N-ethyl-N-nitrosourea Research Articles

Induced cell proliferation in pearl oyster

In shellfish, the mantle epithelium takes charge of the formation of shell or pearl, which is a vital cell in the biomineralization process. However, research on the methods of culture of mantle epithelium has progressed quite slowly, and cell lines have not yet been established in pearl oyster. The present research investigated the growth characteristics of mantle epithelium from Hyriopsis cumingii, and the mantle was processed through free-cell implantation and ethylnitrosourea (ENU) or ultraviolet (UV) light inducement. The results indicated that the cell viability (OD260 ratio of RNA to DNA, i.e., RNA/DNA) of mantle epithelium was maintained at high levels for in vitro culture compared to cell culture (60–108 h) (P < 0.05). After mantle epithelium implantation, proliferation and viability were significantly promoted in the implanted cells when they were selected from previous culture cell at 24 and 108 h (P < 0.05). During the process of ENU or UV inducement, cell viability, proliferation, and cell survival rates were significantly affected (P < 0.05). The cell vitality and proliferation characteristics were significantly decreased with the prolonging of culture time by 0.03% ENU inducement (P < 0.05), and they were significantly promoted at 192 and 240 h compared with other groups by 60 mJ/cm2 UV inducement (P < 0.05). The DNA damage products of ENU and UV were significantly increased under ENU or UV inducement, respectively (P < 0.05). These results suggested that the mantle epithelium of H. cumingii started the repairing process after the damage caused using ENU or UV treatments. The present research provided some basic information for further research of mantle epithelium proliferation and cell line establishment in pearl oyster in vitro.

A proteomic clock for malignant gliomas: The role of the environment in tumorigenesis at the presymptomatic stage.

Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, “clocked”, adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies.

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints.

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days -1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBCCD59- ) or reticulocytes (RETCD59- ) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Genome-wide somatic mutation analysis via Hawk-Seq\u2122 reveals mutation profiles associated with chemical mutagens

It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/107–108 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

Dosage time affects alkylating agents induced micronuclei in mouse peripheral blood reticulocytes through the function of erythropoietin.

Previously, we reported that the frequency of micronucleated reticulocytes (MNRETs) in the peripheral blood of male C3H/He mice intraperitoneally administered ethylnitrosourea (ENU) (25 mg/kg body weight) in the dark period (zeitgeber time, ZT15) was higher than in the light period (ZT3). In this study, to clarify the mechanism underlying this phenomenon, we investigated the differences in micronucleus (MN) induction observed between ZT3 and ZT15 using five chemicals, methylnitrosourea (MNU), ethylmethane sulfonate (EMS), mitomycin C, cyclophosphamide and vincristin. MNU and EMS, monofunctional alkylating agents, showed higher frequencies of MNRETs in the ZT15 than the ZT3 treatment similar to ENU. However, no differences were observed for the other chemicals. In the comet assay, more DNA damage was induced by ENU in the ZT15 than the ZT3 treatment. Furthermore, the plasma erythropoietin (EPO) level, a known effector of MN induction with anti-apoptotic activity mediated by Bcl-xL expression, was higher in the dark than in the light period. EPO did not increase the frequency of MNRETs. However, in the ENU treatment group at ZT3 following EPO injection a significant increase of MNRETs was observed similar to the ZT15 treatment. Higher expression of apoptosis-related genes such as Bcl-xL was induced in bone marrow cells from mice treated with ENU at ZT15 compared with ZT3. From these results, it was speculated that the differences in MN induction in the peripheral blood of mice exposed to monofunctional alkylating agents such as ENU depend on apoptotic or anti-apoptotic conditions related to the circadian rhythms of EPO in bone marrow.

Abstract 16895: Lrp1, an Endocytic Vesicle Trafficking Protein, is Associated With Congenital Heart Diseases - Through a "non-Autonomous" Action

Backgrounds: Congenital heart diseases affect nearly 1% of neonates per year in the United States and are the leading cause of neonatal death. Despite a strong indication of genetic contribution to congenital heart diseases, the developmental processes and genetic components are poorly understood. From the large-scale forward genetic screen using inbred mice chemically mutagenized with ethyl-nitrosourea (ENU), a novel finding of endocytic trafficking gene mutations, including Lrp1, were recovered as causing congenital heart disease phenotypes. Endocytosis plays roles in modulating cell signaling by regulating the trafficking of the internalized endocytic vesicles to different endosomal compartments, some destined for fusion with lysosomes and degradation while others are recycled to the surface by recycling endosomes. Mutations in these genes causes a spectrum of congenital heart disease comprising of outflow tract abnormalities and alignment defects. Methods and Results: The LRP1 [line 1554(MGI 96828)] mutant mouse line recovered from a forward genetic screen results from a missense (C4232R) mutation in the region encoding the epidermal growth factor (EGF) repeat domain of LRP1. The mutants exhibit a spectrum of septation and outflow tract defects including endocardial cushion defects, double outlet of right ventricle with normal related great arteries as well as DORV with D-malposition of great arteries (Taussig-Bing anomalies). Mouse embryonic fibroblasts (MEF) from LRP1 m/m mutants demonstrated decreased cell-surface expression and increased retention in the endosomes. Migration assay of MEF demonstrated decreased migration of LRP1 m/m compared with control with normal Golgi orientation. The decreasing migration LRP1 m/m also observed in the endocardial cushion tissue from E9.5-10.5 embryos. Different cell lineages crucial to cardiac development with tissue-specific genetic manipulations were used to define the roles of the LRP1 pathway in these three fields of cells during outflow tract development and septation. Cardiac neural crest cell (Wnt1-Cre) knockout of LRP1 recapitulate LRP1 m/m cardiac phenotypes as well as Nkx2.5/Tie2-cre double knockout of LRP1. RNA expression profile suggests Notch pathway and integrin pathway are associated with LRP1 during cardiac development. Conclusions: We provide the first evidence of endocytic vesicle trafficking pathway associated with the development of congenital heart diseases with malalignment/septation and outflow tract defects

A genome-wide mutation analysis method enabling high-throughput identification of chemical mutagen signatures

Trinucleotide mutational signatures extracted from cancer genomes provide clues useful in understanding the roles of mutagens and mutagenic mechanisms in cancer development. The lack of a simple method for genome-wide analysis of alterations induced by mutagens hampers the identification of trinucleotide signatures of mutagen exposure and evaluation of their relationships with human cancers. Here, we describe a novel approach to facilitate analysis of chemically induced mutations in bacterial cells by detection of increased frequencies of base substitutions after mutagen exposure, using paired-end overlapping next-generation sequencing. DNA samples from Salmonella typhimurium strain TA100, exposed to three alkylating agents, ethylnitrosourea (ENU), methylnitrosourea (MNU), and ethyl methansulphonate (EMS), were analysed. The G:C > A:T mutation frequency was increased in all samples, whereas A:T base pair substitution frequencies were increased specifically in samples exposed to ENU, consistent with previous reports. Mutation patterns in the context of 96 possible trinucleotide formats in these samples exhibited a sharp peak corresponding to an NpCpY consensus sequence, which is similar to the mutational signature of alkylating agents in human cancer. These results indicate that our approach can be useful in facilitating the understanding of mechanisms underlying chemical mutagenicity and for identification of unknown causal mutagens in human cancer.

Large-scale forward genetics screening identifies Trpa1 as a chemosensor for predator odor-evoked innate fear behaviors

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.

Model Legumes: Functional Genomics Tools in Medicago truncatula.

Many researchers have sought along the last two decades a legume species that could serve as a model system for genetic studies to resolve specific developmental or metabolic processes that cannot be studied in other model plants. Nitrogen fixation, nodulation, compound leaf, inflorescence and plant architecture, floral development, pod formation, secondary metabolite biosynthesis, and other developmental and metabolic aspects are legume-specific or show important differences with those described in Arabidopsis thaliana, the most studied model plant. Mainly Medicago truncatula and Lotus japonicus were proposed in the 1990s as model systems due to their key attributes, diploid genome, autogamous nature, short generation times, small genome sizes, and both species can be readily transformed. After more than decade-long, the genome sequences of both species are essentially complete, and a series of functional genomics tools have been successfully developed and applied. Mutagens that cause insertions or deletions are being used in these model systems because these kinds of DNA rearrangements are expected to assist in the isolation of the corresponding genes by Target-Induced Local Lesions IN Genomes (TILLING) approaches. Different M. truncatula mutants have been obtained following γ-irradiation or fast neutron bombardment (FNB), ethyl-nitrosourea (ENU) or ethyl-methanesulfonate (EMS) treatments, T-DNA and activation tagging, use of the tobacco retrotransposon Tnt1 to produce insertional mutants, gene silencing by RNAi, and transient post-transcriptional gene silencing by virus-induced gene silencing (VIGS). Emerging technologies of targeted mutagenesis and gene editing, such as the CRISPR-Cas9 system, could open a new era in this field. Functional genomics tools and phenotypic analyses of several mutants generated in M. truncatula have been essential to better understand differential aspects of legumes development and metabolism.

Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis

AbstractCongenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9 was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9 in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

Genetic manipulation in zebrafish

The increasing number of genetic manipulation approaches and high-resolution live imaging technique applied in zebrafish have propelled the rise of this organism as a mainstream model for developmental biology and human diseases studies. Zebrafish has many advantages for functional genomics analysis, allowing for easy, cheap and fast functional characterization of novel genes in the vertebrate genome. Here we provide an overview of the principles of genetic manipulation in zebrafish, such as Ethylnitrosourea (ENU) mutagenesis, insertional mutagenesis, gene trapping mutagenesis, Morpholino mediated gene knockdown, targeting induced local lesions in genomes (TILLING), genome editing with engineered nucleases ZFN (Zinc finger nuclease), TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 system, and transgenic methods used in zebrafish.

The influence of mutagenic factors on F1M1 and M1 plants of bread winter wheat

Purpose. To determine the influence of mutagens on plant growth and development of hybrid progenies (F1M1) and varieties (M1) of winter wheat obtained from seeds treated. Methods. In 2006-2013, seeds of F1 hybrids and varieties were treated with chemical mutagens N-nitroso-N-ethyl-urea (NEU), N-nitroso-N-methyl urea (NMU), 1,4-bisdiazoacetylbutane (DAB), dimethyl sulfate (DMS) at various concentrations. Dry seeds were also irradiated with gamma rays at dose of 100 Gy. Results. In 2007, when treating seeds with NMU 0.025 %, germination in 52.2 % of hybrid combinations was significantly over control. In 2011, when treating seeds with mutagens (NEU 0.01 %, NMU 0.0125 %, DMS 0.0125 %), 57.8 % of hybrid combinations exceeded the control for field germination under predominantly stimulating action of the two mutagens (NEU 0.01 % and NMU 0.0125 %). A significantly high stimulating effect the mutagen DMS 0.05 % (in 2012) on field germination of hybrid seeds was detected in 67 % of combinations, while when treating seeds with DMS 0.0125 % (in 2011), it was only in 33 % of combinations. In 2012, among eight varieties which seeds were treated with DAB 0.05 % six of them showed the stimulating effect of the mutagen on field germination level. In the winter of 2007–2008, excess over the control overwintering level in hybrids after treatment with NEU 0.005 % and NMU 0.025% was noted (in 47.7 % and 54.5% of combinations resp.). In 2011–2012, when treating seeds with NEU 0.01 %, the overwintering level exceeded the control in 27 % of combinations, while when treating with the mutagens NMU 0.0125 % and DMS 0.0125 %, it did in 20 % of combinations. In 2012–2013, when treating hybrid seeds with the mutagens NEU 0.05 % and DMU 0.05 %, overwintering level of plants significantly exceeded the control in hybrid combinations Romantika / TAM 107 and Yermak / Demetra (the latter was submitted to State Variety Testing as the variety MIP Valensiia) and also in the varieties Demetra when treating with DMS 0.05 %, and Voloshkova (DAB 0.05 %). Phenological observations conducted in 2008, 2011 and 2012 showed that for the most of F1M1 hybrids and M1 varieties heading dates did not exceed 1–3 days earlier or later than in the control. However, in some cases there are deviations from the control for 7 and 5 days which are rather significantly. Conclusions. It was revealed either inhibiting or stimulating effects of chemical and physical mutagens on winter wheat hybrids and varieties. Depending on kind of mutagen, concentration and genotype, these effects occurred in the parameters of field germination, overwintering level and heading date of F1M1 and M1 plants grown from seeds treated with the mutagens.

Hematopathological alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic niche components in experimental myelodysplasia

Myelodysplastic syndrome (MDS) is a poorly understood dreadful hematopoietic disorder that involves maturational defect and abnormalities in blood cell production leading to dysplastic changes and peripheral blood pancytopenia. The present work aims in establishing the mechanistic relationship of the expressional alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic microenvironmental components with the disease pathophysiologies. The study involves the development of N-N′ Ethylnitrosourea (ENU) induced mouse model of MDS, characterization of the disease with blood film and bone marrow smear studies, scanning electron microscopic observation, mitochondrial membrane potential determination, flowcytometric analysis of osteoblastic and vascular niche components along with the expressional study of cleaved caspase-3, PCNA, Chk-2, p53, Ndn, Gfi-1, Tie-2, Sdf-1, Gsk-3β, p18 and Myt-1 in the bone marrow compartment. Dysplastic features were found in peripheral blood of MDS mice which seemed to be the consequence of three marrow pathophysiological conditions viz; aberrant rise of cellular proliferation, increased apoptosis and crowding of abnormal blast population. Expressional decline of the p53 cascade involving Chk-2, p53, Ndn, Gfi-1 along with the downregulation of major cell cycle inhibitors seemed to be associated with the hyper-proliferative nature of bone marrow cells during MDS. Moreover the disruption of osteoblastic niche components added to the decreased hematopoietic quiescency. Increased marrow vascular niche components signified the pre-malignant state of MDS. Elevated cellular apoptosis and rise in the blast burden were also found to be associated with the p53 expression dependent collapsing of mitochondrial membrane potential and upregulation of Tie-2 respectively. The study established the mechanistic correlation between the alterations of the mentioned signaling components and hematopoietic anomalies during MDS which may be beneficial for the development of therapeutic strategies for the disease.

The inhibiting activity of meadowsweet extract on neurocarcinogenesis induced transplacentally in rats by ethylnitrosourea.

Inhibitory activity of a decoction of meadowsweet, given postnatally, was studied in rats at risk for neurogenic and renal tumors initiated by transplacental exposure to ethylnitrosourea (ENU). Chemical analysis of ethanol and aqueous extracts of meadowsweet has shown high content of biologically active flavonoids and tannins. Pregnant rats of LIO strain were given a single i.v. injection of ENU, 75mg/kg, оn the 21st day of gestation. After weaning at 3 weeks after birth, the offspring were divided into two groups: the first was a positive control (ENU), while rats in the second group (ENU + meadowsweet) were given daily a decoction of meadowsweet as drinking water throughout their lifetime. All rats of the first group (ENU) developed multiple malignant tumors, which occurred in brain (86%), spinal cord (43%), peripheral and cranial nerves (29%) and in kidney (31%). More than one-third of CNS tumors were oligodendrogliomas. Mixed gliomas (oligoastrocytomas) occurred less frequently. All other types including astrocytomas, glioblastomas, and ependymomas were rare. All PNS tumors were neurinomas (schwannomas). The spectrum of tumors was similar in rats of the second group. Postnatal consumption of meadowsweet significantly reduced number of tumor-bearing rats (by 1.2 times), the incidence and multiplicity of CNS tumors (brain-by 2.0 and 2.1 times, respectively; spinal cord-by 3.1 and 3.0 times, respectively) and significantly increased latency period, compared to rats of the first group. No significant reduction in PNS or renal tumors was seen in rats given meadowsweet. Meadowsweet extract can be considered an effective cancer preventive agent especially as a neurocarcinogenesis inhibitor.

Collaborative studies in toxicogenomics in rodent liver in JEMS·MMS; a useful application of principal component analysis on toxicogenomics.

Toxicogenomics is a rapidly developing discipline focused on the elucidation of the molecular and cellular effects of chemicals on biological systems. As a collaborative study group of Toxicogenomics/JEMS·MMS, we conducted studies on hepatocarcinogens in rodent liver in which 100 candidate marker genes were selected to discriminate genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens. Differential gene expression induced by 13 chemicals were examined using DNA microarray and quantitative real-time PCR (qPCR), including eight genotoxic hepatocarcinogens [o-aminoazotoluene, chrysene, dibenzo[a,l]pyrene, diethylnitrosamine (DEN), 7,12-dimethylbenz[a]anthracene, dimethylnitrosamine, dipropylnitrosamine and ethylnitrosourea (ENU)], four non-genotoxic hepatocarcinogens [carbon tetrachloride, di(2-ethylhexyl)phthalate (DEHP), phenobarbital and trichloroethylene] and a non-genotoxic non-hepatocarcinogen [ethanol]. Using qPCR, 30 key genes were extracted from mouse livers at 4 h and 28 days following dose-dependent gene expression alteration induced by DEN and ENU: the most significant changes in gene expression were observed at 4 h. Next, we selected key point times at 4 and 48 h from changes in time-dependent gene expression during the acute phase following administration of chrysene by qPCR. We successfully showed discrimination of eight genotoxic hepatocarcinogens [2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitsosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane] from four non-genotoxic hepatocarcinogens [1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, DEHP and furan] using qPCR and principal component analysis. Additionally, we successfully identified two rat genotoxic hepatocarcinogens [DEN and 2,6-dinitrotoluene] from a nongenotoxic-hepatocarcinogen [DEHP] and a non-genotoxic non-hepatocarcinogen [phenacetin] at 4 and 48 h. The subsequent gene pathway analysis by Ingenuity Pathway Analysis extracted the DNA damage response, resulting from the signal transduction of a p53-class mediator leading to the induction of apoptosis. The present review of these studies suggests that application of principal component analysis on the gene expression profile in rodent liver during the acute phase is useful to predict genotoxic hepatocarcinogens in comparison to non-genotoxic hepatocarcinogens and/or non-carcinogenic hepatotoxins.

Alteration of classical and hematopoiesis specific p53 pathway in the bone marrow hematopoietic stem/progenitor compartment facilitates leukemia progression in experimental mice

Downregulation of p53 is associated with most of the neoplasms, however it claims additional significance for hematopoietic malignancy due to its supplementary role during hematopoiesis. Apart from the classical role as tumor suppressor, p53 during steady state hematopoiesis is associated with the maintenance of quiescent cell population in bone marrow by upregulating necdin (Ndn) and Gfi-1. We felt, it is necessary to delineate its attribution towards malignant conversion of hematopoietic system during leukemogenesis from all the possible angles. The present study deals with the characterization of N-N′ Ethylnitrosourea (ENU) induced mouse model of leukemia by peripheral blood hemogram, bone marrow cytology, histology, cytochemical staining (MPO) and scanning electron microscopic study. We further investigated the alteration of conventional and hematopoiesis specific p53 pathways by flowcytometric expressional analysis of ATM, Chk-2, p53, p21, Ndn, Gfi-1 and Tie-2. The disruption of classical p53 pathway was observed in leukemic hematopoietic stem/progenitor population which involved downregulation of ATM, Chk-2, p53 and p21. Moreover, the expressional decline of Ndn and Gfi-1 hinted towards the mechanism of hindrance of hematopoietic quiescency in leukemic bone marrow. Increased expression of Tie-2 due to reverse correlation with p53 was found to be responsible for pathological angiogenesis in bone marrow together with increased blast burden in bone marrow during leukemia. The study presents the mechanistic scenario of the alteration of both classical as well as hematopoiesis specific p53 pathways in HSPC compartment triggering leukemic pathophysiology.

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.

Key points Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Pyrene did not induce gene mutation in red blood cell Pig-a assay and PIGRET assay in rats

A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents) as an endogenous reporter. Although a large number of chemicals have been evaluated in the rat Pig-a assay in 28-day repeat dose regimens, there was limited reporting of rat Pig-a assay after a single dose. A collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, was conducted to verify the usefulness of the rat Pig-a assay after a single dose as a short-term genotoxicity test. As a part of this collaborative study, the in vivo mutagenicity of a single dose of pyrene (Pyr) was investigated in the red blood cell (RBC Pig-a assay) and in reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed with Pyr at 500, 1000, and 2000 mg/kg or ethylnitrosourea (ENU) at 10 and 40 mg/kg as a positive control. The animals in each group were examined for Pig-a mutant frequencies (MF) except for animals in the 2000mg/kg group because of mortality or severe toxicity. The Pig-a MF in RBCs and reticulocytes, as CD59 negative cells, were evaluated once a week for 4 weeks after the dosing. With a single exposure to ENU, the Pig-a MF in both RBCs and reticulocytes increased in a time- and dose-dependent manner. In contrast, no statistically significant effect was observed in rats dosed with Pyr at 500 and 1000 mg/kg. Therefore, Pyr was concluded to be negative in the RBC Pig-a assay and the PIGRET assay after a single oral administration in rats. The result was consistent with previously reported Pig-a assays with repeat dose regimens.

Nestin+cells forming spheroids aggregates resembling tumorspheres in experimental ENU-induced gliomas.

Nestin+cells from spheroid aggregates display typical histopathological features compatible with cell stemness. Nestin and CD133+cells found in glioblastomas, distributed frequently around aberrant vessels, are considered as potential cancer stem cells. They are possible targets for antitumoral therapy because they lead the tumorigenesis, invasiveness and angiogenesis. However, little is known about their role and presence in low-grade gliomas. The aim of this work is to localize and characterize the distribution of these cells inside tumors during the development of experimental endogenous glioma. For this study, a single dose of Ethyl-nitrosourea was injected into pregnant rats. Double immunofluorescences were performed in order to identify stem-like and differentiated cells. Low-grade gliomas display Nestin+cells distributed throughout the tumor. More malignant gliomas show, in addition to that, a perivascular location with some Nestin+cells co-expressing CD133 or VEGF, and the intratumoral spheroid aggregates of Nestin/CD133+cells. These structures are encapsulated by well-differentiated VEGF/GFAP+cells. Spheroid aggregates increase in size in the most malignant stages. Spheroid aggregates have morphological and phenotypic similarities to in vitro neurospheres and could be an in vivo analogue of them. These arrangements could be a reservoir of undifferentiated cells formed to escape adverse microenvironments.