(-)-Hinokinin causes antigenotoxicity but not genotoxicity in peripheral blood of Wistar rats

Article abstract of DOI:10.1016/j.fct.2006.10.012

(-)-Hinokinin, a dibenzylbutyrolactone lignan, exhibits significant trypanocidal activity both in vitro and in vivo, and was obtained by partial synthesis from (-)-cubebin isolated from the dry seeds of Piper cubeba. Considering the good trypanocidal activity of (-)-hinokinin, as well as its potential for the development of new drugs, it is extremely important to evaluate its possible mutagenic activity to allow its safe use in humans. In the present study, we evaluated the antimutagenic effect of (-)-hinokinin on the chromosome damage induced by the chemotherapeutic agent doxorubicin (DXR). The test system employed was the analysis of micronucleated polychromatic erythrocytes in peripheral blood of Wistar rats. Additionally, the antioxidant activity of (-)-hinokinin was evaluated in in vitro experiments by measuring the production of hydrogen peroxide and other peroxides. Our results showed that animals treated with different doses of (-)-hinokinin (10, 20, and 40 mg/kg b.w.) exhibited micronucleated cell frequencies similar to that of the negative control. In addition, treatment with combinations of (-)-hinokinin and DXR resulted in lower micronucleated cell frequencies than those observed for animals treated with DXR alone. The present study shows that (-)-hinokinin not only has no genotoxic effect, but is also effective in reducing the chromosome damage induced by DXR. (-)-Hinokinin exerted a significant antioxidant effect on parasite mitochondria in the protocol used, which might be one possible mechanism by which this compound may exert a protective effect on the chromosome damage induced by the free radicals generated by DXR.

Full text of DOI:10.1016/j.fct.2006.10.012

Food and Chemical Toxicology 45 (2007) 638–642
www.elsevier.com/locate/foodchemtox
(
ꢀ)-Hinokinin causes antigenotoxicity but not genotoxicity in
peripheral blood of Wistar rats
a
a
a
´
Jamile Ferraris Medola , V ´ı vian Pedigone Cintra , Erika Patr ´ı cia Pesqueira e Silva ,
b
a
b
b
Vanessa de Andrade Royo , Ros aˆ ngela da Silva , Juliana Saraiva , S e´ rgio Albuquerque ,
b
a
a,
*
Jairo Kenupp Bastos , M a´ rcio Lu ´ı s Andrade e Silva , Denise Crispim Tavares
a
Universidade de Franca, Av. Dr. Armando Salles Oliveira, 201-Parque Universit a´ rio, 14404-600 Franca, S a˜ o Paulo, Brazil
Faculdade de Ci eˆ ncias Farmac eˆ uticas de Ribeir a˜ o Preto, Universidade de S a˜ o Paulo, Av. Caf e´ s/n, 14040-903 Ribeir a˜ o Preto, S a˜ o Paulo, Brazil
b
Received 30 January 2006; accepted 21 October 2006
Abstract
(ꢀ)-Hinokinin, a dibenzylbutyrolactone lignan, exhibits significant trypanocidal activity both in vitro and in vivo, and was obtained by
partial synthesis from (ꢀ)-cubebin isolated from the dry seeds of Piper cubeba. Considering the good trypanocidal activity of (ꢀ)-hino-
kinin, as well as its potential for the development of new drugs, it is extremely important to evaluate its possible mutagenic activity to
allow its safe use in humans. In the present study, we evaluated the antimutagenic effect of (ꢀ)-hinokinin on the chromosome damage
induced by the chemotherapeutic agent doxorubicin (DXR). The test system employed was the analysis of micronucleated polychromatic
erythrocytes in peripheral blood of Wistar rats. Additionally, the antioxidant activity of (ꢀ)-hinokinin was evaluated in in vitro exper-
iments by measuring the production of hydrogen peroxide and other peroxides. Our results showed that animals treated with different
doses of (ꢀ)-hinokinin (10, 20, and 40 mg/kg b.w.) exhibited micronucleated cell frequencies similar to that of the negative control. In
addition, treatment with combinations of (ꢀ)-hinokinin and DXR resulted in lower micronucleated cell frequencies than those observed
for animals treated with DXR alone. The present study shows that (ꢀ)-hinokinin not only has no genotoxic effect, but is also effective in
reducing the chromosome damage induced by DXR. (ꢀ)-Hinokinin exerted a significant antioxidant effect on parasite mitochondria in
the protocol used, which might be one possible mechanism by which this compound may exert a protective effect on the chromosome
damage induced by the free radicals generated by DXR.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Micronuclei; Peripheral blood polychromatic erythrocytes; (ꢀ)-Hinokinin
1
. Introduction
At present, treatment of Chagas’ disease is based on two
nitroheterocyclic drugs, nitrofuran nifurtimox (Lampit ),
ꢁ
Chagas’ disease is transmitted by the protozoan Trypa-
whose production has now been discontinued, and 2-
nitroimidazole benznidazole (Rochagan ) (Croft, 1997;
ꢁ
nosoma cruzi and affects more than 18 million people in
Latin America, causing approximately 400,000 deaths per
year (Pozas et al., 2005). In Brazil, five to six million people
are estimated to be infected, 300,000 of them living in the
state of S a˜ o Paulo (Da Silva Filho et al., 2004).
Docampo and Schmu n˜ is, 1997; Paulino et al., 2005). How-
ever, these drugs have marked side effects and, because of
their low effectiveness, are only used for the treatment of
the acute phase of the disease. Blood transfusion is the
most important mechanism of transmission, and gentian
violet is the only effective compound able to eliminate the
parasite from blood prior to transfusion. Despite its good
activity, gentian violet alters blood color, is mutagenic,
*
Corresponding author. Tel.: +55 16 3711 8871; fax: +55 16 3711 8878.
E-mail address: denisecrispim2001@yahoo.com (D.C. Tavares).
0
278-6915/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2006.10.012

J.F. Medola et al. / Food and Chemical Toxicology 45 (2007) 638–642
639
Preto, SP, Brazil, were used. The animals were kept in plastic boxes in an
experimental room under controlled conditions of temperature
O
O
O
O
(
22 ± 2 ꢂC) and humidity (50 ± 10%) on a 12 h light–dark cycle, with
chow and water being available ad libitum. The study protocol was
approved by the Ethics Committee for Animal Care of the University of
Franca, Brazil (Process no. 001/05).
O
OH
O
2.2. Drugs, reagents, solvents and compound identification
procedures
O
Doxorubicin (DXR) was purchased from Pharmacia Brasil Ltd., S a˜ o
Paulo, Brazil. CDCl
Germany. 2 ,7 -Dichlorodihydrofluorescein diacetate (H DCFDA),
3
and KCl were supplied by Merck Co., Darmstadt,
0
0
2
t-butyl hydroperoxide, dimethyl sulfoxide (DMSO), and Hepes-KOH
were purchased from Sigma–Aldrich Co., St. Louis, MO, USA. Methylene
chloride and pyridinium chlorochromate were purchased from Acros Co.,
Pittsburgh, PA, USA. Acetone, chloroform, ethyl acetate, hexane, and
methanol (MeOH) were supplied by Mallinckrodt Co., Xalostoc, Mexico.
Ethanol was bought from a local distillery and purified by distillation.
Silica gel was bought from Merck Co. Optical rotations were measured at
k = 589 nm on a Schmidt–Haensch polartronic HH8 polarimeter (Berlin,
Germany) using a 1.0 cell. Infra Red (IR) spectra were recorded with a
Nicolet FT-IR Proteg e´ 520 instrument. Nuclear magnetic resonance
O
O
O
O
(
-)-Cubebin
(-)-Hinokinin
Fig. 1. Chemical structures of (ꢀ)-cubebin, isolated from Piper cubeba,
and (ꢀ)-hinokinin, obtained by partial synthesis.
(
NMR) spectra were recorded with a Brucker DRX 400 spectrometer.
and is rejected by the patients (Rodrigues Coura and de
Castro, 2002). Thus, the search for new chemoprophylactic
trypanocidal agents has been the main goal in the preven-
tion of Chagas’ disease.
Samples were dissolved in CDCl and the spectra were calibrated at sol-
3
1
13
vent signals of d = 7.26 ( H) or d = 77.0 ( C). A high-performance liquid
chromatography (HPLC) apparatus equipped with an LC-10ADVP
pump, an SPD-M10AVP diode array detector and a DGU-14A degasser
(Shimadzu, Kyoto, Japan) was used for purity determination. Analysis
was performed on a Shimadzu C-18 CLC-ODS column using an
increasing gradient, starting with 50% MeOH:H O and ending with 100%
2
MeOH, for 30 min. The solvents used in the reactions were generally
distilled and dried before use.
On the basis of these data, a comprehensive review of
natural active principles with trypanocidal activity was
published in 1996 (Sepulveda-Boza and Cassels, 1996).
Many classes of secondary metabolites were described in
this review, but there was no mention of a metabolite
belonging to the lignan group. Many dibenzylbutyrolac-
tone lignans exhibit a broad range of biological activities
such as inhibition of platelet-aggregating factor, as well
as antitumor, antiviral, and anti-inflammatory activities.
These compounds also show significant trypanocidal activ-
ity, which makes the trypanocidal evaluation of derivatives
of (ꢀ)-cubebin, isolated from the dry seeds of Piper cubeba,
vitally important (Bastos et al., 1999). On the basis of this
finding, (ꢀ)-hinokinin, an oxidized derivative of (ꢀ)-cube-
bin, was obtained by partial synthesis (Fig. 1) (Souza et al.,
2
.3. Chromosome damage-inducing agent
DXR dissolved in distilled water was used as the inducer of micro-
nuclei in rat peripheral blood cells (positive control).
2
.4. Isolation of (ꢀ)-cubebin
Powdered seeds of commercially available Piper cubeba L. were
exhaustively extracted by maceration with 96% ethanol. The concentrated
crude extract was partitioned between the hexane and methanol/water
(
mass was submitted to repeated column chromatography on 1.0 kg silica
gel (12 · 120 cm). Elution with increasing ratios of hexane, dichloro-
methane, and ethyl acetate yielded six fractions. The cubebin-rich frac-
tions (hexane/dichloromethane 1:1 and 100% dichloromethane) were
submitted to repeated crystallization in hexane/acetone to provide crys-
9:1) phases, providing 430 g of the dried methanol/water fraction. This
2
005).
Because of the promising trypanocidal activity of
(
ꢀ)-hinokinin, as well as its potential use as an antichagasic
drug, it is necessary to evaluate its possible mutagenic activ-
ity for safe use in humans. Therefore, the present study
investigated in vivo the potential mutagenic and/or antimu-
tagenic effects of (ꢀ)-hinokinin using the rat peripheral
blood micronucleus test. Additional in vitro experiments
were performed to understand the in vivo presumed antiox-
idant mechanism of action by measuring the production of
hydrogen peroxide (H O ) and other peroxides.
2
D
6
talline (ꢀ)-cubebin (37 g), mp 130–131 ꢂC, ½aꢁ ꢀ8.12ꢂ (c 0.46, CHCl
3
).
1
The chemical structure was confirmed by H NMR and IR by comparison
with data published in the literature (Koul et al., 1983). Purity was esti-
mated to be 99% by both HPLC and spectral data analysis.
2
.5. Preparation of (ꢀ)-hinokinin by partial synthesis
(ꢀ)-Cubebin (0.5004 g, in 10 ml dichloromethane) was treated with
2
2
two equivalents (2.32 mM) of pyridinium chlorochromate at room tem-
perature and the reaction mixture was stirred for 12 h. The solvent was
removed under vacuum and the residue was chromatographed on silica gel
in hexane–ethyl acetate (4:1), yielding 0.4926 g (98%) of an oily product
2
. Materials and methods
.1. Animals
2
D
6
1
2
(½aꢁ ꢀ30 (26 ꢂC) (c 0.99, CHCl
3 3
)): H NMR d(CDCl ) 6.8–6.4 (m, 1 H),
5
.9 (sl, 2H), 4.15 (dd, 1H, J = 7.1 Hz and J = 9.3 Hz), 3.85 (dd, 1H,
For this experiment, four-week-old Wistar rats with a body weight
b.w.) of approximately 100 g obtained from the animal house of the
Faculty of Pharmaceutical Sciences, University of S a˜ o Paulo, Ribeir a˜ o
J = 7.1 Hz and J = 9.1 Hz), 3.0 (dd, 1H, J = 5.1 Hz and J = 14.2 Hz), 2.85
(dd, 1H, J = 7.3 Hz and J = 14.2 Hz), 2.6 (d, 1H, J = 7.1 Hz), 2.55 (m,
1H), 2.45(d, 1H, J = 8,6 Hz), 2.4 (m, 1H); C NMR d(CDCl ) 178.4,
3
(
1
3

640
J.F. Medola et al. / Food and Chemical Toxicology 45 (2007) 638–642
1
1
47.9, 147.8, 146.5, 146.4, 131.6, 131.3, 122.2, 121.55, 109.4, 108.8, 108.4,
08.3, 101.0, 71.2, 46.4, 41.3, 38.4, 34.8 (Souza et al., 2005).
microscopy under oil immersion. The nuclear division index (NDI) was
calculated by analysis of 400 erythrocytes (PCE/PCE + NCE [normo-
chromatic erythrocytes]), in order to determine the cytotoxicity of
(
ꢀ)-hinokinin (Mersch-Sundermann et al., 2004).
2
.6. Animal treatment
The percent reduction in the frequency of MNPCEs (Waters et al.,
1
990) was calculated by the number of MNPCEs obtained after treatment
The doses of (ꢀ)-hinokinin were selected on the basis of its effective-
with DXR minus the number found for the antigenotoxic treatments (HK
I + DXR; HK II + DXR; HK III + DXR), divided by the number
observed with DXR minus the number obtained for the negative control.
ness in trypanocidal assays (Saraiva et al., 2006). The animals were divided
into 10 treatment groups of six animals each (three males and three
females) as follows: animals treated with three different doses of
(
4
ꢀ)-hinokinin (HK I = 10 mg/kg b.w.; HK II = 20 mg/kg b.w.; HK III =
0 mg/kg b.w.); DXR (90 mg/kg b.w.); DMSO (0.6 g/kg b.w.) and DXR;
2.8. Antioxidant activity of (ꢀ)-hinokinin
HK I and DXR; HK II and DXR; HK III and DXR, and negative (water)
and solvent (DMSO) controls according to the protocol shown in Table 1.
The doses of (ꢀ)-hinokinin were administered to the animals by gavage
This activity was evaluated by measuring the production of H O and
other reactive oxygen species through the quantification of the oxidation
2
2
(
(
1 ml/100 g b.w.), followed by the intraperitoneal (i.p.) injection of DXR
product of H DCFDA, a dichlorofluorescein compound (Carthcart et al.,
2
0.5 ml/100 g b.w.). The different doses of (ꢀ)-hinokinin were obtained
1983). For this, trypomastigote forms of Trypanosoma cruzi cultured in the
LLC-MK2 cell line (Hull et al., 1962) were suspended (1 · 10 parasites/
ml) in a medium containing 125 mM sucrose, 65 mM KCl, and 10 mM
Hepes-KOH, pH 7.2. Next, (ꢀ)-hinokinin was added to the medium at a
concentration of 8, 32 and 64 lM, followed by the addition of 5 lM
6
from a stock solution of 4 mg/ml in 5.6% DMSO in water. The solvent
control group was treated by gavage (1 ml/100 g b.w.) with the same
DMSO dose as the animals that received 40 mg (ꢀ)-hinokinin/kg b.w.
Peripheral blood samples were collected 24 h after the treatments.
2
H DCFDA, and the system was incubated at room temperature for
3
1
5 min. The parasites were then removed by centrifugation (8000g, 4 ꢂC,
0 min), and the fluorescence of the supernatant was determined at 485 nm
2
.7. Micronucleus test
as described by Fang and Beattie (2003).
The peripheral blood micronucleus assay was performed according to
t-Butyl hydroperoxide and a parasite-containing solution of 10%
DMSO were used as positive and negative controls, respectively. The
assays were performed in triplicate.
the protocol described by MacGregor et al. (1987). For determination of
the frequency of micronucleated polychromatic erythrocytes (MNPCEs),
2000 polychromatic erythrocytes (PCEs) per animal were analyzed by light
2
.9. Statistical analysis
Table 1
Experimental groups and treatment protocol
The data were analyzed statistically by analysis of variance for com-
a
Treatment
Group
Dose
pletely randomized experiments, with calculation of the F statistics and
respective P values. When P < 0.05, treatment means were compared by
the Tukey test and the minimum significant difference was calculated for
a = 0.05.
Control
DMSO
1
2
Water
0.6 g/kg b.w.
HK I
3
10 mg/kg b.w.
20 mg/kg b.w.
40 mg/kg b.w.
90 mg/kg b.w. (i.p.)
As in (2) and (6)
As in (3) and (6)
As in (4) and (6)
As in (5) and (6)
HK II
4
HK III
5
3
. Results
DXR
6
DMSO + DXR
HK I + DXR
HK II + DXR
HK III + DXR
7
8
9
10
The frequencies of MNPCEs in peripheral blood cells of
Wistar rats treated with (ꢀ)-hinokinin and/or DXR are
presented in Table 2. Analysis of animals treated with
different doses of (ꢀ)-hinokinin showed no significant
difference in the frequencies of micronucleated cells
DMSO = dimethyl sulfoxide; HK = (ꢀ)-hinokinin; DXR = doxorubicin.
a
Number of animals in each group = 6.
Table 2
Frequency of micronucleated polychromatic erythrocytes (MNPCEs) and nuclear division indexes (NDI) of peripheral blood cells of animals submitted to
treatment with different doses of (ꢀ)-hinokinin and/or DXR, and their respective controls
a
b
Treatment
NDI (mean ± SD)
MNPCEs
Reduction (%)
Number
%
Control
DMSO
HK I
HK II
HK III
DXR
DMSO + DXR
HK I + DXR
HK II + DXR
HK III + DXR
a
0.13 ± 0.03
0.13 ± 0.02
0.13 ± 0.02
0.17 ± 0.03
0.19 ± 0.02
0.10 ± 0.02
0.11 ± 0.02
0.12 ± 0.03
0.10 ± 0.01
0.09 ± 0.01
19
12
14
10
4
0.16
0.10
0.12
0.08
0.03
1.32
0.80
0.42
0.42
0.32
–
–
–
–
–
–
–
76.9
77.7
86.3
c
158
c
95
d
51
d
50
d
38
Four hundred erythrocytes were analyzed per animal, for a total of 2400 per treatment (PCE/PCE + NCE).
Two thousand polychromatic erythrocytes (PCEs) were analyzed per animal, for a total of 12,000 per treatment.
Significantly different from the control (P < 0.05).
b
c
d
Significantly different from the DXR group (P < 0.05).

J.F. Medola et al. / Food and Chemical Toxicology 45 (2007) 638–642
641
be advantageous to use the micronucleus test in parallel as
an indication of the genotoxic effect in rats. In the case of
prolonged exposure of rats to drugs, which is common in
toxicological tests, it should be possible to collect several
peripheral blood samples from the same animal for the
micronucleus test. The advantages of the possibility to
use rat peripheral blood erythrocytes have encouraged sev-
eral studies. The peripheral blood micronucleus test used in
this study has been employed in many studies on the assess-
ment of mutagenicity (Henderson et al., 1993; Sewerynek
et al., 1996; Vijayalaxmi et al., 2001; Trosic et al., 2002;
Hamanda et al., 2003). In addition, the increased frequency
of MNPCEs observed in animals treated with the well-
known clastogenic agent DXR shows that this test system
may reveal an increase in the frequency of MNPCEs in
animals treated with (ꢀ)-hinokinin.
Fig. 2. EC50 value of (ꢀ)-hinokinin in the inhibition of H
2 2
O produced by
Trypanosoma cruzi mitochondria.
(
HK I = 0.12 ± 1.63%; HK II = 0.08 ± 1.86%; HK
III = 0.03 ± 0.82%) compared to the control group
0.16 ± 1.60%), thus demonstrating the absence of a geno-
In the in vivo experiments, a DXR dose of 90 mg/kg b.w.
was chosen because this dose induces the increase in the fre-
quency of MNPCEs necessary for the investigation of the
antimutagenic potential of (ꢀ)-hinokinin. According to
Chatterjee and Raman (1993), the full protective efficiency
of an agent is elicited when the frequency of aberrations
induced is high enough and the concentration of the protec-
tive agent administered is sufficient. On this basis, in the
present study we decided that the minimum frequency of
MNPCEs induced by DXR should be 1% or higher.
The present results also show that (ꢀ)-hinokinin causes
a significant reduction in the frequency of MNPCEs
induced by DXR. The antimutagenic effect of (ꢀ)-hinoki-
nin might be due to its the ability to scavenge free radicals,
acting as an antioxidant agent, since the genotoxic activity
of the chemotherapeutic agent DXR has been attributed to
its ability to produce free radicals (Keizer et al., 1990),
which cause different types of damage to the cell including
DNA cleavage. The chemical structure of DXR favors the
generation of free radicals which can bind to iron and form
complexes with DNA, inducing double-strand breaks
(Eliot et al., 1984). Some studies have demonstrated that
this oxidative damage is probably related to the formation
of free radicals, with a concomitant reduction in anti-
oxidant capacity (Myers et al., 1977).
(
toxic effect. In contrast, animals treated with (ꢀ)-hinokinin
and DXR presented a significant reduction in the frequen-
cies of MNPCEs (HK I = 0.42 ± 4.32%; HK II = 0.42 ±
1
.63%; HK III = 0.32 ± 3.78%) when compared to those
treated with DXR only (1.32 ± 11.82%). However, this
protective effect of (ꢀ)-hinokinin was not dose dependent.
The frequency of MNPCEs was lower in animals treated
with DMSO and DXR (0.80 ± 5.23%) than in those treated
with DXR alone (1.32 ± 11.82%), but this difference was
not statistically significant.
Comparative analysis by the Tukey test showed no
significant differences between male and female rats.
Mean NDI values and the respective standard devia-
tions are presented in Table 2. No significant differences
in NDI were observed between any treatment and the neg-
ative control group, demonstrating the absence of cytotox-
icity in the different treatments under the conditions
employed in this study.
Regarding the antioxidant assay, (ꢀ)-hinokinin inhib-
ited the accumulation of H O produced by mitochondria
2
2
in the presence of the pro-oxidant compound, t-butyl
hydroperoxide, at concentrations of 10, 25 and 50 lM by
4
6%, 57% and 72%, respectively. Fig. 2 shows the EC50
value for (ꢀ)-hinokinin obtained in the antioxidant assay.
In an attempt to better understand the mechanisms
underlying the observed antimutagenic effect of (ꢀ)-hinok-
inin, we evaluated the antioxidant activity of (ꢀ)-hinokinin
by measuring the production of H O and other peroxides.
The positive and negative controls displayed 100% and
0
% of oxidative effects, respectively.
2
2
(
ꢀ)-Hinokinin displayed a significant antioxidant activity
4
. Discussion
on the parasite mitochondria in the protocol used, suggest-
ing that one possible mechanism by which this compound
may exert a protective effect is by acting as a chemoprotec-
tive agent, preventing the DNA damage produced by the
reactive oxygen species generated by DXR.
The aim of the present study was to assess the mutage-
nicity and/or antimutagenicity of (ꢀ)-hinokinin in Wistar
rat peripheral blood. The results obtained here show that
(
ꢀ)-hinokinin does not increase the frequency of micro-
The observation that (ꢀ)-hinokinin does not exert a
significant dose-dependent protection might be due to its
erratic absorption by the cell membrane, leading to an
inconstant bioavailability of the compound in the cell.
The lower frequency of MNPCEs observed for ani-
mals treated with DMSO and DXR compared to the group
nucleated cells; therefore, this compound has no genotoxic
effect.
Regarding the test protocol employed here, Abramsson-
Zetterberg et al. (1999) showed that because rats have been
used as a model in conventional toxicological tests, it might

642
J.F. Medola et al. / Food and Chemical Toxicology 45 (2007) 638–642
treated only with DXR might be due to the ability of
DMSO to scavenge reactive oxygen species (Waldren
et al., 2004). However, this result was not statistically
significant.
acute sub chronic administration of azathioprine. Mutat. Res. 291, 79–
5.
Hull, R.N., Cherry, W.R., Tritch, O.J., 1962. Growth characteristics of
monkey kidney cell strains LLC-MK1, LLC-MK2, and LLC-MK2
8
(
NCTC-3196) and their utility in virus research. J. Exp. Med. 115, 903–
The present study shows that (ꢀ)-hinokinin not only
had no genotoxic effect, but was also effective in reducing
the chromosome damage induced by DXR. The exact
mechanism of action of (ꢀ)-hinokinin is still unclear, but
its anticlastogenic role observed here might be due to its
antioxidant effect on the chromosome damage induced by
the free radicals generated by DXR. Finally, our results
contribute to a better understanding of the mechanism of
action of this drug in the human body, which permits its
more effective and safer use in clinical administrations.
918.
Keizer, H.G., Pinedo, H.M., Schuurhuis, G.J., Joenje, H., 1990. Doxo-
rubicin (Adriamycin): a critical review of free radical-dependent
mechanisms of citotoxicity. Pharmacol. Ther. 47, 219–231.
Koul, S.K., Taneja, S.C., Dhar, K.L., Atal, C.K., 1983. Lignans of Piper
clusii. Phytochemistry 22, 999–1000.
MacGregor, J.T., Heddle, J.A., Hite, M., Margolin, B.M., Ramel, C.,
Salamote, M.F., Tice, R.R., Wild, D., 1987. Guidelines for the conduct
of micronucleus assay in mammalian bone marrow erythrocytes.
Mutat. Res. 189, 103–112.
Mersch-Sundermann, V., Kassie, F., Bohmer, S., Lu, W.Q., Wohlfahrth,
R., Sobel, R., Brunn, H.E., ElSohly, M.A., Ross, S.A., Stahl, T., 2004.
Extract of Toxicodendron quercifolium caused genotoxicity and anti-
genotoxicity in bone marrow cells of CD1 mice. Food Chem. Toxicol.
Acknowledgement
4
2, 1611–1617.
Myers, C.E., MacGuire, W.P., Liss, R.H., Ifrim, I., Grotzinger, K.,
Young, R.C., 1977. Adriamycin: The role of lipid peroxidation in
cardiac toxicity and tumor response. Science 197, 165–167.
Paulino, M., Iribarne, F., Dubin, M., Aguilera-Morales, S., Tapia, O.,
Stoppani, A.O., 2005. The chemotherapy of chagas’ disease: an
overview. Mini Rev. Med. Chem. 5, 499–519.
This study was supported by Funda c¸ a˜ o de Amparo a`
Pesquisa do Estado de S a˜ o Paulo (FAPESP), Brazil.
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