±0.02
V79 0.03 ±0.01 0.39 ±0.04 0.30 ±0.04 0.20 ±0.02 0.12 ±0.05
EMT6 0.05 ±0.01 1.04 ±0.05 0.77 ±0.13 0.36 ±0.11 0.17 ±0.02
* The NADPH-regeneratingsystem was omitted or replaced with 1 mw reduced
pyridine nudeotjde.
Mean ±S.E. with n > 4.
The rate of generation of a reactive species in the absence of
cofactors was markedly faster than the rate in the presence of
oxygen, indicating that there were significant levels of endoge
nous reducing equivalents in the sonicates. These results quali
tatively agree with earlier data from this laboratory using EMT6
and S180 cells (11, 27) and with data obtained using liver
homogenates (28).
The presence of activities representative of enzymes impli
cated in the bioactivation of MC was measured in the cell lines
under investigation. Enzyme activities characteristic of 3 liver
microsomal electron transport proteins and one liver cytosolic
enzyme were present in the cultured cell lines. In addition,
cytochrome os, but not cytochrome P-450, was detected in the
cellular microsomal fraction (Table 2). NADPHxytochrome c
reductase activity and DT-diaphorase activity were higher in
EMT6 cells than in CHO and V79 cells. NADH:cytochrome c
reductase activity (a measurement of NADH:cytochrome i>5re
ductase plus cytochrome i>5), NADH:cytochrome u5 reductase
activity, and cytochrome 05 were not significantly different in the
cell lines.
NADHxytochrome 65 reductase, NADHxytochrome c reduc
tase, and DT-diaphorase activities were markedly variable, as
evidenced by the large standard errors. This variation reflected
increases in the enzyme activities in the cells as the cultures
entered plateau phase (data not shown). In contrast,
NADPHxytochrome c reductase activity was identical in expo
nentially growing and plateau-phase cells. All studies involving
cell survival, inhibitors, and reactive metabolites were performed
with cells in exponential growth.
Inhibitors of various enzymes implicated in the biotransforma-
tion of MC were used as probes to estimate the contribution of
specific oxidoreductases to the reductive activation of MC. Pre
vious papers from this laboratory (11, 12) reported inhibition of
reactive metabolite formation by CO, suggesting a role for cy
tochrome P-450 in the activation of MC. However, anaerobic
metabolism of MC with purified NADPHxytochrome c reductase
and cytochrome P-450 was unaffected by CO, and no cyto
chrome P-450 could be detected in the microsomal fraction of
the cultured cells.
In an attempt to resolve these discrepancies, EMT6 cells were
treated with CO and MC. If P-450, which may have been present
activation of MC, CO should protect cells from MC toxicity by
VOL. 44 DECEMBER 1984
5640
 BIOTRANSFORMATION OF MITOMYCIN C

Table2
Oxidoreductaseactivities in cultured cell lines
All enzyme activities or concentrations were determined by standard assays described in "Materials and Methods.'
Cytochrome P-450 pathway Cytochrome b, pathway
NADPHicytochrome c reducÃ-ase one-insensit!ve
inCell activity (nmol/min/mg protein) reducÃ-ase NADHrcytochromec
P-450 (pmol/ activity OÃ-mol/min/ reducÃ-ase activity b, (pmol/mg activity
mg protein) in mg protein) in (nmol/min/mg protein) protein) in (nmol/min/mg protein)
microsomes*<0.5 microsomes*34.2
lineCHO sonicates1.6 sonicates42.6
in insonicates10.0±
±0.16 ±0.6 ±0.2" ±10.7* 3.2e
V79 1.1 ±0.1 3.6 ±1.0 <0.5 1.0 ±0.1 34.8 ± 5.6 37.5 13.7± 2.1
EMT6Sonicates0.9 14.0 ±1.6Cytochrome
4.6 ±0.6Microsomes*2.0 <0.5NADHrcytochromeus
1.5 ±0.3Roter 60.1 ±20.3Cytochrome 31.1OT-diaphorase 151.4 ±48.7
" Cytochrome P-450, Cytochromei>5,and NADPHicytochrome c reducÃ-ase
activities in microsomes were the average of one, 2, and 3 determinations, respectively.
Mean ±S.E. of 12 determinations.
Mean ±S.E. of 7 independent experiments.

Tables
I.CH
Effects of inhibitors of NADPH:cytochromec reductase activity and DT-
diaphorase activity on the formation of reactive metabolites of mitomycin C
The reaction mixtures were as described in Tables 1 and 2, with the addition of
inhibitors as indicated. NADP* was dissolved in 100 mw Tris-HCI (pH 7.4), and
IO"
mersalyl and dicumarol were added in 0.5 M NaOH. Neither vehicle affected the
reaction rates.
% of control activity*

Cytochromec
Inhibitor
(HIM)Dicumarol0.10.51.0NADP*1.05.010.0Mersalyl0.10.250.5NADPH:
reductase
activity100 activity2± metabolites98

1*10083
± 10018 ±13143±13185
IO"'-
±1021 ±2274

83±
± 94945
± ±1069
3088 ±1233±
O 1.0 2O 3O 271
Time of Exposure to CO (hr)
Chart 4. Effect of carbon monoxide on the survival of MC-treated cells. The ±1265 319± 922±
experimentalconditions were as described in Chart 3 and 'Materials and Methods.'
±1033±13DT-diaphorase
413±13Reactive
± ±106±
Ninety-five% CO:5% CO2replaced95% N2:5%C02 where indicated. MC or vehicle 3
treatments were for 1 hr. A, aerobic cell survival, vehicle; O, aerobic cell survival, * The effects of inhibitors are expressed as percentages of the control activities
1.5 IM MC; A, hypoxic cell survival, vehicle with CO; •, hypoxic ce«survival, 1.5
„MMC with CO. which are given in previous tables.
Mean ±S.E. of at least 3 determinations where applicable.

inhibiting the formation of cytotoxic species. As shown in Chart
4, CO, which as a single agent was not toxic, failed to protect
EMT6 cells against the lethal effects of MC but, instead, in
creased hypoxic cell susceptibility to the antibiotic. In addition,
measurement of reactive produces) from MC demonstrated that
the formation of a reactive electrophile by cell sonicates was not
inhibited by CO (data not shown). The contradiction between
previous results (11,12) and the present studies may be attrib
utable to an oxygen leak in the experimental apparatus used for
the earlier investigations. These findings indicate that cyto-
chrome P-450 is not involved in bioactivation of MC in EMT6
cells and, furthermore, that Cytochrome P-450, if present, would
only function to modify the activity of NADPHicytochrome c
reductase.
Additional experiments using inhibitors of NADPH:cytochrome
c reductase and DT-diaphorase examined the role of these
enzymes in the production of a reactive product from MC by
EMT6 cell sonicates (Table 3). Dicumarol effectively blocked the
activity of DT-diaphorase at 0.1 HIM but did not decrease
NADPHrcytochrome c reductase activity at concentrations up to
1 (TIM. In addition, dicumarol did not decrease but instead en
hanced the formation of a reactive product from MC.
Both mersalyl and NADP+ significantly lowered the
NADPH:cytochrome c reductase and DT-diaphorase activities
and decreased the rate of formation of reactive MC metabolites.
Since studies with dicumarol showed that DT-diaphorase did not
catalyze the production of an electrophile(s) from MC, the results
of studies with NADP* and mersalyl can be used to assess the
role of NADPHrcytochrome c reductase in the formation of
reactive products from MC. There was no quantitative relation
ship between the degree of inhibition of NADPH:cytochrome c
reductase activity by NADP* or mersalyl and the rate of produc
tion of reactive metabolites over the entire range of inhibitor
concentrations used. NADPHxytochrome c reductase was more
sensitive to inhibition by NADP+ than was the generation of
reactive metabolites; the reverse was true with mersalyl. Similar
results were found using V79 and CHO cell sonicates (data not
shown). These results support the concept that NADPH:
Cytochrome c reductase is involved in the activation of MC under
hypoxic conditions but indicate that other factors are also in
volved in this reaction.
DISCUSSION
The biochemical mechanism of MC transformation has been
widely investigated using bacterial and liver systems, but few

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 BIOTRANSFORMATION
studies have used tumors or other mammalian cell lines. Previous
work in our laboratories demonstrated that EMT6 mouse mam
mary tumor cells and Sarcoma 180 ascites cells are more sen
sitive to the cytotoxic action of MC under hypoxic conditions
than in air (11, 27), and that sonicates of these cell lines are
capable of consuming MC to generate a reactive species. Exten
sions of these findings to other cell lines demonstrated that MC
is essentially equitoxic to aerobic and hypoxic CHO cells under
the experimental conditions used in these studies, whereas it is
preferentially cytotoxic to V79 cells under hypoxic conditions. In
addition, sonicates of EMT6, V79, and CHO cells are capable of
generating reactive species from MC, with the greatest alkylating
activity occurring in EMT6 sonicates (Table 1). NADPH is more
efficient than NADH as an electron donor for the generation of
the reaction product(s). These findings are in agreement with
data derived using liver homogenates (12, 28). Enzyme activities
characteristic of NADPH:cytochrome c reductase and DT-dia-
phorase were present in all 3 cell lines, but cytochrome P-450
was undetectable. Both the measured amounts of enzyme activ
ity and the rate of formation of reactive metabolites were highest
in the EMT6 cells. However, neither the measured oxidoreduc-
tase activities (Table 2) nor the rate of formation of reactive
metabolites (Table 1) in the different cell lines con-elated with the
extent of cytotoxicity under hypoxic conditions. These findings
do not preclude bioactivation of MC as a critical event responsible
for cytotoxicity, because studies with sonicates cannot include
the effects of such factors as the rate of transport of drug into
cells, the extent of repair of cytotoxic lesions, and changes in
intracellular pH. However, the lack of a correlation between the
rate at which sonicated cells generate reactive species and the
cytotoxicity of MC to intact cells indicates either that interaction
between activated MC and 4-(p-nitrobenzyl)pyridine does not
reflect accurately the reactions leading to cytotoxicity or that the
rate of bioactivation of MC is not the sole determinant of cyto
toxicity.
In assessing the involvement of mammalian cell enzymes in
the activation of MC, cytochrome P-450 did not appear to be a
primary reductant. This enzyme was not present in any of the
cell lines at levels within the limits of detectability (<0.5 pmol/
mg). Moreover, CO neither inhibited the formation of MC-derived
reactive metabolites by either cell sonicates or purified
NADPHxytochrome c reductase and P-450 (Chart 2) nor pro
tected EMT6 cells from MC-induced cytotoxicity (Chart 4).
When purified enzymes were used, the combination of ana-
erobiosis, NADPH:cytochrome c reductase, and NADPH was
sufficient for MC activation; however, addition of P-450 increased
the reaction rate (Chart 1). The 100-fold difference between the
rate of P-450 reoxidation and the rate of MC disappearance
(Chart 2) indicates, however, that P-450 does not participate
directly in the metabolism of MC to either a toxic or a nontoxic
metabolite. Instead, P-450 appears to modulate NADPH:
cytochrome c reductase activity by increasing the efficiency of
electron transfer from NADPH :cytochrome c reductase to MC.
Potter and Reed (22) reported recently a similar phenomenon for
the anaerobic reduction of 1-(2-chloroethyl)-3-(cyclohexyl)-1-ni-
trosourea.
EMT6 cells contained more NADPHxytochrome c reductase
and DT-diaphorase activity than CHO and V79 cells; EMT6 cells
also formed reactive metabolites from MC under anaerobic con
ditions faster than did either CHO or V79 cells. To determine the
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OF MITOMYCIN C
relative importance of NADPH:cytochrome c reductase and DT-
diaphorase in the biotransformation of MC, the effects of NADP+,
mersalyl, and dicumarol on enzyme activities and reactive me
tabolites were examined. Dicumarol markedly inhibited DT-dia
phorase without decreasing either NADPH:cytochrome c reduc
tase activity or the generation of reactive metabolites. This
finding indicates that DT-diaphorase is not involved in the bioac
tivation of MC. Indeed, the reactive product(s) generated from
MC increased significantly in the presence of dicumarol, sug
gesting that DT-diaphorase may metabolize MC to a nontoxic
substance, possibly by the pathways reported by Thor eÃ-al. (30)
for the metabolism of menadione in rat liver hepatocytes. These
results suggest that treatment of whole cells with both MC and
dicumarol in combination might be used to increase the toxicity
of MC to hypoxic cells. In addition, they imply that the activation
of MC under hypoxic conditions may proceed through an inter
mediate semiquinone radical, as well as through an intermediate
hydroquinone (18). The semiquinone radical intermediate has
been suggested by Bachur eÃ-al. (1, 21) and Tomasz eÃ-al. (32).
The nonspecific NADPHxytochrome c reductase inhibitors,
NADP+ and mersalyl, also yielded interesting results. NADP+
completely inhibited NADPHxytochrome c activity but had less
effect on the generation of reactive metabolites from MC. Con
versely, mersalyl at 0.25 mw inhibited the generation of reactive
metabolites of MC to a greater extent than it inhibited
NADPHxytochrome c reductase activity. The inhibition of both
alkylating activity and NADPHxytochrome c reductase activity
by NADP+ and mersalyl implies that NADPHxytochrome c re
ductase is responsible for at least some of the bioactivation of
MC in EMT6 cells, but the different degrees of inhibition of these
processes suggest that at least one other enzyme may be
involved.
In conclusion, the data in this paper demonstrate that, under
hypoxic conditions, MC is differentially toxic to EMT6 and V79
cells but has less differential toxicity to CHO cells, and that all 3
cell types can generate an electrophile from MC which can be
measured with 4-(p-nitrobenzyl)pyridine. Both NADPHxyto
chrome c reductase and DT-diaphorase were present in all 3 cell
lines. Although there was a qualitative relationship between
enzyme activity and the generation of reactive metabolites from
MC, these parameters did not correlate quantitatively with the
extent of MC-induced cytotoxicity to hypoxic cells. Experiments
with enzyme inhibitors suggested that DT-diaphorase and cyto
chrome P-450 are not involved in the bioactivation of MC,
although they may modulate its activation. On the other hand,
NADPHxytochrome c reductase was capable of activating MC
but did not appear to be the sole enzyme involved in the reductive
activation of the antibiotic.
ACKNOWLEDGMENTS
The authors would like to thank Carmen Arroyo for her excellent technical
assistance.
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CANCER RESEARCH VOL. 44 DECEMBER 1984

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Role of NADPH:Cytochrome c Reductase and DT-Diaphorase in
the Biotransformation of Mitomycin C
Susan R. Keyes, Paula M. Fracasso, David C. Heimbrook, et al.

Cancer Res 1984;44:5638-5643.

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