C3HA Mice Research Articles

Iron oxide nanoparticles as potential agents for combined radiotherapy

Background. Iron oxide nanoparticles (NP) represent a promising theranostic platform for combined radiotherapy: the reactivity of iron oxide enhances oxidative stress of tumor cells associated with irradiation while magnetic properties may provide additional feature as controlled delivery.Aim. To study the potency of heparinized iron oxide NP in experimental antitumor therapy.Materials and methods. The synthesis of iron oxide NP was carried out by chemical precipitation followed by magnetic separation, the resulting sol was stabilized with heparin. For each batch of newly synthesized particles, the hydrodynamic diameter was determined, IR spectrometry, X-ray diffraction analysis, and scanning electron microscopy were performed. The MX-7 tumor model of rhabdomyosarcoma chosen for the study was transplanted into female C3HA mice; NP were administered intratumorally or intravenously, once a day, according to the “5–2–5” scheme. Fractional irradiation (1–2 Gy / fraction; 1.3±0.15 Gy / min) was carried out after NP administration.Increasing life expectancy (ILE), the degree of tumor growth inhibition (TGI), a pathomorphological assessment of the lung, liver, spleen and tumor node was carried out for all experimental mice.Results. As a result of the study, it was found that when administered intratumorally, heparinized iron oxide NP are retained inside the tumor, providing a moderate additive effect, compared with isolated radiotherapy in the first week of irradiation (TGI = 40 % (day 6), TFD = 10 Gy, p <0.05), however, with an increase in tumor volume by the end of the second week, the treatment regimen was not more effective than radiotherapy. with a combination of radiotherapy and intravenous administration of NP, the effect was observed within two weeks (TGI = 43 % (day 6), TGI = 29 % (day 14), TFD = 10 Gy; p <0.05; ILE = 54 %, TFD = 20Gy; p <0.05).Conclusion. The studied iron oxide nanopreparation enhanced capacity of radiation therapy to inhibit tumor growth when administered intravenouslyin experimental mice with rhabdomyosarcoma and irradiated subsequently.

Zinc supplementation prevents the development of thymic involution induced by tumor growth in mice

Aim. The aim of this study was to investigate the influence of zinc supplementation on the development of thymic involution during the growth of transplantable tumor in mice. Materials and methods. Inbred C3HA mice after subcutaneous syngeneic hepatoma 22a inoculation received zinc sulfate with drinking water. After 3 weeks of tumor growth animals were killed, and evaluated for zinc content in serum blood samples, thymus status and parameters of tumor growth. Results. At the 21 day of tumor 22a growth weight and cellular content of the thymus were decreased by 3 times and endogenous zinc content in samples of blood serum was decreased by 1,9 times. Oral zinc sulfate supplementation at concentration of 22 mkg/ml in drinking water during 3 weeks, started from the first day after tumor inoculation, increased thymus weight and cellularity as well as serum zinc content. At the same time, it did not influence tumor size and survival rate of mice with hepatoma 22a. As a possible mechanism of zinc action, we studied the influence of zinc supplementation on the activity of two anti-oxidative defense enzymes: Cu,Zn-superoxide dismutase and catalase in the thymus. Zinc supplementation had no influence on these parameters, both remained at a higher level in tumor-bearing mice. Conclusion. Oral zinc supplementation in hepatoma 22a bearing mice induced retards the development of thymic involution with no influence on the tumor growth per se. The study allows to suggest that oral zinc administration may be considered as a prospective strategy for thymus reconstitution in oncology patients.

Содержание моноаминов в симметричных зонах переднего мозга при длительном изменении тиреоидного статуса

In male C3H-A mice the effect of prolonged (44 weeks) thyroxine hyper- and propylthiourаcyl hypothyroidism on the hormonal status and content of monoamines and their metabolites in the symmetric parts of the forebrain was studied. The level of hormones in the blood serum was determined by enzyme-linked immunosorbent analysis and the content of monoamines (MA) and their metabolites in the brain was determined by HPLC. Experimental hyper- and hypothyroidism did not cause opposite changes in the MA content. So, with any change in thyroid status in the hippocampus and tuberculum olfactorium the level of serotonin (5-HT) increased. At the same time the content of norepinephrine (NA) in the striatum decreased and in the tuberculum olfactorium increased. The level of dopamine (DA) in the striatum and tuberculum olfactorium in animals of both experimental groups increased, however, in hypothyroid mice this effect was observed bilaterally and in hyperthyroid mice it was unilateral; only in the left striatum and right tuberculum olfactorium. In addition to these changes in experimental hyperthyroidism there was a decrease in the level of DA in the cerebral cortex, and in hypothyroidism, an increase in 5-HT in the right striatum and in the right cortex. Most characteristics of monoaminergic systems did not correlate with the content of thyroxin (T4) but correlated with the levels of thyrotropin (TSH) and prolactin (PL) in the blood. At the same time no correlation was found between the content of these hormones and T4, although TSH and PL levels correlated with each other. It can be assumed that the leading role in the mechanisms of the influence of a prolonged change in thyroid status on the monoaminergic systems of the brain is mediated not by T4 but by TSH.

Host antigen modulated anti-influenza immunity with antigen level dependent distinct mechanisms of severe influenza

Abstract Some healthy individuals suffer from severe disease and even mortality with the same strain of influenza virus that causes self-limited illnesses in the majority of people. This fact exemplifies the importance of host factors in influenza disease manifestation. Our C3-HA transgenic mice express hemagglutinin (HA) as a self-antigen that is identical to the HA of PR8 influenza virus. Compared to wild type mice, C3-HA mice incurred severe disease with PR8, but not with HA-mismatched influenza virus. Self-HA in C3-HA mice altered the immune response to the infection and impaired clearance of the influenza virus. However, different mechanisms contribute for exacerbated disease in C3-HA mice with different level of HA. In C3-HALow mice, low-level self-HA modulated HA specific immunity upon infection with less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both HA-specific CD4+ and CD8+ T cells on day 4- post infection. The T cell activation status contracted further on day 7- post infection. Impaired immunity resulted in delayed virus clearance leading to severe disease and death. In C3-HAHigh mice, high-level self-HA also modulated impaired immunity and virus clearance on day 4- post infection. In contrast to further reduction on day 7- post infection in C3-HALow mice, the HA specific T cell immunity was higher on day 7- than day 4- post infection in C3-HAHigh mice. The boosted HA specific immunity resulted in severe disease with autoimmunity as a predominant feature in pathology. Our animal model exemplified the complicated interaction between cross-reactive self-antigen and influenza virus with severe disease as the results of antigen level dependent distinct mechanisms.

Natural killer cell activity irreversibly decreases after Cryptosporidium gastroenteritis in neonatal mice.

Cryptosporidiosis causes persistent diarrhoea in infants, immunocompromised patients and elderly persons. Long-term consequences of the disease include increased risk of malignancy, cardiomyopathy and gastrointestinal inflammation. This study aimed to investigate prolonged effects of cryptosporidiosis on innate immunity and growth in neonatal C3HA mice. The disease was challenged by Cryptosporidium parvum oocyst inoculation into 7-day-old animals. The mice whose intestine smears contained 3-5 or 6 and more oocysts per microscopic field at the day 5 after infection were considered as mildly or severely infected, correspondingly. To determine natural killer cell (NK) activity, we applied 3 H-uridine cytotoxic assay to the animals at 5-68days after infection using K562 cells as targets. At severe infection, there was a statistically significant 1.5-2.0 fold decline of body mass, spleen mass and spleen cellularity that persisted in animals of all ages. Accordingly, NK cytotoxicity showed even more drastic drop reaching 2.7-3.0 folds that was statistically significant in all animals. At mild infection, the discovered effects were less pronounced and reached significance only in some age groups. Thus, our study provides evidence that NK cells show long-term cytotoxic activity decrease following Cryptosporidium infection in neonatal mice, particularly in severe disease.

Pre-existing matched antigen exacerbates the disease of influenza virus infection by attenuating antigen-specific T cell immunity

Abstract Influenza virus infection causes mild and self-limiting illness in most of the healthy individuals, but some healthy people develops complications as a result of the infection and suffers from severe disease and eventually dies. Here we report that pre-existing matched antigens attenuate antigen-specific immunity to the infection. High virus load and associated increase of pathogenic innate response in the lungs exacerbates the disease. The C3-HAHigh and C3-HALow transgenic mice are with inherent expression of Hemagglutinin (HA) antigen from PR8 strain of H1N1 influenza virus. Compared to wild type mice, these mice suffered from severe disease and higher mortality upon infection of wild type PR8 virus but not upon infection of reverse genetically raised PR8 virus with replaced HA from H3N2 influenza virus. Adoptively transferred PR8 virus HA-specific 6.5 CD4+ and Clone-4 CD8+ T cells responded minimally to PR8 virus infection in C3-HA mice. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both 6.5 CD4+ and Clone-4 CD8+ T cells. Virus neutralizing antibodies were less as well. The attenuation was associated with increased LAG-3+ but not Foxp-3+ population of HA-specific T cells in the lungs. In contrast to the instigated weak antigen-specific immunity, PR8 virus infection caused neutrophils more efficient for inflammatory IL-6 secretion and robust bronchus infiltration in C3-HA mice. These results suggest that a host factor cross-reactive to the antigens of influenza virus may exacerbate the disease with similar attenuated antigen-specific adaptive immunity accompanied by augmented innate immunity to influenza virus infection.

A99: The influence of live Streptococcus pyogenes on the growth of solid murine tumors

Ability of Streptococcus pyogenes to fight cancer was discovered and clinically tested more than 100 years ago (Coley, 1897), but the mechanisms of these effects are unknown. Recently several murine models demonstrating the anti-cancer activity of Group A Streptococcus (GAS) M49 were established (Maletzki, 2008). The present study is devoted to investigation of anti-cancer activity of GAS M39 strains. Materials and Methods Tumor cells were injected subcutaneously in concentration of 200,000 cells per animal. Two solid tumor models were used: hepatoma 22a in C3HA mice and sarcoma 37 in Swiss mice. GAS (wild and mutant strains) were injected into the tumor 2 × 104 per animal twice with a 5-day-interval. The influence of GAS on tumor growth was evaluated by measurement of tumor diameter and animal survival. Inactivation of M protein (emm) gene was generated by insertional mutagenesis after cloning the fragment of the emm gene in suicidal plasmid pT7ermB.The plasmid was introduced into GAS by electroporation. Insertion had been proved by DNA sequencing. Results Intratumoral injections of wild GAS M39 strain did not influence on tumor size in mice bearing hepatoma 22a and sarcoma 37. The second injection of GAS enhanced inhibition of the tumor growth, but increased the death rates. The treatment by emm mutant strain caused the inhibition of tumor growth and showed less mortality rate both tumor models. The average number of completely cured animals was 10%. Conclusion Inactivation of emm gene in GAS M39 strain resulted in increased anti-tumor activity and decreased lethality rate. We hypothesized that the absence of anti-phagocytic M protein may increase phagocytosis of GAS by macrophages and thus enhance their antitumor activity.

Sex differences of the content on the monoamines levels in symmetrical structures of the C3H-A mice brain

The Sex differenses in the content and metabolism of dopamine and serotonin were studied in symmetrical brain structures of C3H-A mice. With HPLC the contents of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their metabolites, such as dihydroxyphenylacetic acid (DOPAC), homovanillinic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), were measured in the cortex, tuberculum olfactorium, hippocampus and striatum of both the right and the left hemispheres of the brain in male and female mice. The following sex differences in monoamines and their metabolites in brain areas were found: the NE content was higher in the male striatum and in the female tuberculum olfactorium; in males the DA content in cortex and hippocampus was higher, but in tuberculum olfactorium and striatum was lower than that in females; in females the 5-HT and 5-HIAA levels in hippocampus and tuberculum olfactorium were hither than that in males. In the female left striatum the 5-HIAA content was higher than in males. In males three cases of neurochemical cerebral hemisphere asymmetries were found: 1) the NE content is higher in the right tuberculum olfactorium, 2) the DA level is higher in the right hippocampus, 3) the 5-HIAA content is higher in the left hippocampus. In females the only one case of cerebral asymmetry was found, i. g. the 5-HT level was higher in the right tuberculum olfactorium.

Morphological changes in the breast gland and ovary of mice with experimentally induced hyperthyroid condition

The frequency of breast tumors has been studied in inbreed C3H-A mice with affected thyroid status. It has been shown that frequency of breast tumors was significantly higher in the animals with hyperthyroid condition versus the hypothyroid group (р<0.01). The development of tumors was found on the 21, 29, and 36 weeks of the experiment for hyperthyroid, euthyroid, and hypothyroid, groups correspondingly. The frequency of breast tumors was 86.6, 62.5 and 30.0 % for hyperthyroid, euthyroid, and hypothyroid, correspondingly, at the end the experiment. Throughout the experiment the frequency of breast tumors in hypothyroid group was significantly lower as compared to the frequency of breast tumors in euthyroid and hyperthyroid as well. Morphological study elucidated the changes in ovary of mice with hyperthyroid status. The profound atrophic changes of the gland structures, absence of generative function, chronic inflammation, and development tubular adenoma were detected in the ovary of the hyperthyroid mice. In some animals the mammary glands were affected by malignant transformation characterized by development of glandular epithelial complexes as clear outlined tubes. It could be suggested that those tumors represent ductal adenocarcinoma. The data clear prove the idea that hyperthyroid condition triggers development of breast tumors.

The Incidence of Breast Tumor during Experimental Hyperthyroidism

The incidence of breast tumor in inbred C3H-A mice (virgin female) from hyperthyroid, hypothyroid, and euthyroid control groups at week 44 of the experiment was 86.67, 30, and 62.5%, respectively. The percentage of metastatic lymph nodes in these groups was 65.9±16.7, 35.9±5.9, and 53.9±7.2%, respectively (p<0.005). It was shown that hyperthyroidism was associated with increased incidence and aggressiveness of breast tumor, while hypothyroidism was associated with lower incidence of breast tumor in comparison with the control.

The influence of experimentally changedthyroid status on cognitive activity and angiogenesis in the brain of female inbred С3Н-А mice

The study was performed using 33 virgin females of inbred C3H-A mice. The animals were randomized as follows 3 : 2 : 2. The first group was hyperthyroid (n1 = 15), the second hypothyroid (n2 = 10) and the third euthyroid (control) (n3 = 8). The results of individual behavior investigation as to 18 and 40 weeks of our experiment in “open field” test clarified that quantitative traces of almost whole components of cognitive activity was much higher in hyperthyroid mice as compared to other groups (control and hypothyroid ones). The cognitive behavior activity has gradually been decreasing in hypothyroid group especially in connection to burrow reflex. As to those elements of behavior which are of characteristics of emotions the results were a bit different. The emotional characteristics were more expressed in hyperthyroid group as compared to control and hypothyroid ones. The immunohistochemical investigations clearly showed that expression of vascular endothelial growth factor (VEGF) in neocortex and hyppocampus prevails in hyperthyroid mice and less expressed in animals deprived of thyroid hormones (p &lt; 0.05). The level of glial fibrillar acidic protein (GFAP) expression was significantly lower in brain of hypothyroid animals (p &lt; 0.05). Otherwise the level of platelet derived growth factor receptor-α (PDGF-Rα) expression was much higher in brain of hypothyroid mice (p &lt; 0.05). Our results presented for this paper confirmed the key role of thyroid hormones in regulation of cell interaction not only for developing neural cells but also for adult central nerve system.

Inhibitor of Indoleamine-2,3-Dioxygenase 1-Methyl-D-Tryptophan Can Stimulate the Growth of Immunogenic Tumors

We studied the effect of 1-methyl-D-tryptophan, an inhibitor of indoleamine-2,3-dioxygenase, on the growth of transplanted hepatocarcinoma-29 in C3HA mice. Hepatocarcinoma-29 transplanted into the thigh muscles undergoes immunological rejection in more than 50% non-syngeneic recipients. Chronic local administration of 1-methyl-D-tryptophan promotes progressive growth of the tumor in recipient mice leading to 100% animal death. The stimulating effect of 1-methyl-D-tryptophan on tumor growth is discussed.

PCR-detected genome polymorphism in malignant cell growth

In this chapter, we analyze the problem of genetic polymorphism in tumorigenesis, which determines basic capacities of tumors. The study of genome polymorphism with modified PCR methods allows the detection of various forms of polymorphism in tumor cells. This method has made it possible to determine association of DNA polymorphism with conditions of oncogenes, antioncogenes, and genes of apoptosis and with their allelic states. A special type of nonspecific DNA polymorphism that resulted from an increase in the mutation number in the cancer cell genome was discovered. This phenomenon was called the microsatellite mutator phenotype. Because the type of DNA polymorphism correlates with various biological capacities of malignant tumors and has an important prognostic significance, the analysis of DNA polymorphism in benign and malignant tumors of different histogenesis will play an important role both in theoretical studies of cancer and in oncological practice. A modified B1-PCR was used to study the genome polymorphism in the mouse tumor cells. The gain of the band 470 bp and the loss of the band 600 bp were revealed in the hepatoma cell line MH-22a as compared with liver cells of C3HA mice. The differentiation of teratocarcinoma EC F9 cells to endoderm-like cells was not accompanied by any changes in the B1-AF DNA fingerprint.

Binding and the nature of Cu(II) ion interaction with nucleosomes

The energetics of Cu (II) ion binding to mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells was determined from their binding isotherms by equilibrium dialysis and pulse high frequency inductively coupled plasma atomic emission spectroscopy. Anticooperative binding of copper ions with normal and tumor mononucleosomes were observed under various NaCl concentrations (0.002; 0.02; 0.2 M). The binding constants of Cu(II) ion with normal mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.10×10 4,5.22×10 4,4.31×10 4 respectively. The binding constants of Cu(II) ion with tumor mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.68×10 4,6.12×10 4,4.82×10 4 respectively.

Binding and the nature of Co(II), Ni(II), Zn(II) ion interaction with nucleosomes

The energetics of Co(II), Ni(II), and Zn(II) ions binding to mononucleosomes from C3HA mice liver and the ascitic hepatoma 22A cells was determined from their binding isotherms by equilibrium dialysis and pulse high-frequency inductively coupled plasma atomic emission spectroscopy. Negative interactions of the metal ions in normal and tumor mononucleosomes were observed. The affinity of metal ions to both types of mononucleosomes follows the Irving-Williams stability sequence: Ni(II) > Co(II) > Zn(II). Investigation of the interaction of the first row transition metals with normal and tumor mononucleosomes has revealed that Ni(II) shows significant differences in the binding constants.

1,2-dimethylhydrazine carcinogenesis in C3HA and CBA female mice prenatally treated with diethylstilbestrol

C3HA and CBA female mice received a single intraperitoneal (i.p.) injection of 0.1 or 0.3 mg/kg body weight (b.w.) of diethylstilbestrol (DES) at day 17 of pregnancy. The descendants, starting from the age of 2-3 months, were receiving weekly subcutaneous (s.c.) injections of 1,2-dimethylhydrazine (DMH) (8 mg/kg b.w.), total 20 injections. The survival of C3HA mice treated with DMH together with prenatal DES was considerably better than in mice treated with DMH alone, this being due to the strong inhibiting effect of DES on the induction by DMH of the hemorrhagic ovarian lesions (78.4% in DMH alone vs. 53.3 and 43.7% in groups with DES plus DMH), which frequently were the cause of animal death. Prenatal DES also inhibited the induction by DMH of clitoral gland tumors: 51.4% in the group with DMH alone vs. 26.6 and 28.1% in two groups of DES plus DMH, respectively. DES treatment, at the above doses, did not influence significantly the DMH carcinogenesis in CBA mice. Prenatal DES given to CBA mice at the dose of 1 mg/kg b.w. significantly increased the incidence of DMH-induced uterine sarcomas (42.8% vs. 73.3% in the group with DMH alone and the group receiving DMH together with prenatal DES, respectively) and accelerated their growth. The effects of prenatal DES on DMH-induced carcinogenesis correlated with the degree of hyperestrogenization produced in both strains of mice by DES.

1,2‐dimethylhydrazine carcinogenesis in C3HA and CBA female mice prenatally treated with diethylstilbestrol

C3HA and CBA female mice received a single intraperitoneal (i.p.) Injection of 0.1 or 0.3 mg/kg body weight (b.w.) Of diethylstilbestrol (DES) at day 17 of pregnancy. The descendants, starting from the age of 2–3 months, were receiving weekly subcutaneous (s.c.) Injections of 1,2-dimethylhydrazine (DMH) (8 mg/kg b.w.), total 20 injections. The survival of C3HA mice treated with DMH together with prenatal DES was considerably better than in mice treated with DMH alone, this being due to the strong inhibiting effect of DES on the induction by DMH of the hemorrhagic ovarian lesions (78.4% in DMH alone vs. 53.3 and 43.7% in groups with DES plus DMH), which frequently were the cause of animal death. Prenatal DES also inhibited the induction by DMH of clitoral gland tumors: 51.4% in the group with DMH alone vs. 26.6 and 28.1% in two groups of DES plus DMH, respectively. DES treatment, at the above doses, did not influence significantly the DMH carcinogenesis in CBA mice. Prenatal DES given to CBA mice at the dose of 1 mg/kg b.w. significantly increased the incidence of DMH-induced uterine sarcomas (42.8% vs. 73.3% in the group with DMH alone and the group receiving DMH together with prenatal DES, respectively) and accelerated their growth. The effects of prenatal DES on DMH-induced carcinogenesis correlated with the degree of hyperestrogenization produced in both strains of mice by DES. Teratogenesis Carcinog. Mutagen. 17:19–28, 1997. © 1997 Wiley-Liss, Inc.

Erratum to “estrogen modification of 1,2-dimethylhydrazine carcinogenesis in C3HA mice”

Erratum to “estrogen modification of 1,2-dimethylhydrazine carcinogenesis in C3HA mice”

Estrogen modification of 1,2-dimethylhydrazine carcinogenesis in C3HA mice.

Considerable strain differences were reported in our previous studies in the induction by 1,2-dimethylhydrasine (DMH) of uterine sarcoma and hemorrhagic ovarian lesion (HOL) in mice: the incidence of the former was high and that of the latter was low in CBA mice while the reverse was observed in C3HA mice. The present study confirmed the above observation in that C3HA mice treated with DMH developed high incidence (59%) of HOL and no uterine sarcoma. Combined treatment of C3HA mice with DMH and estradiol dipropionate (EP) completely inhibited the induction of HOL and increased the incidence of uterine sarcomas from naught to 28%. EP significantly decreased (from 61 to 19%) the incidence of clitoral gland tumors and somewhat increased the frequency of liver epithelial tumors and colon tumors. It is suggested that the strain differences in the induction by DMH of uterine sarcoma and HOL may be due rather to differences in the strain hormonal status than to the differences in the susceptibility to carcinogen.

Retinyl acetate-induced arthritis in C3H-A(vy) mice.

Severely impaired musculoskeletal mobility in C3H-A(vy) mice was noted during a pharmacologic trial evaluating the antitumorigenic properties of retinyl acetate (RAc). To determine the etiology of this impairment, we studied 103 female C3H-A(vy) mice that were fed RAc in daily doses of 75-300 micrograms or placebo and were killed after 3-16 months. Whole-body radiographs and histologic sections of the hindlimbs were scored for presence and severity of arthritis. C3H-A(vy) mice treated with RAc in any dose had a significantly higher incidence of arthritis than placebo-treated mice. Histologic evidence of enthesopathic disease closely paralleled the radiographic changes and ranged from small enthesophytes at tendinous and capsular insertions to complete periarticular bony bridging. Articular cartilage was not grossly affected. The incidence and severity of arthritis were significantly correlated with the total dose of RAc administered. The bony metaplasia induced by RAc was similar to the pathologic changes caused by other retinoids. This model may be useful for studying the pathogenesis of periarticular bone formation in diffuse idiopathic skeletal hyperostosis and related syndromes.