Chemopreventive effects of the aromatase inhibitors vorozole (R-83842) and 4-hydroxyandrostenedione in the methylnitrosourea (MNU)-induced mammary tumor model in Sprague-Dawley rats

The chemopreventive activity of the aromatase inhibitors vorozoleand 4-hydroxyandrostenedione were determined in the methylnitrosourea(MNU)-induced model of rat mammary tumorigenesis. Vorozole (5and 2.5 mg/kg body wt) and 4-hydroxyandrostenedione (15 and6 mg/rat) were administered daily (by gavage) to virgin femaleSprague-Dawley rats starting at an age of 43 days. Seven dayslater animals were given a single dose of MNU. Following treatmentwith MNU, animals continued to be treated with vorozole and4-hydroxyandrostenedione daily until the end of the experiment(100 days post MNU treatment). Vorozole at either dose provedto be a profound inhibitor of MNU-Induced mammary tumors. Vorozoledecreased tumor incidence from 100% to 10%, while simultaneouslydecreasing tumor multiplicity from 5 tumors per annual to 0.1tumors per animal. This chemopreventive effect was accompaniedby significant increases In body weight gain in the animalstreated with vorozole when compared with control rats. In contrast,neither dose of 4-hydroxyandrostenedione had any effect on tumorincidence and only the higher dose slightly decreased tumormultiplicity.     

Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone.

The chemopreventive efficacy of p.o. administered dehydroepiandrosterone (DHEA), DHEA plus N-(4-hydroxyphenyl)retinamide (4-HPR), or 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354) was examined in rats treated with N-methyl-N-nitrosourea (MNU; 50 mg/kg body weight, i.v.) at 50 days of age. Semipurified diet (AIN-76A) containing each steroid alone, or DHEA plus 4-HPR, was administered during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or both phases (-1 week post-MNU to termination) of the carcinogenic process. Neither DHEA nor DHEA analogue 8354 (0.2%, w/w) significantly affected the initiation of mammary cancer when administered alone; however, DHEA (0.2%, w/w) plus 4-HPR (1 mmol/kg diet) significantly reduced cancer multiplicity (26%) when given during initiation. All three treatments were strongly effective when given during promotion/progression, significantly reducing mammary cancer multiplicity by 77% (DHEA), 84% (DHEA/4-HPR), and 66% (DHEA analogue 8354), relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA (33% inhibition) and DHEA/4-HPR (24% reduction) during promotion/progression. However, the most effective chemopreventive treatment encompassed both phases of carcinogenesis. Thus, under these conditions, DHEA (0.2% or 0.1%, w/w) reduced cancer incidence (52% and 32% reductions, respectively) and multiplicity (91% and 86% reductions, respectively). Further reduction in mammary cancer incidence was observed in animals that received DHEA (both doses) plus 4-HPR (1 and 0.5 mmol/kg diet, respectively). DHEA analogue 8354 (0.2% or 0.1%, w/w) given for the duration of the study reduced only cancer multiplicity (61% and 56% reductions, respectively). Tumor-related mortality was significantly lower in rats that received long-term treatment with DHEA or DHEA/4-HPR, when compared with carcinogen controls. Except for a slight, but significant, postcarcinogen decrease in the mean body weights of rats treated concomitantly with DHEA (plus or minus 4-HPR) and MNU, additional gross manifestations of steroid-induced toxicity were not observed.     

Effects of combined and sequential treatment with tamoxifen and the aromatase inhibitor vorozole on 7,12-dimethylbenz(a) anthracene-induced mammary carcinoma in the rat

 The aromatase inhibitor vorozole dose-dependently inhibited the growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. An oral dose of 5 mg/kg per day brought about growth inhibition and reduction of tumor multiplicity similar to that produced by ovariectomy. Tamoxifen (8 mg/kg per day) also reduced tumor growth, albeit to a lesser extent than did ovariectomy. Concomitant administration of varying doses of tamoxifen with the fully effective dose of vorozole (5 mg/kg per day) tended to be less effective than ovariectomy or vorozole alone. This is likely to be due to the estrogen-agonistic effects of tamoxifen. Combination of tamoxifen with the partially effective dose of vorozole (1 mg/kg per day) gave results comparable with those obtained for either of these compounds used in monotherapy. Combining tamoxifen with a marginally active low dose of vorozole (0.2 mg/kg per day) resulted in a minor additional growth inhibition as compared with that obtained with this dose of vorozole alone. Sequential treatment with tamoxifen (8 mg/kg per day) for 42 days and vorozole (5 mg/kg per day) for 42 days, and vice-versa, was performed with a drug-free interval of 14 days between treatments. Tumors regressing under vorozole therapy relapsed when subsequently treated with tamoxifen. In contrast, vorozole further reduced tumor volumes in rats previously treated with tamoxifen. Furthermore, monotherapy with tamoxifen as well as the two sequential tamoxifen-vorozole treatment schedules were in most cases less effective than vorozole monotherapy in inhibiting both tumor growth and tumor multiplicity. Although extrapolation of these findings in cycling animals to the clinical situation, involving postmenopausal women, is not straightforward, these results warrant further studies in preclinical models. Moreover, clinical trials comparing the most effective aromatase inhibitors with tamoxifen in previously untreated postmenopausal patients with breast cancer may also be warranted. Received: 7 April 1985/Accepted: 4 August 1985     

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.     

Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.     

Antitumoral and endocrine effects of (+)-vorozole in rats bearing dimethylbenzanthracene-induced mammary tumors.

The antitumoral activity of vorozole, a potent and specific nonsteroidal aromatase inhibitor, against 7,12-dimethylbenz(a)anthracene-induced estrogen-dependent mammary adenocarcinoma was evaluated in 257 Sprague-Dawley rats. Twice daily p.o. administration of 1 and 5 mg/kg of the racemate R 76713 for 42 days induced almost complete regression of tumors, inhibited the appearance of new tumors, and reduced multiplicity of the remaining tumors. Antitumoral effects observed after ovariectomy or treatment with 5 mg/kg twice a day were not significantly different. R 76713, the racemate, (+)-vorozole (both at 2.5 mg/kg twice a day), and ovariectomy all similarly reduced tumor growth at 42 days by 90% or more, lowered the number of existing tumors, and prevented the appearance of new tumors. The less active levo-enantiomer (-)-vorozole at the same dose did not alter tumor growth. Vorozole reduced serum estradiol to the levels measured in ovariectomized animals. Serum progesterone levels were lowered, but to a much lesser extent than after ovariectomy, while serum luteinizing hormone and follicle-stimulating hormone concentrations increased, but also much less than after ovariectomy. On the other hand, the androgen levels, which remained undetectable or decreased after ovariectomy, markedly rose after vorozole treatment. These endocrine changes, observed in intact female rats, were not detected in ovariectomized animals demonstrating the ovarian origin of the endocrine changes induced by vorozole.     

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.     

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.     

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.     

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.     

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.     

Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by N-methyl-N-nitrosourea

The activity of the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), as a cancer chemopreventive agent in the rat mammary gland was determined. Beginning 1 week after a single i.v. dose of 20 or 50 mg N-methyl-N-nitrosourea (MNU)/kg body wt., female Sprague-Dawley rats were fed a semi-purified diet supplemented with 0 or 1000 mg NDGA/kg diet. At both MNU dose levels, groups receiving NDGA developed significantly fewer mammary cancers than did dietary controls; NDGA induced no gross or organ-specific toxicity. These data implicate lipoxygenase products in the process of mammary cancer induction, and suggest an additional enzymatic target for the design of chemopreventive drugs.     

The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate

Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2-20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus 'deprotect' rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.     

Anticarcinogenic activity of quinacrine in the rat mammary gland

Mammary carcinogenesis studies were conducted to determine thechemopreventive activity of quinacrine, an antimalarial drugwhich suppresses the production of arachidonic acid from phospholipidthrough inhibition of phospholipase A₂. Beginning 1 week aftera single i.v. dose of N-methyl-N-nitrosourea (MNU), female Sprague—Dawleyrats were fed a semi-purified diet supplemented with 0 or 75mg quinacrine/kg diet. Quinacrine reduced cancer incidence andcarcinoma multiplicity in rats administered 20 mg MNU/kg bodywt, but had no inhibitory activity in rats treated with 50 mgMNU/kg. These data suggest that pathways of arachldonic acidmeta-bolism in addition to cyclooxygenase present useful targetsfor mammary cancer chemoprevention. However, the chemo-preventiveactivity of quinacrine may be limited by toxicity.     

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.     

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)     

A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer

In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet.     

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.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Carcinogenesis by Soy Foods or Biochanin A

We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N -nitroso- N -methylurea (MNU)-induced rat mammary carcinogenesis. Seven-week-old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy-supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) ( P <0.05, respectively). In the biochanin A-supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) ( P <0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) ( P <0.01 and P <0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A-supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.     

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.