Influence of orange juice over the genotoxicity induced by alkylating
agents: an in vivo analysis
Silvia Isabel Rech Franke1,2, Daniel Pr? a a2, Bernardo
Erdtmann2,3, Jo~ a ao Antonio Pe ˆgas Henriques2,3,4and
Juliana da Silva2,4,?
1Curso de Nutric ¸~ a ao, Departamento de Educac ¸~ a ao Fı ´sica e Sa? u ude, Universidade
de Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil,2Genotox/
Centro de Biotecnologia/Programa de P? o os-Graduac ¸~ a ao em Biologia Celular e
Molecular/Programa de P? o os-Graduac ¸~ a ao em Gen? e etica e Biologia Molecular,
Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, R S,
Brazil,3Instituto de Biotecnologia, Universidade de Caxias do Sul (UCS),
Caxias do Sul, RS, Brazil and4Curso de Biologia/Curso de Farm? a acia,
Universidade Luterana do Brasil (ULBRA), Canoas, RS, Brazil
association between diets rich in fresh fruit and vegetables
and a decreased incidence of cancers. Methyl methanesulf-
onate (MMS) and cyclophosphamide (CP) are alkylating
agents that differ in their mode of action. MMS is a
directly-acting, monofunctional agent, while CP is a bifunc-
tional agent that requires metabolic activation to a reactive
metabolite. To evaluate if orange juice could reduce DNA
damage induced by these alkylating agents, mice were trea-
ted orally (by gavage) with MMS and CP, prior to and after
treatment with orange juice. DNA damage was evaluated
by the comet assay in peripheral white blood cells. Under
these experimental conditions, orange juice reduced the
extent of DNA damage caused by both mutagens. For
MMS, the antigenotoxic effect of the orange juice was
both protective (orange juice pre-treatment) and repara-
tive (orange juice post-treatment); for CP, the effect was
reparative only. The components of orange juice can have
several biological effects, including acting as targets of tox-
icants and modulating metabolization/detoxification routes.
Considering the different mechanisms of the action of the
two drugs, different protective effects are suggested. These
results demonstated the ability of the in vivo comet assay to
detect in vivo modulation of MMS and CP mutagenicity by
Diet represents a major influence on the promotion and pro-
gression of cancer. A micronutrient-equilibrated diet can con-
tribute to genomic stability. Deficiencies in vitamins and
minerals in the human diet are thought to generate DNA dam-
age by enhancing the occurrence of breaks and oxidative
lesions (1--3). Since mutations are key elements in neoplasic
processes, there is a considerable amount of epidemiological
evidence relating diets rich in fresh fruit and vegetables and a
decrease in cancer incidence (2,4).
Methylmethanesulfonate (MMS) and cyclophosphamide
(CP) induce neoplasic processes by different mechanisms.
MMS and CP alkylate nucleophilic organic macromolecules,
including DNA. They can induce depurination and depyrim-
idation as well as monoadduct formation. CP can also induce
DNA--DNA and DNA--protein crosslinks. Both substances
have been shown to induce gene mutation (prokaryotes,
fungi, insects, plants and mammalian cells), chromosome
effects (plants, insects and mammalian cells in vitro and
in vivo), unscheduled DNA synthesis (UDS) (mammalian
cells in vitro and in vivo) and sister chromatid exchange
(SCE) (mammalian cells in vitro and in vivo), as well as other
genotoxic effects (http://toxnet.nlm.nh.gov). MMS is a mono-
functional sulfur-containing compound commonly used as a
solvent and as a catalyst for polymerization, alkylation and
esterification reactions (5). It possesses weak mutagenic and
carcinogenic activity (6). CP is a widely used and well-
documented reference mutagen that expresses its genotoxicity
when metabolically activated. The International Agency for
Research on Cancer (IARC) has concluded that there is suffi-
cient evidence to classify CP as carcinogenic for animals and
humans (7). Since CP needs metabolic activation (8), in vivo
studies are the most appropriate method for addressing the
complex action of CP.
Orange juice is a complex mixture with, among other
things, macro and micronutrients. Its chemopreventative and
antimutagenic property is attributed to some vitamins,
pro-vitamins and other compounds such as phenolics. How-
ever, the same phytochemicals have been characterized as
mutagenic (9--11). Most studies conducted to evaluate the bio-
logical activity of fruit and vegetable juices and extracts have
focused onisolated phytochemicals.Moreover, whole mixtures
have been mainly evaluated by in vitro test systems. Thus, this
work aims to evaluate, in vivo, the effect of orange juice on the
genotoxicity of the alkylating agents MMS, a direct acting
mutagen, and CP, which requires metabolism to a reactive
form, using the comet assay.
Materials and methods
Phosphate buffered saline (calcium- and magnesium-free), Tris [tris (hydroxy-
methyl) aminomethanehydrochloride], disodium ethylenediamine-tetra-acetate
(EDTA), dimethylsulfoxide (DMSO), ethidium bromide (EtBr), MMS, CP and
Triton X-100 were purchased from Sigma (St Louis, MO). Low melting point
(LMP) agarose and normal agarose (electrophoresis grade) were obtained from
Gibco-BRL (Grand Island, NY). Sodium heparin was purchased from Roche
(Brazil) under the commercial name Liquemine?.
Swiss Webster mice, aged between 5--7 weeks and weighing between 20 and
40 g, were obtained from the Agriculture Ministry, Laboratory of Animal
Reference, in Porto Alegre, RS, Brazil. Prior to tests, mice were acclimatized
to the laboratory conditions for 7 days (22?C 6 3?C and 60% humidity).
During acclimatization and tests, mice received commercial standard mouse
cube diet (Nuvilab, CR1, Moinho Nuvipal Ltda., Curitiba, PR, Brazil) and
?To whom correspondence should be addressed. Laborat? o orio de Gen? e etica Toxicol? o ogica, Departamento de Biologia- PPGECIM, Universidade Luterana do Brasil
(ULBRA); R. Miguel tostes, 101, Pr? e edio 14, Sala 230; CEP 92420-280, Canoas, RS, Brasil. Tel/Fax: +55 51 3387 1138; Email: email@example.com
# The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please email: firstname.lastname@example.org
Mutagenesis vol. 20 no. 4 pp. 279--283, 2005
Advance Access publication 14 June 2005
water ad libitum. After acclimatization, they were divided into treatment
groups, each containing 3 males and 3 females. All procedures were accomp-
lished according to the international practices for animal use and care under
the control of an internal committee of the Universidade Federal do Rio Grande
Juice (in natura) was preparedimmediately beforethe test using Citrus sinensis
(Linn.) Osbeck organic oranges (free of agrochemicals). Glass recipients
containing the juice were covered to avoid light exposure.
Treatments and test substances
The treatment groups received by gavage 0.1 ml/10 g body wt of: (a) water,
(b) juice, (c) MMS and (d) CP. Dose levels of the latter were MMS of 40 mg/kg
body wt and CP of 25 mg/kg body wt (Table I). For CP, a dose equivalent
to 18.2% of the LD50dose [25 mg/kg body wt (LD505 137 mg/kg body wt
(12)] was used. For MMS, the dose was equal to 13.8% of the LD50 dose
[40 mg/kg body wt (LD505 290 mg/kg body wt (13)]. All substances were
prepared just before treatment and protected from light. The dose of MMS
was selected after a preliminary study (data not shown). The dose of CP was
based on the historical data of our laboratory obtained from routine use of
the compound. We used the same doses for testing the repair action of
vitamin C (14).
Blood sample collection
One or two drops of blood were collected from mouse tail tips by means of
a small incision (15). Animals were sampled 24 and/or 48 h after treatment
(Table I). Drug administration and blood sampling were performed as
described previously (14). Peripheral white blood cells are among the most
used cells for genotoxicity studies, mainly with the comet assay. They circulate
through the entire body and are easily obtained.
The alkaline comet assay was performed, as described by Singh et al. (16),
according to guidelines proposed by Tice et al. (17) with a slight modification
developed by Da Silva et al. (18). From each mouse, ~15 ml of blood were
sampled and mixed with 7 ml of heparin (anticoagulant). Seven microliters of
cell/heparin mixture were then embedded in 93 ml of LMP agarose (0.75 g/
100 ml). The resulting mixture was spread over a pre-coated microscope slide
(1.5 g/100 ml agarose), a cover glass was gently placed over it and the slide
placed at 4?C for 5 min to allow gel solidification. The cells were lysed in high
salt and detergent solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris,
(pH 10--10.5), plus 1% Triton X-100 and 10% dimetil sulfoxide-DMSO added
just before use) and placed in a horizontal electrophoresis box. Subsequently,
the cells were exposed to alkali [300 mM NaOH and 1 mM Na2EDTA
(pH 413)] for 20 min at 4?C, to allow DNA unwinding. Electrophoresis
was performed using an electric current of 300 mA at 25 V (0.9 V/cm) for
15 min at 4?C. After electrophoresis, the slides were neutralized and stained
with ethidium bromide. Negative and positive (5 3 10?5M MMS, 1--2 h)
human blood controls were included in each electrophoresis run. The runs were
accepted only when the human blood internal controls showed the appropriate
negative and positive responses, respectively.
One hundred cells per animal (two slides of 50 cells each) were analyzed at
2003 using a fluorescence microscope equipped with an excitation filter (BP
546/12 nm) and a barrier filter (590 nm). One scorer was used throughout
the study and all slides were scored in a blinded way. International guidelines
and recommendations for the comet assay consider that visual scoring of
‘comet cells’ is a well-validated evaluation method, since it correlates well
with computer-based image analyses (17,19,20). Comet assay parameters
were calculated, according to Da Silva et al. (18). The damage index (DI)
was calculated for each sample, ranging from 0 (no damage: 100 cells 3 0)
to 400 (maximum damage: 100 cells 3 4), where 0 5 no tail and 4 5 largest
tail (see 18 for pictures of the classes). The damage frequency (DF) was
calculated based on the number of cells with damage (classes 1--4).
Student’s t test was used to compare DNA damage values between the different
times (24 h versus 48 h, 24 h versus orange juice pre-treatment and 48 h versus
orange juice post-treatment). Analysis of Variance (ANOVA) was used to
compare DNA damage induced by different substances at the same time
(24 or 48 h). A parametric ANOVA was used when data showed normal
distribution and were homogeneous in variance. In this case, the Tukey
post hoc test was applied for multiple comparisons. When homocedasticity
or normality was not present, the Kruskal--Wallis non-parametric ANOVA
was used. In this case, Dunn’s post-hoc test was applied to compare
groups. Statistical significance was considered at a level of P ? 0.05. All
statistical analyses were performed independently for the two parameters
The internal controls for the comet assay (human blood)
demonstrated low damage in the negative control (DI 5 0--10)
in agreement with the historical values of our laboratory. In a
preliminary experiment, differences in MMS- or CP-induced
DNA damage between male and female mice were tested
(Figure 1). However, no significant differences in sensitivity
of males and females were detected. Thus male and female data
were grouped for subsequent analyses.
In the comet assay, little damage was seen in mouse peri-
pheral white blood cells sampled at 24 h for the animals
that received only orange juice or water (Table II). The
animals that received water showed less DNA damage than
the mice that were treated with orange juice, although not
significantly (Table II). A slight increase in DNA damage
was observed in these groups at 48 h of exposure (Table II).
This increase was only significant for the water treatment
(for DI, P ? 0.001). However, at 48 h, the extent of DNA
damage did not differ between mice treated with water and
Table I. Experimental procedures
0 h24 h48 h
ControlWater treatment1st Blood sampling
2nd Water treatment
1st Blood sampling
2nd Juice treatment
1st Blood samplinga
1st Blood samplingb
2nd Orange juice
1st Blood sampling
aIndividuals also used as orange juice controls at 24 h.
bIndividuals also used as MMS and CP at 24 h.
Damage Index (0-400)
Fig. 1. Damage index (DI) in white blood cells induced by water, orange
juice, MMS and CP, as evaluated in male and female mice with the
S.I.R.Franke et al.
At 24 h, MMS and CP were genotoxic according to both
parameters evaluated in comparison to both the water and the
orange juice treatments (Table II). A reduction in DNA damage
was observed for both MMS and CP at 48 h. However, the
reduction was significant only in DI for CP and in DF for MMS
(P ? 0.05). Although decreasing in relation to 24 h values, the
DNA damage levels for MMS at 48 h remained significantly
higher in relation to water (P ? 0.001) and orange juice (for DI
and DF, P ? 0.05) treatments. For CP at 48 h, the level of
DNA damage remained higher than for water (for DI and DF,
P ? 0.05) (Table II).
When the level of DNA damage in white blood cells of mice
treated with MMS and sampled at 24 h was compared with
mice pre-treated with orange juice, orange juice induced a
significant reduction in DNA damage in both evaluated comet
assay parameters (P ? 0.001) (Figure 2 and Table II).
Post-treatment with orange juice induced significant reduc-
tion in DNA damage in both parameters in white blood cells for
both mutagens in mice sampled at 48 h (Figure 2 and Table II).
In a previous study, orange juice samples prepared in the same
way and of oranges of the same region were mutagenic by the
Ames test (11). In the present study, no significant differences
in DNA damage between mice treated with water or orange
juice were detected by the comet assay. Orange juice is con-
sumed worldwide. The risk for humans consuming orange
juice may be low, due to enzymatic activities and pH changes
in the digestive tract (11).
The slight DNA damage increase seen at 48 h in the water
treated cases might be associated with the stress of manipula-
tion, gavage and blood sampling procedure. It is likely that
there is an equal influence in all treatments, since water was the
Table II. Detection of DNA damage by the comet assay in white blood cells of mice exposed to water, orange juice, and/or MMS or CP and sampled
at 24 h (with and without pre-treatment with orange juice) or 48 h (with and without post-treatment with orange juice)
(mg/kg body wt)
Scheduleaand comet assay parameters
24 h 48 h Pre-treatment with
Damage index (DI)
DI 6 SD
11.33 6 2.73
22.00 6 5.36
170.63 6 20.21f???; g???
114.54 6 39.59f???; g???
DI 6 SD
19.00 6 3.29e???
26.67 6 4.03
157.67 6 15.28f???; g?
74.83 6 16.68e?; f?
DI 6 SD
56.17 6 42.60e???
97.83 6 12.38
DI 6 SD
95.17 6 16.34h???
32.33 6 20.16h??
Damage frequency (DF)
DF 6 SD
9.33 6 2.34
17.71 6 3.93
98.75 6 2.71f???; g???
73.96 6 22.04f??; g???
nDF 6 SD
11.17 6 3.66
20.50 6 3.33
87.00 6 8.74e?; f???;g?
62.33 6 15.27f?
DF 6 SD
38.50 6 21.99e???
64.50 6 16.54
DF 6 SD
64.33 6 11.59h??
28.67 6 18.12h??
Significance with respect to water and orange juice refers to significance in the same column and was tested using Parametric or non-parametric ANOVA.
All other significances refer to the same row and were tested using Student’s t-test.
aFor more details see Table I.
bGroup sampled 24 h after treatment with an alkylating agent.
cGroup sampled 48 h after treatment with an alkylating agent.
dn, Number of individuals obtained from sum of independent experiments.
eSignificant in relation to 24 h at?P ? 0.05;??P ? 0.01;???P ? 0.001.
fSignificant in relation to water at?P ? 0.05;??P ? 0.01;???P ? 0.001.
gSignificant in relation to orange juice at?P ? 0.05;??P ? 0.01;???P ? 0.001.
hSignificant in relation to 48 h at?P ? 0.05;??P ? 0.01;???P ? 0.001.
Damage Index (DI)
24 h 48 hPre-treat. Post-treat.
Damage Index (DI)
Fig. 2. Peripheral white blood cells damage index (DI) modulation induced
by orange juice in mice treated with MMS (A) and CP (B), evaluated by
comet assay. a: significant in relation to 24 h. b: significant in relation to 48 h.
?P ? 0.05,??P ? 0.01 and???P ? 0.001.
Influence of orange juice over genotoxicity
medium for all substances. Similar results were found in a
previous study (11).
The decrease in DNA damage (likely due to repair of DNA
damage) at 48 h was more pronounced for CP than for MMS.
Vitamin C is an important micronutrient mainly required as
a co-factor for enzymes involved in oxi-reduction reactions
(3,4,21). It has been studied for its protective action against
different diseases (22,23). The mechanisms by which ascorbic
acid acts include bio-antimutagenic (24,25) and desmutagenic
activities (26). Vitamin C can compete with DNA as a target
for alkylation, reducing the genotoxicity of alkylating agents
(22). Moreover, vitamin C has a role in the regulation of DNA
repair enzymes (27) and high concentrations of vitamin C can
also induce apoptotic cell death (28). Vitamin C is not protein-
bound and is eliminated with an elimination half-life of
10 h (29).
Phenolic compounds are another constituent of fruit juices.
They can protect biological systems in different ways
(23,30--32). Phenolic compounds have a dual effect on phase I
and phase II enzymes, repressing some enzymes (mainly in
phase I) and stimulating others (mainly in phase II) (33). Some
flavonids, like hesperetin, can selectively inhibit human
Cytochrome P450 (34), reducing the absorption/elimination
of toxic compounds. Other phenolic compounds, such as
limonoids are inducers of the detoxifying enzyme gluthatione
S-transferase (32). The stimulation of detoxifying enzymes can
facilitate the elimination of toxic compounds, significantly
affecting the toxic potential of endogenous and exogenous
chemicals (32). Moreover, phenolic compounds such as
myricetin can stimulate DNA repair pathways, through tran-
scription regulation or mRNA stabilization (35).
The pharmacokinetics of polyphenols is diverse. It depends
on the chemical structure of polyphenols (29). Naringenin and
hesperitin are among the most prevalent polyphenols in orange
juice. They can be detected in urine up to 38 h after adminis-
tration (36). In liver, polyphenols are subjected to three main
types of conjugation after absorption: methylation, sulfation
and glucuronidation (37). It is likely that phenolic compounds
can be methylated by alkylating agents, instead of the conjuga-
tion enzymes, protecting/reducing DNA from alkylation.
MMS can methylate nucleophilic regions of DNA and
amino acid molecules, particularly at nitrogen atoms. Methyla-
tion of the phosphate groups accounts for a minor percentage
of the total methylation by MMS (51%). MMS’ genotoxicity
is mediated by base modifications, which weaken the
N-glycosylic bond, leading to depurination/depyrimidination
of DNA strands and the appearance of alkali-labile abasic
sites (AP sites). The removal of AP sites by AP endonucleases
cleaves DNA adjacent to these sites and generates DNA strand
breaks in DNA (6,38--40). To a minor extent MMS can also act
as a weak oxidative stress inducer, as observed by Horv? a athov? a a
et al. (6), who tested the effect of a synthetic antioxidant
(stobadine, SBT) on MMS genotoxicity.
Pre-treatment and post-treatment with orange juice reduced
MMS’s genotoxicity about 67 and 40% in DI (61 and 26% in
DF), respectively. Thus, orange juice was both preventive and
reparative for MMS. In pre-treatment, phenolic compounds
and, to a minor extent, vitamin C (due to the shorter half-life)
could have competed as target site for alkylation. With respect
to post-treatment, both phenolic compounds and vitamin C
could have influenced the kinetics of repair.
CP is absorbed well after oral administration. The parent
compound is widely distributed throughout the body with a low
degree of plasma protein binding (20%). The half-life of CP
is between 6 and 9 h (41). Once activated, CP can, besides
monoadducts, also induce the formation of DNA--DNA
and DNA--protein crosslinks (5). CP has the ability to
generate free radicals that cause endothelial and epithelial
cell damage (41).
Pre-treatment with orange juice slightly reduced the level of
DNA damage induced by CP (15 and 13% reduction in DI and
DF, respectively), while orange juice post-treatment induced
a significantly higher reduction in DNA damage (57 and 71%
reduction in DI and DF, respectively). Since CP requires meta-
bolic activation before inducing DNA damage, it is likely that
juice components, such as phenolics, alter the rate of metabol-
ization and/or detoxification. In pre-treatment, only the phen-
olics could have acted as a scavenger, since vitamin C has a
short half-life. Despite acting as scavengers, phenolics could
have blocked CYP 450 and increased the half-life of the CP.
In post-treatment, damage reduction was higher because both
compounds could act as reactive species quenchers and DNA
repair pathways modulators. Moreover, phenolics could have
stimulated phase II enzymes and eliminated CP metabolites. It
is important to consider the kind of DNA damage generated by
CP, particularly crosslinks. Such lesions can retard the migra-
tion of DNA fragments and lead to a wrong evaluation of the
extent of DNA damage (5,17,20).
In conclusion, consumption of orange juice can be both
protective (MMS) and reparative (MMS and CP) of DNA
damage induced in mouse white blood cells by alkylating
agents. Such protective effects of orange juice differ depending
on the mode of action of the mutagen and may be mediated by,
among other things, (1) modulation of phase I and II enzymes;
(2) substrate competition for the nucleophilic action of CP and
MMS or quenching of CP metabolites and side-products (react-
ive species); and (3) enhancement of DNA repair. Our results
demonstate the ability of the in vivo comet assay to detect
in vivo modulation of MMS and CP mutagenicity by orange
The authors thank Christine C.Gaylarde and Dr Raymond Tice for critically
reading the manuscript. UNISC, CNPq, CAPES and GENOTOX-UFRGS sup-
ported the research.
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Received on July 15, 2004; revised on March 30, 2005;
accepted on April 15, 2005
Influence of orange juice over genotoxicity