Genetic Control of Resistance to Chemically ... - Cancer Research

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(CANCER RESEARCH 46, 3958-3963, August 1986] Genetic Control of Resistance to Chemically Induced Mammary Adenocarcinogenesis in the Rat1 John T. Isaacs Oncology Center and Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ABSTRACT Fifty-day-old female rats of a series of outbred (i.e., SD) and inbred (i.e., NSD, WF, LEW, F344, ACI, and COP) strains were exposed to a single dose of either of two highly effective mammary chemical carcino gens, 7,12-dimethylbenzfa|anthracene (DMBA) or 1-methyl-1-nitrosourea (MM), to determine the characteristic number of mammary adenocarcinomas induced/rat for each strain. Female rats of the inbred NSD, WF, and LEW strains were found to be as highly susceptible to DMBA exposure as the randomly outbred SD strain (i.e., >2 mammary adenocarcinomas/rat develop). Inbred female F344 and ACI rats were found to be much less susceptible to DMBA induced mammary adenocarcinogenesis (i.e., 2 years). Inbred female BN and F344 rats are less susceptible, with only 6% of either of these strains developing mammary cancer during their lifetimes (6-8). The inbred ACI female is even less suscep tible with only 4% of these animals developing mammary cancer if untreated (9). Inbred female COP rats are the most resistant to developing mammary cancer with not a single mammary cancer appearing among over 1500 of these female rats allowed to live out their normal lifetimes without any exogenous treat ment (10). This same gradation in susceptibility is observed with regard to mammary cancer induction by AAF2 (11, 12). When SD, inbred OM, or inbred BUF female rats are chronically fed AAF, more than two-thirds of the animals develop mammary cancer with less than 10% developing liver cancer (11). When F344 (11) or inbred AUG (12) rats are fed AAF, 10-20% of animals develop mammary cancer and 10-20% develop liver cancer. Only 8% of ACI rats (11, 12) fed AAF develop mam mary cancer while liver cancer develops in 34% of these animals. Female COP rats were completely resistant to AAF induction Received 12/10/85; revised 4/11/86; accepted 5/2/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'Supported by Grants from the American Cancer Society (IN-I1U) and Department of Health and Human Services (CA 42954). 1The abbreviations used are: AAF, 2-acetylaminofluorene; DMBA, dimethylbenz[a]anthracene; MNU,l-methyl-l-nitrosourea. of mammary cancer (12). Female COP rats did develop, how ever, a 20% incidence of liver cancer demonstrating that not all tissues in this strain are resistant to AAF. Chronic treatment of female rats with exogenous estrogens, like AAF feeding, is an efficient inducer of mammary cancer development in some strains of rats and not others (13, 14). Chronic (i.e., >1 year) high dose estrogen treatment of SD (13), AUG (14), or ACI (14) rats results in more than 40% of all treated animals developing mammary cancers. Inbred LEW (13) and F344 (13) rats are less susceptible to exogenous estrogen treatment, developing only a 31 and 16% incidence of mammary cancer, respectively. Again, the mammary glands of the female COP are completely resistant (i.e., 0% incidence) to such high dose estrogen treatment (14). This chronic estrogen treatment does produce, however, a 20% incidence of bladder cancer in these female COP rats (14), again demonstrating that not all tissues in this strain are of the resistant phenotype. These background data demonstrate that various strains of female rats are not equally susceptible to the induction of mammary cancer and that the female COP rat might be genet ically unique in its resistance to mammary adenocarcinogenesis, regardless of what type of carcinogenic treatment is used. There are a series of chemical carcinogens (e.g., DMBA and MNU) which are known to be highly effective in inducing the devel opment of mammary cancers in female rats of certain strains following a single exposure (5, 15). Therefore, female rats from a series of inbred strains including the COP were separately treated with DMBA and MNU to determine the resistance versus susceptibility of each strain to mammary adenocarcinoma development. MATERIALS AND METHODS Animals. Randomly outbred Sprague-Dawley (SD/HsdBr), inbred ACI/Seg (ACI/SegHsdBR), inbred Fischer (F344/NHsdBr), inbred Wistar-Furth (WF/NHsd3R), inbred COP (COP/NHsdBr), and inbred Lewis (LEW/NHsdBr) rats were obtained from HarÃanSprague-Daw ley, Inc. (Indianapolis, IN). The inbred Sprague-Dawley (NSD/N) animals were derived from an in-house colony established from a breeding nucleus of NSD rats obtained at the 54th brother x sister generation through the generosity of the Veterinary Resources Branch of the NIH Genetic Resource. Following exposure to carcinogen, as described below, all animals were housed in a Vickers Total Contain ment Isolation Unit (London, United Kingdom) for 1 month before being returned to normal animal rooms. Animals were housed in a room lighted 12 h/day and maintained at a temperature of 23Â°C.All rats were palpated for mammary tumors twice weekly with the time of detection post-carcinogen exposure being individually recorded for each tumor which developed in each rat. The rats in each grouping underwent a complete autopsy at the time of spontaneous death or at 365 days post-carcinogen exposure. At the time of autopsy all grossly detectable mammary tumors (i.e., >2 mm in diameter) were removed and fixed in 10% buffered formalin, and paraffin sections were prepared and stained with hematoxylin and eosin. Each mammary tumor was individually classified histopathologically according to the criteria of Van Zwieten (16). This histolÃ³gica!evalu ation thus allowed the number of detectable mammary adenocarcino mas, sarcomas, and fibroadenomas for each rat to be individually 3958 Downloaded from cancerres.aacrjournals.org on February 22, 2013 Copyright © 1986 American Association for Cancer Research
assigned; therefore, the mean number of mammary adenocarcinomas per rat for each experimental treatment group could be determined at various times post-carcinogen exposure. The mean values Â±SE for the mammary adenocarcinomas per animal for each group were determined by dividing the number of new mammary adenocarcinomas observed within the group in each 10-day interval by the number of rats at risk in that interval and then summing these values by the method of McCormick et al. (5). This calculation thus takes into consideration intercurrent mortality. Carcinogenic Exposure. For the systemic exposure to DMBA, groups of animals were given either a single or multiple doses of sesame oil containing varying amounts of DMBA by means of gastric intubation according to the method of Huggins et al. (15). For the direct in vivo exposure of mammary glands to DMBA. the technique of Sinha and Dao (17) was used. This involves anesthetizing rats with Metofane (Pittman-Moore, Inc., Washington Crossing, NJ), exposing the right inguinal mammary gland, dusting 3mg of a 2:1 mixture of cholesterol and DMBA (i.e., 1 mg DMBA total) directly onto the exposed gland, and then closing the skin incision with stainless steel autoclips. For the MNU experiments, groups of rats were given either a single or multiple injections of MNU via the femoral vein according to the method of Cullino et al. (18). Some rats were also given s.c. injections of a single dose of MNU according to the method of Thompson and Meeker (19). For i.v. or s.c. injections, MNU was dissolved in 0.85% NaCl solution adjusted to pH 5.0 with 3% acetic acid. DMBA and MNU were obtained from Sigma Chemical Co. (St. Louis, MO). RESULTS DMBA Experiments. When 20 mg of DMBA are adminis tered systemically via a single stomach tube feeding to 50-dayold female SD rats, greater than 90% of the animals develop multiple mammary adenocarcinomas within 1 year (Fig. 1). Like the randomly outbred SD rat, 50-day-old inbred NSD, inbred WF, and inbred LEW female rats are all equally highly susceptible to multiple mammary adenocarcinoma development following DMBA exposure (Fig. 1). Following a single feeding of 20 mg of DMBA, female F344 rats are less susceptible to development of mammary adenocarcinoma and female ACI rats are even less susceptible (Fig. 1). In direct contrast to the other strains, the mammary glands of female COP rats are resistant to p.o. DMBA feeding. None of a series of twenty five 50-day-old female COP rats fed 20 mg of DMBA developed any detectable mammary adenocarcinomas even if these treated rats were allowed to live for more than 2 years following dosing. At 50 days of age, there are differences in the body weights 3.0! 5O 100 150 200 250 300 350 GENETIC RESISTANCE TO MAMMARY CANCER FISCHER Doys Posi DMBA Feeding Fig. 1. Temporal pattern of mammary adenocarcinoma development in var ious strains of female rats following a single p.o. feeding of 20 mg of DMBA. A total of twenty-five 50-day-old female rats were dosed for each strain. between the various strains of female rats tested. Since each strain of rat received a single feeding of 20 mg of DMBA, this resulted in differences in the dose of DMBA (mg/kg body weight) when normalized on a body weight basis (i.e., SD, 133; NSD, 133; WF, 160; LEW, 148; F344, 200; ACI, 200; COP, 200 mg). The fact that the high susceptibility SD, NSD, WF, and LEW strains actually received a lower dose of DMBA, when expressed on a body weight basis, as compared to the low susceptibility F344 and ACI and resistant COP strains, dem onstrates that the rank order of susceptibility or resistance to DMBA cannot be trivially explained by differences in body weights at time of carcinogen exposure. When female SD rats are exposed multiple times to p.o. feeding of DMBA, the number of mammary adenocarcinomas induced per rat increases synergistically, not merely additively, as compared to a single dose exposure (20). Therefore, 10 female COP rats were fed 10 mg of DMBA 3 times, at weekly intervals, starting at 36 days of life. The animals were followed for up to 1 year of life, or until moribund, during which time no mammary adenocarcinomas developed although 50% of the treated groups did develop leukemia, which was transplantable by means of inoculation of donor blood into recipient untreated COP rats. An additional group of 10 COP female rats was fed 5 mg of DMBA four times at weekly intervals starting at 36 days of life with the same result (i.e., no mammary cancers, 60% incidence of leukemia). As a point of comparison, NSD rats given the same 5-mg, four-dose feeding schedule developed 4.0 Â±0.5 mammary adenocarcinomas/rat within 200 days. Since pregnancy commencing after carcinogen exposure has been shown to sharply increase mammary cancer development and growth in certain strains (21), 20 female COP rats were fed 20 mg of DMBA p.o. at 50 days of life and then 1 week later were bred. These animals were allowed to deliver their litters and then were rebred for a minimum of four pregnancies. These animals were followed for over 1 year and only 1 mammary adenocarcinoma developed in the 20 treated rats (i.e., average of 0.05 Â±0.05 mammary adenocarcinomas/rat). If 1 mg of DMBA is directly applied to an exposed inguinal mammary gland of SD rat, mammary adenocarcinomas develop in 100% of the directly exposed glands within 200 days but in 0% of the unexposed mammary glands of the treated hosts (17). A series of inbred rat strains were tested using this direct application technique. The right inguinal mammary gland of 10 rats/strain were directly exposed to 1 mg of DMBA. In the NSD group, all exposed glands (i.e., 100%) developed mam mary adenocarcinomas within 200 days; for the WF strain, also 100% incidence; for the F344 strain, 40% incidence; for the ACI, 25% incidence; and for the COP, 10% incidence of mam mary adenocarcinoma with a 40% incidence of fibrosarcoma formation. If 50-day-old SD rats are given Â¡.p.injections of an oil emulsion containing 5 mg of DMBA, all animals develop mammary adenocarcinomas (22). Therefore, ten 50-day-old female NSD rats were given injections of an oil emulsion of 5 mg of DMBA and all animals developed mammary adenocar cinomas (i.e., 1.2 Â±0.1 mammary adenocarcinomas/rat) and one animal (i.e., 10%) developed an abdominal sarcoma. In contrast, when ten 50-day-old female COP rats were similarly given i.p. injections of DMBA, no animal developed mammary cancer; however, 40% of these COP animals developed i.p. sarcomas. MNU Experiments. DMBA is known to require metabolic conversion to its ultimate carcinogen (23). The mammary epi thelial cells of female COP rats might be unable to properly 3959 Downloaded from cancerres.aacrjournals.org on February 22, 2013 Copyright © 1986 American Association for Cancer Research
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