Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut.

Male mice of inbred strains Swiss and C17 were fed daily 5 times a week by intragastric tube 0.1 ml of betel-nut aqueous extract, betel-leaf aqueous extract and the polyphenol fraction of betel nut. Male mice of corresponding strains fed 0.1 ml of distilled water served as controls. Treated and control mice were kept under observation and killed when moribund. Betel-nut aqueous extract induced tumours of the gastrointestinal tract in 58% Swiss mice and 25% C17 mice. The polyphenol fraction by the same route induced tumours at other sites in 17% of the mice. Betel-leaf aqueous extract failed to induce any tumour in the treated mice, which supports an earlier report of the lack of any carcinogenic principle in betel leaf, an essential constituent of betel quid. Results are discussed in relation to the relevant literature.

IN INDIA and the Far East the habit of betel chewing is a major factor in the cause of oral cancer. Many attempts have been made to develop a theory of the origin of betel-chewer's cancer, based on the chemical constituents of the chew.
The chew or betel quid consists primarily of a few pieces of areca nut wrapped in the leaf of the betel vine, together with some lime. According to Tenneckoon & Bartlett (I 969), lime might have an irritant action, but was used in such small quantities that dilution by saliva rendered it innocuous. In some localities certain other ingredients such as catechu and tobacco may be added, but they are not essential constituents of betel quid. In any case, similar pathological changes have been found in the absence of these ingredients (Pindborg et al., 1968). Our preliminary studies on the aqueous extract of betel nut and its polyphenol fraction have shown that both produce a high percentage of fibrosarcomas at the site of injection in Swiss mice. Betel-leaf aqueous extract by s.c. injection, however, failed to produce any tumours (Ranadive et al., 1976;Ranadive & Gotboskar 1.978). To simulate human conditions more closely we have tested betel-leaf aqueous extract, betel-nut aqueous extract and polyphenol extract of betel nut by gavage feeding.

MATERIALS AND METHODS
Male mice of two inbred strains, Swiss and C17, were fed by intragastric tube 0-1 ml of aqueous extracts of betel nut and leaf, and also 0-1 ml of the polyphenol extract of betel nut, daily 5 times a week. Feeding -,A,as started at the age of 8-10 weeks and continued throuo,hout the life-span of the treated animals. The following experimental groups, were maintained. Betel-nut aqueous extract and the polyplienol extract (Shivapurkar et al., 1978)  Betel-nut aqueous extract Of the 21 Swiss mice in this group, 7 developed liver tumours (33%), out of which 5 were hepatocellular carcinomas ( Fig. 1) and 2 haemangiomas. Five other mice developed tumotirs at other sites, 2 being lung adenocarcinomas, I a squamouscell carcinoma and I an adenocarcinoma of the stomach (Fig. 2), and I leukaemia.
Of the 30 C, 1 7 mice fed betel-nut aqueous extract, 3 developed squamouscell carcinoma of the fore-stomach (Figs. 3 and 4) and 2 adenocarcinomas of the glandular stomach. In addition I developed lung adenocarcinoma and 2 leukaemia.
repeatedly with 100ml aliquots of distilled -xA,ater on an automatic shaker. The combined extract was lyophilized and the dry residue -%A,as dissolved in 10 ml distilled -vN-ater. For quantitation of the extract, arecoline content ,%A,,as measured by the method described by Sharp (1931) and polyphenol content by the method described by SAan & Hills (1959).
0-1 ml of the aqueous extract was found to contain 1-5 mg of arecoline and 1-9 mg of polyphenol (measured as tannic acid). The polyphenol fraction was prepared by vigorously shaking 100 g of betel-nut powderwith 150 ml of ethyl acetate (containing 8 ml of ethanol/100 ml of ethyl acetate) for 4 h with an automatic shaker. The extraction was repeated several times and the combined extracts A%,ere treated NN-ith 0-IN HCI to reinove anv alkaloid impurities. The purified fraction N%,as lyophilized and the dry residue dissolved in 10 ml of distilled water. The purity of this preparation was checked by silica-gel thin-layer chromatography NNrith arecoline as the reference substance. I't was then diluted 10 times, for treatment. The amount of total polyphenols, measured as tannic acid, was 1-9 mg in 0-1 ml of diluted extract.
Pre,parations of betel-leaf extract.-100 g of bete] leaves were ground with 150 ml distilled AN-ater in a grinder and kept at 4'C for 24 h.

Polyphenolfraction
Of 18 Swiss male mice fed the polyphenol fraction, 2 developed tumours of the salivary gland and I haemangioma of the liver.

DISCUSSION
The present studies attempt to simulate the situation in humans, in which the oral and oesophageal squamous epithelium is in contact with betel nut before it reaches the glandular stomach. Rodent gastric mucosa is pre 'sumed to be the counterpart of the human oesophagus, in which large numbers of tumours are reported in betel-nut chewers (Jussawala & Deshpande, 1971).
The above data have shown that betelnut aqueous extract (BN) induced a sub-stantial number of tumours of visceral organs such as liver, lung and GI tract in treated mice. However, treated mice of the C17 strain failed to develop any liver tumours, whereas 33% of betel-nutextract-treated Swiss mice developed liver tumours. This may be because the liver tissue of Swiss mice is more susceptible to even weak carcinogenic activity than that of C 1 7 mice. We have reported a significant number of liver tumours in Swiss mice treated with relatively weak carcinogens, such as thioacetamide (Date et al., 1976). It is possible that C17 mice lack the necessary enzymes for activation of the carcinogens, or for the formation of proximal carcinogens from the betel-nut aqueous extract.
The tumorigenic effect of betel-nut extract injected s.e. in Swiss mice has already been reported from this laboratory (Randive et al., 1976). By contrast the feeding of aqueous betel-leaf extract was not able to induce any tumours in the present experiments. These observations support those of an earlier report from this group, in which it waa shown that betelleaf extract injected s.e. in Swiss mice failed to induce any tumours (Randive & Gothoskar, 1978). Further studies on these extracts have shown that betel-leaf extract even exerts a protective effect in Swiss mice when injected simultaneously with betel-nut extract (unpublished data). It is also interesting to note that feeding of betel-nut extract produced a significant number of tumours of the gastrointestinal could not detect any phenol degradation products in the urine of rats fed sericea grape tannins, and they tentatively concluded that there was little if any absorption of the anthocyanidin polymer per se or its degradation products from the intestinal tract. Tumours observed in the betel-nut-fed Swiss and C17 mice could be attributed to some constituents in betel nut other than tannins, e.g. alkaloids.
Alkaloids from different plants which are consumed either as food or folk medicine by the natives of various regions in the world are reported to be carcinogenic. Indepth studies on the alkaloids in betel nut (viz. arecoline) are under way using the oral route, and will be reported later.