Genotoxic activity of betel nut on germinal cell in Sarcoma 180 ascites tumour bearing male mice

The genotoxicity of the ethanolic extract of betel nut was evaluated using sarcoma 180 tumour bearing mouse considering sperm motility, sperm viability, biochemical estimation of fructose in seminal fluid and sperm head morphology assays. Sperm head morphology was studied by H-E staining and Toluidine blue staining method. But Toluidine blue staining method is a reliable method to evaluate the DNA damage of sperms. Ethanolic BNE (betel nut extract) can suppress the percentage of sperm motility, sperm viability and seminal fructose level. In addition, it can also enhance the percentage of DNA damaged sperms. Moreover, histological sections of testes have been studied in control and BNE treated sarcoma 180 tumour bearing mice to highlight the potential toxic effect of BNE. The significant decreasing rate of seminal fructose concentration, sperm motility as well as viability and increasing rate of sperm head abnormality in different doses of treated series may be as a result of different toxic alkaloid ingredients present in BNE. Therefore, the results showed the potential of the BNE to induce different types of germ cell abnormalities in tumour bearing male mice.


Introduction
The analysis of semen and sperm chromatin abnormalities has been studied extensively for predicting male fertility [1]. Different types of biochemical components have been found in seminal fluid [2]. Fructose -the most important carbohydrate is found among different biochemical substances that acts as a donor of energy to the spermatozoa [3,4]. Low level of seminal fructose is positively correlated with low seminal fluid volume and low sperm motility [5]. So, fructose is essential for normal growth of sperm head morphology, sperm viability and motility. It is well known that exposure to some plant products cause different physiological as well as cytological abnormalities in experimental animals [6]. Areca nut or betel nut is used to treat different types of disorders such as leprosy, cold, worm infection and leukoderma in ancient period [7]. Now a day, betel nut is very popular as it is consumed by people of India and other Asian countries. The genotoxicity and carcinogenicity of betel nut extract was reported by earlier researchers [8]. Research since last four decades has generated enough evidence to involve betel nut, as a carcinogen in human [9,10,11]. Moreover, the high incidence of oral, oesophagus, liver, stomach and pancreas cancer has been associated with the habit of betel nut chewing [11,12,13,14]. Several studies have also suggested that betel nut extract can enhance the chromosomal aberration rate of mice in vivo system [15,16]. Betel nut treated experimental male mice showed low percentage of sperm viability and motility [17]. Our earlier studies have demonstrated that betel nut significantly enhanced the sperm head abnormality with steady decrease of sperm motility, haemoglobin percentage, and total count of RBC and survival rate of normal Swiss albino mice [17]. But detailed study about the effect of betel nut extracts in sarcoma 180 tumour bearing mouse is insufficient with respect to semen and germinal cell abnormality. The present study has therefore, been oriented to evaluate the dose dependent effect of BNEs on seminal fluid and germinal cell of in vivo sarcoma 180 tumour bearing mouse considering fructose concentration of seminal fluid, sperm motility, sperm viability, sperm head abnormality test by H E (haematoxylin and eosin) staining and toluidine blue staining methods.
Moreover, BNE treated histomorphology of testis in Sarcoma 180 tumour bearing mice were evaluated as betel nut acts as potential deleterious toxic component and its interaction with histological parameters in such tumour model has not been thoroughly evaluated.

Collection, Identification and Extraction of betel nut
Fresh betel was purchased from local market of Kolkata, India and authenticated by the Botanical survey of India, Botanical Garden, Howrah, West Bengal, India. BNE was prepared by slight modifications of the technique as described by earlier researchers [6,15,18,19]. 100gm betel nut seeds were cut into small pieces and kept in 90% ethanol for overnight. Then ethanol-soaked betel nut pieces were placed in thimble of Soxhlet apparatus for extraction. After 48 hours of extraction, ethanolic betel nut solution was collected and filtered. Then this solution was placed in the incubator at 50°C to dry. Sticky betel nut extract was kept for future experiment.
Different concentrations of aqueous solutions of BNE (1 gm /100 ml, 2 gm /100 ml and 3 gm /100 ml) were prepared for intraperitoneal injection to tumour bearing male mice [15]. A parallel group for positive control was done using sterilized distilled water (because BNE solution was prepared in distilled water). S-180 tumour bearing mouse was used as negative control group during the experiment.

Experimental animals
Male Swiss albino mice (Mus musculus) were purchased from registered animal supplier in Kolkata. All the male mice (with average 20 gm weight) were kept in laboratory for acclimatization for seven days. The animals were provided dry animal pellet and water ad libitum. The animals were also maintained hygienic condition with suitable temperature in animal house for acclimatization.

Selection of animal tumour model and tumour transplantation
Murine specific S -180 ascitic cell line was selected for the present experiments. The ascitic Sarcoma -180 cells were inoculated (1 x 10 6 cells / animal) by intraperitoneal injection and maintained in vivo. The transplantation process was done aseptically. The average time for appearance of ascitic tumour was 6 -7 days (log phase) as standardized by the earlier researchers [20,21,22,23].
All experiments were strictly regulated by the rules and guidelines of IAEC (Institutional Animal Ethics Committee) of Rammohan College, Kolkata, and Animal House Registration No. -1795/PO/ERe/S/14 CPCSEA, for maintenance and care of laboratory animals.

Standardization of dose and acute toxicity study
All experiments of Male Swiss albino mice were divided into 5 groups, each contains 6 healthy mice. Three different concentrations of BNE (i.e., 100 mg/kg, 200 mg/kg and 300 mg/kg of body weight) were prepared. The different doses of BNE and sterilized distilled water (vehicle or positive control group) were administered intraperitoneally for the determination of acute toxicity of experimental mice.

Seminal fructose analysis
Fructose concentration in seminal fluid of control, vehicle and BNE treated mice was determined by the Resorcinol method [3,24]. Diluted seminal fluid concentrations were analysed on the basis of the optical density by PC based double beam spectrophotometer (Systronics 2202) on 546 nm wavelength and seminal fructose value was measured on the basis of different fructose standards [25].

Sperm motility analysis
Sperms were separated from cauda of epididymis of different control and treated mice, diluted with 0.3 ml 0.9% normal saline and kept for 15 minutes at 37º C. Then 0.02 ml of diluted sperm containing seminal fluid was charged to Neubauer chamber of Haemocytometer for counting. The motile and immotile sperms were manually counted in each square of WBC counting chamber and total 16 squares counted by 10 x 40 magnifications under binocular research microscope [17,26,27].

Sperm viability analysis
Sperms were collected from caudal part of epididymis of control and treated mice, mixed with 0.3 ml of 0.9% normal saline and kept for 15 minutes at 37° C to allow them to become immotile. Number of living and dead sperms were analysed in haemocytometer by conventional Eosin Nigrosin staining method [26,27].

Sperm head abnormality test by Haematoxylin and Eosin stain
Sperms head morphology analysis by using Hematoxylin and Eosin staining is an important parameter to analyze the male germinal cell toxicity. Sperms were collected from caudal part of the epididymis of control and treated mice, kept in 0.9% normal saline for 30 min. The fluid containing sperms was smeared, dried and stained by Hematoxylin and Eosin. The slides were then washed and studied under binocular research microscope at 10 x 100 magnifications. Different types of sperms i. e. normal, big head, pin head, amorphous head, banana shaped etc. were observed. Total 1500 number of sperm heads of each treatment was analyzed [17,28,29].

Sperm DNA damage analysis by toluidine blue
Toluidine blue (TB) is a basic nuclear dye and is used as a sensitive structural probe for DNA structure and packaging. This staining method is an established conventional method to analyse the DNA packaging of spermatozoa [1]. This method also indicates poor sperm integrity with severe DNA damage and morphological assessment of sperm cells. Sperms of control and treated mice were collected from cauda of epididymis and kept in 0.9% normal saline for 15 minutes. The solution containing sperms was smeared, dried and dipped in pre-chilled 95% ethanol for 5 minutes for fixation. Then fixed slides were transferred to 0.1N HCl for 5 minutes at 4°C for hydrolysis. The slides were then washed in distilled water twice and stained in 1% TB solution (pH 4) for 12 minutes. Finally, the TB-stained slides were gently washed in distilled water and observed under binocular research microscope at 10 x 100 magnifications [30].

Testicular Histology analysis
Testis of sacrificed mice in controlled and treated groups was collected and testicular tissue was fixed in Bouin' s fixative for 1 hour at 4ºC. Then Bouin' s fixed small pieces of tissues were kept in the same fixative for 24 hours. The tissues were dehydrated in graded alcohols, cleared in xylene and embedded in paraffin wax. The paraffin block was sectioned into 0.5 mm thickness and stained by Haematoxylin and Eosin by the following the method of earlier researchers [31,32,33]. The stained testis tissue slides were examined under binocular research microscope in different magnifications.
Seminiferous tubules of control and treated series were analysed by software -open CASA plugin of image J [33] from the haematoxylin and eosin-stained tissue sections of testis.

Seminal fructose
The mean seminal fructose concentration in BNE treated mice was lower in comparison with control as well as vehicle. It is interesting to note that the mean seminal fructose concentration gradually decreased with increasing concentrations of BNE treatment which were highly significant (p**>0.001) in both 200mg BNE /kg and 300mg BNE /kg body weight treated mice but in 100mg BNE /kg body weight treated tumour bearing mice it was slightly lower than control and vehicle series. (Table: 1 and Figure 1A).

Sperm motility
The percentage of motile sperms gradually decreased with increasing concentrations of BNE which was highly significant (p**>0.001) in both 200mg BNE /kg and 300mg BNE /kg body weight treated mice and significant (p*>0.05) in 100mg BNE /kg body weight treated tumour bearing mice (Table:1 and Figure 1B) in comparison to control and vehicle series.

Sperm viability analysis
The percentage of viable sperms gradually decreased and non-viable sperms increased with increasing concentrations of BNE which were significant (p*>0.05) in both 200mg BNE /kg and 300mg BNE /kg body weight treated mice (Table: 1 and Figure 2) in comparison to control, vehicle and 100mg BNE /kg body weight treated series.

Sperm head abnormality analysis by Haematoxylin and Eosin stain
Different types of sperm head abnormalities such as amorphous head, big head etc. were observed from the haematoxylin-eosin-stained slides in control, vehicle and BNE treated tumour bearing mice (Figure 3 A, B). The percentage of abnormal sperms in BNE treated tumour bearing mice was more and highly significant (p**>0.001) in comparison to control and vehicle series ( Table 2 and Figure 3C).

Sperm DNA damage analysis by toluidine blue staining
Damaged or distorted sperm head abnormalities were also observed by toluidine blue staining methodology in control, vehicle and BNE treated tumour bearing mice (Figure 4 A, B, C). The abnormal features of sperm head were high in BNE treated tumour bearing mice which were significant (p*>0.05) in 100 mg BNE / kg body weight treated mice and highly significant (p**>0.001) in both 200 mg BNE /kg and 300mg BNE / kg body weight treated mice (Table 2 and

Testicular Histology analysis
It was observed that the average number of spermatozoa of seminiferous tubules are more in control and vehicle series than in different treated groups (Table: 3). In addition, the percentages of damaged or abnormal seminiferous tubules (i.e., separation of germinal epithelium, vacuolization, presence of detached cellular material in the tubule lumen, atrophied tubules etc) were gradually increased with increasing concentrations of BNE which was highly significant (p**>0.001) in 300mg BNE /kg body weight treated mice (Table: 3

Discussion
Different genetical, physiological, environmental and lifestyle factors are responsible for cancer initiation and progression [34]. However, exposure of some plant species and their bioactive components are also responsible for initiation and progression of cancer. Betel nut chewing not only causes different types of cancers such as oral, oesophagus, liver, pancreas but also induces cardiovascular, neurological and psychoactive disorders [35]. Our earlier studies showed that, ethanolic betel nut extract on in vivo sarcoma -180 tumour bearing mice induced the rate of cell proliferation with steady increase of chromosomal aberrations and hematological toxicities [15]. Stich (1991) demonstrated that presence of high phenolic components in diet can cause chromosomal aberrations as well as DNA breaks in animals [36]. According to our earlier observations [15], BNE contains large amounts of phenolic components that can induce toxic effects which support the concept of Stich [36]. Seminal fructose concentration not only helps in the assessment of seminal vesicle dysfunction but also could give a useful indication of male reproductive function [25]. The Sarcoma 180 cell line is a good murine specific cell line to study the effects of carcinogenic and toxicological potentiality of different compounds [37].
Biochemical, cytogenetical and histopathological studies of germinal cells in BNE treated sarcoma 180 tumour bearing male mice are important to evaluate reproductive cytotoxicity. So, in the present study, the relationship between sperm motility and sperm head abnormality with seminal fructose level was evaluated by cytogenetical and biochemical tests and the clinical usefulness of these tests were evaluated. It was observed that the BNE affected germinal cells of tumour bearing mice to a significant extent and cytotoxicity was manifested in the form of different types of sperm head abnormalities. In our results, the percentage of abnormal sperm head abnormality or sperm chromatin damage was significantly higher in case of Toluidine blue staining method in comparison to H E staining method. So, it is suggested that Toluidine blue staining method is a valuable, reliable parameter. It was also observed that both sperm motility and viability were equally sensitive to the cytotoxic action of BNE. Furthermore, BNE exposure induces some undesirable toxic effects in respect to quality of seminal fructose concentration and abnormal, damaged seminiferous tubules in tumour bearing mice.

Conclusion
Present study focused to analyse the biochemical, cytogenetical and histopathological abnormalities considering the male germinal cell parameters of betel nut extract treated Sarcoma 180 ascitic tumour bearing mouse. On the basis of above results and discussion the present authors may conclude that higher doses such as 200 mg BNE / kg and 300mg BNE /kg body weight treatment cause more adverse effects than 100mg BNE /kg body weight and control groups on seminal fructose concentrations, sperm motility, viability, sperm head morphology, sperm DNA organization and seminiferous tubule morphology.