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  • The Influence of Dose of Carcinogen, Emptiness of Stomach,

    The experimental study of gastric carcino genesis in the mouse is complicated by the dual structure of its stomach—the proximal two-thirds (forestomach) being lined by a stratified squa mous epithelium, while the distal third is lined by glandular epithelium. While interest has generally been focused on the possibility of inducing tumors of the glandular mucosa, most of the experimental results have, in fact, been concerned with the ef fects on the forestomach (see reviews : 3, 16, 18, 19, 47).

    Spontaneous gastric tumors in mice, whether of the forestomach or the glandular portion, are rare (3, 11, 19, 28, 44, 50), though adenomas have been reported in strain I mice and adenocar cinomas in sublines of NHO (28, 46), the subline Br-s displaying an exceptionally high incidence (45). In C3H and Swiss mice (used in the present investigation), spontaneous papillomas of the forestomach are extremely rare, while carcinomas of that portion or of the glandular portion are ap parently never encountered.

    The literature on gastric carcinogenesis in duced by carcinogens added to the diet includes references to the following variations in method ology:

    a) The use of different carcinogens, e.g., 1,2,5,6-dibenzanthracene (2, 21—25), 3,4-benz pyrene (4, 7, 18, 29, 81, 49), 20-methylcholan threne (13, 14, 22—24, 81), 9,10-dimethyl-1,2- benzanthracene (38, 39), 3,4,5,6-dibenzcarbazole (2), 2-acetylaminofluorene (1), p-dimethylamino benzene-1-azo-naphthalene (27), as well as crude products, e.g., heated fats and other materials (18, 20, 26, 30, 35, 48); of these, acetylamino fluorene and some of the crude products produced only papillomas, while the others usually yielded both papillomas and squamous carcinomas.

    * This work was supported in part by grants from the

    Damon Runyon Fund (1951—53).

    Received for publication January 12, 1955.

    b) Tests carried out on different strains of mice, including CSH, C57BL, C57BR, A, DBA, I, CBA, NH, CHI, DB, and certain backcrosses (2, 4, 7, 13, 14, 22—24),as well as stock mice (929,31, 35), rats (20, 26, 29), and rabbits (8, 17) ; of these, CSH (7)appearedto bethemostresponsivefortumor production in the forestomach and I mice the least (24), though other factors (see below) were not al ways comparable.

    c) The influence of dosage, concentration, and frequency of administration of the carcinogen. These differed greatly in the various reported in vestigations.

    d) Different media in which the carcinogens were suspended and the manners of administra tion. These included simple addition to the diet (18, 26, 27, 29, 81, 85, 49), as oily solution into the mouth or pharynx (7) or by stomach tube (2), in “associatedcolloids― by stomach tube (37—39), or as oil-water emulsions added to drinking water (4, 22—24); the most striking results were that, whereas the oil-water emulsions stabilized with Aerosol OT were carcinogenic to the forestomach (22—24),aswasthecasewithmostothermethods, similar emulsions stabilized with NaOH (or not stabilized at all) induced tumors mainly in the intestine (21, 28).

    e) The addition of bile (18) or eugenol (14), injury to the mucosa by surgery (8, 17), double vagotomy (8), heat (14), or x-rays (37)—all de signed to overcome the “mucus barrier― (16) or otherwise to facilitate penetration into the glandu lar mucosa; none of these methods led to tumor induction in the glandular mucosa.

    That the glandular mucosa of the mouse stomach is potentially responsive to carcinogenic hydrocarbons was effectively demonstrated by the production of adenocarcinomas following their intramural injection or by the introduction into the submucosa of crystals or threads impregnated with such carcinogens (12, 15, 36, 42, 43).

    Perhaps the most logical approach to the whole

    504

    and Other Factors on Tumor Induction in the

    Forestomach of the Mouse*

    I. BERENBLUMANDNECHAMAHARAN

    (Department of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel)

    Research. on August 10, 2021. © 1955 American Association for Cancercancerres.aacrjournals.org Downloaded from

    http://cancerres.aacrjournals.org/

  • BERENBLUM AND HAJL&N—Some Factors Influencing Gastric (Jarcinogenesis 505

    RESULTS In the first series of experiments, duplicate tests

    were carried out on Swiss and C3H mice, with 0.5 per cent 9,10-dimethyl-1,2-benzanthracene as the carcinogen. A dose of 0.3 ml. of this solution was given by stomach tube once, 3 times, and 6 times, respectively, at weekly intervals, with no further treatment till the end of the experiment (30 weeks from the time of the first treatment). The sur vivors were then killed and their stomachs ex amined grossly and histologically, as were, also, those mice dying during the course of the experi ment.

    Tumors of the forestomach were obtained in all six groups (Table 1), but with differences accord ing to the number of feedings of carcinogen, and with minor differences in the respective strains: After a single dose of carcinogen, 14/30 Swiss mice and 5/25 C3H mice developed papillomas of the forestomach, in many cases multiple, and most of them easily visible to the naked eye. However, none showed evidence of malignancy. The oh served latent period in these two groups was longer than in the other groups (see below), no tumors being found before the 30th week. After three doses of carcinogen, at weekly intervals, the tumor yields were higher, with 18/25 in Swiss and 10/20 in C3H mice; and after six weekly doses of carcinogen, the yields were higher still. This cor relation is more effectively demonstrated in the corrected figures (last column of Table 1), where all deaths during the first 10 weeks are excluded. Even more striking was the increase in the number of malignant tumors, especially in C3H mice, which rose from zero to twelve as the dose in creased from one to six feedings. Regarding the latent period, the earliest papilloma was observed in a Swiss mouse 4 weeks after the commencement of the experiment (i.e., 1 week after the third feeding) ; the earliest observed malignant tumor was in a C311 mouse receiving six feedings—this appeared after 12 weeks (see Figs. 2 and 5).

    Two subsidiary groups were included in the above experiment, in which identical doses of 3,4- benzpyrene and 20-methylcholanthrene were fed to C3H mice at weekly intervals but continued through the experiment (i.e., for 30 weeks). While the total number of tumors was somewhat lower than that following six doses of dimethylbenzan thracene, the proportion of malignant tumors was higher, especially with benzpyrene, which pro.. duced one papilloma and sixteen carcinomas out of 30 test animals. Thirteen of the mice fed con tinuously with benzpyrene and ten of those fed continuously with methylcholanthrene were tu mor-free after 80 weeks, while none were tumor

    problem was to cause the carcinogen to pass through the glandular cells in the process of secre tion into the stomach, using certain derivatives of carcinogens which have properties similar to cer thin basic dyes known to be secreted by the gastric glands (33). No tumors of the glandular mucosa resulted, however, when these compounds were injected intraperitoneally (32, 34).

    In a series of investigations undertaken in our laboratories, further attempts were made to study the factors influencing gastric carcinogenesis in the mouse, particularly with the hope of overcoming the difficulty in inducing tumors of the glandular mucosa, and partly to study the “two-stage mechanism of carcinogenesis― (5) as it affects the stomach. The present communication deals with gastric carcinogenesis per se, while the follow ing communication (6) is concerned with the at tempted modification of the separate stages.

    The primary objective, in the present experi ments, was to determine how far the number of feedings of carcinogen could be reduced for tumors still to be elicited, with the use of high concentra tions of carcinogen in polyethylene glycol-400, since for the study of the “two-stagemechanism― it was desirable to administer only a single dose, if possible.

    MATERIALS AND METHODS

    The animals used in these experiments were male mice of C8H and Swiss strains, about 3 months old, from colonies bred by brother-to-sister matings in this laboratory. The ex perimental animals were housed in transparent plastic cages and kept in air-conditioned rooms at an average temperature of 21°C. Ten to twenty mice were kept in a cage. The diet was Purina Laboratory Chow (Ralston Purina Co., U.S.A.) and water ad libitum.

    The carcinogens—9,10-dimethyl-1,2.benzanthracene, 20- methylcholanthrene, 3,4-benzpyrene, and 1,2,5,6-dibenzan thracene—obtained from standard commercial firms, were made up as 0.5 per cent solutions in polyethylene glycol-400 and administered to the mice once weekly by stomach tube. These stomach tubes were prepared from polyethylene tubing, of about a 1-mm. bore, as used by Saxen (37), but with one end closed with Duco cement and a lateral opening made, to reduce to a minimum the danger of mechanical injury to the stomach. (Small acute ulcers, observed in some of the early tests, were almost entirely eliminated after the technic of pass ing the tube became, with practice, an easy procedure.)

    Before each feeding of carcinogen, the mice were starved for 18 hours, receiving only drinking water. The solid food was re turned to the cages 2 hours after the treatment. Other modi fications (e.g., milk diet for short periods) will be described be low in the appropriate section.

    At the