Modulatory Effect of Aegle marmelos against Benzo (a) Pyrene B(a)P Induced Genotoxicity in Swiss Albino Mice
Tajdar Husain Khan
Dietary factors are considered important environmental risk determinants for various diseases. Present study shows the modulatory influence of methanolic extract of Aegle marmelos (AM) on Benzo(a)Pyrene (B(a)P) (125 mg kg-1) induced genotoxicity in Swiss albino mice. The effect of AM was studied by in vivo bone marrow chromosomal aberration and micronuclei induction test. Two doses of AM (25 and 50 mg kg-1 b.wt.) were given orally for seven days prior to the administration of B[a]P. The efficacy of plant extract was also evaluated by an in vitro cytochrome P450 (CYP), DNA-sugar damage in calf thymus DNA and Fe++/ascorbate induced lipid peroxidation (p<0.001) in microsome of mice. Significant increase in the antioxidant activity (p<0.001) and a concomitant decrease (p<0.001) in the malondialdehyde level were observed at three concentrations of plant extract (2.5, 5.0 and 7.5 mg kg-1 b. wt.). Administration of AM, showed inhibitory effects on the genotoxicity of B[a]P in terms of chromosomal aberration and micronucleus formation. The chemopreventive effect of AM on the inhibition of CYP activity and DNA integrity mediate the possible mechanism of inhibition of genotoxicity.
Received: February 18, 2012;
Accepted: March 12, 2012;
Published: June 13, 2012
Reactive Oxygen Species (ROS) can cause cell damage and initiate various degradation
processes (Momtaz and Abdollahi, 2012). Oxidative stress
is the result of imbalance between the formation of reactive oxygen species
(ROS) and antioxidants in the body which can lead to oxidative damage to biomolecules
resulting in lipid peroxidation, mutagenesis and carcinogenesis. Oxidative damage
to the DNA plays an important role in carcinogenesis (Hussain
et al., 2003; Khan, 2012). Free radical
induced lipid peroxidation has been involved in pathogenesis of numerous pathological
disorders including cancer (Hristozov et al., 2001).
ROS has been associated in numerous diseases, including malignancy, aging and
neurodegenerative disorders (Balsano and Alisi, 2009;
Momtaz and Abdollahi, 2012).
A promising way to control carcinogenesis or mutagenesis is to prevent the
formation of free radical i.e., reactive metabolite from the inert compounds
which on interacting or binding covalently with the cellular macromolecule like
DNA, lipids or protein may lead to its damage and if not checked or not control
may lead to cancer (Ziech et al., 2011; Aggarwal
et al., 2009). The peroxidation of lipids, the cross-linking and
inactivation of proteins and mutations in DNA are typical consequences of free
radicals. Membranes lipids are the main targets of free radicals and they initiate
lipid peroxidation chain reaction. A result of lipid peroxidation, membrane
fluidity decreased which alters membrane properties (Aggarwal
et al., 2009).
Herbs are gaining additional focus because of their less toxicity and high
efficacy against a number of ailments. Epidemiological studies have shown that
fruits, vegetables, spices, tea and medicinal herbs rich in antioxidants and
other micronutrients protect against diverse forms of chemically induced carcinogenesis,
inhibit DNA-damage, mutagenesis and lipid peroxidation (Ziech
et al., 2011; Birt et al., 2001) Aegle
marmelos, known as bael grows in tropical and subtropical parts of the world.
Various parts of the AM are used in Indian system of medicine for treatment
of many diseases, including diarrhoea, dysentery and dyspeptic symptoms (Shoba
and Thomas, 2001; Sharma et al., 2007). Marmelosin,
isolated from the AM, has been reported to have anti-helminthic, anti-bacterial,
antioxidant activity and anticarcinogenic (Khan and Sultana,
2009; Patil et al., 2010; Khan
and Sultana, 2011).
The exposures to genotoxicants have been associated to the expression of biological
effects and to increased risk for cancer (Holland et
al., 2011). Genotoxicity may cause a long-term effect on the sustainability
of particular populations (Bhattacharya, 2011). Genotoxic
substances may bring about changes in normal DNA integrity leading to pathological
conditions (Holland et al., 2011). DNA damage
is unequivocally the main cause of mutagenesis (Preston and
Hoffmann, 2001). A frequently occurring form of DNA damage is the DNA strand
breaks, divided into single strand breaks (ssBs) and double-strand breaks (dsBs).
B(a)P is prevalent in environment, food, ambient and indoor air. Some of its
metabolites produced are highly reactive to DNA and may lead to covalent binding
causing DNA adduct formation. It has been reported to cause mutations, chromosome
aberrations, chromatid exchanges and cancer. Some B(a)P reactive intermediates
form alkali-labile sites on DNA (Holland et al.,
2011; Paget et al., 2012).
MATERIALS AND METHODS
Chemicals: Calf thymus DNA and Benzo(a)pyrene were obtained from Sigma chemicals Co (St Louis, MO) and all other chemicals were of the highest purity and commercially available.
Animals: Swiss albino mice (20-25 g) were obtained from Central Animal House of Hamdard University, New Delhi, India. They were allowed to acclimatize for one week before the experiments and were given free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India).
Plant material: The plant material was procured from wholesale spice
and herbs market Khari Baoli, old Delhi. Professor Mohammed Iqbal, Medicinal
Plant Division, Department of Environmental Botany, Hamdard University, New
Delhi verified the identity of plant material. The plant material was chopped
and coarsely powdered to a mesh size of 1 mm as described by Antonio
and Brito (1998).
Preparation of extract: Powdered plant material was repeatedly extracted in 4000 mL round bottom flask with 2000 mL methanol. The methanolic extracts was cooled at room temperature, filtered and evaporated to dryness under reduced pressure in a rotatory evaporator (Buchi Rotavapor).
In vitro lipid peroxidation: The assay of microsomal lipid peroxidation
was done according to the method of Wright et al.
(1981). To assess the potential of plant extracts to inhibit lipid peroxidation
four different concentration of plant extract were selected. Group I served
as a control group in which the reaction mixture consisted of 0.58 mL phosphate
buffer (0.1 M, pH 7.4), 0.2 mL ascorbic acid (100 mM) and 0.02 mL ferric chloride
(100 mM). In-group II, III, IV, V and IV in addition to the complete control
reaction mixture different concentration of plant extract were also added. This
reaction mixture was then incubated at 37°C in a shaking water bath for
1 h. The reaction was stopped by the addition of 1 mL of TCA (10%). Following
addition of 1.0 mL TBA (0.67%), all the tubes were placed in a boiling water
bath for a period of 20 min. The tubes were then centrifuged at 2500 xg for
10 min. The amount of malondialdehyde (MDA) formed in each of the sample was
assessed by measuring the optical density of the supernatant at 535 nm. The
results were expressed as the nmol MDA formed h-1 g-1
tissue at 37°C by using a molar extinction coefficient of 1.56x105
M-1 cm -1.
DNA sugar damage assay: The DNA sugar damage was assayed by the method
of Halliwell and Gutteridge (1981). Group I served as
a control group. In control the reaction mixture consisted of 0.5 mL calf thymus
DNA (1 mg mL-1 of 0.15 M NaCl), 0.5 mL phosphate buffer (0.1 M, pH
7.4) and 0.05 mL of FeCl3 (100 μM in final concentration). In
other groups i.e., Group II, III, IV, V and VI in addition to the above mixture
different concentrations of plant extracts were added. The reaction mixture
was incubated for 1 h at 37°C in a water bath shaker. After the incubation
was over, 1 mL TBA (0.67%) was added to the reaction mixture and then it was
kept in boiling water bath for 25 min. The TBA reacting species so generated
forms an adduct showing a characteristic absorption at 535 nm which was monitored
Cytochrome P450: Assay of Cyt - P450 content was done by the method
of Omura and Sato (1964). To assess the efficacy of
AM to inhibition of cytochrome P450 content, 4 groups were chosen. Group I served
as control, in which a pinch of sodium dithionate was added to 2 mL of sample.
This was then divided equally between matched cuvettes. In addition to the above-mentioned
reaction mixture different concentration of plant extract was also added. The
contents of the test cuvette were gently bubbled with carbon monoxide for about
one minute and the OD was taken at 450 and 490 nm.
Treatment schedule: AM extract was suspended in normal saline and B(a)P in corn oil. B(a)P and Aegle marmelos was administered orally. Each group consisted of five animals. In group I (vehicle control) animals were given normal saline (0.9%) orally. The animals of group II served as positive control and were administered single oral dose of B(a) P (125 mg kg-1 b.wt.). Animals of group III and V were pretreated with 50 mg kg-1 b.wt. of Aegle marmelos while group IV were pretreated with 25 mg kg-1 b.wt. of Aegle marmelos for seven consecutive days. The above-mentioned doses of Aegle marmelos were selected based on the preliminary studies by the investigator. On day 8, the animals of group II, IV and V were administered a single oral dose of B(a)P (125 mg kg-1).
Chromosomal aberration test: Mice were sacrificed by light ether anesthesia
30 h after treatment with B(a)P. About 90 min prior to killing, a single i.p.
dose of colchicine (4 mg kg-1) was administered to the animals. The
time of sacrifice was decided based on preliminary experiments as optimal for
scoring of aberrations. The slides of bone marrow cells were prepared and stained
according to the routine schedule for metaphase plate analysis i.e., hypotonic-acetic
acid-methanol-flame-drying-Giemsa (Preston et al.,
1987). At least 100 well-spread intact metaphases were scored per animal
under 100xoil immersion using a light microscope (Olympus BX 50). The type of
chromosomal aberration (CA) included chromatid and chromosome breaks and chromosomal
rearrangements. All aberrations were considered as equal regardless of the number
of breaks involved, gaps were not included. A single observer did blind scoring.
Micronucleus test: For this test, mice were sacrificed 28 h, after treatment
with B(a)P. The mouse bone marrow micronucleus test was carried out according
to the method of Schmid (1975). The air-dried slides
were stained with May-Grunwald and Giemsa as described by Schmid
(1975) made permanent and coded. A total of 2500 polychromatic erythrocytes
(PCEs) were scored per animal by the same observer for determining the frequencies
of micronucleated polychromatic erythrocytes (MnPCEs).
In vitro: Free radical generated by the iron/ascorbate system was inhibited by the addition of plant extracts doses. This inhibition in the formation of malondialdehyde was dose dependent. By the addition of AM extract there was an inhibition in iron ascorbate induced microsomal lipid peroxidation starting from 23-53% respectively as comparison to control group as shown in Fig. 1 and Table 1.
||Percent inhibition values of A. marmelos on lipid peroxidation,
cytochrome P-450 content and DNA sugar damage
||Efficacy of Aegle marmelos in inhibiting iron/ascorbate
induced microsomal LPO AM concentration; D1 = 20 μg, D2 = 40 μg,
D3 = 60 μg, D4 = 80 μg, D5 = 100 μg, p<0.005 when compared
||Efficacy of Aegle marmelos towards hydroxyl radical
induced DNA-sugar damage AM concentration, D1 = 20 μg, D2 = 40 μg,
D3 = 60 μg, D4 = 80 μg, D5 = 100 μg, p<0.005 when compared
All the test groups inhibited DNA sugar damage dose dependently. There was a dose dependent inhibition in DNA sugar damage ranging from 18-54%, respectively. The complete control was assumed to possess 100% DNA damage and 0% inhibition capacity as shown in Fig. 2 and Table 1. The maximum inhibition was observed in-group having highest concentration of plant extract.
||Efficacy of Aegle marmelos on in vitro cytochrome P-450
contents AM concentration, D1 = 20 μg, D2 = 40 μg, D3 = 60 μg,
D4 = 80 μg, D5 = 100 μg, p<0.005 when compared with control
||Effect of Aegle marmelos on chromosomal aberrations
induced by B(a)P in mice
|B: Chromatid breaks, B: Chromosomal breaks, R:
Rearrangements, aTotal 500 metaphase plates were observed per
group (n = 5 animals) for scoring chromosomal aberrations, *Significantly
different from the control group (p<0.001), #Significantly
different from the only B(a)P treated group (p<0.05)
|| Effect of Aegle marmelos on micronuclei induced by
B(a)P in mice
|PCEs: Polychromatic erythrocytes, NCEs: Normochromatic erythrocytes,
MnPCEs: micronucleated polychromatic erythrocytes, bMean±SE
of 2500 PCEs, *Significantly different from the control group (p<0.001),
#Significantly different from the B(a)P treated group (p<0.05),
##Significantly different from the B[a]P treated group (p<0.001)
According to our data with AM there is a dose dependent inhibition in the level of cytochrome P450 activity starting from 22-51%. Maximum inhibition was seen in plant having 100 μg concentration of plant extract as shown in Fig. 3 and Table 1.
In vivo: B(a)P treatment alone produced gaps and DNA strand breaks
in the cells, However cells with multiple chromosomal aberration and exchanges
were observed infrequently as they are not considered as good indicator of chromosomal
damage. Table 2 shows the reduction in frequency of chromosomal
aberrations induced in mouse bone marrow cells following oral administration
of the two doses of Aegle marmelos to the mice for 7 days before B(a)P
treatment as compared with the group treated with clastogen, i.e., B(a)P alone.
Significant reduction in the frequency of chromosomal aberrations was observed
at 25 and 50 mg kg-1 b.wt. doses of AM treatment. Significantly higher
incidence of chromosomal aberrations was observed when we compare control group
with only B(a)P treated group. In all cases, however, the aberration frequency
was lower in the animals given the clastogen alone.
The data presented in Table 3 shows the protective effect of AM against B(a)P induced mutagenicity as assessed by the bone marrow micronucleus test. B(a)P produced significant micronuclei formation when compared with the control group (p<.001) There was 37-57% inhibition of the B(a)P-induced micronuclei by the pretreatment with Aegle marmelos (p<0.001). B(a)P produces micronucleus formation and found significant difference when compared to the control (p<0.001).
Cancer and other chronic diseases may be related with mutations produced by
environmental agents; therefore, minimizing the exposure to harmful agents has
been recommended as a way to prevent these diseases (Aggarwal
et al., 2009). Unfortunately, it is not easy to eliminate the source
of genotoxic agent completely in modern society. Therefore, the identification
and application of well-known antimutagens is essential for improving human
health (Siddique and Afzal, 2008). Considerable emphasis
had been laid down on the use of dietary constituents to prevent mutagen induced
cytogenic damage and DNA damage due to their non-toxic effects. Many plants
and their isolated compounds have been tested to determine their antimutagenic
potential; like green and black teas are known to be strong mutagenic and carcinogenic
inhibitors (Bitiren et al., 2010; Santana-Rios
et al., 2001).
Oxidative stress causes a series of chain reaction which is known as lipid
peroxidation. Lipid peroxidation causes serious membrane damage and may even
lead to cell death (Yassa et al., 2008). It is
now widely known that the mutagenic capacity of free radical is due to the direct
interaction of hydroxyl radicals (. OH) with DNA. Such interactions induce numerous
lesions in DNA that cause deletions, mutations and other genetic effects. Characterization
of this damage to DNA has indicated that both the sugar and the base moieties
are susceptible to oxidation, causing base degradation, single strand breakage
and cross linking to protein (Aggarwal et al., 2009;
Momtaz and Abdollahi, 2012). In this study, in vitro
lipid peroxidation assay exposes that AM suppress free radical induced lipid
peroxidation dose dependently which further may inhibit the process of free
radical induce DNA damage.
The deoxyribose assay showed that on interaction of these free radicals with
transition metals the hydroxyl radical is produced by Fentons reaction
which is responsible for DNA damage (Ayene et al.,
2007). The dose dependent decrease in the calf thymus DNA sugar damage strengthens
our investigation. Our investigation further reveals that AM block the activation
of carcinogens by inhibiting Cyt P450 activity which further may inhibit the
process of tumorigenesis. Cyt P450 is the major enzyme involved in metabolism
of many drugs and xenobiotics. CYP activates Polycyclic Aromatic Hydrocarbon
(PAHs) into ultimate metabolites which covalently bind to DNA, a key event in
the initiation of carcinogenesis (Srinivasan et al.,
2008; Abdel-Latif and Sadek, 1999).
The chromosomal aberrations are produced by error in DNA molecule. Chromosomal aberrations are analyzed in mitotic metaphases from proliferative tissue such as bone marrow tissue. In micronuclei induction test, the clastogenic effects were measured indirectly by counting small nuclei in interphase cells formed by acentric chromosomal fragment or whole chromosomes. In this study, Aegle marmelos have been proven to reduce the micronuclei induction and chromosomal aberration dose dependently.
Thus, the results from the in vitro and in vivo experiments are
quite promising for the use of AM as a protective agent. The anti-clastogenic
effects of Aegle marmelos can be attributed to inhibition of lipid peroxidation,
DNA sugar damage and cytochrome p450 content in dose dependent manner. It is
concluded that Aegle marmelos can be used as a major chemopreventive
agent against B[a]P. induced mutagenicity.
Dr. Tajdar Husain Khan is thankful to the Indian Council for Medical Research (ICMR), New Delhi, India, for providing the funds to carry out this study.
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