The effect of Benzylisothfocyanate on epithelial changes

induced by trauma and DMBA in the hamster tongue

 
The purpose of this study was to investigate the effect of benzylisothiocyanate (BIT) on the epithelial changes induced by trauma and dimethylbenzanthracin (DMBA). Eighty male Syrian golden hamsters, five weeks old, were randomly divided into four groups. Group I received repeated trauma and mineral oil painting to the middle third of the right lateral border of the tongue. Group II was traumatized in the same way as Group I and received painting of the wound with BIT. Group III received similar trauma and painting of the wound with DMBA. Group IV received trauma plus DMBA and BIT painting in alternate dose.
Gross findings showed reduction in the development of persistent ulcers and papillary lesions in the animals that received BIT painting. Microscopic examination revealed epithelial changes ranging from mild dysplasia to invasive carcinoma in all animals. In Groups II and IV, fewer animals showed these changes, and the carcinomas seen in the two groups tended to be well differentiated when compared to the others.The results indicated that BIT retarded the development of neoplastic changes induced by trauma or trauma plus DMBA.

 
For many years chewing sticks were used as a natural tooth brush in different parts of the world especially in Islamic countries.1,2 Many studies have been published about such natural toothbrush and its importance in oral health. Most of these studies concerned the anticariogenic effect of the different types of chewing sticks and their role in reducing cariogenic bacteria and limiting inflammatory changes in the oral soft tissue.3-7
Some investigators have pointed out that some chewing sticks like Dialium guiveenes, Diospyros tricolor, Fagara zanthoxyloides, Garcinia kola, Massularia and Rhus glabra have anticarcinogenic activity.7-8 Recent studies have shown the presence of benzylisothiocyanate (BIT) in the roots of Saivadora persica, the most popular chewing stick in Saudi Arabia.9 BIT is classified as one of the chemo-preventive agents that are thought to prevent carcinogenic and other genotoxic compounds from reaching or reacting with the target sites on the treated tissues.10 BIT and closely related compounds, such as phenethylisothio- cyanate and benzylthiocyanate, are found naturally in cruciferous vegetables like cabbage, brussels sprouts, broccoli, and cauliflower.11,12
Wattenberg13 reported that the systemic admin­ istration of BIT to Sprague-Dawley rats which were exposed to dimethylbenzanthracin (DMBA) resulted in a lower incidence of mammary neop­ lasms and a decrease in the average number of tumors per animal. In another study, the addition of BIT to a diet containing benzo (a)pyrene inhi­ bited carcinogenesis of the mouse's fores-tomach.14
A search of the dental literature did not reveal any previous research on the effect of BIT on the tissues of the oral cavity. The present study was designed to test the effect of BIT on epithelial changes induced in the tongue of hamsters by repeated trauma and DMBA application.
For many years chewing sticks were used as a natural tooth brush in different parts of the world especially in Islamic countries.1,2 Many studies have been published about such natural toothbrush and its importance in oral health. Most of these studies concerned the anticariogenic effect of the different types of chewing sticks and their role in reducing cariogenic bacteria and limiting inflammatory changes in the oral soft tissue.3-7
Some investigators have pointed out that some chewing sticks like Dialium guiveenes, Diospyros tricolor, Fagara zanthoxyloides, Garcinia kola, Massularia and Rhus glabra have anticarcinogenic activity.7-8 Recent studies have shown the presence of benzylisothiocyanate (BIT) in the roots of Saivadora persica, the most popular chewing stick in Saudi Arabia.9 BIT is classified as one of the chemo-preventive agents that are thought to prevent carcinogenic and other genotoxic compounds from reaching or reacting with the target sites on the treated tissues.10 BIT and closely related compounds, such as phenethylisothio- cyanate and benzylthiocyanate, are found naturally in cruciferous vegetables like cabbage, brussels sprouts, broccoli, and cauliflower.11,12
Wattenberg13 reported that the systemic admin­ istration of BIT to Sprague-Dawley rats which were exposed to dimethylbenzanthracin (DMBA) resulted in a lower incidence of mammary neop­ lasms and a decrease in the average number of tumors per animal. In another study, the addition of BIT to a diet containing benzo (a)pyrene inhi­ bited carcinogenesis of the mouse's fores-tomach.14
A search of the dental literature did not reveal any previous research on the effect of BIT on the tissues of the oral cavity. The present study was designed to test the effect of BIT on epithelial changes induced in the tongue of hamsters by repeated trauma and DMBA application.

 
Eighty male Syrian golden hamsters, five weeks old, were used. They were caged individually, weighed weekly, and fed rat chow and water ad libitum. They were divided into four equal groups as follows:
Group I:       Animals in this group were traumatized at the middle third of the right lateral border of the tongue three times a week with a barbed broach. The traumatized part, as well as the middle third of the lateral border of the left side the tongue, was then painted with mineral oil.
Group II:        In   this   group,    the   animals   were traumatized as in Group I, and received painting of the wound and the middle third of the lateral border of the left side of the tongue with BIT* in mineral oil vehicle.
Group III:       The middle third of the lateral border of the right   side of the tongue was traumatized with barbed broach followed by a painting with DMBA** three times a week.
Group IV:       Animals of this group were traumatized in the same way as group III. The traumatized area was painted with BIT and DMBA three times a week in alternate dose. DMBA was applied 48 hours before the application of BIT
BIT was dissolved in heavy mineral oil at a concentration of 50 mg of BIT per 1 ml of mineral oil. DMBA was dissolved in a heavy mineral oil to a final concentration of 0.5% solution. Both chemical agents were applied to the treated sites with a No. 4 camel's hair brush. The solution was in contact with area of application for at least five seconds. All precautions were followed while handling the biohazardous materials, including the wearing of disposable gloves, masks, and laboratory coats.
Prior to traumatization, the animals were sedated by intramuscular injection of xylazine 20 mg/ml at a dose of 0.6 mg (0.03 ml)/100 g body weight. The injections were given in the thigh, alternating sides at successive injections. Following induction of surgical anesthesia, the hamster tongue was pulled forward with a pair of pincers. The tips of the pincers were covered with rubber tubing to minimize non-experimental trauma to the tongue. The tongues were rotated to the left side so that the middle third of the right lateral border became horizontally positioned. A barbed broach was used to abrade the tongue by passing it several times in parallel strokes on the mucosa until the epithelium was visually damaged and/or cutting wounds were noted
The tongues were observed every other day throughout the experiment for gross changes, and selected examples were photographed. After 12 weeks of the first treatment, ten of the 20 hamsters in each group were randomly selected for sacrifice by C02 inhalation and opening of the thoracic cavity to assure death. Each animal was dissected, and the tongues were removed for microscopic examination. Treatment of the remaining 10 animals was continued for another 12 weeks. Gross tissue changes were periodically observed and recorded. At the end of the 24th week, all remaining animals were sacrificed and the tongues were removed for microscopic examination.
The histologic criteria evaluated in this study included those changes described as disturbances in polarity, drop-shaped rete ridges, polymorphism, hyperchromatism, basal cell hyperplasia, alteration in the nuclear/cytoplasmic ratio, increased mitoses, and intraepithelial keratinization. The epithelial alterations from normal were divided into three main types: dysplasia, carcinoma-in-situ, and invasive carcinoma. Dysplasia, defined as alteration of development, was classified as mild, moderate or severe, depending on the degree of the above mentioned changes. Carcinoma-in-situ indicated a dyspiastic "top to bottom" change, with the basement membrane still intact. In cases where the basement membrane was disrupted, a diagnosis of invasive carcinoma was assigned.

During both study periods, all animals were observed before each treatment for changes at the treated sites or adjacent areas. Changes seen grossly were recorded according to one or more of the following categories:

Distribution of the gross findings are summarized in Table 1 [Figs. 1,2]. Results of histological examination of the tongues for all groups are summarized in Table 2. Some examples of such changes are shown in Figures 3-5
Results were analyzed using the variance and subsequent studentized range "q" tests for arcsine transformation of percentage data. No significant differences were detected between the four groups in the development of leukoplakia and papillary lesions during both study periods. A significant difference in the development of persistent ulcers and delayed healing was seen between Groups I and III, and Groups II and IV (P<0.05) during the first study period. The differences were not significant during the second part of the study.
Analysis of the microscopic changes with invasive carcinoma indicated that the only significant difference between groups was between Groups II and III during the first part of the study (P<0.05). Comparing the study periods within each group showed a significant difference in Group I (P<O.05). No significant differences could be detected within the other groups.