Bioprotective Efficacy of Erucin Against 7 , 12-Dimethylbenz ( α ) Anthracene-Induced Microstructural Changes in Male Wistar Rats

Environmental pollutants have always been known to induce various physiological and biochemical alterations in the living system (1). Among the various pollutants, 7,12-dimethylbenz(α)anthracene(DMBA) is the most potent mutagen released due to incomplete combustion (2). It has been known to induce microstructural changes (histopathological) in the liver tissue of male Wistar rats (3). 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 often accompanied by serious side effects (4,5). 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 agents (6,7). Among these, glucosinolates (GSLs) and especially their hydrolytic products have comparatively higher bioactivity with limited side effects (8). 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.

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 method (3,9).

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.

RESULTS
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/ protection.

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 obviating DMBA induced damage.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

A B C
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 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 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).  of stomach.Among the different changes, chronic inflammation was observed in the stomach lining of rats.this inflammation is caused by the action of 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 by the transformation of 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 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 caused 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 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 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).
In conclusion, the current study was designed to investigate the bioprotective role of erucin against DMBA-induced microstructural changes in male Wistar rats.It was observed