ACCELERATING ACTION OF TEA ON MOUSE SKIN CARCINOGENESIS. P. BOGOVSKI* and N. DAY with the technical assistance of M. CHVEDOFF and F.

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1 Cancer Letters,3 (1977) Elsevier/North-Holland Scientific Publishers Ltd. ACCELERATING ACTION OF TEA ON MOUSE SKIN CARCINOGENESIS P. BOGOVSKI* and N. DAY with the technical assistance of M. CHVEDOFF and F. LAFAVERGES International Agency for Research on Cancer, Lyon (France) (Received 25 January 1977) (Accepted 2 February 1977) SUMMARY The effect of different tannin-containing aqueous solutions on the carcinogenic action of benzo(a)pyrene (BP) on the mouse skin was investigated. It was found that the appearance of tumours was accelerated in mice treated with either tea or an oak extract after a single application of BP. The time of appearance of tumours in mice treated with gallic acid or tannic acid after treatment with BP was similar to that in the group treated with BP alone. The overall incidence of tumours was similar in all five groups. INTRODUCTION Tea drinking has been considered as a possible contributing factor in the aetiology of oesophageal cancer. The temperature of tea could be of importance in causing burns. Alternatively, the chemical components of tea could be suspect. Tannins of various origins, for example, have been shown to induce liver tumours in rats [7] and sarcomas at the site of subcutaneous injections in rats and mice [6] ; and Korpassy [7] demonstrated a marked promoting action of tannic acid on the hepatocarcinogenicity of 2-acetylaminofluorene (2-AAF) when tannic acid was applied parenterally and 2-AAF in the food. In skin painting experiments in mice, after one local administration of benzo(a)pyrene (BP) followed by 55 paintings of a brew of fermented (black) tea, Kaiser [5] found a promoting action of the tea infusion, which he attributed to volatile phenolic compounds. As the available data appear inconclusive, we decided to test the modifying action, in conjunction with benzo(a)pyrene, of a black tea infusion, an extract of oak wood, tannic acid and gallic acid. MATERIAL AND METHODS Fifty pl of a 1% solution of BP in acetone was dropped from a microsyringe * Present address: Institute of Experimental and Clinical Medicine, 42 Hiiu Street, Tallinn, Estonia , U.S.S.R.

2 10 on the shaved interscapular area of 250, 6-week-old Charles River CD-1 random-bred albino mice. Each mouse received mg BP. The mice were divided into 5 groups (25 males and 25 females in each). One group (I) received no further treatment and was used as the control group. The remaining mice were painted 80 times (3 times weekly) with the following solutions: Group II, Black Chinese (Keemun) tea (Camellia sinensis) infusion containing 1% tannin; Group III, aqueous oak (Querqus robur) sawdust extract, containing 1% of tannin; Group IV, Commercial tannic acid 1% aqueous solution (Tannin ~ l'eau, Fabriques de produits chimiques, Billault, Aubervilliers, Seine); Group V, 1% aqueous solution of gallic acid (Fabriques de produits chimiques, Billault, Aubervilliers, Seine). The mice were observed for up to days, then all animals still alive were killed. The animals were inspected weekly and local changes recorded. Tumours that did not regress within 4 weeks and had a diameter of not less than 1 mm were considered papillomas, although a number of them regressed and disappeared at a later stage. The mice that died or were killed were necropsied. All skin tumours and some internal organs were fixed in 10% formol and sections were stained with haematoxylin and eosin. RESULTS The mortality in all groups was similar. As the majority of animals ( in each group) were sacrificed after the 567th day the mortality rates could not be followed through the whole life-span. The treatment with the tea infusion and related material in groups II--IV was carried out inless than 200 days, the observation period after the end of treatment lasted more than 365 days. Various spontaneous turnouts were found in almost equal proportions in all groups. The most frequent tumour was lung adenoma (20--25% of mice). Hepatomas occurred in about 6--18% of the mice, a number of them as second primary tumours. Lymphomas were found in 5--15% of the mice. Adenomatous hyperplasia was found in the glandular stomach in % of the mice, in about 1% of the cases adenocarcinoma of the stomach was present and with the same frequency adenocarcinoma of the colon. Hepatomas seemed to occur more frequently in group IV, treatment with tannic acid (18% of the cases). Differences with other groups (BP, 10%; tea, 6%; oak tannins, 8%; gallic acid, 6%) were, however, statistically insignificant. In Table 1 skin tumours arising in the interscapular area are considered. In a few mice skin tumours on the lip, eyelid or inguinal area were observed. They are not included in the table, although these tumours might have been induced by contamination from the BP-treated area of the same mouse or of cagemates. The incidence of skin tumours in the treated area was similar in all groups, varying from 30% to 36% of the initial number of mice. The malignancy of tumours, however, differed in various groups. In the BP + tea group 3 squamous cell carcinomas were found, 2 of them appearing earlier than any tumours in the BP group. In the BP + gallic acid group no malignant turnouts were found

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4 12 and only! in each of the BP + oak extract and BP + tannic acid groups. There were differences among the groups in the ages at which tumours were observed. Comparison of the group treated with BP + tea with the group treated only with BP shows that, of the 35 tumour-bearing animals in the two groups (see Table 1), the first 7 animals to develop tumours (before 180 days) were all in the BP+ tea group (P < 0.02). There was thus a significantly earlier appearance of tumours in the BP + tea group. After 180 days, there was no significant difference between the two groups. Comparison of the group treated with BP + oak tannins with the BP-treated group shows that, of the 32 tumour-bearing animals in the two groups (Table 1), the first 10 (before 100) were in the BP + oak tannin group (P < 0.01), and the succeeding 11 were in the BP group ( days). There was thus an early period with more tumours in the BP + oak tannin group, an intermediate period with more tumours in the BP group, and a late period (after 360 days) where no difference existed in the two groups. The earlier appearance of tumours in the BP + tea and the BP + oak tannin groups was also true for carcinomas (see Table 1), but for carcinomas the effect was not in itself significant. The two remaining groups, BP + gallic acid and BP + tannic acid, had an agespecific incidence of tumour appearance similar to the BP-treated group. DISCUSSION Our results do not confirmthe findings of Kaiser [5] that, out of 15 mice painted 55 times with brewed tea after a single dose of BP, 6 mice developed carcinomas and all other mice precancerous or cancerous stages of squamous cell tumour, whereas after a single application of BP the mice did not develop pathologic lesions. In all our groups the number of tumours was approximately equal. There was, however, a statistically significant shortening of the latency of skin tumours in mice treated with tea infusion or oak tea extract after the initial application of BP. This effect could not be observed in mice treated with tannic acid or gallic acid. Tannic acid from different sources and tannins found in aqueous extracts of various material of plant origin represent complex, often unstable, chemical compounds. It is difficult to speculate which components of the tea infusion or oak extract are responsible for the modifying action we observed. Tannins produce hepatomas [7] and sarcomas at the injection site [6]. Tannic acid inhibits enzyme activity. At low concentration the inhibition is reversible, at high concentration it is irreversible since the proteins are precipitated [2]. In plant extracts other biologically active components can be present together with tannins. It has been found that some flavonols (ionol, 4-(h-anisyl)-phenol, propylgallate) exert an antileukaemic effect in experiments and inhibit the growth of transplanted tumours [1,4]. Wattenberg and Leong [8] tested several flavones, which are potent BP-hydroxylase activity inducers, and found that these compounds inhibited

5 13 the induction of pulmonary adenomas and skin tumoral. These data indicate that the action of tea infusion and oak extract can be rather complex and may be dependent upon the relationships between components acting in relation to carcinogenesis in opposite ways, and probably by means of different biochemical mechanisms. In our experiments we could demonstrate an accelerating action on the induction of skin tumours in mice by BP, when BP was followed by treatment with brewed tea and aqueous extract of oak wood. REFERENCES 1 Bogdanov, G.N., Burlakova, E.B., Konovalova, N.P. and Kruglyakova, K.E. (1968) Antitumorous and radioprotective properties of phenolic compounds. In: Phenolic Compounds and Their Biological Functions, pp Editors: A.L. Kursanov and M.N. Zaprometov. Nauka, Moscow (in Russian). 2 Firenzuoli, A.M., Mastronuzzi, E. and Vanni, P. (1970) Effect of tannic acid on glucose-6- phosphate dehydrogenase. A possible model of mixed-type inhibition. Physiol. Chem. Phys., 2, Fong, M.H.S., Bhath, W. and Farnsworth, N.R. (1972) Antitumor activity of certain plants due to tannins. J. Pharm. Sci., 61, Gorbatsheva, L.B., Kukushkina, T.V. and Petrov, O.E. (1968) Inhibition of turnout growth and biosynthesis of RNA by administration of phenolic compounds to turnout-bearing animals. In: Phenolic Compounds and Their Biological Functions, pp Editors: A.L. Kursanov and M.N. Zaprometov. Nauka, Moscow (in Russian). 5 Kaiser, H.E. (1967) Cancer-promoting effects of phenols in tea. Cancer, 14, Kirby, K.S. (1960) Induction of tumours by tannin extracts. Brit. J. Cancer, 14, Korpassy, B. (1959) The hepatocarcinogenicity of tannic acid. Brit. J. Cancer, 19, Wattenberg, L.W. and Leong, J.L. (1970) Inhibition of the carcinogenic action of benzo(a)pyrene by flavones. Cancer Res., 30,