Bioprotective Efficacy of Erucin Against 7,12-Dimethylbenz(α)anthracene-Induced Microstructural Changes in Male Wistar Rats
Rahul MANNAN1, Rohit ARORA2, Sakshi BHUSHAN3, Sonam SHARMA4, Tejinder BHASIN1, Saroj ARORA2
1Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, AMRITSAR, INDIA
2Department of Biochemistry, Sri Guru Ram Das Institute of Medical Sciences and Research, AMRITSAR, INDIA
3Department of Botanical and Environmental Sciences, Guru Nanak Dev University, AMRITSAR, INDIA
4Department of Pathology, Vardhman Mahavir Medical College and Safdarjung Hospital, NEW DELHI, INDIA
Keywords: Isothiocyanate, Erucin, Polyaromatic hydrocarbons, DMBA, Histological analysis
Environmental pollutants are responsible for inducing a number of biochemical and physical changes in the various organs of an
organism. The most potent pollutants is7,12-dimethylbenz(α)anthracene (DMBA) (a genotoxic carcinogen), a polyaromatic hydrocarbon. It
is known to induce a number of genetic changes including the formation of DNA adducts. These genotypic changes in turn are responsible for
inducing microstructural changes in the vital organs viz. kidneys, stomach and lungs of an organism. These changes in turn cause biochemical
alterations in an animal and hence alter the normal functioning. Synthetic drugs provide a sigh of relief but a large number of side effects in
addition to the growing resistance has made it necessary to find an efficient alternative. Plant secondary metabolites, especially glucosinolate
hydrolytic products, are known for their high bioprotective activity. The current study was therefore designed to analyze the bioprotective role of
erucin against DMBA-induced microstructural changes in kidneys, lungs and stomach of male Wistar rats using histological analysis.
Material and Method: The bioprotective efficacy of erucin was evaluated in male Wistar rats against DMBA induced microstructural changes.
The rats were divided into five groups where group I was untreated control, group II was DMBA treated and group III-V were treatment (DMBA
+ erucin) groups.
Results: Erucin was able to protect the rats against DMBA-induced histopathological changes in the lungs and stomach. No changes were
observed in the kidney following treatment with DMBA or erucin.
Conclusion: Erucin has bioprotective activity against genotoxic carcinogen.
Environmental pollutants have always been known to
induce various physiological and biochemical alterations
in the living system1
. Among the various pollutants,
7,12-dimethylbenz(α)anthracene(DMBA) is the most
potent mutagen released due to incomplete combustion2
. It has been known to induce microstructural changes
(histopathological) in the liver tissue of male Wistar rats3
. These changes in turn are responsible for inducing
biochemical changes in the animals and thus lead to the
onset of various damaging effects. A number of drugs are
available for countering these environmental pollutants but
these they are often accompanied by serious side effects4,5
In addition, they mostly have limited efficacy and are prone
to resistance development after a certain time period. These
limitations of the synthetic drugs demand the use of natural
plant products with diverse biological activities. Plant secondary metabolites such as alkaloids, phenols, flavonoids
and glucosinolates have shown immense biological activity
and are often used as an antioxidant, antitumor, anticancer,
bioherbicide and insecticide agents6,7
. Among these,
glucosinolates (GSLs) and especially their hydrolytic
products have comparatively higher bioactivity with limited
. The current study was therefore designed to
evaluate the protective activity of a glucosinolate hydrolytic
product (GHP), erucin (4-methylthiobutyl isothiocyanate),
against the microstructural changes induced by DMBA in
the extrahepatic organs (lungs, stomach and kidneys) of
male Wistar rats using histopathological analysis.
|Animals and Treatment
Male albino rats of Wistar strains aged 40-80 days and
weighing 0.15-0.30 kg were chosen for the current study. The rats were kept in polypropylene cages with paddy husk
bedding and maintained at a temperature of 25 ± 2 °C and a
12 h light: 12 h dark condition in the animal house of Guru
Nanak Dev University, Amritsar. They were allowed water
and pellet diets at ad libitum. The study was approved by the
animal ethical committee of GNDU, Amritsar with the file
number 226/CPCSEA. The animal experimental analysis
was done using the guidelines given by Committee for the
Purpose of Control and Supervision of Experiments on
Animals (CPCSE), Ministry of Environment and Forests,
Government of India. The rats were divided in five groups
and each group had a total of six rats. The first group was
a negative control receiving corn oil (vehicle). The second
group received DMBA (20 mg/kg bw), and the third to fifth
groups received DMBA + erucin (20, 35 and 50 mg/kg bw).
All the doses were given for five days through intraperitoneal
injection and the experiment was terminated on the sixth
day. The animals were sacrificed using cervical dislocation
and their liver was removed under sterile conditions. The
test compound erucin was isolated and characterized as per
the earlier given method3,9
The rats were euthanized and their lungs, stomach and
kidneys were removed under sterile conditions and
transported in 10 % formalin to the histopathological
unit. The organs were individually inspected and sections
were taken from the representative areas. The sections
were processed as per the standardized protocol and
were embedded in paraffin blocks. Two to three micron
sections were cut and then stained using haematoxylin
and eosin. The slides thus prepared were segregated
according to the specific organ system and then labeled in
a blinded way to prevent any biasing. The slides themselves
were observed on light microscope and scored by two
histopathologists in a blinded manner. The observations
were noted after a mutual consensus was reached by both
the histopathologists. The scoring systems were designed
before the start of the study. For the kidney, the European
Vasculitis Study Group (EUVAS) classification system was
chosen for characterisation of injury patterns. For gastric
biopsies, the Baylor modification of “The Sydney System”
for gastropathies was employed whereas for the lung, a
novel injury score pattern was developed.
The histological studies were conducted in the three
extrahepatic organs viz. lung, stomach and kidney and
the results were presented as per the observed damage/
Extensive study revealed the absence of any damage of
either DMBA or erucin on the kidney of male Wistar rats
(Figure 1A-C). A normal kidney tissue with intact structural
characteristics was observed. No microstructural changes
were observed following the treatment. The study therefore
indicates absence of any role of the kidney in the obviation
of damage incurred by DMBA. The mutagen was not able
to cause any deleterious change in the structure and hence
a normal physiological role of the organ was maintained, as
observed by histological studies.
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|Figure 1: Figure representing normal kidney histopathology
following the treatment with DMBA alone and in combination
with erucin. A) Unremarkable renal parenchyma showing
glomerulus, tubules, blood vessels and interstitium (H&E;
x100). B) Normal glomerulus with thin walled capillaries and
surrounding normal tubules (H&E; x400). C) Unremarkable
proximal and distal convoluted tubules (H&Ex; x100).
The changes noted in the gastric biopsies were graded and
scored according to the four main parameters: types of
damage viz. chronic inflammation, activity, atrophy and
intestinal metaplasia. These changes were further subdivided
as no change, mild change, moderate and severe change.
The scoring was then done as per the damage observed
and cumulative score was considered as the total damage
incurred in the organ of the animal following the treatment.
The current study showed that the highest damage was in
the stomach of DMBA-treated rats with a cumulative score
of 8 out of 12. Moderate chronic inflammation, activity,
atrophy and intestinal metaplasia was seen in this treatment
group. The untreated control group had mild chronic
inflammation and a damage score of 1/12 was recorded. In
contrast, the treatment (DMBA + erucin) groups showed
a dose-dependent result. The highest damage was in the
DMBA + 20 mg/kg bw erucin group, with a score of 5/12.
This damage was further reduced to 3/12 and 1/ 12 in the
group treated with 35 and 50 mg/kg bw erucin, respectively
(Table I, Figure 2A-D).
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|Table I: Gastric abnormalities scored using Baylor Modification of “The Sydney System”
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|Figure 2: Figure representing gastric histopathology following the treatment with DMBA alone and in combination with erucin.
A) Mucosal ulceration with moderate degree of chronic inflammation (H&E; x400) [DMBA + erucin (50 mg/kg bw)]. B) Neutrophilic
infiltrate in the biopsy showing evidence of moderate activity (H&E; x1000) [DMBA + erucin (35 mg/kg bw)]. C) Mucosal flattening
and moderate degree of gastric atrophy with focal evidence of intestinal metaplasia (H&E; x200) [DMBA + erucin (20 mg/kg bw)].
D) Intestinal metaplasia (goblet cells)(H&E; x400) [DMBA alone].
The effect of DMBA and erucin alone and in combination
on the lungs of male Wistar rats was also analyzed in the
current study. The histological changes in the lungs of the
rat were categorized in six different parameters with further
subtypes. These changes were disease pattern (absent,
localized and generalized), intra-alveolar congestion
(absent and present), intra-alveolar infiltration by
inflammatory cells (mild, moderate and severe), alveolar
hyperplasia (absent and present), interstitial inflammation
(mild, moderate and severe) and interstitial fibrosis (mild,
moderate and severe). The changes were recorded as per the
damage observed in the prepared slides. It was seen that the
rats treated with DMBA alone showed the highest damage
with a score of 9/13. All the damaging characteristics were observed in the tissue, suggesting severe microstructural
changes in this group. In contrast, a low damage of 3/13
was observed in the untreated control. The slight damage
observed in this group might be due to the continuous
delivery of corn oil to the intraperitoneal cavity of the
animal. As seen in the stomach, a dose-dependent result
was observed in the lungs of rats treated with DMBA in
addition to erucin (20, 35 and 50 mg/kg bw). sThe erucin
treatment ameliorated the damage incurred on the lungs as
seen by a reduction in damage score to 5/13, 4/13 and 3/13
in the low, medium and high erucin treated group (Table II,
Click Here to Zoom
|Table II: Method for scoring the damage incurred by lung tissue of the animal
Click Here to Zoom
|Figure 3: Figure representing
lung histopathology following
the treatment with DMBA
alone and in combination
A) Generalised inflammatory
response in lung parenchyma
(H&E; x400) [DMBA +
erucin (50 mg/kg bw)].
B) Mild intra-alveolar
congestion (H&E; x1000)
[DMBA + erucin (35 mg/kg
C) Dense inflammatory
infiltrate and interstitial
fibrosis around the
terminal bronchiole in lung
parenchyma (H&E; x1000)
[DMBA + erucin (20 mg/kg
D) Alveolar hyperplasia in
lung parenchyma (H&E;
x1000) [DMBA alone].
E) Mild to moderate
in lung parenchyma (H&E;
F) Interstitial fibrosis in lung
parenchyma (H&E; x1000)
Increasing pollution and the drastic change in the lifestyle
these days have made us susceptible to various deleterious
. These pollutants are also responsible for
inducing various microstructural changes in the vital organs of the body11
. The xenobiotic compounds are
metabolized by liver but other organs such as the lungs,
stomach and kidneys play a key role in their metabolism
. These xenobiotic compounds are
countered efficiently by plant secondary metabolites such
. Among the various isothiocyanates,
erucin (an analogue of sulforaphane) has been isolated and
evaluated for its ability to protect the rats against the toxic
effect of DMBA. The current study was therefore designed
to investigate the role of erucin against the deleterious effect
of the pollutant DMBA on the lungs, stomach and kidneys
of the male Wistar rats. The mutagen and test compound
were unable to induce any histopathological changes in
the kidney of the rats suggesting that DMBA is completely
metabolized by liver and other organs and thus it has no
negative effect on kidneys. In addition, the test compound
erucin had no toxicity as observed by an absence of
Since the mutagen (DMBA) was administered in the
intraperitoneal cavity, it increased the susceptibility of
the stomach to its toxicity. This was confirmed by marked
microstructural changes in the tissue of experimental animals. DMBA acts as a genotoxic mutagen and causes
the formation of DNA adducts16. These adducts alter
the genetic makeup of the rats and thus cause a number
of changes in the physiological and physical makeup of stomach. Among the different changes, chronic
inflammation was observed in the stomach lining of rats.
This inflammation is caused by the action of the drug on the
lining of stomach and a prolonged contact results in further
degradation17. A study by Coussens and Werb has
proved a link between inflammation and onset of cancer18. The DMBA administration was also responsible
for further degradation of the stomach as observed by
increased atrophy. The activity of stomach is also reduced
by the action of DMBA. This reduced activity develops due
to lower enzyme function, altered by the action of drug. The
combined damaging effects of DMBA in the stomach caused
the transformation of the gastric epithelium. This intestinal
metaplasia further degraded the normal functioning of
rat tissue. In contrast, the treatment with erucin was able
to counter the deleterious role of DMBA and helped in
protecting the stomach microstructural makeup of male
Wistar rats. A thorough literature survey has shown the
bioprotective role of isothiocyanates (ITCs)19,20. These
metabolites protect the various organs of an organism viz.
lungs, liver and stomach against different carcinogens. They
are known to inhibit DNA adduct formation and counter
phase I and phase II xenobiotic enzymes3,21.
The administration of DMBA induced a generalized disease
pattern in the lungs of rats. Intra-alveolar congestion and
infiltration were seen in the tissues of the animal. The
alveoli were readily swollen and filled with blood due to the
prolonged action of DMBA. This alveolar swelling caused
increased opacity and density of the tissue, hampering the
normal functioning of the lungs of rats. The volatile nature
of this compound further added to an increased irritation
in the tissue and thus increased damage22. The combined
effect of the above damage is responsible for atypical
alveolar hyperplasia. The size of the organ was enlarged
due to the activity of DMBA. The mutagen causes the
formation of DNA adducts and thus elevated inflammation
and thickening or scarring of the pulmonary tissue. The
treatment with erucin on the other hand prevented the
formation of DNA adducts and protected the lungs of
the rats from the deleterious effects of DMBA. A study
by Hecht et al. has shown the bioprotective role of ITCs
against polyaromatic hydrocarbons-induced toxicity in A/J
mouse23. The ability of isothiocyanates to ameliorate the
carcinogen metabolism has been shown in a number of
The current study was designed to investigate the
bioprotective role of erucin against DMBA-induced
microstructural changes in male Wistar rats. It was observed that DMBA caused severe changes in the physical makeup
of the lungs and stomach of the animal. The test compound
erucin, on the other hand, protected the animal against
these severe changes and is thus a suitable candidate for
further studies. No alteration was observed in the kidneys
of the animals following the treatment with DMBA, erucin
or their combination.
The authors would like to acknowledge the financial
assistance by Department of Science and Technology (DST),
New Delhi and University with Potential for Excellence
(UPE) scheme of University Grants Commission (UGC),
CONFLICT of INTEREST
The authors pose no conflict of interest.
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