[CANCER RESEARCH 49, 3168-3172, June 15, 1989] Differential Effects of Tranylcypromine and Imidazole on Mammary Carcinogenesis in Rats Fed Low and High Fat Diets1 David L. McCormick,2 Ann M. Spicer, and Jacqueline L. Hollister Life Sciences Department, IIT Research Institute, Chicago, Illinois 60616 ABSTRACT studies with this class of compounds used inhibitors of the cyclooxygenase pathway of arachidonic acid catabolism; exper Neoplastic development in the rat mammary gland can be suppressed iments performed in our laboratory and by Ip and coworkers by inhibition of the activity of several enzymes involved in eicosanoid biosynthesis. In order to investigate the potential utility of prostacyclin demonstrated that the postcarcinogen phase of rat mammary and thromboxane synthetases as targets for mammary cancer chemopre- carcinogenesis can be suppressed by dietary administration of vention, experiments were conducted to determine the influence of tran- indomethacin (7, 8) or flurbiprofen (9). However, although the ylcypromine (TCP), an inhibitor of prostacyclin synthetase, and ¡mida/ole anticarcinogenic activity of indomethacin is similar to that of (IMI), an inhibitor of thromboxane synthetase, on mammary carcinogen- more widely studied inhibitors of mammary carcinogenesis such esis induced in rats by 7V-methyl-/V-nitrosourea. Fifty-day-old female as retinyl acetate (10) and selenium (11), the dose levels of Sprague-Dawley |Hsd:SD(BR)l rats received a single s.c. dose of 0 or 40 indomethacin required for chemopreventive efficacy in rats are mg of .V-mcth>l-.Y-nitrosoiirea per kg of body weight. Beginning 7 days close to the threshold of lethal toxicity (12). For this reason, after carcinogen administration, groups of rats were fed isoenergetic, studies are ongoing to identify additional modifiers of arachi casein-based diets containing 3 or 20% corn oil (w/w), supplemented with donic acid metabolism whose administration provides an effec (per kg of diet) 10 mg of TCP, 1000 mg of IMI, or sucrose carrier only. TCP reduced mammary carcinoma multiplicity in rats fed the 20% corn tive means for the suppression of mammary cancer induction, yet which possess improved therapeutic ratios, i.e., greater oil diet, but had no effect in rats fed the diet containing 3% fat. By "margins of safety" between the dose levels required for anti- contrast, supplementation with IMI increased mammary cancer incidence in the group fed the 20% fat diet and increased carcinoma multiplicity in carcinogenic efficacy and those which induce significant toxic the 3% fat group to the levels seen in rats fed the 20% fat diet. These ity. data suggest that inhibition of prostacyclin synthetase, but not thrombox The oxidation of arachidonic acid by cyclooxygenase is an ane synthetase, may present a useful mechanism for mammary cancer early step in the biosynthesis of a number of eicosanoids, chemoprevention in animals consuming a diet high in fat. Furthermore, including PGA2,3 PGD2, PGE2, PGF2o, PGI2, and TXB2. Be the differential effects of TCP and IMI in rats fed low and high fat diets cause cyclooxygenase is involved in the synthesis of a large suggest that the action of dietary fat in mammary cancer induction may number of biologically active compounds, the possibility exists involve influences on the arachidonic acid cascade. that the chemopreventive and toxic effects of cyclooxygenase inhibition are a result of alterations in the synthesis of different INTRODUCTION eicosanoids. In such a circumstance, the chemopreventive effi Chemical carcinogenesis in the rat mammary gland is subject cacy and toxicity of cyclooxygenase inhibition might be disso to modulation through a variety of endocrine, pharmacological, ciable through the use of a more narrowly targeted approach and nutritional manipulations (1). Because of the close biolog designed to influence levels of fewer eicosanoids. The enzymes ical and histopathological correspondence between mammary prostacyclin synthetase and TX synthetase utilize a common substrate in the post-cyclooxygenase portion of the arachidonic neoplasia in rats and in humans, modification of mammary cancer response in the rat model may provide useful insights acid cascade; a negative interaction between the products of into the regulation of neoplastic development in the human these two enzymes has been proposed (13, 14). The present breast. For example, the identification of factors which enhance study was designed to determine the efficacy of TCP, an inhib mammary cancer induction in rats [e.g., dietary fat (2, 3)] can itor of prostacyclin synthetase (15, 16), and IMI, an inhibitor aid in the elucidation of risk factors for human breast cancer. of TX synthetase (17, 18), as chemopreventive agents in the rat Conversely, a number of factors which suppress neoplastic mammary gland, and to determine if the activity of these development in the rat mammary gland [e.g., early full-term compounds as modifiers of mammary cancer induction is influ pregnancy (4, 5)] appear to play a similar role in reducing the enced by dietary fat intake. risk of breast cancer in humans (6). Finally, the experimental use of modifiers of carcinogenesis can provide data concerning MATERIALS AND METHODS the mechanisms through which neoplastic development in the mammary gland is regulated; such data can be applied to both Experimental Animals. Virgin female Sprague-Dawley [Hsd:SD(BR)J cancer risk assessment and the identification of appropriate rats were received at 28 days of age from Harlan/Sprague-Dawley, targets for the design of anticarcinogenic drugs. Indianapolis, IN. Rats were housed in groups of three in polycarbonate Modifiers of arachidonic acid metabolism are one class of cages on hardwood bedding, and they were held in a temperature- and agents with significant activity as inhibitors of rat mammary humidity-controlled room maintained on a daily cycle of 14 h of light carcinogenesis. The initial mammary cancer chemoprevention and 10 h of dark. During the quarantine period, rats were allowed free access to a standard laboratory chow diet (Wayne Lab Chow; Allied Received 12/5/88; revised 2/16/89; accepted 3/17/89. Mills, Chicago, IL) and drinking water; 5 days prior to the initiation The costs of publication of this article were defrayed in part by the payment of the study, the chow diet was replaced with a semipurified, casein- of page charges. This article must therefore be hereby marked advertisement in based diet containing 3% fat. All food and bedding materials were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Grant R01-CA-40874 from the National Cancer Institute, changed twice weekly. Department of Health and Human Services. Presented in part at the Annual Experimental Diets. Basal diets used in the experiment were isoener- Meeting of the American Association for Cancer Research, New Orleans, LA, May 25-28, 1988. 3The abbreviations used are: PG, prostaglandin(s); TX, thromboxane(s); TCP, 2To whom requests for reprints should be addressed, at Life Sciences Depart tranylcypromine; IMI, imidazole; MNU, A'-methyl-iV-nitrosourea; T50, time to ment, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616. 50% cancer incidence (median cancer induction time). 3168 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. TRANYLCYPROMINE AND IM1DAZOLE EFFECTS ON RAT MAMMARY CARCINOGENESIS getic, semipurified, casein-based diets containing either 3% or 20% fat mammary cancer incidence at 180 days post-MNU were compared (w/w), as added corn oil. Diets were manufactured to our specifications using x2 analysis; comparisons of T$owere made using the median test by Teklad Test Diets, Madison, WI. The composition of the basal diets (21). Group body weights were compared using analysis of variance. is provided in Table 1. As required by the protocol, diets were supplemented with either 10 mg of TCP (99% purity; Sigma Chemical Co., St. Louis, MO) or 1000 RESULTS mg of IMI (99+% purity; Sigma) per kg; dietary levels of TCP and IMI were selected on the basis of preliminary subchronic toxicity studies. TCP and IMI had opposite effects on mammary cancer To facilitate homogeneous distribution of these agents in the diets, induction by MNU. Significant differences between these two TCP and IMI were mixed into diets using a sucrose carrier (10 g/kg of agents were found in comparisons of their influence on mam diet). Dietary controls received the 3% or 20% fat diets supplemented mary carcinoma multiplicity and tumor latency and in the with sucrose carrier only. effects of dietary fat on their activity as modifiers of mammary Experimental Protocol. MNU was purchased from Ash-Stevens (De carcinogenesis. troit, MI) and was dissolved in sterile saline (pH 5.0) immediately prior Administration of a supplement of 10 mg of TCP per kg of to administration. At 50 days of age, all rats received a single s.c. injection of 0 or 40 mg of MNU per kg of body weight. Seven days diet conferred significant protection against mammary carci after carcinogen administration, rats were randomized by weight into nogenesis in rats fed the diet containing 20% corn oil (Table groups according to the protocol (Table 2), and administration of 2). As illustrated in Fig. 1, TCP decreased the mean number of experimental diets was begun. mammary cancers per rat by approximately 40%, from 2.67 in Beginning 4 wk after MNU administration, rats were palpated twice controls to 1.64 (P < 0.01). The compound also increased T5o weekly to monitor mammary tumor appearance; the location and date from 98 days in the 20% fat control group to 130 days and of appearance of all palpable lesions were recorded. Animals were decreased tumor-related mortality from 12% in controls to 0% observed twice daily throughout the study to monitor their overall (0.05 < P < 0.10). However, although exposure to TCP in health status, and they were weighed weekly.