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The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs).
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Modelos Animais de Doenças , Cardiopatias Congênitas , Coração , Animais , Cardiopatias Congênitas/fisiopatologia , Cardiopatias Congênitas/patologia , Coração/embriologia , Coração/fisiopatologia , Coração/crescimento & desenvolvimento , Humanos , Camundongos , MorfogêneseRESUMO
Background: The present study aimed to investigate the protective effect of icaritin (ICT) on ENU-induced leukemia in male mice. Methods: The mice received intraperitoneal injections of 80 mg/kg ENU twice a week for three months for induction of leukemia. Blood smears from these mice showed blast cells, confirming the presence of leukemia. After confirming leukemia, mice were divided into control, ENU-induced leukemia, and leukemia groups (10 mg/kg bw and 20 mg/kg bw) were treated with ICT for 35 days. Blood, spleen, and liver samples were collected for analysis. The expression of IL-6, JAK2, STAT3, as well as inflammatory, pro-apoptotic (Bax), and anti-apoptotic (Bcl-2) proteins were evaluated using qPCR, immunohistochemistry, and immunofluorescence techniques. Results: The study found that ICT inhibited inflammation and the IL-6/JAK2/STAT3 pathway in ENU-induced mice. ICT treatment induced apoptosis in the spleen and liver by activating Bax and downregulating Bcl-2. The findings provide novel evidence that ICT acts as a dual inhibitor of IL-6/JAK2/STAT3 signaling, promoting apoptosis and playing an essential role in anti-leukemic activity. Conclusion: These results suggest that ICT has potential as a therapeutic target for treating leukemia, offering a novel approach to leukemia treatment through inhibiting the IL-6/JAK2/STAT3 pathway and induction of apoptosis.
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Direct DNA sequencing can be used for characterizing mutagenicity in simple and complex biological models. Recently we described a method of whole-genome sequencing for detecting mutations in simple models of cultured bacteria, mammalian cells, and nematode. In the current proof-of-concept study, we expand and improve our method for evaluating a more complex mammalian biological model in outbred mice. We detail the method by applying it to a small set of animals treated with a mutagen with known mutagenicity profiles, N-ethyl-N-nitrosourea (ENU), for consistency with the known data. Whole-genome high-fidelity sequencing (HiFi Sequencing) showed frequencies and spectra of background mutations in tissues of untreated mice that were consistent with normal ageing and characterized by spontaneous or enzymatic deamination of 5-methylcytosine. In mice treated with a single 40 mg/kg dose of ENU, the frequency of mutations in the genomic DNA of solid tissues increased up to 7-fold, with the greatest increase observed in the spleen and the smallest increase in the liver. The most common mutations detected in ENU-treated mice were T > A transitions and T > C transversions, consistent with the types of mutations caused by alkylating agents. The data suggest that HiFi Sequencing may be useful for characterizing mutagenicity of novel compounds in various biological models.
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Alquilantes , Mutagênicos , Camundongos , Animais , Mutagênicos/toxicidade , Testes de Mutagenicidade , Mutagênese , Mutação , Etilnitrosoureia/toxicidade , DNA , MamíferosRESUMO
Intestinal immunity is dependent on barrier function to maintain quiescence. The mechanisms for the maintenance of this barrier are not fully understood. Delta 4-desaturase, sphingolipid 2 (DEGS2) is a lipid desaturase and hydroxylase that catalyzes the synthesis of ceramide and phytoceramide from dihydroceramide. Using a forward genetic approach, we found and validated a mutation in Degs2 as causative of increasing susceptibility to colitis and altering the phytoceramide balance in the colon. DEGS2 is expressed in the intestinal epithelium, and the colitis phenotype is dependent on the non-hematopoietic compartment of the mouse. In the absence of DEGS2, the colon lacks phytoceramides and accumulates large amounts of the precursor lipid dihydroceramide. In response to dextran sodium sulfate (DSS)-induced colitis, colonic epithelial cells in DEGS2-deficient mice had increased cell death and decreased proliferation compared to those in wild-type mice. These findings demonstrate that DEGS2 is needed to maintain epithelial integrity, protect against DSS-induced colitis and maintain lipid balance in vivo.
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Colite , Animais , Camundongos , Ceramidas , Oxigenases de Função Mista , Inflamação , Ácidos Graxos DessaturasesRESUMO
Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.
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DNA , Mutagênicos , Humanos , Reprodutibilidade dos Testes , Mutação , Mutagênicos/toxicidade , Mutagênese , EtilnitrosoureiaRESUMO
Mouse models of red blood cell abnormalities are important for understanding the underlying molecular mechanisms of human erythrocytic diseases. DBA.B6-Mha (Microcytic hypochromic anemia) congenic mice were generated from the cross between N-ethyl-N-nitrosourea (ENU)-mutagenized male C57BL/6J and female DBA/2J mice as part of the RIKEN large-scale ENU mutagenesis project. The mice were established by backcrossing with DBA/2J mice for more than 20 generations. These mice showed autosomal-dominant microcytic hypochromic anemia with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels and increased red blood cell distribution width (RDW) and plasma ferritin levels. Linkage analysis indicated that the Mha locus was located within an interval of approximately 1.95-Mb between D16Nut1 (58.35 Mb) and D16Mit185 (60.30 Mb) on mouse chromosome 16. Mutation analysis revealed that DBA.B6-Mha mice had a point mutation (c.921-2A>G) at the acceptor site of intron 4 in the coproporphyrinogen oxidase (Cpox) gene, a heme-synthesizing gene. RT-PCR revealed that the Cpox mRNA in DBA.B6-Mha mice caused splicing errors. Our results suggest that microcytic hypochromic anemia in DBA.B6-Mha mice is owing to impaired heme synthesis caused by splice mutations in Cpox. Therefore, the DBA.B6-Mha mice may be used to elucidate the molecular mechanisms underlying microcytic hypochromic anemia caused by mutations in Cpox. Although low MCV levels are known to confer malarial resistance to the host, there were no marked changes in the susceptibility of DBA.B6-Mha mice to rodent malarial (Plasmodium yoelii 17XL) infection.
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Anemia Hipocrômica , Coproporfirinogênio Oxidase , Animais , Feminino , Masculino , Camundongos , Anemia Hipocrômica/induzido quimicamente , Anemia Hipocrômica/genética , Coproporfirinogênio Oxidase/genética , Heme , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , MutaçãoRESUMO
This review is intended to summarize the existing literature on the mutagenicity of N-ethyl-N-nitrosourea (ENU) in inducing hematological malignancies, including acute myeloid leukemia (AML) in mice. Blood or hematological malignancies are the most common malignant disorders seen in people of all age groups. Driven by a number of genetic alterations, leukemia rule out the normal proliferation and differentiation of hematopoietic stem cells (HSCs) and their progenitors in the bone marrow (BM) and severely affects blood functions. Out of all hematological malignancies, AML is the most aggressive type, with a high incidence and mortality rate. AML is found as either de novo or secondary therapeutic AML (t-AML). t-AML is a serious adverse consequence of alkylator chemotherapy to the cancer patient and alone constitutes about 10%-20% of all reported AML cases. Cancer patients who received alkylator chemotherapy are at an elevated risk of developing t-AML. ENU has a long history of use as a potent carcinogen that induces blood malignancies in mice and rats that are pathologically similar to human AML and t-AML. ENU, once entered into the body, circulates all over the body tissues and reaches BM. It creates an overall state of suppression within the BM by damaging the marrow cells, alkylating the DNA, and forming DNA adducts within the early and late hematopoietic stem and progenitor cells. The BM holds a weak DNA repair mechanism due to low alkyltransferase, and poly [ADP-ribose] polymerase (PARP) enzyme content often fails to obliterate those adducts, acting as a catalyst to bring genetic abnormalities, including point gene mutations as well as chromosomal alterations, for example, translocation and inversion. Taking advantage of ENU-induced immune-suppressed state and weak immune surveillance, these mutations remain viable and slowly give rise to transformed HSCs. This review also highlights the carcinogenic nature of ENU and the complex relation between the ENU's overall toxicity in the induction of hematological malignancies.
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Neoplasias Hematológicas , Leucemia Mieloide Aguda , Alquilantes , Animais , Carcinógenos , Etilnitrosoureia/toxicidade , Neoplasias Hematológicas/induzido quimicamente , Neoplasias Hematológicas/genética , Humanos , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/genética , Camundongos , Mutagênicos , Poli(ADP-Ribose) Polimerases , RatosRESUMO
BACKGROUND: The Ayurvedic system of medicine mentions the use of seeds of Bryonopsis laciniosa (L.) Naud. (also known as Shivlingi due to their unique structure resembling a 'Shivling') for treating sexual dysfunction, impaired fertility, and as a general virility-booster in both males and females. To investigate the scientific basis for such claims, the current study was designed for the chemical characterization of the super critical fluid extracted Shivlingi seed oil (SLSO), and subsequent evaluation of its reproductive fecundity in the zebrafish model of N-ethyl-N-nitrosourea induced infertility. RESULTS: Linoleic and linolenic acids were the major fatty acids in the SLSO, with trace amounts of ß-sitosterol and stigmasterol. Both male and female zebrafish, when fed orally with the SLSO infused pallets, showed a dose-dependent increase in fertility and fecundity rates. Microscopic observations revealed recovery in the gross ovarian anatomy and consequential improvement in egg production in infertile female zebrafish. Similarly, cytological studies exhibited increased sperm counts and motility in male zebrafish. SLSO exhibited effects similar to the human equivalent dose of Letrozole. CONCLUSION: Taken together, these observations demonstrated the fertility-boosting potentials of SLSO comparable to the widely used infertility drugs. As a whole, this research work has provided scientific evidence for the rationale behind the use of Shivlingi seeds in Ayurvedic treatment for infertility in humans. Finally, but importantly, this study warrants further scientific investigations into different aspects of SLSO on human reproductive health.
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Infertilidade , Peixe-Zebra , Animais , Feminino , Fertilidade , Masculino , Extratos Vegetais/farmacologia , SementesRESUMO
BACKGROUND: Several rodent models with chemically induced colon cancer have been developed. Among these models, dextran sulfate sodium (DSS), a colitis inducer, combined with azoxymethane as a colon mutagenic carcinogen, is commonly used. We previously reported that although benzo [a] pyrene (BP) is mutagenic but not carcinogenic in the colon, it rapidly develops colon tumors at a high incidence/multiplicity after treatment with DSS. In the present study, we examined whether other colon-mutagenic non-carcinogens (CMNCs) induced colon tumors after treatment with DSS. RESULTS: o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea were selected as CMNCs. Male CD2F1 mice were orally administered CMNC for 5 consecutive days. After a 9-day dose-free period, mice were treated with 4% DSS in drinking water for 1 week. Three months after DSS treatment, colon samples were collected for histopathology and ß-catenin immunohistochemistry analyses. All CMNCs in combination with DSS induced colonic adenocarcinomas at a high incidence/multiplicity in the distal and middle parts of the colon, coinciding with the location of colitis. Unlike in normal cells where ß-catenin is exclusively located on the cell membrane, in adenocarcinoma cells, it was translocated to both the nucleus and cytoplasm or only to cytoplasm. The translocation of ß-catenin is closely associated with colon carcinogenesis in rodents and humans. No colonic tumors or dysplastic lesions were found after exposure to either CMNC or DSS alone. CONCLUSION: We provided further evidence clearly showing that CMNCs can rapidly induce colonic tumors in mice with DSS-induced colitis, even if they are not colonic carcinogens.
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Embryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular and nervous systems, which may result in deformities, such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated N-ethyl-N-nitrosourea alleles in 21% of pedigrees. Our study provides proof of concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced transactivation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.
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Mutagênese , Coluna Vertebral/anormalidades , Animais , Proteínas de Ligação ao Cálcio/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Linhagem , Índice de Gravidade de DoençaRESUMO
In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with "eye open at birth" (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.
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Movimento Celular , Etilnitrosoureia/efeitos adversos , Pálpebras/crescimento & desenvolvimento , MAP Quinase Quinase Quinase 1/genética , Mutação , Animais , Células Epiteliais/fisiologia , MAP Quinase Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Exposure to N-nitroso compounds (NOCs) during pregnancy has been associated with an increase in brain tumors in the progeny. This study investigated the brain tumorigenic effect of N-ethyl N-nitrosourea (ENU) after differential exposure of rats during pregnancy. Sprague Dawley rats were exposed to a single dose of ENU (80 mg/kg) in three different circumstances: 1) at first, second or third week of gestation; 2) at the 15th embryonic day (E15) in consecutive litters and 3) at E15 in three successive generations. Location and characterization of the offspring's brain tumors were performed by magnetic resonance imaging and histopathological studies. Finally, tumor incidence and latency and the animals' survival were recorded. ENU-exposure in the last two weeks of pregnancy induced intracranial tumors in over 70% of the offspring rats, these being mainly gliomas with some peripheral nerve sheath tumors (PNSTs). Tumors appeared in young adults; glioma-like small multifocal neoplasias converged on large glioblastomas in senescence and PNSTs in the sheath of the trigeminal nerve, extending to cover the brain convexity. ENU-exposure at E15 in subsequent pregnancies lead to an increase in glioma and PNST incidence. However, consecutive generational ENU-exposure (E15) decreased the animals' survival due to an early onset of both types of tumors. Moreover, PNST presented an inheritable component because progeny, which were not themselves exposed to ENU but whose progenitors were, developed PNSTs. Our results suggest that repeated exposure to ENU later in pregnancy and in successive generations favours the development of intracranial gliomas and PNSTs in the offspring.
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Alquilantes/toxicidade , Neoplasias Encefálicas/induzido quimicamente , Etilnitrosoureia/toxicidade , Glioma/induzido quimicamente , Neoplasias de Bainha Neural/induzido quimicamente , Envelhecimento , Animais , Feminino , Idade Gestacional , Glioblastoma/induzido quimicamente , Glioblastoma/patologia , Glioma/diagnóstico por imagem , Glioma/patologia , Estimativa de Kaplan-Meier , Imageamento por Ressonância Magnética , Neoplasias de Bainha Neural/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Sprague-Dawley , Análise de SobrevidaRESUMO
Putrajeevak (Putranjiva roxburghii Wall.; synonym Drypetes roxburghii (Wall.) Hurus) seeds have been used since ancient times in the treatment of infertility in the Ayurvedic system of medicine in India. In this study, the oil component of Putrajeevak seeds (PJSO) was extracted using the supercritical fluid extraction (SCFE) method using liquid CO2 and the constituents were analyzed using gas chromatography-flame ionized detectorand high-performance thin-layer chromatography. PJSO contained trace amounts of ß-sitosterol with oleic and linoleic acids as the major fatty acid constituents. Male and female zebrafish were mutagenized with N-ethyl-N-nitrosourea (ENU) and fish that produced less than 20 viable embryos were selected for the study. SCFE oil extracts from the P. roxburghii seeds were used in this study to reverse fertility impairment. The mutant fish were fed with PJSO for a period of 14 days and the rates of fertility, conception, and fecundity were determined with wild-type healthy fish as a breeding partner. Treatment with PJSO increased the ovarian follicle count as well as the number of mature eggs, while reducing the number of ovarian cysts. Sperm count as well as sperm motility were greatly enhanced in the ENU-mutagenized male zebrafish when treated with PJSO. The results obtained in this study demonstrate the effectiveness of P. roxburghii seed oil in reversing impaired fertility in both male and female zebrafish models.
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Cromatografia com Fluido Supercrítico/métodos , Euphorbiaceae/química , Fertilidade/fisiologia , Sementes/química , Peixe-Zebra/fisiologia , Animais , Modelos Animais de Doenças , Ácidos Graxos/análise , Ácidos Graxos/química , Feminino , Fertilidade/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Ovário/fisiologia , Óvulo/fisiologia , Pelve/patologia , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Óleos de Plantas/uso terapêutico , Sitosteroides/análise , Sitosteroides/química , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
Chemical carcinogens are commonly used to investigate the biology and prognoses of various cancers. This study investigated the mechanism of leukaemogenic effects of n-ethyl-n-nitrosourea (ENU) in a mouse model. A total of 14 3-week-old male Institute of Cancer Research (ICR)-mice were used for the study. The mice were divided into groups A and B with seven mice each. Group A served as the control while group B received intraperitoneal (IP) injections of 80 mg/kg ENU twice with a one-week interval and were monitored monthly for 3 months for the development of leukaemia via blood smear examination. The mice were sacrificed humanely using a CO2 chamber. Blood, spleen, lymph nodes, liver, kidney and lung samples were collected for blood smear examination and histopathological evaluation. The expression of angiogenic protein (VEGF), and pro and anti-apoptotic proteins (BCL2 and BAX), was detected and quantified using Western blot technique. Leukaemia was confirmed by the presence of numerous blast cells in the peripheral blood smear in group B. Similarly, the VEGF and BCL2 proteins were significantly (p < 0.05) upregulated in group B compared to A. It is concluded that IP administration of 80 mg/kg ENU induced leukaemia in ICR-mice 12 weeks post administration through upregulation of angiogenic and anti-apoptotic proteins: VEGF and BCL2.
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Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe As opposed to many other alkylating agents, ENU only induces an S N 1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.
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Etilnitrosoureia/farmacologia , Mutagênicos/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Análise Mutacional de DNA , Metanossulfonato de Etila/farmacologia , Mutagênese , Mutação , Schizosaccharomyces/genéticaRESUMO
Ip3r1 encodes an inositol 1,4,5-trisphosphate-responsive calcium channel. Mutations in the IP3R1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation in Ip3r1 gene (NM_010585), which is predicted to result in a C1976Y amino acid change in the open reading frame of IP3R1 (NP_034715). We named this novel Ip3r1 allele Ip3r1C1976Y. Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Ip3r1C1976Y. The dilated pupil phenotype in Ip3r1C1976Y was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Ip3r1C1976Y mice that did not recover. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Ip3r1C1976Y mice represent a self-healing model that may be used to study the therapeutic approach for Ip3r1-related diseases.
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Receptores de Inositol 1,4,5-Trifosfato/genética , Iris/fisiopatologia , Mutação de Sentido Incorreto , Midríase/genética , Músculos Oculomotores/fisiologia , Resposta a Proteínas não Dobradas/genética , Animais , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , CamundongosRESUMO
Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused significant losses in both yield and economy to the aquaculture. Host factors involved in NNV infection remain to be identified due to the lack of ideal model for the study of NNV and host interaction. Haploid stem cells have proven to be ideal materials in genetic screens. Here, we generated a cell line HX1G1 (simply named G1) with the activity against red-spotted grouper nervous necrosis virus (RGNNV) by N-ethyl-N-nitrosourea (ENU)-mediated whole genome random mutagenesis from the haploid embryonic stem cell HX1a, a cell clone from haploid cell line HX1 that we previously derived from the medaka fish. G1 cells retained the characteristics of haploidy and pluripotency as indicated by the EBs differentiation ability after genetic mutagenesis. Compared with HX1a cells, no typical cytopathic effects were observed, and the expression of RNA-dependent RNA polymerase (RDRP) was significantly reduced in G1 cells post RGNNV infection, indicating the enhanced anti-RGNNV activity of G1. Furthermore, we demonstrated that RGNNV entry into G1 cells was partially inhibited, and this inhibition might be relevant to the induced mutation of heat shock cognate protein 70 (HSC70) which was decisive for NNV entry. Interestingly, G1 cells were to some extent permissive to RGNNV infection, but RGNNV was spontaneously cleared in G1 cells during serial passage. In addition, we also found that the expression levels of interferon (IFN)-related genes were higher in G1 cells than those in HX1a cells, suggesting that viral clearance might be associated with the elevated expression of IFN-related genes in G1 cells.
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Células-Tronco Embrionárias , Mutagênese , Oryzias/genética , Oryzias/virologia , Animais , Aquicultura , Linhagem Celular/virologia , Etilnitrosoureia , Doenças dos Peixes/virologia , Expressão Gênica , Haploidia , Interações entre Hospedeiro e Microrganismos , Interferons/genética , Nodaviridae , Infecções por Vírus de RNA , RNA Polimerase Dependente de RNARESUMO
BACKGROUND: The model of lung tumors transplacentally induced by N-ethyl-N-nitrosourea (ENU) in inbred BALB/c and C57BL/6J mice was used to investigate the impact of a high sucrose-high fat (HSHF) diet on lung tumorigenesis. METHODS: The offspring was separated by gender and randomly divided into 2 subgroups in both ENU- and buffer-treated groups at the time of weaning. One subgroup was put on the standard diet and the other on the HSHF diet from weaning to the age of 24 weeks. The entire lungs went through a standard process of paraffin-embedded blocks. Every lung block was cut in serial sections but one in every five sections was saved to generate step sections that were stained by hematoxylin and eosin. The tumor histology was assessed on the step sections. RESULTS: At 24-week checkpoint, a spectrum of histological changes was observed in the mice on both diets. Specifically, they presented as alveolar hyperplasia, adenomas and adenomas with nuclear dysplasia at various degrees. Those tumors were actually at different developmental stages. Lung adenocarcinomas were developed in mice on the HSHF diet. A cluster of tumor cells with wide foamy or clear or signet-ring shaped cytoplasm (fatty changes) appeared in a low frequency on the HSHF diet. CONCLUSIONS: The observed histological changes indicated that lung tumors were developed at different times and evolved at different paces. The HSHF diet accelerated the course of tumor evolvement. Tumor cells with fatty changes might be induced by the HSHF diet.
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Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) transcription factor family. ARNT2 heterodimerizes with several members of the family, including single-minded homolog-1 (SIM1) and neuronal PAS domain protein 4 (NPAS4), primarily in neurons of the central nervous system. We screened 64,424 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea (ENU)-mutagenized great-grandsires for weight abnormalities. Among 17 elevated body weight phenotypes identified and mapped, one strongly correlated with an induced missense mutation in Arnt2 using a semidominant model of inheritance. Causation was confirmed by CRISPR/Cas9 gene targeting to recapitulate the original ENU allele, specifying Arg74Cys (R74C). The CRISPR/Cas9-targeted (Arnt2R74C/R74C) mice demonstrated hyperphagia and increased adiposity as well as hepatic steatosis and abnormalities in glucose homeostasis. The mutant ARNT2 protein showed decreased transcriptional activity when coexpressed with SIM1. These findings establish a requirement for ARNT2-dependent genes in the maintenance of the homeostatic feeding response, necessary for prevention of obesity and obesity-related diseases.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diabetes Mellitus/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença , Hiperfagia/genética , Mutação/genética , Obesidade/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peso Corporal , Etilnitrosoureia , Feminino , Glucose/metabolismo , Células HEK293 , Homeostase , Homozigoto , Humanos , Hipotálamo/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
N-ethyl-N-nitrosourea (ENU) is highly used in rodent models of tumerogenesis/carcinogenesis. Xenografting human-cancer tissues/cells with estradiol (E2) treatment is also used to generate rodent-models of gynaecological cancers. The altered metabolic-redox environment leading to establishment of pre-tumorigenesis condition and their mechanism are less studied. Here, female Wister rats were treated with these drugs at their pre-tumerogenic dosage (one group ENU single intra-peritoneal dose of 90 mg/kg b.w. and another group were implanted with human breast tumor (stage-IIIB) and fed with 2.5 mg of 17ß-estradiol once in a week for 4 months). After 4 months, animals were sacrificed; their serum and liver tissues were tested. A brief comparison was made with a rat model (regarded as positive control) of toxicity induced by mutagenic environmental pollutant arsenic (0.6 ppm daily/4 weeks). The increase in serum alkaline phosphatase and glutamate-pyruvate transaminase suggests the possible organ toxicity is favoured by the increase in hepatic/systemic free radicals and oxidative stress in all drug application models. But the increase in the serum E2 level as noted in the ELISA data with impairment in the hepatic estrogen sulfotransferase (SULT1E1) protein expression (immuno-blot data) were noticed with interfered hepatic free-thiols only in ENU and xenograft-E2 group compared to arsenic group. It is also evident in the in vitro result from E2/GSH/NAC added hepatic slices with altered antioxidant regulations. Moreover, impairment in hepatic SOD1, catalase and glutathiole peroxidase activities (PAGEzymographic data), especially in the ENU-treated group makes them more vulnerable to the oxidative threat in creating pre-tumerogenic microenvironment. This is evident in the result of their higher DNA-damage and histological abnormalities. The Bioinformatics study revealed an important role of rSULT1E1 in the regulations of E2 metabolism. This study is important for the exploration of the pre-tumerogenic condition by ENU and E2 by impairing SULT1E1 expression and E2 regulations via oxidant-stress signalling. The finding may help to find new therapeutic-targets to treat gynaecological-cancers more effectively.