At 50 days of age, normal female rats were fed a single, dose of 3-MC (100 mgm.) and divided into groups. This feeding was the only experimental variable in the control rats which were maintained as virgins, and all developed mammary cancer (Table 2). Indeed, we have shown above that a single feeding of 3-MC (100 mgm.) induced mammary cancer without exception in normal female rats of this age, and that the tumours were demonstrated although not visible to the unaided eye after two weeks. Accordingly, at 65 days of age, the endocrine status of rats in many of the groups was modified drastically by breeding or by administration of steroid hormones.

Table 2. Suppression Or Mammary Cancer In Pregnancy And In Rats Injected Daily For 50 Days Beginning 15 Days After A Single Intragastric Instillation Of 3-Mc (100 Mgm.)

There were 10 rats in each group and 3-MC (100 mgm.) was administered to every rat at age 60 days. All the animals were virgin females except one group which were bred between age 65 and 08 days; steroids were not administered to these groups. In the others, hormones dissolved in sesame oil (0-2 ml.), were injected intramuscularly 6 days each week for 50 days beginning at 65 days of age. The rats were observed for 150 days.

The expected incidence of mammary cancer was lessened notably in rats bred 15 days after their solitary feeding (Table 2). Moreover, the daily injection of progesterone (4 mgm.) or of cestradiol-17β (10 µgm.) beginning at age 65 days, caused a similar decrease in the rate of incidence. The combined administration of progesterone (4 mgm.) with cestradiol-17p (1 or 10 µgm.) each day for 50 days induced the characteristic gestational type of hyperplasia of tho mammary glands, which resembles that found in rats on day 15 of pregnancy, but few mammary cancers ever developed in these rats (Table 2); the neoplasms had been suppressed by tho hormonal treatments.

In earlier work11 the repeated feeding of 3-MC to rats at the onset of pregnancy, or to othor animals injected with large quantities of equine gonadotro-phins, seldom induced mammary cancer. The present experiments demonstrate an effect of a different kind since they show that cestradiol with progesterone, while inciting brisk growth of normal mammary glands, often destroyed the young cancers induced within the previous two weeks by a large dose of 3-MC.

Discussion

Induction of cancer by a solitary feeding of poly-nuclear carcinogens emphasizes the high importance of the alimentary tract as a portal for their entry and, indeed, of a single meal for tho development of mammary cancer in the rat. This method has advantages over multiple feeding in its extreme simplicity and in saving of labour and of rare or costly compounds associated with equal rapidity, and invariability of development of multiple mammary cancers. A stoichiometric relationship exists between the dose of effective carcinogens and the rate of incidence of the tumours, together with their number. The mammary cancers arise under highly restricted conditions, but these are easily fulfilled. All young adult female rats of the Sprague-Dawley strain fed an optimal amount of DMBA developed mammary cancer under the favourable hormonal conditions which prevail in them. Pregnancy, or the administration of progesterone with cestradiol, frequently destroyed many young cancers that were obtaining a foothold, and rats in this class never suffered a recrudescence of their mammary cancers.

Not all the colls of the body were injured by a single moderately large feeding of DMBA and lesions developed selectively. The haematopoietic and lymphatic systems were damaged and two types of cancer were induced, while the production of gonadotropin by the hypophysis, and of phenolic cestrogens by the ovary, were undisturbed.

Not all tho cells of the responding animals became malignant after feeding the effective polynuclear hydrocarbons ; cancer developed only in the mamma and in special sebaceous glands, while sarcoma was never encounterod. All the mammary cancers were adonocarcinomata and squamous carcinoma was not observed in tho breast. Yet in companion rats the intramuscular injection of tho effective carcinogens ovoked sarcoma, locally, in most of them when these carcinogens were injected in muscle. The properties which endow special cells of the body with selective vulnerability to trace amounts of polynuclear hydrocarbons arc unknown.

Not all the many millions of cells of tho mammary glands became malignant after feeding effective amounts of the carcinogens. The maximal number of mammary cancers which has been observed in this laboratory after a single feeding is 21. A threshold response to the hyperconjugated hydrocarbons is involved in certain cells of the mammary gland in the induction of cancer of the breast by hydrocarbons, since a minimal effective dose gives rise to only one active centre.

Berenblum12 has postulated that carcinogenesis develops in multiple independent stages. The present experiments demonstrate that mammary cancer, under the stated conditions, arises in a single step.

This work was aided by grants from the American Cancer Society and from the Jane Coffin Childs Memorial Fund for Medical Research.

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