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Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide induced genotoxicity in human lymphocyte culture

Authors:
  • CuraTeQ Biologics (Formerly Aurobindo Biologics)
  • Jawaharlal Nehru Cancer Hospital & Research Centre

Abstract and Figures

Diets rich in fruits and vegetables have been associated with reduced risks for many types of cancers. Avocado (Persea americana Mill.) is a widely consumed fruit containing many cancer preventing nutrients, vitamins and phytochemicals. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with 50% Methanol from avocado fruits help in proliferation of human lymphocyte cells and decrease chromosomal aberrations induced by cyclophosphamide. Among three concentrations (100 mg, 150 mg and 200 mg per Kg Body Weight), the most effective conc. of extract was 200 mg/Kg Body Wt. It decreased significant level of numerical and structural aberrations (breaks, premature centromeric division etc. up to 88%, p < 0.0001)), and accrocentric associtation within D & G group (up to 78%, p = 0.0008). These studies suggest that phytochemicals from the avocado fruit can be utilized for making active chemoprotective ingredient for lowering the side effect of chemotherapy like cyclophosphamide in cancer therapy.
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Avocado fruit (Persea americana Mill) exhibits chemo-protective
potentiality against cyclophosphamide induced genotoxicity in
human Lymphocyte culture
Rajkumar Paul1,2, Paresh Kulkarni1,3 and Narayan Ganesh1
1Department of Research, Jawaharlal Nehru Cancer Hospital and Research Centre, Idgah Hills, Bhopal, India
2School of Biotechnology, Rajiv Gandhi Technological University, Bhopal, India
3Bangalore Medical College & Research Institute, Fort, K. R. Road, Bangalore-560002 India
Correspondence to: Rajkumar Paul, Department of Research, Jawaharlal Nehru Cancer Hospital & Research Centre,
Idgah Hills, P. B. No. 32, Bhopal - 462001, Madhya Pradesh, India; Telephone: +91 755 2665720, +919634447622,
Fax: +91 755 2738325, E-mail: rajkumarpaul81@gmail.com
(Received December 30, 2010; revised June 16, 2011; accepted June 28, 2011)
Diets rich in fruits and vegetables have been associated with
reduced risks for many types of cancers. Avocado (Persea
americana Mill.) is a widely consumed fruit containing many
cancer preventing nutrients, vitamins and phytochemicals.
Studies have shown that phytochemicals extracted from
the avocado fruit selectively induce cell cycle arrest, inhibit
growth, and induce apoptosis in precancerous and cancer
cell lines. Our recent studies indicate that phytochemicals
extracted with 50% Methanol from avocado fruits help
in proliferation of human lymphocyte cells and decrease
chromosomal aberrations induced by cyclophosphamide.
Among three concentrations (100 mg, 150 mg and 200 mg
per Kg Body Weight), the most effective conc. of extract
was 200 mg/Kg Body Wt. It decreased significant level of
numerical and structural aberrations (breaks, premature
centromeric division etc. up to 88%, p < 0.0001)), and
accrocentric associtation within D & G group (up to 78%,
p = 0.0008). These studies suggest that phytochemicals
from the avocado fruit can be utilized for making active
chemoprotective ingredient for lowering the side effect of
chemotherapy like cyclophosphamide in cancer therapy.
Key words: Cyclophosphamide, Anti-cancer drug, Ge-
notoxicity, Avocado fruit, Persea americana, Chemo-
protective
Abbreviation: CP: Cyclophosphamide, PA: Persea
Americana, RPMI: Rosewell Park Memorial Institute,
PHA: Phytohaematoagglutinin, AML: Acute Myeloid
Leukemia, ALL: Acute Lymphoid Leukemia, CML: Chronic
Myeloid Leukemia, CLL: Chronic Lymphoid Leukemia
INTRODUCTION
Chemotherapy targets cancer cells, which are
unhealthy cells that divide and reproduce quickly in the
stomach, mouth, skin, hair and bone marrow (1-2). Some
chemotherapy drugs can reduce the number of white
blood cells in the blood, which can make a child prone
to infection causing “lowered immunity”. Alterations
of lymphocyte subpopulations by chemotherapy and
chemoimmunotherapy (Thymostimulin) in patients
with head and neck cancer were observed (3). Chromo-
some aberrations were seen after etoposide containing
cisplatin-based chemotherapy for malignant germ-cell
tumours (4). Thiotepa, etoposide, and paclitaxel pro-
duced a statistically significant higher percentage of
abnormal metaphases in the rhesus monkey (Macaca
mulatta) following bolus chemotherapy (p<0.0001,
p=0.0015 and p<0.0001 respectively) (5).
Cyclophosphamide is an inactive cyclic phosphamide
ester of mechlorethamine. It is converted by hepatic and
intracellular enzymes to active alkylating metabolites, 4-
hydroxycyclophophosphamide, aldophosphamide, acro-
lein and phosphoramide mustard. Cyclophosphamide
causes prevention of cell division primarily by cross-
linking DNA and RNA strands leading to an increasing
inhibition of DNA polymerase activity (6). Cyclophos-
phamide is a potent immunosuppressive agent that exerts
its effect through metabolic degradation of products
(7-8). Cyclophosphamide exhibits antimetabolic effect
on hepatic cells after activation by hepatic enzymes.
Rajkumar Paul et al.
222 Journal of Experimental Therapeutics and Oncology Vol. 9 2011
It also accelerates the degeneration of cells in vitro by
inhibiting growth rate (9). CP is not metabolized within
pulmonary artery endothelial cells and acrolein may be
the principal CP metabolite involved in mediating direct
injury to pulmonary artery endothelial cells (10). 4-hy-
droxycyclophosphamide, a product of CP causes dena-
turation of microsomal cytochrome P-450 which has
role in in the oxidative detoxification and activation of
carcinogens, teratogens, mutagens, and toxicants (11).
Cyclophosphamide-induced chromosomal aberrations
and associated congenital malformations were seen in
rats (12). Many scientists think about the involvement of
CP in bladder cancer development (13) and, this is due to
mutation in p53 gene (14). More over, it was seen that CP
induced transitional cell carcinoma in a renal transplant
patient (15).
Epidemiological studies showing a protective effect
of diets rich in fruits and vegetables against cancer have
focused attention on the possibility that biologically-ac-
tive plant secondary metabolites exert anti-carcinogenic
activity (16). This huge group of compounds, now col-
lectively termed ‘phytochemicals’, provides much of
the flavour and colour of edible plants and the beverages
derived from them (17-18). Many of these compounds
also exert anti-carcinogenic effects in animal models of
cancer, and much investigation has been performed in
defining their many biological activities at the molecu-
lar level (19). Glucobrassicin derivative seems to have a
preventive potential against cyclophosphamide induced
chromosomal aberrations in Swiss mouse bone marrow
cells at the doses tested (20). Chemoprevention using
pharmacological doses of isolated compounds, or the
development of ‘customised’ vegetables, may prove
valuable but such strategies require a full risk–benefit
analysis based on a thorough understanding of the long-
term biological effects of what are often surprisingly
active compounds (21). Various morphological parts
of Persea americana Mill (avocado) are widely used
in African traditional medicines (22) for the treatment,
management and/or control of a variety of human ail-
ments, including childhood convulsions and epilepsy
(23-26). The combination of pressurized liquid extrac-
tion with LC-MS/MS provides a sensitive and selective
method for determining nine benzoylureas (BUs) in
fruit, vegetable, cereals, and animal products (27).
Studies have shown that phytochemicals extracted
from the avocado fruit selectively induce cell cycle arrest,
inhibit growth, and induce apoptosis in precancerous and
cancer cell lines (28). Phytochemicals e.g. persin, perse-
nones A and B extracted with chloroform from avocado
fruits target multiple signaling pathways and increase
intracellular reactive oxygen leading to apoptosis refer-
ring the avocado fruit an advantageous dietary strategy
in cancer prevention (29-30). High levels of reactive
oxygen (ROS) in human oral cancer cell lines may be
a key factor in selective apoptosis and molecular target-
ing for chemoprevention by phytochemicals (31). Alpha-
lipoic acid, an antioxidant, induces apoptosis through
the intrinsic pathway in hepatoma cells by increasing
the levels of ROS followed by p53 and Bax and by
down regulating cell cycle regulatory proteins (32). In
vitro studies in a panel of human breast cancer cell lines
showed that persin selectively induces a G2-M cell cycle
arrest and caspase-dependent apoptosis in sensitive cells
and persin acts as a microtubule-stabilizing agent (33).
Luteolin inhibits xanthine oxidase-generated superoxide
formation and reduces LPS-induced hydroxyl radical
formation (34). An acetone extract of avocado containing
carotenoids and tocopherols also inhibits the growth of
both androgen-dependent (LNCaP) and androgen-inde-
pendent (PC-3) prostate cancer cell lines in vitro (35). A
marked fall in mean arterial blood pressure which lasted
2–3 min after intravenous administration of constituents
of the leaves of P. americana in anaesthetized normoten-
sive rats was due to rapid metabolism (36). The effect
of aqueous and methanolic leaf extracts of P. americana
lower plasma glucose and influence lipid metabolism
in hypercholesterolemic rats with consequent lower-
ing of T-CHOL and LDL-CHOL and a restoration of
HDL-CHOL levels. This could represent a protective
mechanism against the development of atherosclerosis
(37). The methanol leaf extract of PA dose-dependently
protected against acute hepatotoxicity induced by Para-
cetamol (PCM) by increasing the activity of the antioxi-
dant enzymes and preventing GSH depletion (38). The P.
americana Mill (Lauraceae) aqueous leaf extract (PAE)
causes bradycardia, vasorelaxation and hypotension in
the mammalian experimental models used. P americana
leaf could be used as a natural supplementary remedy in
essential hypertension and certain cases of cardiac dys-
functions in some rural Africa communities (39). Persin
causes Bim dependent apoptosis in human breast can-
cer cells (33). Where as, Quercetin down-regulates the
expression of bcl-2 in the xenograft B16M-F10 cells
which facilitates endothelium-induced tumor cytotoxic-
ity in B16M-F10 cells (40).
MATERIALS AND METHOD
The project was sanctioned by Board of ethical
committee of our Institute (JNCHRC, Bhopal)”. The
approval ref no is 1777/225/19.10.2005. This work was
a part of this project.
1. Plant Extract preparation
Fresh fruits of Persea americana were collected from
the campus of Kidwai Memorial Institute of Oncology,
Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide …
Journal of Experimental Therapeutics and Oncology Vol. 9 2011 223
Bangalore. The fruits were shade-dried at room temper-
ature. The fruit pulp was macerated with 50% metha-
nol and extracted once with 300 ml of 50% methanol at
room temperature for one week with occasional shak-
ing. The methanol extract of fruit was concentrated to
dryness at 60 ± 1ºC in a rotary evaporator. Freeze drying
and solvent elimination under reduced pressure finally
gave a light-brown, oily, viscous extract. These crude
extract were used in our study as solutions/suspensions
in double distilled water after removing the particulate
matter by centrifugation and sterilization using syringe
filters.
2. Culture Establishment
The peripheral blood was taken from a healthy nor-
mal volunteer at department of Pathology, JNCHRC,
Bhopal. Each Experimental culture bottle contained
5 ml of RPMI-1640 culture media, 0.5 ml of blood, 800
µl (10% of culture medium) of donor’s blood serum and
300 µl of PHA (40 µg/ml). 28 µl of Cyclophosphamide
(20 mg/ml) was added in each culture (560 µg/5 ml cul-
ture as 28 ml required for a patient i.e. 5 L blood). Fruit
extracts (100, 200, and 300mg/kg of blood culture) was
taken in experimental culture along with control (No
extract). The culture was maintained at 37ºC and 5%
CO2 in a CO2 incubator for 70 hrs.
3. Pretreatment and Harvesting of Culture
After 70 hrs, 50 µl of colchicine (200 µg/ml) was
added in each culture and then transferred in CO2 incu-
bator for 45 minutes. The cultures were centrifuged
at 700 rpm for 10 minutes and the supernatant was
discarded. 0.56% KCl solution was added in cultures
up to 1/3 of the tube while cyclomixing. The tubes
were kept at 37ºC and 5% CO2 in a CO2 incubator for
45 minutes. The tubes were taken out and centrifuged
again at 700 rpm for 10 minutes and the supernatant
was discarded. Freshly prepared and Chilled Cornu-
oy’s fixative (Acetic acid: Methanol = 1:3) was added
to the pellets drop by drop up to 1/3 of the tube while
cyclomixing. Then, centrifugation was done again at
700 rpm for 10 minutes and the supernatant was dis-
carded. The process was repeated thrice. White colour
pellets were cyclomixed with the above mentioned
fixative and kept at 4ºC in a refrigerator. Lymphocytes
solution was dropped down (3-4 drops for each slide)
on slide from a height by air-drop method. The excess
fixative was allowed to run off by tilting. The slides
were allowed to air-dry for half-an-hour and then,
transferred at 56ºC in an incubator for 2 hrs. The slides
were dipped in Giemsa stain for 10 minutes followed
by dip once in double distilled water.
4. Microscopy
The slides were observed under ordinary light micro-
scope with lowered condenser and reduced illumina-
tion. 100 well-spread second division metaphases were
scored for chromosomal analysis. The number of total
aberrant metaphases, metaphases with numerical aber-
rations, metaphases with structural aberrations (exclud-
ing gaps) and metaphases with acrocentric associations
were scored. The number and type of chromosomes
involved in the association were also scored.
5. Scoring and Statistical Analysis
Statistical analysis of the data was performed using
one-tailed Student’s t-test.
RESULT
(A) Fruit extract significantly reduced cyclophos-
phamide-induced chromosomal aberrations in
a concentration dependent manner
In this experiment, the means of total aberrations per
100 metaphases in the culture groups exposed to both
cyclophosphamide and fruit extract were found to be 68
± 13.23, 36 ± 7.43 and 20 ± 7.00 at fruit extract concen-
trations of 100mg/kg, 150mg/kg and 200mg/kg respec-
tively (Table 1, Fig. 2-4,9). The mean total aberrations
per 100 metaphases in the culture group exposed to
cyclophosphamide alone was found to be 118 ± 17.77,
which is significantly higher than those found in groups
also exposed to fruit extract concentrations of 100mg/
kg (p = 0.0131), 150mg/kg (p < 0.0001) and 200mg/
kg (p < 0.0001). Also, the mean total aberrations per
100 metaphases in the culture group exposed to 100mg/
kg of fruit extract along with cyclophosphamide was
found to be significantly higher than those exposed to
150mg/kg (p = 0.0188) and 200mg/kg (p = 0.0009)
fruit extract. This shows that the fruit extract containing
active components reduced cyclophosphamide-induced
chromosomal aberrations in a concentration dependent
manner.
(B) Fruit extract completely neutralized cyclophos-
phamide-induced numerical aberrations
The culture group exposed to cyclophosphamide
alone showed 12 ± 4.64 mean percentage aberrant met-
aphases with numerical aberrations. The most com-
mon numerical aberration was found to be aneuploidy
(Fig 10, Table 2). However, the culture groups exposed
Rajkumar Paul et al.
224 Journal of Experimental Therapeutics and Oncology Vol. 9 2011
Table 1. Mean aberrant Metaphases in different culture conditions
Experiment
No.
Cultures
Mean aberrations per hundred metaphases
Numerical
aberrations
Structural
aberrations
Premature
centromeric
division
Acrocentric
associations
Total
aberrations
PA‘CC Control 0 0 0 4±2.80 4 ± 2.80
PACD Control with CP
(112 mg/L)
12 ± 4.64 34 ± 7.34 14 ± 4.96 58 ± 12.15 118 ± 17.77
PA‘CE1 Fruit Ext
(100 mg/Kg Body Wt.)
0 0 2 ± 2 8 ± 3.88 10 ± 4.29
PA‘CE2 Fruit Ext
(150 mg/Kg Body Wt.)
0 0 2 ± 2 14 ± 5.72 16 ± 5.97
PA‘CE3 Fruit Ext
(200 mg/Kg Body Wt.)
0 0 2 ± 2 16 ± 5.97 18 ± 6.82
PA‘DE1 Fruit Ext
(100 mg/Kg Body Wt.)
+ CP (112 mg/L)
0 14 ± 5.72 12 ± 4.64 42 ± 9.93 68 ± 13.23
PA‘DE2 Fruit Ext
(150 mg/Kg Body Wt.)
+ CP (112 mg/L)
0 8 ± 3.88 6 ± 3.39 22 ± 6.57 36 ± 7.43
PA‘DE3 Fruit Ext
(200 mg/Kg Body Wt.)
+ CP (112 mg/L)
0 2 ± 2 4 ± 2.80 14 ± 5.72 20 ± 7.00
RT
1 (A) 1 (B)
PCD
TD
1 (C) 1 (D)
Figure 1. (A) - 1(D): Photographs of Metaphasic spreads showing (A) Normal Metaphase (B) Robertsonian
Translocation (C) Terminal Deletion and (D) Premature Centromeric division. (PCD – Premature Centromeric
division, TD - Terminal deletion, RT – Robertsonian translocation).
Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide …
Journal of Experimental Therapeutics and Oncology Vol. 9 2011 225
Table 2. Mean numerical aberrations in different culture conditions
Exp. No.
Mean numerical aberrations (per hundred metaphases counted)
Chromosome
Number 42
Chromosome
Number 43
Chromosome
Number 44
Chromosome
Number 45
Chromosome
Number 47
Chromosome
Number 52
Ploidy Total
PA‘CC 0 0 0 0 0 0 0 0
PACD 0 3 1 2 0 1 0 6
PA‘CE1 0 0 0 0 0 0 0 0
PA‘CE2 0 0 0 0 0 0 0 0
PA‘CE3 0 0 0 0 0 0 0 0
PA‘DE1 0 0 0 0 0 0 0 0
PA‘DE2 0 0 0 0 0 0 0 0
PA‘DE3 0 0 0 0 0 0 0 0
AA
(
DG
)
AA
(
DD
)
2 (A) 2 (B)
AA
(
DGG
)
Rosette
2 (C) 2 (D)
Figure 2. (A) – 2(D): Photographs of Metaphasic spreads showing different Acrocentric associations (A)
Acrocentric association within chromosomes of DD group (B) Acrocentric association between chromosome of
D group and chromosome of G group (C) Acrocentric association between one chromosome of D group and two
chromosomes of G group and (D) Acrocentric association forming Rosette. (AA – acrocentric association, DD - Two
chromosomes of D group, DG - one chromosome of D group and another of G group, DGG - one chromosome
of D group and other two of G group, Rosette – Association of chromosomes of D and G group forming Rosette
structure).
to fruit extract along with cyclophosphamide showed
no numerical aberrations. This indicates that the fruit
extract completely neutralized the cyclophosphamide
induced numerical aberrations. These compounds
might act in this experiment through their antioxidant
activity.
(C) Fruit extract significantly reduced cyclophos-
phamide-induced clastogenic effect
In this experiment, the mean percentage of struc-
tural aberrations excluding acrocentric association and
premature centromeric separation in the culture groups
Rajkumar Paul et al.
226 Journal of Experimental Therapeutics and Oncology Vol. 9 2011
exposed to both cyclophosphamide and fruit extract were
found to be 14 ± 5.72, 8 ± 3.88 and 2 ± 2 at fruit extract
concentrations of 100mg/kg, 150mg/kg and 200mg/kg
respectively (Table 3, Fig. 2,3,4,11). The mean percent-
age structural aberrations in the culture group exposed
to cyclophosphamide alone was found to be 34 ± 7.35,
which is significantly higher than those found in groups
also exposed to fruit extract concentrations of 100mg/
Table 3. Mean structural aberrations in different culture conditions
Experiment No.
Mean structural aberrations (per hundred metaphases counted)
Dicentrics Breaks Single
Minute
Robertsonian
Translocation
Terminal
deletion
Total
PA‘CC 0 0 0 0 0 0
PACD 4 16 6 2 6 34
PA‘CE1 0 0 0 0 0 0
PA‘CE2 0 0 0 0 0 0
PA‘CE3 0 0 0 0 0 0
PA‘DE1 0 8 2 0 4 14
PA‘DE2 0 2 2 0 4 8
PA‘DE3 0 0 0 0 2 2
0
10
20
30
40
50
60
70
Numerical aberrations Structural ab errations Acrocent ric
associat ions
Chromosomal A berrations
Aberrant Metaphases (%)
PA‘CC
PACD
PA‘CE1
PA‘CE2
PA‘CE3
PA‘DE1
PA‘DE2
PA‘DE3
3(A)
0
0.5
1
1.5
2
2.5
3
3.5
PA‘CC
PACD
PA‘CE1
PA‘CE2
PA‘CE3
PA‘DE1
PA‘DE2
PA‘DE3
Ex pe ri mental setups
Numerical chromosomal aberration (%)
Chromosome Number 42
Chromosome Number 43
Chromosome Number 44
Chromosome Number 45
Chromosome Number 47
Chromosome Number 52
Ploidy
3(B)
0
2
4
6
8
10
12
14
16
18
Dicentrics Breaks Single
Minute
RT Termina l
deletion
Type s of Structural Ab erra tions
Aberrant metaphases (%)
PA‘CC
PACD
PA‘CE1
PA‘CE2
PA‘CE3
PA‘DE1
PA‘DE2
PA‘DE3
3(C)
0
2
4
6
8
10
12
14
16
18
20
DD
DG
GG
DDG
DGG
DDD
GGG
Rosette
Type s of Acroce ntric associations
Aberrant metaphases (%)
PA‘CC
PACD
PA‘CE1
PA‘CE2
PA‘CE3
PA‘DE1
PA‘DE2
PA‘DE3
3(D)
Figure 3. (A) - 3(D). Showing graphical representation of different chromosomal abnormalities: (3.A) Aberrant
metaphases with different chromosomal aberration in different culture conditions (3.B) Numerical Aberrant
metaphases with different chromosome numbers in different culture conditions (3.C) Structural Aberrant metaphases
with different types of aberrations in different culture conditions (3.D) Aberrant metaphases with different types of
Acrocentric association in different culture conditions.
Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide …
Journal of Experimental Therapeutics and Oncology Vol. 9 2011 227
kg (p = 0.0171), 150mg/kg (p = 0.0011) and 200mg/
kg (p < 0.0001). Thus, the fruit extract was found to
significantly reduce the clastogenic effect of cyclophos-
phamide. The various types of structural aberrations in
different culture groups have been described in Table 3.
The antioxidants and chemoprotective chemicals of
avocado may help in reduction of clastogenic effect of
cyclophosphamide induced genotoxicity.
(D) Fruit extract at higher concentrations sig-
nificantly reduced cyclophosphamide-induced
acrocentric associations
The mean number of acrocentric associations per
100 metaphases in the culture groups exposed to both
cyclophosphamide and fruit extract were found to be 42
± 9.93, 22 ± 6.57 and 14 ± 5.72 at fruit extract concen-
trations of 100mg/kg, 150mg/kg and 200mg/kg respec-
tively (Table 4, Fig. 5,6,7,8,12). The mean number of
acrocentric associations per 100 metaphases in the
culture group exposed to cyclophosphamide alone was
found to be 58 ± 12.15, which is significantly higher
than those found in groups also exposed to fruit extract
concentrations of 150mg/kg (p = 0.0053) and 200mg/
kg (p = 0.0008), but not 100mg/kg (p = 0.1553). This
shows that the fruit extract at higher concentrations
reduced cyclophosphamide induced acrocentric asso-
ciations. The mean number of different types of acro-
centric associations is detailed in Table 4.
(E) Fruit extract had significant effect on cyclo-
phosphamide-induced premature centromeric
division only at very high concentrations
The mean percentage metaphases with premature
centromeric division in the culture groups exposed to
both cyclophosphamide and fruit extract were found
to be 12 ± 4.64, 6 ± 3.39 and 4 ± 2.80 at fruit extract
concentrations of 100mg/kg, 150mg/kg and 200mg/
kg respectively. The mean percentage metaphases with
premature centromeric division in the culture group
exposed to cyclophosphamide alone was found to be 14
± 4.96, which was significantly higher than those found
in groups also exposed to fruit extract concentration of
200mg/kg (p = 0.0411), but not 100mg/kg (p = 0.3845)
and 150mg/kg (p = 0.0930). This shows that the fruit
extract had no significant effect on cyclophosphamide-
induced premature centromeric division at lower fruit
extract concentrations. However, higher concentrations
of fruit extract showed a significant effect.
(F) Fruit extract itself exhibited some genotoxic
effect predominantly acrocentric associations
In this experiment, the means of total aberrations
per 100 metaphases in the culture groups exposed to
fruit extract alone were found to be 10 ± 4.29, 16 ±
5.97 and 18 ± 6.82 at fruit extract concentrations of
100mg/kg, 150mg/kg and 200mg/kg respectively (Fig
12). The mean total aberrations per 100 metaphases in
the control group was found to be 4 ± 2.80, which is
significantly lower than those found in groups exposed
to fruit extract concentrations of 150mg/kg (p = 0.0358)
and 200mg/kg (p = 0.0302), but not 100mg/kg (p =
0.1220). Hence, the fruit extract showed some genoto-
xic effect. The only major chromosomal abnormality
induced was acrocentric association. The mean number
of acrocentric associations per 100 metaphases in the
culture groups exposed to fruit extract alone were found
to be 8 ± 3.88, 14 ± 5.72 and 16 ± 5.79 at fruit extract
concentrations of 100mg/kg, 150mg/kg and 200mg/kg
respectively.
Table 4. Mean Acrocentric associations in different culture conditions
Experiment No.
Mean Acrocentric associations (per hundred metaphases counted)
DD DG GG DDG DGG DDD GGG Rosette Total
PACC 220000004
PACD 10 14 6 6 18 0 0 4 58
PACE1 422000008
PA‘CE2 4 4 6 0 0 0 0 0 14
PA‘CE3 2 8 4 0 2 0 0 0 16
PA‘DE1 6 10 4 6 14 0 0 0 21
PA‘DE2 4 4 2 2 10 0 0 0 22
PA‘DE3 6 4 0 0 4 0 0 0 14
Rajkumar Paul et al.
228 Journal of Experimental Therapeutics and Oncology Vol. 9 2011
DISCUSSION
Cyclophosphamide (CP) is extensively used in treat-
ment of different types of blood cancer. It is well charac-
terized for its beneficial effect as well as Genotoxicity.
Therefore, the use of chemoprotective agent with CP
has become necessary. Avocado is being used all over
world for its nutritional and medicinal properties. Here,
chromosomal aberration assay was adopted to evaluate
the potential effect of 50% Methanolic extract of Avo-
cado fruit as a chemoprotective agent on CP induced
genotoxicity.
A methanol extract of avocado fruits (2-hydroxy-4-
oxoheneicosa-5,12,15-trienyl, 2-hydroxy-4-oxoheneicosa-
12,15-dienyl), 2,4-dihydroxyheptadec-16-enyl and 2,4-
dihydroxyheptadec-16-ynyl acetates) showed potent
inhibitory activity against acetyl-CoA carboxylase, a
key enzyme in fatty acid biosynthesis (41). Previous
study showed that Persin selectively induced a G2-M
cell cycle arrest and caspase-dependent apoptosis in
sensitive cells dependending on expression of the BH3-
only protein Bim. Persin could represent a novel class
of microtubule-targeting agent with potential specifi-
city for breast cancers (42). From this experiment, it
is well characterized that persin and persin like com-
pounds present in avocado for which the extract reduced
cyclophosphamide-induced chromosomal aberrations
in a concentration dependent manner. Fruit extract
completely neutralized cyclophosphamide-induced
numerical aberrations. This may be due to the antioxi-
dant activity of phytochemicals from avocado extracts.
Persin and three further analogs namely 2,4 dihydrox-
yheptadec-16-enyl acetate, 2,4 dihydroxyheptadec-
16-ynyl acetate and Persenone A showed antioxidant
activity by inhibition of acetyl CoA carboxylase (ACC)
activity, in the IC50 value range 4.0–9.4 M (41).
Single doses of 40, 80 and 160 mg of cyclophos-
phamide per kg body weight of C57BL/6 male mice
induced structural chromosomal aberrations including
translocations and interfered with the normal develop-
ment of bivalents. The dose of 80 mg/kg yielded 0.6%
spermatocytes with translocations and 3% transloca-
tion carriers in F1 males. Robertsonian translocations
were also detected in 3 males (43). The antioxidants
and chemoprotective chemicals of avocado extract
may reduce the clastogenic effect of cyclophosphamide
induced genotoxicity as seen in this experiment. Perse-
none A at a concentration of 20 µM almost completely
suppressed both lipopolysaccharide and Interferon-γ
induced inducible Nitric oxide synthase and (iNOS)
and Cyclooxygenase (COX-2) in a mouse macrophase
cell line (34). Fruit extract at higher concentrations also
significantly reduced cyclophosphamide-induced acro-
centric associations.
Ethanol extract of avocado has potent inhibitory
effect on the proliferation and selective killing of
human oral malignant cells mediated via ROS induced
apoptosis (44). Thus, the premature centromeric divi-
sion was also successfully reduced by the phyto-
chemicals present in the avocado extract due to their
antioxidant properties. Although, the fruit extract had
no significant effect, however, higher concentrations
of fruit extract showed a significant effect on cyclo-
phosphamide-induced premature centromeric divi-
sion at lower fruit extract concentrations. As, it was
seen that Quercetin down-regulated the expression of
the Cdc6, CDK4 and cyclin D1 cell cycle genes, in
concert with growth inhibition and cell cycle arrest in
Caco-2 cells (45). Chloroform extract decreased the
levels of cyclin D, cyclin A, and cdk2, while increas-
ing the levels of p21WAF1/Cip1 indicating the phy-
tochemicals in the chloroform extract inhibit growth
by targeting cell cycle regulatory proteins (46). Evi-
dently, few compounds of avocado itself are involved
in inhibition of normal cell development. Therefore,
it is essential to exclude those compounds from avo-
cado fruits extract before using it as chemoprotective
agent.
CONCLUSION
Cyclophosphamide is being used as potent anti-
cancer drug in treatment of cancer like AML, ALL,
CML and CLL for long time. Some studies show that
it also exhibits gentoxic effects on normal blood cells
via structural and numerical aberrations. On the other
hand, Avocado is well known for its medicinal value.
This experiment was performed to evaluate its poten-
tiality against CP-induced genotoxicity. Interestingly,
reduction of PCD and acrocentric associations indi-
cates the presence of microtubule stabilizing agent in
the extract like persin (33), which act on centromere
directly or indirectly. Lowering of breaks, Terminal
deletion and numerical aberration strongly suggest
the existence of active components which work on
DNA repair and proper chromosome seggregation.
Avocado contains constituents like persin, persenones
A and B having anti-oxidant properties, which might
help to prevent formation of these breaks rather than
inducing repair. The chemopreventive effect was con-
centration dependent with 200 mg/Kg body weight
showing highest efficacy among the concentrations
used. It is recommended to do further experiment
with higher concentrations of fruit extract and with
purified active components before recommending its
use as chemopreventive agent as part of a cancer pre-
vention diet (29).
Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide …
Journal of Experimental Therapeutics and Oncology Vol. 9 2011 229
ACKNOWLEDGEMENTS
We heartily thankful to Mr. Tahir Molla and Mrs.
Samina Begum, Jawaharlal Nehru Cancer Hospital and
Research centre, Idgah Hills, Bhopal, India for giv-
ing their continuous support during the experimental
works. This project was completely financed by the
above Institute.
CONFLICT OF INTEREST
We don’t have any conflict of interest for this study.
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Publisher's Note: Products purchased from 3rd Party sellers are not guaranteed by the Publisher for quality, authenticity, or access to any online entitlements included with the product. Updated to include the newest drugs and those currently in development, Cancer Chemotherapy and Biotherapy, Fifth Edition is a comprehensive reference on the preclinical and clinical pharmacology of anticancer agents. Organized by drug class, the book provides the latest information on all drugs and biological agents-their mechanisms of action, interactions with other agents, toxicities, side effects, and mechanisms of resistance. Chapters emphasize pharmacology and mechanisms of action at the molecular and cellular levels, followed by clinical activity and toxicity, both acute and delayed. The authors explain the rationale for use of drugs in specific schedules and combinations and offer guidelines for dose adjustment in particular situations. This edition's introduction includes timely information on general strategies for drug usage, the science of drug discovery and development, economic and regulatory aspects of cancer drug development, and principles of pharmacokinetics. Eight new chapters have been added and more than twenty have been significantly revised. A companion website includes the fully searchable text and an image bank. © 2011 By Lippincott Williams & Wilkins, A Wolters Kluwer Business. All Rights Reserved.
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