Science of the Total Environment 364 (2006) 200 214

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Antimutagenic effect of neem leaves extract in freshwater fish,

Channa punctatus evaluated by cytogenetic tests
M. Abul Farah *, Bushra Ateeq, Waseem Ahmad
Gene-Tox Lab, Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh-202002, India
Received 20 March 2005; received in revised form 6 July 2005; accepted 8 July 2005
Available online 16 September 2005

Abstract
Neem (Azadirachta indica), an indigenous plant commonly grown in India and its sub-continent is a multipurpose plant well
known for its insecticidal and biomedical properties, however, its antimutagenic effects in vertebrate organisms are lacking. The
present work is therefore, focused on possible antimutagenic potential of ethanolic extract of neem leaves evaluated on the
clastogenicity induced by Pentachlorophenol (PCP) and 2, 4-dichlorophenoxyacetic acid (2,4-D) in freshwater fish, Channa
punctatus used as a vertebrate model, by cytogenetic endpoints: chromosome aberration (CA) and micronucleus (MN) test. In
the first set of experiment, fish were exposed by medium treatment to a single treatment of each chemical (PCP, 0.6 ppm; 2,4-D,
75 ppm; neem extract, 3 ppm) along with the controls. The chromosome preparations were made after processing kidney cells
and micronucleus slides were prepared from peripheral blood at multiple duration (48, 72 and 96 h). PCP and 2,4-D when used
alone, induced significant CA and MN in a time dependent manner. Neem extract did not show genotoxic potential in both
assays. The maximum frequency of CA were recorded as 18.58% and 15.17%, while frequency of MN reached to 8.08% and
4.62% by PCP and 2,4-D respectively, after 96 h exposure. In the second set of experiment, three concentrations of neem extract
(1, 2 and 3 ppm) were run simultaneously with the same concentration of PCP (0.6 ppm) and 2,4-D (75 ppm) for
antimutagenicity estimates. In mixed treatment, neem extract significantly reduced the frequency of CA and MN. The reduction
in the frequency of CA ranged from 4075% and 45.483.3% and similar values for MN were 40.275.3% and 44.165.8% for
PCP and 2,4-D respectively. Although the reductions were significant but not dependent on concentration and time intervals
employed. Results suggested that under present experimental conditions, neem extract exhibit strong antimutagenic activity in
this fish model, which could further contribute to study its benefit in humans.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Antimutagenicity; Neem extract; Chromosome aberrations; Micronucleus; Channa punctatus

1. Introduction
* Corresponding author. Present address: Proteonik Inc.,
Gyeonggi Technopark, Rm. 911, 1271-11, Sa-1-dong, Sangnokgu, Ansan-city, Gyeonggi-do, South Korea 425-170. Tel.: +82 10
6632 4070; fax: +82 31 500 4074.
E-mail address: abulfarah@lycos.com (M.A. Farah).
0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2005.07.008

Environmental pollution as a consequence of the

vigorous growth of industry and development of
human civilization leads to a rise in undesirable

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

genetic mutations and increase in cancer risk (Odin,

1997). Therefore, the discovery and exploration of
compounds possessing antimutagenic and anticarcinogenic properties are of great importance. Many indigenous substances possess some inhibitory activity
towards natural and man made environmental genotoxic agents (Grover and Bala, 1992; Brockman et al.,
1992). A diverse range of materials are being studied
as antimutagens such as vitamins and minerals.
Numerous studies continue to occur on extracts
from either common edible constituents, such as broccoli, brussel sprout, green pepper, bamboo shoots and
various mushrooms or various traditional medicines
from countries such as Brazil and India (Lohman et
al., 2001). Antimutagenic and anticarcinogenic potential of commonly consumed vegetables in Thailand
has been reported against direct and indirect acting
mutagen using Ames Salmonella mutagenicity test
(Kusamran et al., 1998). Nowadays, medicinal plants
have received growing attention as a chemopreventive
agent. Various Indian medicinal plant extracts were
tested for their antimutagenic activities by employing
Ames Salmonella test (Arora et al., 2003). Recently, a
wide range of South African medicinal plant extracts
were investigated for mutagenic and antimutagenic
effects in Salmonella/ microsome and micronucleus
test (Verschaeve et al., 2004).
There are many reports showing the rising trends
of antimutagenicity studies with extracts of plant. For
example, the aqueous extracts of fermented and unfermented tea (Asparathus linearts) and honeybush tea
(Cyclopta intermedia), were found to possess antimutagenic activity against 2-acetylaminofluorine and
aflatoxin B1 (Marnewick et al., 2000). Lemon grass
(Cymbopogon citratus Stapf) extract also found to
have antimutagenic effect towards various known
mutagens in Salmonella typhimurium strains TA98
and TA100 (Vinitketkumnuen et al., 1994). Vitamin
C and E against rifamipicin (an anti-tuberculosis drug)
in mouse bone marrow cells reduced the CA frequency significantly (Aly and Donya, 2002). In a
similar observation the protective effect of Agaricus
blazei tea was demonstrated in vivo against the clastogenicity induced by cyclophosphamide. Mice treated with three different teas of mushroom, also
significantly reduced the frequencies of micronuclei
in polychromatic erythrocytes and in reticulocytes, in
cyclophosphamide exposures (Danadai et al., 1998).

201

Neem (Azadirachta indica A. Juss), is an indigenous plant commonly grown in India and its subcontinent. It is one of the most versatile multipurpose plant species well known for its insecticidal and various types of biomedical properties
(Govindachari, 1992; ICAR, 1993). Almost every
part of neem tree have been known to possess a
wide range of pharmacological properties (Biswas
et al., 2002), and hence, traditionally used to treat
large number of diseases including malignancies
(Van Der Nat et al., 1991). This ecofriendly native
tree of India is perhaps most researched tree in the
world. Water soluble part of alcoholic extract of A.
indica leaves found to possess significant hypoglycemic, hypolipidemic, hepatoprotective, antifertility
and hypotensive activities. Further, it also shown to
exert significant anti-inflammatory and antiulcer
effects in rats (Chattopadhyay, 1998; Garg et al.,
1992). Methanol extract of Thai species of neem
leaves (A. indica var. siamensis) has been shown to
contain weak antimutagen capable of inhibiting the
mutagenicity of direct and indirect acting mutagens
in Ames Salmonella mutagenicity test (Kusamran et
al., 1998). The chemopreventive potential of dietary
neem flowers has also been demonstrated in inhibiting Aflatoxin B1 and 9,10 dimethyl-1,2-benzanthracene (DMBA) induced liver and mammary
gland carcinogenesis in rats (Tepsuwan et al.,
2002). Moreover, leaves of A. indica have been
shown to effectively suppress hamster buccal
pouch carcinogenesis initiated by DMBA and also
induce glutathione-s-transferase (GST) level in the
oral mucosa of animals bearing tumors (Balasenthil
et al., 1999). More recently, reports on the protective effects of ethanolic and aqueous neem leaf
extract against N-methyl-N-nitro-N-nitrosoguanidine
(MNNG) induced oxidative stress, gastric carcinogenesis, clastogenicity and genotoxicity in rats and
mice has been appeared (Subapriya et al., 2003,
2004; Subapriya and Nagini, 2003; Arivazhagan et
al., 2003). On the contrary, some reports of genotoxicity of crude extract of neem also been
appeared in terms of chromosome breakages in
bone marrow and male germ cells in mice
(Awasthy et al., 1999; Awasthy, 2001).
Although less commonly used than in the past,
bacterial and mammalian assays are still playing
role in antimutagenicity specially in relation to

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M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

establishing mechanistic effects (Lohman et al.,

2001). However, mechanistic studies probably
become increasingly more important, both in mutagenic and antimutagenic evaluation in vertebrate
organisms, because they are not only preferable
but necessary to provide a better understanding of
biochemical interactions and the consequences of
those interactions. Furthermore, for testing chemical
of concern for human health, vertebrate assays has
advantages as they are metabolically and physiologically more closely related to the humans. Fish,
living in different water bodies also carry the risk of
direct or indirect exposure to various toxicants,
industrial and agricultural chemicals. The advantage
of using fish as a vertebrate model organism
includes easy procuring, handling, acclimatizing in
the laboratory, exposing to the chemicals and costeffective. In fish, antimutagenic studies are still in
preliminary stage and only few reports are available. One such report concerns about the antimutagenic and anticarcinogenic activity of chlorophyllin
towards Aflatoxin B1 in rainbow trout (Dashwood et
al., 1998). Recently, the ameliorating effect of vitamin C, h-carotene and azadirachtin (principle compound of neem) against genotoxicity of ethyl
methanesulfonate (EMS) and cadmium chloride
(CdCl2) has been demonstrated in a fish, Oreochromis mossambicus (Guha and Khuda-Bukhsh, 2002,
2003; Chandra and Khuda-Bukhsh, 2004).
A large group of mutagenic chemicals comprises
herbicides and pesticides. One such group of compound is phenoxy acid derivatives, viz. pentachlorophenol (PCP) and 2,4-dichlorophenoxyacetic acid
(2,4-D). These are widely used as wood preservative
and herbicide and noted for their strong genotoxic
effects in various organisms (Seiler, 1991; Garabrant
and Philbert, 2002). In our laboratory, we have previously demonstrated the genotoxicity, clastogenicity
and toxicity stress of PCP and 2,4-D in plant system
(Ateeq et al., 2002a) and different species of freshwater fish such as, Heteropneustes fossilis, Clarias
batrachus and Channa punctatus (Ali and Ahmad,
1998; Ateeq et al., 2002b; Ahmad et al., 2002; Farah
et al., 2003, 2004). On the other hand, there is little
information available regarding the antimutagenic
potential of neem. Therefore, it is of particular interest to explore possible antimutagenicity of neem
extract in vertebrate organism using fish as a

model to study its corresponding benefit to humans.

Fish may act as sentinel organism for indicating the
potential for exposure of human population to genotoxic chemicals and subsequently can be used to
screen the natural substances to evaluate their protective affects.
In the present investigation, the possible antimutagenic potential of ethanolic extract of neem leaves
was proposed by cytogenetic endpoints: chromosome
aberration (CA) and micronucleus (MN) formation
as against the genotoxicity of PCP and 2,4-D in a
freshwater fish Channa punctatus, used as a vertebrate model. The above two parameters are considered as very sensitive genetic assays for detecting
genotoxic chemicals and identifying antimutagenic
agents as well, and being used extensively in cytogenetic investigations. Micronucleus test is much
favoured in fish because unlike mammals, erythrocytes are nucleated fascilitating easy scoring.

2. Materials and methods

2.1. Chemicals
PCP (CAS No. 87-86-5, 99% purity) and 2,4-D
(CAS No. 94-75-7, 98% purity) were purchased from
Fluka Chemika (Switzerland) and Loba Chemie
(India), respectively. All other reagents used in various formulations were of analytical grade and commercially available.
2.2. Specimen
Freshwater fish Channa punctatus (Channidae)
were obtained from a local fish market. Fish were
kept in polypropylene troughs containing fresh tap
water and acclimatized for two weeks to the laboratory conditions following the procedure of Clesceri et
al. (1998). Fish were fed with minced calf liver ad
libitum during acclimatization. Only healthy and
active individuals with average body weight of
50 F 5 g and measuring 18 F 2 cm were randomly
selected and used in the experiments. Water quality
like temperature (22 F 2 8C), pH (6.58.5), dissolved
oxygen (N 60% of saturation) and hardness (140160
mg/l as CaCO3) were kept constant throughout the
experiments.

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

2.3. Preparation of ethanolic extract of neem leaves

Fresh neem leaves were collected from the university campus and identified by a pharmacognosy
expert. The ethanolic extract of neem leaves was
prepared by the procedure described by Chattopadhyay (1998). Briefly, the leaves were cleaned by
washing in tap water; air dried and made semi
powder by crushing. About 400 gm of powdered
leaves was dissolved in 2 L of ethyl alcohol and
kept at room temperature for 48 h. The mixture was
filtered and the filtrate was concentrated under
reduced pressure in a water bath (temperature 50
8C) and finally dried in a vacuum dessicator. The
residue so obtained was a thick green paste which
was suitably diluted with normal saline and used for
the experiments.
2.4. Experimental design
Since PCP and 2,4-D have low solubility
in water, a stock solution was prepared by dissolving
desired amount in ethanol. Treatment was given
through aqueous medium, exposure was continued
for 96 h and samples were collected at the intervals
of 48, 72 and 96 h. In the first set of experiment, fish
were exposed to each of single sub-lethal concentration of PCP (0.6 ppm), 2,4-D (75 ppm) and neem
extract (3 ppm). These concentrations were selected
on the basis of previous studies and LC50 values
(PCP, 0.77 ppm and 2,4-D, 107 ppm) determined
by probit analysis in this fish (Farah et al., 2004).
Optimally low concentration of neem extract (LC50
28 ppm determined in Channa punctatus) is used to
ascertain if it has any genotoxic effect. Beside that,
negative control (tap water) and solvent control
(0.2% ethanol) were also carried out. In the second
set of experiment, three concentration of neem
extract (1, 2 and 3 ppm) were used simultaneously
with PCP (0.6 ppm) and 2,4-D (75 ppm). Series 1
(S1) contains PCP + neem extract (0.6 + 1 ppm) and
2,4-D + neem extract (75 + 1 ppm), Series 2 (S2)
contains PCP + neem extract (0.6 + 2 ppm) and 2,4D + neem extract (75 + 2 ppm) and Series 3 (S3)
contains PCP + neem extract (0.6 + 3 ppm) and 2,4D + neem extract (75 + 3 ppm). After the termination
of desired duration, 4 fishes were utilized for chromosome preparation and 4 fishes were utilized

203

for micronucleus slide preparation (n = 4 for each

group).
2.5. Chromosome preparation
Fish kidney was used for chromosome preparations because it is haemapoietic and mitotically
active organ. The technique of Al-Sabti et al.
(1983) and Cucchi and Baruffaldi (1990) were followed for preparation of chromosomes. Before 24 h
of completion of desired duration, fish were injected
intraperitonially with CoCl2 solution (as mitogen)
at the dosage of 2 mg/100 g body weight. 19 h
after the first injection, specimen were injected with
freshly prepared colchicine solution at the dosage of
1 mg/100 g body weight. 5 h after the second
injection, fish were sacrificed and kidneys were
removed, macerated and suspended in 0.075M KCl
and incubated for 3040 min at 32 8C. The cell
suspension was fixed in chilled CornoyTs fixative
(methanol : glacial acetic acid, 3 : 1), mixed gently
with Pasteur pipette, centrifuged at 1500 rpm for
10 min and supernatant was discarded. The pellet
was resuspended in chilled CornoyTs fixative and the
above process was repeated twice. Chromosome
slides were prepared by dropping the few drops of
cell suspension onto pre-cold slides in 70% alcohol.
Immediately thereafter, the fixative was burned off
using flame drying. The slides were stained in 10%
GiemsaTs stain prepared in Sorensens buffer (pH6.8) for 2025 min. Finally, the slides were cleared
in xylene and permanently mounted in DPX.
Brightly stained having well spread metaphase chromosomes slides were independently coded and
observed in the light microscope under oil immersion (10  100 magnification). A minimum of 25
metaphases per fish in each group including control
were analysed (since n = 4 per group, minimum 100
metaphases). Not withstanding the conventional
method of scoring, the CA was recorded under
two broad categories i.e. classical aberration and
non classical aberration. In the classical aberrations,
both chromatid and chromosome type breaks,
including acentric fragments, exchanges, and multiple
aberrations (polyploidy, aneuploidy, rings etc) were
counted and non-classical aberration comprised of
stickiness, pulverization and c-metaphases. Regardless of whether it was a mono or poly aberration

204

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

in one metaphase, it was counted as one aberrant

metaphase.
2.6. Micronucleus slide preparation
After the termination of desired duration, blood
smear were drawn on clean grease free microscopic
slides by making a caudal cut. Slides were air-dried,
fixed in methanol and stained with filtered haematoxylin (10 min). Then the slides were rinsed in ScottTs
tap water substitute (0.2% potassium bicarbonate and
2% magnesium sulphate) until the nuclear color changed from red to blue. It was followed by dehydration
in ascending grades of alcohol (30%, 50%, 70%, 90%
and absolute) and counter stained in filtered eosin
(made in 70% alcohol) for staining the cytoplasm
(Pascoe and Gatehouse, 1986). Lastly, slides were
cleared in xylene and made permanent by mounting
in DPX. A minimum of 12,000 erythrocytes were

scored for the occurrence of MN in each treatment

groups including controls (three slides per fish, 1000
1200 erythrocytes per slide) under the light microscope at 60 objective and 15 eye piece. The established criteria for scoring MN were followed (Schmid,
1975). Micronuclei are cytoplasmic chromatin masses
resembling minute nuclei, formed when acentric chromosome fragments or chromosomes lag during anaphase and fail to become incorporated into daughter
cell nuclei during cell division. Non-refractile particles
that resembled nuclei in all respects such as similar
color and staining intensity were considered to be
micronuclei (Das and Nanda, 1986).
2.7. Data evaluation and statistical analysis
The reduction percentage in number of chromosome aberration and micronuclei in the treatments
with the neem leaves extract was calculated accord-

Table 1
Frequency profiles of chromosomal aberrations (CA) induced alone by PCP (0.6 ppm) and neem extract (NE, 3 ppm) and simultaneous
exposures at variable concentrations of NE with PCP for different intervals to evaluate antimutagenicity in Channa punctatus
Duration (h)

48

72

96

Conc. (ppm)

Cont.1
Cont.2
PCP
NE
S1
S2
S3
Cont.1
Cont.2
PCP
NE
S1
S2
S3
Cont.1
Cont.2
PCP
NE
S1
S2
S3

Total
metaph
studied

Classical aberrations
B

MA

Non-classical aberrations
SP

CM

104
101
117
117
105
107
110
111
106
110
120
107
112
104
105
108
113
115
115
108
110

2
3
8
4
7
6
7
4
2
9
3
7
7
8
4
3
10
4
8
7
5

1
0
1
0
1
1
0
0
0
3
1
2
0
1
0
0
2
0
0
1
2

0
0
3
1
1
2
1
0
1
3
0
2
1
1
1
0
3
1
2
1
1

1
1
3
0
1
0
2
0
1
2
0
2
1
0
0
1
4
1
1
0
1

0
0
3
0
0
0
1
0
0
2
1
0
1
0
0
1
2
0
1
1
0

Total aberr. mean

(%) F S. D.
3.84 F 0.26
3.96 F 0.19
15.38 F 0.40*
4.27 F 0.28
9.52 F 0.32#
8.41 F 0.51#
10.00 F 0.35#
3.60 F 0.24
3.77 F 0.26
17.27 F 0.50*
4.16 F 0.29
12.14 F 0.45#
8.92 F 0.25#
7.69 F 0.28#
4.76 F 0.25
4.62 F 0.35
18.58 F 0.40*
5.21 F 0.48
10.45 F 0.26#
9.25 F 0.38#
8.18 F 0.25#

Cont.1; negative control (tap water), Cont.2; solvent control (ethanol, 0.2%), S1 (series 1, 0.6 ppm PCP + 1 ppm NE,); S2 (series 2, 0.6 ppm
PCP + 2 ppm NE); S3 (series 3, 0.6 ppm PCP + 3 ppm NE) B, break; E, exchanges; MA, multiple aberration; SP, stickiness and pulverization;
CM, c-metaphase.
*Significant from controls, #Significant from PCP, (ANOVA, p b 0.05).

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

ing to Manoharan and Banerjee (1985) and Waters

et al. (1990) by the following formula:
Reduction %
Frequency of CA or MN in A 

Frequency of CA or MN in A 

Frequency of CA or MN in B
 100
Frequency of CA or MN in C

Where,
A
B
C

= PCP or 2,4-D alone

= Neem extract mixed with PCP or 2,4-D
= Negative control (tap water).

Frequency of CA and MN are expressed as mean

percentage F standard deviation. The data obtained
from both the experimental set were presented
together under the results of CA and MN, respectively. Statistically significant differences between
groups were determined by performing ANOVA followed by Tukey HSD post hoc comparison. The
significance level considered was P b 0.05.

205

3. Results
3.1. Antimutagenicity of neem leaves extract in
chromosome aberration
The typical diploid metaphase complements of
Channa punctatus were found to consist of 32 chromosomes and three mixed types of chromosomes such
as metacentric, submetacentric and subtelocentric
were distinguished. Tables 1 and 2 are showing the
frequency of CA observed alone by the test compounds and effect of neem extract in simultaneous
treatment of PCP and 2,4-D respectively. Fig. 1 (a)
shows the increase in CA frequency by PCP and 2,4D. A comparative account of reduction of CA frequency is presented in Fig. 2 (a, b). Both PCP and 2,4D were able to produce aberration of chromatid and
chromosome types in a significant ( p b 0.05) manner
when used alone. Various form of chromosome
damage recorded were chromosome and chromatid

Table 2
Frequency profiles of chromosomal aberrations (CA) induced alone by 2,4-D (75 ppm) and neem extract (NE, 3 ppm) and simultaneous
exposures at variable concentrations of NE with 2,4-D for different intervals to evaluate antimutagenicity in Channa punctatus
Duration (h)

48

72

96

Conc. (ppm)

Cont.1
Cont.2
2,4-D
NE
S1
S2
S3
Cont.1
Cont.2
2,4-D
NE
S1
S2
S3
Cont.1
Cont.2
2,4-D
NE
S1
S2
S3

Total
metaph
studied
104
101
107
117
110
115
107
111
106
110
120
112
109
111
105
108
112
115
106
110
109

Classical aberrations

Non-classical aberrations

MA

SP

CM

2
3
9
4
6
5
6
4
2
8
3
7
6
6
4
3
10
4
7
5
5

1
0
1
0
0
1
0
0
0
2
1
1
1
0
0
0
3
0
2
1
0

0
0
2
1
0
1
1
0
1
2
0
1
0
1
1
0
2
1
1
1
1

1
1
2
0
1
2
1
0
1
2
0
0
1
0
0
1
1
1
1
1
1

0
0
0
0
0
0
1
0
0
1
1
1
1
0
0
1
1
0
0
1
0

Total aberr. mean

(%) F S. D.
3.84 F 0.26
3.96 F 0.19
13.08 F 0.45*
4.27 F 0.28
6.36 F 0.42#
7.82 F 0.30#
8.41 F 0.36#
3.60 F 0.24
3.77 F 0.26
13.63 F 0.40*
4.16 F 0.29
8.92 F 0.50#
8.25 F 0.33#
6.30 F 0.35#
4.76 F 0.25
4.62 F 0.35
15.17 F 0.45*
5.21 F 0.48
10.37 F 0.28#
8.18 F 0.45#
6.42 F 0.32#

Cont.1; negative control (tap water), Cont.2; solvent control (ethanol, 0.2%), S1 (series 1, 75 ppm 2,4-D + 1 ppm NE); S2 (series 2, 75 ppm 2,4D + 2 ppm NE); S3 (series 3, 75 ppm 2,4-D + 3 ppm NE) B, break; E, exchanges; MA, multiple aberration; SP, stickiness and pulverization; CM,
c-metaphase.
*Significant from controls, #significant from 2,4-D, (ANOVA, p b 0.05).

206

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

(a)
20

% CA

15

48 h
72 h
96 h
n=4, each bar

*
*

*
**

10

0
C1

C2

NE

PCP

2,4-D

Experimental groups

(b)
10

48 h
72 h
96 h

n=4, each bar

% MN

*
4

*
* *

0
C1

C2

NE

PCP

2,4-D

Experimental groups
Fig. 1. Bars showing the frequency of chromosome aberration (a) and micronucleus (b) by PCP, 2,4-D and neem extract used alone. C1-negative
control (tap water) C2-solvent control (0.2% ethanol), NE-neem extract.

breaks, acentric, rings, polyploidy, aneuploidy, stickiness, pulverization and c-metaphases, whereas gaps
were excluded. The frequency of CA induced by PCP
reaches to 15.38 F 0.40, 17.27 F 0.50 and 18.58 F
0.40 after 48, 72 and 96 h respectively. The similar
values for 2,4-D were recorded as 13.08 F0.45,
13.63 F 0.40 and 15.17 F 0.45 (48 96 h). However,

the frequency of CA by neem leaves extract (3 ppm)

were recorded as 4.27 F 0.28, 4.16 F 0.29 and
5.21 F 0.48 (48 96 h) which were found not significant with controls.
All concentrations of neem extract reduced the CA
frequency significantly ( p b 0.05) in simultaneous
treatment with PCP and 2,4-D. The reduction profiles

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

(a)

207

20

48 h
72 h
96 h
n=4, each bar

15

% CA

#
#
#

10

#
#

0
PCP

S1

S2

S3

Experimental groups

(b)

18
16
48 h
72 h
96 h

14
12

n=4, each bar

% CA

#
10

#
#

#
# #

#
6
4
2
0
2,4-D

S1

S2

S3

Experimental groups
Fig. 2. Bars depicting the reduction of chromosome aberration frequencies by neem extract and PCP (a) and neem extract and 2,4-D (b) after
variable exposures. S1; (series 1, 0.6 ppm PCP or 75 ppm 2,4-D and 1 ppm neem extract); S2; (series 2, 0.6 ppm PCP or 75 ppm 2,4-D and 2
ppm neem extract) S3; (series 3, 0.6 ppm PCP or 75 ppm 2,4-D and 3 ppm neem extract).

by all the concentrations of neem extract (1, 2 and

3 ppm) with PCP were estimated as 57.1%, 64.2%
and 50% (48 h), 40%, 60% and 60% (72 h) and
56.2%, 68.7% and 75% (96 h), respectively. In

the 2,4-D treated groups the reduction profiles were

70%, 50% and 50% (48 h), 45.4%, 54.5% and 72.7%
(72 h) and 50%, 66.6% and 83.3% (96 h), respectively. The highest reduction of 75% after 96 h expo-

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M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

sure was recorded by frequency of 8.18 F 0.25 (0.6

ppm PCP + 3 ppm neem extract) as compared to
18.58 F 0.40 of PCP (0.6 ppm) alone. In the 2,4-D
treated group, neem extract was found to be more
effective as the highest reduction of 83.3% (75
ppm + 3 ppm neem extract) was recorded after 96 h
registering the frequency of 6.42 F 0.32 compared to
15.17 F 0.45 by 2,4-D (75 ppm) alone.
3.2. Antimutagenicity of neem leaves extract in
micronucleus test
The erythrocytes of Channa punctatus were generally observed as elliptical with a centrally located
oval nucleus and a considerable amount of cytoplasm,
any abnormality could therefore, be seen easily. The
size and position of micronucleus in the cytoplasm
showed slight variation and normally one MN per cell
was observed, though in some instances 2 or 3 MN
was also observed at longer duration. The frequency
of MN recorded alone and simultaneous treatment of

neem extract with PCP and 2,4-D are summarized in

Tables 3 and 4 respectively. Increase in MN frequency
by test chemicals were shown in Fig. 1 b. A comparative account showing decrease in the frequency of
MN is depicted in Fig. 3 (a, b).
In accordance with the results obtained in CA test,
PCP and 2,4-D induced MN significantly ( p b 0.05) in
all duration when used alone while, a clear negative
effect on induction of MN by neem extract (3 ppm)
was found in all exposure. The frequency of MN
induced by PCP was recorded as 2.89 F 1.02,
4.16 F 1.83 and 8.08 F 2.32 after 48, 72 and 96 h
respectively. Whereas, the MN frequency in the 2,4D treated groups were observed as 1.96 F 0.65,
2.15 F 0.58 and 4.62 F 1.23 (4896 h). The MN frequencies observed by neem extract (3 ppm) were
0.74 F 0.35, 0.83 F 0.76 and 0.95 F 0.55 (4896 h),
found not significant when contrasted with controls.
However, neem extract significantly ( p b 0.05)
reduced the frequency of MN by all three concentrations tested (1, 2 and 3 ppm), compared to the MN

Table 3
Frequency profiles of micronuclei (MN) induced alone by PCP (0.6 ppm) and neem extract (NE, 3 ppm) and simultaneous exposure at variable
concentrations of NE with PCP for different intervals to evaluate antimutagenicity in Channa punctatus
Exposure (h)

Conc. (ppm)

Total cells scored

48

Cont.
Cont.
PCP
NE
S1
S2
S3
Cont.
Cont.
PCP
NE
S1
S2
S3
Cont.
Cont.
PCP
NE
S1
S2
S3

12,225
12,180
12,214
12,080
12,250
12,300
12,200
12,550
12,350
14,048
12,600
12,250
12,200
12,000
12,280
12,150
13,440
12,300
12,200
12,350
12,100

72

96

1
2

1
2

1
2

Number of MN
1

93
96
338
93
147
160
179
100
99
411
101
289
270
272
113
106
802
110
625
534
463

1
2
7
0
3
4
3
1
2
66
2
2
3
3
1
2
116
2
25
28
20

0
0
1
0
2
1
0
0
1
12
0
1
0
1
0
0
18
1
7
5
6

Total no. of MN

Frequency of MN
mean (%) F S.D

95
100
355
93
159
171
185
102
106
589
105
296
276
281
115
110
1088
117
696
605
521

0.75 F 0.42
0.79 F 0.39
2.89 F 1.02*
0.74 F 0.35
1.28 F 0.40#
1.37 F 0.29#
1.49 F 0.52#
0.79 F 0.51
0.83 F 0.60
4.16 F 1.83*
0.83 F 0.76
2.39 F 0.90#
2.25 F 0.68#
2.31 F 0.62#
0.91 F 0.70
0.88 F 0.61
8.08 F 2.32*
0.95 F 0.55
5.66 F 0.95#
4.87 F 1.02#
4.26 F 0.85#

Cont.1 (negative, tap water), Cont.2 (solvent, 0.2% ethanol), S1 (series 1, 0.6 ppm PCP + 1 ppm NE); S2 (series 2, 0.6 ppm PCP + 2 ppm NE);
S3 (series 3, 0.6 ppm PCP + 3 ppm NE).
*Significant from controls, #significant from PCP, (ANOVA, p b 0.05).

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

209

Table 4
Frequency profiles of micronuclei (MN) induced alone by 2,4-D (75 ppm) and neem extract (NE, 3 ppm) and simultaneous exposure at variable
concentrations of NE with 2,4-D for different intervals to evaluate antimutagenicity in Channa punctatus
Exposure (h)

48

72

96

Conc. (ppm)

Cont. 1
Cont. 2
2,4-D
NE
S1
S2
S3
Cont. 1
Cont. 2
2,4-D
NE
S1
S2
S3
Cont. 1
Cont. 2
2,4-D
NE
S1
S2
S3

Total cells scored

12,225
12,180
12,096
12,080
12,150
12,225
12,050
12,550
12,350
12,512
12,600
12,300
12,350
12,200
12,280
12,150
12,984
12,300
12,250
12,000
12,150

Number of MN
1

Total no.
of MN

Frequency of MN
mean (%) F S.D

93
96
226
93
170
150
158
100
99
251
101
164
150
150
113
106
536
110
282
269
280

1
2
4
0
3
2
5
1
2
6
2
5
3
2
1
2
25
2
5
7
10

0
0
2
0
0
2
1
0
1
2
0
2
1
2
0
0
6
1
2
1
3

95
100
240
93
176
160
171
102
106
269
105
180
159
160
115
110
604
117
298
286
309

0.75 F 0.42
0.79 F 0.39
1.96 F 0.65*
0.74 F 0.35
1.41 F 0.66#
1.29 F 0.54#
1.39 F 0.40#
0.79 F 0.51
0.83 F 0.60
2.15 F 0.58*
0.83 F 0.76
1.45 F 0.81#
1.25 F 0.65#
1.29 F 0.55#
0.91 F 0.70
0.88 F 0.61
4.62 F 1.23*
0.95 F 0.55
2.41 F 0.80#
2.35 F 0.95#
2.53 F 0.72#

Cont.1 (negative, tap water), Cont.2 (solvent, 0.2% ethanol), S1 (series 1, 75 ppm 2,4-D + 1 ppm NE); S2 (series 2, 75 ppm 2,4-D + 2 ppm NE);
S3 (series 3, 75 ppm 2,4-D + 3 ppm NE).
*Significant from controls, #significant from 2,4-D, (ANOVA, p b 0.05).

induced by PCP and 2,4-D alone. The reduction profiles in the MN incidence at the concentrations of
neem extract (1, 2 and 3 ppm) with PCP were estimated as 75.3%, 70.7% and 65.3% (48 h), 60.1%,
64.2% and 63.2% (72 h) and 40.2%, 49.6% and
58.2% (96 h), respectively. In the 2,4-D treated groups
the reduction profiles were 44.1%, 55.1% and 47.5%
(48 h), 53.2%, 65.8% and 65.2% (72 h) and 62.5%,
65% and 60.3% (96 h), respectively. The maximum
reduction in MN frequency when used simultaneous
with PCP and neem extract (0.6 ppm + 1 ppm) was
75.3% after 48 h registering a frequency of
1.28 F 0.40 as against PCP alone 2.89 F 1.02. Within
the 2,4-D and neem extract treated groups, the highest
reduction was 65.8% after 72 h (75 ppm + 2 ppm),
recording the frequency of 1.25 F 0.65 as against the
2.15 F 0.58 by 2,4-D alone.
Overall, the results revealed that PCP was more
potent genotoxic agent than 2,4-D at the concentrations used in this study in terms of CA and MN
induction when used alone. Neem extract effectively

reduced the frequency of CA and MN when used

simultaneously with PCP or 2,4-D. It was found
more effective in reducing the frequency of CA
induced by 2,4-D and frequency of MN induced by
PCP. For the durations of exposure and the range of
neem concentrations used in this study, the reduction
percentage recorded was not dependent on concentrations of neem extract used in any duration.

4. Discussions
The occurrence of cytogenetic damage in the fish
Channa punctatus exposed to PCP and 2,4-D has
been demonstrated by an enhanced frequency of CA
in kidney cells and MN in the peripheral blood
erythrocytes in a time dependent manner. These
results are more environmentally relevant than previous studies, which have typically used injection as
the route of exposure, because waterborne exposure
is more realistic of what occurs in nature. Presum-

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M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

(a)
10
48 h
72 h
96 h
n=4, each bar

8
#
6

% MN

#
#

4
#
2

#
#

0
PCP

(b)

S1

S2

S3

Experimental groups
6
48 h
72 h
96 h
n=4, each bar

% MN

#
#

0
2,4-D

S1

S2

S3

Experimental groups
Fig. 3. Bars depicting the reduction of micronucleus frequencies by neem extract and PCP (a) and neem extract and 2,4-D (b) after variable
exposures. S1; (series 1, 0.6 ppm PCP or 75 ppm 2,4-D and 1 ppm neem extract); S2; (series 2, 0.6 ppm PCP or 75 ppm 2,4-D and 2 ppm neem
extract) S3; (series 3, 0.6 ppm PCP or 75 ppm 2,4-D and 3 ppm neem extract).

ably, both PCP and 2,4-D has affected the genetic

material by absorption through the gill epithelium.
Earlier, it has been emphasized that exposure of fish
to genotoxic chemicals for various interval of time by

the respiratory route following the absorption of chemicals through gill epithelium could be occurred (De
Flora et al., 1993; Rishi and Grewal, 1995). Further,
the fish Channa punctatus was found to be well

M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

suited organism for cytogenetic analysis because of

their nearly ideal karyotype and relatively less number of chromosomes (2n = 32).
Considering the world wide use of these chemicals and due to the conflicting results documented,
PCP and 2,4-D have encouraged scientific investigation in many organisms. Apart from some negative
results for both the tested compounds, many confirmed the toxicity of PCP to the aquatic organisms
(Stephenson et al., 1991; Klobucor et al., 1997), as
well as to mammalian cells (Jansson and Jansson,
1991). PCP was able to induce DNA strand breaks
and micronuclei in Zebra mussel and snail (Pavlica
et al., 2000, 2001). Various forms of structural chromosome damage and significant increase in MN
frequency by manual and automated method in H.
fossilis were also observed (Ali and Ahmad, 1998;
Ahmad et al., 2002). On the other hand, 2,4-D was
evidently showed increased CA frequency in mammalian lymphocyte culture (Mustonen et al., 1986)
and genotoxic effects in rat bone marrow was also
observed (Adhikari and Grover, 1988). Recently,
significant increase in the frequency of CA and sister
chromatid exchanges (SCE) by 2,4-D in mouse bone
marrow were documented (Amer and Ali, 2001;
Madrigal-Bujaidar et al., 2002). Furthermore, significant increase in MN frequency along with various
forms of erythrocyte alterations were recorded due to
2,4-D exposure in freshwater fish, Clarias batrachus
(Ateeq et al., 2002b). Beside this, increase in MN
frequencies have already been evaluated due to 2,4D and PCP exposure in other fish species like
Channa punctatus and Heteropneustes fossilis
(Farah et al., 2003; Ahmad et al., 2002). Nevertheless, the antagonist role of any natural compound
against these genotoxic compounds was waiting to
be unraveled. And the present study fills the lacunae
in this field.
Most of the toxic chemicals that produce genotoxic effects have been known to form reactive oxygen species as well as electrophilic free radical
metabolites that interact with DNA to cause disruptive changes. Tetrachlorohydroquinone being the
principle metabolite of PCP, reported to be real
genotoxic agent capable of binding to DNA and
producing DNA strand breaks. This activity is probably due to semiquinone radical formation and partly
mediated through reactive oxygen species (Seiler,

211

1991). Similarly, azadirachtin as one of the active

principle present in the extract of the neem, reported
to have mitotic poisoning effect on mouse bone
marrow and spermatocyte chromosome (Awasthy et
al., 1995; Khan and Awasthy, 2003). However, a
recent study showed the ameliorating potential of
azadirachtin against the CdCl2 induced clastogenic
changes in fish O. mossambicus by various endpoints (Chandra and Khuda-Bukhsh, 2004).
The results of the present investigation clearly
showed that the neem extract had an antimutagenic
and anticlastogenic potential in the fish. The neem
leaves extract suppressed the action of PCP and 2,4-D
as measured in CA and MN tests. The simultaneous
treatment showed protective activity against two end
points. The antimutagenic effect in the CA and MNT
could be due to reduced induction of damage or
increased repair.
Three experimental designs (simultaneous, preand post treatment) when applied in relation to
MMS for a better investigation of the possible antimutagenicity or antigenotoxicity mechanisms of the
A. blazei extracts on Chinese hamster V79 cells by
comet assay and MNT, showed positive results
(Menoli et al., 2001). The post treatment could
show its antimutagenic potential, by playing a role
in optimization of DNA repair. The conclusion
obtained in the pre-treatment could reflect the
effects on the prevention of DNA damage by affecting metabolic pathways, being antioxidant or acting
on DNA replication. These action mechanisms,
occurring in both pre- and post treatments, could
be called bio-antimutagenicity (Morita et al., 1978;
Kada, 1983) or fidelogenesis. The use of a simultaneous treatment appeared to identify mechanisms
with direct action on the mutagen by inactivating
it, which may be classified as desmutagenicity
effects (Morita et al., 1978; Kada, 1983). Since
during present investigations the fish were simultaneously treated with neem extract in the aqueous
medium, that may be simply preventing the uptake
of PCP and 2,4-D from the water and reducing their
subsequent toxicity. Previously, it has been reported
that some drugs, dietary components and endogenous biochemicals can function as antimutagen by
altering the rates of mutagen absorption and uptake
(Waters et al., 1998). However, the present experimental design does not rule out the possibility of

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M.A. Farah et al. / Science of the Total Environment 364 (2006) 200214

indirect effects of neem during PCP and 2,4-D

exposure.
The exact mechanism by which neem leaves
extract reduced or minimizes the genotoxicity of
PCP and 2,4-D is not understood. However, the protective effect of neem leaf could be ascribed to the
antioxidant property, as it is known that neem leaf
contains a number of potent antioxidants and anticarcinogens including carotenes, terpenoids, limonoids,
quercetin and sitosterol (Govindachari, 1992; Schaaf
et al., 2000), that might be inhibited the genotoxic
effect of tested chemicals in the present investigation.
Neem flower has been shown to contain some sterols
and flavonoids glycosides (Subramanian and Nair,
1972). These glycosides exhibit antioxidant activity
whereas, the corresponding flavonoids posses many
activity in chemopreventive assay, including inhibition of cytochrome p450 dependent activity (Beecher,
1995; Siess et al., 1995). The radical scavenging
properties of neem preparations has been demonstrated by many workers (Balasenthil et al., 1999;
Chattopadhyay et al., 1993). The chemopreventive
potential of neem leaf extract may be related to one
or more of the constituent phytochemical that are
potentially anticarcinogenic (Van Der Nat et al.,
1991). Our results were in good agreement with
those of Arivazhagan and colleagues (Arivazhagan
et al., 2003) who reported pretreatment of aqueous
extract of neem leaf significantly reduced MNNG
induced increase in MN and CA in bone marrow of
rat. Furthermore, ethanolic neem leaf extract also
reduced MNNG induced macronuclei and lipid peroxides and enhanced GSH-dependent antioxidant
activity in mice (Subapriya et al., 2004).
Results in this study therefore, can be concluded
that simultaneous treatment with ethanolic extract of
neem leaves under certain circumstances exert significant antimutagenicity against PCP and 2,4-D
induced CA and incidence of MN. The obtained
data indicated that natural products such as neem
extracts may yield a wealth of commercially viable
antimutagenic agents. The precise characterization of
the antimutagenic activity of neem extracts and to
exactly identify the active compounds and their
modes of action is recommended. Further studies
on different vertebrate models are needed to determine whether administration of neem extract is a
practical approach to antimutagenesis in humans.

Acknowledgements
The Council of Scientific and Industrial Research
(CSIR), New Delhi, India is gratefully acknowledged
for providing financial support in the form of Senior
Research Fellowship to the authors MAF and BA
(Award no. 9/112 (339) 2002, EMR-I; 9/112 (340)
2002, EMR-I). Authors are thankful to the Chairman,
Department of Zoology, AMU, Aligarh, India, for
providing necessary facilities.

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