Modulation of Methylnitrosourea-induced Breast Cancer in Sprague Dawley Rats by Dehydroepiandrosterone: Dose-dependent Inhibition, Effects of Limited Exposure, Effects on Peroxisomal Enzymes, and Lack of Effects on Levels of Ha-Ras Mutations1

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, is an effective chemopreventive agent in the A/-methyl-A/-nitrosourea (MNU)induced rat mammary tumor model. Dietary 1)111 \ (5-600 ppm; 600 mg/kg diet) was administered beginning 1 week before MM and admin istered continually throughout the duration of the experiment. The highest dose of DHEA (600 ppm) significantly decreased tumor incidence from 95 to 45% and increased tumor latency and decreased tumor multiplicity from 4.1 to 0.5 tumors/rat. Lower doses of DHEA (5, 24, and 120 ppm) were also effective, decreasing tumor multiplicity by 28, 40, and 55%, respectively, increasing tumor latency in a dose-dependent manner but only minimally affecting final tumor incidence. DHEA in the diet caused a dose-dependent increase in serum levels of DHEA. The 120-ppm dietary dose of DHEA resulted in serum levels of DHEA of -42 pmol/ml levels, similar to those seen in young humans. When we examined whole mounts of mammary glands derived from rats exposed to higher levels of DHEA (600 ppm), we observed a striking increase in lobular development. The doses of DHEA used in these studies (£600 ppm) had minimal effects on the induction of fatty acid CoA synthetase, a peroxisome-associated en zyme. In contrast, a dose of 2000 ppm substantially increased levels of peroxisome-associated fatty acid CoA synthetase. The varied and striking efficacy of DHEA was achieved in the absence of any significant effect on body weight gain in the treated rats. Furthermore, tumors from rats treated with MNU alone or rats treated with MNLI plus DHEA were examined for the presence of mutations in the \\-.\-Kn\ oncogene. There was a slight decrease in the percentage of tumors bearing \\-.\-Kn\ muta tions in tumors derived from MM -control rats as contrasted with tumors from MNL-DHEA (120 and 600 ppm »-treatedrats. Based on the striking chemopreventive efficacy of continual exposure to 1)1II A. we examined the effects of more limited exposure to DHEA. Rats were treated with DHEA for a period of 7 weeks immediately before and after MNU injection. Rats were then placed on the control diet for the ensuing 15 weeks. Even this limited exposure to DHEA for a period of 7 weeks profoundly decreased final tumor incidence and multiplicity. Addition ally, we examined the effects of intermittent dosing with DHEA. Rats were treated alternatively at 3-week intervals either with diet containing DHEA or with control diet. It was found that this intermittent dosing with DHEA also substantially inhibited the formation of mammary tumors. Received 9/12/97; accepted 1/5/98. The costs of publication of this article were defrayed in part hy 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. ' These studies were supported in part by National Cancer Institute Contracts NO1CN-35535-02 and NOI-CN-35535-03 from the National Cancer Institute (to R. D. M.i. These studies were further supported by NIH Grant CA43839 (to R. A. P.). X. D. L. was supported hy the Humana Findowment for Hxcellence. James Graham Brown Cancer Center. University of Louisville School of Medicine. 2 To whom requests for reprints should be addressed, at National Cancer InstituteDCPC, Chemoprevenlion Branch. EPN-201. 9000 Rockville Pike. Bethesda. MD 20892. INTRODUCTION DHEA,' which is produced at high levels in the adrenals of primates, is the primar)' steroid precursor to androstenedione. DHEA is readily converted to androstenedione. which is itself converted into androgens and estrogens in peripheral tissues (1, 2). Interestingly, serum levels of DHEA and its sulfate conjugate decrease markedly throughout the life span of a human (3). There is epidemiolÃ3gica! evidence in humans associating higher levels of DHEA/DHEA sulfate with decreased inci dence of breast cancer in premenopausal females (4). However, further epidemiolÃ3gica! studies on breast cancer in postmenopausal individuals do not support this association between high incidence of DHEA/DHEA sulfate and low levels of breast cancer (5, 6). DHEA has proven to be an effective chemopreventive agent in a variety of mouse tumor models including the 7.12-dimethylben/anthracene/12-O-tetradecanoylphorbol-13-acetate-induced mouse skin model (7); the 1,2-dimethylhydra/ine-induced mouse colon model (8): the P-53 transgenie mouse lymphoma model (9); and the 7,12-dimethylben/anthracene and urethane-induced mouse lung model (10). In addition. DHEA has been shown previously to have chemopreventive activity in a variety of carcinogen-induced mammary cancer models in rats: (a) the 7.12-dimethylben/.anthracene-induced rat mammary tumor model (11); (/>)the MNU-induced rat mammary tumor model ( 12). In a recent set of experiments using the MNU-induced model, we found that even rela tively low-dose levels of DHEA (400 or SOOppm in diet) had striking chemopreventive efficacy (13).; and (r) the radiation-induced, estrogenenhanced rat mammary tumor model ( 14). A wide variety of potential mechanisms have been postulated to explain the ehemopreventive efficacy of DHEA including effects on cellular proliferation, effects on body weight, peroxisomal proliferation, effects on Ras famesylation, effect on the immune response, and others (reviewed in Ref. 15). Furthermore, the striking efficacy of DHEA. even at relatively low dietary doses (13). allows one to address various ques tions regarding optimal treatment with DHEA in a chemopreventive setting. We therefore performed various studies to address questions dealing both with optimal dosing of DHEA as well as a potential mechanism of action of DHEA. Specific questions included: (a) What are the lowest doses levels of DHEA that effect MNU-induced rat mammary tumorigenesis? (/>)Do the preventive effects of DHEA relate to altered serum levels of DHEA? (r) Does DHEA induce peroxisomeal en/.ymes at effective preventive doses? (d) Does DHEA treatment alter the per centage of breast tumors bearing a mutation in the Ha-/fc;.voncogene? (e) Does DHEA alter breast morphology and lobule development? (ft Is limited treatment with DHEA sufficient to cause substantial long-term effects on mammary tumor induction by DHEA? and (,i;) Is intermittent dosing with DHEA effective in decreasing final tumor multiplicity? 1The abbreviations used are: DHEA. dehydroepiandroslerone: MNU. /V-meihyl-/vnilrosourea: dNTP. deoxynucleotide triphosphate; PACO, fatty acid CoA synthelase. on April 28, 2017. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from MNU-INDUCED BREAST CANCER AND DEHYDROEPIANDROSTERONE MATERIALS AND METHODS Rats. Virgin, female Sprague Dawley rats were received from HarÃ-an/ Sprague Dawley at 35 days of age. After a quarantine period of 1 week, animals were randomized by weight into various treatment groups. The stand ard diet used was Teklad 4% Rat/Mouse Chow. DHEA was added to the diet at the doses indicated in the individual figures. Carcinogen Treatment (MNU). The basic model used here was devel oped by Guillino et al. (16) and revised by Moon and coworkers (17). When the rats were 50 days of age, a single dose of MNU was administered i.v. in acidified saline (pH 5.0) at a dose of 50 mg/kg body weight. Dose Response to DHEA. Rats were placed into groups receiving 0, 5, 24, 120, or 600 ppm DHEA in the diet. Rats were administered DHEA in the diet beginning on day 43. 1 week before the day that they were administered MNU (day 50), and remained on the DHEA diet until the time of sacrifice ( 102 days after MNU injection; Fig. 1 and Table 1). DHEA was obtained from Sigma Chemical Co. The dietary concentrations of DHEA were determined at three time points during the course of the experiment and was found to be within 5% of the nominal dose. The effects of DHEA on the latency of palpable tumors were determined using log-rank analysis (18). Tumor multiplicity was com pared using Armitage analysis (19). Necropsy. Rats that were moribund during the study were sacrificed by CO, asphyxiation. At the termination of the experiment (102 days after MNU treatment), all animals were anesthetized, and blood was collected for serum preparation and determination of DHEA levels (see below). Animals were sacrificed by asphyxiation, and all mammary masses were located and matched with palpation data as to location. Tumors from each group were weighed to determine a mean tumor weight. Tumors from each of the groups were frozen to isolate DNA and examined for Ha-Rns mutations (see below). Serum Levels of DHEA. At the end of the initial experiment examining the chemopreventive efficacy of various doses of DHEA, serum was taken from at least 12 individual rats in each of the MNU/DHEA treatment groups (Table 2). The sera from these rats were used to determine serum levels of DHEA using methods published previously (20). Effects of DHEA Treatment on Ila-AVv Mutations: DNA Isolation. In general, the methods used here were similar to those used previously by Wang et al. (21 ). Pieces of tumors were either processed for histopathology or frozen in liquid nitrogen and subsequently used to determine the presence or absence of mutations in the Ha-Ac/i oncogene. High molecular weight DNA was isolated from tumors obtained from rats treated with MNU alone or MNU plus DHEA using Pronase-SDS lysis. Following phenol-chloroform extraction and ethanol precipitation, the DNA samples were treated with RNase and addi tional phenol-chloroform extractions and ethanol precipitation. The size of the DNA was checked in a 0.7% agarose gel. DNA Amplification. Amplification reactions were carried out in a 100-/¿I reaction mixture containing I fig of genomic DNA in 50 itiM KCI, 10 irmi Tris (pH 8.4), 2.5 mM MgCU, each primer at 1 /AM,each dNTP (dATP, dCTP, dTTP. and dGTP) at 200 /J.M. gelatin at 200 /ig/ml. and 2 units of Taq polymerase (Perkin-Elmer). The samples were overlaid with several drops of paraffin oil to prevent evaporation and subjected to 25 cycles of amplification as follows. The samples were heated to 95°Cfor 1 min, cooled to 50°Cfor 2 min, and incubated at 72°Cfor 3 min. The primers used to generate products for the PCR analysis were: RHU1, 5'-GCAGGAGCTCCTGGTTTGGC-3'; and RHL1, 5'-CAGAGCTCACCTCTATAGT-3'. Direct Sequencing of Amplified DNAs. PCR-amplified DNA was de salted, and excess oligonucleotides and dNTPs were removed by spin-dialysis Table 1 Effects of dietary DHEA on MNU-induced mammary tumori^enesis" Table 2 Levels of DHEA in the serum of rats treated with dietary DHEA in a longterm chetnoprevention experiment Dose of DHEA(ppm)0524120600Tumor incidence (%)1008980SO40*Tumor multiplicity4.6 0.7*3.6 0.52.6 0.5'1.9 0.4'0.5 O.I'Time (days) to first tumor54 3.269 5.4r71 6.5'73 6.7C99 6.5'Final body weight (g)246 ± 4.2247 ± 2.9252 ± 5.5252 ± 3.7257 ±3.5 Length of exposure (days)102"102102102102Dose of DHEA(mg/kg of diet)(I524120 600Serum levels of DHEA (nmol/1)0.89 ±0.4 lh-'3.1 ±0.508.7 ± 2.2342.3 ±4.3 104'' " Administration of MNU at 50 mg/kg BW to 50-day-old virgin female Sprague Dawley rats. h Group means ±SE. ' P < 0.05. " All of the rats treated for 102 days were 152 days old at the time of sacrifice. All of the rats were treated with MNU at day 50, and these rats had mammary tumors at the time of sacrifice. These rats are derived from the animals described in Table I. h Results are presented as means ±SE. ' These results are based on determining mean ±SE from three pools of rats, each pool containing four to five rats who had been treated with the indicated dose of DHEA. d Results presented are based on the single available pool of sera at this specific dose. on a Centrifucon 30 (Amicon, Danvers. ME), concentrating the volume from 100 to 40 /J.1.Sequencing primers (2 pmol). end-labeled with [y-32P]ATP and T4 polynucleotide kinase, were annealed to one-tenth of the amplified \\a-Ras DNA by heat denaturing the strands at 95°Cfor 5 min in 40 mM Tris (pH 7.5), 20 mM MgCl2, and 50 mM NaCl. The sequence of the sequencing primer is 5'-TCCTGGTTTGGCAACCCCTG-3'. The reaction was spun briefly to col lect the condensate, equilibrated at 37°Cfor 2 min, and divided into four tubes containing 3 ¡L\ of 80 /xM dNTPs and 8 yxMddNTPs. Five units of T7 DNA polymerase (Sequenase; USB, Cleveland. OH) were added to each tube, and the reaction was allowed to proceed for 10 min at 37°C.Samples were heated to 65°Cfor 5 min in formamide-dye-stop mix and subjected to electrophoresis on 8% acrylamide gel. Gels were dried and exposed to X-ray film for 24 h (21). DHEA Effects on Peroxisomal Enzymes. Liver tissue was obtained from animals in experiment I. Specifically, female rats were treated with DHEA until 43 days of age. 7 days before dosing with MNU, until day 152 of age. Alternatively, rats were treated with DHEA for a period of 28 days in the absence of MNU. At the time that individual rats were sacrificed by CO2 asphyxiation, livers were removed and quickly frozen in liquid nitrogen. KCN-insensitive peroxisomal ß-oxidation of fatty acids was determined with post-5000 rpm liver supernatants by measuring NADH produced from the oxidation of the C-3 hydroxyl group of palmitoyl CoA catalyzed by 3-hydroxyl-CoA-dehydrogenase, the rate-limiting step in ß-oxidationusing the methods described by Wu et al (22). Limited Exposure to DHEA and Mammary Gland Morphology. Rats, 50 days of age, were exposed to DHEA for 28 days. At that time, the animals were sacrificed by CO, asphyxiation. Mammary glands were removed, and abdominal inguinal glands were processed for whole mounts using methodol ogies described previously. Limited Exposure to DHEA and Chemopreventive Effects. Certain groups of rats received continual administration of DHEA (600 or 2000 ppm) beginning 1 week before MNU administration and then continually thereafter (Fig. 5). Alternatively, rats were administered DHEA continually from 7 days before MNU administration until 42 days after MNU (7 weeks) administration, after which animals were placed on standard Teklad basal diet for the remain der of the experiment. Using the methods described in experiment I. rats were initiated with MNU on day 50. The experiment was followed for 135 days after MNU on the rationale that the limited exposure regimen used might only serve to increase the latency of tumorigenesis. Intermittent Exposure to DHEA and Chemopreventive Effects. Rats received either continual administration of DHEA (600 or 2000 ppm) or were administered these chemopreventives on an intermittent basis (e.g., DHEA from 7 days before until 14 days after MNU treatment, 35-56, 77-98, and 119-135 days at other times animals received basal Teklad diet; Fig. 6]. Using methods as described in experiment I, animals were initiated with MNU on day 50. The experiment was followed for 135 days after MNU rather than 100 days after MNU, like experiment I, because the intermittent exposure regimen used might only have served to increase the latency of tumorigenesis.