Effects of H2O2, Fe2+ and Fe3+ on curcumin-induced chromosomal aberrations in CHO cells

Genetics and Molecular Biology, 28, 1, 161-164 (2005)
Copyright by the Brazilian Society of Genetics. Printed in Brazil
www.sbg.org.br
Short Communication
Effects of H O , Fe2+ and Fe3+ on curcumin-induced chromosomal
2
2
aberrations in CHO cells
Lusânia Maria Greggi Antunes1, Maria Cristina Paiva Araújo2†, Francisca da Luz Dias1
and Catarina Satie Takahashi2,3
1Faculdade de Medicina do Triângulo Mineiro, Departamento de Ciências Biológicas, Uberaba,
MG, Brazil.
2Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética,
Ribeirão Preto, SP, Brazil.
3Universidade de São Paulo, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,
Departamento de Biologia, Ribeirão Preto, SP, Brazil.
Abstract
The effects of H O , Fe2+ and Fe3+ on curcumin-induced clastogenicity were evaluated in CHO cells. Curcumin
2
2
combined with H O did not increase the chromosomal aberrations more than expected based on a simple additive
2
2
effect. In contrast, the combination of curcumin-Fe significantly decreased the total number of chromosomal
aberrations and the number of abnormal metaphases. The clastogenicity of curcumin may be related to its
pro-oxidant properties and its ability to generate free radicals.
Key words: CHO, chromosomal aberrations, curcumin, hydrogen peroxide, mutagenesis.
Received: April 26, 2004; Accepted: August 18, 2004.
Experiments in vitro and epidemiological studies
mutagenic in cultured CHO cells and, when combined with
have shown that some compounds present in the diet have
the antitumor drugs bleomycin or doxorubicin, the fre-
antimutagenic and anticarcinogenic properties (Surh and
quency of chromosomal aberrations is markedly increased
Ferguson, 2003). Turmeric, a spice obtained from the rhi-
(Araújo et al., 1999; Antunes et al., 1999).
zome of Curcuma longa Linn (Zingiberaceae), and its ac-
The clastogenicity of curcumin in mammalian cells in
tive principle curcumin, have been studied for their ability
vitro has been attributed to a pro-oxidant action of this com-
to protect cells from DNA damage (Polasa et al., 2004).
pound (Araújo et al., 1999). In agreement with this pro-
Curcumin, widely used as a coloring for foods, has antioxi-
posed mechanism, thiourea, a hydroxyl radical scavenger,
dant, antimutagenic and anticarcinogenic properties
significantly inhibited the increase in chromosomal aberra-
(Nagabhushan and Bhide, 1992; Aggarwal et al., 2003).
tions in CHO cells treated with curcumin (Araújo et al.,
Curcumin reduces chromosomal aberrations in ro-
2001). Thus, hydroxyl radicals generated by curcumin
dent bone marrow cells exposed to gamma-radiation or
could contribute to clastogenicity in vitro.
treated with the antitumor drug cisplatin (Abraham et al.,
1993; Antunes et al., 2000). However, clastogenic effects
A combination of Fe accessibility and high produc-
-
of curcumin have also been reported. An increase in the fre-
tion of H2O2 and O2 results in a pro-oxidant state within
quency of chromosomal damage has been seen in mice and
cells (Meneghini, 1997). The molecular structure of
rats treated acutely and chronically with curcumin (Giri et
curcumin is susceptible to auto-oxidation in the presence of
al., 1990; Mukhopadhyay et al., 1998), and curcumin is
oxygen and transition metal ions (Sahu and Washington,
mutagenic in cultured Chinese hamster fibroblasts (Ishidate
1992). Indeed, curcumin is a good scavenger of H2O2 at
et al., 1984). At concentrations up to 10
high concentrations, but at low concentrations it activates
µg/mL, curcumin is
the Fenton reaction to increase the production of H2O2
(Kuchandy and Rao, 1990).
Send correspondence to Lusânia Maria Greggi Antunes. Facul-
Although, there is evidence indicating that metal ions
dade de Medicina do Triângulo Mineiro, Departamento de Ciências
are involved in curcumin-induced mutagenicity, there is
Biológicas, Praça Manoel Terra 330, 38015-050 Uberaba, MG,
Brazil. E-mail: lusaantunes@yahoo.com.br.
still little on this subject in the literature. There have been
†Deceased June 21, 2001.
no studies of the effects of H2O2 on curcumin-induced chro-

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162
Antunes et al.
mosomal aberrations in CHO cells. To examine the hypoth-
15 µg/mL. However, as expected, there was a significant
esis that the generation of free radicals is involved in
(p < 0.05) increase in the total number of chromosomal ab-
curcumin-induced clastogenicity, CHO cells were treated
errations and in the number of abnormal metaphases after
with a pre-determined clastogenic concentration of
treatment with curcumin.
curcumin alone or in combination with H2O2, Fe2+ or Fe3+.
Curcumin readily penetrates into the cytoplasm and
Curcumin (diferuloylmethane; CAS No. 458-37-7)
can accumulate in membrane structures (Joe et al., 2004),
was purchased from Sigma Chemicals Co. (St. Louis, MO).
primarily because of the molecule’s structure, which con-
DMSO and FeCl3 were purchased from Merck (Darmstadt, sists of two isomers, i.e. the ?-diketone and enol forms. The
F.R.G.). FeSO4 and H2O2 were obtained from Reagen diketone form is a potent ligand for metals such as iron (Sun
(Brazil). Dulbecco’s modified Eagle’s medium (DMEM)
et al., 2002). In the presence of copper or chromium,
and HAM-F10 were purchased from Gibco (Invitrogen
curcumin becomes a pro-oxidant and damages DNA
Corporation, USA). Fetal calf serum (FCS) was obtained
(Ahsan and Hadi, 1998). The antioxidant/pro-oxidant ac-
from Cultilab (Campinas, SP, Brazil). Chinese hamster
tion of phenolic compounds depends on factors such as
ovary cells (CHO-9) were grown as monolayers at 37 °C in
metal reducing potential and chelating behavior (Decker,
25-cm2 flasks containing HAM-F10 plus DMEM (1:1 ra-
1997).
tio), supplemented with 10% FCS, penicillin (0.06 mg/mL)
and streptomycin (0.1 mg/mL). For all experiments, expo-
Cultured CHO cells treated with different concentra-
nentially growing cells were seeded at a density of 1 x
tions of H2O2 alone (Table 1) showed a significant
106/5 mL flask. Curcumin was dissolved in 0.5% DMSO.
(p < 0.05) increase in chromosomal aberrations at all con-
CHO cells were incubated for 14 h with H
centrations tested. There was a concentration-dependent re-
2O2 (1.7, 3.4 or
6.8
sponse in the total number of chromosomal aberrations and
µg/mL), FeCl3 (1.25, 2.5 or 5.0 µg/mL) or FeSO4 (1.25,
in the number of abnormal metaphases in cultures treated
2.5 or 5.0 µg/mL) in the absence or presence of curcumin.
with H2O2. The most frequently detected aberrations were
Colcemid (0.1 µg/mL) was added to the culture me-
chromatid breaks followed by chromosomal breaks. At the
dium 2 h before fixation of the cells. Each experiment was
highest H2O2 concentration, a significant (p < 0.05) de-
repeated three times and 300 metaphases (100 in each ex-
crease in the mitotic index was also observed when com-
periment) were analyzed per treatment to assess the fre-
pared to control cultures. In combined treatments of
quencies of chromosomal aberrations. The mitotic index
curcumin and H2O2, all of the concentrations of H2O2tested
(MI) was defined as the percentage of metaphases in 3000
increased the total number of chromosomal aberrations in-
cells analyzed per treatment. The differences in the number
duced by curcumin (Table 1). However, this combination
of abnormal metaphases, total number of chromosomal ab-
did not increase the frequency of chromosomal aberrations
errations, and mitotic indices in the absence and presence of
beyond what was expected from a simple additive effect
curcumin were compared by analysis of variance
when compared to H2O2 and curcumin alone (H2O2: 144 at
(ANOVA).
the highest concentration; curcumin: 68, and curcumin +
The effects of H
H
2O2, Fe2+ and Fe3+ on curcumin-
2O2: 225).
induced chromosomal aberrations in CHO cells are shown
The influence of iron on the effects of curcumin has
in Tables 1 and 2. Curcumin was not cytotoxic at up to
been investigated because of this metal’s important role in
Table 1 - Effects of different concentrations of H2O2 on chromosomal aberrations induced by curcumin (CMN) in CHO cells.
Concentrations (µg/mL)
MI (%)
Chromosomal aberrations
Total
Abnormal cells
(Mean ± SD)
H2O2
CMN
Gaps
B’
B”
0
0
11.0
1
5
0
5
1.6 ± 1.0
1.7
0
10.6
2
41
8
49a
13.3a ± 2.8
3.4
0
10.3
5
101
16
117a
21.3a ± 3.2
6.8
0
7.0a
3
122
22
144a
24.6a ± 2.5
0
15
10.4
11
56
12
68a
17.0a ± 4.0
1.7
15
8.1
5
117
11
128a
29.6a ± 3.2
3.4
15
9.6
4
153
17
170a
35.6a ± 3.0
6.8
15
8.6
8
198
27
225a
46.0a ± 4.9
Three hundred cells per treatment were analyzed for chromosomal aberrations, and 3000 cells were scored for MI. Gaps were not included in the total
number of abnormal metaphases or in the total number of chromosomal aberrations.
B’ - chromatid break; B” - chromosomal break; SD - standard deviation.
ap < 0.05 for treated vs. control cultures.

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Curcumin-induced chromosomal damage
163
Table 2 - Effects of different concentrations of Fe2+ and Fe3+ on chromosomal aberrations induced by curcumin (CMN) in CHO cells.
Concentrations (µg/mL)
MI (%)
Chromosomal aberrations
Total
Abnormal cells
(Mean ± SD)
Fe2+
CMN
Gaps
B’
B”
0
0
11.7
1
2
1
3
1.0 ± 0.5
1.25
0
12.6
1
2
5
7
2.3 ± 0.5
2.5
0
11.7
3
4
2
6
2.0 ± 1.0
5.0
0
10.8
1
3
2
5
1.6 ± 0.6
0
15
9.2
11
51
12
63a
16.6a ± 4.0
1.25
15
8.5
16
47
8
55a
13.6a ± 4.6
2.5
15
8.2
3
21
8
29b
9.0b ± 4.0
5.0
15
9.7
2
8
0
8b
2.6b ± 2.8
Fe3+
CMN
0
0
11.6
3
3
2
5
1.6 ± 1.1
1.25
0
8.9
3
5
2
7
2.3 ± 1.0
2.5
0
10.2
2
3
0
3
1.0 ± 0.5
5.0
0
9.9
1
4
3
7
2.3 ± 1.1
0
15
7.8
12
71
7
78a
18.3a ± 6.0
1.25
15
7.1
4
35
4
39b
10.0b ± 3.5
2.5
15
10.5
0
11
4
15b
5.3b ± 2.5
5.0
15
7.8
0
9
4
13b
4.3b ± 1.5
Three hundred cells per treatment were analyzed for chromosomal aberrations, and 3000 cells were scored for MI. Gaps were not included in the total
number of abnormal metaphases or in the total number of chromosomal aberrations.
B’ - chromatid break; B” - chromosomal break; SD - standard deviation.
ap < 0.05 for treated vs. control cultures.
bp < 0.05 for combined treatment vs. curcumin alone.
biological processes such as oxygen transfer and DNA syn-
to generate DNA damaging species, in contrast to the data
thesis (Bernabé-Pineda et al., 2004). Table 2 shows that
obtained with a curcumin-Cu2- combination in cell-free
neither Fe2+ nor Fe3+ alone was clastogenic in any of the
systems (Ahsan and Hadi, 1998). These authors suggested
concentrations tested. The mitotic indices were also unaf-
that the inhibitory effect of Cu2- ions could be partly attrib-
fected when compared to control cultures. The combined
uted to the presence of protein in the medium since the
treatments with curcumin and Fe caused a significant
binding of copper to proteins would influence the activity
(p < 0.05), concentration-dependent decrease in the total
of curcumin (Verma and Goldin, 2003). In the experiments
number of chromosomal aberrations and in the number of
described here, the presence of serum proteins in the me-
abnormal metaphases. At 2.5 or 5.0 µg of Fe2+/mL, there
dium could have influenced the activity of the curcumin-Fe
was a significant reduction in the total number of chromo-
complex. Although the exact mechanism by which
somal aberrations induced by curcumin from 63 to 29
curcumin induces chromosomal aberrations remains to be
(54%) and 8 (87%), respectively. A similar response was
elucidated, the clastogenic activity of this compound is ap-
seen in CHO cells treated with Fe3+ (Table 2).
parently related to its pro-oxidant properties and to its abil-
The simultaneous addition of Fe2+ or Fe3+ signifi-
ity to generate free radicals.
cantly protected CHO cells against curcumin-induced chro-
mosomal damage in a concentration-dependent manner
Acknowledgments
compared to treatment with curcumin alone. This sug-
gested the possibility that curcumin bound strongly to Fe.
The authors thank Dr. Sérgio N. Kronka (UNESP -
The reaction between the complexes curcumin-Fe2+ and
Campus Jaboticabal) for helping with the statistical analy-
curcumin-Fe3+ studied in aqueous media using UV
sis, and Sueli A. Neves and Luis Augusto da Costa Jr. for
spectrophotometry and cyclic voltammetry also showed a
technical assistance. This work was supported by CAPES
similar behavior, and indicated that a chemical reaction had
and CNPq.
occurred between the curcumin and Fe before the forma-
tion of the complexes (Bernabé-Pineda et al., 2004).
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Associate Editor: Carlos F.M. Menck

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