J Appl Genet 48(4), 2007, pp. 389–396
Protective in vivo effect of curcumin on copper genotoxicity
evaluated by comet and micronucleus assays
Alfredo Corona-Rivera1,4, Patricia Urbina-Cano1, Lucina Bobadilla-Morales1,
José de Jesús Vargas-Lares1, Mario Alberto Ramírez-Herrera2, María Luisa Mendoza-MagaZa2,
Rogelio Troyo-Sanromán1, Pedro Díaz-Esquivel3, Jorge Román Corona-Rivera1,4
1Laboratorio de Citogenética Genotoxicidad y Biomonitoreo, Instituto de Genética Humana “Dr. Enrique Corona Rivera”,
Departamento de Biología Molecular y Genómica, Universidad de Guadalajara, Guadalajara, Jalisco, México
2Laboratorio de Neurofisiología, Departamento de Fisiología, Universidad de Guadalajara, Jalisco, México
3Bioterio, Coordinación de Investigación, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara,
4Laboratorio de Citogenética, OPD, Hospitales Civiles de Guadalajara, Guadalajara, Jalisco, México
Abstract. Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Ap-
parently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage.
The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and
micronucleus assays in the presence of curcumin. We tested 0.2% curcumin in Balb-C mice at normal (13 ppm)
and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed
48 hr after chemical application. Comet tail length in animals treated with 0.2% curcumin was not significantly
different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA
damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant
increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was
lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to
390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically signifi-
cant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm cop-
per versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic
erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attrib-
uted to curcumin. In vivo exposure to 0.2% curcumin for 48 hr did not cause genomic damage, while 390 ppm
copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin
seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations.
The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of
Keywords: comet assay, copper, curcumin, genotoxicity, genoprotection, micronuclei, mouse.
anticarcinogenic effects (Kawamori et al. 1999;
Murray and Pizzorno 1999; Conney 2003).
Curcumin is a phytochemical extracted from the
In vivo curcumin exposure studies suggest no
rhizome of Curcuma longa L. (Eigner and Scholz
genotoxicity or clastogenicity due to its antioxidant
1999; Araujo and León 2001), and has been re-
activity, evaluated on the basis of chromosome ab-
ported to have antiinflammatory, antioxidant and
errations (Vijayalaxmi 1980; Mukhopadhyay and
Received: April 6, 2007. Accepted: May 15, 2007.
Correspondence: A. Corona-Rivera, Universidad de Guadalajara, Centro Universitario de Ciencias de la Salud, Departamento
de Biología Molecular y Genómica, Instituto de Genética Humana “Dr. Enrique Corona Rivera”, Laboratorio de Citogenética
Genotoxicidad y Biomonitoreo, Edificio P, 2do Nivel, Sierra Mojada # 950, Colonia Independencia, S.L., CP-44340,
Guadalajara, Jalisco, México; e- mail: firstname.lastname@example.org
A. Corona-Rivera et al.
Mukherjee 1998; Shukla et al. 2003), micronuclei
Cell sample preparation
(Vijayalaxmi 1980), or somatic mutations and re-
combination assays (el Hamss et al. 1999).
Blood was obtained from a cut at the tail tip and
Despite reported benefits, in vitro studies in
cultured cells have demonstrated that curcumin in-
and quantitative comet assay (300 m L) and
duces chromosomal or DNA damage (Antunes
a micronucleus assay (one drop per slide), accord-
et al. 1999; Araujo et al. 1999), detected by the
ing to the following protocols.
comet assay (Blasiak et al. 1999a,b; Kelly et al.
2001; Urbina-Cano et al. 2006). To explain this in
vitro effect, it was proposed that curcumin can
It was performed under alkaline conditions, essen-
also act as pro-oxidant. It was observed that
tially as described by Singh et al. (1988) with some
curcumin interacts with transition metals, such as
modifications (Urbina-Cano et al. 2006), briefly as
excess of exogenous copper, which causes DNA
follows. Lymphocytes (2.5 × 104) were pelleted by
breaks in plasmid pB322 (Ahsan and Hadi 1998;
centrifugation at 1200 rpm for 10 min. Cell pellets
Ahsan et al. 1999) or strand breakage detected by
were washed and suspended in 85 m L of PBS or
the comet assay (Urbina-Cano et al. 2006). This
RPMI medium. The cell suspension was mixed
corresponds to other biological antioxidants, such
(1:1) with 85 m L of 1% low-melting-point agarose
as flavonoids, ascorbic acid, tannic acid or uric
acid, which also generate reactive oxygen species
(Type VII), and next 75 m L of the cell-agarose mix
(ROS) in the presence of transition metals, such as
was dropped on 1% normal-melting-point agarose
iron and copper ions, causing DNA strand breaks
(Type I) precoated slides. Cover slips were placed
(Gaetke and Chow 2003). The aim of this study is
on the second layer until it solidified at 4°C. Then,
to evaluate whether exposure to increasing con-
cells were lysed for 1 hr in a chilled buffer lysis so-
centrations of copper induces DNA damage (mea-
lution (2.5M NaCl, 0.1 M EDTA, 10mM Tris,
sured by the comet and micronucleus assays) in
0.3 M NaOH; pH adjusted to 10, 1% Tritón X-100,
the presence of curcumin in a murine model.
and 10% DMSO, were added just before use). Af-
terwards, slides were placed in a horizontal gel
electrophoresis chamber filled with alkaline solu-
Materials and methods
tion of 0.3 M NaOH/1mM EDTA (pH > 13), al-
lowing DNA unwinding for 20 minutes. Then,
20 V/300 mA electrophoresis was conducted for
20 min at room temperature with a chilled buffer.
The slides were drained and neutralized by using
4–6-week-old male Balb-C mice weighing ap-
0.4 M Tris, pH 7.5, for 5 min. Finally, the obtained
proximately 20 g. We established 8 groups of 6 in-
slides with 2 layers of agarose microgels were de-
hydrated with 80% methanol at room temperature
distributed, and exposed for 48 hr to the following
and stored until analysis (Belpaeme et al. 1998;
experimental treatments: no treatment (control),
Urbina-Cano et al. 2006). Before microscopic ex-
65 ppm copper, 130 ppm copper, 390 ppm copper,
amination, slides were rehydrated and propidium
0.2% curcumin, 0.2% curcumin + 65 ppm copper
iodide stained (Sigma, CAS No. 25535–16–4,
(5X), 0.2% curcumin + 130 ppm copper (10X),
10 m g mL–1). Observations were made under a flu-
and 0.2% curcumin + 390 ppm copper (30X).
orescence microscope (Zeiss Axiolab 4FL-HBO
Mice were fed ad libitum with Rodent Chow
50/AC). Fifty nucleoids (nuclei after lysis) or
(Purina), which contains a regular concentration
comets were measured (mm) in each sample. Mea-
of copper (13 ppm, 1X). Additional copper (CuCl2
surements were performed by one observer in a
salt, Sigma, CAS No. 10125–13–0), was adminis-
blind manner, with the aid of a micrometer
tered in deionized water as a daily source adjusted
adapted to the microscope eye-piece (Avishai et
to the mentioned concentrations. Curcumin
al. 2003). Nucleoids that were overlapping or lo-
(Sigma, CAS No. 458–37–7) was added to Rodent
cated at the edge of the microgels were excluded.
Chow pellets from an alcoholic solution to fit a fi-
A photographic record was created for nucleoids
nal concentration of 0.2%, allowing alcohol evap-
or comets observed with a digital camera (Zeiss
oration before consumption. The dose of curcumin
Axiocam MRc) attached to the microscope.
was selected on the basis of literature data, as such
The extent of DNA damage was measured by
a dose is well known to exert a suitable biological
means of 2 complementary methods, a tail length
response (Rao et al. 1999).
quantitative method (Collins 2004) and a qualita-
In vivo curcumin and copper by comet and micronuclei
tive method of damage distribution (Avishai et al.
sion objective (100×) under a fluorescence
2003; Urbina-Cano et al. 2006).
microscope (Zeiss Axiolab 4FL-HBO 50/AC).
Tail length, quantitative method
Tail length of comets was used to measure DNA
For comet analysis, average tail length and standard
damage. It was considered as the distance in mm
deviation per treatment were obtained. Variance
from the end of the head to the end of the formed
analysis was performed by using 1-way ANOVA.
comet tail (Collins 2004).
Variance homogeneity and data distribution was
determined with Levene and Kolmogorov-Smirnov
tests, respectively. Post-hoc comparison between
control (untreated) and treated groups was per-
Each nucleoid was assigned to one of the spe-
formed with 2-tailed T3 Dunnett test, which was
cific damage categories (0–4) based on the tail
also used to evaluate results on micronuclei. Statis-
size relative to the head general appearance,
tical program SPSS version 11.0 for Windows was
modified from Avishai et al. (2003), and previ-
performed; P < 0.05 was regarded as statistically
ously reported (Urbina-Cano et al. 2006),
as follows: 0 = nucleoids without tail or undam-
aged, 1 = tail length equal to or shorter than nu-
cleus head diameter, 2 = tail length 1.1 to 3.5
times longer than the head diameter, 3 = tail
length more than 3.5 times the head diameter;
The average comet tail length ± standard deviation
and 4 = absence of head (all DNA migrates to
(SD), and data on micronuclei are shown in Table 1.
Table 1. Average (±SD) comet tail length (in m m) and frequencies of micronucleated and polychromatic
erythrocytes 48 hr after in vivo exposure to curcumin and/or copper
Treatment- in vivo
Tail length ± SD
MNE/10000TE ± SD
MNPE/1000PE ± SD
PE/1000TE ± SD
Control (13 ppm copper)
14.60 ± 4.00
2.83 ± 1.83
1.00 ± 1.26
21.83 ± 5.19 b
Copper 65 ppm
14.51 ± 1.99
10.00 ± 8.60
0.50 ± 0.84
41.66 ± 11.86
Copper 130 ppm
21.51 ± 6.68
1.66 ± 2.66
0.33 ± 0.52
23.66 ± 3.67 b
Copper 390 ppm
38.39 ± 4.00 a, b
16.00 ± 6.60 a, b
1.50 ± 1.64
9.33 ± 5.43 b
16.06 ± 3.22
0.50 ± 0.84
0.16 ± 0.41
42.16 ± 8.28 a
Curcumin + copper 65 ppm
19.88 ± 3.89
14.83 ± 11.14
1.16 ± 1.47
34.66 ± 9.61
Curcumin + copper 130 ppm
23.36 ± 3.03
9.60 ± 6.19
0.40 ± 0.55
25.00 ± 5.61
Curcumin + copper 390 ppm
19.54 ± 2.04c
6.50 ± 5.28
0.16 ± 0.41
18.50 ± 11.17
aP < 0.05, compared to the control (multiple comparisons T3 Dunnett test)
bP < 0.05, compared to curcumin alone (the same test)
cP < 0.05, compared to curcumin + corresponding copper concentration (the same test)
Comet tail length, quantitative method
Obtained blood smears were air-dried, fixed in
The group treated with 0.2% curcumin was not
80% ethanol for 10 min, and stained with an
significantly different from the control group.
acridine orange supravital stain method. Genetic
In vivo exposure for 48 hr to curcumin did not in-
duce DNA damage. We observed a statistically sig-
micronucleated erythrocytes (MNE), considering
nificant damage increase in individuals exposed to
3 parameters: (a) MNE in 10 000 total erythro-
390 ppm of copper cations versus the control or
cytes (TE) [MNE/10000TE]; (b) micronucleated
curcumin alone. A statistically significant differ-
polychromatic erythrocytes (MNPE) in 1000 poly-
ence was observed in 390 ppm copper versus
chromatic erythrocytes (PE) [MNPE/1000PE],
390 ppm copper + curcumin, when comparing each
and (c) PE/1000TE (ZuZiga-González et al. 2000).
copper concentration versus the corresponding cop-
The samples were observed by using an oil immer-
per concentration + curcumin. This suggests that
A. Corona-Rivera et al.
130 ppm 0.2% +
390 pmm 0.2% +
Figure 1. Effect of 48-hr in vivo curcumin/or and copper exposure in mouse lymphocytes assessed by the comet assay.
Comet tail length (m m) means (±SE) per treatment show the toxicity of 390 ppm copper and its reduction due to curcumin
damage induced by 390 ppm copper was decreased
by curcumin (Table 1, Figure 1). Thus DNA dam-
age against copper cations was decreased by
curcumin and its effect could be considered protec-
MNE/10000TE was observed only in the 390 ppm
copper group versus the control and curcumin-
treated groups. This is interpreted as: (1) after
Comets, qualitative method
48 hr of exposure, curcumin does not induce
MNE, (2) 390 ppm copper induces MNE, and
Continuous in vivo exposure to copper and
(3) considering that curcumin + copper in any con-
curcumin allowed us to analyze genetic damage
ditions was not different from control or curcumin
distribution at 48 hr, as shown in Table 2. Most of
groups, curcumin protects cells against copper
the comets in the control group are in categories
0 (undamaged) and 1 (minimum damage). Most of
Acute damage tested with MNPE/1000PE fre-
the comets from the 390 ppm copper concentration
quencies provided no differences in any condi-
exposure, were classified into category 4 (highest
tions. Cytotoxicity measured by PE/1000TE
DNA damage), but damage values decreased in
provided the following results: curcumin PE val-
the presence of curcumin (Figure 2). Thus the pro-
ues were significantly higher than the values for
cedure demonstrates that the maximum DNA
the control, 130 ppm copper, and 390 ppm copper.
damage observed at the 390 ppm copper by tail
The decreased cytotoxic effect observed was at-
length is characterized by longer comet tails as ad-
ditional evidence of the severity of damage, which
cytotoxic effect could be due to copper. The grad-
was reduced by the presence of curcumin.
ual increase in cytotoxicity related to copper con-
Table 2. Percentage of comets assigned to damage categories 0–4 in mouse lymphocytes 48 hr after in vivo
exposure to curcumin and/or copper
Copper added (ppm)
Curcumin 0.2% ± copper added (ppm)
0 (no damage)
1 (min. damage)
2 (medium damage)
3 (severe damage)
4 (max. damage)
aP < 0.05, compared to the control (chi-square test)
bP < 0.05, 390 ppm copper versus curcumin + 390 ppm copper (chi-square test)
In vivo curcumin and copper by comet and micronuclei
% of comets per category
10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Control (13 ppm copper)
Copper 65 ppm
Curcumin 0.2% + copper 65 ppm
Copper 130 ppm
Curcumin 0.2% + copper 130 ppm
Copper 390 ppm
Curcumin 0.2% + copper 390 ppm
Figure 2. Comet percentage distribution per category (from 0, undamaged, to 4, maximum DNA damage), 48 hr after
in vivo curcumin and/or copper exposure in mouse lymphocytes
Control (13 Curcumin
Copper 65 Curcumin
Figure 3. Cytotoxicity of copper and/or curcumin measured as the frequency of polychromatic erythrocytes (PE/1000TE
means ± SE) in mouse lymphocytes 48 hr after in vivo curcumin and copper exposure
centration can be observed in Figure 3, which was
ently the first in vivo study in which the effect of
not modified by the presence of curcumin. A mod-
curcumin in the presence of copper was assessed
erate protective effect of curcumin can be noticed,
by such methods. In vivo exposure to 0.2%
particularly in 390 ppm copper.
curcumin for 48 hr was not genotoxic. Moreover,
0.2% curcumin seems to exert a genoprotective ef-
fect against DNA damage induced by high con-
centrations of copper. The previous was evidenced
in the analysis of the group exposed to 390 ppm
The alkaline comet assay (single-cell gel electro-
copper (30X), which induced severe genomic cel-
phoresis analysis) is a sensitive method for mea-
lular DNA damage that was in turn diminished by
suring DNA strand breaks (Hayashi et al. 2000;
Urbina-Cano et al. 2006). In the present study, we
Consumption of 0.2% curcumin in the mouse
evaluated the extent of DNA damage by determin-
diet for 48 hours did not induce genomic damage.
ing average tail length, analysis of qualitative dis-
The previous result is in agreement with other in
tribution of tail length of comet images, as well as
vivo studies (Vijayalaxmi 1980; Mukhopadhyay
a micronucleus assay. The present work is appar-
and Mukherjee 1998; el Hamss et al. 1999; Polasa
A. Corona-Rivera et al.
et al. 2004; Shukla et al. 2003). Some of these
ished in the presence of curcumin. Apparently,
studies even reported on genoprotection by
in vivo cellular and metabolic conditions contrib-
curcumin (Polasa et al. 2004; Sukla et al. 2003).
ute to the protective effect of curcumin. Baum and
Ng (2004) showed that copper ions bound to the
benzo(a)pyrene-induced damage in the presence
curcumin molecule exhibited a cooperative pro-
of curcumin by the comet assay. Shukla et al.
tective effect. Curcumin activates detoxifying en-
(2003) revealed the antimutagenic potential of
zymes, such as antioxidant and phase II enzymes
curcumin towards benzo(a)pyrene- and cyclo-
in mouse liver and kidneys, acting as a protective
phosphamide-induced genotoxicity in microbial
agent against chemical carcinogenesis and other
and mammalian test systems (chromosomal aber-
forms of electrophilic toxicity (Iqbal et al. 2003).
rations, micronuclei, and sister chromatid ex-
Also, curcumin can be metabolized to tetra-
hydrocurcumin, which may have greater antioxi-
DNA damage was observed in lymphocytes
dant, antimutagenic or antitumor effects than
from individuals exposed to 390 ppm copper.
curcumin (Sugiyama et al. 1996). Barik et al.
Two previous studies reported DNA damage by
(2005) demonstrated that copper-curcumin com-
high copper concentrations (Hayashi et al. 2000;
plexes show superoxide dismutase-like activity,
Saleha Banu et al. 2004). Saleha Banu et al. (2004)
acting as free radical scavengers and antioxidants.
tested DNA damage in leukocytes of orally treated
Curcumin in vivo exerts a global antioxidant ef-
mice with various copper sulfate concentrations
fect, but in vitro increased genomic damage has
(0, 1.25, 2.50, 5.00, 7.50, 10.00 and 12.50 mg kg–1
been reported (Antunes et al. 1999; Araujo et al.
body weight). Significant DNA damage was
1999; Blasiak et al. 1999a,b; Kelly et al. 2001).
found at all copper doses by the comet assay after
The same phenomenon seems to occur with
1–2 weeks of treatment, showing a clear
curcumin and copper exposure (Ahsan and Hadi
dose-dependent response. Long-Evans Cinnamon
1998; Ahsan et al. 1999; Yoshino et al. 2004;
rats accumulate hepatic copper as in human Wil-
Urbina-Cano et al. 2006).
son’s disease; copper accumulation in the liver in-
The applied comet and micronucleus assays
duces DNA strand breaks detected by the comet
have been used simultaneously to test genotoxic
assay (Hayashi et al. 2000). Our work provides ev-
agents (Zhong et al. 2001; Moraes et al. 2004;
idence of acute copper DNA damage detected by
Andrighetti-Fröhner et al. 2006). Different mecha-
the comet assay.
nisms are involved in genotoxicity tests. Essen-
In vivo copper exposure involves complex in-
tially, the micronucleus assay detects damage
teractions. The amount of ingested copper in food
persisting for at least one mitotic cycle, while the
and water is usually relatively low, and most hu-
comet assay identifies repairable DNA damage or
mans and animals are able to control excessive
alkali-labile sites (Zhong et al. 2001). It is neces-
amounts of copper in the body by either decreased
sary to combine the comet assay with other
absorption or increased excretion. Usually, copper
genotoxicity assays, such as the micronucleus as-
is linked to prosthetic groups or tightly bound in
say, to obtain a more comprehensive understand-
storage or transport proteins (like ceruloplasmin)
ing (Zhong et al. 2001). In this work, both methods
and is, therefore, not available for oxidation reac-
were able to detect DNA damage due to high cop-
tions (Gaetke and Chow 2003; Nair et al. 2005).
per concentrations. Both methods evidenced pro-
DNA copper damage might be limited or not evi-
tection by curcumin, and this effect was better
dent at low copper concentrations but at high con-
supported by the comet assay. The extent of dam-
centrations free copper may exert a profound
age was more accurately measured by the comet
genotoxic effect, as observed in this work. Al-
assay qualitatively, but the micronucleus assay
though rodents can tolerate chronic excessive cop-
provided evidence of cytotoxicity. This study re-
per intake (Gaetke and Chow 2003), DNA damage
veals that the comet assay is a sensitive and rapid
appears before clinical effects. Free copper in-
method for detection of DNA damage caused by
duces ROS production and several types of DNA
trace metals, such as copper, in agreement with
damage, such as base alteration and DNA strand
others (Saleha Banu et al. 2004; Urbina-Cano et al.
breaks, which may cause extensive cell death
2006). Although comet assay is a more sensitive
(Hayashi et al. 2000). Such damage was decreased
method to assess genotoxicity (Zhong et al. 2001;
by curcumin, indicating its genoprotective effect.
Moraes et al. 2004), the micronucleus assay was
DNA damage observed in lymphocytes from
also informative and its usefulness should be con-
individuals exposed to excess copper was dimin-
In vivo curcumin and copper by comet and micronuclei
action in Alzheimer’s disease animal models. J Alz-
heimer’s Dis 6: 367–377; discussion 443–449.
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