N-nitrosomethylurea Research Articles

Реакция образцов озимого ячменя на обработку семян химическими мутагенами

In nature, a huge variety of traits is created in the systems of living organisms due to the occurrence of mutations. The use of ionizing radiation, ultraviolet rays, and many chemical compounds makes it possible to significantly increase the frequency of gene mutations, the genotypic and phenotypic variability of traits, and to achieve a sharp increase in the efficiency of artificial selection and the intensity of the selection process. The genotypic flexibility of the population and selection ensure adaptation to long-term changes in environmental factors. In the present work, the specificity of the mutagenic effect of some chemical mutagens on the winter barley variety Ardak on the variability of population and individual traits was studied for the first time. Our goal was to study the effect of carbolite and N-nitrosomethylurea in different concentrations on the agrobiological characteristics of winter barley. The scientific novelty of the conducted research lies in the study of the effect of carbolite and N-nmm in different concentrations on the agrobiological characteristics of winter barley, as well as on the content of total nitrogen and proteins in barley grains at previously unused or little studied doses. The object of the study was the seeds of barley variety Ardak. Carbolite compounds, N-nitrosomethyl urea were used as mutagenic factors. Carbolite was used at a concentration of 2n, the seeds were treated with carbolite solution for 6, 12, 18 and 24 hours. In the present studies, N-nitrosomethyl urea was used at concentrations of 0.012% and 0.025%. Barley seeds were treated with aqueous solutions of the mutagen for three hours, and then washed in running water at a temperature of 22 °C for 30 minutes. Control seeds were soaked in water. After treatment, the seeds were dried on filter paper and sown in the soil after 10 days. Each option contains 250 seeds. Seed germination was counted after two weeks. Subsequently, the linear parameters of plants were measured every 10 days. After harvesting, the plants were analyzed for morphological changes and the total percentage of changed plants was calculated. In addition, the sign was studied — the weight of 1000 grains. Seeds of the first generation for the next year were sown in the soil according to the treatment options (250 pieces). Second generation plants were analyzed in the same way as M1 plants. Next, a biochemical analysis of the seeds was carried out to determine the total amount of nitrogen and protein according to the treatment options. The Kjeldahl micro-method was used to determine total nitrogen. As a result of the research, it was found that carbolite at a concentration of 2n in 6- and 18-hour exposures stimulates the content of total nitrogen and protein in the first generation in grains of barley variety Ardak. It has an overwhelming effect on the biochemical processes of barley seeds in the second generation. At the same time, carbolite inhibits the development of barley and causes the appearance of a large number of speltoid and sterile plants. But at the same time, it should be emphasized that a large number of speltoids and sterile plants in the variants treated with carbolite decreases in the second generation. N-nmm at concentrations of 0.012% and 0.025% contribute to an increase in the content of total nitrogen and protein in barley grains. The increased content of nitrogen and protein in the treated N-nmm variant is preserved in the second generation. It should be noted that N-nmm at a concentration of 0.012% is more effective for this variety than at a concentration of 0.025%. Also, N-nmm inhibits the development of barley and causes a large number of morphological changes. In a comparative study of the effect of mutagens N-nmm and carbolite on the content of the total amount of nitrogen and protein, it was found that N-nmm contributes to an increase in the amount of nitrogen and protein, while carbolite decreases.

Calliandra portoricensis ameliorates ovarian and uterine oxido-inflammatory responses in N-methyl-N-nitrosourea and benzo[a]pyrene-treated rats.

Infertility resulting from reproductive impairment is traumatic in families. Exposure to chemicals may play insidious roles not easily connected to infertility. We examined benzo[a]pyrene (BaP), and N-methyl nitrosourea (NMU)-induced ovarian and uterine toxicity and the role of Calliandra portoricensis in mitigating toxicity. In a bid to illuminate folk medical claims cloaked in mystery, unearthing lost knowledge, advance natural chemopreventive agents, and report new evidence lacking in the literature attributed to CP. Although CP is known to exhibit anticonvulsant, antidiarrheal, antipyretic, antirheumatic, and analgesic effects in humans, its possible roles for mitigating toxicity stemming from inadvertent chemical exposures are reported here. Our findings affirm and further show that CP abates toxic response incumbent on oxidative damage and inflammatory responses associated with NMU and BaP exposure. Development of phytochemical derived from CP may serve as a potential natural therapy against chemical toxicities in individuals inadvertently exposed, and promote human health and reproductive satiety.

Deregulation of the SRC Family Tyrosine Kinases in Gastric Carcinogenesis in Non-human Primates.

The evolution of gastric carcinogenesis remains largely unknown, as the regulatory mechanisms involved in the aggressiveness of gastric cancer are still poorly understood. Kinases are downstream modulators and effectors of various cell signaling cascades and play key roles in the development of neoplastic diseases. The objective of this study was to evaluate the expression of genes and proteins of the SRC family, including FYN, YES, BLK, FGR, LYN and SRC, in a model of intestinal gastric carcinogenesis generated by treating Cebus apella, a New World non-human primate, with N-methyl nitrosourea (MNU). mRNA expression of genes was measured by real-time reverse transcription quantitative PCR (RT-qPCR) and protein expression was measured by western blotting in six Cebus apella treated with N-methyl-nitrosourea (MNU) for about 2.5 years. Elevated mRNA and protein expression mainly of the SRC and LYN kinases was observed. Their expression was gradually increasing as non-atrophic gastritis was evolving to gastric cancer. SRC family kinases play a key role in tumor progression and metastasis and may be a promising target for the treatment of gastric cancer.

Effects of Dietary Fat on Mammary Gland Pyroglutamyl Aminopeptidase on Experimental Breast Cancer

Aim: To analyze the relationship between dietary fat and breast cancer mediated by the local functions of gonadotropin-releasing hormone (GnRH), analyzed by its regulating peptidase. The presence of GnRH and its receptors has been demonstrated in tumor tissue of the breast, although the functions of this peptide remain unclear. Pyroglutamyl aminopeptidase (PAP) is the enzyme involved in the local regulation of GnRH, and changes in its soluble and membrane-bound activities have been described in both women with breast cancer and animals with mammary tumors induced by N-methyl nitrosourea (NMU). Methods: We analyze here the effects of different normolipidic (4%) dietary fats (soybean oil (commercial), extra virgin olive oil (EVOO), refined sunflower oil (SO) and refined sunflower oil enriched with 50% oleic acid (OAESO)) on soluble and membrane-bound PAP specific activities in breast tissue of rats with mammary tumors induced by N-methyl-nitrosourea (NMU) administration. Results: We found that animals with breast cancer showed higher levels of soluble PAP than control healthy animals, but in a different degree depending on the type of dietary fat. On the contrary, membrane-bound mammary PAP specific activity was modified only in animals fed on EVOO. Conclusion: Dietary fat is involved in the regulation of GnRH functions mediated by PAP at mammary tumor tissue in different degree depending on its localization and the type of fat administrated.

Abstract 1269: Preventive potentials of the fruits of Detarium microcarpum on N-methyl nitrosourea (MNU) induced colon carcinogenesis in rats

Abstract Detaruim microcarpum Guill. & Perr is a leguminous African plant with significant nutritional and medicinal uses. In an experimental N-methyl nitroso urea (MNU) induced colon carcinogenesis in rats, various concentrations of pulverized D. microcarpum fruits were included in diets of rats and the chemopreventive effects of these supplemented diets were investigated. The rats were divided into 10 groups (n=7) and fed for 10 weeks with diets containing 2.5%, 5.0% and 10.0% D. microcarpum. Subsequently, the feeding continues for another 12 weeks but with concomitant intra-rectal administration of MNU at every 72 hour interval. Respective control groups fed similar concentrations of supplemented diets, normal diet with or without MNU intoxication were also included. All rats were sacrificed at the end of the 22 weeks experiment; blood collected for hematological analysis and serum was separated for carcinoembryogenic antigen (CEA) assay and other biochemical tests. Liver, kidney and colon were also collected for antioxidant and tissue peroxidation assays. Sections of the colon were subjected to histopathological studies and immunohistochemical (IHC) staining using mutL homolog 1 (MLH1) antibody. A significant (p<0.05) elevation was observed in levels of CEA (210 ± 18 pg/ml) in the MNU positive control group when compared with the test groups and the negative control group (70 ± 6 to 140 ±11 pg/ml). There were significant decreases (p<0.05) in the major hematological parameters (PCV, hemoglobin, WBC etc) levels, catalase and superoxide dismutase (SOD) activities in the MNU control group when compared to other groups. A concomitant significant increases (p<0.05) was also observed in the levels of liver function enzymes and kidney function biomarkers as well as thiobarbituric reactive substance (maliondealdehyde) in the MNU control group when compared to the test groups and other controls . It was further observed that these variations in the CEA and antioxidant status markers were dose dependent in many instances. Histopathological and IHC staining of the colons in the treated groups showed mild changes in tissues with no expression of the MLH1 antibody when compared with the MNU control group having moderate damage and expression of the antibody. Hence it was concluded that the inclusion of fruits of D. microcarpum in the diet protected the organs and tissues of the rats from induced MNU toxicity; ameliorated oxidative stress in colon carcinogenesis model and possibly prevented the initiation of the process of carcinogenesis implying that the fruits possess significant health benefits which may have warranted their usage in foods and also in traditional medicine in Africa. Citation Format: Mubarak L. Liman, Sunday E. Atawodi. Preventive potentials of the fruits of Detarium microcarpum on N-methyl nitrosourea (MNU) induced colon carcinogenesis in rats [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 1269. doi:10.1158/1538-7445.AM2017-1269

Design, synthesis and anticancer screening of 3-(3-(substituted phenyl) acryloyl)-2H-chromen-2ones as selective anti-breast cancer agent.

By utilizing concept of molecular hybridization, involving combination of various Pharmacophore, novel substituted coumarin-chalcone hybrids was synthesized and evaluated for anti-proliferative activity against estrogen receptor-positive MCF-7 and negative MDA-MB-435 breast cancer cell lines. In-vivo study was carried out by N-methyl nitrosourea (MNU) induced mammary carcinoma in virgin female Spraque Dawly (SD) rats. The compound 5b has highest potential than standard drug Adriamycin, comparable against Tamoxifen against ER-positive MCF-7 breast cancer cell lines. Docking study was performed to study the binding orientation and affinity of synthesized compounds on the ER-α enzyme.

Protective effect of Chuquiraga spinosa extract on N-methyl-nitrosourea (NMU) induced prostate cancer in rats.

BackgroundThe main objective was to evaluate the possible protective effect of Chuquiraga spinosa extract on N-methyl nitrosourea (NMU)-induced prostate cancer in rats and DU-145 cell line.Materials and methodsProstate carcinogenesis was induced in 30 male Holtzman rats by providing cyproterone acetate, testosterone, and NMU. The tumors were monitored and hematological and biochemical parameters and frequency of micronucleated polychromatic erythrocytes were recorded. The cell line was assessed by a cytotoxicity assay.ResultsOral administration of C. spinosa extract significantly lowered superoxide dismutase malondialdehyde, NO, C-reactive protein, and prostate-specific antigen levels (all P < 0.01 compared with Inductor Group). There was a significant decrease in the frequency of micronucleated polychromatic erythrocytes (P < 0.05). C. spinosa presented a selectivity index of 17.24 in the cytotoxicity assay.ConclusionsConsidering its anti-inflammatory, antioxidant, and antigenotoxic effects, and important variations on biochemical and hematological parameters, including prostate-specific antigen of C. spinosa extract, we conclude that it has a protective effect on NMU-induced prostate cancer in rats and cytotoxicity in the DU-145 cell line.

α-Chymotrypsin regulates free fatty acids and UCHL-1 to ameliorate N-methyl nitrosourea induced mammary gland carcinoma in albino wistar rats.

This study was undertaken to investigate the effect of α-chymotrypsin on methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats. Animals were randomized into four groups (six animals in each). Group I (sham control 0.9% normal saline p.o.); Group II (toxic control, MNU 47mg/kg, i.v.); Group III (α-chymotrypsin, 5mg/kg, p.o.); Group IV (α-chymotrypsin, 10mg/kg p.o.). Toxicity was induced by single i.v. injection of MNU followed by α-chymotrypsin supplementation therapy for 100days. MNU treatment was evident with increased alveolar bud count, differentiation score, upregulated inflammatory enzymes markers (COX, LOX and NO) antioxidative stress markers (TBARs, SOD, catalase and GSH).MNU associated toxicity was also ascertained by PGP 9.5 and NF-κB expression in the mammary gland tissue followed by FAME analysis for fatty acid profiling. α-chymotrypsin afforded significant protection against the deleterious effects of MNU.

Anticancer and Cancer Prevention Effects of Piperine-Free Piper nigrum Extract on N-nitrosomethylurea-Induced Mammary Tumorigenesis in Rats.

Piper nigrum (P. nigrum) is commonly used in traditional medicine. This current study aimed to investigate the anticancer and cancer preventive activity of a piperine-free P. nigrum extract (PFPE) against breast cancer cells and N-nitrosomethylurea (NMU)-induced mammary tumorigenesis in rats. The cytotoxic effects and the mechanism of action were investigated in breast cancer cells using the MTT assay and Western blot analysis, respectively. An acute toxicity study was conducted according to the Organization for Economic Co-operation and Development guideline. Female Sprague-Dawley rats with NMU-induced mammary tumors were used in preventive and anticancer studies. The results showed that PFPE inhibited the growth of luminal-like breast cancer cells more so than the basal-like ones by induction of apoptosis. In addition, PFPE exhibited greater selectivity against breast cancer cells than colorectal cancer, lung cancer, and neuroblastoma cells. In an acute toxicity study, a single oral administration of PFPE at a dose of 5,000 mg/kg body weight resulted in no mortality and morbidity during a 14-day observation period. For the cancer preventive study, the incidence of tumor-bearing rats was 10% to 20% in rats treated with PFPE. For the anticancer activity study, the growth rate of tumors in the presence of PFPE-treated groups was much slower when compared with the control and vehicle groups. The extract itself caused no changes to the biochemical and hematologic parameters when compared with the control and vehicle groups. In conclusion, PFPE had a low toxicity and a potent antitumor effect on mammary tumorigenesis in rats.

Biochemical role of phytoestrogen (Chrysin) against N-methyl nitrosourea (NMU)- induced proliferative lesions in mammary glands of postmenopausal female rat

This study aimed to investigate the role of the phytoestrogen (chrysin) in the treatment or protection of NMU-induced preneoplastic lesions in mammary glands of postmenopausal female rats. Rats (n=105) were divided into seven groups (n=15/group); group1(negative control group).Groups 2&3 (positive control groups), rats were injected intraperitoneally (i.p.) with 4 doses of NMU (75 mg/kg b.w.,once/5days) before or after oral administration of glycofurol. Groups 4 &5(treatment groups),rats received 4 doses of NMU (75 mg/kg b.w., once/5days,i. p.), left for 6 weeks then followed by 3 doses, per week, of chrysin (125 or 250 mg/kg b.w., orally; p.o), respectively, for another 6 weeks. Groups 6 & 7(protective groups), rats received 3 doses, per week, of chrysin(125 or 250 mg/kg b.w.,p.o.), for 6 weeks then followed by 4 doses of NMU (75 mg/kg b.w., once/5days, i.p.) then left for another 6 weeks. The NMU injection caused significant decrease in PLT count, lymphocytes %, Hb, MCH, MCHC, E2, TAC levels as well as GST and CAT activities. The results also showed that NMU significantly elevated WBC count, granulocytes%, monocytes %, MCV, RDW%, CRP, CEA levels and arginase activity. Either treatment or protection with chrysin modulated the adverse effects of NMU and ameliorated the change in the biochemical parameters. The biochemical observations were also confirmed by histological studies. In conclusion, phytoestrogens may relieve the severity of postmenopausal pre-cancerous disorders, especially when consumed in high dose for a while before the incidence of lesions.

Protective Effect of Helix aspersa Extract against Spleen and Liver Injuries Induced by N-Nitroso Methyl Urea in Mice

Objective: This study was carried out to investigate the protective effect of an aqueous extract, from the garden snail Helix aspersa (Mollusca, pulmonate), against N-nitrosomethylurea (NMU) induced damage in mice. Methods: Haematoxylin/ Eosin based histology was performed to evaluate the morphological changes in liver and spleen. Results: our results show that NMU causes different damages in liver and spleen. It induces alterations of blood system of the two organs with the apparition of hemangioma. The histological assessment shows that H. aspersa extract markedly reduces the damage induced by the carcinogen and preserved the histo-architecture of these tissues. Furthermore, histopathological finding demonstrates that H. aspersa extract increase immune system activity through the stimulation of immune cell penetration in liver and differentiation of macrophages into foreign body giant cells. Conclusion: These results suggest that H. aspersa extract acts as a potent hepatic and spleen protective agent against NMU carcinogenesis.

Normolipidic dietary fat modifies circulating Renin-Angiotensin system-regulating aminopeptidase activities in rat with breast cancer.

Renin-angiotensin system (RAS) has been considered not only as a regulator of systemic volume and electrolyte balance but also has been recently involved in various pathological processes such as cancer. In the etiology of breast cancer, dietary factors have been analyzed and especially the influence of dietary fat has been studied, but the underlying mechanisms remain unclear. In this study, we analyzed RAS-regulating enzymes in serum of rats with N-methyl nitrosourea (NMU)-induced breast cancer fed with different diets. Four groups of rats were injected intraperitoneally with 3 doses of 50 mg/kg body weight of NMU at different days after birth and were fed with an AIN-93 commercial diet or AIN-93 diets with 4% fat constituted respectively by extra virgin olive oil, refined sunflower oil, and refined sunflower oil enriched to 50% with oleic acid. After sacrifice, blood and tumor samples were collected by spectrophotometric determinations of RAS-regulating enzymes in plasma and histopathology studies. We show that the type of dietary fat does not influence latency period, incidence of animals with tumors, incidence of mortality, or tumor yield per rat. However, changes were observed in tumor volume and the histopathology. The type of dietary fat also differently modified the enzymes involved in RAS regulation. It might suggest that one of the mechanisms by which dietary fat affects breast cancer is the modification of the RAS system, which may be consider as a new target for integrative therapies.

An Anthocyanin-Rich Extract from Hibiscus sabdariffa Linnaeus Inhibits N-Nitrosomethylurea-Induced Leukemia in Rats

A previous study reported that anthocyanins from roselle (Hibiscus sabdariffa L.) showed significant anticancer activity in human promyelocytic leukemia cells. To explore the antitumor effect of anthocyanin, a roselle bioactive polyphenol in a rat model of chemical-induced leukemia was assayed. Anthocyanin extract of roselle (Hibiscus anthocyanins, HAs) was supplemented in the diet (0.1 and 0.2%). This study was carried out to evaluate the protective effect of HAs on N-nitrosomethylurea (NMU)-induced leukemia of rats. The study employed male Sprague-Dawley rats (n = 48), and leukemia was induced by intravenous injection of 35 mg kg(-1) body weight of NMU dissolved in physiologic saline solution. The rats were divided into four groups (n = 12): control, NMU only, and HAs groups that received different doses of HAs (0.1 and 0.2%) daily, orally, after NMU injection. After 220 days, the animals were killed, and the following parameters were assessed: morphological observation, hematology examination, histopathological assessment, and biochemical assay. When compared with the NMU-only group, HAs significantly prevented loss of organ weight and ameliorated the impairment of morphology, hematology, and histopathology. Treatment with HAs caused reduction in the levels of AST, ALT, uric acid, and MPO. Also, the results showed that oral administration of HAs (0.2%) remarkably inhibited progression of NMU-induced leukemia by approximately 33.3% in rats. This is the first report to demonstrate that the sequential administration of HAs followed by NMU resulted in an antileukemic activity in vivo.

Evaluation of apoptosis using TUNEL assay and caspase-3 immunostaining in NMU induced rat mammary tumours

Avoidance of apoptosis is an important event of tumourigenesis. Tumour progression is dependent on balance between cell proliferation and apoptosis. The present study was carried out to evaluate apoptosis in N-methyl nitrosourea (NMU) induced rat mammary tumours by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL) assay in correlation with caspase-3 and PCNA immunostaining. A total of 22 female virgin Wistar rats of 28–30 days of age were selected for the experiment. NMU was injected at 50th, 80th and 110th days of age in 16 rats and 6 control group rats received the vehicle only. Six numbers of mammary tumours were successfully induced in 4 (25%) rats with average latency period of 87±2.63 days. The mean counts of PCNA and caspase-3 immunostaining were 57.67±9.45 and 1.82±0.21, respectively. The mean count of the TUNEL positive cells were 2.44±0.69. It was observed that apoptosis was evident by TUNEL assay and caspase-3 immunostaining in highly proliferative and more aggressive NMU induced rat mammary tumours, similar to that observed in human breast tumours.

Upregulated Myc Expression in N-Methyl Nitrosourea (MNU)-induced Rat Mammary Tumours

The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75- 91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

Intestinal Formation of N-Nitroso Compounds in the Pig Cecum Model

N-Nitroso compounds (NOC) are a group of compounds including N-nitrosamines and N-nitrosamides, which are well-known for their carcinogenic, mutagenic, and teratogenic properties. Humans can be exposed to NOC through the diet and environmentally, or NOC can be formed endogenously in the stomach and intestine. In the intestine, the formation of NOC is supposed to be afforded by the gut microbiota. In this study, the formation of the N-nitrosamines, N-nitrosomorpholine (NMOR) and N-nitrosopyrrolidine (NPYR), and the N-nitrosamides, N-nitrosomethylurea (NMU) and N-nitrosoethylurea (NEU), was investigated in the pig cecum model after the incubation of the corresponding precursor amine or amide with nitrite or nitrate. Following the incubation with nitrate, the formation of NMOR, NPYR, NMU, and NEU was detectable with the microbiota being responsible for the reduction of nitrate to nitrite. After the incubation of nitrite a chemical formation of NOC was shown.

Chemopreventive and anti‐angiogenic effects of dietary phenethyl isothiocyanate in an N‐methyl nitrosourea‐induced breast cancer animal model

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.

Hypothalamus–pituitary–thyroid axis disruption in rats with breast cancer is related to an altered endogenous oxytocin/insulin-regulated aminopeptidase (IRAP) system

Associations of breast cancer with diseases of the thyroid have been repeatedly reported, but the mechanism underlying this association remains to be elucidated. It has been reported that oxytocin (OXT) attenuates the thyroid-stimulating hormone (TSH) release in response to thyrotrophin-releasing hormone (TRH) and decreased plasma levels of TSH as well as the thyroid hormones by an effect mediated by the central nervous system. Oxytocinase (IRAP) is the regulatory proteolytic enzyme reported to hydrolyze OXT. Changes in IRAP activity have been reported in both human breast cancer and N-methyl-nitrosourea (NMU)-induced rat mammary tumours. Here, we measure IRAP activity fluorometrically using cystyl-β-naphthylamide as the substrate, in the hypothalamus-pituitary-thyroid axis together with the circulating levels of OXT, and its relationship with circulating levels of TSH and free thyroxine (fT4), as markers of thyroid function in control rats and rats with breast cancer induced by NMU. We found decreased thyroid function in rats with breast cancer induced by NMU, supported by the existence of lower serum circulating levels of both TSH and fT4 than their corresponding controls. Concomitantly, we found a decrease of hypothalamic IRAP activity and an increase in circulating levels of OXT. We propose that breast cancer increases OXT pituitary release by decreasing its hypothalamic catabolism through IRAP activity, probably due to the alteration of the estrogenic endocrine status. Thus, high circulating levels of OXT decreased TSH release from the pituitary, and therefore, of thyroid hormones from the thyroid, supporting the association between breast cancer and thyroid function disruption.

Cyanine dyes as contrast agents for near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluorescence imaging

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1(')-bis-(4-sulfobutyl) indotricarbocyanine-5,5(')-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

Mammary renin–angiotensin system-regulating aminopeptidase activities are modified in rats with breast cancer

Angiotensin II in particular and/or the local renin-angiotensin system in general could have an important role in epithelial tissue growth and modelling; therefore, it is possible that it may be involved in breast cancer. In this sense, previous works of our group showed a predominating role of angiotensin II in tumoral tissue obtained from women with breast cancer. However, although classically angiotensin II has been considered the main effector peptide of the renin-angiotensin system cascade, several of its catabolism products such as angiotensin III and angiotensin IV also possess biological functions. These peptides are formed through the activity of several proteolytic regulatory enzymes of the aminopeptidase type, also called angiotensinases. The aim of this work was to analyse several specific angiotensinase activities involved in the renin-angiotensin system cascade in mammary tissue from control rats and from rats with mammary tumours induced by N-methyl-nitrosourea (NMU), which may reflect the functional status of their target peptides under the specific conditions brought about by the tumoural process. The results show that soluble and membrane-bound specific aspartyl aminopeptidase activities and membrane-bound glutamyl aminopeptidase activity increased in mammary tissue from NMU-treated animals and soluble aminopeptidase N and aminopeptidase B activities significantly decreased in mammary tissue from NMU-treated rats. These changes support the existence of a local mammary renin-angiotensin system and that this system and its putative functions in breast tissue could be altered by the tumour process, in which we suggest a predominant role of angiotensin III. All described data about the renin-angiotensin system in mammary tissue support the idea that it must be involved in normal breast tissue functions, and its disruption could be involved in one or more steps of the carcinogenesis process.