DNA damage induced by sulfite autoxidation catalyzed by copper(II) tetraglycine complexes

Article abstract of DOI:10.1039/b416299d

Copper(II)/(III) tetraglycine complexes were investigated for their ability to catalyze the autoxidation of sulfite resulting in oxidative DNA damage. The focus of this work is on DNA damage by Cu(III) and oxysulfur radicals formed by the oxidation of S(IV) oxides by dissolved oxygen in the presence of Cu(II) tetraglycine complexes. The results suggest that sulfite is rapidly oxidized by oxygen in the presence of Cu(II) complexes producing Cu(III) tetraglycine, which can be monitored spectrophotometrically at 365 nm. A synergistic effect of Cu(II) with a second metal ion (Ni(II), Co(II) or Mn(II) traces) was observed. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b416299d

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F U L L P A P E R
DNA damage induced by sulfite autoxidation catalyzed by copper(II)
tetraglycine complexes
Ruben G. M. Moreno, Mar ´ı a V. Alip a´ zaga, Marisa H. G. Medeiros and Nina Coichev*
Instituto de Qu ´ı mica, Universidade de S a˜ o Paulo, CP 26077, CEP 05513-970, S a˜ o Paulo,
SP-Brazil. E-mail: ncoichev@iq.usp.br
Received 21st October 2004, Accepted 2nd February 2005
First published as an Advance Article on the web 21st February 2005
Copper(II)/(III) tetraglycine complexes were investigated for their ability to catalyze the autoxidation of sulfite
resulting in oxidative DNA damage. The focus of this work is on DNA damage by Cu(III) and oxysulfur radicals
formed by the oxidation of S(IV) oxides by dissolved oxygen in the presence of Cu(II) tetraglycine complexes. The
results suggest that sulfite is rapidly oxidized by oxygen in the presence of Cu(II) complexes producing Cu(III)
tetraglycine, which can be monitored spectrophotometrically at 365 nm. A synergistic effect of Cu(II) with a second
metal ion (Ni(II), Co(II) or Mn(II) traces) was observed.
Introduction
oxidation of sulfite and in the horseradish peroxidase–H
2
O
2
45,46
oxidation of sulfite.
oxidase/H
It has been reported that the xanthine
The use of sulfite in foods, alcoholic beverages and drugs for
many important technical purposes, including the control of
enzymatic and non-enzymatic browning, antimicrobial action,
as antioxidant and reducing agent, and in a variety of processing
•−
2
O
2
47
system is also capable of generating SO
3
radical
from sulfite.
In the present study oxidative DNA damage of sulfite in the
II
II
presence of Cu G
4
, traces of M G
4
(M = Ni, Co or Mn), S(IV)
1
,2
aid uses is well known. However, the safety of the continued
use of sulfite in foods has been questioned on the basis of its
role in the initiation of asthmatic reactions in certain sensitive
and oxygen was investigated. By using tetraglycine as a ligand,
it was possible to follow the formation of Cu G
III
4
complex by
the appearance of an intense band with maximum absorbance
2
individuals, its mutagenic effect and the ability to act as a
III
at 250 and 365 nm (at this wavelength only the Cu G complex
4
3,4
cocarcinogen.
absorbs). The chemistry of copper tetraglycine complexes with
sulfite is of interest as a model of copper and sulfite cotoxicity.
−
2−
The interaction of S(IV) oxides (SO
2
, HSO
3
and SO
3
), a
◦
reducing agent (E S(VI)/S(IV) = 0.17 V in acidic medium), with traces
◦
of Cu(III) tetraglycine (G
4
) complex (E Cu(III)/Cu(II)/tetraglycine = 0.63 V,
pH = 9) or other metallic ions in the +3 oxidation state (Fe(III),
Results and discussion
Mn(III), Co(III) or Ni(III)), results in the oxidation of sulfite to
•
−
(a) Spectrophotometric studies in the absence of DNA
the SO
2 oxidation state.
Several studies in the presence of a ligand have been accom-
3
radical and the reduction of the metallic species to the
5–12
II
+
The reaction of dissolved oxygen with Cu G
4
, is very slow
at room temperature and pH = 7–10, and sulfite has no
plished to verify the interaction between sulfite and metal ions,
in order to elucidate the mechanism and the kinetics, such as in
influence on the kinetics of this oxidation. However, in a detailed
II
kinetic study, we have found that Cu G
4
complexes (1.0 ×
13
−3
−3
the systems Fe(III)/1,10-phenanthroline, Cu(II)/Cu(III)/tetra-
10 mol dm in borate buffer) are rapidly oxidized in the
5,6,10–12
14
15
−5
glycine,
Co(II)/Co(III)/NH
3
,
Fe(II)/Fe(III)/EDTA,
presence of oxygen (air-saturated solutions), sulfite (1.0 × 10 –
−
5
−3
Fe(II), Mn(II), Co(II), Ni(II), Cu(II) and V(IV) with phthalo-
9.0 × 10 mol dm ) with marked synergistic effect of Ni(II),
1
6
17
−
18
−6
cyanines,
Mn(III)/N
tides,
Mn(II)/CDTA,
,
Co(II)/Co(III)/N
3
,
Mn(II)/
Co(II) and Mn(II), present at low concentrations (1.0 × 10 –
−
19
−
20
−5
−3 6,10
3
Mn(II)/Mn(III)/Ac , Ni(II)/Ni(III) with pep-
2.0 × 10 mol dm ). In the presence of traces of Ni(II) and
Co(II), the Cu(III) formation at pH = 9.0 becomes faster (being
complete in 5 s, with simultaneous oxidation of S(IV) to S(VI)
and oxygen consumption). The reaction can be followed by the
absorbance changes at 365 nm, the maximum absorbance peak
7
,8,21
9
22
23
cyclam, hydroxide and Fe(II)/(III)/H O.
2
Most studies involving DNA damage mediated by transition
metal ions were carried out in the presence of strong oxidants
agents as KHSO5.
24–26
However, studies on the DNA damage
III
48,49
induced by autoxidation of S(IV), a reducing agent, in the
of the Cu G
4
complex.
The Cu(III) formation is also higher
27,28
29
30,31
32
33
presence of Mn(II),
Cr(VI), Fe(III),
Co(II), Ni(II), and
at pH = 9.0. The latter event can be explained by the different
31
II
Cu(II) combined with mechanistic and kinetic data, showed the
importance of the balance of oxygen and S(IV) concentrations.
The DNA damage studies were carried out in most cases at S(IV)
concentrations higher than 1 mM. Oxysulfur radicals, formed
as intermediates in sulfite autoxidation catalyzed by metal ions,
reactivity of Cu G
4
complexes due to the different degree of
11,12
protonation of the coordinated ligand.
Fig. 1(a) shows the absorbance changes for Cu G
III
4
formation
II
−
3
−3
after addition of sulfite to a 1.0 × 10 mol dm Cu G
4
−
6
−3
solution (containing 1.0 × 10 mol dm Ni(II)) at pH =
7. The kinetic traces clearly show an induction period, an
evidence of autocatalytic behavior. The Cu(III) formation is
significantly faster and the induction period decreases as the
sulfite concentration increases.
24,28–35
have been proposed as key species in DNA damage.
While the detailed mechanism of sulfite toxicity is still unclear,
it is believed that an event of primary importance involves the
•
−
sulfur trioxide anion radical SO
3
, generated during sulfite
6–8,18,36,37
•−
10
oxidation.
For example, SO
3
radicals are known to
As reported in our previous work for solutions of
II
cause many adverse reactions with biological molecules, includ-
ing methionine and diphosphopyridine nucleotide oxidation,
b-carotene and tryptophan destruction,
Cu G
4
containing higher Ni(II) concentrations (up to 2.0 ×
38,39
−5
−3
10 mol dm ) at pH = 9, the reaction is faster and more
38,40
II
double bond ad-
efficient. Initially, the sulfite induced autoxidation of Cu G
4
4
1
42
dition in alkenes, lipid fatty acid peroxidation, and DNA
occurs with simultaneous oxidation of sulfite to sulfate. After
43,44
•−
damage.
In cellular systems, SO
3
has been reported to
the sulfite consumption, the absorbance increases slowly due to
II
be generated in the prostaglandin hydroperoxidase-catalyzed
the oxidation of Cu G
4
by the oxygen still remaining in solution.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
D a l t o n T r a n s . , 2 0 0 5 , 1 1 0 1 – 1 1 0 7
1 1 0 1

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Fig. 1 (a) Absorbance changes at 365 nm after addition of sulfite to
II
II
II
−3
−3
Scheme 1 Mechanism of the sulfite induced autoxidation of Cu G
4
;
a Cu G
4
solution: [Cu G
4
] = 1.0 × 10 mol dm (containing 1.0 ×
II
−
6
−3
◦
4
synergistic effect of M G .
1
[
1
1
0
mol dm Ni(II)); pH = 7.0; T = 25.0 C. Reference solution: water;
−
6
−5
−5
S(IV)]: (A) 0; (B) 5.0 × 10 ; (C) 1.0 × 10 ; (D) 5.0 × 10 and (E) 10.0 ×
−
−
5
4
−3
0
0
mol dm . (b) Absorbance changes at 365 nm after addition of 2.0 ×
is slow to generate enough of Cu(III) to initiate the process.
Low concentrations of Fe(III) (10 –10 mol dm ), present as
unavoidable impurities in reagents (buffers, metal ion solution,
−3
II
4
mol dm sulfite to a Cu G solution (A) purged with nitrogen, (B)
−
8
−7
−3
air-saturated and (C) oxygen-saturated.
20,53
etc.), have also been proposed as initiator (eqn. (4)).
The results represented in Fig. 1(b) show the influence of
∼
−4
−3
The observed synergistic effect of two metal ions depends
on the type and concentration of the second metal ion. The
synergism can not be described as the sum of the individual
dissolved oxygen. When [S(IV)] = [O
2
] (2.0 × 10 mol dm ),
III
the Cu G
4
formation is less effective (compare Fig. 1(a)(E)
−
4
−3
[
S(IV)] = 1.0 × 10 mol dm and Fig. 1(b)(B) [S(IV)] = 2.0 ×
II
4
−
4
−3
sulfite induced autoxidation reactions of Cu G and the second
10
mol dm ). At higher [S(IV)] concentration, due to the lack
10
metal ion (M(II) = Ni(II), Co(II) or Mn(II)).
of oxygen, the Cu(III) formed can be partially reduced by S(IV)
still in solution.
On a longer time scale (up to 3600 s) the absorbance continues
The synergism effect of M(II) can be explained by the faster
II
oxidation of M G
4
by O
2
(eqn. (3)) originating the metal ion in
the 3+ oxidation state (M G ), which reacts with sulfite to form
III
II
4
increasing due to the spontaneous oxidation of Cu G
4
by oxygen
•
−
III
the SO
3
radical (eqn. (4)).
still remaining in solution. This process is followed by Cu G
decomposition, which is more pronounced at higher O concen-
tration (Fig. 1(b)(C)). The rate of subsequent decomposition of
4
Reactions (2) and (3) (in absence of sulfite) are very slow,
however, M G (generated at trace level) acts as initiator. In the
2
III
4
III
presence of sulfite, the subsequent reactions (eqns. (4)–(11)) are
very fast, with the generation of sulfite radical (eqns. (4) and (5))
Cu G
4
is dependent on the pH and oxygen concentration and
48
was studied in details by Kurtz et al.
1
0,18,52
as the rate-limiting step, as pointed out in previous work.
Due to the lack of sufficiently detailed experimental infor-
mation, the proposed kinetic data treatment yields a semi-
quantitative description. Therefore, in the present work only
the main pathways in the intrinsic mechanism are described.
II
2−
II
Our studies with M G
4
/O
2
/SO
3
systems (M = Ni and Cu)
II
−4
−3
showed that the oxidation of Ni G
4
(1.0 × 10 mol dm ) in
the presence of sulfite and oxygen (air-saturated solutions) at
II
pH 9, is much faster than the oxidation of Cu G
4
at the same
8,10
III
Mechanism. The proposed mechanism in the Scheme 1 is
conditions. The first-order Cu G
the S(IV) concentration ((1.0–8.0) × 10 mol dm ; pH 7 and
9 ). In the case of Ni G there is evidence for the formation of a
4
formation is dependent on
−
5
−3
similar to those proposed in earlier studies for the Co(II)/
−
18
−
19
50,51
10
II
Co(III)/N
3
,
Mn(II)/Mn(III)/N
3
,
Fe(II)/Fe(III)/H
2
O,
4
9,52
5,10
II
Ni(II)/Ni(III)/cyclam,
Mn(III)/acetate systems, where the discussion was based on
the available literature and includes several steps considering
Cu(II)/Cu(III)/G
4
and Mn(II)/
mixed-ligand complex (Ni G
4
·SO
3
·O
2
) or a bipeptide complex
20
8
prior to the oxidation step.
II
In an autocatalytic process with synergistic effect, Cu G
4
•
−
•−
•−
−3
−3
the different radicals formed as: SO
3
, SO
5
and SO
4
.
(in large excess, 1.0 × 10 mol dm over M(II)) is oxidized
III
•−
◦
•
Scheme 1 describes only the main reactions involving the
to Cu G
4
by SO
5
(E SO −/HSO5
−
= 1.10 vs. NHE) (eqn. (7)),
5
II
III
•−
redox cycling of the Cu /Cu /G
4
complexes promoted by
produced by oxidation of SO
3
with dissolved oxygen (eqn. (6)).
−
◦
•−
◦
•
strong oxidizing agents, generated in the reaction. The auto-
HSO
5
(E HSO5−/SO42− = 1.75 vs. NHE) and SO
4
(E SO4 −/SO42−
in subsequent
=
III
II
catalytic nature of Cu G
and a second metal ion) can be observed by the induction
period, which decreases with the sulfite, M(II) and M(III)
4
formation, in the presence of S(IV)
2.43–3.08 vs. NHE) can also oxidize Cu G
4
III
•−
(
steps (eqns. (9)–(11)); Cu G
4
then can be reduced by SO
(eqn. (5)) continuing the chain reaction. This redox cycling is
active as long as sulfite and oxygen are present in the solution
species. The metal redox
cycling depends on the balance of both sulfite and oxygen
concentrations in solution, as clearly demonstrated on the sulfite
3
10
concentrations.
Some initial Cu G
process, which could be produced by the disproportion of Cu G
III
•−
−
•−
4
, at time zero, is necessary to initiate the
to generate SO
5
, HSO
5
or SO
4
II
4
5
(
eqn. (1), Scheme 1), as suggested by Anast and Margerum, or
II 48
18
by the direct oxidation of Cu G
4
by oxygen (eqn. (2)). The
induced autoxidation of Co(II) and Mn(II) in azide medium,
Ni (cyclam), and Ni(II) hydroxide medium. M G can be
II
II
9,52
22
II
H
2
O
2
produced can also oxidize Cu G
4
. However, this reaction
4
1
1 0 2
D a l t o n T r a n s . , 2 0 0 5 , 1 1 0 1 – 1 1 0 7

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III
•−
−
•−
also oxidized to M G
4
by SO
5
, HSO
5
and SO
4
in a
DNA. It is important to note that both Cu(II) (in large excess)
and DNA can be oxidized by the generated radicals.
Liu et al. studied the interaction of a variety of copper(II)
similar way to Cu(II) reaction. The chain propagation, the
product formation and termination reactions involving sulfite
56
18,19
are described in the literature.
complexes (with different structural features) with DNA to
promote double-strand DNA damage upon reductant/H
2
O
2
(
b) Spectrophotometric studies in the presence of DNA
activation. According to those authors, the interaction of the
copper complex with DNA results in hyperchromism and
shifts to longer wavelengths the strongest transitions of the
copper complexes, as well as striking hyperchromism of DNA
absorption at 260 nm.
III
In order to verify the interaction of DNA, Cu(II) and Cu G
complexes, spectrophotometric studies were carried out fol-
lowing the spectra changes after mixing the DNA solution
4
−
1
3
−1 54
II
(
k
max = 260 nm, e = 6600 mol dm cm ) with the Cu G
4
Fig. 2 also shows that absorbance band around 260 nm (the
−
1
3
−1
solution (kmax = 250 nm, e = 2300 mol dm cm and 520 nm,
III
sum of Cu G and DNA) is affected by interaction between
4
−
1
3
−1 55
III
e = 530 mol dm cm ) also containing Cu G
4
(kmax
=
DNA and Cu(II)/Cu(III) coordination compounds, resulting
−
1
3
−1
2
7
50 nm, e = 9000 mol dm cm and kmax = 365 nm, e =
200 mol dm cm ). The absorption spectra represented in
in hyperchromism (Fig. 2(B)) and shifts this band to longer
−
1
3
−1 55
III
wavelengths. From these data it can be concluded that Cu G
4
−
3
−3
II
the Fig. 2 were recorded using 1.0 × 10 mol dm Cu G
4
remains stable (during the monitored time, at 365 nm) in the
presence of DNA. When similar experiments were carried out
II
solution as reference (blank), such as the contribution of Cu G
4
complexes on the absorbance values (especially at 260 nm) does
not overlap with the strongest absorption of DNA at 260 nm.
with Cu(II), added as Cu(ClO
4
)
2
solution, the absorbance at
2
60 nm increased, with hyperchromism, and no absorbance
changes at 365 nm (since G is not present). So it is important to
4
emphasize that the use of tetraglycine, as a ligand, made possible
to follow spectrophotometrically the variation of the oxidation
II
state of Cu G
4
complex.
II
III
The Cu G
4
and Cu G complexes, with square-planar struc-
4
ture can bind to the double-helical DNA in different ways
depending on the pH of the medium, since the charge of the
complex may change. The degree of protonation of Cu(II) and
Cu(III) tetraglycine complexes depends on the medium acidity.
II
II
−
At pH = 7.0, [Cu (H−1
G
4
)] and [Cu (H−2
G
4
)] at the ratio
refers
0
.4 : 0.6, are the species present in solution (where H−x
G
4
to the G ligand with x deprotonated nitrogens coordinated to
4
III
−
the copper ion). [Cu (H−3
G )] is the predominant species in
49
4
solution from pH 4.0 to 12.0.
Some spectrophotometric studies were carried out to follow
the Cu(III) formation (k = 365 nm), in a short time scale, just after
II
the mixing of Cu G
4
solution with S(IV). Fig. 3(A) and (B) show
III
that Cu G
4
formation is faster and more efficient at pH = 9.0
Fig. 2 Absorption spectra changes as function of the addition order of
(borate buffer) than pH = 7.0. The same experiments, at pH =
7.0 (Fig. 3(C)) and pH = 9.0 (buffered and unbuffered medium,
Fig. 3(D) and (E)) were repeated in the presence of DNA. At
pH = 7.0 and 9.0 the Cu(III) formation is slower in the presence
of DNA (Fig. 3(C) and (D)). In the borate buffer (Fig. 3(E))
II
the reagents: (A) DNA solution; (B) after DNA addition to a Cu G
4
+
S(IV) solution (600 s after the addition); (C) after addition of sulfite to a
II
−6
Cu G
4
+ DNA solution (600 s after the addition): [DNA] = 3.4 × 10
g
−
1
II
−3
−3
−6
−3
mL ; [Cu G
4
] = 1.0 × 10 mol dm (containing 1.0 × 10 mol dm
−5 −3
◦
Ni(II); pH = 7.0); [S(IV)] = 5.0 × 10 mol dm ; T = 25.0 C. Reference
II
−3
−3
solution: [Cu G
4
] = 1.0 × 10 mol dm
.
II
After 3600 s of mixing Cu G
4
(containing traces of Ni(II)
−
5
and 5.0 × 10 M S(IV), the resulting solution Fig. 1(a)(D)
II
∼
−3
−3
III
∼
may contain Cu G
0
4
= 1.0 × 10 mol dm , Cu G
4
= 4.0 ×
−
5
−3
1
7
mol dm (calculated by the absorbance value (0.3) and e =
−
1
3
−1
200 mol dm cm ) and no S(IV). To this solution a small
volume (to avoid dilution effect) of CT-DNA was added. The
spectrum obtained is represented in Fig. 2(B) and gives some
III
information about the interaction of Cu G with DNA. By
4
comparing the spectra in Fig. 2(A) (solution of DNA) and
III
Fig. 2(B), it can be concluded that some Cu G (absorbance
4
at 365 nm) remains in solution after the mixture. However the
absorbance at 260 nm is much smaller than the sum of DNA (e =
−
1
3
−1
III
−1
3
−1
6
600 mol dm cm ) and Cu G
4
(e = 9000 mol dm cm ),
showing some interaction of Cu(II) or Cu(III) with DNA.
Several addition orders of the reagents were considered since
the redox cycle with radical generation (Scheme 1) is relatively
fast (10–60 s), therefore the order of reagents mixture was shown
to be an important factor in our studies. The spectrum in
Fig. 2(C) refers to the final solution obtained by mixing S(IV)
III
4
Fig. 3 Cu G formation, followed by absorbance change at 365 nm,
after addition of S(IV) to a (A) CuIIG4 solution, [borate buffer] =
−
5
−3
−3
II
(
5.0 × 10 mol dm ) with a solution containing DNA (3.4 ×
0.05 mol dm (pH = 9.0); (B) Cu G
4
solution (unbuffered medium,
−
6
−1
II
−3
−3
II
10
g mL ) and Cu G
4
(1.0 × 10 mol dm ) (600 s after the
pH = 7.0); (C) Cu G
4
/DNA solution (unbuffered medium, pH =
II
7
.0); (D) Cu G
4
/DNA solution (unbuffered medium, pH = 9.0); (E)
mixture). The absorbance at 365 nm shows that Cu(III) remains
in solution (compare Fig. 2(B) and (C)). The absorbance at
II
−3
Cu G
4
/DNA solution, [borate buffer] = 0.05 mol dm (pH = 9.0).
II
−3
−3
−6
−3
[
Cu G
4
] = 1.0 × 10 mol dm (containing 1.0 × 10 mol dm
2
60 nm (Fig. 2(C)) decreased instead of increasing, as expected
−5 −3 −6 −1
Ni(II)); [S(IV)] = 5.0 × 10 mol dm ; [DNA] = 3.4 × 10 g mL
;
III
◦
just considering the sum of DNA, and Cu G
4
contribution)
T = 25.0 C. Reference solution to (A), (B) and (C): CuIIG ; reference
4
showing that Cu(III) and the formed radicals interacted with
2
solution to D and E: H O.
D a l t o n T r a n s . , 2 0 0 5 , 1 1 0 1 – 1 1 0 7
1 1 0 3

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the reaction is slower than in unbuffered medium (Fig. 3(D)),
The intensities of the positive band of DNA increased and
shifted to higher wavelengths, with the negative band being more
showing that borate buffer may have some influence.
The absorbance changes at 260 nm (sum of DNA and Cu G
III
4
)
affected than the positive band, these changes strongly suggest
II
followed the same profile with longer induction period (about
0 s) in the presence of DNA (pH = 7.0) (compare Figs. 3(C)
and 4(D)). Fig. 4(B) and (C) refer to experiments with different
that some binding of Cu G
4
complexes induces some type of
1
structural change within the DNA molecule.
In contrast, introduction of Cu(II) solution (as Cu(ClO
induced only a small change in the CD (Fig. 5(B)).
4
2
) )
II
addition orders of the reagents. When Cu G is initially mixed
4
with DNA (Fig. 4(B)) the initial absorbance value is smaller
showing hypochromism, it does not occurs in borate buffer
(c) Gel electrophoresis studies
II
medium (Fig. 4(C)) which may reduce the interaction of Cu G
4
4
According to literature sulfite ion itself is responsible for some
of the damaging effect to proteins and nucleic acids including
deamination of cytosine and addition of bisulfite to thymine and
uracil.
with DNA. In fact the electrophoresis experiments in borate
buffer medium, showed no DNA damage as will be further
discussed. This shows that the structure of copper complexes
bound to DNA not only governs the DNA recognition but also
determines the DNA damage chemistry.
29
EPR studies by Shi and Mao showed that the autoxidation
•
−
of sulfite (in the absence of metal ions) generates both SO
3
and
•
2−
3
OH ; and the incubation for 4 h of DNA with SO
caused little
DNA damage. In the present work, by electrophoresis studies,
no DNA damage was observed in the presence of sulfite alone
−
3
−3
(
0.1–1.0) × 10 mol dm ) even after incubation for 8 h.
Agarose gel electrophoresis using pUC19 plasmid DNA in the
presence of Ni(II) or Ni G
carried out. The results did not show evidence of DNA damage
II
−4
−3
4
((0.01–1.0) × 10 mol dm ) were
−
3
−3
in the absence or presence of sulfite ((0.1–10) × 10 mol dm ).
II
However, exposure of pUC19 plasmid DNA to Cu G
4
(contain-
ing or not a second metal ion) in the presence of sulfite leads to
the formation of DNA strand breaks in significant yield.
A remarkable difference in the damage was observed by
changing the addition order of the reagents. The higher ratio of
open circular (OC): super coiled (SC) percentage was observed
II
with the following order: DNA + Cu G
4
(or Cu(ClO
4
)
2
) + S(IV).
−
6
Fig. 6 shows the experiments carried out with 5.0 × 10
g
−
1
−3
mL pUC19 plasmid DNA + 0.1 mmol dm Cu(ClO
4
)
2
or
−
3
II
−7
−3
0
.1 mmol dm Cu G
4
(also containing 1.0 × 10 mol dm
Ni(II)) (pH = 7.0) and S(IV).
Fig. 4 Absorbance changes at 260 nm after addition of (B) S(IV) to a
II
Due to the synergistic effect of Ni(II), Co(II) and Mn(II) on
the oxidation of Cu G in the presence of S(IV) and oxygen,
Cu G
4
/DNA solution (unbuffered medium, pH = 9.0); (C) S(IV)/DNA
II
4
6,10
II
−3
to a Cu G
S(IV)/DNA to a Cu G
4
solution, [borate buffer] = 0.05 mol dm (pH = 9.0); (D)
II
4
solution (unbuffered medium, pH = 7.0); (A)
some experiments were carried out to evaluate the DNA strand
breakage as reported in Fig. 7. The percentages are the average
absorbance changes at 260 nm of a solution containing only DNA.
II
−3
−3
−6
−3
[
Cu G
4
] = 1.0 × 10 mol dm (containing 1.0 × 10 mol dm Ni(II));
−5 −3 −5 −1
[
2
S(IV)] = 5.0 × 10 mol dm ; [DNA] = 3.4 × 10 g mL ; T =
◦
II
4
5.0 C. Reference solution: Cu G .
II
Additional evidences of DNA and Cu G
4
interaction were
II
obtained by circular dichroism measurements. As Cu G
4
has
solution was used as
into a solution of CT-DNA
II
an absorption band at 250 nm, the Cu G
4
II
reference. Introduction of Cu G
4
induced change in the UV circular dichroic spectrum (Fig. 5(C)).
Fig. 6 Gel electrophoresis of pUC19 plasmid DNA. The DNA is
present in two conformations SC (native) and OC (resulting from sin-
−
3
gle-strand breaks). Lane 1: DNA alone; lane 2: DNA + 0.1 mmol dm
−
3
II
Cu(ClO
4
)
2
; lane 3: DNA + 0.1 mmol dm Cu G ; lane 4: lane 3 +
−3 2−
3
4
−
3
2−
0
6
SO
0
1
SO
.0010 mmol dm SO
3
; lane 5: lane 3 + 0.0025 mmol dm SO
; lane
−
3
2−
−3
: lane 3 + 0.0050 mmol dm SO
3
; lane 7: lane 3 + 0.0075 mmol dm
−3 2−
3
2−
3
; lane 8: lane 3 + 0.0100 mmol dm SO
. lane 9: lane 3 +
−3 2−
3
−
3
2−
.025 mmol dm SO
3
; lane 10: lane 3 + 0.050 mmol dm SO
; lane
−
3
2−
−3
1: lane 3 + 0.075 mmol dm SO
3
; lane 12: lane 3 + 0.100 mmol dm
−3 2−
3
2−
Fig. 5 UV CD spectra: (A) CT-DNA solution: (B) DNA + Cu(II)
3
; lane 13: lane 3 + 0.250 mmol dm SO
; lane 14: lane 3 +
II
−3
2−
−3
2−
−1
solution; (C) DNA + Cu G
4
−3
solution, unbuffered medium, pH = 7.0;
0.500 mmol dm SO
3
; lane 15: lane 3 + 0.750 mmol dm SO
3
;
;
II
−3
−6
−3
−3
2−
−6
[
[
Cu G
4
] = 1.0 × 10 mol dm (containing 1.0 × 10 mol dm Ni(II));
lane 16: lane 3 + 1.0 mmol dm SO
3
. [pUC19] = 5.0 × 10 g mL
−
6
−1
◦
◦
II
−3
CT-DNA] = 3.4 × 10 g mL ; T = 25.0 C. Reference solution to (A)
pH = 7.0; T = 25.0 C; [Cu G
4
] = 0.1 mmol dm (containing 1.0 ×
II
−7
−3
and (C): Cu G
4
; reference solution to (B): H
2
O.
10 mol dm Ni(II)).
1
1 0 4
D a l t o n T r a n s . , 2 0 0 5 , 1 1 0 1 – 1 1 0 7

View Article Online
between the different species depends only on the pH and the
total borate concentration.
For any particular sugar molecule, the complex stability
depends strongly on the configuration, the number of hydroxyl
59
groups and the presence of substituents. The conversion of
ribonucleosides to anions by borate complexation provided the
60
separation on anion-exchange resins. When hydroxyl groups
are in cis position, the formation of a borate complex is more
59–61
favored than in trans.
The sugar phosphates were also
investigated, showing that the presence of phosphate group, on
C(1) in glucose, eliminates the possibility of a strongly ionized
60
borate complex being formed.
In the case of DNA molecule the ribonucleosides with sub-
stituents (phosphate and the base) have no free sugar hydroxyl
groups. On the other hand, borate may affect the phosphodiester
bridges between the phosphate and C(3) and C(5) from pentose.
II
−3
In excess of Cu G (1.0 mmol dm ) in unbuffered medium,
4
−3
−
5
−6
−1
5
.0 × 10 mol dm S(IV) and DNA (5.0 × 10 g mL ), no
II
DNA strand breaks were observed, since the Cu G
4
complex in
Fig. 7 Percentage of single-strand break formation upon exposure
excess is preferably oxidized to Cu(III) by the oxysulfur radical.
According to Liu et al., the substantial amount of free metal
complex in the solution phase cannot damage the DNA because
DNA cleavage chemistry is diffusion-controlled by the hydroxyl
radical mediated by metal complex. The freely diffusing Cu(II)
complex binds reversibly to DNA.
II
62
of pUC19 plasmid DNA + Cu G
4
(also containing Ni(II), Co(II) or
−
6
Mn(II)) and different concentrations of S(IV): [pUC19] = 5.0 × 10
g
−
1
◦
−4
−3
−3
II
mL , pH = 7.0, T = 25.0 C; (A) 1.0 × 10 mol dm Cu G
4
/1.0 ×
/1.0 ×
/1.0 ×
−
−
−
7
7
6
−3
II
−4
II
1
1
1
0
0
0
mol dm Ni G
4
; (B) 1.0 × 10 mol dm Cu G
4
−3
II
−4
−3
II
mol dm Co G
4
and (C) 1.0 × 10 mol dm Cu G
4
−3
II
mol dm Mn G
4
.
An important aspect to consider is the reduction potential of
5
5
III
the copper species, 1.1 and 0.66 V (vs. NHE) for [Cu (H−2
G
4
)]
III
−
of three trials. By comparing Fig. 7(A) and (B), where the second
metal ion (Ni(II) and Co(II)) is at the same concentration, it is
possible to conclude that when Ni(II) is present the percentage
and [Cu (H−3
G
4
)] respectively. As can be seen in Fig. 3(A)
and (B) in the absence of DNA, the oxidation of Cu(II) in
the presence of oxygen and S(IV) is more effective as the pH
6
,8,10
12
III
of OC DNA form increases. As reported before,
the S(IV)
increases. Considering the reduction potentials of Cu species
and the oxysulfur radicals, formed in the presence of DNA,
II
induced oxidation of Cu G
4
is more effective in the presence
◦
63
of traces of Ni(II) than Co(II) and Mn(II). The percentage of
DNA strand breaks increases with S(IV) concentration up to
they can oxidize guanine (E = 1.29 V vs. NHE), which has
the lowest redox potential of the four nucleosides. On the other
hand, the guanine reduction potential can be lower in DNA
−
3
[
S(IV)] = 0.5 mmol dm . In a higher S(IV) concentration range,
63
the percentage of OC decreases, showing a less effective damage.
This can be explained by the lack of oxygen dissolved when S(IV)
is added in large excess over oxygen (2.0 × 10 mol dm , air-
saturated solution), the formed Cu(III) can be reduced by S(IV)
still remaining in the solution (eqn. (4)), and the redox cycling
molecule.
Sulfite radical, SO
•
−
◦
•
64
(E SO3 −/SO32− = 0.76 vs. NHE), is the
3
−
4
−3
initial product (eqns. (4) and (5)); in the presence of O it reacts
with oxygen (eqn. (6)) to form SO
2
•
−
◦
•
(E SO5 −/HSO5
−
64
= 1.10 vs.
5
64
•−
◦
•
NHE). SO
4
(E SO4 −/SO42− = 2.43–3.08 vs. NHE) (eqn. (9))
9,18
−
◦
64
(
Scheme 1) is no longer observed.
and HSO
5
(E HSO5−/SO42− = 1.75 vs. NHE), the most potent
III
Cu G
4
alone induced little or no DNA damage in the absence
oxidants, can also oxidize the four nucleosides in DNA. The
reaction of SO
of Cu(II) and M(II) complexes with HSO
and OH (eqns. (9) and (10)), which can also oxidize the four
nucleosides.
•
−
of S(IV). Another control experiment with plasmid DNA added
5
with nucleosides is unknown, but the reaction
−
4
−3
II
−
•−
to a 1.0 × 10 mol dm Cu G
4
solution containing 6.0 ×
5
may produce SO
4
−
5
−3
•
5,8
10
mol dm of Cu(III) (formed by spontaneous oxidation of
with oxygen dissolved, after 5 h of preparation), yielded
II
Cu G
4
only 28% OC DNA (data not shown), showing that Cu(III)
induced little DNA damage in the absence of sulfite.
The results demonstrate that the loss of native SC DNA are
not controlled by the tetraglycine coordinated to Cu(II). The
Our experiments bygelelectrophoresis studies showed thatthe
exposure of pUC19 plasmid DNA to the strong oxidizing agent
−
−3
−3
HSO
5
(1.0 × 10 mol dm ) lead to the significant formation
of DNA strand breaks (data not shown).
The literature
24,25,65,66
−
DNA damage occurs in the presence of Cu(II) and S(IV), when
reports that the use of HSO , in the
5
II
Cu(II) is added as Cu(ClO
4
)
2
or as Cu G
4
complex (even when
presence of metal ion complexes, enhance the DNA oxidation.
Systematic studies of different macrocyclic ligands coordinated
to Ni(II), showed the importance of ring size, degree of unsatu-
G was kept in large excess as 100% over Cu(II)). However, as
4
III
showed by the spectrophotometric data Cu G
4
complex exists
in solution (Fig. 3). This result may be attributed to the proximity
ration, steric bulk, redox potential, in-plane ligand field strength
II
•−
of Cu(II) or Cu G
4
to DNA deoxyl ribose rings.
for binding to DNA, affecting its reactivity. For instance SO
4
,
The DNA damage at pH = 7.0 and 9.0 (unbuffered medium)
possibly generated, may react rapidly in solution with the ligand
or a guanine base; as such, the radical may be quenched by some
macrocyclic ligands (such as cyclam), leading to a diminished
are about the same with 35 ± 4 OC (data not shown) DNA
−
3
II
−3
−3
25
(
0.1 mmol dm Cu G
4
, 0.1 × 10 mmol dm Ni(II) and
−
3
1
9
.0 mmol dm S(IV)). However in borate buffer medium, pH =
lifetime in solution.
.0, no damage was observed, although the Cu(III) formation
When S(IV), a reducing agent, and the metal ion complex are
exposed to DNA, the oxygen concentration is very important
(eqn. (6)), since the generation of oxysulfur radicals and
metal ion (in the 3+ oxidation state) occurs via redox cycling
(Scheme 1).
occurs (see Figs. 3 and 4(C)). This difference implies a different
II
binding of Cu G to DNA (also shown by spectrophotometric
4
data, compare Fig. 4(B) and 4(C)).
In the literature the formation of a carbohydrate–borate
57
28
complex has been used for separation of sugars by capillary
In fact, according to Muller and co-workers the DNA mod-
5
8,59
60
+
electrophoresis
droxyborate ([B(OH)
range 8.0–12.0 aqueous borate solutions also contain condensed
or by ion exchange resin. The tetrahy-
4
ification by [Ni(KGH–NH
2
)] (where KGH = lysylglycylhisti-
−
59
] ) is complexed by polyols. In a pH
dine) catalyzed sulfite autoxidation was highly O
2
−
dependent,
were much
while DNA oxidation involving preformed HSO
less so.
5
−
2−
polyanions ([B
3
O
3
(OH)
5
]
2
and [B
4
O
5
(OH) ] ); the equilibrium
4
D a l t o n T r a n s . , 2 0 0 5 , 1 1 0 1 – 1 1 0 7
1 1 0 5

View Article Online
The use of a copper(II) famotidine complex (an anti-ulcer
Circular dichroism (CD) spectroscopy
•
−
•−
drug) as a catalyst showed that SO
important role in the DNA damage, while HSO
free or metal-bounded) are not relevant.
In the present work, it is difficult to exactly identify the
3
and SO
4
may have an
II
•−
CD spectra of CT-DNA in the absence and presence of Cu G
complex were recorded on a JASCO J-720 spectropolarimeter
operating at room temperature (ca. 25 C), using 0.5 cm path
4
−
5
and SO
4
31
(
◦
quartz cells. The results are displayed as the change in molar
oxidative intermediates, generated in the S(IV) autoxidation
catalyzed by the metal ion complex, which are responsible for
−
1
−3
−1
absorptivity (De/mol dm cm ).
6–12
the DNA damage. Our previous work about the mechanism
II
Gel electrophoresis experiments
of S(IV) autoxidation, showed that Cu G itself is a very poor
4
catalyst (at pH = 9.0), and the oxidation is extremely rapid in
Treatment of pUC19 plasmid DNA with sulfite in the presence
of Cu(II) tetraglycine or Cu(II) perchlorate solution. Stock
solutions of Cu(II) (free of nickel) were prepared by direct
reaction of Cu wire (99.99% Sigma) with double distilled nitric
acid. In some experiments small traces of Ni(II), Co(II) or Mn(II)
were added.
the presence of traces of Ni(II) or Co(II) ions. The stoichiometry
III
of [Cu G ] : [S(IV)] depends on the Ni(II) (or Co(II)) and the
4
initial S(IV) concentrations.
Experimental
The highest percentages of DNA strand breaks were observed
Reagents
when the freshly prepared solution of the reagents were mixed
II
in the following order: DNA + Cu(ClO
or 9.0, adjusted with NaOH or HClO
In most of the experiments 100 ng of pUC19 DNA were
4
)
2
(or Cu G
4
, pH = 7.0
All reagents were of analytical grade (Merck or Sigma). All
solutions were prepared by using deionised water purified with
a Milli-Q Plus Water system (Millipore).
4
solutions) + S(IV).
II
exposed to Cu G
Cu(ClO
4
(also containing Ni(II), Co(II) or Mn(II)) or
−
2
−3
Stock solutions of sulfite (1.00 × 10 mol dm ) were daily
4
)
2
solutions at pH = 7.0, followed by the addition of
prepared by dissolving Na in water previously purged with
2
S
2
O
5
sulfite solution. The concentrations of the final solutions are
indicated in the legends of the figures. The temperature was kept
nitrogen. Water was flushed with nitrogen for at least 30 min to
remove dissolved oxygen. To prepare diluted solutions of sulfite,
small volumes of the stock solutions were accurately added to
air-saturated water. The sulfite content of the stock solution was
◦
at 25.0 C.
Quantification of DNA strand breaks induced by sulfite in
the presence of Cu(II) tetraglycine. Separations of the different
conformations of pUC19 plasmid DNA, SC (supercoiled DNA,
native conformation) and OC form (open circular DNA,
resulting from single-strand breaks), were performed by gel
67
determined by iodometric titration.
Ethidium bromide, Ficoll type 400, Bromophenol Blue and
other reagents used for gel electrophoresis were obtained from
Plusone (Pharmacia Biotech, Uppsala, Sweden).
CT-DNA (Calf Thymus) solution was prepared by dissolving
the disodium salt (Sigma) in deionised water.
−3
electrophoresis, using 0.8% agarose/1.8 lmol dm ethidium
bromide in a horizontal gel electrophoresis chamber at 30 mA
−
3
−3
for 2 h in 90 mmol dm . Tris-borate/2 mmol dm EDTA buffer
−
3
−1
II
pUC19 plasmid DNA preparation. Cells of the Escherichia
coli strain HB101 were transformed with pUC19 plasmid DNA
using the calcium chloride procedure. Bacteria were plated on
agar plates and then incubated overnight at 37 C. Transformed
E. coli cells were grown in LB medium. Plasmid DNA was
at pH 8.0 Plasmid DNA (5.0 × 10 g mL containing Cu G
4
and S(IV)) was mixed with 5.0 lL of gel-loading buffer (15%
Ficoll type 400/0.25% Bromophenol Blue) and applied to the
gel. The bands were visualized under UV light and quantified
with the ImageMaster VDS densitometer (Pharmacia Biotech,
San Francisco, CA, USA).
68
◦
6
9
purified using the diatomaceous earth method.
Spectrophotometric measurements
Acknowledgements
−
3
The metal ion stock solution of 0.2 mol dm Cu(II) was prepared
We gratefully acknowledge the financial support from Funda c¸ a˜ o
de Amparo a` Pesquisa do Estado de S a˜ o Paulo (FAPESP),
Conselho Nacional de Pesquisa e Desenvolvimento Tecnol o´ gico
(CNPq) (Brazilian Agencies).
by dissolving Cu(ClO
4
)
2
(Sigma) in water. This solution also
−
4
−3
contains 2.0 × 10 mol dm Ni(II) as impurity.
In the experiments freshly prepared solutions were em-
ployed. Cu(II) tetraglycine solution was prepared by dissolving
−
3
tetraglycine in water or borate buffer 0.05 mol dm (pH = 9.0)
followed by the addition of Cu(ClO solution (also containing
Ni(II)). The final pH of unbuffered solutions was adjusted with
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