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Toxicology in Vitro 22 (2008) 36–44

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Effects of mixing metal ions on oxidative DNA damage mediated

by a Fenton-type reduction
a,*
Hiroshi Moriwaki , Martin R. Osborne b, David H. Phillips b

a
Osaka City Institute of Public Health & Environmental Sciences, 8-34, Tojo-cho, Tennoji-ku, Osaka 543-0026, Japan
b
Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, UK

Received 31 July 2006; accepted 30 July 2007

Available online 10 August 2007

Abstract

The formation of 8-hydroxy-deoxyguanosine (8-OHdG) and strand breaks in DNA by Fenton-type reactions by mixtures of two of
five metal ions, iron (II), cadmium (II), nickel (II), chromium (III) or copper (II), has been investigated and compared to their formation
by each single metal ion. Salmon sperm DNA and pBluescript K+ plasmid were each incubated with hydrogen peroxide and metal ions.
The formation of 8-OHdG declined in the Fe (II) or Cu (II) Fenton reaction upon addition of Cd (II) or Ni (II) ion. In contrast, the Fe
(II) reaction upon addition of Cr (III) ion showed an additive influence on the formation of 8-OHdG. Furthermore, the Cu (II) plus Cr
(III) reaction showed a synergistic effect. These influences relate to the interaction of metal ions with DNA, the potentials of the metal
ions to generate activated oxygen and electron transfer between metal ions. The formation of DNA strand breaks was investigated in
plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of DNA strand breaks in the Fe (II) or
Cu (II) Fenton reaction decreased upon the addition of Ni (II) ion, as with the formation of 8-OHdG mediated by these metal ions.
On the other hand, the formation of DNA strand breaks in the Fe (II) reaction decreased upon addition of Cr (III) ion, and the Cu
(II) plus Cr (III) reaction did not show the synergistic influence on DNA strand breaks. These results suggest that interactions between
two metal ions can influence the generation of 8-OHdG and the formation of DNA strand breaks and demonstrate that these lesions can
arise by different mechanisms.
 2007 Elsevier Ltd. All rights reserved.

Keywords: DNA oxidative stress; Metal ion; Multiple influence

1. Introduction
Oxidative DNA damage is mediated by reactive oxygen
species (ROS) and is thought to play an important role in
mutagenesis, aging, and carcinogenesis (Bjelland and See-
berg, 2003; Barja, 2004; Olinski et al., 2002). ROS arise
during normal cellular processes such as aerobic metabo-
lism. However, the endogenous amount of ROS can be
enhanced by exposure to environmental pollutants includ-
*
Corresponding author. Present address: Department of Applied
Biology, Faculty of Textile Science & Technology, Shinshu University,
3-15-1 Tokida, Ueda, Nagano 386-8567, Japan. Tel.: +81 268 21 5333; fax:
+81 268 21 5331.
E-mail address: moriwaki@shinshu-u.ac.jp (H. Moriwaki).
ing genotoxic metals (Shi et al., 2004; Ercal et al., 2001),
and elevated levels of ROS in cells have been implicated
in the etiology of various diseases including cancer (Feig
et al., 1994). A possible mechanism of oxidative DNA
damage induced by metals is thought to be the formation
of hydroxyl radicals through Fenton-type reduction with
hydrogen peroxide, which is naturally present in cells.
Iron salts are known to cause the formation of 8-
hydroxydeoxyguanosine (8-OHdG) in DNA, which is
the production of a single base modification by a damage
to DNA following hydroxyl radical attack, and DNA sin-
gle- and double-strand breaks when incubated with DNA
and hydrogen peroxide in the Fenton systems (Tenopou-
lou et al., 2005; Glei et al., 2002). Furthermore, Cu (II)
and Cr (III) ions, both recognized as human carcinogens,

0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2007.07.011
 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44
also produce 8-OHdG and strand breaks in DNA, when
their salts are incubated with DNA in vitro in the presence
of hydrogen peroxide (Lee et al., 2002; Qi et al., 2000). In
general, the formation of oxidative DNA damage needs
to generate a hydroxyl radical near to DNA, because
the lifetime of the hydroxyl radical is very short. There-
fore, the interaction between DNA and metal ion plays
a significant role of the formation of oxidative DNA dam-
age mediated by a Fenton-type reduction. In fact, in a
previous study (Lloyd and Phillips, 1999), the Fenton-
type reactions in the presence of EDTA were investigated,
and the results suggested that interaction between DNA
and the metal ion influenced formation of DNA oxidative
damage.
It has been reported by many investigators that oxida-
tive DNA damage can result from exposure to air pollution
particles or diesel exhaust particles (Tsurudome et al.,
1999; Hirano et al., 2003; Risom et al., 2003; Prahalad
et al., 2001). Most environmental exposure profiles are het-
erogeneous with co-exposure occurring coincident with
multiple toxic metal species, and this co-exposure to metals
in complex mixtures can result in an additive, suppressive
or synergistic toxic response (Nygren et al., 1992; Cui
et al., 2005). The interaction of a metal ion with DNA
could be changed by the addition of the other metal ions.
Therefore, mixing transition metal ions could influence
the oxidative DNA damage mediated by a Fenton-type
reduction. It is very important for assessments of the geno-
toxicity induced by environmental samples to understand
the influence of interactions between several heavy metals
on oxidative DNA stress. However, little is known about
such effects (Sugden et al., 2004).
In this report, salmon sperm DNA and pBluescript K+
plasmid were each incubated with hydrogen peroxide and
mixtures of two of five genotoxic metal ions, Fe (II), Cd
(II), Ni (II), Cr (III) or Cu (II). Cr (III) is unable to pass
easily through the cell membrane. However, within the cell,
Cr (VI) is metabolically reduced by ascorbic acid, glutathi-
one or cysteine to Cr (III), the most stable form of Cr
(O’Brien et al., 2002). Therefore, Cr (III) ion was selected
as a metal ion for the Fenton-type reaction in this study.
The formation of 8-OHdG and strand breaks in DNA
mediated by Fenton-type reactions has been investigated
and compared to their formation by each single metal ion.
2. Experimental
2.1. Materials
Salmon sperm DNA, deoxyribonuclease I, alkaline
phosphatase, nd immobilized catalase (4% beaded agarose)
were obtained from Sigma Chemical Co. (Poole, Dorset,
UK). Snake venom phosphodiesterase was purchased from
USB Corporation (Cleveland, Ohio, USA). pBluescript
K+ (SK+) plasmid DNA was obtained according to a
method previously described (Lloyd and Phillips, 1999).
Chemicals used in the Fenton-type treatment of DNA,
37
copper (II) sulfate, iron (II) sulfate, nickel (II) sulfate, cad-
mium (II) chloride and chromium (III) chloride were all
analytical grade, and were freshly made up as stock solu-
tions (1.0 mM) in de-ionized water.
2.2. Treatment of salmon sperm DNA
Salmon sperm DNA was dissolved in de-ionized water
(0.5 mg/mL). The aliquots of salmon sperm DNA
(500 lL) were incubated with hydrogen peroxide (50 mM)
and each single metal ion (0.010 or 0.10 mM) or mixtures
of two of five metal ions, Fe (II), Cd (II), Ni (II), Cr
(III), and Cu (II) (the metal stock solution (1.0 mM) was
added 10 or 100 lL and de-ionized water was added 490
or 400 lL for metal concentrations 0.010 or 0.10 mM,
respectively), for 1 h at 37 C. Fenton-type reactions were
terminated using immobilized catalase (100 lL) which
was removed by centrifugation after 30 min at ambient
temperature. DNA treated with Fenton-type reactions
was precipitated from the supernatant with sodium chlo-
ride and ethanol.
2.3. LC-ECD analysis of salmon sperm DNA
DNA samples in 900 lL of water were digested over-
night with deoxyribonuclease I (150 lg, pH 7) followed
by a 6-h incubation with snake venom phosphodiesterase
(12 lg, pH 9). The resulting nucleotides were converted
to nucleosides with alkaline phosphatase (1.2 units).
Hydrolysate was applied to a Nucleosil ODS column and
eluted with ammonium acetate (50 mM) and acetic acid
(50 mM) in 5% methanol at a flow rate of 1.0 mL/min. 8-
OHdG was eluted after 18–21 min and detected using an
ESA Coulochem II electrochemical detector. Unmodified
deoxyguanosine (dG) was detected by UV absorption using
a Pharmacia UV-1 detector. The amounts of 8-OHdG and
dG present in the DNA samples were calculated by mea-
suring the height of the peaks obtained from both electro-
chemical and UV traces and comparing those obtained
from synthetic standards.
2.4. Treatment and analysis of pBluescript K+ DNA
Plasmid DNA was dissolved in de-ionized water (10 ng/
mL). Aliquots of plasmid DNA (10 lL) were incubated
with hydrogen peroxide (1 mM) and each single metal
ion (final concentration at 0.4 or 4.0 lM) or equivalent
mixtures of two of five metal ions for 15 min at ambient
temperature. Samples were immediately loaded, without
further treatment or purification, onto a 1% agarose gel
containing ethidium bromide. Following electrophoresis
(1 h, 100 V) in Tris acetate buffer, gels were viewed under
UV light and photographed using a UVP Imagestore
5000 video camera (Ultraviolet Products) and images were
stored in TIFF format on floppy disk. Densitometry anal-
ysis of individual DNA bands was achieved using Image-
Quant software.
38 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44

3. Results 0.010 mM and Cd (II) and Ni (II) at 0.10 mM did not gen-
erate levels of 8-OHdG that were significantly higher than
3.1. Formation of 8-OHdG the yield found in DNA treated with hydrogen peroxide
alone (p < 0.05, Mann–Whitney test). On the other hand,
8-OHdG is an important oxidative DNA adduct leading incubation of DNA with the Fe (II) and Cu (II) Fenton-
to mutagenesis and carcinogenesis. The ability of single type reactions at 0.010 mM generated a yield of 8-OHdG
metal ion or a mixture of metal ions in the presence of higher than that observed with peroxide only. The highest
hydrogen peroxide to induce 8-OHdG formation in salmon yield of 8-OHdG through the Fenton-type reaction by sin-
sperm was determined using HPLC/ECD/UV. The forma- gle metal ion was generated in the Fe (II) reaction
tions of 8-OHdG induced by metal ions through Fenton- (0.10 mM), and a yield of 250/104 deoxyguanosine was
type reactions are summarized in Table 1. observed. In the case of the Fenton-type reactions with a
In the case of single metal ion Fenton-type reactions, mixture of metal ions, Cd (II) plus Ni (II), Cd (II) plus
treatment of DNA with Cd (II), Ni (II) and Cr (III) at Cr (III), Ni (II) plus Cr (III) at 0.010 mM and Cd (II) plus
Ni (II) at 0.10 mM did not generate levels of 8-OHdG that
were significantly higher than that with peroxide only
Table 1
(p > 0.05, Mann–Whitney test).
8-OHdG formation mediated by Fenton-like reaction with salmon sperm
DNA In order to evaluate the influence of mixing metal ion on
the formation of 8-OHdG, the influence of the interaction
Metal ions 0.010 mM 0.10 mM
of metal ions on the formation of 8-OHdG was evaluated
8-OHdG per Recovery of 8-OHdG per Recovery of
by Eq. (1).
10e4 dGa dG (%)b 10e4 dGa dG (%)b
Untreated 7.5 ± 3.6c 100 7.5 ± 3.6c 100 8-OHdG ðmetal A plus BÞ
Ratio ¼
H2O2 only 20 ± 6.3d 86 ± 14j 20 ± 6.3d 86 ± 14j 8-OHdG ðmetal AÞ þ 8-OHdG ðmetal BÞ  8-OHdG ðuntreatedÞ
Fe 51 ± 5.1e,h 80 ± 3.0k 250 ± 37.5c,h 56 ± 3.1l ð1Þ
Cd 10 ± 1.5e,i 71 ± 19k 24 ± 8.4f,i 100 ± 7.2m
Ni 8.9 ± 3.8e,i 110 ± 16k 26 ± 9.4f,i 92 ± 9.3m
Cr 14 ± 2.5e,i 73 ± 16k 87 ± 23g,h 78 ± 12n ‘‘8-OHdG (metal A)’’ in Eq. (1) means the value of the for-
Cu 180 ± 40e,h 53 ± 9.0k 200 ± 50g,h 7.3 ± 3.5n mation of 8-OHdG (per 104 dG) through Fenton-type reac-
Fe + Cd 44 ± 4.4e,h 67 ± 12k 150 ± 48f,h 47 ± 8.2m tion of metal A. The results were summarized in Table 2.
Fe + Ni 51 ± 7.7e,h 66 ± 12k 140 ± 35f,h 47 ± 13m The differences between the 8-OHdG formation treated
Fe + Cr 67 ± 11e,h 68 ± 10k 320 ± 21e,h 36 ± 5.8k
Fe + Cu 180 ± 27e,h 56 ± 8.0k 330 ± 18e,h 21 ± 14k
with the mixture of two ions (numerator in Eq. (1)) and
Cd + Ni 13 ± 3.3e,i 75 ± 17k 16 ± 5.9e,i 96 ± 4.0k that calculated by the sum of the 8-OHdG formation with
Cd + Cr 15 ± 3.9e,i 68 ± 6.6k 69 ± 3.5e,h 73 ± 11k
Cd + Cu 180 ± 38e,h 54 ± 8.0k 160 ± 42f,h 7.3 ± 1.5m
Table 2
Ni + Cr 19 ± 8.7e,i 69 ± 14k 88 ± 19e,h 72 ± 12k
Effects of mixture of metal ion on the formation of 8-OHdG
Ni + Cu 180 ± 38e,h 50 ± 5.1k 150 ± 38e,h 10 ± 6.8k
Cr + Cu 220 ± 17e,h 53 ± 5.9k 620 ± 93e,h 12 ± 2.3k Mixture of metal ion Values of ratioa,b
a
The values are the number of 8-OHdG per 10e4 of unmodified 0.010 mM 0.10 mM
deoxyguanosine. Fe + Cd 0.82 ± 0.10 ()c,d 0.57 ± 0.18 ()c
b
The recovery of deoxyguanosine are calculated by the equation as Fe + Ni 0.97 ± 0.16e 0.53 ± 0.14 ()c
follow: recovery (%) = (dG concentration under the metal concentration/ Fe + Cr 0.89 ± 0.31e 0.98 ± 0.14 (+)f
dG concentration without addition of metal and H2O2) · 100. Fe + Cu 0.78 ± 0.15 (+)f,g 0.76 ± 0.12 ()c
c
The values are the average (n = 4), and the standard deviation. Cd + Ni 1.2 ± 0.42e 0.40 ± 0.16 ()c
d
The values are the average (n = 7), and the standard deviation. Cd + Cr 0.91 ± 0.27e 0.70 ± 0.18 ()c
e
The values are the average (n = 3), and the standard deviation. Cd + Cu 0.98 ± 0.24e 0.79 ± 0.28 ()c
f
The values are the average (n = 5), and the standard deviation. Ni + Cr 1.3 ± 0.59e 0.88 ± 0.28e,f
g
The values are the average (n = 6), and the standard deviation. Ni + Cu 1.0 ± 0.24e 0.71 ± 0.24 ()c
h
The difference between the level of 8-OHdG treated with hydrogen Cr + Cu 1.2 ± 0.21e 2.3 ± 0.57 (·)c,h
peroxide alone and that treated with the metal was significant (p < 0.05,
a
Mann–Witney test). The values are calculated by Eq. (1).
b
i
The difference between the level of 8-OHdG treated with hydrogen The values are averages and standard deviations of the ratio.
c
peroxide alone and that treated with the metal was not significant The difference between the 8-OHdG formation treated with the mix-
(p > 0.05, Mann–Witney test). ture of two ions and those calculated by the sum of the 8-OHdG forma-
j
The values are the average and standard deviation (n = 7) and tion treated with each single ions was significant (p < 0.05, Mann–Witney
calculated the dG concentration in untreated DNA as 100%. test).
d
k
The values are the average and standard deviation (n = 3) and calcu- () Suppressive effect (ratio < 1, p < 0.05).
e
lated the dG concentration in untreated DNA as 100%. The effect of interaction of metal ions was not evaluated.
f
l
The values are the average and standard deviation (n = 4) and The difference between the 8-OHdG formation treated with the mix-
calculated the dG concentration in untreated DNA as 100%. ture of two ions and those calculated by the sum of the 8-OHdG forma-
m
The values are the average and standard deviation (n = 5) and calcu- tion treated with each single ions was not significant (p > 0.05, Mann–
lated the dG concentration in untreated DNA as 100%. Witney test).
g
n
The values are the average and standard deviation (n = 6) and (+) Additive effect (p > 0.05).
h
calculated the dG concentration in untreated DNA as 100%. (·) Synergistic effect (ratio >1, p < 0.05).
 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44 39

each single ions (denominator in Eq. (1)) were evaluated Table 3

using Mann–Whitney test. As to the denominator in Eq. The formation of 8-OHdG through Cu (II) plus Cr (III) Fenton reaction
(1), the 8-OHdG formation with each single ion were Concentration 8-OHdG per 10e4 Ratio Recovery of dG
summed for every pairs of the values of 8-OHdG formation (mM) dGa (%)b
treated with metals A and B, and their average and stan- Cu Cr
dard deviation are calculated. The relationship between 0.010 0 180 ± 40c – 53 ± 9.0d
the values of ratios and influences of the interaction of me- 0.010 0.010 220 ± 17 1.2 ± 0.21e,f 58 ± 5.0
tal ions is defined as follows: p < 0.05 (the 8-OHdG forma- 0.010 0.020 356 ± 53 1.6 ± 0.36 (·)g,h 44 ± 12
0.010 0.050 405 ± 110 1.6 ± 0.42 (·)g 50 ± 6.0
tion treated with the mixture of two ions and that 0.010 0.10 400 ± 200 1.5 ± 0.64f 55 ± 10
calculated by the sum of the 8-OHdG formation with each 0.10 0.10 620 ± 93 2.3 ± 0.57 (·)g
single ions is significantly different.), and the average of the 0 0.010 14 ± 2.5 – 73 ± 16
ratio < 1; suppressive effect, p > 0.05; additive effect, 0 0.020 50 ± 27 – 75 ± 18
p < 0.05 and the average of the ratio >1; synergistic effect. 0 0.050 82 ± 7.6 – 62 ± 19
0 0.10 87 ± 23 – 74 ± 4.0
The influence of interaction of metal ions were not evalu-
a
ated, when the 8-OHdG level in DNA treated with both The values are the number of 8-OHdG per 10e4 of unmodified
deoxyguanosine.
or either metal ions were not significantly different from b
The recovery of deoxyguanosine are calculated by the equation as
those of control (Table 1), and the difference between the follow: recovery (%) = (dG concentration under the metal concentration/
values of the numerator and those of the denominator in dG concentration without addition of metal and H2O2) · 100.
c
Eq. (1) were not significant (p > 0.05). The values are the average and the standard deviation (n = 3).
d
Fig. 1 shows the changes of the formation of 8-OHdG The values are the average and standard deviation (n = 3) and calcu-
lated the dG concentration in untreated DNA at the same batch as 100%.
by mixing metal ions for Fe (II) plus Cd (II), Fe (II) plus e
The values (average and standard deviation) are calculated by Eq. (1).
Cr (III) and Cu (II) plus Cr (III) at 0.10 mM. Additions f
The difference between the 8-OHdG formation treated with the mix-
of Cd (II) and Ni (II) ion (0.10 mM) showed suppressive ture of Cu(II) and Cr(III) and those calculated by the sum of the 8-OHdG
effects on Fenton-type reaction induced by other metal formation treated with each single ions was not significant (p > 0.05,
ions. In the case of Fe (II) and Cu (II) mixture Mann–Witney test).
g
The difference between the 8-OHdG formation treated with the mix-
(0.10 mM), a suppressive effect on the formation of 8- ture of Cu(II) and Cr(III) and those calculated by the sum of the 8-OHdG
OHdG was observed. The pair of Cr (III) and Cu (II) formation treated with each single ions was significant (p < 0.05, Mann–
shows a synergistic effect on the formation of 8-OHdG at Witney test).
h
0.10 mM. (·) Synergistic effect.
Next, the Cu (II) plus Cr (III) Fenton-type reaction at
several concentrations was observed (the concentration of formation in DNA by Fenton-type reactions with Cu (II)
Cu was set at 0.010 mM). Table 3 summarizes 8-OHdG and Cr (III) ions, singly and as mixtures. The effects of

Fig. 1. 8-OHdG level in the DNA of salmon sperm treated with Fenton-type reactions at 0.1 mM. (a) Fe (II) and Cd (II), (b) Fe (II) and Cr (III), (c) Cu
(II) and Cr (III).
40 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44

mixing Cu (II) (0.010 mM) and Cr (III) ions (0.02 and
0.05 mM) on the formation of 8-OHdG showed a synergis-
tic effect (maximum 1.6 times for Cu (II) at 0.010 mM and
Cr (III) at 0.05 mM). The results showed a mixture of Cu
(II) and Cr (III) shows synergistic influence on the forma-
tion of 8-OHdG under various ratio of Cu (II)/Cr (III).
3.2. Generation of single- and double-strand breaks in DNA
Strand break induction in DNA can be monitored using
supercoiled plasmid DNA. The double-stranded SK+ plas-
mid exists in a tightly compact supercoiled conformation in
its native form, and as such it has a relatively high electro-
phoretic mobility. When treated with an agent that induces
DNA strand breaks, supercoiled DNA is converted to the
open-circle conformation by a single-strand break and to
linear DNA by a double-strand break. Extensive double-
strand breakages lead to DNA degradation. These effects
can be monitored by gel electrophoresis. The migration
pattern of a plasmid under the gel electrophoresis condi-
tions is: supercoiled > linear > open circular DNA. Hence
analysis by agarose gel electrophoresis yields information
regarding the type of strand breakage occurring in each
Fenton-type reaction by separation of the different confor-
mations of plasmid DNA (Fig. 2).
Table 4 summarizes the results of an incubation of plas-
mid DNA with hydrogen peroxide in the presence of a sin-
gle metal ion or a mixture of two ions. With 0.4 lM metal
ion, over 85% of the DNA remained supercoiled, as in the
absence of metal ion (p > 0.05), except in two cases: with Fe
(II) (where 25% became open circular) and Fe (II) plus Cu
(II) (where 21% became open circular). With 4.0 lM metal

Fig. 2. Agarose gel electrophoresis of SK+ plasmid DNA following treatment with Fenton-type reactions by mixtures of metal ions at 4.0 lM.

Table 4
SK+ DNA strand breaks mediated by Fenton-type reaction
0.40 lM 4.0 lM
Supercoiled (%) Linear (%) Open circular (%) Supercoiled (%) Linear (%) Open circular (%)
Untreateda 96 ± 3.0 0.68 ± 1.5 3.0 ± 2.3 96 ± 3.0 0.68 ± 1.5 3.0 ± 2.3
H2O2 onlya 96 ± 3.5 2.1 ± 2.1 2.2 ± 2.3 96 ± 3.5 2.1 ± 2.1 2.2 ± 2.3
Feb 74 ± 2.0c 1.2 ± 1.2d 25 ± 1.7c 39 ± 2.5c 3.4 ± 2.3 57 ± 3.5c
Cdb 95 ± 4.0d 0.86 ± 1.0d 4.5 ± 3.6d 95 ± 4.0d 0.9 ± 1.0 4.5 ± 3.6d
Nib 94 ± 4.0d 0.54 ± 0.48d 5.3 ± 3.7d 93 ± 1.2d 1.0 ± 0.12 6.5 ± 0.95c
Crb 91 ± 5.0d 1.7 ± 1.9d 7.5 ± 4.3d 73 ± 4.0c 2.2 ± 0.42 25 ± 4.0c
Cub 91 ± 1.2d 2.0 ± 1.2d 6.8 ± 0.32c 87 ± 2.6c 1.4 ± 1.2 12 ± 3.7d
Fe + Cdb 94 ± 4.0d 2.6 ± 2.4d 3.6 ± 3.2d 47 ± 2.6c 1.1 ± 0.66d 52 ± 3.8c
Fe + Nib 89 ± 11d 2.6 ± 3.0d 8.5 ± 8.3d 50 ± 3.6c 1.4 ± 0.68d 48 ± 2.5c
Fe + Crb 92 ± 6.7d 0.77 ± 0.86d 7.5 ± 5.7d 42 ± 5.5c 11 ± 4.4c 47 ± 1.2c
Fe + Cub 79 ± 16c 1.1 ± 0.57d 21 ± 17c 45 ± 1.5c 5.5 ± 0.87c 49 ± 1.7c
Cd + Nib 94 ± 1.5d 1.4 ± 0.78d 4.4 ± 0.87d 85 ± 0.58c 3.8 ± 2.0d 10 ± 0.72c
Cd + Crb 91 ± 7.2d 0.40 ± 0.70d 8.3 ± 6.1d 86 ± 4.0c 1.6 ± 0.78d 13 ± 3.2c
Cd + Cub 95 ± 3.5d 0.40 ± 0.61d 4.4 ± 2.9d 95 ± 1.0d 0.80 ± 0.70d 4.0 ± 0.76d
Ni + Crb 93 ± 4.6d 0.43 ± 0.75d 6.2 ± 3.4d 80 ± 2.6c 2.0 ± 0.62d 18 ± 2.6c
Ni + Cub 95 ± 2.5d 0.90 ± 1.1d 3.9 ± 3.6d 89 ± 2.3c 2.0 ± 1.5d 9.5 ± 0.55c
Cr + Cub 96 ± 2.6d 0.63 ± 1.1d 3.5 ± 3.5d 77 ± 3.5c 2.8 ± 0.38d 20 ± 2.9c
a
Mean value and standard deviation (n = 5).
b
Mean value and standard deviation (n = 3).
c
The difference between the percentage of SK + DNA strand breaks treated with hydrogen peroxide alone and that treated with the metal was
significant (p < 0.05, Mann–Witney test).
d
The difference between the percentage of DNA strand breaks treated with hydrogen peroxide alone and that treated with the metal was not significant
(p > 0.05, Mann–Witney test).
 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44 41

ion, however, conversion to open circular DNA was at
least 10% in all the incubations containing Fe (II) and Cr
(III). Conversion to linear DNA by double-strand break-
age was negligible except with 4.0 lM Fe (II) plus Cr
(III) and Fe (II) plus Cu (II), when it was 11% and 5.5%.
The difference between the percentages of linear form trea-
ted with these metal ions and those treated with peroxide
only was significant (p < 0.05).
The percentage of DNA strand breaks (break/plasmid)
was calculated by Eq. (2).
break=plasmid ¼ ln ðsupercoiled ð%Þ=100Þ
The influence of the interaction of metal ions on the
strand breaks in DNA was evaluated by Eq. (3), and the
results are summarized in Table 5.
ðbreak=plasmidÞmetal A plus B
Ratio ¼
ðbreak=plasmidÞmetal A þ ðbreak=plasmidÞmetal B  ðbreak=plasmidÞuntreated
The difference between the percentage of break/plasmid
treated with the mixture of two metal ions (numerator in
Eq. (3)) and those calculated by the sum of the percentages
treated with each single ions (denominator in Eq. (3)) were
tested using Mann–Whitney test. As to the denominator in
Eq. (3), the percentages treated with each single ion were
summed for every pairs of the values of 8-OHdG formation
treated with metal A and B, and their average and standard
deviation are calculated. In the same way as the formation
of 8-OHdG, the relationships between the values of ratios
and influences of the interaction between metal ions is
defined as follows: p < 0.05, and the average of the ratio
< 1; suppressive effect, p > 0.05; additive effect, p < 0.05
and the average of the ratio >1; synergistic effect. The
effects of a mixture of metal ions at 0.40 lM (except for
ð2Þ Fe (II) plus Cu (II), Ni (II) plus Cu (II) and Cr (III) plus
Cu (II)) were not evaluated, because the percentage of
break/plasmid treated with both or either metal ions were
not significantly different from those of control, and the
values of the numerator and those of the denominator in
Eq. (3) were not significant (p > 0.05).
The calculated values of ratio, except for Fe (II) plus Cr
ð3Þ (III) and Fe (II) plus Cu (II) at 4.0 lM, reflect the effects of
mixing metal ions on single-strand break formation in
DNA, because significant double-strand breaks in DNA
were not observed by the Fenton-type reactions. Most
cases (at 4.0 lM) showed the suppressive effects of the
interaction of metal ions on DNA strand breaks.

Table 5
Effects of mixing metal ions on strand breaks in DNA
0.40 lM 4.0 lM
a b
Break/plasmid Ratio Break/plasmid Ratio
Untreated 0.037 ± 0.031 – 0.037 ± 0.031 –
H2O2 only 0.046 ± 0.037 – 0.046 ± 0.037 –
Fe 0.30 ± 0.027c – 0.93 ± 0.064c –
Cd 0.055 ± 0.043d – 0.055 ± 0.043c –
Ni 0.062 ± 0.043d – 0.076 ± 0.012d –
Cr 0.099 ± 0.055d – 0.32 ± 0.055c –
Cu 0.091 ± 0.013c – 0.14 ± 0.030c –
Fe + Cd 0.066 ± 0.042 0.21 ± 0.12 ()e,f 0.76 ± 0.057 0.80 ± 0.073 ()f
Fe + Ni 0.12 ± 0.13 0.38 ± 0.34 ()f 0.70 ± 0.073 0.72 ± 0.075 ()f
Fe + Cr 0.089 ± 0.074 0.26 ± 0.19 ()f 0.88 ± 0.13 0.73 ± 0.075 ()f
Fe + Cu 0.26 ± 0.22 0.72 ± 0.52 (+)g,h 0.79 ± 0.034 0.77 ± 0.052 ()f
Cd + Ni 0.058 ± 0.016 0.17 ± 2.9i 0.17 ± 0.0068 2.2 ± 1.1(·)f,j
Cd + Cr 0.096 ± 0.081 1.6 ± 2.8i 0.15 ± 0.047 0.46 ± 0.15 ()f
i
Cd + Cu 0.048 ± 0.026 0.53 ± 0.42 0.051 ± 0.011 0.36 ± 0.14 ()f
Ni + Cr 0.073 ± 0.050 0.88 ± 1.1i 0.22 ± 0.033 0.64 ± 0.12 ()f
Ni + Cu 0.048 ± 0.026 0.46 ± 0.28 ()f 0.12 ± 0.026 0.70 ± 0.17 ()f
Cr + Cu 0.041 ± 0.028 0.30 ± 0.21 ()f 0.26 ± 0.044 0.64 ± 0.12 ()f
a
The values are calculated according to Eq. (2).
b
The values (average and standard deviation) are calculated by Eq. (3).
c
The difference between the percentages of break/plasmid treated with hydrogen peroxide alone and those treated the metal ion was significant
(p < 0.05, Mann–Witney test).
d
The difference between the percentages of break/plasmid treated with hydrogen peroxide alone and those treated the metal ion was not significant
(p > 0.05, Mann–Witney test).
e
() Suppressive effect.
f
The difference between the percentages of break/plasmid treated with the mixture of two ions and those calculated by the sum of the percentages
treated with each single ions was significant (p < 0.05, Mann–Witney test).
g
(+) Additive effect.
h
The difference between the percentages of break/plasmid treated with the mixture of two ions and those calculated by the sum of the percentages
treated with each single ions was not significant (p > 0.05, Mann–Witney test).
i
The effect of interaction of metal ions was not evaluated.
j
(·) Synergistic effect.
42 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44
4. Discussion
4.1. Formation of 8-OHdG by Fenton-type reaction
In order to confirm the hypothesis that the interaction of
metal ions with DNA influences DNA oxidative stress via a
Fenton-type reaction, we examined the ability of mixtures
of two of five metal ions, Fe (II), Cd (II), Ni (II), Cr (III)
or Cu (II) to induce the formation of 8-OHdG in DNA,
an important oxidative DNA adduct leading to mutagene-
sis and carcinogenesis. First, the formation of 8-OHdG
mediated by a Fenton-type reaction of the single metal
ion with DNA was observed. When the concentration of
Fe (II) and Cr (III) was at 1.0 mM, the peak of dG was
not detected (the detection limit of injected dG calculated
as three times the baseline noise was 0.5 nmol, and the
recovery (%) of dG was about 0.5%). This result was likely
due to the extensive DNA strand breakage by the Fenton-
type reactions at 1.0 mM. For the comparison with the
Fenton-type reaction between mixing metal ion and
DNA, it was thought that the recoveries of dG would need
to be higher (i.e. >5%). Then, the concentrations of metal
ions were set at 0.010 and 0.10 mM, and the recovery of
dG and the formation of 8-OHdG through the Fenton-
type reaction were observed (Table 1). At 0.010 or
0.10 mM, the recoveries of dG were over 5% under all con-
ditions. The highest yield of 8-OHdG through the Fenton-
type reaction of the single metal ion was obtained following
treatment of DNA with the Fe (II) Fenton reaction (250
per 104 dG at 0.10 mM), followed by Cu (II) (200) and
Cr (III) (87). There is a tendency that the formation of 8-
OHdG at 0.10 mM of the metal concentration was higher
than that of at 0.010 mM in every case for the Fe (II),
Cu (II) and Cr (III) Fenton-type reaction. It is well known
that the Cu (II) Fenton-type reaction proceeds through the
electron transfer reaction as shown in Eqs. (4)–(6) (Perez-
Benito, 2004).
Cu ðIIÞ þ H2 O2 ! CuOOHþ þ Hþ
þ
CuOOH ! Cu ðIÞ þ O
2 þH þ
 
Cu ðIÞ þ H2 O2 ! Cu ðIIÞ þ OH þ OH
Cu (II) ion can interact strongly with DNA (Eichhorn
and Shin, 1968). Therefore, the Cu (II) Fenton-type reac-
tion with DNA would be influenced by the interaction
between copper ion and DNA. The difference between
the recoveries of dG through Cu (II) Fenton-type reaction
and those treated peroxide only was significant (p < 0.05,
Mann–Whitney test). In addition, the recoveries of dG
through the Cu (II) Fenton-type reaction with DNA
(53% at 0.010 mM, 5.1% at 0.10 mM) were low compared
to those by the Fenton-type reactions of the other metal
ions. The results could be due to the DNA strand breakage
induced by the Cu (II) Fenton-type reaction or the influ-
ence of Cu (II) ion on the precipitation of DNA.
On the other hand, in the case of the Fenton-type reac-
tions by single metal ion, treatment of DNA with Cd (II),
Ni (II) and Cr (III) at 0.010 mM and Cd (II) and Ni (II) at
0.10 mM did not generate levels of 8-OHdG that were sig-
nificantly higher than the yield found in DNA treated with
hydrogen peroxide alone (p > 0.05, Mann–Whitney test).
The reason may be that there is no formation of ROS by
the Cd (II) and Ni (II) Fenton-type reaction, because Cd
(II) and Ni (II) ions do not mediate the electron transfer
to hydrogen peroxide. Mikhailova et al. reported that Cd
(II) induced the formation of 8-OHdG in human lympho-
blastoid cells (Mikhailova et al., 1997). The result could
be due not to the formation of ROS by Cd (II) but to
the influence of Cd (II) on the DNA repair system (Wang
et al., 2004).
Next, we studied the formation of 8-OHdG mediated by
the Fenton-type reaction with a mixture of metal ions.
Effects on the formation of 8-OHdG by mixing metal ions
were observed, and depend on the kind of the pair of metal
ions (Fig. 1).
Additions of Cd (II) and Ni (II) ion (0.10 mM) showed
suppressive effects on Fenton-type reaction induced by
other metal ions, except for the case of Cr (III) plus Ni
(II). It was reported that Cd (II) interacts with guanine
bases (Moriwaki, 2003). In addition, it is well known that
Ni (II) ion interacts with DNA phosphate group and the
interaction stabilizes the structure of DNA (Eichhorn and
Shin, 1968). Furthermore, these ions do not form OH rad-
ical with hydrogen peroxide. Therefore, it is thought that
the interaction of Cd (II) or Ni (II) with DNA will suppress
DNA oxidation by Fe (II), Cu (II) and Cr (III) by interfer-
ing with their interaction with DNA.
In the case of Fe (II) plus Cu (II) mixture (0.010 mM),
an additive effect on the formation of 8-OHdG was
observed. In contrast, Fe (II) plus Cu (II) Fenton-type
reaction at 0.10 mM showed a suppressive effect. Fe and
Cu ions interact with DNA bases. Therefore, the competi-
tion of the interactions between DNA base and metal ions
would make a suppressive effect on 8-OHdG formation at
0.10 mM.
ð4Þ The Fe (II) plus Cr (III) Fenton-type reaction
ð5Þ (0.10 mM) shows an additive effect on the formation of
8-OHdG. This result suggests that Fe (II) and Cr (III)
ð6Þ
interact with different sites in DNA, and independently oxi-
dize DNA through Fenton-type reaction. In fact, it was
reported that Cr (III) interacts with both base and phos-
phate groups of DNA, and Fe interacts with DNA bases
(Eichhorn and Shin, 1968; Levina et al., 2001).
The pair of Cr (III) and Cu (II) shows a synergistic effect
on the formation of 8-OHdG at 0.10 mM. In the case of the
Cu (II) plus Cr (III) Fenton-type reaction at 0.010 mM, an
additive effect, not a synergistic effect, was observed. The
Cu (II) and Cr (III) Fenton-type reaction at other concen-
trations (the concentration of Cu was set at 0.010 mM)
showed a synergistic effect on the formation of 8-OHdG
(Table 3). Cu (II) has been proposed to generate OH radi-
cal through the Fenton-type reaction (Eqs. (4) and (5)).
The preferential binding of Cu (I) to the N7 guanine would
result in the formation of 8-OHdG.
 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44
It was reported by Lay et al. that Cr (V) species are
interacted DNA and highly reactive intermediates, Cr
(VI), induced oxidative damage of DNA (Pattison et al.,
2001). Therefore, a synergistic effect of the mixture of Cu
(II) plus Cr (III) ion would be due to the electron transfer
between formed Cu (I) and Cr (VI) to generate Cr (V) (Eqs.
(6)–(8)).
Cr ðIIIÞ þ 3H2 O2 ! Cr ðVIÞ þ 3OH þ 3OH ð7Þ
Cu ðIÞ þ Cr ðVIÞ ! Cu ðIIÞ þ Cr ðVÞ ð8Þ
The Cr (V) ion from the electron transfer reaction can
undergo Fenton-type reaction with hydrogen peroxide
(Eq. (9)), and the electron transfer reaction to form Cr
(V) would accelerate the formation of 8-OHdG.
Cr ðVÞ þ H2 O2 ! Cr ðVIÞ þ OH þ OH ð9Þ
There are several examples that the presence of reducing
agent, such as o-quinone and benz[a]anthracene dihydrodi-
ols, activates DNA oxidative stress induced by Cu (II)
(Flowers et al., 1997; Seike et al., 2004). Furthermore, the
electron transfer between copper and chromium ion in
the presence of hydrogen peroxide has been reported
(Perez-Benito and Arias, 1999). In view of these facts, the
synergistic effect would be induced by the interaction
between Cu (II), Cr (III), Cr (V), and DNA and electron
transfer between Cu, Cr, and hydrogen peroxide.
Consideration of these synergistic effects is needed for
evaluating the risk of pollution from plating industries,
because metal plating wastes often contain copper and
chromium together (Vijay and Sihorwala, 2003).
4.2. DNA strand breaks
In order to observe single- and double-strand breaks in
DNA by Fenton-type reaction with a mixture of two kinds
of metal ions, pBluescript K+ plasmid was incubated with
hydrogen peroxide and mixtures of two kinds of metal
ions. The concentration of metal ions were set at 0.40
and 4.0 lM conforming to the ratio between DNA and
heavy metal in 8-OHdG study. The single-strand breaks
in DNA through Fenton- type reactions of single metal
ions at 0.40 lM (Cd (II), Ni (II), Cr (III) and Cu (II))
and 4.0 lM (Cd (II) and Ni (II)) were similar to that occur-
ring with peroxide only (p > 0.05, Table 4). Double-strand
breaks in DNA were not observed except for the Fe (II)
plus Cr (III) and Fe (II) plus Cu (II) Fenton-type reaction
at 4.0 lM. The percentages of the linear form under other
conditions were similar to that of blank.
Most cases (at 4.0 lM) showed the suppressive effects of
the interaction of metal ions on DNA strand breaks. There
were some cases that the influences of the interaction of
metal ions on strand breaks in DNA were different from
those on the formation of 8-OHdG (For example, Fe (II)
plus Cr (III), Cd (II) plus Ni (II) and Cr (III) plus Cu
(II) in Tables 2 and 3). In an earlier study (Lloyd and Phil-
lips, 1999), Fenton-type reactions with EDTA were
43
observed. The results suggested that the interaction
between DNA and metal ion was influenced on DNA oxi-
dative stress, and a site-specific mechanism is involved in
the formation of 8-OHdG, while the formation of single-
strand breaks is more likely to involve generation of hydro-
xyl radicals in solution. Therefore, influence of mixing
metal ions on the formation of 8-OHdG would be due to
the change of the interactions between DNA and metal
ions, while that of single-strand breaks would owed the
change of reactivity between DNA and the formed hydroxy
radical in solution. The difference of the mechanisms would
cause the several inconsistency of the influence of mixing
metal ions on strand breaks in DNA and on the formation
of 8-OHdG.
The case of the Cd (II) plus Ni (II) Fenton-type reaction
at 4.0 lM with DNA showed the synergistic effect. The
mechanism is not clear at this stage. The reaction between
the plasmid and the OH radical formed by reactions other
than Fenton-type reaction may be accelerated by the inter-
action between the DNA and Cd (II) plus Ni (II).
In the case of the Fe (II) plus Cr (III) Fenton-type
reaction with DNA, the percentage of the linear form,
containing double-strand breaks in DNA, was increased
compared to those of the single Fe (II) or Cr (III) Fen-
ton-type reaction (Table 4). However, the difference
between the percentage of linear form treated with Fe
(II) plus Cr (III) and the sum of the percentages of linear
form treated Fe (II) and Cr (III) was not statistically sig-
nificant (p > 0.05, Mann–Whitney test). It was reported
that double-strand breaks in DNA through a Fenton-type
reaction was related to the interaction between metal ion,
such as iron, and DNA (Lloyd and Phillips, 1999). For-
mation of linear form SK+ plasmid would be initiated
by the interaction between DNA and two metal ions. Lin-
ear DNA could result from the formation of a true dou-
ble-strand break (i.e., scission of both strands of DNA in
one single event). It might also be due to the formation of
many single-strand breaks such that two are formed inde-
pendently but closely on opposing strands. Therefore,
there is a possibility that the formation of ROS was
increased by mixing Fe (II) and Cr (III), and that influ-
enced the formation of the linear DNA.
In summary, we have demonstrated effects of mixing
metal ions on oxidative DNA damage, the formation of
8-OHdG and single- and double-strand breaks in DNA,
mediated by a Fenton-type reduction, and found interac-
tions between metal ions had various influences (suppres-
sive, additive and synergistic effects) on DNA oxidative
stress. The in vitro tests in this study showed the model
of DNA reaction with activated oxygen species. However,
it is noted that the models used in this study do not reflect
DNA structures in cell and the interactions between metal
ions and proteins in chromatin. It is well known that these
factors influence on DNA oxidative stresses by metal ions
(Kasprzak et al., 2003), and next step of this study is to
evaluate the effects of mixing metal ions on DNA oxidative
stress by in vivo system.
44 H. Moriwaki et al. / Toxicology in Vitro 22 (2008) 36–44
Acknowledgements
H.M. thanks the Section of Molecular Carcinogenesis at
the Institute of Cancer Research for receiving him as a vis-
iting researcher from September 2004 to September 2005.
Supported by Cancer Research UK.
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