Pharmacological Research, Vol. 43, No.

6, 2001
doi:10.1006/phrs.2001.0822, available online at http://www.idealibrary.com on

ANTIOXIDANT ACTION OF BIXIN AGAINST CISPLATIN-INDUCED

CHROMOSOME ABERRATIONS AND LIPID PEROXIDATION IN RATS

CECÍLIA RODRIGUES SILVA, LUSÂNIA M. GREGGI ANTUNES and MARIA DE LOURDES P.

BIANCHI∗
Depto Análises Clı́nicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, Brasil. Av. do Café s/n, 14040-903, Ribeirão Preto, SP,
Brasil

Accepted 23 March 2001

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer, but has serious side
effects, inducing nephrotoxicity and chromosome aberrations. In this study we evaluated the role
of the carotenoid bixin on cisplatin-induced oxidative stress in Wistar rats through three markers
of oxidative damage: chromosome aberrations, glutathione depletion and lipid peroxidation. The
animals were divided into six treatment groups with six rats in each (n = 6). The dose of cisplatin
(5.0 mg kg−1 body wt.) was injected i.p. and bixin (2.5 or 5.0 mg kg−1 body wt.) was given by
gavage at 48, 24 h and 10 min before the cisplatin injection. The treatment with the highest dose of
bixin resulted in a statistically significant reduction, by about 33%, in cisplatin-induced abnormal
metaphases (P< 0.05). A single dose of cisplatin enhanced the formation of lipid peroxides in 29%
and resulted in a 29% depletion in renal glutathione 24 h after cisplatin administration (P< 0.05).
The pretreatment with bixin reduced the total number of chromosome aberrations, inhibited the
increase in lipid peroxidation, and inhibited renal glutathione depletion induced by cisplatin. Since
the pretreatment with bixin alone was safe, under the present experimental conditions, the results
c 2001 Academic Press
suggest that bixin may have future clinical application after further studies.

K EY WORDS : bixin, annatto, lipid peroxidation, chromosome aberrations.

INTRODUCTION induces a fall in plasma antioxidant levels, which

Cisplatin (cis-diamminedichloroplatinum II) is one of the
most potent antitumor agents. Activity has been demon-
strated against a variety of tumors, particularly for head
and neck, testicular, ovarian, bladder and small cell lung
cancers [1]. However, cisplatin has serious side effects
on non-tumor cells and causes nephrotoxicity. The alter-
ations in kidney function induced by cisplatin are charac-
terized by signs of injury, such as changes in urine volume
and in glutathione status, and cisplatin-induced nephro-
toxicity is closely associated with an increase in lipid per-
oxidation [2, 3]. Cisplatin is highly mutagenic, inducing
chromosome aberrations in peripheral blood lymphocytes
in patients and in rat bone marrow cells [4–6].
This antitumoral was able to generate active oxygen
species, such as superoxide anions and hydroxyl
radicals [7–9], and to inhibit the activity of antioxidant
enzymes in renal tissue [3]. Cisplatin chemotherapy
∗ Corresponding author. Dr Maria de Lourdes P. Bianchi, Faculdade de
Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
Laboratório de Bromatologia e Nutrição, Av. do Café s/n. 14040-903,
Ribeirão Preto, São Paulo, Brasil. E-mail: mdlpbian@usp.br
may reflect a failure of the antioxidant defense
mechanism against oxidative damage induced by
commonly used antitumor drugs [10]. Much attention
has been given to the possible role that dietary
antioxidants play in protecting against cisplatin-induced
nephrotoxicity [11–13]. We have previously investigated
the protective effects of the antioxidants vitamin C,
curcumin and selenium against the nephrotoxicity
and lipid peroxidation induced by cisplatin [14–16].
Nevertheless, there are no studies of the effects of
carotenoids on cisplatin nephrotoxicity in the literature.
Carotenoids are a family of fat-soluble pigments
found in fruits and vegetables and many studies
involving epidemiological and animal models have
investigated their potential in the carcinogenic process.
A rational mechanism for potential anticarcinogenic
and antimutagenic effects of β-carotene and other
carotenoids is its ability to scavenge free radicals that
cause oxidative DNA damage [17, 18].
Bixin, a carotenoid extracted from the seeds of the
tropical Annatto plant Bixa orellana, is unique among

1043–6618/01/060561–06/$35.00/0 c 2001 Academic Press

562
naturally occurring carotenoids because of its two
carboxylic groups, and is one of the few cis carotenoids.
Indigenous tribes from the Rainforest have used the
Annatto seeds as a body paint and the entire plant
as a medicine for centuries. In an oil suspension it is
used as a folk remedy in the West Indies [19]. The
red powdery extract from the Annatto seeds is a well
known food coloring. Among the natural carotenoids,
bixin is one of the more effective biological singlet
molecular-oxygen quenchers and may contribute to the
protection of cells and tissues against deleterious effects
of free radicals [20]. Bixin is also an effective inhibitor
of lipid peroxidation [21]. Therefore, the present study
was undertaken to investigate the effects of the dietary
carotenoid bixin on cisplatin-induced chromosome
aberrations and lipid peroxidation in adult Wistar rats.
MATERIALS AND METHODS
Chemical agents
Bixin was a gift from Corantec, Corantes Naturais
LTDA (Brazil). Cisplatin (Platiranr ) was purchased
from Bristol-Myers Squibb (Brazil). Bixin was dissolved
in ethanol plus distilled water (1 : 9) just before the
experiments. The reagents were of analytical grade.
Animals and experimental design
Experiments were carried out on male Wistar rats (Rat-
tus norvegicus). The animals were supplied by the Ani-
mal House of the Prefeitura do Campus Administrativo
de Ribeirão Preto, USP. Rats, weighing 200 ± 10 g, had
free access to standard rat chow and water ad libitum, and
were housed in plastic cages, kept in a room maintained
at 23 ± 2 ◦ C with a 12 h light/dark cycle.
Preliminary experiments were conducted to select the
bixin dose, which ranged between 0.5 and 5.0 mg kg−1
body wt. The highest dose of bixin was selected after
ensuring that the administration of the carotenoid alone
did not induce chromosome aberrations compared to
the control group. The administration of ethanol plus
distilled water (1 : 9) did not induce any significant
change in the parameters investigated when compared to
controls that received only distilled water. In the definitive
experiments, the animals were divided into six treatment
groups with six rats in each (n = 6). The cisplatin dose
(5.0 mg kg−1 body wt.) was injected intraperitoneally
(1.0 ml 100 g−1 body wt.). The animals received three
pretreatments with bixin (2.5 or 5.0 mg kg−1 body wt.).
Bixin (1 ml 100 g−1 body wt.) was given by gavage for
three consecutive days, at 48, 24 h and 10 min before
cisplatin intraperitoneal injection. The control group was
given the same volume of water by gavage and saline
intraperitoneally as the treated animals.
Group I animals received three doses of distilled water
by gavage and were injected with saline intraperitoneally.
This group served as negative control. Groups II and
III received three doses of bixin by gavage (2.5 or
5.0 mg kg−1 body wt., respectively) and saline intraperi-
Pharmacological Research, Vol. 43, No. 6, 2001
toneally. Group IV received three doses of distilled water
by gavage and were injected with cisplatin (5.0 mg kg−1
body wt.) intraperitoneally. Groups V and VI received
the respective doses of bixin by gavage and each animal
was injected with cisplatin intraperitoneally. At the end
of the experiments, animals of each group were killed by
decapitation. The femurs and kidneys were removed and
the kidneys kept in an ice bath until homogenization.
Wistar rat bone marrow cell system
All animals were injected intraperitoneally with
2.0 mM colchicine 90 min before being killed by
decapitation, which occurred 24 h after cisplatin
treatment. Bone marrow preparations for the analysis
of chromosome aberrations in metaphase cells were
obtained by the technique of Ford and Hamerton [22].
One hundred metaphases per animal were analysed
in order to determine the frequencies of chromosome
aberrations in a blind test. The mitotic index was obtained
by counting the number of mitotic cells in 1000 cells
analysed per animal.
Biochemical assays
Reduced glutathione was determined as non-protein
and total sulphydryl contents in rat kidneys using the
method of Sedlak and Lindsay [23]. Briefly, 0.25 g
of tissue was homogenized in 5.0 ml of cold KCl
(1.15%) solution using motor-driven tissue homogenizers
(Marconir ). The samples were precipitated with 50%
trichloroacetic acid and then centrifuged at 1200 g
for 10 min. The reaction mixture contained 0.5 ml of
supernatant, 2.0 ml of Tris-EDTA buffer (0.2 M; pH 8.9),
and 0.1 ml of 0.01 M 5,50 -dithio-bis-2-nitrobenzoic acid.
The solution was kept at room temperature for 15 min,
and then was read at 412 nm on a spectrophotometer
(Genesys, Spectronic Instruments). The determination of
protein was measured by the modification of the Lowry
method that gives a linear photometric response, and the
samples were read in triplicate at 650 nm [24]. The results
were expressed in µmol glutathione g−1 of protein.
The production of thiobarbituric acid-reactive
substances (TBARS) was measured by the method of
Uchiyama and Mihara [25] to assess lipid peroxidation.
0.5 ml of the same homogenized tissue, 3.0 ml of 1%
H3 PO4 and 1.0 ml of thiobarbituric acid (0.6%) were
added to the tubes. The tubes were heated for 45 min in
a boiling water bath and the reaction mixture was then
cooled in an ice bath followed by the addition of 4.0 ml
of n-butanol. The contents were mixed for 40 s with a
vortex mixer, centrifuged at 1200 g for 10 min, and the
absorption of the organic layer was measured at 520 and
535 nm. The results were expressed in nmol of lipid
peroxidation g−1 of tissue. All assays were made using
samples in triplicate from each animal.
Statistical analysis
The differences in the number of cells with chromo-
some aberrations, abnormal metaphases and mitotic in-
Pharmacological Research, Vol. 43, No. 6, 2001 563

Table I
Mitotic index, distribution of the different types of chromosome aberrations and abnormal metaphases observed in Wistar rat bone marrow
cells pretreated with bixin (BXN; 2.5 or 5.0 mg kg−1 body wt.) alone or in combination with cisplatin (DDP; 5.0 mg kg−1 body wt.). Animals
were killed 24 h after cisplatin treatment

Treatment MI Types of chromosome aberrations AM

(%) Gaps Breaks E T Q N o. (%)
C IC
Control 2.6 3 7 0 0 0 0 10 (1.7)
BXN 2.5 2.3 6 7 0 0 0 0 13 (2.0)
BXN 5.0 2.7 2 6 0 0 0 0 8 (1.3)
DDP 1.5a 15 112 13 9 2 0 111a (18.5)
BXN 2.5 + DDP 2.0 12 94 13 6 1 0 88a (14.7)
BXN 5.0 + DDP 2.0 6 81 7 12 0 1 74b (12.3)

One-hundred cells were analysed per animal for chromosome aberrations, for a total of 600 metaphases per treatment. The mitotic index was
obtained by counting 6000 mitotic cells per treatment. MI = mitotic index, C = chromatid type, IC = isochromatid type, E = complex exchange,
T = triradial figure, Q = quadriradial figure, AM = abnormal metaphase. a Significantly different from the control group (P< 0.05). b Significantly
different from the DDP group (P< 0.05).

Table II
Effects of bixin (BXN) on the total of chromosome aberrations induced by cisplatin (DDP) in Wistar bone marrow cells. Animals were killed
24 h after cisplatin treatment

Treatment Cells with aberrations Total of

0 1 2 3 4 5 aberrations
Control 590 10 0 0 0 0 10
BXN 2.5 587 13 0 0 0 0 13
BXN 5.0 592 8 0 0 0 0 8
DDP 489 92 8 5 2 4 151a
BXN 2.5 + DDP 512 68 10 5 2 3 126a
BXN 5.0 + DDP 526 59 7 2 2 4 107b
a Significantly different from the control group (P< 0.05). b Significantly different from the DDP group (P< 0.05).

dex between cells treated with bixin and/or cisplatin were
analysed statistically by the Fisher exact test, with the
level of significance set at α = 0.05. Statistical analysis
of the biochemical assays was performed using analysis
of variance (ANOVA). Differences between treatments
were determined by the Mann–Whitney test. The results
were expressed as the mean ± SD of six values in each
group, and a statistical probability of P< 0.05 was con-
sidered to be significant.
RESULTS
Chromosome aberrations
The results of the chromosome aberrations analysis in
bone marrow cells of male Wistar rats treated with bixin,
cisplatin alone or bixin plus cisplatin are presented in
Table I. The groups treated with bixin (2.5 or 5.0 mg kg−1
body wt.) did not show any significant variation in the
total number of chromosome aberrations or abnormal
metaphases compared to the control values.
As expected, animals treated with a single dose of
cisplatin (5.0 mg kg−1 body wt.) presented a high
frequency in both the total number of chromosome
aberrations and abnormal metaphases (P< 0.05), in
bone marrow cells observed 24 h following treatment
with cisplatin, compared to controls (Tables I and II).
In general, one aberration per cell was observed in
animals treated with cisplatin, but cells with four or
five aberrations were also observed (Table II). The
chromosome aberrations detected at highest frequency
were chromatid-type breaks, followed by isochromatid-
type breaks, gaps, complex exchanges, and other
rearrangements such as triradial and quadriradial figures.
The data showed that the lowest dose of bixin
administered presented a reduction of about 21% in
cisplatin-induced abnormal metaphases, when compared
to the animals treated with cisplatin alone (Table I).
However, the Fisher test showed that this reduction
was not statistically significant. The highest dose of
bixin appeared to be more efficient in decreasing the
frequency of chromosome aberrations and abnormal
metaphases induced by cisplatin in bone marrow cells
(Tables I and II). The treatment with the highest dose of
bixin resulted in a statistically significant reduction, by
about 33%, in cisplatin-induced abnormal metaphases
(P< 0.05) compared to the cisplatin group (Table I).
No significant difference in mitotic index values was
observed between the animals that received bixin alone
compared to the controls. The animals treated with
cisplatin presented a significantly lower mitotic index
compared to the controls (Table I). Both doses of bixin
protected against the slight inhibitory effect of cisplatin
on mitotic index.
Lipid peroxidation and glutathione level
TBARS production in the kidney tissue had been
used as a measure of lipid peroxidation. The TBARS
564
Table III
Effects of bixin (BXN; 2.5 or 5.0 mg kg−1 body wt.) or its combination with a single dose of cisplatin (DDP; 5 mg kg−1 body wt.) on levels of
renal glutathione (GSH) and lipid peroxidation (MDA) in rats killed 24 h after cisplatin treatment
Treatment GSH
(µmol GSHg−1 of protein)
Control 28.59 ± 3.61
BXN 2.5 27.16 ± 5.36
BXN 5.0 26.94 ± 6.00
DDP 20.21 ± 3.03a
BXN 2.5 + DDP 31.75 ± 6.86b
BXN 5.0 + DDP 33.48 ± 3.85b
Each value represents mean ± SD (standard deviation) of six animals. a Significantly different from the control group (P< 0.05). b Significantly
different from the DDP group (P< 0.05).
production was similar in groups treated with bixin and
in the control group. The administration of both doses of
bixin alone did not increase lipid peroxidation compared
to the control group. Table III shows the results for all the
groups. A single dose of cisplatin enhanced the formation
of lipid peroxides in 29% compared to the control
group. This increase was prevented by pretreatment with
bixin at both doses tested (P< 0.05). The values of
lipid peroxidation were restored to approximately normal
levels in the rats pretreated with bixin plus cisplatin
(Table III).
The effect of pretreatment of adult rats with the
carotenoid bixin on cisplatin-mediated changes in the
levels of renal glutathione is shown in Table III. The
cisplatin treatment resulted in a 29% depletion in renal
glutathione 24 h after cisplatin administration compared
to the control group (P< 0.05). The administration of
bixin induced a slight increase of renal glutathione in an-
imals injected with cisplatin. However, this enhancement
on renal glutathione level was not statistically significant
when compared to the control group. The levels of renal
glutathione were also restored to normal in the rats pre-
treated with the carotenoid bixin plus cisplatin compared
to the control group (Table III). The carotenoid bixin was
effective in inhibiting lipid peroxidation and glutathione
renal depletion induced by cisplatin in Wistar rats.
DISCUSSION
The commercial extract of Bixa orellana seed, known
as Annatto, is rich in C24 -apocarotenoid bixin. Purified
bixin is used in a number of formulations in the food
industry, for example in coloring bitter, cheese and ice
cream [26]. In this study we evaluated the role of the
carotenoid bixin on cisplatin-induced oxidative stress in
Wistar rats through three markers of oxidative damage:
chromosome aberrations, glutathione depletion and lipid
peroxidation.
The clastogenic action of cisplatin in somatic cells in
vivo has already been described [27, 28]. Chromatid-type
aberrations were the main type of defects detected among
the chromosomal aberrations induced by cisplatin, and,
Pharmacological Research, Vol. 43, No. 6, 2001
% of MDA % of
control (nmol MDA g−1 of tissue) control
100 130.20 ± 7.35 100
95 126.77 ± 18.02 97
94 123.00 ± 17.64 94
71 167.70 ± 22.19a 129
111 123.70 ± 14.01b 95
117 117.66 ± 34.42b 90
in general, one aberration per cell was observed. This
is in line with data obtained from mice bone marrow
cells [27]. It has been reported that antitumor agents
produce a high frequency of aberrations in rodents 24 h
after a single dose [29].
The administration of bixin per se did not produce any
significant change in the parameters investigated when
compared to controls. The results obtained by the pre-
treatment with the highest dose of bixin showed a sig-
nificant reduction in the total number of chromosomal
aberrations and in the number of abnormal metaphases
induced in bone marrow cells by cisplatin. In the present
study the protective activity of bixin was statistically sig-
nificant when the animals were treated with the highest
dose of bixin plus cisplatin, although a tendency towards
reducing the cisplatin-induced chromosome aberrations
was also observed with the lowest dose of bixin. The pro-
tective effects of bixin and β-carotene against chromo-
some damage induced by radiation or cyclophosphamide
in mice were observed by Salvadori et al. [30] and Thre-
siamma et al. [31]. Although the exact mechanism un-
derlying these protective effects of bixin is still not fully
understood, the antioxidant characteristics of this com-
pound are likely to be involved.
One of the most important intracellular antioxidant
systems is the glutathione redox cycle. Glutathione is one
of the essential compounds for maintaining cell integrity
because of its reducing properties and participation in
the cell metabolism [32]. Furthermore, cisplatin-induced
glutathione depletion is a determinant step in oxidative
stress in kidney tissue that leads to nephrotoxicity [33].
The depletion of levels of renal glutathione has been
observed in rats in response to oxidative stress caused by
cisplatin 7 days after its administration [14, 33].
In the present study, a single dose of cisplatin was
able to induce glutathione renal depletion 24 h after
intraperitoneal treatment. In the groups treated with bixin
plus cisplatin, it was observed that the administration with
both doses of bixin prevented the depletion of renal glu-
tathione caused by cisplatin, resulting in values close to
those observed in the control group. These results suggest
the possible role of bixin-mediated protection, given as a
pretreatment, against cisplatin-induced depletion of renal
Pharmacological Research, Vol. 43, No. 6, 2001
glutathione could be ascribed to its antioxidant properties.
Several investigations have shown that cisplatin
nephrotoxicity is associated with lipid peroxidation [34,
35]. Cisplatin generates reactive oxygen species such
as superoxide anions and hydroxyl radicals that lead to
renal lipid peroxidation [7]. Renal lipid peroxidation,
determined by TBARS production, was increased 3, 5
or 7 days after a single dose of cisplatin [9, 14, 35].
In the present investigation, a significant increase in
lipid peroxidation was observed 24 h after cisplatin
administration. Cisplatin-induced lipid peroxidation in
kidney was inhibited by the pretreatment with both doses
of the carotenoid bixin. Carotenoids, such as β-carotene,
lycopene, zeaxanthin and lutein, exert antioxidant
functions in lipid phases by quenching singlet oxygen or
free radicals [17].
The possible explanation for the protective effects
of bixin against the cisplatin-induced increase in lipid
peroxidation and renal glutathione depletion in this study
is its ability to react with the oxygen metabolites, that is,
scavenging free radicals and reactive oxygen molecules
generated by cisplatin. Di Mascio et al. [20] observed
that the carotenoid bixin was one of the more effective
biological singlet molecular-oxygen quenchers. Bixin
was an active carotenoid that inhibited lipid peroxidation
in C3H/10T1/2 cells [21]. Bixin also inhibited TBARS
production in rat peripheral macrophages, and this
could be the mechanism by which carotenoids in vivo
protect cells and tissues from damage induced by oxygen
metabolites [18].
Other antioxidants studied, such as eugenol, silibinin
and cysteine, showed protection against cisplatin-
induced renal toxicity in vivo [13, 36, 37]. In the present
investigation, the pretreatment with bixin reduced the
total number of chromosome aberrations, and inhibited
the increase in lipid peroxidation and renal glutathione
depletion induced by cisplatin. Since the pretreatment
with bixin alone was safe under the present experimental
conditions, the results suggest that bixin may have a
future clinical application after further study.
ACKNOWLEDGEMENTS
This investigation was supported by FAPESP. The
authors are grateful to Ms Heloı́sa D. C. Francescato
and Ms Luciana Mora for their help, and to Joana D’arc
C. Darin for technical assistance.
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