Effect of letrozole in carcinogen-plus-estrogen-induced endometrial hyperplasia in mice

PURPOSE: To evaluate the effects of letrozole (Ltz) in carcinogen+estrogen-induced endometrial hyperplasia. METHODS: BALB/c female mice were divided into four groups of 12 animals each receiving an intrauterine dose of N-ethyl-N-nitrosourea (ENU) and weekly subcutaneous injections of estradiol hexaidrobenzoate (EHB), except for group I(control). The groups were divided in I (control), II (ENU+EHB), III (ENU+EHB+MPA) and IV (ENU+EHB+Ltz). Group III also received intramuscular injections of MPA (medroxy progesterone acetate) every four weeks, while group IV received oral doses of Ltz daily. At the end of 16 weeks, the animals were sacrificed, and blood samples were collected for the measurement of serum estradiol and progesterone levels. Uterine histological sections were made to evaluate the presence of endometrial proliferative lesions. Differences between groups were evaluated with student's t test, ANOVA and chi-square test. RESULTS: Groups ENU+EHB, ENU+EHB+MPA and ENU+EHB+Ltz showed varying degrees of endometrial hyperplasia. The incidence of hyperplasia in groups ENU+EHB and ENU+EHB+Ltz was higher and more severe than in group ENU+EHB+MPA. Control group showed lower levels of serum estradiol than the other groups. CONCLUSION: There was no evidence that letrozole could act as an antiestrogenic drug in the development of endometrial proliferative lesions.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X−/−) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X−/−) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.

Effect of N-ethyl-N-nitrosourea on the fatality, recovery of fertile period and breeding record in C57BL/6J mice / 동물의과학연구지

N-ethyl-N-nitrosourea (ENU) is a potent mutagen in a mouse model by inducing point mutation in a random manner and, in particular, causing heritable base substitutions in spermatogonia. In this study, systematic development of phenotype-driven mutant mice with large scale was carried out by using ENU. Nine-week-old male mice of C57BL/6J received intraperitoneal injection at three times with 100 mg/kg of ENU at weekly intervals for three weeks. After injections with ENU, the changes of body weight, fatality, recovery of fertile period, and breeding record were measured in these mice. Body weight lost as a result of ENU treatments was reversed after the last ENU injection. Live fertile male mice recovered from infertility from 104 to 165 days after ENU treatments were mated with C57BL/6J female mice for generation of G1 offspring. An average birth rate was 5.9 mice from 1 pair of paternal and maternal mice. All of 231 G1 offspring mice were analyzed by modified-SHIRPA with standard procedure at nine weeks of age. Among G1 mice, 166 mice were identified as mutagenic phenotypes in 20 test items. The changes in mutagenic phenotypes after ENU treatments, for instance, pattern in the region with a different color, touch escape, changes in head morphology, pupil, and teeth, and negative geotaxis etc., were found in these mice. Taken together, these results indicate that ENU may be a trans-generational mutagen in C57BL/6J mice.

Leukemic stromal hematopoietic microenvironment negatively regulates the normal hematopoiesis in mouse model of leukemia / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Leukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term.</p><p><b>METHODS</b>The mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry.</p><p><b>RESULTS</b>Long-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells.</p><p><b>CONCLUSIONS</b>Stromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.</p>

Forward genetic screening for zebrafish mutants defective in erythropoiesis / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen and identify zebrafish mutants with erythropoiesis defects by N-ethyl-N-nitrosourea (ENU) mutagenesis and large-scale forward genetic screening using beta e 1 as the marker.</p><p><b>METHODS</b>The chemical mutagen ENU was used to treat healthy wild-type male fish (AB strain, F0). The surviving ENU-treated fish were mated with wild-type female fish to generate F1, and further F2 family was generated by F1 family intercross. The adult F2 fish were intercrossed within each F2 family and the resulting F3 embryos from each crossing were subjected to whole mount in situ hybridization (WISH) with the beta e 1 probe. Mutagenesis was performed by treating the male zebrafish with ENU to induce mutations in pre-meiotic germ cells to generate the founders, which were outcrossed to obtained the F1 fish. The F1 fish from different founders were mated to generate the F2 families. F3 embryos from the sibling cross in the F2 family were examined by whole mount in situ hybridization using beta e 1-globin probe. The putative mutants were then characterized with different hematopoiesis markers.</p><p><b>RESULTS AND CONCLUSION</b>We identified 4 beta e 1-deficient mutants with erythropoiesis defects, including two with specific erythiod lineage defects and two with concurrent lymphopoiesis defects.</p>

Positional cloning of a novel allele of zebrafish cloche mutant / 南方医科大学学报

<p><b>OBJECTIVE</b>To perform the genetic identification of cloche(172) mutant zebrafish.</p><p><b>METHODS</b>The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization (3dpf) stage were observed and the cloche(172) gene was identified by mapping and complementation test.</p><p><b>RESULTS</b>We selected 4 lyC-deficient zebrafish by WISH. cloche(172) mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche(172) gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche(172) mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining.</p><p><b>CONCLUSION</b>cloche(172) gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.</p>

Adaptability of zirconia core fabricated by cold isostatic pressing / 대한치과보철학회지

PURPOSE: The purpose of this study is to fabricate the new zirconia block (CNU block) and to evaluate fit of core and porcelain veneered zirconia crown. MATERIAL AND METHODS: The experimental blocks were fabricated from the commercial ytrria-stabilized zirconia powder (KZ-3YE Type A). The powder was uniaxial pressing and the green bodies were conducted using the Cold Isostatic Pressing. The zirconia blocks were presintered at 1040degrees C and the final sintering was performed at 1450degrees C. The Kavo Everest ZS blank(R) (KaVo, Biberach/Ri beta.) was used as a control group. The linear shrinkage of CNU block and Kavo block were compared. Twenty-one cores for porcelain veneered crowns were fabricated with CAD/CAM system (Everest(R), Biberach/Ribeta.). Group I: seven cores fabricated from Kavo blocks, Group II: seven cores fabricated from CNU blocks, Group III: seven cores from CNU blocks and porcelain veneering for crowns. All specimens were cemented and sectioned into two planes: diagonal and bucco-lingual. The measurement of the marginal, internal, and occlusal fit was carried out using SEM (S-4800(R)) at 30 x. The results were analyzed by one-way ANOVA test. RESULTS: The linear shrinkage of the CNU block and the KaVo block was 19.00% and 20.09%. The marginal gap of cores (29.67 +/- 6.58 micrometer) fabricated from CNU blocks showed significantly smaller than that of the cores of Kavo blocks (36.84 +/- 7.18 micrometer) (P < .05). The internal gaps of the porcelain veneered crowns (32.23 +/- 6.33 micrometer) were larger than those of the other two groups (37.57 +/- 6.81 micrometer and 38.14 +/- 6.81 micrometer). CONCLUSION: No statistically significant difference was found in between experimental groups and control group. The experimental groups in marginal gap showed significantly smaller than the control group.

Shear bond strength of veneer ceramic and colored zirconia by using aqueous metal chloride solutions / 대한치과보철학회지

PURPOSE: The purposes of this study was to evaluates shear bond strength between zirconia core and veneer-ceramic in order to examine the clinical practice of colored zirconia block fabricated by infiltration method into the metal chloride solution. MATERIAL AND METHODS: CNU block and Everest(R) ZS blank were used. VITA In-Ceram(R)2000 YZ Coloring liquid (LL1) and 3 aqueous metal chloride solutions containing chromium and molybdenum ingredients were used. 40 zirconia specimens were prepared into cuboid shape (5 x 5 x 10 mm). All specimens were divided into 5 groups by infiltrating into the coloring liquids. After that, porcelain was build up into the shape of 5 x 5 x 4 mm3, followed by sintering. The maximum loading and shear bond strength was measured. Failure patterns and failure sites were examined. RESULTS: 1. There were no statistical differences in shear bond strength between zirconia blocks (P > .05). 2. There were no statistically significant differences in shear bond strength between non-colored and colored zirconia blocks, while shear bond strength of non-colored zirconia blocks is higher than that of colored specimen (P > .05). 3. In the comparison with shear bond strength among colored zirconia blocks, there were no statistical differences according to kinds of coloring liquid (P > .05). 4. Mixed failure patterns were mainly observed in the failure between zirconia and veneering ceramic. The veneering ceramic failure of all specimens was observed in either interface of zirconia or veneering ceramic. CONCLUSION: Shear bond strength between colored zirconia and veneering ceramic shows lower tendency than non-colored zirconia, but there was clinically allowable value.

Evaluation of Tumor Angiogenesis with a Second-Generation US Contrast Medium in a Rat Breast Tumor Model

OBJECTIVE: Tumor angiogenesis is an important factor for tumor growth, treatment response and prognosis. Noninvasive imaging methods for the evaluation of tumor angiogenesis have been studied, but a method for the quantification of tumor angiogenesis has not been established. This study was designed to evaluate tumor angiogenesis in a rat breast tumor model by the use of a contrast-enhanced ultrasound (US) examination with a second-generation US contrast agent. MATERIALS AND METHODS: The alkylating agent 19N-ethyl-N-nitrosourea (ENU) was injected into the intraperitoneal cavity of 30-day-old female Sprague-Dawley rats. Three to four months later, breast tumors were detected along the mammary lines of the rats. A total of 17 breast tumors larger than 1 cm in nine rats were evaluated by gray-scale US, color Doppler US and contrast-enhanced US using SonoVue. The results were recorded as digital video images; time-intensity curves and hemodynamic parameters were analyzed. Pathological breast tumor specimens were obtained just after the US examinations. The tumor specimens were stained with hematoxylin and eosin (H & E) and the expression of CD31, an endothelial cell marker, was determined by immunohistochemical staining. We also evaluated the pathological diagnosis of the tumors and the microvessel density (MVD). Spearman's correlation and the Kruskal-Wallis test were used for the analysis. RESULTS: The pathological diagnoses were 11 invasive ductal carcinomas and six benign intraductal epithelial proliferations. The MVD did not correlate with the pathological diagnosis. However, blood volume (BV) showed a statistically significant correlation with MVD (Spearman's correlation, p < 0.05). CONCLUSION: Contrast-enhanced US using a second-generation US contrast material was useful for the evaluation of tumor angiogenesis of breast tumors in the rat.

Point Mutation of Hoxd12 in Mice

PURPOSE: Genes of the HoxD cluster play a major role in vertebrate limb development, and changes that modify the Hoxd12 locus affect other genes also, suggesting that HoxD function is coordinated by a control mechanism involving multiple genes during limb morphogenesis. In this study, mutant phenotypes were produced by treatment of mice with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed mutant mice exhibiting the specific microdactyly phenotype and examined the genes affected. MATERIALS AND METHODS: We focused on phenotype characteristics including size, bone formation, and digit morphology of ENU-induced microdactyly mice. The expressions of several molecules were analyzed by genome-wide screening and quantitative real-time PCR to define the affected genes. RESULTS: We report on limb phenotypes of an ENU-induced A-to-C mutation in the Hoxd12 gene, resulting in alanine-to-serine conversion. Microdactyly mice exhibited growth defects in the zeugopod and autopod, shortening of digits, a missing tip of digit I, limb growth affected, and dramatic increases in the expressions of Fgf4 and Lmx1b. However, the expression level of Shh was not changed in Hoxd12 point mutated mice. CONCLUSION: These results suggest that point mutation rather than the entire deletion of Hoxd12, such as in knockout and transgenic mice, causes the abnormal limb phenotype in microdactyly mice. The precise nature of the spectrum of differences requires further investigation.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

p16 protein expression in the transplacental induced brain tumor in rat / 中华外科杂志

<p><b>OBJECTIVE</b>To study in series the p16 protein expression on the rat brain tumor induced transplacentally by ENU.</p><p><b>METHODS</b>The p16 protein expression was determined with immuno-histochemistry stain on the offspring's brain at their 60, 90, 120, 150 days after birth.</p><p><b>RESULTS</b>(1) p16 proteins were expressed in all of the brain samples in the 60-day group; occasionally negative in the 90-day group; partly expressed in the 120-day group; significantly less expressed in the 150-day group. (2) The rate of expression in the tissue around tumor was higher than that in the tumor. (3) The p16 protein was mainly orientated in the nuclear of cell and sporadically orientated in the cytoplasm.</p><p><b>CONCLUSION</b>(1) It shows the p16 protein expression decreases with the increase of tumor incidence in the rat brain, which accompanies the start and development of the induced tumor. So we speculate that the dysfunction of p16 gene is one of the factors related to tumor incidence in this animal model. (2) The p16 protein is mainly orientated in the nuclear of cell and sporadically orientated in the cytoplasm.</p>

HUP98-HOXA9 transgenic mice are susceptible to N-ethyl-N-nitrosourea stimulation in leukemogenesis / 中华血液学杂志

<p><b>OBJECTIVE</b>In order to investigate the leukemogenic potential of NUP98-HOXA9 fusion gene in vivo.</p><p><b>METHODS</b>Molecular cloning technology was used to construct NUP98-HOXA9 transgenic plasmid and NUP98-HOXA9 transgenic mice were generated. The genotype and phenotype of the NUP98-HOXA9 transgenic mice were analyzed by PCR, RT-PCR and colony-forming assay. The effect of N-ethyl-N-nitrosourea (ENU) stimulation on the transgenic mice was analyzed by peripheral blood count, bone marrow (BM) cells morphology pathological examination.</p><p><b>RESULTS</b>The transgenic expression was detected in 5 independent lines of NUP98-HOXA9 transgenic mice, but no expected phenotypes was found in 2 year follow-up. Upon ENU stimulation, 2 of 10 transgenic mice developed myeloid leukemia, suggesting that NUP98-HOXA9 transgenic mice have increased susceptibility to ENU mutagenesis in leukemogenesis.</p><p><b>CONCLUSION</b>The fusion gene expressed in BM cells of NUP98-HOXA9 transgenic mice. It seems that the expression of the fusion gene is insufficient to trigger leukemogenesis. However, the increased susceptibility to ENU mutagenesis suggests that NUP98-HOXA9 fusion gene might play a potential role in leukemogenesis.</p>

Brain tumor inhibition in experimental model by restorative immunotherapy with a corpuscular antigen.

In view of the advances in our understanding of anti-tumor immune response, it is now tempting to contemplate the development of immunotherapies for malignant brain tumors, for which no effective treatment exists. Immunotherapy, with agents known as biological response modifiers (BRMs) are thus gaining increasing interest as the fourth modality of treatment. A non-specific BRM, sheep erythrocytes (SRBC) when administered (ip, 7% PCV/V, 0.5 ml) in a group of animals at the end of seventh month of ethylnitrosourea administration, resulted in significant increase in the mean survival time (> 350 days). Studies conducted for growth kinetics pattern with proliferation index and fluorochrome (HO-33342) uptake techniques at the tissue culture level exhibited a regulatory inhibition of the cells isolated from tissue excised from the tumor susceptible area of brain of SRBC treated animals. Moreover, histological examination of brain from animals showed immunomodulatory role of SRBC in experimentally induced brain tumor. Further probe into the mechanisms involving immunological investigations at the cellular level in these animals indicated an augmented and potentiated cell mediated immune response (CMI) as evidenced by enhanced spontaneous rosette forming capacity and cytotoxic activity of lymphocytes and neutrophil (PMN) mediated phagocytosis respectively. The observations suggest that SRBC down regulate malignant growth pattern of experimental brain tumors either by an immunologically enhanced killing of tumor cells and/or by directly inhibiting the tumor growth possibly via a stimulated cytokine network. Thus, a corpuscular antigen, can potentiate CMI response in experimentally induced brain tumor animal model, in which response induced in the periphery are able to mediate anti-tumor effects in the brain.

Effect of ethylnitrosourea on HPRT gene in human promyelocytic leukemia cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene mutation induced by ethyluitrosourea (ENU).</p><p><b>METHODS</b>Single cell cloning culture, two-way screening, multiple PCR amplification and electrophoresis technique were used.</p><p><b>RESULTS</b>With dose of ENU increasing, cell plating efficiency reduced (in the group with 100-200 micro g/ml doses, P < 0.01) and mutation frequency increased (in the group with 12.5-200.0 micro g/ml doses, P < 0.05) significantly. There was no all exons deletion in spontaneous mutations, and only 7.7% of them were detected as single exon deletion. But, deletion was found in 79.7% of ENU-induced mutations (62.5%-89.4%, P < 0.01), and deletion mutations in all nine exons of HPRT gene. Most of ENU-induced mutations were chain deletion with multiple exons (88.1%).</p><p><b>CONCLUSIONS</b>The spectra in spontaneous mutations differed completely from ENU-induced ones. ENU was liable to cause bigger changes in genetic structure, which suggested a stronger ENU's mutagenesis.</p>

Centrilobular Distribution of Ethylnitrosourea-Induced Hepatocellular Foci in the Mouse / 대한간학회지

BACKGROUND/AIMS: Hepatocytes on the hepatic lobule mipate from portal zone to centrilobular mea as the DNA synthesis within it. And also, the xenobiotic reactions reveal characteristic differences associated with zone specific metabolism in the liver acinus. In this study, the zonal distribution of ethylnitrosourea (ENU)-induced hepatic precancerous lesion was stereologically investigated. METHODS: Nine B6C3F1 mices were given I.p. injection of ENU (60 ug/pn body weight) when the pups were 15 days old prior to sacrifices at 8 weeks of life. All the 150 consecutive sections, 3 p m in thickness, were stained with hematoxylin and eosin and identified the basophilic precancerous lesions with 80-165 p m diameter in equatorial plane by the Zeiss microprojector. And then the distances from the center of selected foci to terminal hepatic vein or portal vein branches were estimated under the microscopic fields. As a control group, the same estimations were performed from the random points by the appointments of random digit table. RESULTS: Mean distance between ENU-induced 52 hepatocellular foci and the nearest terminal hepytic vein was 181.15+112.39 p m (Mean+ SD), but that of randomly selected 104 points was 291.73+157.98pm (Mean+5D) (Students t-test, p<0.0005). Substantially, 52.7% of ENU-induced 52 hepatocellular foci were within 300 p m from the terminal hepatic vein, but randomly selected 104 points were only 50.9% (Shapiro Wilk W test, w=0.819857, p=0.048038). Mean distance from ENU-induced 52 foci to portal vein was 398.85+149.98pm (Mean+SD), but that from the randomly selected 104 points was 315.87+145.79 pm (Mean+SD)(Students t-test, p<0.0005). CONCLUSION: Stereologically, ENU-induced mice liver cell foci distribute non-randomly to Zone III, centrilobular zone of mouse hepatic acini where promote invasion toward terminal hepatic veins.