Recent Papers -abstracts-
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Hiraku Y. Murata M. Kawanishi S. Determination
of intracellular glutathione and thiols by high performance liquid chromatography
with a gold electrode at the femtomole level: comparison with a spectroscopic
assay. Biochimica et Biophysica Acta. 1570(1):47-52, 2002 Feb
15.Glutathione (GSH) is an important thiol, which has multiple
functions in human metabolism, including the detoxification of xenobiotics,
radioprotection and antioxidant defense. Here we provide a sensitive and
specific method to quantify intracellular GSH and other thiols using an
electrochemical detector coupled to a high performance liquid chromatograph
(HPLC-ECD). This HPLC-ECD system includes a specially devised gold electrode
with a large surface area and a thin gasket to provide an extremely high
sensitivity to thiols. The standard curve for GSH showed a good linear
relationship at low femtomole levels (r=0.970). We could simultaneously
detect GSH, cysteine, N-acetylcysteine, gamma-glutamyl-cysteine and cysteinyl-glycine
by this method. We compared the specificity and sensitivity of this method
with those of the conventional spectroscopic method by measuring the amounts
of GSH in HL-60 cell extracts. Although the values obtained from these
methods were closely correlated (r=0.984), the electrochemical method was
much more specific for GSH. This method could detect 2 fmol of GSH and
was 6 orders and 2-3 orders of magnitude more sensitive than the spectroscopic
method and previous methods using HPLC, respectively. As an example of
the application of this method, we demonstrated that the time-dependent
alteration in intracellular GSH and cysteine levels could be easily measured
using buthionine sulfoximine, an inhibitor of GSH synthesis. On the basis
of these results, the advantage of this electrochemical method is extremely
sensitive and specific to detect femtomole levels of GSH and other various
thiols.
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Murata M. Kawanishi S.
Oxidation of 5'-site guanine
at GG and GGG sequences induced by a metabolite of carcinogenic heterocyclic
amine PhIP in the presence of Cu(II) and NADH. Carcinogenesis.
23(5):855-60, 2002 May. Adduct formation has been considered
to be a major causal factor of DNA damage by carcinogenic heterocyclic
amines. By means of experiments with an electrochemical detector coupled
to a high-performance liquid chromatograph, we revealed that N-hydroxy
metabolite of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)
induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in the presence
of Cu(II). Addition of an endogenous reductant NADH enhanced the 8-OH-dG
formation. Experiments with 32P-labeled DNA fragments showed
that this metabolite [PhIP(NHOH)] caused 8-hydroxylation of guanines in
the presence of Cu(II) and NADH, and subsequent treatment with formamidopyrimidine-DNA
glycosylase led to chain cleavages at the 5'-site guanine of GG and GGG
sequences. Interestingly, antioxidant enzyme SOD enhanced the intensity
of DNA damage, and thymine residues were appended to its guanine-predominant
cleavage sites. Catalase and bathocuproine, a Cu(I)-specific chelator,
inhibited the DNA damage, suggesting the involvement of H2O2
and Cu(I). A UV-visible spectroscopic study indicated that Cu(II) and SOD
catalyze the autoxidation of PhIP(NHOH). These results suggest that Cu(II)-dependent
autooxidation of PhIP(NHOH) coupled with NADH-mediated reduction of its
oxidized product form redox cycle, resulting in oxidative DNA damage by
low concentrations of PhIP(NHOH). We conclude that in addition to DNA adduct
formation, oxidative DNA damage may be involved in the carcinogenic process
of PhIP.
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Kawanishi S. Sakurai H. Differential anti-lipid
peroxidative activity of melatonin. Naturwissenschaften. 89(1):31-3,
2002 Jan. Scavenging activities of melatonin, which is a
pineal secretory product and functions in circadian biology, and its related
compounds against reactive oxygen species such as superoxide anion radical,
hydrogen peroxide, hydroxyl radical and singlet oxygen as well as organic
peroxide radical (t-BuOO*) were evaluated chemically by using electron
spin resonance-spin trap and chemiluminescence methods. Antioxidative activity
of the compounds was estimated by IC50 value (microM), 50% inhibiting concentration
of a compound against reactive oxygen species formed in each system, and
the second-order rate constants (k2) for the reactions of the
compounds and superoxide anion radical or hydroxyl radical. Because melatonin
has exhibited the highest scavenging activity against t-BuOO*, the biochemical
anti-lipid peroxide radical scavenging activities of melatonin were examined.
We found that melatonin exhibits higher anti-lipid peroxidative activity
in the rat brain microsomes than in the rat liver microsomal and liposomal
systems, suggesting that melatonin may function as a treatment for reactive
oxygen species-related diseases of the brain.
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Sakano K. Oikawa S. Hasegawa K. Kawanishi S. Hydroxyurea
induces site-specific DNA damage via formation of hydrogen peroxide and
nitric oxide.Japanese Journal of Cancer Research. 92(11):1166-74,
2001 Nov. Hydroxyurea is a chemotherapeutic agent used for
the treatment of myeloproliferative disorders (MPD) and solid tumors. The
mutagenic and carcinogenic potential of hydroxyurea has not been established,
although hydroxyurea has been associated with an increased risk of leukemia
in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity,
we examined site-specific DNA damage induced by hydroxyurea using 32P-5'-end-labeled
DNA fragments obtained from the human p53 and p16 tumor suppressor
genes and the c-Ha-ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated
DNA damage especially at thymine and cytosine residues. NADH efficiently
enhanced hydroxyurea-induced DNA damage. The DNA damage was almost entirely
inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, suggesting
the involvement of hydrogen peroxide H2O2 and Cu(I).
Typical free hydroxyl radical scavengers did not inhibit DNA damage by
hydroxyurea, but methional did. These results suggest that crypto-hydroxyl
radicals such as Cu(I)-hydroperoxo complex (Cu(I)-OOH) cause DNA damage.
Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea
in the presence of Cu(II). An electron spin resonance spectroscopic study
using N-(dithiocarboxy)sarcosine as a nitric oxide (NO)-trapping reagent
demonstrated that NO was generated from hydroxyurea in the presence and
absence of catalase. In addition, the generation of formamide was detected
by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass
spectrometry (TOF-MS). A high concentration of hydroxyurea induced depurination
at DNA bases in an H2O2-independent manner, and endonuclease
IV treatment led to chain cleavages. These results suggest that hydroxyurea
could induce base oxidation as the major pathway of DNA modification and
depurination as a minor pathway. Therefore, it is considered that DNA damage
by hydroxyurea participates in not only anti-cancer activity, but also
carcinogenesis.
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Ohnishi S. Murata M. Kawanishi S. DNA
damage induced by hypochlorite and hypobromite with reference to inflammation-associated
carcinogenesis. Cancer Letters. 178(1):37-42,
2002 Apr 8.Hypohalites (OCl-,
OBr-) are formed at inflammation sites as antimicrobial agents.
OCl- is also used for the disinfection of water supplies and
the association of drinking chlorinated water with cancer risk is pointed
out. In this study, OCl- itself induced 8-oxo-7,8-dihydro-2'-deoxyguanosine
(8-oxodG) formation, while OBr- damaged DNA only when glutathione
(GSH) was added. OCl- caused oxidative DNA damage more efficiently
than OBr-/GSH. In experiment with 32P-labeled DNA
fragments, OCl- strongly caused piperidine-labile sites at guanine
residues than piperidine-inert 8-oxodG, whereas OBr-/GSH caused
no piperidine-labile sites. Endogenous OCl- may play a role
in genotoxicity close to the site of inflammation.
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Ohnishi S. Kawanishi S. Double
base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.
Biochemical
& Biophysical Research Communications. 290(2):778-82, 2002 Jan 18.
Carcinogenic
benzo[a]pyrene (BP) is generally considered to show genotoxicity
by forming DNA adducts of its metabolite, BP-7,8-diol-9,10-epoxide. We
investigated oxidative DNA damage and its sequence specificity induced
by BP-7,8-dione, another metabolite of BP, using 32P-5'-end-labeled
DNA. Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites
mainly at G residues of 5'-TG-3' sequence and at poly(C) sequences, in
DNA incubated with BP-7,8-dione in the presence of NADH and Cu(II), whereas
piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. BP-7,8-dione
strongly damaged the G and C of the ACG sequence complementary to codon
273 of the p53 gene. Catalase and a Cu(I)-specific chelator attenuated
the DNA damage, indicating the involvement of H2O2
and Cu(I). BP-7,8-dione with NADH and Cu(II) also increased 8-oxo-7,8-dihydro-2'-deoxyguanosine
formation. We conclude that oxidative DNA damage, especially double base
lesions, may participate in the expression of carcinogenicity of BP in
addition to DNA adduct formation.
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Hirakawa K. Oikawa S. Hiraku Y. Hirosawa I. Kawanishi S. Catechol
and hydroquinone have different redox properties responsible for their
differential DNA-damaging ability. Chemical Research in Toxicology.
15(1):76-82, 2002 Jan.We examined the redox properties of the "carcinogenic"
catechol and the "noncarcinogenic" hydroquinone in relation to different
DNA damaging activities and carcinogenicity using 32P-labeled
DNA fragments obtained from the human genes. In the presence of endogenous
NADH and Cu2+, catechol induces stronger DNA damage than hydroquinone,
although the magnitudes of their DNA damaging activities were reversed
in the absence of NADH. In both cases, DNA damage resulted from base modification
at guanine and thymine residues in addition to strand breakage induced
by Cu+ and H2O2, generated during the
oxidation of catechol and hydroquinone into 1,2-benzoquinone and 1,4-benzoquinone,
respectively. EPR and 1H NMR studies indicated that 1,2-benzoquinone
is converted directly into catechol through a nonenzymatic two-electron
reduction by NADH whereas 1,4-benzoquinone is reduced into hydroquinone
through a semiquinone radical intermediate through two cycles of one-electron
reduction. The reduction of 1,2-benzoquinone by NADH proceeds more rapidly
than that of 1,4-benzoquinone. This study demonstrates that the rapid 1,2-benzoquinone
two-electron reduction accelerates the redox reaction turnover between
catechol and 1,2-benzoquinone, resulting in the enhancement of DNA damage.
These results suggest that the differences in NADH-mediated redox properties
of catechol and hydroquinone contribute to their different carcinogenicities.
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Ohkuma Y. Hiraku Y. Kawanishi S. Sequence-specific
DNA damage induced by carcinogenic danthron and anthraquinone in the presence
of Cu(II), cytochrome P450 reductase and NADPH. Free Radical
Research. 34(6):595-604, 2001 Jun.The mechanism of metal-mediated
DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone
was investigated by the DNA sequencing technique using 32P-labeled
human DNA fragments obtained from the human c-Ha-ras-1 protooncogene
and the p53 tumor suppressor gene. Danthron caused DNA damage particularly
at guanines in the 5'-GG-3', 5'-GGGG-3', 5'-GGGGG-3'
sequences (damaged bases are bold-faced) in the presence of Cu(II), cytochrome
P450 reductase and the NADPH-generating system. The DNA damage was inhibited
by catalase and bathocuproine, suggesting the involvement of H2O2
and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine increased
with increasing concentration of danthron. On the other hand, carcinogenic
anthraquinone induced less oxidative DNA damage than danthron. Electron
spin resonance study showed that the semiquinone radical could be produced
by P450 reductase plus NADPH-mediated reduction of danthron, while little
signal was observed with anthraquinone. These results suggest that danthron
is much more likely to be reduced by P450 reductase and generate reactive
oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated
DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated
metabolites with similar reactivity to danthron may participate in DNA
damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone
seems to be relevant for the expression of their carcinogenicity.
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Midorikawa K. Murata M. Oikawa S. Hiraku Y. Kawanishi S. Protective
effect of phytic acid on oxidative DNA damage with reference to cancer
chemoprevention.Biochemical & Biophysical Research Communications.
288(3):552-7, 2001 Nov 2.Phytic acid (myo-inositol hexaphosphate)
is one of the most promising cancer chemopreventive agents. We investigated
the mechanism by which phytic acid expresses preventive action to cancer.
Phytic acid inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine
in cultured cells treated with an H2O2-generating
system, although it did not scavenge H2O2. Site-specific
DNA damage by H2O2 and Cu(II) at GG and GGG sequences
was inhibited by phytic acid, but not by myo-inositol. Phytic acid alone
did not cause DNA damage and thus, it should not act as a prooxidant. We
conclude that phytic acid acts as an antioxidant to inhibit the generation
of reactive oxygen species from H2O2 by chelating
metals, resulting in chemoprevention of cancer. Copyright 2001 Academic
Press.
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Murata M. Ohnishi S. Kawanishi S. Acrylonitrile
enhances H2O2-mediated DNA damage via nitrogen-centered
radical formation.Chemical Research in Toxicology. 14(10):1421-7,
2001 Oct.Acrylonitrile (ACN) is widely used as a monomer in the
polymer industry. Studies on carcinogenicity in rats exposed to ACN showed
increased incidences of tumors including glial cell tumors of central nervous
system and increased production of 8-oxo-7,8-dihydro-2'-deoxyguanosine
(8-oxo-dG) in glial cells. Using a high performance liquid chromatograph
equipped with an electrochemical detector, we revealed that ACN enhanced
the formation of 8-oxo-dG induced by H2O2 and Cu(II)
whereas ACN itself did not cause DNA damage. The enhancing effect of ACN
was much more efficient in the double-stranded DNA than that in the single-stranded
DNA. Experiments with 32P-labeled DNA revealed that addition
of ACN enhanced the site-specific DNA damage at guanines, particularly
at 5'-site of the GG and GGG sequences while H2O2/Cu(II)
induced piperidine-labile sites at thymine, cytosine, and guanine residues.
An electron spin resonance spectroscopy using alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone
showed that a nitrogen-centered radical was generated from ACN in the presence
of H2O2 and Cu(II). It is considered that ACN enhances
H2O2-mediated DNA damage via nitrogen-centered radical
formation. We will discuss the mechanism of the enhancing effect on oxidative
DNA damage in relation to expression of ACN carcinogenicity.
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Sakano K. Oikawa S. Murata M. Hiraku Y. Kojima N. Kawanishi S.
Mechanism
of metal-mediated DNA damage induced by metabolites of carcinogenic 2-nitropropane.Mutation
Research. 479(1-2):101-11, 2001 Aug 8.2-Nitropropane (2-NP), a
widely used industrial solvent, is carcinogenic to rats. To clarify the
mechanism of carcinogenesis by 2-NP, we investigated DNA damage by 2-NP
metabolites, N-isopropylhydroxylamine (IPHA) and hydroxylamine-O-sulfonic
acid (HAS), using 32P-5'-end-labelled DNA fragments obtained
from genes that are relevant to human cancer. In the presence of Fe(III)
EDTA, both IPHA and HAS caused DNA damage at every nucleotide position
without marked site preference. The damage was inhibited by free hydroxyl
radical (-*OH) scavengers, catalase and deferoxamine mesilate, an iron
chelating agent. These results suggest that the DNA damage was caused by
-*OH generated via H2O2 by both IPHA and HAS. In
contrast, in the presence of Cu(II), IPHA frequently caused DNA damage
at thymine. The Cu(II)-mediated DNA damage caused by IPHA was inhibited
by catalase, methional and bathocuproine, a Cu(I)-specific chelator, suggesting
the involvement of H2O2 and Cu(I). These results
suggest that the DNA damage induced by IPHA in the presence of Cu(II) was
caused by a reactive oxygen species like the Cu(I)-hydroperoxo complex.
On the other hand, HAS most frequently induced DNA damage at 5'-TG-3',
5'-GG-3' and 5'-GGG-3' sequences. Catalase and methional only partly inhibited
the Cu(II)-mediated DNA damage caused by HAS, suggesting that the reactive
oxygen species and another reactive species participate in this process.
Formation of 8-oxodG by IPHA or HAS increased in the presence of metal
ions. This study suggests that metal-mediated DNA damage caused by 2-NP
metabolites plays an important role in the mutagenicity and the carcinogenicity
of 2-NP.
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Oikawa S. Hirosawa I. Hirakawa K. Kawanishi S. Site
specificity and mechanism of oxidative DNA damage induced by carcinogenic
catechol. Carcinogenesis. 22(8):1239-45, 2001 Aug.Catechol,
a naturally occurring and an important industrial chemical, has been shown
to have strong promotion activity and induce glandular stomach tumors in
rodents. In addition, catechol is a major metabolite of carcinogenic benzene.
To clarify the carcinogenic mechanism of catechol, we investigated DNA
damage using human cultured cell lines and 32P-labeled DNA fragments
obtained from the human p53 and p16 tumor suppressor genes
and the c-Ha-ras-1 proto-oncogene. Catechol increased the amount
of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is known to be
correlated with the incidence of cancer, in a human leukemia cell line
HL-60, whereas the amount of 8-oxodG in its hydrogen peroxide (H2O2)-resistant
clone HP100 was not increased. The formation of 8-oxodG in calf thymus
DNA was increased by catechol in the presence of Cu2+. Catechol
caused damage to 32P-labeled DNA fragments in the presence of
Cu2+. When NADH was added, DNA damage was markedly enhanced
and clearly observed at relatively low concentrations of catechol (<1
microM). DNA cleavage was enhanced by piperidine treatment, suggesting
that catechol plus NADH caused not only deoxyribose phosphate backbone
breakage but also base modification. Catechol plus NADH frequently modified
thymine residues. Bathocuproine, a specific Cu+ chelator and
catalase inhibited the DNA damage, indicating the participation of Cu+
and H2O2 in DNA damage. Typical hydroxyl radical
scavengers did not inhibit catechol plus Cu2+-induced DNA damage,
whereas methional completely inhibited it. These results suggest that reactive
species derived from the reaction of H2O2 with Cu+
participates in catechol-induced DNA damage. Therefore, we conclude that
oxidative DNA damage by catechol through the generation of H2O2
plays an important role in the carcinogenic process of catechol and benzene.
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Kawanishi S. Inoue S. Oikawa S. Yamashita N. Toyokuni S. Kawanishi M.
Nishino K. Oxidative DNA damage in cultured cells
and rat lungs by carcinogenic nickel compounds.Free Radical Biology
& Medicine. 31(1):108-16, 2001 Jul 1.DNA damage in cultured
cells and in lungs of rats induced by nickel compounds was investigated
to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in
cultured cells exposed to nickel compounds were measured by using a pulsed
field gel electrophoresis technique. Among nickel compounds (Ni3S2,
NiO (black), NiO (green), and NiSO4), only Ni3S2,
which is highly carcinogenic, induced lesions of both double- and single-stranded
DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured
HeLa cells with Ni3S2 (10 microg/ml) induced a 1.5-fold
increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) compared with control,
whereas NiO (black), NiO (green), and NiSO4 did not enhance
the generation of 8-OH-dG. Intratracheal instillation of Ni3S2,
NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs
significantly. NiSO4 induced a smaller but significant increase
in 8-OH-dG. Histological studies showed that all the nickel compounds used
induced inflammation in lungs of the rats. Nitric oxide (NO) generation
in phagocytic cells induced by Ni3S2, NiO(black),
and NiO(green) was examined using macrophage cell line RAW 264.7 cells.
NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was
enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative
DNA damage have been proposed as follows: all the nickel compounds used
induced indirect damage through inflammation, and Ni3S2
also showed direct oxidative DNA damage through H2O2
formation. This double action may explain relatively high carcinogenic
risk of Ni3S2.
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Ohnishi S. Murata M. Oikawa S. Totsuka Y. Takamura T. Wakabayashi K.
Kawanishi S. Oxidative DNA damage by an N-hydroxy
metabolite of the mutagenic compound formed from norharman and aniline.Mutation
Research. 494(1-2):63-72, 2001 Jul 25.Norharman (9H-pyrido[3,4-b]indole),
which is a heterocyclic amine included in cigarette smoke or cooked foodstuffs,
is not mutagenic itself. However, norharman reacts with non-mutagenic aniline
to form mutagenic aminophenylnorharman (APNH), of which DNA adducts formation
and hepatocarcinogenic potential are pointed out. We investigated whether
N-OH-APNH, an N-hydroxy metabolite of APNH, can cause oxidative DNA damage
or not, using 32P-labeled DNA fragments. N-OH-APNH caused Cu(II)-mediated
DNA damage. When an endogenous reductant, beta-nicotinamide adenine dinucleotide
(NADH) was added, the DNA damage was greatly enhanced. Catalase and a Cu(I)-specific
chelator inhibited DNA damage, suggesting the involvement of H2O2
and Cu(I). Typical -*OH scavenger did not inhibit DNA damage. These results
suggest that the main reactive species are probably copper-hydroperoxo
complexes with DNA. We also measured 8-oxo-7,8-dihydro-2'-deoxyguanosine
(8-oxodG) formation by N-OH-APNH in the presence of Cu(II), using an electrochemical
detector coupled to a high-pressure liquid chromatograph. Addition of NADH
greatly enhanced 8-oxodG formation. UV-VIS spectra and mass spectra suggested
that N-OH-APNH was autoxidized to nitrosophenylnorharman (NO-PNH). We speculated
that NO-PNH was reduced by NADH. Cu(II) facilitated the redox cycle. In
the presence of NADH and Cu(II), very low concentrations of N-OH-APNH could
induce DNA damage via redox reactions. We conclude that oxidative DNA damage,
in addition to DNA adduct formation, may play an important role in the
expression of genotoxicity of APNH.
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Murata M. Bansho Y. Inoue S. Ito K. Ohnishi S. Midorikawa K. Kawanishi
S. Requirement of glutathione and cysteine in guanine-specific
oxidation of DNA by carcinogenic potassium bromate.Chemical Research
in Toxicology. 14(6):678-85, 2001 Jun.Potassium bromate (KBrO3),
a food additive, induces renal-cell tumors in rats. KBrO3 induced
8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human leukemia
cell line HL-60 as well as in its H2O2-resistant
clone, HP100, suggesting no involvement of H2O2.
Depletion of GSH by buthionine sulfoximine (BSO) had a little inhibitory
effect on KBrO3-induced 8-oxodG formation. However, the amount
of 8-oxodG was still significantly higher than that in control, suggesting
that intracellular Cys can affect KBrO3 to oxidize DNA, when
GSH decreased. KBrO3 caused 8-oxodG in isolated DNA in the presence
of GSH (tripeptide; gamma-GluCysGly), gamma-GluCys, CysGly, or Cys. Methional
completely inhibited 8-oxodG formation induced by KBrO3 plus
GSH, but typical hydroxyl radical scavengers, SOD and catalase, had little
or no inhibitory effects. When bromine solution (BrO-) was used
instead of BrO3-, similar scavenger effects were
observed. Experiments with 32P-labeled DNA fragments obtained
from the human p53 tumor suppressor gene and the c-Ha-ras-1
protooncogene suggested that KBrO3 induced 8-oxodG formation
at 5'-site guanine of GG and GGG sequences of double-stranded DNA in the
presence of GSH and that treatment of formamidopyrimidine-DNA glycosylase
led to chain cleavages at the guanine residues. ESR spin-trapping studies
showed that 1:2:2:1 quartet DMPO (5,5-dimethyl-1-pyrroline N-oxide) spectrum
similar to DMPO/hydroxy radical (*OH) adduct, but the signals were not
inhibited by ethanol. Therefore, the signal seemed not to be due to *OH
but byproduct due to oxidation of DMPO by the reactive species. The signals
were suppressed by the addition of dGMP, but not by other mononucleotides,
suggesting the specific reactivity with guanine. On the basis of our results
and previous literature, it is speculated that reduction of KBrO3
by SH compounds in renal proximal tubular cells yields bromine oxides and
bromine radicals, which are the reactive species that cause guanine oxidation,
leading to renal carcinogenesis of KBrO3.
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Ohkuma Y. Kawanishi S. Oxidative DNA damage induced
by a metabolite of carcinogenic o-anisidine: enhancement of DNA
damage and alteration in its sequence specificity by superoxide dismutase.Archives
of Biochemistry & Biophysics. 389(1):49-56, 2001 May 1.The
mechanism of DNA damage by a metabolite of the carcinogen o-anisidine
in the presence of metals was investigated by the DNA sequencing technique
using 32P-labeled human DNA fragments. The o-anisidine
metabolite, o-aminophenol, caused DNA damage in the presence of
Cu(II). The DNA damage was inhibited by catalase and bathocuproine, suggesting
the involvement of H2O2 and Cu(I). The formation
of 8-oxo-7,8-dihydro-2'-deoxyguanosine by o-aminophenol increased
in the presence of Cu(II). We conclude that Cu(II)-mediated oxidative DNA
damage by this o-anisidine metabolite seems to be relevant for the
expression of the carcinogenicity of o-anisidine. o-Aminophenol
plus Cu(II) caused preferential DNA damage at the 5'-site guanine of GG
and GGG sequences. When CuZn-SOD or Mn-SOD was added, the DNA damage was
enhanced and its predominant cleavage sites were changed into thymine and
cytosine residues. We consider that SOD may increase the frequency of mutations
due to DNA damage induced by o-aminophenol and thus increase its
carcinogenic potential.
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Midorikawa K. Kawanishi S. Superoxide dismutases
enhance H2O2-induced DNA damage and alter its site
specificity.FEBS Letters. 495(3):187-90, 2001 Apr 27.Superoxide
dismutases (SODs) are involved in the protection of cells from oxygen toxicity.
However, several papers have reported that the overexpression of CuZn-SOD
causes oxidative damage to cells. We investigated a mechanism by which
an excess of SODs accelerates oxidative stress. The presence of CuZn-SOD,
Mn-SOD or Mn(II) enhanced the frequency of DNA damage induced by hydrogen
peroxide (H2O2) and Cu(II), and altered the site
specificity of the latter: H2O2 induced Cu(II)-dependent
DNA damage with high frequency at the 5'-guanine of poly G sequences; when
SODs were added, the frequency of cleavages at thymine and cytosine residues
increased. SODs also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine
by H2O2 and Cu(II). We conclude that SODs may increase
carcinogenic risks, e.g. of tumors in Down syndrome.
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Hishita T. Tada-Oikawa S. Tohyama K. Miura Y. Nishihara T. Tohyama Y.
Yoshida Y. Uchiyama T. Kawanishi S. Caspase-3 activation
by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic
syndrome-derived cell line P39.Cancer Research. 61(7):2878-84,
2001 Apr 1.In most cases, apoptosis is considered to involve mitochondrial
dysfunction with sequential release of cytochrome c from mitochondria,
resulting in activation of caspase-3. However, we found that etoposide
induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell
line, without the release of cytochrome c. Furthermore, in etoposide-treated
P39 cells, no changes in mitochondrial membrane potential (delta psi m)
were detected by flow cytometry. Flow cytometry using a pH-sensitive probe
demonstrated that lysosomal pH increased during early apoptosis in P39
cells treated with etoposide. A reduction in the ATP level preceded the
elevation of lysosomal pH. In addition, specific inhibitors of vacuolar
H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although
etoposide-induced activation of caspase-3 was followed by DNA ladder formation
in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically
suppressed etoposide-induced activation of caspase-3. Western blotting
analysis provided direct evidence for the involvement of a lysosomal enzyme,
cathepsin L. These findings indicate that lysosomal dysfunction induced
by a reduction in ATP results in leakage of lysosomal enzymes into the
cytosolic compartment and that lysosomal enzyme(s) may be involved in activation
of caspase-3 during apoptosis in P39 cells treated with etoposide.
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Oikawa S. Tada-Oikawa S. Kawanishi S. Site-specific
DNA damage at the GGG sequence by UVA involves acceleration of telomere
shortening.Biochemistry. 40(15):4763-8, 2001 Apr 17.Telomere
shortening is associated with cellular senescence. We investigated whether
UVA, which contributes to photoaging, accelerates telomere shortening in
human cultured cells. The terminal restriction fragment (TRF) from WI-38
fibroblasts irradiated with UVA (365-nm light) decreased with increasing
irradiation dose. Furthermore, UVA irradiation dose-dependently increased
the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both
WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration
of telomere shortening, we investigated site-specific DNA damage induced
by UVA irradiation in the presence of endogenous photosensitizers using
32P
5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)4.
UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments
containing telomeric sequence, and Fpg protein treatment led to chain cleavages
at the central guanine of 5'-GGG-3' in telomere sequence. The amount of
8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)7CGC-3']
was approximately 5 times more than that in DNA fragment containing nontelomere
sequence [5'-CGC(TGTGAG)7CGC-3']. Catalase did not inhibit this
oxidative DNA damage, indicating no or little participation of H2O2
in DNA damage. These results indicate that the photoexcited endogenous
photosensitizer specifically oxidizes the central guanine of 5'-GGG-3'
in telomere sequence to produce 8-oxodG probably through an electron-transfer
reaction. It is concluded that the site-specific damage in telomere sequence
induced by UVA irradiation may participate in the increase of telomere
shortening rate.
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Hiraku Y. Yamashita N. Nishiguchi M. Kawanishi S. Catechol
estrogens induce oxidative DNA damage and estradiol enhances cell proliferation.International
Journal of Cancer. 92(3):333-7, 2001 May 1.Estrogen-induced carcinogenesis
involves enhanced cell proliferation (promotion) and genotoxic effects
(initiation). To investigate the contribution of estrogens and their metabolites
to tumor initiation, we examined DNA damage induced by estradiol and its
metabolites, the catechol estrogens 2-hydroxyestradiol (2-OHE2)
and 4-hydroxyestradiol (4-OHE2). In the presence of Cu(II),
catechol estrogens formed piperidine-labile sites at thymine and cytosine
residues in 32P 5'-end-labeled DNA fragments and induced the
formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. NADH markedly enhanced
Cu(II)-dependent DNA damage mediated by nanomolar concentrations of catechol
estrogens. Catalase and bathocuproine inhibited the DNA damage, suggesting
the involvement of H2O2 and Cu(I). These results
suggest that H2O2, generated during Cu(II)-catalyzed
autoxidation of catechol estrogens, reacts with Cu(I) to form the Cu(I)-peroxide
complex, leading to oxidative DNA damage, and that NADH enhanced DNA damage
through the formation of redox cycle. To investigate the role of estrogens
and their metabolites in tumor promotion, we examined their effects on
proliferation of estrogen-dependent MCF-7 cells. Estradiol enhanced the
proliferation of MCF-7 cells at much lower concentrations than catechol
estrogens. These findings indicate that catechol estrogens play a role
in tumor initiation through oxidative DNA damage, whereas estrogens themselves
induce tumor promotion and/or progression by enhancing cell proliferation
in estrogen-induced carcinogenesis. Copyright 2001 Wiley-Liss, Inc.
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Murata M. Tamura A. Tada M. Kawanishi S. Mechanism
of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl. Free
Radical Biology & Medicine. 30(7):765-73, 2001 Apr 1.DNA adduct
formation is thought to be a major cause of DNA damage by carcinogenic
aromatic amines. We investigated the ability of an aromatic amine, 4-aminobiphenyl
(4-ABP) and its N-hydroxy metabolite (4-ABP(NHOH)) to cause oxidative DNA
damage, using 32P-labeled human DNA fragments from the p53
tumor suppressor gene and the c-Ha-ras-1 protooncogene. 4-ABP(NHOH)
was found to cause Cu(II)-mediated DNA damage, especially at thymine residues.
Addition of the endogenous reductant NADH led to dramatic enhancement of
this process. Catalase and bathocuproine, a Cu(I)-specific chelator, reduced
the amount of DNA damage, suggesting the involvement of H2O2
and Cu(I). 4-ABP(NHOH) dose-dependently induced 8-hydroxy-2'-deoxyguanosine
(8-OHdG) formation in the presence of Cu(ll) and NADH. 4-ABP(NHOH) conversion
to nitrosobiphenyl, as measured by UV-visible spectroscopy, occurred rapidly
in the presence of Cu(II), suggesting Cu(II)-mediated autoxidation. Increased
amounts of 8-OHdG were found in HL-60 cells compared to the H2O2-resistant
clone HP100 following 4-ABP(NHOH) treatment, further supporting the involvement
of H2O2. The present study demonstrates that an N-hydroxy
derivative of 4-ABP induces oxidative DNA damage through H2O2
in both a cell-free system and in cultured human cells. We conclude that,
in addition to DNA adduct formation, oxidative DNA damage may play an important
role in the carcinogenic process of 4-ABP.
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Kawanishi S. Hiraku Y. Oikawa S. Mechanism of
guanine-specific DNA damage by oxidative stress and its role in carcinogenesis
and aging. [Review] [85 refs] Mutation Research. 488(1):65-76,
2001 Mar.Reactive species generated by chemicals and UV radiation
can cause sequence-specific DNA damage and play important roles in mutagenesis,
carcinogenesis and aging. We have investigated sequence specificity of
oxidative stress-mediated DNA damage by using 32P-labeled DNA
fragments obtained from the human c-Ha-ras-1 and p53 genes.
Free hydroxyl radical causes DNA damage with no marked site specificity.
Reactive nitrogen species, sulfate radicals, nitrogen-centered radicals,
benzoyloxyl radical and alkoxyl radical show different sequence specificity.
Benzoyloxyl radical specifically causes damage to the 5'-G in GG sequence.
UVA radiation also causes DNA damage at this site through electron transfer
in the presence of certain photosensitizers. The 5'-G in GG sequence is
easily oxidized because a large part of the highest occupied molecular
orbital is distributed on this site. On the basis of these findings, the
sequence specificity of DNA damage is presumably determined by (a) redox
potential of reactive species; (b) ionization potential of DNA bases; and
(c) site-specific binding of metal ion to DNA. Here we discuss the mechanisms
of sequence-specific DNA damage in relation to carcinogenesis and aging.
[References: 85]
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Kawanishi S. Hiraku Y. Sequence-specific DNA damage
induced by UVA radiation in the presence of endogenous and exogenous photosensitizers.
[Review] [20 refs] Current Problems in Dermatology. 29:74-82,
2001.
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Ohnishi S. Murata M. Degawa M. Kawanishi S. Oxidative
DNA damage induced by an N-hydroxy metabolite of carcinogenic 4-dimethylaminoazobenzene.Japanese
Journal of Cancer Research. 92(1):23-9, 2001 Jan.Formation of adducts
has been considered to be a major causal factor of DNA damage by carcinogenic
aminoazo dyes. We investigated whether a metabolite of hepatocarcinogenic
4-dimethylaminoazobenzene (DAB) can cause oxidative DNA damage or not,
using 32P-5'-end-labeled DNA fragments. The DAB metabolite N-hydroxy-4-aminoazobenzene
(N-OH-AAB) was found to cause Cu(II)-mediated DNA damage, including 8-oxo-7,8-dihydro-2'-deoxyguanosine
(8-oxodG) formation. When an endogenous reductant, beta-nicotinamide adenine
dinucleotide (NADH) was added, the DNA damage was greatly enhanced. Very
low concentrations of N-OH-AAB could induce DNA damage via redox reactions.
Catalase and a Cu(I)-specific chelator inhibited the DNA damage, suggesting
the involvement of H2O2 and Cu(I). A typical.OH scavenger
did not inhibit the DNA damage. The main reactive species are probably
DNA-copper-hydroperoxo complexes. We conclude that oxidative DNA damage
may play an important role in the carcinogenic processes of DAB, in addition
to DNA adduct formation.
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