Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea

Dose-response relationships for the induction of mammary tumors by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) were studied. Groups of 30 female Sprague-Dawley rats were given i.p. injections of 50, 37.5, 25, 12.5, or 0 mg MNU/kg body weight at 50 days of age. Animals were palpated for tumor detection twice weekly throughout a 28-week observation period. Administration of MNU i.p. caused no acute toxicity. Both benign and malignant mammary tumors were induced by MNU, but malignant tumors appeared earlier and at a faster rate than benign tumors. The incidence and numbers of mammary carcinomas increased whereas median cancer-free time decreased with increasing dose of MNU. Approximately twice as many mammary cancers were observed in the cervical-thoracic as in the abdominal-inguinal mammary gland chains irrespective of carcinogen dose, while the frequency of tumor occurrence in the left versus right chains was similar. Tumor latency decreased with increasing dose of MNU, but the quartiles for time to detection of all tumors within each carcinogen dose group were similar irrespective of anatomical region in which the tumors occurred. The mammary tumor response attained via i.p. injection was similar but the coefficient of variation for tumor multiplicity within a carcinogen dose group was lower in comparison to that observed when MNU was administered i.v. or s.c. Among these techniques for carcinogen injection, the i.p. route is the most rapid method and offers an added advantage of ease of administration with quantitative, reproducible delivery of the desired amount of carcinogen and a decrease in variability of tumor response among animals within a treatment group. This method is well suited for the technically less experienced investigator and for those in need for a rapid method of injecting MNU to large numbers of animals.     

Effect of D,L-2-difluoromethylornithine and endocrine manipulation on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea

The purpose of this investigation was to establish an efficientroute and dose regime for the long-term administration of tamoxifenin the study of mammary tumorigenesis in the rat. The secondobjective of this work was to determine whether treatment withD, L-2-difluoromethylornithine (DFMO), a synthetic inhibitorof the enzyme ornithine decarboxylase, would reduce the occurrenceof mammary cancers in tamoxi-fen-treated or ovariectomized rats.A total of 265 female Sprague-Dawley rats were assigned to oneof two experimental protocols. All animals were injected with50 mg 1-methyl-1-nitrosourea (MNU) per kg body wt at 50 daysof age. In experiment 1, beginning 7 days after the injectionof the carcinogen, animals were assigned to one of six groupswhich received either 0, 1 or 5 mg tamoxifen citrate per kgAIN-76A purified diet in addition to either no DFMO or a 0.125%w/v solution of DFMO as the drinking water. The experiment wasterminated 180 days following carcinogen treatment. Treatmentwith tamoxifen resulted in a dose-dependent reduction in cancerincidence, and the number of cancers induced and significantlyprolonged the median cancer-free time. This effect was alsoaccompanied by a decrease in the rate of body weight gain. Treatmentwith DFMO delayed latency and reduced tumor number. DFMO inaddition to tamoxifen (1 mg/kg diet) further prolonged latency.In experiment 2 each animal was assigned to one or four treatmentgroups when its first palpable mammary tumor was detected. Atthat time each was either ovariectomized or sham-operated. Inaddition, the rats were either provided no DFMO or a 0.5% w/vsolution of DFMO as the drinking water. The study was terminated35 weeks following carcinogen injection. Ovariec-tomy significantlyinhibited the occurrence of additional mammary tumors. Ovariectomyplus DFMO was more effective than ovariectomy alone in reducingtumor number. Collectively, these observations indicate thatsuppression of polyamine biosynthesis via the systemic administrationof DFMO inhibits the development of ovarian hormone insensitivemammary tumors.     

Influence of perinatal genistein exposure on the development of MNU-induced mammary carcinoma in female Sprague-Dawley rats

Genistein, a phytoestrogen, was subcutaneously (s.c.) injected to pregnant Sprague-Dawley CD rats on gestational days 16-20 at either 25 mg (Group 1) or 5 mg/day (Group 2). Female offspring of mothers not exposed to genistein during pregnancy received 12.5 mg genistein s.c. at neonatal days 15 and 18 (Group 3), or received vehicle only (Group 4). At 35 days of age, 4-9 female offspring from each group were autopsied to observe the influence of genistein, and remainder of female offspring received 50 mg/kg N-methyl-N-nitrosourea (MNU) intraperitoneally and were sacrificed when mammary tumors were larger than 1 cm in size or when they reached 35 weeks of age. Genistein treatment during the perinatal period resulted in lower body weight and lower relative uterine-ovarian weight at 35 days, and a prolonged estrus cycle with a long estrus phase at 12-16 weeks. However, at 35 days (time at MNU administration), mammary gland development, cell proliferation rate (PCNA labeling index), and the number of estrogen receptor (ER)- and progesterone receptor (PgR)-positive cells were similar between genistein-treated and untreated rats. Twenty-five or 5 mg genistein/day in utero (between days 16 and 20 of gestation) or 12.5 mg genistein/day on neonatal days 15 and 18 did not affect the incidence of mammary tumors > 1 cm or the latency but did increase the number of mammary cancer lesions when MNU was administered at the time when the mammary gland growth in genistein-treated and untreated rats was similar. Thus, perinatal genistein is an endocrine disrupter and increases the multiplicity of MNU-induced mammary carcinoma in rats.     

Estradiol and progesterone can prevent rat mammary cancer when administered concomitantly with carcinogen but do not modify surviving tumor histology,estrogen receptor alpha status or Ha-ras mutation frequency

An early full-term pregnancy is protective against mammary cancer in both humans and rodents. Treating rats with two hormones of pregnancy, estradiol and progesterone, for 5 weeks renders the rat mammary glands refractory to carcinogenesis. Our objectives was to determine if a shortened regimen (3 weeks) would be as effective as the 5-week regimen and to determine if the mammary gland was vulnerable to carcinogenic insult during the hormone treatments. We also examined cancers that survived the chemopreventive regimen to see if those tumors were particularly aggressive compared to control tumors (i.e., less differentiated, estrogen receptor alpha (ER alpha)-negative or harbored mutations in Ha-ras). In the first experiment, Lewis rats were injected with N-methyl-N-nitrosourea (MNU, 50 mg/kg) at 50 days of age. At 60 days of age, the rats were either mated and allowed to nurse their young for 3 weeks, treated with hormone vehicle for 5 weeks, or 17 beta-estradiol (E, 20 micrograms) and progesterone (P, 4 mg) 5 times per week for 3 or 5 weeks. All the rats exposed to MNU but not estradiol and progesterone developed multiple mammary cancers. Pregnancy reduced multiplicity to 0.40 cancers/rat. Treatments of estradiol and progesterone for 3 or 5 weeks reduced cancer multiplicity and increased latency to a similar degree as pregnancy. Mammary cancers from each group displayed a similar spectra of histologic class, estrogen receptor alpha (ER alpha) content and Ha-ras mutation status. In the second experiment, 50-day-old rats were treated for five weeks with either estradiol and progesterone or vehicle as above beginning at 60 days of age and treated with MNU at 50, 64, 78 or 92 days of age. In each case, estradiol and progesterone treatments resulted in significantly reduced mammary tumor frequency. These results demonstrate that a three-week regimen of estradiol and progesterone can protect the mammary gland from chemically-induced carcinogenesis even when carcinogen exposure occurs concomitant with estradiol and progesterone stimulation.     

Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats.

The Copenhagen rat is completely resistant to mammary cancer induction by N-methyl-N-nitrosourea (MNU) when the carcinogen is administered during sexual development, a period when other strains of rats are normally susceptible to mammary gland carcinogenesis. Here we administered 30 mg/kg MNU i.p. to two groups of neonatal (2-3-day-old) Copenhagen rats. One group (group B, 18 animals) received no further treatment, while the other group (group C, 17 animals) received a second dose of 30 mg/kg MNU via the tail vein at 50 days of age. About 30% of the rats in group B and about 70% of those in group C developed mammary carcinomas before they were 1 year of age. About one-half of the tumors in both groups were cribriform adenocarcinomas and one-half were adenosquamous carcinomas. The latter tumor type has not been observed previously in susceptible rat strains. The ability to induce these mammary tumors in the Copenhagen rat suggests that the putative mammary carcinoma suppressor gene is functionally inactive in neonatal animals or is inactivated when these animals are treated with MNU.     

Loss of chromosomal integrity drives rat mammary tumorigenesis

Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of carcinogens on cell growth control pathways are poorly understood. Here, we have examined processes that are activated in the mammary glands of rats treated with 1-methyl-1-nitrosourea (MNU). This synthetic carcinogen was used to study events occurring during mammary tumor initiation and development. In female Wistar-Furth rats, given 1 dose of MNU beginning at 50 days of age, 84% of the rats developed tumors by 46 weeks of age (latency 13-15 weeks). Changes in the gland occurred as early as 1-day post-MNU. Cells exhibited DNA damage, leading to chromosomal instability, supernumerary centrosomes and higher levels of Aurora A; these events correlated with the appearance of preneoplasia in the glands. In mammary tumors, elevated numbers of centrosomes coincided with genomic instability. Tumors were transplanted into syngeneic hosts and subsequent tumor generations displayed the same marker chromosomes in mostly aneuploid metaphases with hyperdiploid numbers of chromosomes, suggesting that clonality and aneuploidy were passed on from one generation to the next. Collectively, these data suggest that the carcinogen MNU induces changes resulting in genetic instability detectable before hyperplasia and tumors develop in the rat mammary gland.     

Influence of caffeine on development of benign and carcinomatous mammary gland tumors in female rats treated with the carcinogens 7,12-dimethylbenz(a)anthracene and N-methyl-N-nitrosourea

The effect of chronic caffeine consumption (500 mg/liter of drinking water) on the initiation and promotion stages of 7,12-dimethylbenz(a)anthracene (DMBA) (a low dose, 0.5 mg/100 g body weight, i.v.) and N-methyl-N-nitrosourea (MNU) (a standard dose, 2.5 mg/100 g body weight, i.v.) induced mammary gland tumorigenesis in female Sprague-Dawley rats was determined. In the initiation studies, caffeine was administered for 30 days prior to and for 3-4 days after carcinogen treatment (carcinogens administered at 55-57 days of age); in the promotion studies, caffeine was administered beginning 3-4 days after carcinogen treatment and until experiment termination (DMBA study and MNU study, 48 and 26 weeks after carcinogen treatment, respectively). In the DMBA study, there were 62-73 rats/group, in the MNU study, 40 rats/group. Eighty-nine % of the mammary tumors induced by DMBA were benign (adenomas, fibroadenomas, often with cystic secretory activity), 11% were carcinomas (intraductal and invasive); virtually all of the MNU-induced mammary tumors were carcinomas (approximately 99%). Caffeine consumption during the initiation stage in the DMBA-treated rats resulted in a significant decrease in the mean number of mammary carcinomas per rat (50% reduction, P less than 0.01) and mean number of benign mammary tumors per rat (28% reduction, P less than 0.05); caffeine consumption during the promotion stage significantly decreased the mean number of benign mammary tumors per rat (57% reduction, P less than 0.001) while not significantly influencing mammary carcinoma number. In contrast, caffeine consumption during either the initiation or promotion stages of MNU-treated rats did not significantly influence this tumorigenic process. The influence of caffeine on urinary and fecal excretion of tritiated DMBA and on rat mammary gland development at the time of carcinogen treatment also was determined. Slightly reduced levels of tritium in 24-h urinary samples were observed in caffeine-treated animals (P = 0.06). No significant effect of caffeine on 24- to 96-h fecal or 48- to 96-h urinary excretion of the isotope was observed. No apparent effect of caffeine on rat mammary gland development (number of ducts, degree of lobuloalveolar development) was observed. That caffeine significantly suppresses the initiation stage of DMBA-induced rat mammary gland tumorigenesis, while not influencing this stage when MNU is used as a carcinogen, suggests that caffeine acts via an alteration in carcinogen (DMBA) activation. The lack of a pronounced effect of caffeine on tritiated DMBA excretion, however, does cast some doubt on this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immuno-neutralization of circulating relaxin does not alter the breast cancer-protective action of parity in MNU-treated rats

Early pregnancy and childbirth protects women against future development of breast cancer by an unknown mechanism. Parity likewise reduces mammary cancer incidence in rats exposed to the carcinogen, N-methyl-N-nitrosourea (MNU), providing a model for the human phenomenon. We hypothesized that relaxin, a 6KD luteal mammotropic hormone of pregnancy, might be the anti-cancer pregnancy factor, and that induced relaxin deficiency during rat gestation would restore carcinogen sensitivity. Forty-one pregnant (age 50 days) and 25 age-matched virgin Sprague-Dawley rats were used. Relaxin deficiency was induced by injecting mouse monoclonal anti-rat relaxin antibody (MCA1) days 12-18 of gestation. Pregnant controls were injected with vehicle or mouse IgG on the same schedule. Because MCA1 disrupts parturition, all rats underwent cesarean section on day 22. At age 100 days, all rats were injected i.v. with MNU (50mg/Kg) and examined daily for tumors until euthanized at age 240 days.Mammary tumor incidence and frequency were significantly (p<0.01) reduced and tumor latency was increased (p<0.001) in primiparous as compared with virgin rats. However, tumor incidence, type, size and latency were similar in MCA1-treated and control primiparous rats. Thus, luteal relaxin does not appear to be the factor responsible for resistance to breast cancer.     

Temporal sequence of mammary intraductal proliferations, ductal carcinomas in situ and adenocarcinomas induced by 1-methyl-1- nitrosourea in rats

An experimental model for mammary carcinogenesis has been described in which intraductal proliferations, ductal carcinomas in situ and adenocarcinomas can be readily detected and the frequency of their occurrence quantified. The objective of the experiment reported in this study was to determine the latency period between carcinogen administration and the occurrence of each of these types of lesion. A total of 150 female Sprague-Dawley rats were injected i.p. with 50 mg 1- methyl-1-nitrosourea (MNU)/kg body wt at 21 days of age. Groups of 30 rats each were killed at 7, 14, 21, 28 and 35 days post-carcinogen. Mammary intraductal proliferations were the first detected lesions and were observed in 20% of the animals at 14 days following carcinogen administration. At 21 days post-carcinogen ductal carcinomas in situ and adenocarcinomas were observed. The number of each type of lesion increased with time post-carcinogen, but the temporal pattern of occurrence was different among lesion types. The pattern of lesion occurrence was consistent with intraductal proliferations being a precursor lesion for ductal carcinomas in situ and adenocarcinomas. Furthermore, the data imply that ductal carcinomas in situ represent one pathway of morphological progression by which intraductal proliferations evolve into invasive carcinomas, but that this lesion type, as currently defined histologically, may not be an obligatory intermediate in morphologic progression. These findings are consistent with emerging evidence of multiple but distinct pathogenetic pathways leading to mammary carcinomas that display different morphological patterns and biological activities.      

Dietary effects of conjugated docosahexaenoic acid on N-methyl-N-nitrosourea-induced mammary carcinogenesis in female Sprague-Dawley rats

The dietary effects of conjugated docosahexaenoic acid (CDHA) were examined in an N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis model. Female Sprague-Dawley rats were administered 50 mg/kg MNU intraperitoneally at 49 days of age. A powdered AIN-76A diet containing 0, 0.2 or 1.0% CDHA was fed to the rats from 21 to 49 days of age (before MNU; pre-initiation phase) or from 49 days to 40 weeks of age (after MNU; post-initiation phase). Rats were sacrificed when their largest mammary tumor was > or =1 cm in size or when they reached 40 weeks of age. All histologically detected mammary carcinomas were evaluated. In rats that received CDHA after MNU, development of mammary carcinoma > or =1 cm was inhibited, and there was a significant decrease in the final mammary cancer incidence and multiplicity, compared with rats that did not receive CDHA. Consumption of the 0.2% CDHA diet after MNU significantly prolonged latency. Suppression of mammary cancer yield by consumption of a CDHA diet after MNU administration was not dose-dependent. In rats that received CDHA before MNU, suppression of mammary cancer was not observed. These results indicate that CDHA administration in the post-initiation period suppressed mammary carcinogenesis, whereas CDHA administration in the pre-initiation period was ineffective.     

Exercise during adolescence and its effects on mammary gland development,proliferation, and nitrosomethylurea (NMU) induced tumorigenesis in rats

The purpose of this investigation was to determine the effects of moderate intensity exercise training during peripuberty on nitrosomethylurea (NMU) induced mammary tumorigenesis and to assess the effects of this activity on mammary epithelial cell proliferation and mammary gland development in rats. Female Sprague Dawley rats were divided into two groups, sedentary and exercised. The rats were exercised five times per week from 21 to 50 days of age on a progressive treadmill training program with a final workload of 18 m/min at 15% incline for 60 min a day. At fifty days of age eight rats from each group were sacrificed to determine the effects of exercise on mammary gland labelling index and development scores. The remaining animals were given NMU at a concentration of 50 mg/kg body weight. The experiment was terminated 24 weeks post carcinogen administration, and cancer incidence, multiplicity, and latency were analyzed. The total tumor number per group was reduced by exercise (58 vs. 33 carcinomas p < 0.05). This corresponded to a significantly higher number of tumors/animal in the sedentary rats compared to those that were exercised prior to NMU administration. However latency period (124.9 ± 4.3 vs. 125.2 ± 6.4) and final incidence (68.9% vs. 61.5%) were not significantly different in sedentary vs. exercised rats. There were no significant differences in mammary gland developmental scores (4.1 ± 0.24 vs. 4.4 ± 0.26) or labelling index of mammary epithelial cells in either ducts or lobuloalveolar units (ductal 41.5 ± 4.7 vs. 39.4 ± 5.7; lobuloalveolar 38.5 ± 4.1 vs. 47.7 ± 5.7) between the two groups. The results of this study suggest that although exercise prior to carcinogen administration has an effect on mammary tumorigenesis, these effects do not appear to be related to exercise-induced changes in mammary gland development.     

Effect of concentration of D,L-2-difluoromethylornithine on murine mammary carcinogenesis

The appearance of chemically induced mammary gland carcinomas in virgin female Sprague-Dawley rats was blocked by the administration of D,L-2-difluoromethylornithine (DFMO) in drinking water during the stage of tumor promotion. Rats were given injections s.c. at 50 days of age with either 35 mg of 1-methyl-1-nitrosourea (MNU) per kg of body weight or the 0.9% NaCl solution in which the carcinogen was dissolved. At 57 days of age, the rats were each randomly allocated to one of 14 treatment groups. Ten groups (five solvent treated and five MNU treated) were assigned to treatments consisting of 0.00, 0.0625, 0.125, 0.25, or 0.50% (w/v) solution of DFMO in their drinking water; two MNU-treated groups were placed on or removed from DFMO treatment (0.5%; w/v) at 90 days post-carcinogen exposure; and two carcinogen-treated groups received either putrescine (0.5-g/kg diet) or putrescine and DFMO (0.5%; w/v) throughout the experiment. The study was terminated 183 days after carcinogen treatment. All doses of DFMO exerted a protective effect against the induction of mammary cancer; however, only the feeding of the 0.125% and the 0.5% solutions of DFMO resulted in a significant reduction in cancer incidence. The average number of cancers per rat was reduced, and cancer-free time was extended at all concentrations of DFMO. The protective effect of DFMO was sustained following withdrawal of treatment at 90 days post-MNU injection. Feeding putrescine in conjunction with DFMO treatment partially blocked the inhibitory activity of DFMO. DFMO treatment did not affect food or water intake; body weight gain; the weight of ovaries, uterus, adrenal glands, liver, kidney, or spleen; or the periodicity of the estrous cycle. These data provide evidence of an inhibitory effect of DFMO against mammary cancer induced by MNU which cannot be attributed to a systemic toxic effect of this compound.     

Chemoprevention of MNU-induced mammary tumors in the mature rat by 4-HPR and tamoxifen.

The chemopreventive efficacies of the retinoid all-trans-N-(4-hydroxyphenyl)-retinamide (4-HPR) and the anti-estrogen tamoxifen citrate were evaluated against N-methyl-N'-nitrosourea (MNU) induced mammary cancer in 120-day old female Sprague-Dawley rats. The agents were tested alone and in combination. They were administered in a modified AIN-76A diet, beginning 60 days prior to a single i.v. dose of 50 mg MNU/kg-bw and continuing until the end of the study, 180 days post-carcinogen treatment. At 782 mg/kg diet, 4-HPR alone significantly inhibited the induction of mammary adenocarcinomas compared with carcinogen controls. At 0.250 mg/kg diet, tamoxifen alone reduced tumor incidence compared with carcinogen controls. At 0.125 mg/kg diet, tamoxifen was ineffective. Combinations of 782 mg 4-HPR/kg diet with either 0.250 or 0.125 mg tamoxifen/kg diet were effective in inhibiting MNU-induced adenocarcinomas. The reductions in tumor incidence were greater for these combinations than for either agent alone. 4-HPR and 0.250 mg tamoxifen/kg diet decreased tumor incidence 81% (p less than 0.005), whereas 4-HPR and 0.125 mg tamoxifen/kg diet decreased tumor incidence 72% (p less than 0.005) compared with carcinogen controls. The combination of 391 mg 4-HPR/kg diet and 0.500 mg tamoxifen/kg diet was also tested and was effective in reducing tumor incidence.     

Fasting during promotion, but not during initiation, enhances the growth of methylnitrosourea-induced mammary tumours

The purpose of this work was to investigate the effect of fasting on the induction and growth of chemically-induced mammary carcinogenesis. Female Sprague-Dawley rats were given methylnitrosourea (MNU) i.p. (50 mg/kg) at 50 days of age; a group of rats were exposed to 4 day fasting followed by 1 day of refeeding before the administration of the carcinogen, while another group was exposed to three cycles of 3 days fasting in 10 days, beginning 1 week after MNU injection. Fasting enhanced the development of mammary tumours only in rats fasted after carcinogen damage, while it did not affect the induction of tumours in rats fasted before MNU, if compared with full-fed controls. The enhanced growth of mammary tumours sustained by fasting during promotion was observed in the cervical-thoracic region. In addition, exposure to fasting made rats susceptible to the development of MNU- induced extra-mammary cancers. Different from the preventive effect of caloric restriction on tumor development, these data demonstrate that fasting affects the promotion phase of carcinogenesis by enhancing the growth of MNU-induced mammary tumours.      

Moderate exercise training slows mammary tumour growth in adolescent rats.

Adolescence and young adulthood may be critical windows in establishing risk for breast cancer development in humans. Epidemiological data suggest that exercise during this life stage is associated with decreased breast cancer risk yet few experimental studies to elucidate the mechanism have been performed. The purpose of these studies was to evaluate the effects of moderate exercise training on mammary tumour development in adolescent rats using the 1-methyl 1-nitrosourea (MNU) chemical carcinogen model. Exercise (EX) consisted of moderate-intensity treadmill running 30 min/day, 5 days a week. A total of 274 animals were used: 94 in study 1 and 180 in study 2. Animals were injected with MNU (50 and 25 mg/kg body weight in studies 1 and 2, respectively) at 21 days of age and began training at 28 days of age. Groups of animals (n=10-30 depending on the study and time point) were sacrificed every 2 weeks for 8 weeks to evaluate tumour development. No difference in median tumour-free survival time was observed in the EX versus sham-exercise (SHAM), nor were there any differences in multiplicity at either a high or moderate dose of MNU. Latency to first tumour palpated was increased in both studies by 3-4 days. Consistent across both studies, tumour weights were less and the growth rates of the tumours, defined as tumour weight divided by the number of days elapsed since the tumour was first palpated, were reduced in the EX group. The data suggest that latency is increased and tumour growth is retarded in response to moderate exercise training.     

DNA methyl-adduct dosimetry and O6-alkylguanine-DNA alkyl transferase activity determinations in rat mammary carcinogenesis by procarbazine and N-methylnitrosourea

The metabolism of the carcinogenic antitumor drug procarbazine (PCZ) is complex with the ultimate production, among other metabolites, of a methyldiazonium ion which is also the ultimate carcinogenic species of the DNA-methylating N-nitroso compounds including N-methylnitrosourea (MNU). This suggests a similar mechanism of carcinogenic action. Following a single oral dose of [14C]PCZ (50 mg/rat) to 50 day old female Sprague-Dawley rats under the reported conditions of mammary gland carcinogenicity, the DNA adducts 7-methylguanine (7-meG) and O6-methylguanine (O6-meG) were determined in target (mammary gland) and non-target organs. The degree of DNA methylation was similar in all the organs considered. In the mammary gland, lung, spleen, small intestine and stomach the O6-meG/7-meG ratio was close to 0.11. At a lower dose of PCZ (26 mg/rat), the levels of 7-meG in the tissues were 40-60% of those produced by the higher dose. Eighty percent of the rats given the higher dose versus 37% of those given the lower dose developed mammary tumors after 20 weeks. With the higher dose of MNU (50 mg/kg body wt) DNA methylation was more or less uniform in all the organs including the mammary gland, with slightly greater yields in the liver. At a lower MNU dose (25 mg/kg) the levels of 7-meG were 40-48% of those produced by the higher dose. Fifty seven percent of the rats given the higher dose versus 21% of the animals given the lower dose developed mammary gland tumors after 20 weeks. On a mol/kg body wt basis, PCZ was approximately 5-times less active than MNU in the production of 7-meG in mammary gland but only approximately 2-times less active than MNU in the production of mammary gland tumors. The O6-alkylguanine-DNA alkyltransferase (AGT) levels in the liver, kidney, spleen and lung of PCZ or MNU treated rats were approximately 9-28% (expressed relative to protein content) and 10-33% (expressed relative to homogenate DNA content) of those in the corresponding organs of the saline-treated controls. However, the AGT levels of the mammary gland and brain were in the range of 45-61% (expressed relative to protein content) and 39-54% (expressed relative to homogenate DNA content) of those of the saline-treated controls. Also the mammary gland of the 50 day old female rats has the lowest AGT activity (expressed relative to DNA content).(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of Mammary Tumors by Pretreatment with 17β-Estradiol in F344 Rats Induced with N-Methyl-N-nitrosourea

The influence of 17β-estradiol (E2) and prolactin was studied on N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas (MCAs) in rats. MNU was intravenously injected once into seven-week-old female F344 rats at a dose of 50 mg/kg body weight. Groups of rats also received either 2.5 mg of E2 or a continuous supply of prolactin and/or growth hormone via transplanted MtT/F84 (mammo-somatotropic pituitary tumor). Rats were observed for up to 36 weeks after MNU administration. Although simultaneous administration of MNU and E2 did not much affect the occurrence of MCAs as compared to administration of MNU alone, rats treated with 2.5 mg of E2 for two weeks before MNU administration had significantly reduced occurrence of MCAs compared to those given MNU alone. In contrast, rats with MNU plus MtT/F84 showed high incidence and shortened latency of MCAs and they also had a high incidence of clitorial gland hyperplasias. Average pituitary weights and serum prolactin levels in E2-treated rats were greatly increased compared to those of MNU-alone rats. Average serum E2 levels were about 100 ng/ml in E2-treated rats and 0.05 ng/ml in rats without E2 treatment. Serum prolactin levels were greatly increased in rats with MtT/F84. The results indicated that pretreatment with E2 before MNU administration was inhibitory while increased prolactin caused by grafting MtT/F84 after MNU injection was promotive for the occurrence of MCAs in female F344 rats.     

Prevention of methylnitrosourea-induced mammary cancers by 9-cis-retinoic acid and/or vitamin D3

Two cancer chemopreventive agents, vitamin D3 and 9-cis-retinoic acid (9-cis-RA), were evaluated alone and in combination in the methylnitrosourea (MNU)-induced mammary cancer model. In this study, female Sprague-Dawley rats received MNU (50 mg/kg BW) at 50 days of age. Vitamin D3 and 9-cis-RA were administered in the diet beginning three days later. The groups were: Group 1, vehicle only; Group 2, 9-cis-RA (60 mg/kg diet); Group 3, vitamin D3 (10 microg/kg diet); Group 4, vitamin D3 (3.3 microg/kg diet); Group 5, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (10 microg/kg diet); and Group 6, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (3.3 microg/kg diet). Animals were observed daily for signs of toxicity and were palpated 2x/week for mammary tumors. The study was terminated 150 days after treatment with MNU. The average number of mammary cancers was 6.7 in the animals receiving only the carcinogen. 9-cis-RA alone caused a 23% decrease in mammary cancer multiplicity, while vitamin D3 alone actually caused slight increases of 17 and 16% at 10 and 3.3 microg/kg diet dose levels, respectively. When the agents were given in combination, however, the 9-cis-RA plus the high dose of vitamin D caused a statistically significant decrease (44%) in mammary cancer number, while the 9-cis-RA plus the low dose resulted in a 37% decrease. Thus, low doses of these agents that were not effective in preventing mammary cancer when given alone appeared to be active when given in combinations. Possible interactions between the retinoic acid receptors and vitamin D receptor may be responsible for the observed inhibition of mammary carcinogenesis.     

Mammary tumorigenesis in growth hormone deficient spontaneous dwarf rats; effects of hormonal treatments

This study was carried out to investigate mammary tumorigenesis in growth hormone (GH) deficient spontaneous dwarf rats (SDR). At 50–60 days of age, the rats were divided into five groups. Group 1 received bovine (b) GH (prolonged release formulation) administered at a dose of 40–50 mg/kg body wt. in 50 l weekly injections; group 2 received recombinant human insulin-like growth factor-I (IGF-I) at a dose of 1 mg/kg body wt./day administered via osmotic pumps; animals in group 3 were fitted with subcutaneous silastic capsule containing 30 g 17-estradiol (E2) plus 30 mg progesterone (P4), replaced every 2 months; group 4 received both bGH and E2 plus P4 treatments at the same doses as above, and control animals (group 5) received sham treatments (vegetable oil injection, silastic capsules containing cellulose). After 1week of treatment, all animals were injected intraperitoneally with the carcinogen N-methyl-N-nitrosourea (MNU) at a dose of 50 mg/kg body wt. Other groups of animals, receiving identical hormonal treatment to those exposed to MNU, were treated for 10 days only and then sacrificed for assessment of circulating concentrations of hormones and mammary gland characteristics at the time of carcinogen exposure. The hormonal treatments of the animals exposed to the MNU were continued for an additional 20 weeks and mammary tumor development monitored by weekly palpation and tumors collected as necessary. The rats were weighed weekly. At the end of the treatment period, all animals were sacrificed and remaining tumors were collected. Rats in all groups continued to gain weight throughout the experimental period, but the largest weight gain was see in animals receiving GH either alone or with E2 and P4. Animals treated with IGF-I also gained weight compared to controls, but this weight gain was less than that seen in GH-treated rats. GH treatment alone increased mammary tumor incidence from 4.8% in controls to 100%. Average tumor load and latency in the GH-treated rats were 7.0 ± 0.8 tumors/tumor-bearing rat (mean±SEM) and 57.3 ± 2.7 days (mean±SEM), respectively. As in intact Sprague–Dawley rats, approximately 90% of the tumors that developed in the GH-treated rats were ovarian dependent for growth. IGF-I treatment also increased mammary tumor development to 62.5%. Average tumor load and latency in the IGF-I-treated rats were 1.6 ± 0.4 tumors/tumor-bearing rat (mean±SEM) and 96.2 ± 14.5 days (mean±SEM), respectively. However E2+P4 treatments did not significantly alter tumorigenesis and, surprisingly, simultaneous treatment with E2+P4 and GH obliterated the GH-stimulated increase in tumor development. Prolactin (PRL) did not appear to influence mammary tumorigenesis in the SDRs, as untreated SDRs had significantly elevated serum concentration of PRL as compared with normal Sprague–Dawley (SD) rats, whereas GH-treated SDRs had PRL levels similar to that of normal SD rats. No obvious structural characteristics were associated with high or low susceptibility to mammary tumorigenesis, as assessed by mammary gland whole mounts from the different animal groups sacrificed at the time of carcinogen administration.Enhanced expression of the extracellular signal-regulated kinase 1/2 (ERK1/2), and activation (phosphorylation) of ERK1/2 were associated with an increase in mammary tumorigenesis. Similarly, the expression of the estrogen receptor- (ER) was significantly elevated in animal groups with the highest susceptibility to tumorigenesis, whereas the levels of cyclin D1 expression were not related to mammary tumorigenesis.     

Modulation of DNA damage by vitamin A in developing Sprague-Dawley rats.

Prenatal and neonatal Sprague-Dawley rats were given a diet deficient in or with an excess of Vitamin A and at the age of 55 days female progeny were treated with a single i.g. dose of 80 mg/kg DMBA or 50 mg/kg MNU. Under these experimental conditions it was found that the exposure of perinatal rats to a diet containing an excess of Vitamin A caused a decrease in the amount of DMBA- and MNU-induced DNA damage in the mammary gland and the liver of the female offspring. When diets were deficient in Vitamin A there was a dual effect in terms of DNA damage detected in the same organs, namely DMBA caused an amount of DNA damage comparable to controls, while the extent of DNA damage induced by MNU greatly increased in both organs. These results indicate that Vitamin A can permanently change the sensitivity of adult progeny to chemically induced DNA damage when it is given to pregnant and lactating females.