Estrous cycle modification of rat mammary gland DNA alkylation by N-methyl-N-nitrosourea

Virgin Sprague-Dawley rats exhibiting regular estrous cycles were used as a model system to determine whether the level of circulating estrogen modifies the alkylation pattern of mammary gland DNA by a direct-acting carcinogen, N-methyl-N-nitrosourea (NMU). The concentration of 7-methylguanine and O6-methylguanine were similar in mammary epithelial DNA 0.25, 0.50, and 1.0 h after i.v. injection of 50 mg/kg body weight NMU on different days of the rat estrous cycle. However, O6-methylguanine was significantly higher in mammary gland DNA 8 and 24 h after a single i.v. dose of carcinogen on proestrus or estrus, compared to rats receiving carcinogen on diestrus. There was no difference in the 7-methylguanine levels at 8 h in any group, but this adduct was higher in estrous-treated rats at 24 h. The ratio of O6-methylguanine to 7-methylguanine was significantly lower at 8 h in mammary gland DNA from diestrous-injected rats, and this difference reflected the lower level of O6-methylguanine adducts in this group. In contrast, O6-methylguanine concentrations in DNA extracted from the liver of the same animals were virtually identical at all time periods examined. 7-Methylguanine levels were higher in the liver at 0.5, 1, 8, and 24 h post-NMU in proestrus as compared with diestrous-injected rats. The observed adduct clearance suggests that rat mammary epithelium may contain repair systems capable of removing O6-methylguanine. These results also suggest that the initial removal of the O6-methylguanine lesions in mammary epithelial DNA (rather than the initial rate of alkylation) is affected by the hormonal environment during carcinogen exposure. This effect may be tissue specific since removal of O6-methylguanine from liver DNA is apparently not altered by the stage of the estrous cycle at which NMU is administered.     

Key stages in mammary gland development: The mammary end bud as a motile organ

In the rodent, epithelial end buds define the tips of elongating mammary ducts. These highly motile structures undergo repeated dichotomous branching as they aggressively advance through fatty stroma and, turning to avoid other ducts, they finally cease growth leaving behind the open, tree-like framework on which secretory alveoli develop during pregnancy. This review identifies the motility of end buds as a unique developmental marker that represents the successful integration of systemic and local mammotrophic influences, and covers relevant advances in ductal growth regulation, extracellular matrix (ECM) remodeling, and cell adhesion in the inner end bud. An unexpected growth-promoting synergy between insulin-like growth factor-1 and progesterone, in which ducts elongate without forming new end buds, is described as well as evidence strongly supporting self-inhibition of ductal elongation by end-bud-secreted transforming growth factor-β acting on stromal targets. The influence of the matrix metalloproteinase ECM-remodeling enzymes, notably matrix metalloproteinase-2, on end bud growth is discussed in the broader context of enzymes that regulate the polysaccharide-rich glycosaminoglycan elements of the ECM. Finally, a critical, motility-enabling role for the cellular architecture of the end bud is identified and the contribution of cadherins, the netrin/neogenin system, and ErbB2 to the structure and motility of end buds is discussed.     

Ultrastructure of a murine mammary carcinoma exposed to hyperthermia in vivo.

The ultrastructural changes following local hyperthermic therapy was studied in a solid murine mammary carcinoma. A few hr after treatment, a pronounced lysosomal activity was observed in the cytoplasm of the tumor cells, together with mitochondrial destruction and disaggregation of the polyribosomes. Later, more destructive changes with intense cell shrinkage and cytoplasmic lysis occurred, and within 24 hr the entire cytoplasm of all the tumor cells was completely destroyed. In the nuclei shrinkage and condensation of heterochromatin were early features. The nucleoli were gradually degranulated, but with preservation of the fibrillary component. However, here, too, complete destruction occurred within the first few days after treatment. Cells in mitosis were also arrested and destroyed in the same way. The nonmalignant cells of the tumor tissue presented only minor reversible morphological changes, and within a few days the tumor area was replaced by ingrowth of fibroblasts and macrophages. The morphological changes are discussed in the light of our present knowledge of the hyperthermic effect on tumor cells. As regards the mechanism of hyperthermic destruction of solid tumors in vivo, the hypothesis is advanced that a primary, lysosomally conditioned, selective destruction of the malignant cells occurs and that this reaction is intensified by a high acidity in the tumor milieu.     

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias. © 1996 Wiley-Liss, Inc.     

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.     

Hyperplastic alveolar nodules of the rat mammary gland: tumor-producing capability in vivo and in vitro.

Carcinogen-induced hyperplastic alveolar nodules failed to form tumors after transplantation into isologous female rats; instead they developed ductal or ductal-alveolar outgrowths. Mammary glands from mid-pregnant female rats showed similar outgrowths following transplantation to the isologous hosts. In vitro and in vivo studies failed to demonstrate that subcarcinogenic doses of 7,12-dimethylbenz(a)anthracene can induce mammary tumorigenesis in the hyperplastic alveolar nodules. Our observations suggest that carcinogen-induced hyperplastic alveolar nodules are not 'preneoplastic' lesions in mammary carcinogenesis in the rat.     

X-radiation induces 8-hydroxy-2'-deoxyguanosine formation in vivo in rat mammary gland DNA

Ionizing radiation is a carcinogen that induces oxidative DNA damage. 8- Hydroxy-2'-deoxyguanosine (8-OHdG) is a relatively abundant, mutagenic lesion that is widely regarded as a reliable index of oxidative DNA damage. The purpose of this study was to examine the effects of X- radiation on levels of 8-OHdG in the context of an experimental model for breast cancer in which chronic radiation exposure has been shown to be carcinogenic in Sprague-Dawley rats. A secondary objective of this study was to determine if the use of phenol during DNA isolation affected the concentration of 8-OHdG subsequently measured. Our results indicate that a profoundly carcinogenic dose of radiation induced a small but significant increase in 8-OHdG concentration in mammary gland DNA, and that the use of a phenol-based versus a salt-based method of DNA isolation had no significant impact on the levels of 8-OHdG detected in either control or irradiated tissue.      

Luminal plasma membrane organization in rat urinary bladder urothelium after direct exposure in vivo to N-methyl-N-nitrosourea.

Adult female rat urinary bladders were exposed to N-methyl-N-nitrosourea in vivo. After 12 weeks, urothelial hyperplasia was established. Ultrastructural changes of the luminal plasma membrane were studied with thin sectioning and freeze-fracturing techniques. The mosaic pattern which is a characteristic specialization of the normal luminal plasma membrane and of cytoplasmic vesicles disappeared. The plasma membrane of the transformed urothelium was indistinguishable from an undifferentiated membrane in the following respects: thickness; apparent symmetry in cross-section; and distribution of intramembranous particles. Zonulae occludentes became permeable to colloidal lanthanum and appeared disassembled, while desmosomes developed concomitantly. These membrane changes implicate increased permeability and less adaptability of urothelial function to mechanical stress caused by bladder volume changes. The increased desmosome formation may indicate that squamous metaplasia is a feature of early malignant urothelium transformation. It is proposed that N-methyl-N-nitrosourea treatment induces a change in the plasma membrane assembly and specialization.     

Cellular retinol-binding protein in normal and neoplastic human mammary gland

The concentration of cellular retinol-binding protein (CRBP) was determined in samples of normal and neoplastic mammary gland, using a specific and sensitive radioimmunoassay. The CRBP concentration was significantly higher in neoplastic tissue, but detectable levels were also present in all samples of normal gland. Tubulo-ductal cancers had significantly lower CRBP levels than other cancer types. The CRBP concentration of the neoplastic tissue showed no correlation with the concentration of progesterone or estrogen receptor.     

Growth kinetics of transplantable mammary gland tumors in mice]

The function of: (see article), fits the tumors weight growth for all studied generations in both control and treated animals. Morphological studies show the transplantable tumors in A mice to lose their similarity to a spontaneous tumor--low differentiated adenocarcinoma. Meanwhile, the tumors in C3H mice have a high differentiation level which is typical for the original tumor. 1-nitroso-1-methylurea is very effective against the first generation of tumors in both strains of mice. The tumors response to the drug becomes weaker with further passages. 1-nitroso-1-methyl urea is superior to other nitrous derivatives when used in such tumor models as breast adenocarcinoma in C3H mice.     

Thymic lymphomas in Wistar rats exposed to N-methyl-N-nitrosourea (MNU)

The Brazilian Agency for the Environment (IBAMA) recently adopted an alternative medium-term multiple-organ assay system with the Wistar rat strain for detection of the carcinogenic potential of pesticides. Originally, this initiation-promotion protocol was established in Japan with the isogenic Fischer 344 male rat. Among the initiating agents used in that assay, N-methyl-N-nitrosourea (MNU) rapidly induces malignant lymphoma and leukemia and early mortality of rats from different strains. This study was developed to evaluate whether the outbred Wistar rats are also similarly susceptible to MNU. Particularly, it aimed to evaluate the dose-response relationship and to register the MNU-induced pre-neoplasia and neoplasia that may develop in the lympho-hematopoietic system (LHS) of the Wistar rat within a medium-term period. Four groups of male Wistar rats were treated during 2 weeks with vehicle or with MNU (80, 160 or 240 mg/kg body weight, i.p.). After sacrifice at the 12th and 20th weeks, the thymus, spleen, bone marrow, cervical and mesenteric lymph nodes and liver were collected for analysis. At the 20th week, LHS malignant tumors and benign vascular tumors occurred only in the high- and intermediate-dose MNU-treated animals. Four animals treated with 240 mg/kg developed diffuse thymic lymphomas; two others, treated respectively with 240 mg/kg and 160 mg/kg, developed spleen hemangiomas. The present observations indicate that the Wistar strain is as susceptible as other strains to the early development of MNU-induced LHS (pre)neoplasia. Therefore, this strain seems suitable to be used as test system in bioassay protocols that adopt MNU as an initiating agent for carcinogenesis. (Cancer Sci 2003; 94: 240–243)     

Estrous cycle modification of rat mammary tumor induction by a single dose of N-methyl-N-nitrosourea

The potential of individual stages of the rat estrous cycle to alter the incidence and subsequent behavior of mammary carcinomas induced by a single dose of N-methyl-N-nitrosourea on diestrus, proestrus, or estrus was examined. Mean latencies to first tumor appearance in diestrous, proestrous, and estrous groups were 86, 71, and 69 days, respectively (P less than 0.05 diestrus versus proestrus, estrus). Tumor incidence in diestrous rats given injections (73%) was significantly lower than in proestrous (87%) or estrous (89%) animals given injections, as was the mean number of tumors per rat. However, the number of days required for tumors to reach 1 cm in diameter in diestrous animals given injections (13.0) was significantly lower as compared with tumors in rats given injections during proestrus (19.3) or estrus (22.2). In later growth stages, the diestrous tumor doubling time was one-half that of tumors in proestrous rats given injections. Flow cytometric analysis of tumor tissues during midlog and later growth phases did not reveal any significant changes in ploidy or growth fractions between groups. Further, there was no significant difference in tumor cytosol estrogen receptor incidence, affinity (Kd), or content between groups, although tumor cell nuclear receptor for estrogen was higher (38.3 fmol/mg DNA; P less than 0.05) in proestrous rats given injections than in diestrous (21.6) or estrous (21.8) animals given injections. These data support the concept that the prevailing hormonal profile of the estrous cycle at the time of tumor initiation modulates the subsequent induction of mammary tumors. Further, the absence of any observed difference in tumor behavior between proestrous and estrous rats given injections suggests that prolactin does not impose an additive or synergistic effect on the initial stage of tumor induction when mammary gland epithelial cell DNA is previously stimulated by estrogen.     

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.     

O6-methylguanine accumulates in DNA of mammary glands after administration of N-methyl-N-nitrosourea to rats.

N-Methyl-N-nitrosourea (MNU) induces mammary carcinoma in female rats when given intravenously. After a single intravenous dose of N-methyl-N-nitrosourea (5 mg/100 g body wt.), we were unable to detect a shift of rat mammary gland DNA on an alkaline sucrose gradient. However, the alkylated products in DNA, 7-methylguanine and O6-methylguanine, were determined at various times following treatment with N-methyl-N-nitrosourea. O6-Methylguanine was removed from the DNA at a slower rate than 7-methylguanine and increased in the DNA with a second injection of N-methyl-N-nitrosourea. 3-Methyladenine was not detected in DNA from the mammary gland of the rat. These data support previous work with brain and bladder that suggest the persistence of O6-methylguanine in DNA might be involved in the induction of cancer by N-methyl-N-nitrosourea.     

ras oncogene activation in mammary carcinomas induced by N-methyl-N-nitrosourea in Copenhagen rats.

The occurrence of Ha-ras and Ki-ras oncogenes was investigated in mammary tumors produced by treating genetically resistant Copenhagen (Cop) rats with N-methyl-N-nitrosourea. G35-->A codon 12 mutations in both Ha-ras and Ki-ras genes were analyzed by a polymerase chain reaction/liquid hybridization and gel retardation assay. More than half of the adenocarcinomas analyzed contained an activated Ha-ras gene. This was also the predominant mutation in similar tumors from susceptible Buf/N rats, suggesting a common mechanism of initiation. In contrast, only two of 15 mammary adenosquamous carcinomas from the Cop rats contained an activated Ha-ras gene, suggesting a different initiation mechanism for most of these tumors. Ki-ras activation was found in none of five and one of five adenocarcinomas from Buf/N and Cop rats, respectively, and in none of 13 adenosquamous carcinomas from Cop rats. These results suggest that Ki-ras activation does not play a major role in the initiation of the mammary tumors.     

Acquisition of angiogenic capacity and neoplastic transformation in the rat mammary gland.

The ability to induce formation of new vessels was tested in fragments of rat mammary tissue transplanted onto the rabbit iris and observed through the transparent cornea. Virgin, pregnant, and lactating glands showed an angiogenic capacity in about 5% of implants. In contrast mammary carcinomas induced angiogenesis in 75 to 100% of implants. Fragments of mammary gland previously treated with 7,12-dimethylbenz[alpha]anthracene of N-nitrosomethylurea but without histological evidence of neoplastic transformation showed an angiogenic response in about 5% of implants. The same low angiogenic response was detected in primary hyperplastic alveolar nodules. However, angiogenesis was observed 2 to 3 times more frequently in implants from hyperplastic outgrowths that acquired of continuous transplantability and showed a high degree of neoplastic transformation. These data on the rat mammary gland confirm previous findings on mouse mammary gland, indicating that: (a) neoplastic epithelium has a higher angiogenic capacity than does normal epithelium; and (b) hyperplastic epithelium at high risk of undergoing neoplastic transformation induces angiogenesis more frequently than does hyperplastic epithelium with low tumor potential.