Oxidative DNA damage by an N-hydroxy metabolite of the mutagenic compound formed from norharman and aniline

Article abstract of DOI:10.1016/S1383-5718(01)00177-2

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, β-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.

Full text of DOI:10.1016/S1383-5718(01)00177-2

Mutation Research 494 (2001) 63–72
Oxidative DNA damage by an N-hydroxy metabolite of the
mutagenic compound formed from norharman and aniline
Shiho Ohnishia, Mariko Murataa, Shinji Oikawaa, Yukari Totsukab,
Takeji Takamurab, Keiji Wakabayashib, Shosuke Kawanishia,∗
Department of Hygiene, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
a
b
Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
Received 20 February 2001; received in revised form 10 April 2001; accepted 10 April 2001
Abstract
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, ␤-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 reac-
tive species are probably copper-hydroperoxo complexes with DNA. We also measured 8-oxo-7,8-dihydro-2ꢀ-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 forma-
tion, may play an important role in the expression of genotoxicity of APNH. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Norharman; N-hydroxy metabolite; DNA damage; Copper; Hydrogen peroxide; Redox cycle
Abbreviations: Norharman, 9H-pyrido[3,4-b]indole; APNH (aminophenylnorharman), 9-(4ꢀ-aminophenyl)-9H-pyrido[3,4-b]indole;
N-OH-APNH (N-hydroxy-aminophenylnorharman), 9-(N-hydoroxy-4ꢀ-aminophenyl)-9H-pyrido[3,4-b]indole; NO-PNH (nitrosophenyl-
norharman), 9-(4ꢀ-nitrosophenyl)-9H-pyrido[3,4-b]indole; NO2-PNH (nitrophenylnorharman), 9-(4ꢀ-nitrophenyl)-9H-pyrido[3,4-b]indole;
MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; 8-oxodG, 8-oxo-7,8-dihydro-2ꢀ-deoxyguanosine; ROS, reactive oxygen
−
species; O2
, superoxide; NADH, ␤-nicotinamide adenine dinucleotide (reduced form); SOD, superoxide dismutase; DTPA,
diethylenetriamine-N,N,Nꢀ,Nꢀꢀ,Nꢀꢀ-pentaacetic acid; DMSO, dimethylsulfoxide; HPLC–ECD, an electrochemical detector coupled to a high-
pressure liquid chromatography; MS, mass spectrometry
∗Corresponding author. Tel.: +81-59-231-5011; fax: +81-59-231-5011.
E-mail address: kawanisi@doc.medic.mie-u.ac.jp (S. Kawanishi).
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00177-2

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S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
1. Introduction
traced the autoxidation of N-OH-APNH, and the redox
reaction between NADH and oxidized N-OH-APNH,
using UV–VIS spectroscopy and mass spectrometry
(MS).
Norharman(9H-pyrido[3,4-b]indole), aheterocyclic
amine, exists in cigarette smoke and cooked meat and
fish at much higher levels than those of other hetero-
cyclic amines [1]. Norharman was detected in all urine
samples from healthy volunteers eating ordinary diet
[2]. Interestingly, norharman becomes mutagenic in
the presence of non-mutagenic aniline with S9 mix,
although norharman itself is not mutagenic either with
or without S9 mix [3–5]. It is reported that norhar-
man reacts with aniline to form aminophenylnorhar-
man (APNH), and that APNH is metabolized to
N-hydroxy-aminophenylnorharman (N-OH-APNH) in
metabolic process [6]. APNH induces glutathione S-
transferase placental form (GST-P) positive foci in the
livers of F344 male rats in a dose-dependent manner,
suggesting its carcinogenicity [7].
Regarding the mechanisms of DNA damage by car-
cinogenic heterocyclic amines, DNA adduct formation
has been considered to be a major causal factor [8–10].
Both APNH and N-OH-APNH formed the same DNA
adducts in Salmonella typhimurium YG1024, suggest-
ing that APNH is N-hydroxylated to N-OH-APNH fol-
lowed by formation of DNA adducts [6]. On the other
hand, Hayatsu et al. showed that N-hydroxy deriva-
tive of 3-amino-1-methyl-5H-pyrido[4,3-b]indole
(Trp-P-2)producesintracellularreactiveoxygenspecies
(ROS) that can damage DNA in mouse cultured cells
[11,12]. It is also reported that an N-hydroxy metabo-
lite of heterocyclic amine, 2-amino-3,8-dimethylimi-
dazo[4,5-f]quinoxaline (MeIQx), induced oxidative
damage to isolated DNA fragments [13]. Maeda et al.
reportedO2− generationfromheterocyclicamineswith
cytochrome P450 reductase and NADPH [14,15].
There remains a possibility that oxidative DNA
damage also plays a role in genotoxicity induced by
APNH.
2. Materials and methods
2.1. Materials
Restriction enzymes (HindIII, StyI, AvaI, EcoRI, and
XbaI) and T4 polynucleotide kinase were purchased
from New England Biolabs (Beverly, MA, USA). The
[␥-32P]ATP (222 TBq/mmol) was obtained from New
England Nuclear. Alkaline phosphatase from calf intes-
tine was purchased from Roche Molecular Biochemi-
cals (Mannheim, Germany). Piperidine, methional
(3-(methylthio)propionaldehyde)werepurchasedfrom
Wako Chemical Industries Ltd. (Osaka, Japan). Cu(II)
chloridedihydratewaspurchasedfromNacalaiTesque,
Inc. (Kyoto, Japan). Diethylenetriamine-N,N,Nꢀ,Nꢀꢀ,Nꢀꢀ-
pentaacetic acid (DTPA) and bathocuproinedisulfonic
acid were purchased from Dojin Chemicals Co. (Ku-
mamoto, Japan). Calf thymus DNA, superoxide dismu-
tase (SOD) (3000 units/mg from bovine erythrocytes)
and catalase (45,000 units/mg from bovine liver) were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Nuclease P1 (400 units/mg) was purchased from
Yamasa Shoyu Co. (Chiba, Japan).
2.2. Synthesis of 9-(N-hydoroxy-4ꢀ-aminophenyl)-9H-
pyrido[3,4-b]indole (N-OH-APNH)
9-(4ꢀ-Nitrophenyl)-9H-pyrido[3,4-b]indole (Nard.
Co. Hyogo, Japan) (275 mg) was suspended in tetrahy-
drofuran (20 ml) cooled to 0◦C, and then 5% palla-
dium/carbon (50 mg) and hydrazine monohydrate
(0.2 ml) were added with stirring [6]. The progress
of the reaction was monitored by thin-layer chromato-
graph(Merckprecoatedsilicagelsheets60F-254, chlo-
roform:ethanol 10:1). After the reduction was
completed (within 30 min), the reaction mixture was
filtered and diluted with N2-purged water. Solid mate-
rial was collected by centrifugation and lyophilized to
give pure 9-(N-hydoroxy-4ꢀ-aminophenyl)-9H-pyrido-
[3,4-b]indole: a yellow powder (yield 80%). 1H-NMR
(Varian Gemini-400) (CDCl3): δ 8.72 ppm (d, J =
0.9 Hz, 1H), 8.50 (d, J = 5.3, 1H), 8.20 (dd, J =
In this study, we investigated whether N-OH-APNH
can cause oxidative DNA damage or not in the pres-
enceofCu(II)and␤-nicotinamideadeninedinucleotide
(NADH), using32P-5ꢀ-end-labeledDNAfragmentsob-
tained from the human p53 tumor suppressor gene and
thec-Ha-ras-1protooncogene. Weanalyzed8-oxo-7,8-
dihydro-2ꢀ-deoxyguanosine (8-oxodG) formation, a
marker of oxidative DNA damage, in calf thymus DNA
treated with N-OH-APNH. Furthermore, in order to
clarify the mechanism of oxidative DNA damage, we

S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
65
7.9, 1.0, 1H), 8.1 (dd, J = 5.3, 0.9, 1H), 7.56 (ddd,
J = 8.0, 7.0, 1.1, 1H), 7.44 (d, J = 9.0, 2H), 7.42
(dd, J = 7.9, 1.0, 1H), 7.35 (ddd, J = 8.0, 7.0, 1.1,
1H), 7.25 (d, J = 9.0, 2H). Electron ionization MS
(Shimadzu GC–MS QP-5000 with DI-50); m/z (%) =
275 (M+, 24), 154 (100), 136 (62). FAB-HRMS (fast
atom bombardment-high resolution MS, recorded with
a JEOL JMS-SX 102A QQ apparatus with direct injec-
tion); calculated for C17H14N3O [M + 1]+ 276.1137,
found 276.1132. From the results of direct injection
FAB-HRMS measurements in addition to the observa-
tion of the only single peak obtainable by two different
HPLC systems, the compound was quite pure enough
for following experiments.
procedure [21] using a DNA-sequencing system (LKB
2010 Macrophor). A laser densitometer (LKB 2222 Ul-
troScan XL) was used for the measurement of the rela-
tive amounts of oligonucleotides from the treated DNA
fragments.
2.5. Analysis of 8-oxodG formation by N-OH-APNH
Calf thymus DNA fragment was incubated with
N-OH-APNH, NADH, and CuCl2. After ethanol pre-
cipitation, DNA was digested to its component nucle-
osides with nuclease P1 and calf intestine phosphatase,
and analyzed by an electrochemical detector coupled to
a high-pressure liquid chromatography (HPLC–ECD),
as described by Kasai et al. [22,23].
2.3. Preparation of 32P-5ꢀ-end-labeled DNA
fragments obtained from the p53 gene and the
c-Ha-ras-1 gene
2.6. Measurement of UV–VIS spectra
UV–VIS spectra were measured with UV–VIS spec-
trometer (UV-2500PC Shimadzu, Kyoto, Japan). To
observe the autoxidation of N-OH-APNH, the spec-
trum was measured every 5 min at 37◦C. The sample
was 10 ␮M N-OH-APNH in 10 mM sodium phosphate
buffer (pH 7.8) containing 20% (v/v) DMSO and 2.5
␮M DTPA. The concentration of nitrophenylnorhar-
man (NO2-PNH) was 10 ␮M. For analysis of NADH
consumption, the absorption at 340 nm (reduced
NADH) was traced every 3 min at 37◦C. The reaction
mixture contained various concentrations of N-OH-
APNH, 100 ␮MNADH, and/or20 ␮MCuCl2 in10 mM
sodium phosphate buffer (pH 7.8) containing 2% (v/v)
DMSO and 2.5 ␮M DTPA.
DNA fragments were obtained from the human p53
tumor suppressor gene [16] and the c-Ha-ras-1 pro-
tooncogene [17]. A singly 32P-5ꢀ-end-labeled double-
stranded 118 bp p53 fragment (HindIII∗ 13038–StyI
13155) and 343 bp p53 fragment (StyI 13160–EcoRI∗
13507) were prepared according to the method de-
scribed previously [18]. A 261 bp c-Ha-ras-1 fragment
(AvaI∗ 1645–XbaI 1905) and 341 bp fragment (XbaI
1906–AvaI∗ 2246) were prepared from plasmid pbcNI,
which carries a 6.6 kb BamHI chromosomal DNA
restriction fragment. The asterisk (∗) indicates
32P-labeling.
2.4. Detection of DNA damage
2.7. Measurement of HPLC–MS spectra of oxidized
N-OH-APNH
A standard reaction mixture (in a microtube; 1.5 ml)
containedCuCl2, NADH, adimethylsulfoxide(DMSO)
solution of N-OH-APNH, 32P-labeled double-stranded
DNA fragments, and calf thymus DNA in 200 ␮l of
10 mM sodium phosphate buffer (pH 7.8) containing
2.5 ␮MDTPA. Afterincubationat37◦C, theDNAfrag-
ments were heated at 90◦C in 1 M piperidine for 20 min
and treated as described previously [19,20]. The treated
DNA fragments were electrophoresed on an 8% poly-
acrylamide/8 Mureagelandanautoradiogramwasob-
tained by exposing X-ray film to the gel. The preferred
cleavage sites were determined by direct comparison
of the positions of the oligonucleotides with those pro-
duced by the chemical reactions of the Maxam–Gilbert
To the solution of 100 ␮M N-OH-APNH in 1.5 ml of
phosphate buffer (pH 7.4) was added 15 ␮l of 2 mM so-
lution of CuCl2. Reaction mixture was immediately an-
alyzed with HPLC–MS. Electrospray ionization mass
spectrometrywasperformedusingaJEOLJMS-7000Q
equipped with HP 1100 HPLC system. Elution condi-
tions were follows. An analytical column of Zorbax
Extend C18 (4.6 mm × 25 cm) was used with a linear
gradient from 10 to 80% acetonitrile in sodium phos-
phate buffer (pH 2) over the course of 30 min at a rate
of 1 ml/min.

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S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
3. Results
cleotide formation, while some oligonucleotides were
observed also without piperidine treatment (data not
shown). This result suggests that N-OH-APNH induces
not only strand breakage but also base modification
and/or liberation.
3.1. Damage to 32P-labeled DNA
Fig. 1 shows an autoradiogram of a DNA fragment
treated with N-OH-APNH in the presence and absence
of NADH and/or Cu(II). Oligonucleotides that formed
after DNA cleavage were detected on the autoradio-
gram. In the presence of Cu(II), N-OH-APNH caused
DNA damage in a dose-dependent manner. When
NADHwasadded, lowconcentrationsofN-OH-APNH
induced Cu(II)-mediated DNA damage efficiently. In
the absence of N-OH-APNH, DNA damage was not
observed with NADH and Cu(II) under the condition
used. N-OH-APNH alone or N-OH-APNH + NADH
did not cause DNA damage. Piperidine treatment
showed approximately five-fold increase of oligonu-
3.2. Effects of scavengers and bathocuproine on DNA
damage
TheeffectsofscavengersandbathocuproineonDNA
damage•by N-OH-APNH were investigated (Fig. 2). A
typical OH scavenger mannitol did not inhibit DNA
damage induced by N-OH-APNH in the presence of
Cu(II), whereas methional inhibited the DNA dam-
age. Bathocuproine, a Cu(I)-specific chelator, inhibited
DNA damage, suggesting the involvement of Cu(I).
DNA damage induced by N-OH-APNH was inhibited
Fig. 1. Autoradiogram of 32P-labeled DNA fragment incubated with N-OH-APNH, NADH, and Cu(II). The reaction mixture contained
32P-5ꢀ-end-labeled 261 bp DNA fragment, 10 ␮M/base calf thymus DNA, various concentrations of N-OH-APNH, 10% (v/v) DMSO, 100 ␮M
NADH, and 20 ␮M CuCl2 in phosphate buffer (pH 7.8). The reaction was incubated at 37◦C for 1 h, followed by a piperidine treatment, as
described in Section 2. The DNA fragments were electrophoresed on an 8% polyacrylamide/8 M urea gel and an autoradiogram was obtained
by exposing an X-ray film to the gel.

S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
67
presence of NADH, same DNA cleavage pattern was
observed (data not shown).
3.4. Formation of 8-oxodG in calf thymus DNA
Using HPLC–ECD, we measured the 8-oxodG con-
tent of calf thymus DNA treated with N-OH-APNH
in the presence of Cu(II) (Fig. 4A). The amount of
8-oxodG increased with the concentrations of N-OH-
APNH. Formation of 8-oxodG increased after DNA
denaturation. When NADH was added, 8-oxodG for-
mation was observed at very low concentrations of
N-OH-APNH (Fig. 4B).
3.5. Autoxidation of N-OH-APNH, and NADH
consumption
Fig. 5A shows spectral changes of N-OH-APNH
(solid lines) compared to a spectrum of NO2-
PNH (a dotted line). The absorption near 400 nm in-
creased gradually with autoxidation of N-OH-APNH,
whereas NO2-PNH does not have maximum peak at
400 nm but has 356 nm. This result shows that the oxi-
dation of N-OH-APNH did not proceed to NO2-PNH.
In the presence of Cu(II), UV–VIS spectrum of oxi-
dized product was similar, but the oxidation of N-OH-
APNH occurred within 1 min (data not shown).
Fig. 5B shows NADH consumption during redox re-
actionbetweenNADHandoxidizedproductsofN-OH-
APNH. In the absence of Cu(II), NADH was slowly
consumed. In the presence of Cu(II), NADH was con-
sumed efficiently, suggesting that the redox reactions
cycled very rapidly. Without N-OH-APNH, the spec-
trum of the reaction mixture of Cu(II)+NADH did not
change under the condition used (data not shown).
The HPLC–MS analyses of the oxidized products
showed four typical chromatogram peaks. The biggest
peak had a molecular ion peak m/z 274 [M + 1]+,
indicating formation of nitroso form of APNH, nitroso-
phenylnorharman (NO-PNH) (data not shown).
Fig. 2. Effects of scavengers and bathocuproine on DNA damage
induced by N-OH-APNH + Cu(II). The reaction mixture contained
32P-5ꢀ-end-labeled 118 bp DNA fragment, 10 ␮M/base calf thymus
DNA, 10 ␮M N-OH-APNH, 10% (v/v) DMSO, and 20 ␮M CuCl2
in phosphate buffer. The reaction was incubated at 37◦C for 1 h,
followed by a piperidine treatment. The DNA fragments were ana-
lyzed as described in Fig. 1. The concentrations of scavengers and
bathocuproine were as follows: 0.1 M mannitol; 0.1 M methional;
100 units of catalase; 100 units of catalase (heat-denatured for 10 min
at 90◦C before use); 30 units of SOD; 50 ␮M bathocuproine.
by catalase, while not inhibited by heated catalase.
SOD showed little inhibitory effect on DNA damage.
In the presence of NADH, same scavenger effects were
observed (data not shown).
3.3. Site specificity of DNA damage by N-OH-APNH
An autoradiogram was obtained and scanned with
a laser densitometer to measure the relative intensities
of DNA cleavage products from the human c-Ha-ras-1
protooncogene (Fig. 3A) and the p53 tumor suppressor
gene (Fig. 3B). N-OH-APNH was observed to prefer-
entially induce piperidine-labile sites at thymine and
cytosine residues in the presence of Cu(II). In the
4. Discussion
The present study showed that N-OH-APNH caused
Cu(II)-mediated DNA damage, which was dramati-
cally enhanced with endogenous reductant NADH. N-
OH-APNH induced Cu(II)-mediated formation of

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S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
Fig. 3. Site specificity of DNA cleavage induced by N-OH-APNH+Cu(II). The reaction mixture contained 32P-5ꢀ-end-labeled DNA, 50 ␮M/base
calf thymus DNA, 20 ␮M N-OH-APNH, 2% (v/v) DMSO, and 10 ␮M CuCl2 in phosphate buffer. The reaction was incubated at 37◦C for 2 h,
followed by a piperidine treatment. The horizontal axis shows the nucleotide number: (A) 341 bp fragment (XbaI 1906–AvaI∗ 2246) of the
c-Ha-ras-1 protooncogene; (B) 343 bp fragment (StyI 13160–EcoRI∗ 13507) of the p53 tumor suppressor gene.
piperidine-labile sites preferentially at thymine and cy-
tosine residues. We detected formation of 8-oxodG, a
piperidine-inert oxidative damage at guanine. NADH
also enhanced the 8-oxodG formation induced by
N-OH-APNH in the presence of Cu(II). To clarify the
reactive species involved in oxidative DNA damage by
N-OH-APNH, we examined the effects of scavengers
onDNAdamage. Bothcatalaseandbathocuproinewere
found to reduce DNA damage, indicating the involve-
ment of H2O2 and Cu(I). Failure to see an effect of
typical •OH scavenger implies that free •OH does not
play a major role in DNA damage. Alternatively, the

S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
69
Fig. 4. Cu(II)-mediated formation of 8-oxodG in calf thymus DNA by N-OH-APNH. Calf thymus DNA (50 ␮M per base) was incubated with the
indicated concentrations of N-OH-APNH and 20 ␮M CuCl2 for 1 h at 37◦C in the absence (A) and presence (B) of 100 ␮M NADH. After ethanol
precipitation, DNA was enzymatically digested into nucleosides, and 8-oxodG formation was measured with an HPLC–ECD, as described in
Section 2. Open symbols indicate that native DNA was used, and closed symbols indicate denatured DNA.
lack of effect of •OH scavengers may be due to DNA
damage induced by •OH which is generated by metal
ions bound in very close proximity to DNA. The in-
hibitory effect of methional on DNA damage can be
explained by assuming that sulfur compounds are re-
active with •OH and less reactive species [24].
A possible mechanism as shown in Fig. 6 can be
envisioned as accounting for most of the observations.
Norharman reacts with aniline to form APNH, which
is N-hydroxylated to N-OH-APNH [6]. N-OH-APNH
induces Cu(II)-mediated oxidative DNA damage.
Autoxidation of N-OH-APNH to nitroso compound
(NO-PNH)viaradicalintermediateoccurscoupledwith
generation of O2−. Formation of NO-PNH was sug-
gested by UV–VIS spectroscopic studies and MS anal-
yses. Relevantly, it is demonstrated that N-hydroxy
compound of Trp-P-2 is autoxidized to form its nitroso
derivative with generation of O2− [25]. O2− is dismu-
tated to H2O2 and reduces Cu(II) to Cu(I). H2O2 in-
teracts with Cu(I) to form DNA–copper–hydroperoxo
complexes, causing DNA damage. Both Cu(I) and ROS
generation during autoxidation of N-OH-APNH are re-
quired for DNA damage. DNA–copper–hydroperoxo
complexes have been suggested in other studies
[26–28].
The biological significance of copper has recently
drawnmuchinterestinconnectionwithcarcinogenicity
and mutagenicity. Copper is an essential component of
chromatine and is known to accumulate preferentially
in the heterochromatic regions [29–31]. Copper sulfate
showed clastogenic effects on the bone marrow chro-
mosomes of mice in vivo [32]. Sone et al. suggested
that excess intracellular copper in LEC rats induces
DNA damage through ROS produced by Cu(II)/Cu(I)
redox cycling and subsequently plays a crucial role in
hepatocarcinogenesis [33]. Therefore, it is reasonable
Fig. 5. Changes in spectrum with the time for autoxidation of
N-OH-APNH and NADH consumption. (A) UV–VIS spectrum of
10 ␮M N-OH-APNH was traced every 5 min (solid lines). The
dotted line shows the spectrum of 10 ␮M NO2-PNH. (B) The con-
centrations of NADH were plotted every 3 min during redox reac-
tion of N-OH-APNH. NADH (100 ␮M) was incubated with 2 ␮M
N-OH-APNH(closedcircles), 0.5 ␮MN-OH-APNH+20 ␮MCuCl2
(crosses), 1 ␮M N-OH-APNH+20 ␮M CuCl2 (triangles), and 2 ␮M
N-OH-APNH + 20 ␮M CuCl2 (open circles).

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S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
Fig. 6. (A) A process of the formation of N-OH-APNH [4]. (B) A proposed mechanism of metal-mediated DNA damage by N-OH-APNH in
the presence of NADH.
to consider a possibility of copper involvement in DNA
damage by N-OH-APNH.
andNADPHasnuclearreductants[37]hasbeendemon-
strated before.
When NADH is added, NO-PNH, the oxidized prod-
uct of N-OH-APNH, may be reduced to the intermedi-
ate or N-OH-APNH, and again autoxidation occurred
with excessive generation of ROS by forming redox
cycle non-enzymatically. This is supported by reports;
NADH reduced nitroso-aromatic compounds to reac-
tive intermediates causing oxidative DNA damage
[34,35]. Relevantly, in the case of a metabolite of car-
cinogenic heterocyclic amine, such as MeIQx, oxida-
tive DNA damage was greatly enhanced by NADH
via redox cycle [13]. The concentration of NAD(P)H
in certain tissue has been estimated to be as high as
100–200 ␮M[36]. ThebiologicalimportanceofNADH
It is considered that N-hydroxy derivatives of car-
cinogenic heterocyclic amines covalently form DNA
adducts to induce mutagenicity and carcinogenicity
[10]. The present study has revealed that N-OH-APNH
has oxidative DNA-damaging ability, and NADH en-
hances the damage through cycling redox reactions.
It is noteworthy that a very low concentration
(0.1 ␮M)ofN-OH-APNHinduced8-oxodGformation,
suggesting that oxidative DNA damage can occur in
vivo. Oxidative DNA damage may play an
important role in carcinogenic process of APNH
through ROS formation, in addition to DNA adduct
formation.

S. Ohnishi et al. / Mutation Research 494 (2001) 63–72
71
Norharman can react with an aniline derivative, such
as toluidine, to form coupling compound like APNH
[3]. Although, aniline and toluidine are industrial ma-
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Acknowledgements
This work was supported by Grants-in-Aid for Sci-
entific Research from the Ministry of Education, Sci-
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