Single Dose Of Methylnitrosourea Research Articles

Abstract 5255: Comparison of high-fructose and standard diets in methylnitrosourea (MNU)-induced ER+ mammary cancers: Altered carcinogenesis, differential effects of metformin, and metabolomic differences

Abstract The MNU-induced model of ER+ mammary cancers in Sprague-Dawley rats has routinely been employed in screening for chemopreventive agents. We recently reported that metformin was ineffective in preventing mammary cancers in rats on a standard diet (Thompson, et al, Cancer Prev. Res, 8, 231-9, 2015). In this study, we evaluated the carcinogenic response in Vehicle-treated rats and rats receiving Metformin (150 mg/Kg BW/day) on standard Teklad or isocaloric High-Fructose diets. In the first study, rats were placed on Teklad or High-Fructose diet at 43 days of age. At 100 days of age, animals were administered a single dose of MNU. Animals were administered Metformin or tamoxifen beginning 5 days later. Rats were palpated weekly for mammary tumors and sacrificed at 300 days of age. Body weights between groups were not different. Tumor multiplicity and tumor weights were: Vehicle-Teklad,1.15 and 1.44g; Vehicle- High-Fructose, 2.10 and 2.94g; Metformin-Teklad, 1.25 and 1.91g; Metformin -High-Fructose, 1.00 and1.99g. Tamoxifen resulted in the prevention of all tumors. High-Fructose alone resulted in a 2X increase in tumor multiplicity (P<0.05) and tumor weights (P<0.05). In the Teklad diet, Metformin marginally increased tumor multiplicity and weights (0.1>P<0.05). In the High-Fructose diet, Metformin decreased tumor multiplicity by 50% (P>0.05). In the second study, which parallels the standard MNU model, rats were also placed on Teklad or High-Fructose diet at 43 days of age. At 50 days of age, rats were administered a single dose of MNU. Rats on High-Fructose diet were administered either Vehicle or Metformin, while rats on Teklad diet were given only Vehicle; beginning 5 days later. Rats were palpated weekly and sacrificed at 170 days of age. Body weights between groups were not different. Tumor multiplicity and tumor weights were: Vehicle-High-Fructose, 6.00 and 7.74g; Metformin High-Fructose, 5.00 and 6.17g; and Vehicle-Teklad, 2.70 and 5.75g. As indicated, Vehicle-treated rats on High-Fructose diet had twice the number of cancers as Vehicle-Teklad diet (P<0.05). Metformin had a minimal effect in rats on a High-Fructose diet; unlike our previous results in rats ( receiving MNU at 50 days of age) on Teklad or High-Fat diet where metformin increased cancer multiplicity and cancer weights. Furthermore, we performed metabolomic studies on both serum and cancers from animals on Teklad and High-Fructose diets. It was observed (based on principal component analysis) that serum from rats on High-Fructose and standard Teklad diet were profoundly different. Furthermore, there appeared to be a metabolic signature in rats bearing cancers. These and other metabolomic changes will be presented. Supported by NCI contract number HHSN261201200021I, Task Order HHSN26100005. Citation Format: Mark S. Miller, Clinton J. Grubbs, Steve Stirdivant, Ronald A. Lubet. Comparison of high-fructose and standard diets in methylnitrosourea (MNU)-induced ER+ mammary cancers: Altered carcinogenesis, differential effects of metformin, and metabolomic differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5255. doi:10.1158/1538-7445.AM2017-5255

Abstract A84: Lack of efficacy of the anti-diabetic drug metformin in the ER+ methylnitrosourea (MNU)-induced and the ER− MMTV-Neu/p53KO models of mammary cancer.

Abstract Metformin, an anti-diabetic drug, activates AMP-activated protein kinase (AMPK), and has generated a great deal of interest as a potential preventive agent for a variety of cancers, including breast. This interest is based both on epidemiologic studies showing that diabetics treated with metformin have a lower incidence of a variety of cancers, and on mechanistic considerations arguing that activation of AMPK is likely to be relevant to cancer treatment and prevention. We examined the preventive efficacy of metformin in ER+ and ER− rodent mammary cancer models. Treatment of Sprague-Dawley female rats at 50 days of age with a single dose of MNU (75 mg/kg BW) resulted in the development of multiple ER+ mammary cancers beginning approximately 7 weeks after MNU treatment. This model has been highly responsive to a wide variety of agents that affect ER+ tumors in humans (SERMs, aromatase inhibitors, pregnancy, ovariectomy). Metformin was administered by gavage (7×/week) at either 150 or 50 mg/kg BW/day beginning 7 days after MNU dosing, and continuing for the duration of the study. Metformin failed to decrease mammary tumor incidence and, in fact, increased tumor multiplicity in this model by approximately 35%. In addition, we are evaluating the ability of metformin to inhibit mammary cancer in MMTV-Neu/p53KO mice (which spontaneously develop ER− cancers). In this study, metformin was administered in the diet at a dose level of 1500 mg/kg diet. Initial supplementation of the diet with this agent began when the mice were 60 days of age, and will continue for 10 months. This study similarly has shown no preventive efficacy of metformin (av. tumors/mice: controls, 2.1; metformin, 2.5). Thus, these initial studies fail to observe any chemopreventive efficacy of metformin in two commonly employed models of ER+ and ER− mammary cancers in rodents. Supported by NCI contract number HHSN261200433001C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A84.

Abstract PD03-07: Evaluation of the Anti-Diabetic Drug Metformin in the Methylnitrosourea (MNU)-Induced Model of ER+ Mammary Cancer and the MMTV-Neu/p53KO Model of ER-Mammary Cancer

Abstract Metformin, an anti-diabetic drug, activates AMP-activated protein kinase (AMPK), and has generated a great deal of interest as a potential preventive agent for a variety of cancers, including breast. This interest is based both on epidemiologic studies showing that diabetics treated with metformin have a lower incidence of a variety of cancers, and on mechanistic considerations arguing that activation of AMPK is likely to be relevant to cancer treatment and prevention. We are examining the preventive efficacy of metformin in ER+ and ER-rodent mammary cancer models. Treatment of Sprague-Dawley female rats at 50 days of age with a single dose of MNU (75 mg/kg BW) resulted in the development of multiple ER+ mammary cancers beginning approximately 7 weeks after MNU treatment. This model has been highly responsive to a wide variety of agents that affect ER+ tumors in humans (SERMs, aromatase inhibitors, pregnancy, ovariectomy). Metformin was administered by gavage (7x/week) at either 150 or 50 mg/kg BW/day beginning 7 days after MNU dosing, and continuing for the duration of the study. Body weights were not altered by these dose levels and other signs of toxicity were not observed. Metformin failed to decrease mammary tumor incidence and, in fact, increased tumor multiplicity in this model by approximately 35%. In addition, we are evaluating the ability of metformin to inhibit mammary cancer in MMTV-Neu/p53KO mice (which spontaneously develop ER-cancers). In this study, metformin is being administered in the diet at a dose level of 1500 mg/kg diet. Initial supplementation of the diet with this agent began when the mice were 60 days of age, and will continue for 10 months. Preliminary results examined at 6 months into the study imply no efficacy of metformin in this model as well (average tumors/mouse: controls, 0.9; metformin, 1.6). Thus, these initial studies fail to observe any chemopreventive efficacy of metformin in two commonly employed models of ER+ or ER-mammary cancer in rodents. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-07.

Abstract B48: Lack of efficacy of the anti-diabetic drug metformin in the ER+ methylnitrosourea (MNU)-induced and the ER- MMTV-Neu/p53KO models of mammary cancer

Abstract Metformin, an anti-diabetic drug, activates AMP-activated protein kinase (AMPK), and has generated a great deal of interest as a potential preventive agent for a variety of cancers, including breast. This interest is based both on epidemiologic studies showing that diabetics treated with metformin have a lower incidence of a variety of cancers, and on mechanistic considerations arguing that activation of AMPK is likely to be relevant to cancer treatment and prevention. We examined the preventive efficacy of metformin in ER+ and ER- rodent mammary cancer models. Treatment of Sprague-Dawley female rats at 50 days of age with a single dose of MNU (75 mg/kg BW) resulted in the development of multiple ER+ mammary cancers beginning approximately 7 weeks after MNU treatment. This model has been highly responsive to a wide variety of agents that affect ER+ tumors in humans (SERMs, aromatase inhibitors, pregnancy, ovariectomy). Metformin was administered by gavage (7x/week) at either 150 or 50 mg/kg BW/day beginning 7 days after MNU dosing, and continuing for the duration of the study. Metformin failed to decrease mammary tumor incidence and, in fact, increased tumor multiplicity in this model by approximately 35%. In addition, we are evaluating the ability of metformin to inhibit mammary cancer in MMTV-Neu/p53KO mice (which spontaneously develop ER- cancers). In this study, metformin was administered in the diet at a dose level of 1500 mg/kg diet. Initial supplementation of the diet with this agent began when the mice were 60 days of age, and will continue for 10 months. This study similarly has shown no preventive efficacy of metformin (av. tumors/mice: controls, 2.1; metformin, 2.5). Thus, these initial studies fail to observe any chemopreventive efficacy of metformin in two commonly employed models of ER+ and ER- mammary cancers in rodents. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B48.

A gene expression signature predicts chemopreventive response of an EGFR inhibitor in a rat mammary cancer model.

Abstract Abstract #1111 Rats treated with the carcinogen methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. The efficacy of the epidermal growth factor receptor (EGFR) inhibitor Gefitinib (Iressa) to prevent MNU-induced mammary cancers in female Sprague-Dawley rats was determined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. Rats bearing MNU-induced small palpable cancers showed variable responses to treatment with Iressa; with approximately 50% having a complete regression and the remaining 50% having a partial or no response. We conducted exon array studies of differentially expressed genes and/or alternative splicing exons related to the responses (complete vs partial or no response). Preliminary studies showed that, although there were genes whose expression was similarly modulated in both groups, a gene expression signature can be identified that is highly predictive of which rat mammary tumors are susceptible to treatment with Iressa. Cross validation results indicated that gene expression signatures may be useful in predicting the response of a rat mammary tumor to treatment with Iressa. Currently, we are conducting validation studies in a separate set of rat mammary tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1111.

Identification of urinary biomarkers to distinguish tumor bearing and control rats in the methylnitrosourea (MNU) – induced model of mammary carcinogenesis: use of label-free, comparative, ultra-high resolution nano-LC mass spectrometry.

Abstract Abstract #1109 In the MNU induced model of mammary cancer, rats given a single dose of MNU (75 mg/kg BW), via the jugular vein, at 50 days of age develop multiple ER+ mammary cancers. These cancers appear similar to highly differentiated ER+ human breast cancers, and have been shown to be sensitive to many of the same chemotherapeutic agents that are effective in women. When a female rat developed its first palpable cancer, it was placed in a metabolic cage and urine collected overnight on dry ice. The urine from these rats were compared to those obtained from control rats. Using a new sample preparation protocol, peptides were prepared from the urines of control (n = 4 individual animals) and rats bearing induced mammary tumors (n = 4). The peptide pools were analyzed using nano-LC-LTQ-FT-MS. The 8 LC-MS analyses were aligned and the integrated accurate mass signals were quantified using Rosetta ElucidatorTM software. The ion currents from 4430 peptides (charge state > +2) were analyzed between the two groups using ANOVA. Two hundred and twenty nine peptides were found to be significantly different (P< 0.01) between the two groups, with clear distinction between the urines from control and experimental animals being observed using hierarchical clustering and principal component analysis. The protein identifications that were significantly increased in the tumor-bearing urines were confirmed by targeted tandem mass spectrometry. Further studies relating the altered expression of these peptides to differences in tumor size, the effects of therapeutic agents, etc will also be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1109.

The combination of genistin and ipriflavone prevents mammary tumorigenesis and modulates lipid profile

Recent reports have indicated that soy isoflavones may be protective against breast cancer. However, the effects of the synthetic isoflavone, ipriflavone, on mammary tumorigenisis, alone or in combination with genistin, a soy isoflavone, have not been investigated. Eighty-eight 36-day-old female Sprague-Dawley rats were divided as follows: Gen (20 mg genistin/kg body weight), Ipr (200 mg ipriflavone/kg body weight), Gen+Ipr (20/200 mg per kg body weight, respectively), and control (solvent vehicle). A week later, animals were injected with a single dose of methylnitrosourea. The isoflavones and solvent vehicle were administered daily via gastric gavage for 84 days post methylnitrosurea injection. The Gen+Ipr group had the lowest number of palpable tumors and adenocarcinomas per group, the least palpable tumors per rat, and the highest serum total and non-HDL cholesterol levels. No changes in circulating levels of indicators of oxidative stress were detected due to treatment. The findings of this study imply that the combination of genistin and ipriflavone is effective in suppressing mammary methylnitrosurea-induced tumorigenesis and also the lipid environment of the tumor cells that impact tumor growth or proliferation. Further studies are needed to establish the optimal dose of genistin and ipriflavone, individually or in combination, for the prevention of mammary tumorigenesis.

Effects of gefitinib (Iressa) on mammary cancers: preventive studies with varied dosages, combinations with vorozole or targretin, and biomarker changes

The ability of the epidermal growth factor receptor inhibitor gefitinib (Iressa) to prevent/treat methylnitrosourea (MNU)-induced mammary cancers and to modulate biomarkers in female Sprague-Dawley rats was examined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. In the prevention studies, continual treatment with Iressa at 10, 3, or 1 mg/kg body weight per day beginning 5 days after MNU reduced tumor multiplicity by 93%, 43%, and 20%, respectively. Treatment of rats bearing small palpable cancers with Iressa (10 mg/kg body weight per day) resulted in the complete regression of 70% of the tumors. Short-term treatment of tumor-bearing rats with Iressa caused decreases in cell proliferation and phosphorylated epidermal growth factor receptor and increases in apoptosis. To examine treatment regimens that might decrease the skin toxicity associated with Iressa, both intermittent treatments and combinations of lower doses of Iressa with other effective agents were evaluated. Treatment with Iressa (10 mg/kg body weight per day) continually or intermittently (either "3 weeks on/3 weeks off" or "4 days on/3 days off") reduced cancer multiplicity by 91%, 24%, and 68%, respectively. However, all regimens reduced tumor weights >85%. Finally, combining suboptimal doses of Iressa with suboptimal doses of vorozole (an aromatase inhibitor) or targretin (a retinoid X receptor agonist) yielded greater chemopreventive efficacy than any of these agents given alone.

Preventive effects of Polyphenon E on urinary bladder and mammary cancers in rats and correlations with serum and urine levels of tea polyphenols

Polyphenon E, a standardized mixture of green tea polyphenols, was examined for its chemopreventive efficacy against chemically induced urinary bladder and mammary cancers. In the present study, Polyphenon E was administered after the last dose of 4-hydroxybutyl(butyl)nitrosamine, or roughly 30% of the way into the experiment. Polyphenon E (100 or 250 mg/kg body weight/d) caused a dose-dependent decrease in palpable urinary bladder tumors [low dose, 14 of 34; high dose, 6 of 35; controls, 20 of 34 (P < 0.01)]. In the mammary cancer model, Polyphenon E [333 or 1,000 mg/kg body weight (BW)/d] was administered beginning 5 days after a single dose of methylnitrosourea. In contrast to its significant efficacy in bladder tumor prevention, Polyphenon E had a minimal effect in the prevention of mammary cancers. Levels of polyphenols were determined in the urine and serum of rats. Relatively high levels of various polyphenols (and metabolites) were found in the urine. However, virtually no epigallocatechin-3-gallate was observed in the urine because of low systemic bioavailability; although it represents almost 65% of the polyphenols in Polyphenon E. Levels of polyphenols in serum were 50 x to 1,000 x less than were observed in urine. The bioavailability of these tea polyphenols to different organ sites may contribute to the differing preventive efficacy of Polyphenon E against urinary bladder and mammary cancers.

Conjugated Linoleic Acid Isomers and Mammary Cancer Prevention

There is increasing evidence that individual isomers of conjugated linoleic acid (CLA) may have unique biological or biochemical effects. A primary objective of this study was to determine whether there might be differences in the anticancer activity of 9,11-CLA and 10,12-CLA. This was achieved by evaluating the reduction in premalignant lesions and carcinomas in the mammary gland of rats that had been treated with a single dose of methylnitrosourea and given 0.5% of either highly purified CLA isomer in the diet. Our results showed that the anticancer efficacies of the two isomers were very similar. At 6 wk after carcinogen administration, the total number of premalignant lesions was reduced by 33-36%. At 24 wk, the total number of mammary carcinomas was reduced by 35-40%. The concentration of each CLA isomer and its respective metabolites was analyzed in the mammary fat pad. Tissue level of 10,12-CLA was much lower than that of 9,11-CLA. The pool of metabolites from each isomer was very similar between the two groups and represented only a small fraction of total conjugated diene fatty acids. Feeding of 9,11-CLA resulted in minimal changes in other unsaturated fatty acids. In contrast, feeding of 10,12-CLA produced a wider spectrum of perturbations. Small but significant increases in 16:1 and 16:2 were detected; these were accompanied by decreases in 20:2, 20:3, 20:4, 22:4, and 22:6. The above observation suggests that 10,12-CLA might be more potent than 9,11-CLA in interfering with elongation and desaturation of linoleic and linolenic acids. In summary, our study showed that, at the 0.5% dose level, the anticancer activity of 9,11-CLA and 10,12-CLA was very similar, even though accumulation of 10,12-CLA in the mammary tissue was considerably less than that of 9,11-CLA. These confounding changes of the other unsaturated fatty acids in contributing to the effect of 10,12-CLA need to be clarified.

Effect of timing and duration of dietary conjugated linoleic acid on mammary cancer prevention.

Conjugated linoleic acid (CLA) is a minor fatty acid found predominantly in the form of triglycerides in beef and dairy products. Previous work by Ip and co-workers showed that free fatty acid-CLA at < or = 1% in the diet is protective against mammary carcinogenesis in rats. The present study verified that the anticancer activities of free fatty acid-CLA and triglyceride-CLA are essentially identical. This is an important finding, because it rules out a nonspecific free fatty acid effect. In terms of practical implication, we can continue the in vivo research with the less-expensive free fatty acid-CLA without compromising the physiological relevance of the data. A primary objective of this report was to investigate how the timing and duration of CLA feeding might affect the development of mammary carcinogenesis in the methylnitrosourea (MNU) model. We found that exposure to 1% CLA during the early postweaning and pubertal period only (from 21 to 42 days of age) was sufficient to reduce subsequent tumorigenesis induced by a single dose of MNU given at 56 days of age. This period incidentally corresponds to a time of active morphological development of the mammary gland to the mature state. In contrast to the above observation, a continuous intake of CLA was required for maximal inhibition of tumorigenesis when CLA feeding was started after MNU administration, suggesting that some active metabolite(s) of CLA might be involved in suppressing the process of neoplastic promotion/progression.

Induction of Congenital Malformations in Mice by Paternal Methylnitrosourea Treatment

ABSTRACTA single dose of methylnitrosourea (MNU, 25–100 mg/kg) was injected intraperitoneally into ICR strain male mice. The males were mated to untreated females of the same strain on days 1–21 and 64–80 after the treatment. On day 18 of pregnancy, the fetuses were examined for external and skeletal abnormalities. MNU treatment of paternal germ cells caused significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 3.0 × 10−4, which is quite similar to the rate previously estimated for the same endpoint at the same germ cell stage with the fractionated doses of MNU (daily doses at 5–25 mg/kg for 5 days). Cleft palate and dwarfism were the most frequent external abnormalities in the MNU‐treated and the control series. Malformed ribs was the most frequent skeletal abnormality in the treated series. It was concluded that congenital malformations induced after treating male mice with a single dose of MNU were quantitatively and qualitatively similar to those induced after treating male mice with the fractionated doses of MNU.

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 vorozole and 4-hydroxyandrostenedione were determined in the methylnitrosourea (MNU)-induced model of rat mammary tumorigenesis. Vorozole (5 and 2.5 mg/kg body wt) and 4-hydroxyandrostenedione (15 and 6 mg/rat) were administered daily (by gavage) to virgin female Sprague-Dawley rats starting at an age of 43 days. Seven days later animals were given a single dose of MNU. Following treatment with MNU, animals continued to be treated with vorozole and 4-hydroxyandrostenedione daily until the end of the experiment (100 days post MNU treatment). Vorozole at either dose proved to be a profound inhibitor of MNU-induced mammary tumors. Vorozole decreased tumor incidence from 100% to 10%, while simultaneously decreasing tumor multiplicity from 5 tumors per animal to 0.1 tumors per animal. This chemopreventive effect was accompanied by significant increases in body weight gain in the animals treated with vorozole when compared with control rats. In contrast, neither dose of 4-hydroxyandrostenedione had any effect on tumor incidence and only the higher dose slightly decreased tumor multiplicity.

Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues.

The kinetics of accumulation of the premutagenic DNA adduct O6-methylguanine (O6-meG) in the liver, blood leukocytes, lymph nodes and bone marrow of rats was examined and compared after single or multiple doses of procarbazine, a methylating cytostatic drug employed in the treatment of Hodgkin's lymphoma patients, and methylnitrosourea (MNU), an experimental methylating agent and carcinogen. Maximal O6-meG levels occurred 1-2 h after administration of single doses of procarbazine (10 mg/kg) or MNU (1 mg/kg), thereafter decreasing with half-lives of approximately 20-45 h, depending on the tissue. A relatively uniform tissue distribution was observed with both agents, with the liver generally showing highest adduct levels, followed by the lymph nodes, bone marrow and blood leukocytes which contained broadly similar amounts of O6-meG. During daily, oral administration to rats of procarbazine for 10 days at dose rates of 2.5, 5, 10 or 20 mg/kg/day (treatment analogous to that of the MOOP chemotherapy protocol for Hodgkin's lymphoma) followed by animal death on different days (in each case 24 h after the last treatment), a biphasic mode of O6-meG induction was observed: an initially steep build-up during the first 3-4 days was followed by a transient decline in the rate of accumulation, in turn followed by a second wave of accumulation and then a further slow-down. During the same treatment, liver O6-methylguanine-DNA alkyltransferase (AGT) declined in a dose-related manner. AGT recovery after the end of treatment was slow, taking nearly 20 days after the end of the high-dose treatment to return to control levels, despite the fact that all detectable adducts had been lost from DNA within 3 days after the end of treatment. A similar depletion and slow recovery of AGT in the liver, blood lymphocytes, bone marrow and lymph nodes was observed after treatment with a single dose of 100 mg/kg procarbazine. In contrast to these observations, O6-meG accumulated smoothly during a 10 day administration of MNU (1 or 10 mg/kg/day) to reach a steady-state within 5-6 days, while liver AGT was partially depleted after the high dose and recovered fully within 72 h of cessation of treatment. Similarly, a single dose of MNU (35 mg/kg) resulted in AGT depletion followed by rapid recovery in all four tissues examined. It is concluded that procarbazine (but not MNU) causes a decrease in cellular AGT concentrations by a mechanism additional to suicide repair of O6-meG.(ABSTRACT TRUNCATED AT 400 WORDS)

Preferential methylation of the Ha‐ras proto‐oncogene by methylnitrosourea in rat mammary glands

Activation of the Ha-ras proto-oncogene, but not the Ki-ras or N-ras genes, has been found in mammary gland carcinomas induced in female rats by a single dose of methylnitrosourea (MNU). Here we show that a 10-kb restriction fragment containing the Ha-ras gene was extensively methylated by MNU in DNA isolated from mammary glands of female rats 4 h after carcinogen treatment. Fragments of similar size containing either the Ki-ras or N-ras genes were methylated less extensively. The extent of methylation of the three ras genes by MNU correlated with their transcriptional activity. These results suggest that the extent of interaction of a carcinogen with an oncogene, which depends on its transcriptional activity, may be a factor in determining whether the gene is mutated during the initiation of carcinogenesis.

Autochthonous Prostate Adenocarcinomas in Lobund-Wistar Rats: A Model System

An experimental model system for autochthonous prostate adenocarcinoma (PA) has been developed in Lobund-Wistar (L-W) rats. Large primary PAs were induced in 31 of 40 (77.5%) L-W rats within an average of 10.7 months after a single dose of methylnitrosourea (MNU) and subcutaneous implants of testosterone. Metastatic tumors had developed in over 60% of the tumor-bearing rats. In addition, localized in situ PAs had developed in 5 of the 40 test rats. At 14 months 50 untreated L-W rats were free of demonstrable PAs. Two of 20 (10%) L-W rats developed PA at 14 months after inoculation of MNU alone. Six of 42 (14%) L-W rats developed PAs within 14 months after s.c. administration of testosterone implants. Thus, testosterone acted as a tumor promoter of PA for cells that had been initiated by MNU. The manifestations of the PAs in the L-W rats resembled many aspects of the counterpart disease in man.

Trisomy 15 as a regular finding in chemically induced murine T-cell leukemogenesis.

Trisomy 15 is described as a common finding in all T-cell leukemias induced by a single dose of methylnitrosourea (MNU) in BDF1 mice and in the leukemias induced by 7 doses of benzo(a)pyrene. Additional trisomies were found in about half of the leukemias. The organ distribution suggests that the leukemic cells with trisomy 15 originate in the thymus. Trisomy 15 was detected in the thymus as early as 6 weeks after the application of MNU, i.e. during the latency period.

Autochthonous prostate adenocarcinomas in Lobund-Wistar rats: a model system.

An experimental model system for autochthonous prostate adenocarcinoma (PA) has been developed in Lobund-Wistar (L-W) rats. Large primary PAs were induced in 31 of 40 (77.5%) L-W rats within an average of 10.7 months after a single dose of methylnitrosourea (MNU) and subcutaneous implants of testosterone. Metastatic tumors had developed in over 60% of the tumor-bearing rats. In addition, localized in situ PAs had developed in 5 of the 40 test rats. At 14 months 50 untreated L-W rats were free of demonstrable PAs. Two of 20 (10%) L-W rats developed PA at 14 months after inoculation of MNU alone. Six of 42 (14%) L-W rats developed PAs within 14 months after s.c. administration of testosterone implants. Thus, testosterone acted as a tumor promoter of PA for cells that had been initiated by MNU. The manifestations of the PAs in the L-W rats resembled many aspects of the counterpart disease in man.

RNA virus expression during and after methylnitrosourea-induced T-cell leukemogenesis in mice.

The expression of RNA tumor virus was studied in BDF 1 mice after leukemogenic treatment with a single dose of methylnitrosourea (MNU) and in leukemic thymuses by a cell ELISA using antibodies against the viral glycoprotein gp 70 and by co-culture for the detection of eco- and xenotropic virus. The majority of the thymomas were positive for gp 70; ecotropic, but not xenotropic infectious virus could be detected in some of them. Early after MNU application the thymus and the bone marrow were positive for gp 70 in some animals. Later, after a phase with positive results with spleen cells, the bone marrow and the spleen were negative again. Only the thymus of some mice were positive during the last weeks before the first leukemias appeared.

Residual toxicity in hematopoietic cells following a single dose of methylnitrosourea

The residual injury to the proliferation capability of hemopoietic stem cells (CFU-S) which results from their exposure to leukemogenic agents was evaluated in mice given a single leukemogenic dose of methol nitrosourea (MNU 50 mg/kg body weight, i.v.). Bone marrow cellularity, splenic weight, number of CFU-S and the proportion of cycling to noncycling CFU-S were measured in an effort to detect acute and residual injury to the CFU-S from mice given MNU 21 and 3 days earlier. Marrow cells were also transferred into lethally irradiated mice to observe the self-renewal capability of the CFU-S in the recipient spleen and bone marrow. The results of these measurements show that the CFU-S in marrow from mice given 50 mg/kg of MNU 21 days earlier still have a defective ability for self-renewal, although the total cellularity, number of CFU-S and proportion of cycling and noncycling CFU-S in the donor have returned to the normal range. The relationship of this self-renewal defect to the development of leukemia after this leukemogenic dose of MNU is not known.