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.
Histological Analysis
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.
Kidney
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.
Stomach
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).
Table I: Gastric abnormalities scored using Baylor Modification of The Sydney System
Lungs
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,
Figure 3A-F).
Table II: Method for scoring the damage incurred by lung tissue of the animal
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 adducts[16]. 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 degradation[17]. A study by Coussens and Werb has proved a link between inflammation and onset of cancer[18]. 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 enzymes[3,21].
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 damage[22]. 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 mouse[23]. The ability of isothiocyanates to ameliorate the carcinogen metabolism has been shown in a number of different studies[24,25].
ACKNOWLEDGEMENT
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),
New Delhi.
CONFLICT of INTEREST
The authors pose no conflict of interest.
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