Assessment of interaction among three carcinogens on rat mammary carcinogenesis in a factorially designed experiment.

Female Sprague-Dawley rats were given by stomach tube 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6] on the 77th day of age at the rate of 1.6 mg/100 g body weight, or procarbazine [(PCZ) CAS: 671-16-9] on the 84th day of age at the rate of 10 mg/100 g body weight, or 0.5 Gy of total-body x-rays on the 91st day of age, singly or in all possible combinations or no treatment. All rats were studied for mammary carcinogenesis for 370 days after the 84th day of age. Three measures of mammary carcinogenesis were studied. These were the incidence of rats with mammary adenocarcinomas, or mammary fibroadenomas, or mammary neoplasia of either type. Each of these measures was studied also for rats with 2 or more or 3 or more mammary neoplasms. Assessment of possible interaction among the three carcinogens with regard to the incidence of neoplasms was done by time-independent or time-dependent methods, both of which gave remarkably consistent results. For rats with 1 or more adenocarcinomas, 1 or more fibroadenomas, or 1 or more adenocarcinomas and/or fibroadenomas, both methods showed no interaction among the carcinogens, which can, therefore, be considered to have produced additive effects. An exception to this finding of additivity was an apparent synergistic interaction between DMBA and PCZ when the measures of rats with 2 or more, or 3 or more mammary neoplasms of either type, or 3 or more fibroadenomas were analyzed; these analyses, however, were based on relatively small numbers of rats with multiple tumors. Since no interactions were found for the usual measure of carcinogenesis, namely, incidence of rats with 1 or more neoplasms, the overall conclusion is that DMBA, PCZ, and x-ray act additively in the induction of mammary neoplasms in the female Sprague-Dawley rat.

Carcinogenic responses to chemicals applied directly to rat mammary glands in situ.

In four experiments, concentrated glycerin suspensions of 7,12-dimethylbenz[a]anthracene (DMBA), or the water-soluble compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and 4-nitroquinoline-N-oxide (4-NQO) were delivered into pockets in the mammary fat pads in female Sprague-Dawley rats. In a 90-day experiment, each of three groups of 20-22 rats were treated with either 25, 50 or 100 micrograms DMBA delivered to each of four sites in each rat per group. Dose-related responses were detected for incidence of rats with mammary adenocarcinoma (MAC), and for number of MAC per treated site. In a 120-day study, each of three groups of 20 animals were treated with either 250, 500 or 1000 micrograms MNU at each of 80 sites per group. Dose-related MAC responses were detected for incidence of rats with MAC, number of MAC per treated site, and time to detection of MAC. In two experiments of 90 days duration, groups of 20 females were treated with either 1000 micrograms ENU or 1000 micrograms 4-NQO at each of 80 sites per group. ENU produced a total of 27 MAC sites in 65% of the rats, and 4-NQO produced a total of 26 MAC in 60% of the rats. A comparison of the time to detection of all MAC data for the three water-soluble compounds at the 1000 micrograms treatment level, or on the basis of moles per treatment, yielded a carcinogenic potency relationship of 4-NQO greater than ENU greater than MNU.

Differences in DMBA-induced mammary neoplastic responses in two lines of Sprague-Dawley rats.

It has been reported that female Sprague-Dawley rats obtained from a U.S. source and studied in the U.S. gave a larger and more rapid mammary neoplastic response to radiation than did female Sprague-Dawley rats obtained from a Dutch source and studied in The Netherlands. To learn if the different mammary neoplastic responses of the two 'lines' of Sprague-Dawley rats are due to inherent differences between the lines of rats or due to differences in experimental conditions, two groups of rats from the American source and one group from the Dutch source were studied for their response to a chemical carcinogen, dimethylbenzanthracene (DMBA), at the same laboratory. When 10 mg of DMBA per 100 g body wt was given by stomach tube to 28 rats from the Dutch source, 367 days later approximately 25% of these rats had developed mammary carcinomas and approximately 18% had developed mammary fibroadenomas. When the same dose of DMBA was given to rats from the U.S. source, 300 days later 90 and 100% had developed mammary carcinomas and 83 and 95% had developed mammary fibroadenomas. Similar trends were found for the number of neoplasms per rat and the mean time of appearance of the neoplasms. It was concluded that there are inherent differences between Sprague-Dawley rats obtained in the U.S. and Sprague-Dawley rats obtained in The Netherlands in regard to their mammary neoplastic responses to DMBA, as well as in their responses to radiation. Genetic differences between the two lines were confirmed by establishing dissimilarities in the expression of erythrocyte antigens coded for by RT1 (major histocompatibility complex).

Effect of interval between neutron radiation and diethylstilbestrol on mammary carcinogenesis in female ACI rats.

Both radiation and diethylstilbestrol (DES) are carcinogens for the mammary gland of ACI female rats. When DES is given at about the same time as radiation, DES and radiation interact in a synergistic fashion particularly in regard to the number of mammary adenocarcinomas per rat. We have studied the effect of increasing the time interval between radiation and DES on the capacity of DES to enhance (promote?) radiation-induced mammary carcinogenesis. DES, in the form of a compressed pellet containing a mixture of cholesterol and DES, formulated to average 1.25 mg of DES/100 gr body weight, was given to groups of approximately 28 rats at 2 days before, or 50, 100 or 200 days after 0.064 Gy of 0.43 MeV neutron radiation. At each time that DES was given to irradiated rats, DES was also given to nonirradiated rats. All rats were studied for 375 days after the date of the DES administration. When the total number of mammary adenocarcinomas was calculated as a percentage of 24 sites per rat at-risk, DES and radiation always produced a response that was larger than the sum of the responses of DES alone plus radiation alone. This result suggests that these two agents can interact in a synergistic fashion. The interaction between radiation and DES did not decline as the time interval between radiation and DES was lengthened. This result suggests that radiation-induced (initiated?) mammary carcinogenesis is not subject to repair since DES enhancement (promotion?) continues to be effective over long time intervals.

Induction of mammary neoplasms in the ACI rat by 430-keV neutrons, X-rays, and diethylstilbestrol.

Mammary tumorigenesis was studied in female ACi rats after treatment with X-irradiation or neutron-irradiation, with or without diethylstilbestrol (DES) treatment. The mortality-corrected cumulative tumor rate based on all mammary neoplasms and the mortality-corrected incidence based on the first neoplasms only have been derived. In non-DES-treated animals, at the relatively high radiation doses studied, all dose-effect relationships were consistent with relative biological effectiveness (RBE) values slightly in excess of 10. In DES-treated rats definite findings were observed at neutron doses as low as 0.01 Gy (1 rad). The dose-effect relationship in DES-treated rats showed a strong sublinearly (dose exponent less than 1) at low neutron doses. RBE values in DES-treated rats increased in inverse proportion to the square root of the neutron dose, and exceeded 100 at a neutron dose of 0.01 Gy (1 rad).

Synergism of estrogens and X-rays in mammary carcinogenesis in female ACI rats.

A previously demonstrated synergistic interaction between diethylstilbestrol (DES) and radiation on rat mammary carcinogenesis was extended to another estrogen, 17-ethinylestradiol (EE2). These newly reported results with EE2 demonstrated that the previously reported synergistic interaction between DES and radiation is not confined to just DES. Instead, these new results implied that the synergistic interaction is a synergistic interaction between the estrogenic activity of DES and radiation on rat mammary carcinogenesis. Female inbred ACI rats were used. By the end of the experiment, no neoplasia was detected in rats bearing cholesterol pellets, with and without X-ray exposure. No significant tumor data were obtained from rats treated with 0.1 me EE2, with and without X-rays. Approximately 50% of the rats treated with DES and approximately 90% of the rats treated with 1 mg EE2 had 1 or more mammary adenocarcinomas (MAC). X-rays synergistically increased the number of MAC per rat in the groups implanted with DES or 1 mg EE2. X-rays also increased the trend toward earlier increased incidence of rats with MAC as compared to rats treated with estrogens only. All rats treated with DES and 1 mg EE2 had pituitary tumors. The mean weight of the pituitary tumors in the groups treated with 1 mg EE2 was approximately 1.5 times that of the groups treated with DES. Mean terminal plasma prolactin levels for rats treated with 1 mg EE2 or DES were, respectively, 17.5 and 9.5 times control values.

Soybean diet lowers breast tumor incidence in irradiated rats.

This paper examines the relationship between feeding a diet rich in protease inhibitors and the reduction of mammary cancer induced by x-irradiation in Sprague-Dawley rats. Of a total of 145 irradiated animals, 44% of the 45 rats fed a raw soybean diet containing a high concentration of protease inhibitor developed mammary tumors as compared to 74% of 50 rats fed a casein diet containing no protease inhibitor. Animals fed Purina rat chow which contained low levels of protease inhibitor exhibited a 70% mammary tumor incidence. No spontaneous neoplasms were found in any of the non-irradiated animals on the raw soybean diet whereas about 10% of the animals on the protease-free diet developed tumors. Thus, soybeans which are rich in protease inhibitors reduced the induction of mammary cancer in x-irradiated rats. This work suggests that diets rich in protease inhibitors may contribute to reducing cancer incidence in man.

Interaction of dimethylbenzanthracene and diethylstilbestrol on mammary adenocarcinoma formation in female ACI rats.

It has been reported that X-irradiation and diethylstilbestrol (DES) act synergistically on mammary adenocarcinoma formation in female ACI rats. The physical carcinogen, X-irratiation, was replaced by a chemical carcinogen, dimethylbenzanthracene (DMBA), and their interaction was studied in this system. Thirty-three female ACI rats were given 13.3 mg of DMBA per 100 grams of body weight. A total of 10 mammary adenocarcinomas were found, 8 in rats with a single mammary adenocarcinoma and 2 in a single rat, over a 266-day study period. Twenty-nine rats were implanted with a cholesterol pellet containing 5 mg of DES, and a total of 47 mammary adenocarcinomas were found, 5 in rats with a single mammary adenocarcinoma and 42 in 5 rats with 2 or more mammary adenocarcinomas. Twenty-four rats were given a combined treatment of both compounds, DES 2 days before DMBA, and a total of 12l mammary adenocarcinomas were found, 2 in rats with a single mammary adenocarcinoma and 124 in 18 rats with 2 or more mammary adenocarcinomas. The interaction between DMBA and DES was interpreted to be synergistic in regard to the proportion of rats with one or more mammary adenocarcinomas, the proportion of rats with two or more mammary adenocarcinomas, and the median times of appearance of both first and second mammary adenocarcinomas. These interactions between DMBA and DES resemble the previously reported synergistic interactions between radiation and DES on mammary adenocarcinoma formation in female ACI rats.

Induction of mammary neoplasms in the Sprague-Dawley rat by 430keV neutrons and X-rays.

Female noninbred Sprague-Dawley rats were exposed to single doses of 0.28, 0.56, and 0.85 gray (Gy = 1 J/kg or 100 rads) of X-rays or 0.001, 0.004, 0.016, and 0.064 Gy of 430-keV neutrons at 62 +/- 1 days of age and were then observed over the rest of their lives for the appearance of mammary neoplasia. As mammary neoplasms were detected, they were removed and given a classification of adenocarcinoma(s) (AC) or fibroadenoma(s) (FA) after microscopic study. All irradiated groups exhibited an increased incidence of mammary neoplasia. The tumor rate increased steeply with age of the animals, and the effect of the irradiation could be adequately described as a forward shift in time of the spontaneous incidence. The cumulative prevalence was derived from first neoplasms only, and a formalism was presented that makes it possible to derive the integral tumor rate from all neoplasms in all animals. Mortality-corrected cumulative prevalences and integral tumor rates as a function of age were given for the different doses and separately for FA and AC. The mammary FA response and the total mammary neoplastic response (including both FA and AC) were approximately proportional to the absorbed dose of X-rays or the square root of the neutron dose. The relative biological effectiveness (RBE) of the neutrons increased with decreasing dose and reached values exceeding 100 at a neutron dose of 1 mGy; the single dose of 1 mGy of neutrons produced a significant increase of the tumor rate that corresponded to a foward shift or roughly 35 days of the spontaneous incidence. The AC, taken separately, were subject to considerable statistical uncertainties due to their small numbers. However, their RBE-dose dependence was consistent with that for the FA and, even at the highest dose studied, the RBE value exceeded 10. The nonrandom development of multiple FA within individual animals appeared to be the result of differences in susceptibility to radiation. However, mammary FA and AC within individual animals were not statistically correlated.

Effect of dietary fat on X-ray-induced mammary cancer in Sprague-Dawley rats.

We studied the effect of dietary fat levels on the induction of mammary cancer by 350 rads total-body X-radiation given to noninbred albino Sprague-Dawley rats at 50 days of age. Compared to rats on a low-fat (LF) diet (5% lard), rats on a high-fat (HF) diet (20% lard) from 30 days of age had more tumors, with a higher multiplicity of carcinomas per rat. LF-fed groups exhibited a longer median tumor latency period thatn did HF-fed groups. A similar trend toward more tumors with an earlier time of death was seen in rats given single iv doses of 50 mg 1-methyl-1-nitrosourea/kg and fed an HF diet as compared to an LF diet.

Synergism of diethylstilbestrol and radiation in mammary carcinogenesis in female F344 rats.

One compressed 20-mg pellet containing cholesterol only or cholesterol mixed with 0.98, 1.6, 2.6, or 3.9 mg of diethylstilbestrol (DES) was implanted into each of 203 female F344 rats. Two days later, half the animals in each group were exposed to 150 R of X-rays, and the other half were sham irradiated. The rats were maintained until 350 days post implantation. Mortality increased with the higher doses of DES, with or without X-rays. DES at all dose levels, with or without X-rays, produced pituitary tumors and pyometritis. Only rats that received both DES and X-rays had mammary adenocarcinomas (AC). A synergistic AC response was found in the group that received 2.6 mg DES plus X-rays. Synergism was defined as a significantly greater incidence of rats with mammary neoplasia resulting from DES plus X-ray treatment when compared to the summed incidence from comparable individual treatments. For all other groups of rats that received both treatments, synergism was detected only when their data were combined. Synergism was not detected among rats that had fibroadenomas (FA). Both types of neoplasms were independent phenomena because no significant relationship was found between the incidences of FA and AC.

Apparent rat strain-related sensitivity to phorbol promotion of mammary carcinogenesis.

It has been reported that twice-weekly i.p. injections of 4 mg phorbol for 10 weeks, after a single feeding of 6 mg dimethylbenz(a)anthracene (DMBA) in female Wistar rats, led to a significant augmentation of mammary adenocarcinoma incidence and of lymphatic leukemia incidence as compared to 6 mg DMBA alone. In an experiment reported here, in female Sprague-Dawley rats, using the same doses of DMBA and phorbol and the same injection schedule, phorbol given after DMBA did not augment mammary adenocarcinoma incidence or lymphatic leukemia incidence as compared to DMBA given alone. It thus appears that there is a strain-related sensitivity between Wistar and Sprague-Dawley rats with regard to the promoting activity of phorbol when phorbol treatment follows DMBA treatment, and mammary adenocarcinoma incidence and lymphatic leukemia incidence are studied. Further, in Sprague-Dawley rats, phorbol did not promote mammary fibroadenoma incidence in DMBA-treated rats, mammary adenocarcinoma incidence in procarbazine-treated rats, and mammary adenocarcinoma incidence or mammary fibroadenoma incidence in X-ray-treated rats. DMBA and procarbazine, with or without phorbol, tended to induce more mammary neoplasms in the anterior (thoracic) than in the posterior (abdominal) mammary glands. X-irradiation tended to induce mammary neoplasms in approximately equal numbers in the anterior and posterior mammary glands. It was suggested that regional differences in chemically induced mammary carcinogenesis were due to a difference in the transport and delivery of the chemical carcinogens to the regions rather than a difference in the amount of mammary gland tissue in the regions. An analysis of the numbers of Sprague-Dawley rats that developed either no mammary neoplasms, or only mammary adenocarcinomas, or only mammary fibroadenomas, or both mammary adenocarcinomas and mammary fibroadenomas in response to DMBA, procarbazine, and X-ray, suggested that the development of a mammary adenocarcinoma or the development of a mammary fibroadenoma are independent processes.

Rat differences in mammary tumor induction with estrogen and neutron radiation.

Young adult female rats of either the Sprague-Dawley stock or the ACI strain were each given an implant of a compressed pellet of 5 mg diethylstilbestrol (DES) and 15 mg cholestrol 2 days before irradiation with 0.4, 1.3, or 4.0 rads of 0.43-MeV neutrons. These rats were studied, along with appropriate irradiated and nonirradiated controls, until death or for a maximum of 48 weeks. Response differences between the strain and stock included the following: DES produced both pituitary tumors and mammary adenocarcinomas (MAC) in ACI rats only. Neutron radiation increased mammary fibroadenoma (MFA) formation in Srague-Dawley rats only. No interactions between DES and radiation on MAC formation in Sprague-Dawley rats or MFA formation in ACI rats were demonstrated. However, when DES and neutron radiation were combined, DES appeared to inhibit the MFA response to radiation in Sprague-Dawley rats. In contrast, DES appeared to act synergistically with neutron radiation on MAC formation in ACI rats. These results clearly demonstrate rat differences in mammary gland carcinogenesis in response to estrogen, to radiation, or to a combination of both agents.