Download prevention of radiation-induced hepatic damge on swiss albino mice

PREVENTION OF RADIATION-INDUCED HEPATIC DAMAGE ON
SWISS ALBINO MICE BY ALOE VERA LEAF EXTRACT
Prashasnika Gehlot and P.K. Goyal*
Radiation and Cancer Biology Laboratory, Department of Zoology,
University of Rajasthan, Jaipur 302004, India
*Corresponding author E-mail; [email protected],
ABSTRACT
The radioprotective effect of Aloe vera leaf extract (AVE) was studied in Swiss albino mice
against radiation-induced changes in the liver. The mice were treated with 1000 mg /kg body
weight of AVE orally once daily for 15 consecutive days before exposure to an acute dose of 6
Gy of gamma radiation after the last administration. Irradiation of mice caused a significant
elevation in lipid peroxidation with the decrease in the glutathione, acid phosphatase and
alkaline phosphatase. Treatment of mice with AVE before irradiation elevated the glutathione,
acid phosphatase and alkaline phosphatase accompanied by a decline in lipid peroxidation. The
recovery and regeneration were faster in AVE pretreated animals than the irradiation alone.
The data clearly indicate that the AVE significantly reduced the deleterious effect of radiation
in the liver and could be a useful agent in reducing the side effects of therapeutic radiation.
Key Words: Aloe vera, liver, gamma radiation, Swiss albino mice, LPO
Corresponding author Email: [email protected]. Phone 091-0141- 2 651199, fax
(1) 0141-2742309 (2) 0141-2740867 Radiation and Cancer Biology Laboratory, Department of
Zoology, Rajasthan University, Jaipur 302004, India.
1
INTRODUCTION
Gamma radiation is the most commonly used source of ionizing radiation to treat neoplastic
disorders in clinical conditions. Clinical success of radiotherapy depends on its ability to
selectively kill tumor cells while sparing the normal surrounding tissues. The response of
mammalian cells to ionizing radiation sat the cellular and molecular levels is complex and is an
molecular levels is complex and is an irreversible process that is dependent on both the
radiation dose and tissue –weighting factor[1].
Due to lack of an effective protective agent, newer compounds are currently under
investigation as possible adjuvant in the radiation treatment of cancer, and herbal medicines
have only recently begun to receive some attention as possible modifiers of the radiation
response [2].
Studies carried out in the past 15 years have shown that herbal preparation such as Liv. 52 [3],
Brahmarasayana [4], Pododphyllum [5], Ocimum sanctum [6], Triphala [7], Emblica
officinalis [8], Rosemarinus officinalis [9] reduced radiation –induced damage in different
body systems.
One such popularly known used plant is Aloe vera barbadensis [10] belonging to family
Liliacae and consists of about more than 250 species [11]. It is commonly called “Guar –patha”
or Ghee-Guar. It is rich in vitamin A, E, C and Zinc and Selenium. It is reported to have
antioxidant, anti-tumor and anti-inflammatory activities [12, 13].
The present investigation has been made to assess the radioprotective efficacy of Aloe vera leaf
extract in the hepatic constituents of Swiss albino mice.
MATERIALS AND METHODS
Animal care and handling: The animal care and handling was done according to the
guidelines set by World Health Organization, Geneva, Switzerland and INSA (Indian National
Science Academy, New Delhi, India). The departmental animal ethical committee approved
this study. Swiss albino mice (Mus musculus), 6-8 weeks old with body weight of 24+2 gm,
from an inbred colony were used for the present study. These animals were maintained under
controlled conditions of temperature and light (light: dark, 10hrs:14 hrs.). Four animals were
housed in a polypropylene a cage containing sterile paddy husk (procured locally) as bedding
throughout the experiment. They were provided standard mice feed (procured from Hindustan
Levers Ltd., India) and water ad libitum. Tetracycline water once a fortnight was given as
preventive measures against infections.
2
Irradiation: Mice were irradiated by 60Co source in the cobalt teletherapy unit (ATC-C9) at
radiation oncology department, Sawai Man Singh Medical College and Hospital, Jaipur, India.
The mice wee kept in ventilated box with a distance of 77.5 cm from the source to deliver a
dose rate of 0.99 Gy min-1.
Plant material: Aloe leaves were collected locally during the whole year. The Aloe vera plant
was identified by the curator at the Herbarium of Botany, University of Rajasthan, and Jaipur,
India. (RUBL Number19886)
AV Extract: To prepare aqueous extract, fresh shade dried leaves of Aloe vera powdered and
refluxed with double distilled water (DDW) for 36 hours at 400 C and vaccum evaporated so as
to get in powder form. The powder of extract was redissolved in DDW just before oral
administration.
Experimental design: Mice were randomly divided into following groups (five per group per
interval:
Group I: Normal / sham-irradiated mice were given distilled water (DDW) through oral
gavage once in a day for 15 consecutive days.
Group II: Mice were treated with 1000 mg/kg body weight of AV dried extract dissolved in
distilled water through oral gavage for 15 consecutive days.
Group III: Mice were given distilled water for 15 days and then exposed to 6 Gy dose of
gamma radiation. This group served as positive control.
Group IV: Extract of Aloe vera was given 1000 mg/ kg body weight of mouse orally for 15
days and after 30 min. of last dose; they were exposed to 6 Gy dose of gamma radiation.
Following various treatments, mice were autopsied by cervical dislocation on days 12hrs. 24
hrs. 3, 5, 10, 20 and 30 days. Liver were surgically removed and fixed in Bouin’s fluid. The
liver was embedded in paraffin block after dehydrating with increasing concentrations of 70,
90 and 100% ethanol. Five micrometer sections were cut using hand microtome, were placed
on glass slide and were stained with Harris hematoxyline and Eosin. Stained liver sections
were observed under light microscope to determine histopathological changes. Homogenate of
liver was prepared and activity of acid and alkaline phosphatase was measured by using
commercially available kits. Spectrophotometer (Systronics UV-VIS-108) was used to measure
the optical densities.
3
Glutathione (GSH) Assay: GSH in liver and blood was measured using the method described
by Moron et al. [14] and Beutler et al. [15]. The absorbance was read at 412 nm using a
systronic UV-VIS-108 Spectrophotometer.
Lipid peroxidation (LPO) Assay: LPO levels in liver and blood were estimated by the
method of Ohkawa et al. [16]
as thiobarbituric acid (TBA) reactive substances. The
absorbance was read at 532 nm with a Systronic UV-VIS-108 Spectrophotometer.
Statistical analysis: The statistical significance of the difference between normal and DDW+
irradiated (control) as well as control and AVE+ irradiated (experimental) set up were
evaluated by using the student’s ‘t’ test.
Results
Acid phosphatase: A significant increase in acid phosphatase activity over normal was
measured up to day 5th after 6 Gy irradiation. On day 5th, the value was found to be 76.26 per
cent more as compared to normal. After this, level of such enzyme declined but remained
higher than the normal even on the last day of experimentation (Fig. 1).
3
a
Acid Phosphatase (KAU)
c
a
Normal
Control
b
Experimental
a
2.5
c
c
c
a
2
1.5
0 hrs.
12 hrs.
24 hrs.
3 days
5 days
10 days
20 days
30 days
Autopsy Interval
Fig.1: Acid Phosphatase level in liver after 6 Gy of γ-irradiation with
(Experimental) or without (Control) AVE
4
Alkaline phosphatase: Alkaline phosphatase values elevated sharply from beginning until day
5th, where the value was 2 folds higher than the normal. After day 5th, a declining trend was
observed but normal value could not be restored even till day 30th (Fig. 2).
3.75
Alkaline Phosphatase (KAU)
Normal
Control
b
Experimental
b
b
3.25
c
b
a
2.75
c
2.25
0 hrs.
12 hrs.
24 hrs.
3 days
5 days
Autopsy Interval
10 days
20 days
30 days
Fig.2: Alkaline Phosphatase level in liver after 6 Gy of γ-irradiation with
(Experimental) or without (Control) AVE
Glutathione (GSH):
No significant variation was observed in hepatic GSH content of normal (Group-I) and AVE
treated animals (Group-II). A dose dependent decrease in GSH content was evident in control
animals (Group-III). The decrease in GSH level was more pronounced at the higher dose than
at the lower doses of gamma radiation. An increase in GSH value was measured in AVE
pretreated irradiated animals i.e. experimental animals.
Blood-GSH:
Blood GSH level also did not show any significant variation in normal (Group-I) and ROE
treated animals (Group-II). However, a dose dependent decline was noticed in control animals
(Group-III). AVE pretreated irradiated animals (Group-IV) showed a significant increase in
GSH level over the control, but the values remained below the normal (Fig. 3).
5
Glutathione Level
GSH Level
10
8
C
6
4
C
2
0
Liver μmole/gm
blood GSH
μg/ml
normal
drug alone
control
experimental
Fig.3: Glutathione level in blood and liver after 6 Gy of γ-irradiation with
(Experimental) or without (Control) AVE
Liver-LPx:
A dose dependent elevation in LPx level in terms of thiobarbituric acid reactive substances
(TBARS) or malondialdehyde (MDA) was measured in the control animals. This increase was
more at higher dose than the lower doses of gamma radiation. AVE pretreatment inhibited such
increase in LPx level as it was found to be significantly lower than the corresponding controls.
Serum-LPx:
No significant alterations in LPx level of serum were observed in normal and AVE treated
animals. In the control animals, a dose dependent increase in TBARS was noted following
irradiation, and such values were found to be significantly higher than the normal. However, in
AVE pretreated irradiated animals (Group-IV), such level was found to be significantly below
the respective controls (Fig. 4).
6
Lipid peroxidation
8
7
6
5
C
4
C
3
2
1
0
Blood
mol/ml
Normal
Liver
mol/mg
Drug alone
Control
Experimental
Fig.4: Variation in LPO level after 6 Gy of γ-irradiation with (Experimental) or
without (Control) AVE
Discussion
In the present study, after exposure to 6 Gy gamma radiations to animals resulted in radiation
sickness within 3-5 days after exposure. The symptoms included reduction in the food and
water intake, weight loss, diarrhea, ruffling of hairs and irritability. The similar symptoms have
been observed in mice after gamma irradiation by others also [17, 18].
Liver is the primary organ of drug metabolism. It plays a key role of detoxification agency in
the body. Any damage to this organ may cause serious disorders in the form of various
diseases, which can be observed in the form of histopathological and biochemical lesions.
Therefore, the present study has been targeted on liver to evaluate radiation induced
biochemical lesions and the radioprotective potential of the leaf extract of plant Aloe vera in
gamma irradiated mice.
An increase in the liver phosphatase activity of mice due to irradiation was observed in the
present study. Similar increase in such enzymes was observed earlier after exposure of mice or
rats with various doses [19]. Aikman and wills [20] suggested that lesions are produced in the
membrane lipids due to irradiation possibly buy peroxides , which lead to the activation of
latent acid hydrolases , that could result in the digestion of the membrane itself, with the
consequent activation and release of the other lysosmal enzymes , in the present study . It was
observed that after AVE administration radiation-induced increase in ACP was found to be
significantly of lower degree. Hamdy et al. [21] observed protection of acid phosphatase, when
solutions of lysosomal enzymes were exposed to high doses of radiation containing cysteine
7
and 4-aminoaphthal. Nam [22] too observed significant protection against rise in of serum acid
phosphatase at 6 and 130 hrs, if methylene blue was injected to rats prior to irradiation with
360 R. Bhartiya and Khan [23] also found an increase in ACP activity in mice after exposure to
different doses of radiation.
A rise in ALP activity in liver after gamma exposure, as evident in present experiment, is quite
similar to early reports Khan et al. [24]. ACP and ALP are the enzymes, concerned with
biosynthesis of fibrous proteins [25] and mucopolysaccharides [26]. They also act as the
hydrolytic enzymes which play an important role in dissolution of dead cells of the body [27].
Radiation –induced cell death may be a possible reason for increased activity of ACP and ALP.
Post-irradiation damage to liver is another reason attributing to increased level of ACP and
ALP. Radiation –induced stress also accounts for an increased activity of these enzymes as
observed in the present study. It has been observed that AVE provided protection by exhibiting
a significant decrease against radiation-induced increase in ACP as well ALP at various
periods of study in experimental animals compared with respective controls. Similar increase
in such enzymes was observed earlier [28].
The ALP activity in KB cells increased upon treatment with aloe-emodin when compared to
controls was observed. Samarth et al. [29] reported that the activity of ACP increased and
ALP decreased in Mentha piperita treated irradiated mice.
Aloe barbadensis Mill has been used in variety of disease in traditional Indian system of
medicine in India and its used for hepatic ailments is also documented. In the present study an
attempt s been made to validate its hepatoprotective activity. This is in good accordance with
the studies of aqueous extract of Aloe barbadensis is significantly capable of restoring integrity
of hepatocytes indicated by improvement in physiological parameters, excretory capacity (BSP
retention) of hepatocytes and also by stimulation of bile flow secretion [30].
Ionizing radiation induces lipid peroxidation (LPx) which can lead to DNA damage and cell
death [31, 32]. Therefore, an agent that protects against such damage can provide protection
against radiation injury. The administration of AVE before irradiation significantly reduced the
amount of LPx compared to the control group. The inhibition of LPx by AVE may also have
been responsible for the observed radioprotection.
Earlier reports with same animal model also proved that Aloe vera is a good radioprotection
against mouse intestinal mucosa after low level (0.5 Gy) exposure to gamma radiation [33].
An increase in the glutathione (GSH) level by AVE may be responsible for the scavenging of
radiation-induced free radicals including LPx and thereby protecting against radiation-induced
mortality. It has been reported that LPx starts to increase as soon as the endogenous GSH is
exhausted, and the addition of GSH promptly stops further peroxidation [34]. The AVE is
found effective in altering the sensitivity to radiation possibly by protecting the biological
molecules against radiation-induced oxidation of water by decreasing the oxygen tension in the
organism.
8
CONCLUSIONS
From the present study, it can be concluded that Aloe vera leaf extract provides a significant
protection against radiation-induced biochemical alterations in liver. The exact mechanism of
its action is not known; however, it may scavenge free radicals produced by radiation and thus
inhibit radiation-induced damage to the cellular DNA. Alternatively, it may also increase GSH
levels and reduce radiation-induced LPx.
ACKNOWLEDGEMENT
Authors are heartily thankful to Prof D.P.Agarwal (Head) and Dr. A.K. Chougule (RSO),
Department of Radiotherapy, SMS Medical College and Hospital, Jaipur, for providing
radiation facility and dosimetry respectively.
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