[CANCER RESEARCH 53. 5470-5474.
November 15. 1993]
Kinetics and Mechanism of Mitomycin C Bioactivation by Xanthine Dehydrogenase
under Aerobic and Hypoxie Conditions1
Daniel L. Gustafson2 and Chris A. Pritsos3
Department of Nutrition and the Cellular ami Molecular Pharmacology and Physiology Program, University of Nevada, Reno, Nevada 89557-0132
ABSTRACT
These studies examined the kinetic and mechanistic parameters of mi
tomycin C (MMC) bioreduction by xanthine dehydrogenase (XDH), an
enzyme recently shown to be capable of MMC activation. The bioreduc
tion of MMC by XDH leads to the formation of 2,7-diaminomitosene
(2,7-DM) under both aerobic and hypoxic conditions, with greater 2,7-DM
formation observed under hypoxic conditions. The XDH-induced forma
tion of 2,7-DM is pH dependent with increasing formation as the pH is
varied from 7.4 to 6.0. In this study, the kinetics of MMC bioreduction by
XDH was assessed under aerobic and hypoxic conditions and at pH 7.4
and 6.0. MMC interaction with XDH was also assessed by monitoring the
ability of MMC to inhibit XDH-mediated uric acid and NADH formation.
The ability of xanthine to serve as reducing equivalents for MMC reduc
tion was also measured. Aerobically but not hypoxically, MMC reduction
by XDH followed Michaelis-Menten kinetics. Kinetic constants calculated
under aerobic conditions suggested that the pH-dependent increase (pH
6.0 > pH 7.4) in MMC activation by XDH is due to an approximately
2-fold decrease in the A'm and a 2-fold increase in the \ ,„.,,,
at pH 6.O.
Stimulation of uric acid formation and decreases in NADH formation by
XDH in the presence of MMC suggest that MMC interaction with XDH
may occur at the NAD*-binding region of the enzyme. The ability of
xanthine to serve as reducing equivalents for MMC conversion to 2,7-DM
also supports the hypothesis that MMC reduction is occurring at the
NAD* site.
ditions by XDH also support the occurrence of both one- and twoeleclron reductions (11).
The ability of XDH to bioreduce MMC at physiological pH (7.4) to
metabolites that are stable to oxygen (11) suggests that XDH may play
an important role in the bioactivation of MMC in vivo. Other studies
have suggested that the ratio of XDH to XO present in a given tissue
may play an important role in the degree of oxidative damage induced
by MMC (15). XO is formed by the proteolytic cleavage or oxidative
modification of XDH (16, 17), but MMC activation by XO is solely
by a one-electron pathway (9, 11), whereas XDH-medialed bioreduc
tion of MMC can be via either a one- or a two-eleclron reduction (11).
Studies on the abilities of the various enzymes capable of MMC'
activalion have shown pH dependence (11, 13) as well as an inabilily
lo salurale the enzymes with MMC as a substrate at least lo Ihe limil
of MMC solubilily in a given solution (3). This inability to salurate Ihe
enzymes at MMC concentrations up to 2 HIM(3) suggests that these
enzymes have a very low affinity for MMC as a substrate. The present
studies look at the kinetics of MMC activation by XDH in an attempt
to further elucidate the role of XDH in the bioaclivalion of MMC in
vivo. These studies also look at the ability of xanthine to serve as
reducing equivalents for MMC bioreduction by XDH, the consump
tion of NADH, as well as examine the interaclion of MMC wilh the
XDH-mediated conversion of xanthine to uric acid and NAD+ to
NADH in an atlempl to investigate the mechanism of MMC bio
reduction by XDH.
INTRODUCTION
Bioreductive activation of the naturally occurring antineoplastic
antibiotic MMC4 is required for this agent to exert a toxic effect.
Following bioreduction, MMC may cause toxicity either through alkylation and cross-linking of DNA (1-3), or through oxygen radical
generation (4-7). The enzymes responsible for the activation of MMC
have been well studied and include NADPHrcytochrome c reducÃ-ase
(8-10), xanthine oxidase (9, 11), DT-diaphorase (10, 12, 13), NADH
bs reducÃ-ase (14), and xanthine dehydrogenase (11). Activation of
MMC by NADPH:cytochrome c reducÃ-ase,xanthine oxidase, NADH
fe5 reducÃ-ase, and xanthine dehydrogenase under aerobic conditions
result in the generation of oxygen radicals (4, 8, 9, 11, 14). Reductive
activation by DT-diaphorase and xanthine dehydrogenase under both
aerobic and hypoxic conditions results in the formation of stable
MMC metaboliles (11, 13), which are also produced by NADPHxytochrome c reducÃ-ase and xanthine oxidase only in the absence of
oxygen (9, 11). Reductive activation of MMC by XDH therefore
appears to occur by both one- and two-eleclron reduction pathways.
Increases in the formation of MMC metabolites under hypoxic con-
Received 5/7/93; accepted 9/14/93.
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.
1 This work was supported by USPHS Grant CA-43660 from the National Cancer
Institute and by the Nevada Agricultural Experiment Station.
- Present address: School of Pharmacy, University of Colorado Health Science Center.
Denver, CO 80262.
-' To whom requests for reprints should be addressed, at Department of Nutrition. Cell
and Molecular Pharmacology Physiology Program, University of Nevada, Mail Stop 142,
Reno, NV 89557-0132.
4 The abbreviations used are: MMC, mitomycin C; XDH, xanthine dehydrogenase;
XO. xanthine oxidase; 2,7-DM, 2.7-diaminomitosene; DT-diaphorase, NAD(P)H:quinone
oxidoreductasc; FAD, flavin adenine dinucleotide.
MATERIALS
AND METHODS
Chemicals. Mitomycin C was kindly provided by Bristol-Myers Squibb
Co. (Wallingford. CT). EDTA (disodium salt), 2-mercaptoethanol,
NAD +,
NADH (grade IV), phenylmethylsulfonyl fluoride, reactive blue 2-Scpharose,
Sephadex G-200, trypsin inhibitor (from soybean), and xanthine were pur
chased from Sigma Chemical Co. (St. Louis, MO). 5-Chloro-2-pyridinol was
purchased from Aldrich Chemical Co. (Milwaukee, WI). All other reagents
were of analytical grade.
Enzyme Preparation. Xanthine dehydrogenase was partially purified from
EMT6 tumors grown in athymic nude mice according to the method of
Suleiman and Stevens (18) as modified for EMT6 tissue by Gustafson and
Pritsos (11).
Enzyme Assays. XDH activity was measured by monitoring the formation
of uric acid at 293 nm and by measuring the xanlhine-dependent formation of
NADH at 340 nm (16). The assay mixture contained 100 mm potassium
phosphate (pH 7.8), 0.2 ITIMxanthine, and 0.4 ITIMNAD*. XDH activity is
expressed as /imol uric acid formed/min. All enzyme assays were done at 25°C
with the use of a Beckman DU-o4 spectrophotometer.
High-Pressure Liquid Chromatography Analysis of Mitomycin C Me
tabolites. High-pressure liquid chromatography analysis of mitomycin C and
its metabolites was done by the method of Pan el al. (9) as modified by Siegel
et al. (13). The reaction mixture contained 100 ITIMpotassium phosphate (pH
7.4 or 6.0), 0.5 ITIMNADH or 0.2 ITIMxanthine, mitomycin C, and approxi
mately 40 milliunits xanthine dehydrogenase and was kept at 37°Cin a total
volume of 1 ml for 45 min. 5-Chloro-2-pyridinol
(10 fig/ml) was used as an
internal standard. Hypoxic conditions were obtained by maintaining the reac
tion mixture under nitrogen gas throughout the incubation.
Measurement of NADH Consumption. NADH consumption was meas
ured by high-pressure liquid chromatography as described by Jones (19).
Samples were run as for the determination of MMC metabolites and MMC"
consumption.
5470
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.
KINETICS
OF MMC ACTIVATION
BY XANTHINE
Protein Determination. Total protein was determined by the method of
Lowry el al. (20), using bovine serum albumin as a standard.
Statistical Analysis. Statistical analysis was performed using Student's t
DEHYDROOENASE
A
80
A pH 6.0
A pH 7.4
70
test. The level of significance was attributed to P < 0.05.
60
I»50
RESULTS
Kinetics of Mitomycin C Activation by Xanthine Dehydroge-
40
nase. A kinetic evaluation of MMC activation by XDH was done at
MMC concentrations of 50, 100, 200, 400, and 500 JJ.M;under aerobic
and hypoxic conditions; and at pH 7.4 and pH 6.0 (Figs. 1 and 2).
Measurements of both 2,7-DM formation and MMC consumption
(data not shown) were performed, and kinetic constants were calcu
lated for each. MMC consumption and 2,7-DM formation under aero
bic conditions as a function of MMC concentration at both pH 7.4 and
£E 30
20
10
100
200
300
400
500
[mitomycin C] uM
A
B
A pH 6.0
pH 7.4
ISO
X
o pH 6.0
*; 70
» pH 7.4
!E 60
if
E
D
I"
50
.«
s40
o
ü 30
I
20
S
Ë 10
100
B
200
300
400
[mitomycin C] uM
500
O
100
200
300
400
[mitomycin C] uM
500
Fig. 2. Kinetic analysis of MMC activation by XDH measuring (A ) 2,7-DM formation
and (B) MMC consumption under hypoxic conditions and at pH 7.4 and 6.0. Points, mean
of at least 3 independent determinations; bars, SE. Linear fit was assessed using least
squares linear regression. (A ) r values for linear fit were: pH 7.4. 0.990; and pH 6.0, 0.956.
(B) r values for linear fil were; pH 7.4, 0.987; and pH 6.0, 0.976.
250 T
200
pH 6.0 gave rise to curves that fit the equation for a rectangular
hyperbola
150-
AX
100
-300-200-100
(Fig. ÃŒA).
Further analysis of these data, using the Hanes-Wolff plot
to calculate Michaelis-Menten constants (Km and Vmax) was done for
MMC activation by XDH under aerobic conditions (Table 1). MMC
consumption and 2,7-DM formation were also measured under hy
poxic conditions as a function of MMC concentration (Fig. 2). Under
hypoxic conditions, MMC consumption and 2,7-DM formation were
O 100 200 300 400 500
[mitomycinC] uM
linear with respect to MMC concentrations up to 500 ¡J.M,
and due to
limitations on the solubility of MMC no higher concentrations were
tested.
NADH Consumption during MMC Reduction by XDH. NADH
consumption during XDH-mediated bioreduction of MMC was meas
Fig. 1. Kinetic analysis of MMC activation by XDH measuring 2.7-DM formation
under aerobic conditions at pH 7.4 and 6.0. Points, mean of at least 3 independent
determinations; bars, SE. A, curves plotted using the equation for a rectangular hyperbola
'-¥«
and "goodness of fit" assessed using actual distances. The r2 values were: pH 7.4, 0.996;
and pH 6.0, 0.998. B, kinetic analysis using Hanes-Wolff plot. Linear fit was assessed
using least squares linear regression and the r values obtained were: pH 7.4, 0.976; and
pH 6.0, 0.998.
ured under both aerobic and hypoxic conditions (Table 2). Under
aerobic and hypoxic conditions, at a MMC concentration of 200 ¡JLM,
NADH consumption was 129.4 ±1.4 (SE) and 32.9 ±2.1 nmol/min/
unit XDH, respectively. This gave a molar ratio of NADH consumed
5471
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.
KINETICS
OF MMC ACTIVATION
BY XANTHINE
(3, 4, 9). NADPHxytochrome c reducÃ-ase,NADH bs reducÃ-ase,DTdiaphorase, xanthine oxidase, and XDH have all been identified as
being capable of bioreducing MMC lo reaclive metabolites (7-14).
The mechanisms by which these enzymes actÃ-valeMMC differ, in
cluding the pH and coenzymes required for bioactivation, as well as
Ihe resulling melabolites. NADPHicytochrome c reducÃ-aseand XO
both actÃ-valeMMC via a one-electron reduction to generate reactive
oxygen species under aerobic conditions (8, 9, 11). This one-electron
reduction under hypoxic conditions leads to the formation of the
metabolile 2,7-DM (9, 11). DT-diaphorase-mediated melabolism of
MMC aerobically does noi give rise to oxygen radicals but does form
the 2,7-DM metabolile aerobically as well as hypoxically (13), sug
gesting lhat DT-diaphorase-activated MMC does noi involve a semiquinone product or intermediates unstable lo oxygen. While NADPH:cylochrome c reducÃ-ase, XO, and DT-diaphorase may be
important in MMC bioactivalion, various inhibitor studies have shown
thai they alone cannot account for all of Ihe bioactivation of MMC and
thai other enzymes must be involved (10, 21-23). XDH has recently
been shown to be capable of bioaclivating MMC, and the resulting
metabolism can be by eilher a one- or a Iwo-eleclron reduclion,
resulling in oxygen radical generation and 2,7-DM formation under
aerobic conditions (11).
Looking at the bioreduction of MMC by XDH over a range of
MMC concentralions (50-500 JAM)under aerobic and hypoxic condi-
Table 1 Kinetic constants of mitomycin C metabolism by xanthine dehydrogenase
under aerobic conditions"
K
"M
b
v
v max
'"
MMC
consumption1'pH
7.4pH
6.0Metabolite
formation''pH
7.4pH6.029916323811433.655.63.27.1
" Kinetic constants were calculated from Hanes-Wolff plots shown in Figs. 10 and 2B.
h M.MMMC.
' nmol MMC consumed/min/unit XDH or 2,7-DM formed/unit XDH.
'' Kinetic constants were calculated using MMC consumption as a measure of MMC
metabolism.
*'Kinetic constants were calculated using 2,7-DM formation as a measure of MMC
metabolism.
Table 2 NADH consumption during MMC metabolism by XDH under aerobic and
hypoxic conditions at pH 6.0
ratio3.8
±1.4e
±1.4
Aerobically
29.5 ±1.8NADHconsumption''129.4
32.9 ±2.1NADH:MMC
HypoxicallyMMCconsumption"34.6
" nmol MMC consumed/min/unit XDH.
h nmol NADH consumed/min/unit XDH.
' Mean ±SE of a! least 3 independent determinations.
1.1
to MMC consumed of 3.8 under aerobic conditions and 1.1 under
hypoxic conditions.
Ability of Xanthine to Serve as Reducing Equivalents for Mi
tomycin C Reduction by Xanthine Dehydrogenase. The formation
of 2,7-DM by XDH in the absence of NADH, but in the presence of
0.2 HIM xanthine, under both aerobic and hypoxic conditions was
tested (Table 3). The formation of 2,7-DM with xanthine using 200 JU.M
MMC at pH 6.0 under aerobic conditions was approximately equal to
that seen with the use of NADH as reducing equivalents. However,
under hypoxic conditions, xanthine-induced formation of 2,7-DM was
increased only approximately 60% (relative to aerobic conditions) as
opposed to the approximately 3-fold increase observed hypoxically
when NADH is used.
Effect of Mitomycin C on Uric Acid and NADH Formation by
Xanthine Dehydrogenase. The effect of MMC on the XDH-medi-
lions and at pH 6.0 and 7.4, kinetic parameters and a better under
standing of the mechanisms by which XDH can bioreduce MMC by
both one and two electrons were obtained. Both MMC consumption
and 2,7-DM formation were monitored as measures of MMC bioreduction by XDH. Under aerobic conditions, both MMC consump
tion and 2,7-DM formalion gave rise to curves that fit the equation for
a rectangular hyperbola
ated conversion of xanthine to uric acid and the concomitant reduction
of NAD+ to NADH was assessed at MMC concentrations of 50, 100,
and 200 /J.M(Fig. 3). The addition of MMC led to a dose-dependent
increase in the formation of uric acid and a dose-dependent decrease
in the formation of NADH. Uric acid formation was increased 1.8,
3.6, and 6.5% above control levels at 50, 100, and 200 LIMMMC,
respectively. NADH formation was decreased 10.2, 13, and 16.5% at
these same MMC concentrations. Further kinetic studies on the effect
of 100 LIMMMC on XDH-mediated formation of NADH following
the oxidation of xanthine to uric acid were performed (Fig. 4). In both
the presence and absence of MMC, reduction of NAD+ to NADH
followed saturation kinetics with respect to differing xanthine con
centrations, with the inclusion of MMC leading to a decrease in
NADH formation. Plotting the data on a Hanes-Wolff plot (Fig. 45)
showed that the inhibition of NADH formation by MMC was consis
tent with competitive inhibition, with the Kms for xanthine being 3.4
JAMin the absence of MMC and 7.4 /LIMin the presence of 100 JAM
MMC. The Vmax was unchanged in the presence of MMC.
MMC is considered lo be Ihe prototype bioreductive alkylating
agent used in cancer chemotherapy. Reductive activalion of MMC is
for eilher
AX
Table 3 2, 7-DM formation by XDH using xanthine as reducing equivalents
2,7-DM formed/unit XDH
Aerobic
Hypoxic
1Mean ±SE of at least 3 independent determinations.
20 T
DNA alkylalion
Or generalion
of oxygen
radicals
5.76 ±0.55°
8.80 ±0.79
acid formation
formation
10-E
O
u
8
«*-10+
01
Q.
-20-1
DISCUSSION
required
DEHYDROGENASE
50
100
200
[mitomycin C] uM
Fig 3 Effectof MMCon xoH-mediateduricacidand NADHformationfromxanthine. Points, mean of at least 3 independent determinations; bars, SE.
5472
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.
KINETICS
A
too
=
OF MMC ACTIVATION
(-) mitomycin C
•¿â€¢â€¢(+)
mitomycin C
nes to form the MMC hydroquinone, which upon electron rearrange
ment forms 2,7-DM.
50
The combination of xanthine and NADH increases the reductive
activity (Vmax) of XDH for a variety of substrates as compared to the
Vmaxs obtained when these electron donors are used individually (24,
25). This suggested that xanthine and NADH donate electrons at
separate sites on the enzyme (24). NADH was presumed to donate
electrons to the FAD of XDH (26), and since the reaction of NADH
with FAD is an obligatory two-electron reduction (27, 28), FADH2
would be the expected product. Analysis of the NADH-reduced flavin
25
40
60
80
[xanthlne] uH
20
B
2.5 T
100
in XDH showed that the FAD existed as both the flavin semiquinone
(FADH ) and the flavin hydroquinone (FADH2), with the FADH
predominating (29). This is consistant with the one- and two-electron
(-) mitomycin C
•¿
•¿
(t) mitomycin C
reduction of MMC by XDH involving the reduction state of the flavin
of XDH.
Further evidence supporting a possible flavin site for MMC reduc
tion by XDH is that if MMC reduction took place at the xanthine site,
then competition between xanthine and MMC for this site would
cause a decrease in the rate of formation of 2,7-DM when xanthine
2.0-
15-
1.0
was used as reducing equivalents, as compared to NADH. This was
not observed experimentally under aerobic conditions (Fig. IA; Table
3). The approximately 60% increase in 2,7-DM formation under hy
0.5
poxic conditions is less than that seen with NADH (11). This suggests
that the one-electron reduction of MMC by XDH is not as great when
l
-20
DEHYDROGENASE
gests that XDH activation by MMC can take place by either a one- or
a two-electron reduction. The increases in 2,7-DM formation seen
under hypoxic conditions are consistent with the one-electron com
ponent of activation. The stability of the one-electron metabolite of
MMC (semiquinone) in the absence of oxygen allows for a second
one-electron addition or disproportionation of two MMC semiquino-
75
i
BY XANTHINE
20
40
60
[xanthlne] uH
Fig. 4. Effect of 100 UM MMC on XDH-mediated
xanthine is used as an electron donor as opposed to NADH. If FADH2
is responsible for the two-electron reduction of MMC and FADH is
responsible for the one-electron reduction, the difference in the for
mation of reduced flavin products (NADH favors FADH' and xan
100
conversion of NAD*to NADH
thine favors FADH2) could account for the differences in 2,7-DM
using varying xanthine concentrations. Points, mean of at leasl 3 independent determi
nations; bars, SE. In A, curves were plotted using the equation for a rectangular hyperbola:
formation hypoxically by the 2 electron donors. This is further sup
ported by our findings that xanthine-reduced XDH generated more
2,7-DM (-28% increase) than NADH-reduced XDH under aerobic
A*X
and "goodness of fit" was assessed using actual distances. The r- values were: control,
0.975; and KHJ»J.M
MMC, 0.987. B, kinetic analysis using Hanes-Wolff plot. Linear fit was
assessed using least squares linear regression and the r values obtained were: control,
0.998 and 100 UM; MMC, 0.999.
which is the same equation attained kinetically by a saturatable en
zyme system. Hanes-Wolff plots of the curves obtained from these
data allowed for calculation of the Michaelis-Menten constants Km
and Vmax of MMC metabolism by XDH. Km values at pH 6.0 were
approximately 2-fold smaller than those calculated for pH 7.4, while
Vmax values were approximately 2-fold elevated from pH 7.4 to pH
6.0, suggesting that the increase in MMC consumption and metabolite
formation seen at pH 6.0 as opposed to pH 7.4 was due to an increase
in XDH affinity for MMC as well as an increase in Vmax at pH 6.O.
Under hypoxic conditions, both MMC consumption and 2,7-DM
formation were linear over the range of MMC concentrations used.
Under these conditions, XDH was unable to be saturated by MMC
over the concentration range used, therefore giving a linear relation
ship between substrate concentration and reaction rate. The inability to
saturate XDH under hypoxic conditions is consistent with data that
have shown XO to be unsaturatable by MMC, up to 2.0 min under
hypoxic conditions, when MMC-DNA adduci formation was used as
a measure of drug activation (3).
The ability of XDH to activate MMC to generate oxygen radicals
and to form stable MMC metabolites under aerobic conditions, sug
conditions.
The efficiency of XDH in catalyzing the two-electron reduction of
MMC was addressed by measuring the ratio of NADH consumed to
MMC consumed by XDH under aerobic and hypoxic conditions. The
ratio (nmol NADH consumed/nmol MMC consumed) was 3.8 under
aerobic conditions and 1.1 under hypoxic conditions. Assuming that
all NADH consumption is due to electron transfers to MMC (NADH
consumption in the absence of MMC was negligible; data not shown),
only about 25% of MMC reductions under aerobic conditions resulted
in the formation of the two-electron metabolite, suggesting that
roughly 75% of the reductive activity resulted in MMC semiquinone
formation, which is unstable to molecular oxygen. Under hypoxic
conditions, the ratio was 1.1, showing that in the absence of oxygen,
the second one-electron addition to the semiquinone takes place in
stead of redox cycling with molecular oxygen.
The effect of MMC on XDH-mediated conversion of xanthine to
uric acid suggests that the ability of MMC to act as an electron
acceptor may facilitate the removal of electrons from the enzyme
itself, thereby allowing for a more rapid oxidation of enzyme com
ponents and a more rapid oxidation of xanthine to uric acid. The
decrease in NADH formation that accompanies this increased uric
acid formation and the fact that this inhibition is competitive are
consistent with the suggestion that MMC is acting as an alternative
electron acceptor at the flavin site within XDH.
5473
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.
KINETICS
OF MMC ACTIVATION
BY XANTHINE
The data presented here support the idea that XDH may be an
important enzyme in the bioactivation of MMC in vivo under both
aerobic and hypoxic conditions. The Km for MMC as a substrate for
reduction by XDH is low enough that it would be expected that
cellular concentrations of MMC approach these levels. These studies
also help to elucidate the mechanism by which MMC accepts elec
trons from XDH. The ability of xanthine to act as an electron donor as
well as increases in xanthine conversion to uric acid and decreases in
NADH formation in the presence of MMC by XDH show that MMC
reduction occurs at a site that interferes with the reduction of NAD+
while potentiating the oxidation of xanthine. This scenario may in
volve the donation of electrons to MMC at the NAD+-binding region
of the enzyme or at a site in the electron transport chain of the enzyme
downstream to where electrons are donated to xanthine but upstream
from where the reduction of NAD+ takes place.
REFERENCES
1. Iyer, V. N., and Szybalski, W. A molecular mechanism of mitomycin action: linking
of complimentary DNA strands. Proc. Nati. Acad. Sci. USA, 50: 355-362, 1963.
2. Iyer, V. N., and Szybalski, W. Mitomycin and porfiromycin: chemical mechanism of
activation and crosslinking of DNA. Science (Washington DC), 145: 55-58, 1964.
3. Pan, S. S., Iracki, T., and Bachur, N. R. DNA alkylation by enzyme-activated mito
mycin C. Mol. Pharmacol., 29: 622-628, 1986.
4. Pritsos, C. A., and Sartorelli, A. C. Generation of reactive oxygen radicals through
bioactivation of mitomycin antibiotics. Cancer Res., 46: 3528-3532, 1986.
5. Doroshow, J. H. Role of hydrogen peroxide and hydroxyl radical in the killing of
Ehrlich tumor cells by anticancer quiñones. Proc. Nati. Acad. Sci. USA, 83:
4514-4518, 1986.
6. Pritsos, C. A., Keyes, S. R., and Sartorelli, A. C. Effect of the Superoxide dismutase
inhibitor, diethyldithiocarbamate, on the cytotoxicity of mitomycin antibiotics. Can
cer Biochem. Biophys., JO: 289-298, 1989.
7. Gustafson, D. L., and Pritsos, C. A. Inhibition of mitomycin C's aerobic toxicity by
the seleno-organic antioxidant PZ-51. Cancer Chemother. Pharmacol., 28: 228-230,
1991.
8. Bachur, N. R., Gordon, S. L., Gee, M. V, and Kon, H. NADPH-cytochrome P-450
reducÃ-aseactivation of quinone anticancer agents to free radicals. Proc. Nati. Acad.
Sci. USA, 76: 954-957, 1979.
9. Pan, S., Andrews, P. A., Glover, C. J., and Bachur, N. R. reductive activation of
mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-45Û
reducÃ-aseand xanthine oxidase. J. Biol. Chem., 259: 959-966, 1984.
10. Keyes, S. R., Fracasso, P. M., Heimbrook, D. C., Rockwell, S., Sugar, S. G., and
Sartorelli, A. C. Role of NADPHicytochrome e reducÃ-aseand DT-diaphorase in thè
biotransformation of mitomycin C. Cancer Res., 44: 5638-5643, 1984.
11. Gustafson, D. L., and Pritsos, C. A. Bioactivation of mitomycin C by xanthine
dehydrogenase from EMT6 mouse mammary carcinoma tumors. J. Nati. Cancer Inst.,
DEHYDROOENASE
84: 1180-1185, 1992.
12. Keyes, S. R., Rockwell, S., and Sartorelli, A. C. Modification of the metabolism and
cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic
murine tumor cells. Cancer Res., 49: 3310-3313, 1989.
13. Siegel, D., Gibson, N. W., Preusch, P. C., and Ross, D. Metabolism of mitomycin C
by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in
human colon carcinoma cells. Cancer Res., SO: 7483-7489, 1990.
14. Hodnick, W. F., and Sartorelli, A. C. Reductive activation of mitomycin C by
NADH-fcs reducÃ-ase.Proc. Am. Assoc. Cancer Res., 32: 397, 1991.
15. Gustafson, D. L., Swanson, J. D., and Pritsos, C. A. Role of xanthine oxidase in the
potentiation of doxorubicin-induced cardiotoxicity by mitomycin C. Cancer Com
mun., 3: 299-304, 1991.
16. Stirpe, F., and Della Corte, E. The regulation of rat liver xanthine oxidase. Conversion.
in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type O). J.
Biol. Chem., 244: 3855-3863, 1969.
17. Della Corte, E., and Stirpe, F. The regulation of rat liver xanthine oxidase. Involve
ment of thiol groups in the conversion of the enzyme activity from dehydrogenase
(type D) into oxidase (type O) and purification of the enzyme. Biochem. J., 726:
739-745, 1972.
18. Suleiman, S. A., and Stevens, J. B. Purification of xanthine dehyydrogenase: a rapid
procedure with high enzyme yields. Arch. Biochem. Biophys., 258: 219—225,1987.
19. Jones, D. P. Determination of pyridine nucleotides in cell extracts by high perfor
mance liquid chromatography. J. Chromatogr., 225: 446-449, 1981.
20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. Protein measurement
with the Polin phenol reagent. J. Biol. Chem., 193: 265-275, 1951.
21. Dulhanty, A. M., and Whilmore, G. F. Chinese hamster ovary cell line resistant to
mitomycin C under aerobic but not hypoxic conditions are deficient in DT-diaphorase.
Cancer Res., 51: 1860-1865, 1991.
22. Tomasz, M., Hughes, C. S., Chowdary, D., Keyes, S. R., Lipman, R., Sartorelli, A. C.,
and Rockwell, S. Isolation, identification, and assay of [H3]-porfiromycin adducts of
EMT6 mouse mammary cell DNA: effects of hypoxia and dicumarol on adduci
patterns. Cancer Commun., 3: 213-223, 1991.
23. Begleiter, A., Robotham, E., and Leith, M. K. Role of NAD(P)H:(quinone acceptor)
oxidoreductase (DT-diaphorase) in activation of mitomycin C under hypoxia. Mol.
Pharmacol., 41: 677-682, 1992.
24. Rajagopalan, K. V., and Handler, P. Purification and properties of chicken liver
xanthine dehydrogenase. J. Biol. Chem., 242: 4097^107,
1967.
25. Waud, W. R., and Rajagopalan, K. V. Purification and properties of the NAD4dependent (type D) and CK-dependent (type O) forms of rat liver xanthine dehydro
genase. Arch. Biochem. Biophys., 772: 354-364, 1976.
26. Kanda, M., Brady, F. O., Rajagopalan, K. V., and Handler, P. Studies on the disso
ciation of flavin adenine dinucleotide from metalloflavoproteins. J. Biol. Chem., 247:
765-770, 1972.
27. Powell, M. F., and Bruice, T. C. Hydride versus electron transfer in the reduction of
flavin and flavin radical by 1,4-dihydropyridines.
J. Am. Chem. Soc., 105:
1014-1021, 1983.
28. Powell, M. F., and Bruice, T. C. Effect of isotope scrambling and tunneling on the
kinetic and product isotope effects for reduced nicotinamide adenine dinucleotide
model hydride transfer reactions. J. Am. Chem. Soc., 705: 7139-7149, 1983.
29. Schöpfer,L. M., Massey, V., and Nishino, T. Rapid reaction studies on the reduction
andoxidation of chicken liver xanthine dehydrogenase by the xanthine/urate and
NAD/NADH couples. J. Biol. Chem., 263: 13528-13538, 1988.
5474
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.
Kinetics and Mechanism of Mitomycin C Bioactivation by
Xanthine Dehydrogenase under Aerobic and Hypoxic
Conditions
Daniel L. Gustafson and Chris A. Pritsos
Cancer Res 1993;53:5470-5474.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/53/22/5470
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1993 American Association for Cancer Research.