[CANCER RESEARCH
45, 6442-6445,
December 1985]
Production of Hydroxyl-free Radical by Reaction of Hydrogen Peroxide with
/V-Methyl-A/'-nitro-W-nitrosoguanidine1
Tomiko Mikuni,2 Masaharu Tatsuta, and Mikiharu Kamachi
Department of Gastrointestinal Oncology, The Center for Adult Diseases, Osaka, 3-3, Nakamichi 1-chome, Higashinari-ku, Osaka 537 [T. M., M. H.], and Department of
Polymer Science, Faculty of Science, Osaka University, Machikaneyama 1-chome, Toyonaka 560 [M. K.J Japan
ABSTRACT
Production of a hydroxyl free radical (-OH) by reaction of
hydrogen peroxide (H2O2)with A/-methyl-/V'-nitro-A/-nitrosoguanidine (MNNG) was examined by electron spin resonance using
the -OH spin trapping agent 5,5-dimethyl-1-pyrroline-1-oxide
(DMPO).
The electron spin resonance spectra of the H202-MNNGDMPO system after exposure to light at an intensity of 0.03 mW/
cm2 for 5 min, and the DMPO-(-OH) spin adduci (2-hydroxy-5,5dimethyl-1-pyrroline-1-oxide) generated by use of Fenton's re
agent showed the same hyperfine structure and g-value. The
signal of the DMPO adduci obtained in the H2O2-MNNG-DMPO
system disappeared on addilion of Ihe •
OH scavenger sodium
benzoato. The addition of another -OH scavenger, ethanol,
resulted in the appearance of a new signal due to trapping of the
a-hydroxyethyl radical. These results show that •
OH was formed
in the H2O2-MNNG-DMPO system. The typical signal of the
DMPO-(-OH) spin adduct was not observed in the system in the
absence of light. The amount of DMPO-(-OH) spin adduct in
creased with increase in the concentration of H2O2 when the
MNNG level was kept constant, and it changed with the concen
tration of MNNG at a constant H2O2 level, indicating that -OH
was produced by the interaction of MNNG with H2O2. In the
absence of H2O2, complicated trapped signals appeared in the
spectrum of the MNNG-DMPO system in the light, but these
signals were not observed when the system was kept in the
dark. In the absence of MNNG, the H2O2-DMPO system did not
show any signal, even in the light.
These results indicate that interaction of free radicals derived
from MNNG with H2O2 on exposure to light resulted in -OH
production.
INTRODUCTION
In 1967, Sugimura and Fujimura (1) reported that when MNNG3
was given continuously to rats in their drinking water, gastric
cancers developed at a high incidence within 1 year. Gastric acid
secretion decreased during cancer development in rats treated
in this way (2), as it does during development of gastric cancer
in humans (3). These findings suggested that MNNG may be
useful for studying the pathogenesis and pathophysiology of
gastric cancer (2).
MNNG directly methylates and deaminates the basic portion
of nucleic acids and reacts with proteins (4-9), but the mecha-
nism of its carcinogenic activity is unknown. We reported previ
ously that the incidence of gastric cancer induced by oral admin
istration of MNNG was significantly reduced by prolonged admin
istration of the antioxidant agent, butylated hydroxytoluene (10).
Since butylated hydroxytoluene is a radical scavenger (11), free
radicals may be related to the carcinogenic activity of MNNG.
Among the free radicals, •
OH is extremely reactive, damaging
nucleic acids (12-15) and the cell membrane (16-18).
In the present work, we examined -OH production in the
reaction of MNNG with H202 by ESR spectrometry using the
spin-trapping technique.
MATERIALS AND METHODS
Production of -OH in the reaction of MNNG with H2O2 was detected
by ESR spectrophotometry
using the spin-trapping technique; -OH
reacts with the nitrone spin trap, DMPO (Sigma Chemical Co., St. Louis,
MO) to yield 2-hydroxy-5,5-dimethyl-1-pyrroline-1-oxide,
which can be
detected by ESR spectrometry (19, 20). The distilled water used in
experiments and stock solutions of 100 TTIMNaH2PO4-H3PO4 buffer (pH
3.5) and 750 HIM NaCI were passed through columns of Chelex-100
resin to reduce the amount of polyvalent metal ion impurities.
A stock solution of DMPO was prepared by dissolving 1 g of DMPO
in 9 ml of distilled water and filtering the solution through water-washed
activated charcoal (21). This stock solution was stored in brown am
poules under N2 at -20°C. The concentration of the DMPO stock solution
was determined from its optical absorbance («;7220 M^crrr1 at 266 nm)
(22). Within 24 h before use, MNNG (Aldrich Chemical Co., Milwaukee,
Wl) was dissolved in distilled water at a concentration of 10 mu in a
beaker covered with aluminum foil to avoid light denaturatoli of the
MNNG. Working solutions of H2O2 were prepared before use from a
stock solution of 30% H2O2(Mitsubishi Gas Chemical Co., Tokyo, Japan)
by dilution with distilled water.
•
OH Production. A typical experiment was carried out as follows. The
reaction mixture (H2O2-MNNG-DMPO system) contained, in a final vol
ume of 200 p\, 50 rnw NaH2PO4-H3PO4 buffer (pH 3.5), 38 mw NaCI, 100
(TIMDMPO, 0.15% H2O2, and 2.5 mw MNNG. Reactions were initiated
by adding MNNG. The reaction mixture was transferred rapidly to a flat
quartz cell and exposed to room light at an intensity of 0.03 mW/cm2 for
5 min from the beginning of the reaction at room temperature. Immedi
ately after exposure to light, the ESR spectrum was recorded in an ESR
spectrometer, model JES-FEIX (Japan Electron Optics), with 100-kHz
field modulation. Typical conditions for the measurement were as follows:
magnetic field, 3288 ±100 G; microwave power, 20 mW; modulation
width, 2 G; sweep time, 2 min; response, 1 s; amplitude (which corre
sponds to "gain"), 6 x 102; and room temperature.
•
OH was generated with Fenton's reagent (23). The spectrum of
Fenton's reagent was recorded as described above, except that the
Received 5/21/84; revised 7/10/85; accepted 8/28/85.
1This work was supported in part by a Grant-in-Aid from the Ministry of Health
and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan.
2 To whom requests for reprints should be addressed.
3The abbreviations used are: MNNG, N-methyl-W'-nitro-W-nitrosoguanidine;
•
OH, hydroxyl free radical; ESR, electron spin resonance; DMPO, 5,5-dimethyl-1pyrroline-1-oxide.
CANCER
RESEARCH
amplitude was 6 x 10, and it was compared with that of the H2O2MNNG-DMPO system. Fenton's reagent contained, in a final volume of
200 iti. 38 rriM NaCI-50 rnw NaH2PO4-H3PO4 buffer (pH 3.5), 100 HIM
DMPO, 0.15% H202, and 50 MM FeCI2. The effects of -OH scavengers
on the spectrum of the H2O;rMNNG-DMPO system were examined by
addition of sodium benzoate
VOL. 45 DECEMBER
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1985
6442
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HYDROXYL-FREE
RADICAL
BY HYDROGEN
PEROXIDE AND MNNG
exposure to light.
lOGauss
The effect of exposure to light on the H2O;rMNNG-DMPO system was
also examined. The spectra of this system after keeping it in the absence
of light for 5 min and after exposure to light were examined to compare
the amounts of the DMPO adduci formed.
The amount of the DMPO adduct was calculated as the relative
intensity of the signal that appeared in the same magnetic field as the
third peak of 2-hydroxy-5,5-dimethyl-1-pyrroline-1
-oxide from the lower
field using Mn2+ as a standard.
Test for Fenton's Reaction. Examination of the MNNG solution with
a polarized Zeeman atomic absorbance spectrophotometer, model 18080 (Hitachi Co., Tokyo, Japan) showed that it contained 1.8 x 10~8 M
iron. Therefore, we examined whether signals from the H2O2-MNNGDMPO system resulted from Fenton's reaction of H2O2with Fe2+contam
inating the MNNG solution. For this, 1.8 x 10~°M FeCI2 was added to
the H2O2-DMPO system in place of MNNG. FeCI2 solution was prepared
just before use by dissolving FeCI2 in 0.0012 N HCI in a brown tube and
bubbling the solution with N2 gas (22).
•
OH Production by the Interaction of H2O2and MNNG. The effect of
H2O2 at concentrations of 0 to 0.20% on the amount of the DMPO
adduct was examined, when the levels of MNNG and DMPO were kept
constant. The effect of MNNG at concentrations of 0 to 3 mw on the
amount of the DMPO adduct was also examined, when the levels of
H2O2and DMPO were kept constant.
Free Radicals Formed from MNNG on Light Exposure. We tested
whether any free radicals were formed from MNNG or H2O2 alone on
exposure to light. For this, the spectra and amounts of trapped signal
obtained after exposure of the systems to light in the absence of H2O2
(MNNG-DMPO system) or MNNG (H2O2-DMPO system) were compared
with those obtained when the systems were kept in the dark.
Absorbance Spectra. The UV absorbance spectra of solutions of
H2O2 (0.0015%), MNNG (0.025 rtiM), and H2O2 (0.0015%) plus MNNG
(0.025 mw), all in 38 HIM NaCI-50 mw NaH2PO4-H3PO4 buffer (pH 3.5),
were recorded at room temperature in a spectrophotometer, model 20020 (Hitachi Co., Tokyo, Japan). In all experiments, recordings were made
immediately after preparation of solutions.
Statistical Analysis. Data were expressed as means ±SE.and results
were analyzed by Student's t-test (24). "Significant" indicates a calculated
P-value of less than 0.05.
RESULTS
•
OH Production. The ESR spectra of the H2O2-MNNG-DMPO
system (Chart 1/1) and Fenton's reagent (Chart 1S) showed the
same hyperfine structure and g-value; namely, a quartet with
1:2:2:1 signal intensity, hyperfine splitting constants of AN = A"
= 14.8 G, and a g-value of 2.006. However, the residual signals
were superimposed on the quartet in the spectrum of the H2O2MNNG-DMPO system.
The effects of •
OH scavengers, sodium benzoate and ethanol,
on the spectrum of the H202-MNNG-DMPO system were ex
amined. On addition of sodium benzoate to the H2O2-MNNGDMPO system, the characteristic quartet disappeared (Chart 1C)
(25), while on addition of ethanol a new signal appeared due to
trapping of the a-hydroxyethyl radical (Chart 1D) (19, 26). These
results showed that -OH was produced in the H2O2-MNNGDMPO system.
As shown in Chart 1E, when the system was kept in the
absence of light, -OH signal was less than in Chart 1A. The
amount of •
OH trapped was significantly (P < 0.001 ) more after
light exposure [1.00 ±0.05 (SE)] than in the absence of light
(0.29 ±0.03).
These results show that free radicals were produced and
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RESEARCH
ÃŽ
g=2.006
Chart 1. Typical ESR spectra of the H2O2-MNNG-DMPO system and Fenton's
reagent containing H202 and Fe2+. A, after light exposure of the H202-MNNGDMPO system; B, Fenton's reagent; C, after light exposure of the H2O¡rMNNGDMPO system with added sodium benzoate; D, after light exposure of the HjCV
MNNG-DMPO system with added ethanol; E, the H2O2-MNNG-DMPO system in
the absence of light; F, after light exposure of DMPO solution alone as a control.
trapped in the H202-MNNG-DMPO system on exposure to light.
Absence of Fenton's Reaction. As shown in Chart 2, A and
B, the spectrum of the H202-DMPO system was not affected by
addition of 1.8 x 10~8 M FeCI2, which is the same concentration
as that contaminating the MNNG solution. This result excludes
the possibility that •
OH was produced by Fenton's reaction of
H2O2with Fe2+ contaminating the MNNG solution.
OH Production by the Interaction of MNNG and H2O2.Chart
3A shows that the amount of trapped signal increased with
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HYDROXYL-FREE
RADICAL
BY HYDROGEN
lOGauss
PEROXIDE AND MNNG
the absence of light (Chart 4B). The amount of trapped signal in
the MNNG-DMPO system after light exposure was 2.6 times as
large as that in the absence of light (Table 1). In the absence of
MNNG, no typical signal was observed in the spectra of the
H2O2-DMPO system with or without light exposure to light
(Charts 4, C and D; Table 1). These results indicate that free
radicals were derived from MNNG by light exposure.
Absorbance Spectra. The UV absorbance spectrum of H2O2
plus MNNG solution did not show any difference with that of the
sum of the absorbance spectra of H2O2 solution and MNNG
'2.006
solution, which suggested that the signals did not result from
Chart 2. Comparison of ESR spectra after light exposure of the H2O2-DMPO direct interaction of MNNG and H2O2.
system without (A) and with (8) added FeCI2(1.8 x 1fr* M).This concentration of
iron is the same as that contaminating the MNNG solution.
DISCUSSION
From the present study we conclude that •
OH is produced by
the interaction of H202 with free radicals derived from MNNG on
its exposure to light.
•
OH is also produced by Fenton's reaction, i.e., the reaction
of H2O2 with Fe2+ (23), and since we did not remove Fe2+ ion
from the MNNG solution completely, -OH might have been
produced by Fenton's reaction in this system. However, we
showed that addition of 1.8 x 10~8 M Fe2+, the amount contam-
p
_
, , 0.03
0.0075 °-015
0.06
Table 1
Effect oÃ-exposure of the MNNG-DMPOand HA-DMPO systems to light on the
amount of trapped signal
The concentrations of MNNG, H2O2,and DMPO were 2.5 mM,0.15%, and 100
mM,respectively.
0.20
0.15
H2O2 Concentration
(%)
Amount of trapped signal
(relative intensity)
System
With light
Without light
0.29 ±0.02s (6)6
0.11 ±0.01°
(5)
MNNG-DMPO
H202-DMPO
0.16 ±0.00(4)
0.13 ±0.00(4)
' Mean ±SE.
3Numbers in parentheses, number of experiments.
: Significantlydifferent from the value after exposure to light (P < 0.001).
1
2
2.5
MNNG Concentration
lOGauss
3
(mM)
Chart 3. Effects of the concentrations of H2O2(A) and MNNG(B)on the amount
of trapped signal. Amounts of trapped signal were measured when the concentra
tions of H2O2were changed between 0 and 0.2%, but the levels of MNNG and
DMPO were kept constant (A). Amounts of trapped signal were also measured
when the concentrations of MNNG were changes between 0 and 3 mw, but the
levels of H202and DMPO were kept constant (ß).
Amounts of trapped signal were
calculated as relative intensities of the signals in ESR spectra.
(A)
increase in H2O2 concentration when the level of MNNG was
kept constant. The amount of trapped signal also increased with
the concentration of MNNG to a maximum at 2.5 mw MNNG
when the level of H2O2 was kept constant (Chart 38). These
results indicate that •
OH production resulted from the interaction
of MNNG and H2O2. These results also show that the relevant
concentrations of H2O2 and MNNG were 0.15% and 2.5 HIM,
respectively, in the H2O2-MNNG-DMPO system, the H2O2-DMPO
system, and the MNNG-DMPO system.
Free Radicals Formed from MNNG on Light Exposure. As
shown in Chart 4/4, in the absence of H2O2, the spectrum of the
MNNG-DMPO system after light exposure showed complicated
signals, which include the residual ones observed in Chart 1/4.
These signals were not observed when this system was kept in
CANCER
RESEARCH
ÃŽ
_g=2.006
Chart 4. Typical ESR spectra of the MNNG-DMPO and H202-DMPOsystems.
A and B, MNNG-DMPOsystem with (A) and without (B) light exposure; C and D,
H202-DMPOsystem with (C) and without (D) light exposure.
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HYDROXYL-FREE
RADICAL
BY HYDROGEN
PEROXIDE AND MNNG
615-620,1969.
5. Lawley, P. D. Methylatton of DMA by N-methyl-N-nitrosourethane and Nmethyl-N-nitroso-N'-nitroguanidine.Nature (Lond.), 278: 580-581, 1968.
6. Lawley, P. D. and Thatcher, C. J. Methylatlon of deoxyribonucleic acid in
cultured mammaliancells by N-methyl-N'-nitro-N-nitrosoguanidine. The influ
We found that the amount of •
OH trapped by DMPO depended
ence of cellular thlol concentrations on the extent of methylation and the 6oxygen atom of guanine as a site of methylatlon. Biochem. J., 776:693-707,
on the concentrations of both H2O2 and MNNG. This finding
1970.
indicated that •
OH was produced by the interaction of H2O2and
7. McCalla,D. R. Reactionof N-methyl-N'-nitro-N-nitrosoguanidineand N-methylMNNG. Moreover we found that, on increasing the MNNG con
N-nitroso-p-toluenesulfonamidewith DNA in vitro. Biochim. Biophys. Acta,
755: 114-120, 1968.
centration, the amount of -OH trapped showed a maximum,
8. Nagao,M., Yokoshima,T., Kosoi, H., and Sugimura,T. Interactionof N-methylwhich suggested that -OH production was not the direct con
N'-nitro-N-nitrosoguanidine with ascites hepatoma cells in vitro. Biochim.
sequence of the reaction of MNNG with H2O2 but possibly that
Biophys. Acta, 792: 191-199,1969.
9. Sugimura, T., Fujimura, S., Nagao, M., Yokoshima, T., and Hasegawa, M.
of some side reaction. The absorbance spectra of H2O2, MNNG,
Reaction of N-methyl-N'-nitro-N-nitrosoguanidine with protein. Biochim. Bio
and H2O2 plus MNNG were consistent with this suggestion.
phys. Acta, Õ70:427-429,1968.
Since in the present work we found that free radicals were
10. Tatsuta, M., Mikuni, T., and Taniguchi, H. Protective effect of butylated
hydroxytoluene against induction of gastric cancer by N-methyl-N'-nitrc-Nproduced in the MNNG-DMPO system, it seems likely that -OH
nitrosoguanidinein Wistar rats. Int. J. Cancer, 32: 253-254,1983.
was produced by the interaction of free radicals derived from
11. Harman,D. Free radicaltheory of aging: effect of free radical reaction inhibitors
on the mortality rate of male LAP, mice. J. Gérant.,
23: 476-482, 1968.
MNNG with H2O2. The exact mechanism of this production is
12. Brawn, K. and Fridovich, I. DNA strand scission by enzymically generated
not clear. However, it is known that NO groups are quite labile,
oxygen radicals. Arch. Biochem. Biophys., 206: 414-419,1981.
split off in light to yield reactive •
NO radical (27). Nagata ef a/.
13. Kuwabara, M., Zhi-Yi, Z., and Yoshii, G. E.S.R. of spin-trapped radicals in
aqueous solutions of pyrimidinenucleosidesand nucleotides. Reactionsof the
(28) showed the formation of the radical from MNNG,
hydroxyl radical. Int. J. Radiât.Biol., 41: 241-259, 1982.
CH3NC(NH)NHNO2, on photoirradiation of MNNG in benzene
14. Lesko, S. A., Lorentzen, R. J., and Ts'o, P. O. P. Role of Superoxide in
deoxyribonucleicacid strand scission. Biochemistry, 79: 3023-3028,1980.
solution and in the solid state, which indicated that MNNG, one
15. Ward, J. F. Some biochemical consequences of the spatial distribution of
of NO groups, splitted off to yield -NO and CH3NC(NH)NHNO2
ionizing radiation-producedfree radicals. Radial. Res., 86:185-195,1981.
radicals on photoirradiation. Consistent with this report, we
16. Freeman, B. A. and Crapo, J. D. Biology of disease. Free radicals and tissue
injury. Lab. Invest., 47: 412-426,1982.
observed residual signals superimposed on the -OH spectrum
17. Lai, C. S. and Piette, L. H. Spin-trappingstudies of hydroxyl radical production
after exposure of the H202-MNNG-DMPO system to light,
involved in lipid peroxidation. Arch. Biochem. Biophys., 790: 27-38,1978.
suggesting that -NO and CH3NC(NH)NHNO2 were formed on
18. Weddle, C. C., Hombrook, R., and McCay, P. B. Lipid peroxidation and
alteration of membrane lipids in isolated hepatocytes exposed to carbon
exposure of MNNG to light. Since Gray ef a/. (29) reported that
tetrachtoride.J. Biol. Chem., 257: 4973-4978,1976.
•
OH is produced by the reaction of H202 with -NO, it is probable
19. Fmkelstein.E., Rosen, G. M., and Rauckman,E. J. Spin trapping of Superoxide
that -OH production resulted from the interaction of H202 with •
and hydroxyl radical: practical aspects. Arch. Biochem. Biophys., 200: 1-16,
1980.
NO derived from MNNG in light in the H202-MNNG-DMPO sys
20. Janzen, E. G., and Liu, J. I-P. Radical addition reactions of 5,5-dimethyl-1tem. There is no report of •
OH production by the interaction of
pyrroline-1-oxide.ESR spin trapping with a cyclic nitrone. J. Magn. Reson., 9:
510-512,1973.
CH3NC(NH)NHNO2 with H202, but further investigations are
21. Buettner, G. R. and Oberley. L. W. Considerations in the spin trapping of
needed to clarify the role of this radical in •
OH production.
Superoxide and hydroxyl radical in aqueous systems using 5,5-dimethyl-1In our system, exposure to light was necessary for -OH
pyrroline-1-oxide.Biochem. Biophys. Res. Commun., 83:69-74,1978.
production, and the H2O2 concentrations used were unphysid22. Floyd, R. A. and Lewis, C. A. Hydroxyl free radical formation from hydrogen
peroxide by ferrous iron-nucleotidecomplexes. Biochemistry, 22:2645-2649,
ogically high (30-32), but Nagata ef a/. (28) reported that
1983.
23.
Walling,
C. Fenton's reagent revisited. Accounts. Chem. Res., 8: 125-131,
CH3NC(NH)NHNO2 radical was also produced by stirring MNNG
inating the MNNG solution, to the H2O2-DMPO system did not
influence the spectrum, that is, that -OH was not produced from
contaminating Fe2+ by Fenton's reaction.
in aqueous solution at pH 3 to 6. Moreover, Ramasarma (33)
observed that H2O2was generated by biomembranes. Therefore,
OH may be produced by the interaction of free radicals derived
from MNNG with H2O2 at biomembranes in the stomach of
animals given MNNG solution orally. We are planning to investi
gate •
OH production in the stomach of animals after oral admin
istration of MNNG.
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VOL. 45 DECEMBER
1985
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Production of Hydroxyl-free Radical by Reaction of Hydrogen
Peroxide with N-Methyl-N′-nitro-N-nitrosoguanidine
Tomiko Mikuni, Masaharu Tatsuta and Mikiharu Kamachi
Cancer Res 1985;45:6442-6445.
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