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Chemical carcinogen-induced oxidative stress has a key role in cell signaling linked to the development of cancer. Oxidative stress leads to oxidative damage to cellular membranes, proteins, chromosomes and genetic material. It is thought that compounds like hesperidin with high antioxidant and anticancer potential can reduce development of cancer induced by chemical carcinogens via neutralizing their oxidative damages. We investigated protective effect of hesperidin against N-Ethyl-N-Nitrosourea (ENU)-induced neurotoxicity, congenital abnormalities and possible brain cancer after exposure of mice during pregnancy as model of glioma. The mice were divided to four groups; control (normal saline), ENU (40 mg/kg daily for three consecutive days from the 17th to the 19th of pregnancy), hesperidin (pretreated with 25 mg/kg for 30 consecutive days, before mating) + ENU and hesperidin alone. Developmental toxicity parameters (the number of pregnant mice, stillbirths, abortion, live and dead offspring), behavioral tests (novel object recognition, open field and elevated plus maze) were performed. Moreover, the activity of butrylcholinesterase and acetylcholinesterase enzymes, oxidative markers and histopathological abnormalities were detected in brain tissue. Our data showed that conversely, the pretreatment of hesperidin reduces various degrees of developmental toxicity, neurobehavioral dysfunction, neurotoxicity, oxidative stress and histopathological abnormalities induced by ENU as a neurotoxic and carcinogenic agent in the next generation. In conclusion, pre-mating exposure with hesperidin may open new avenues for prevention of primary brain cancer in next generation and could be valuable for enhancing the antioxidant defense and minimizing the developmental and neurotoxicity of DNA alkylating agents., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

We used N -ethyl- N -nitrosurea-induced germline mutagenesis combined with automated meiotic mapping to identify specific systolic blood pressure (SBP) and heart rate (HR) determinant loci. We analyzed 43,627 third-generation (G3) mice from 841 pedigrees to assess the effects of 45,378 variant alleles within 15,760 genes, in both heterozygous and homozygous states. We comprehensively tested 23% of all protein-encoding autosomal genes and found 87 SBP and 144 HR (with 7 affecting both) candidates exhibiting detectable hypomorphic characteristics. Unexpectedly, only 18 of the 87 SBP genes were previously known, while 26 of the 144 genes linked to HR were previously identified. Furthermore, we confirmed the influence of two genes on SBP regulation and three genes on HR control through reverse genetics. This underscores the importance of our research in uncovering genes associated with these critical cardiovascular risk factors and illustrate the effectiveness of germline mutagenesis for defining key determinants of polygenic phenotypes that must be studied in an intact organism.

Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc ( socrates , formerly s8-3 ), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca 2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca 2+ -conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays., Competing Interests: Declaration of Competing Interest G.A.P., F.Y.L, J.E.H., E.K.S., L.N.W., D.N., C.C.V., and J.J.S. are employees (or former employees) and equity holders of TwinStrand Biosciences, Inc. F.Y.L, E.K.S., L.N.W., C.C.V., and J.J.S. are authors on one or more duplex sequencing-related patents., (Published by Elsevier B.V.)

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer., (© 2023 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Published by Elsevier B.V.)

The main goal of the study was to investigate the genotoxic response of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) at low doses in a multi-endpoint genotoxicity assessment platform in rats and to derive potential thresholds and related metrics. Male Sprague-Dawley rats were treated by daily oral gavage for 28 consecutive days with ENU (0.25 ~ 8 mg/kg bw) and EMS (5 ~ 160 mg/kg bw), both with six closely spaced dose levels. Pig-a gene mutation assay, micronucleus test, and comet assay were performed in several timepoints. Then, the dose-response relationships were analyzed for possible points of departure (PoD) using the no observed genotoxic effect level and benchmark dose (BMD) protocols with different critical effect sizes (CES, 0.05, 0.1, 0.5, and 1SD). Overall, dose-dependent increases in all investigated endpoints were found for ENU and EMS. PoDs varied across genetic endpoints, timepoints, and statistical methods, and selecting an appropriate lower 95% confidence limit of BMD needs a comprehensive consideration of the mode of action of chemicals, the characteristics of tests, and the model fitting methods. Under the experimental conditions, the PoDs of ENU and EMS were 0.0036 mg/kg bw and 1.7 mg/kg bw, respectively., (© 2022. The Author(s).)

Exposure to N-nitroso compounds (NOCs) in our environment via pesticides, tobacco, and smoked meat can be potentially carcinogenic. The induction of N-N' ethylnitrosourea (ENU), a genotoxic NOC, leads to leukemogenesis. The study aimed to explore the ameliorating effect of the Ayurvedic herb Eclipta alba on the bone marrow cells of ENU-induced leukemic mice. Eclipta alba is investigated for its anti-cancer effect on various cell lines, but never on haematological malignant models. Theefficacy of the extract was explored on leukemia by changes in body weight, survivability, peripheral blood hemogram, bone marrow cytological, histological, and cell culture studies pre-and post-treatment. The treated group revealed significant immunomodulation of the expressional profile of NF-kB family and IL-1β in marrow cells, by flow-cytometry, and immunofluorescence study. Through our experimental endeavour we depicted the cellular mechanism, signaling modality and tried to establish the anti-cancer potency of Eclipta alba on ENU-induced leukemia.

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., (© 2022 Wiley Periodicals LLC.)

Environmental exposure of N-nitroso compounds (NOCs) from various sources like tobacco smoke, pesticides, smoked meat, and rubber manufacturing industries has been an alarming cause of carcinogenesis. Neonatal exposure to the carcinogenic N-N'ethylnitrosourea (ENU), a NOC has been established to cause leukemogenesis. Our world is constantly battling against cancer with consistent investigations of new anti-cancer therapeutics. Plant derived compounds have grasped worldwide attention of researchers for their promising anti-cancer potentials. Eclipta prostrata is one such ayurvedic herb, renowned for its anti-inflammatory properties. Currently, it has been explored in various cancer cell lines to establish its anti-cancer effect, but rarely in in-vivo cancer models. Wedelolactone (WDL), the major coumestan of E. prostrata is recognized as an inhibitor of IKK, a master regulator of the NF-kB inflammatory pathway. As persistent inflammation and activated inflammasome contribute to leukemogenesis, we tried to observe anti-leukemogenic efficacy of E. prostrata and its active compound WDL on the marrow cells of ENU induced experimental leukemic mice. Treatment groups were administered an oral gavage at a dose of 1200 mg/kg and 50 mg/kg b.w of crude extract and WDL respectively for 4 weeks. Various parameters like hemogram, survivability, cytological and histological investigations, migration assay, cell culture, flowcytometry and confocal microscopy were taken into consideration pre- and post-treatment. Interestingly, the plant concoction portrayed maximum effects in comparison to WDL alone. The study suggests E. prostrata and WDL as vital complementary adjuncts for anti-inflammasome mechanism in ENU-induced leukemia., (© 2021 Wiley Periodicals LLC.)

The forward or reverse processes of intragenic recombination (IGR), which occur through the addition or deletion of duplicated homologous exons of the p un allele in Pun mice, was observed in vivo, after introducing an homozygous p un allele in a C57BL/6 background. We assessed the frequency of IGR upon N-ethyl-N-nitrosourea (ENU) treatment of pre-melanocytes (PMCs: somatic cells) and primordial germ cells (PGCs: germ cells) of embryonic mice at 10.5 days of development (E10.5). We simultaneously examined IGR and other mutations at the p locus of PMCs responsible for coat color in the offspring obtained by crossing p un /p un with p un /P mice. The frequencies of both spontaneous and ENU-induced IGR were markedly higher than that of the recessive mutation (RM) in PMCs obtained from crossing C57BL/6 and PW strains (Shibuya et al., 1982). ENU also induces IGR at a higher frequency in PGCs at E10.5, which was observed in the next generation. These results indicate that ENU, which preferentially induces gene mutations through base substitution, also induces IGR at a high frequency in the p un allele in both somatic and germ cells of embryonic mice at the E10.5 developmental stage., (Copyright © 2021. Published by Elsevier B.V.)

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models-features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single-Molecule, Real-Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error-free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone-sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10 -7 mutations per base pair (mut/bp)., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Endometrial cancer (EMC) is one of the complicated gynecological cancers, affecting more than three million women worldwide. Anticancer strategies such as chemotherapy, radiation, and surgery are found to be ineffective and are associated with patient incompliances. The aim of the present study is to repurpose non-oncological drug, i.e., Pioglitazone, a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, in the treatment of endometrial cancer. The study groups consist of 50 female Swiss albino mice, out of which 40 had endometrial cancer induced with N-ethyl-N-nitrosourea (ENU) and estradiol hexadrobenzoate (EHB). The other groups received saline, EHB, paclitaxel, and different test doses of pioglitazones. Different preliminary parameters such as weekly body weight, mean survival time, percentage increase in life span, and uterine tissue weight were analyzed along with histopathological analysis. We observed a significant change in weekly body weight, improvement in percentage life span, and partial restoration of uterine tissue weight to normal compared to a standard drug, paclitaxel. In the present preliminary evaluation, we have identified that pioglitazone exhibited a significant dose-dependent anticancer activity against ENU- and EHB-induced endometrial cancer, compared to the standard paclitaxel.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N -ethyl- N -nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3 -/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3 -/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function., Competing Interests: The authors declare no competing interest.

There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g. , CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license., (Copyright © 2020 Zhu et al.)

Twist1 encodes a basic helix-loop-helix transcription factor (TF), which forms homodimer or heterodimer with other TFs, like E2A, to regulate target genes' expression. Mutations in TWIST1 are associated with Saethre-Chotzen syndrome (SCS), a rare congenital disorder characterized with osteogenesis abnormalities. However, how dysfunction of TWIST1 leads to SCS is still largely unknown. Here, using an unbiased ENU-induced mutagenesis screening, we identified a novel Twist1 mutation and the mutant mouse phenocopies some features of SCS in a dominant manner. Physically, our mutation p.F191S lies at the edge of a predicted α-helix in Twist1 transactivation (TA) domain. Adjacent to F191, a consecutive three-residue (AFS) has been hit by 3 human and 2 mouse disease-associated mutations, including ours. Unlike previously reported mouse null and p.S192P alleles that lead to hindlimb polydactyly with incomplete penetrance but a severe craniofacial malformation, our p.F191S causes the polydactyly (84.2% bilateral and 15.8% unilateral) with complete penetrance but a mild craniofacial malformation. Consistent with the higher penetrance, p.F191S has stronger impairment on E2A-dependent transcription than p.S192P. Although human p.A186T and mouse p.S192P disease mutations are adjacent to ours, these three mutations function differently to impair the E2A-dependent transcription. Unlike p.A186T and p.S192S that disturb local protein conformation and unstabilize the mutant proteins, p.F191S keeps the mutant protein stable and its interaction with E2A entire. Therefore, we argue that p.F191S we identified acts in a dominant-negative manner to impair E2A-dependent transcription and to cause the biological consequences. In addition, the mutant mouse we provided here could be an additional and valuable model for better understanding the disease mechanisms underlying SCS caused by TWIST1 dysfunction.

Human midfacial clefting is a rare subset of orofacial clefting and in severe cases, the cleft separates the nostrils splitting the nose into two independent structures. To begin to understand the morphological and genetic causes of midfacial clefting we recovered the Unicorn mouse line. Unicorn embryos develop a complete midfacial cleft through the lip, and snout closely modelling human midfacial clefting. The Unicorn mouse line has ethylnitrosourea (ENU)-induced missense mutations in Raldh2 and Leo1 . The mutations segregate with the cleft face phenotype. Importantly, the nasal cartilages and surrounding bones are patterned and develop normal morphology, except for the lateral displacement because of the cleft. We conclude that the midfacial cleft arises from the failure of the medial convergence of the paired medial nasal prominences between E10.5 to E11.5 rather than defective cell proliferation and death. Our work uncovers a novel mouse model and mechanism for the etiology of midfacial clefting.

FOXP3 + regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1 + (NRP1 + ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1 - Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4 + T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein., (© 2019 Australian and New Zealand Society for Immunology Inc.)

The primary goals of modern genetics are to identify disease-causing mutations and to define the functions of genes in biological processes. Two complementary approaches, reverse and forward genetics, can be used to achieve this goal. Reverse genetics is a gene-driven approach that comprises specific gene targeting followed by phenotypic assessment. Conversely, forward genetics is a phenotype-driven approach that involves the phenotypic screening of organisms with randomly induced mutations followed by subsequent identification of the causative mutations (i.e., those responsible for phenotype). In this article, we focus on how forward genetics in mice can be used to explore dermatologic disease. We outline mouse mutagenesis with the chemical N-ethyl-N-nitrosourea and the strategy used to instantaneously identify mutations that are causative of specific phenotypes. Furthermore, we summarize the types of phenotypic screens that can be performed to explore various aspects of dermatologic disease., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases., Competing Interests: The authors have declared that no competing interests exist.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2 + / Y265X ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2 +/+ ). The Polg2 +/Y265X and Polg2 + / + mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 + / Y265X kidneys was reduced compared to Polg2 +/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 + / Y265X mice, compared to Polg2 + / + littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc., (© 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.)

Forward genetic screening is an extremely powerful method for identifying novel genes driving a broad range of phenotypes. This protocol describes the complete process for conducting a forward genetic screen in zebrafish, including mutagenesis with N-ethyl-N-nitrosourea (ENU), mating, phenotypic screening, and genetic mapping.

The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4-16, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: Brain tumorigenesis is related to oxidative stress and a decreased response of antioxidant defense systems. As it is well known that gender differences exist in the incidence and survival rates of brain tumors, it is important to recognize and understand the ways in which their biology can differ., Objective: To analyze gender differences in redox status in animals with chemically-induced brain tumors., Methods: Oxidative stress parameters, non-enzyme and enzyme antioxidant defense systems are assayed in animals with brain tumors induced by transplacental N-ethyl-N-nitrosourea (ENU) administration. Both tissue and plasma were analyzed to know if key changes in redox imbalance involved in brain tumor development were reflected systemically and could be used as biomarkers of the disease., Results: Several oxidative stress parameters were modified in tumor tissue of male and female animals, changes that were not reflected at plasma level. Regarding antioxidant defense system, only glutathione (GSH) levels were decreased in both brain tumor tissue and plasma. Superoxide dismutase (SOD) and catalase (CAT) activities were decreased in brain tumor tissue of male and female animals, but plasma levels were only altered in male animals. However, different protein and mRNA expression patterns were found for both enzymes. On the contrary, glutathione peroxidase (GPx) activity showed increased levels in brain tumor tissue without gender differences, being protein and gene expression also increased in both males and female animals. However, these changes in GPx were not reflected at plasma level., Conclusion: We conclude that brain tumorigenesis was related to oxidative stress and changes in brain enzyme and non-enzyme antioxidant defense systems with gender differences, whereas plasma did not reflect the main redox changes that occur at the brain level., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze-thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80-90%. Despite some loss of erythrocytes, Pearson coefficients and Bland-Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze-thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47-55, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of U2af1 (S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1 (S34F)-cooperating changes that promote leukemogenesis, we combined U2af1 (S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N -ethyl- N -nitrosourea (ENU). Overall, 3 of 16 ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss- or gain-of-function mutations in Tet2 , Gata2 , Idh1 , and Ikzf1 However, the engineered U2af1 (S34F) missense mutation reverted to WT in two of the three AML cases, implying that U2af1 (S34F) is dispensable, or even selected against, once leukemia is established., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The in vivo erythrocyte Pig-a gene mutation assay measures the phenotypic loss of GPI-anchored surface markers. Molecular analysis of the marker-deficient erythrocytes cannot provide direct proof that the mutant phenotype is due to mutation in the Pig-a gene because mammalian erythrocytes lack genomic DNA. Granulocytes are nucleated cells that originate from myeloid progenitor cells in bone marrow as is the case for erythrocytes, and thus analysis of Pig-a mutation in bone marrow granulocytes can provide information about the source of mutations detected in the erythrocyte Pig-a assay. We developed a flow cytometric Pig-a assay for bone marrow granulocytes and evaluated granulocyte Pig-a mutant frequencies in bone marrow from male rats treated acutely with N-ethyl-N-nitrosourea (ENU). Bone marrow cells from these rats were stained with anti-CD11b for identifying granulocytes and anti-CD48 for detecting the Pig-a mutant phenotype. The average Pig-a mutant frequency in granulocyte precursors of control rats was 8.42 × 10 -6 , whereas in ENU-treated rats it was 567.13 × 10 -6 . CD11b-positive/CD48-deficient mutant cells were enriched using magnetic separation and sorted into small pools for sequencing. While there were no Pig-a mutations found in sorted CD48-positive wild-type cells, Pig-a mutations were detected in mutant granulocyte precursors. The most frequent mutation observed was T→A transversion, followed by T→C transition and T→G transversion, with the mutated T on the nontranscribed DNA strand. While the spectrum of mutations in bone marrow granulocytes was similar to that of erythroid cells, different Pig-a mutations were found in mutant-phenotype granulocytes and erythroids from the same bone marrow samples, suggesting that most Pig-a mutations were induced in bone marrow cells after commitment to either the granulocyte or erythroid developmental pathway. Environ. Mol. Mutagen. 59:733-741, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

We have established a flow cytometry-based Pig-a assay for rat bone marrow erythroid cells (BMEs). The BME Pig-a assay uses a DNA-specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI-anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N-ethyl-N-nitrosourea (ENU) the frequency of CD59-deficient phenotypically mutant BMEs increased approximately 24-fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59-deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X-linked Pig-a gene of CD59-deficient BMEs revealed that they are Pig-a mutants. The spectrum of ENU-induced Pig-a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59-deficient erythrocytes measured in the flow cytometric erythrocyte Pig-a assay develop from BMEs containing mutations in the Pig-a gene. Thus, the erythrocyte Pig-a assay detects mutation in the Pig-a gene. Environ. Mol. Mutagen. 59:722-732, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01)., Competing Interests: The authors have declared that no competing interests exist.

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018., (© 2018 Her Majesty the Queen in Right of Canada 2018.)

Background: In the East Asia, the genus Acer (Aceraceae) is a herbal medicine that is used to treat various diseases, including hemostasis, hepatic disorders, traumatic bleeding and poor eyesight. However, the effects of Acer palmatum thumb. on retinal degeneration are unknown., Aim: In this study, we investigated whether Acer palmatum thumb.ethanol extract (KIOM-2015E) can protect eyes from retinal degeneration. Our research investigated whether KIOM-2015E could have a protective effect in the retinal degenerating mouse model induced by N-ethyl-N-nitrosourea (ENU)., Materials and Methods: Retinal degeneration was induced by a single intraperitoneal injection of ENU in ICR mice. KIOM-2015E (100, 200 mg/kg) was orally administered once per day. The eyeballs were embedded and lysed after drug administration to examine the histological changed and protein expression levels., Results: The ENU-induced retinal degeneration model exhibited increased photoreceptor cell death and a loss of the outer nuclear layer. Additionally, the expression of PKCα and OPN1SW was reduced, and that of GFAP and Nestin was increased in ENU-treated retinal tissues., Conclusion: KIOM-2015E treatment ameliorated the ENU-induced retinal degeneration. KIOM-2015E prevents ENU-induced retinal degeneration by modulating protein expression and the thickness of the outer nuclear layer in the retina., (Copyright © 2018. Published by Elsevier GmbH.)

Positional cloning of ENU-induced mutations has traditionally relied on analysis of polymorphic variation between two strains. In contrast, the application of whole-genome sequencing (WGS) has enabled gene discovery in mutant lines maintained on an inbred genetic background. This approach utilizes genetic variation derived from ENU-induced variants for mapping and reduces the likelihood of phenotypic variation, making it an ideal method for genetic modifier screening. Here, we describe the results of such a screen, wherein we determined the minimal number of mutant genomic DNA samples to include in our analyses and improved the sensitivity of our screen by individually barcoding each genomic DNA library. We present several unique cases to illustrate this approach's efficacy, including the discovery of two distinct mutations that generate essentially identical mutant phenotypes, the ascertainment of a non-ENU-induced candidate variant through homozygosity mapping, and an approach for the identification of putative dominant genetic modifiers., (Copyright © 2018 Geister et al.)

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.

Computational inference of mutation effects is necessary for genetic studies in which many mutations must be considered as etiologic candidates. Programs such as PolyPhen-2 predict the relative severity of damage caused by missense mutations, but not the actual probability that a mutation will reduce/eliminate protein function. Based on genotype and phenotype data for 116,330 ENU-induced mutations in the Mutagenetix database, we calculate that putative null mutations, and PolyPhen-2-classified "probably damaging", "possibly damaging", or "probably benign" mutations have, respectively, 61%, 17%, 9.8%, and 4.5% probabilities of causing phenotypically detectable damage in the homozygous state. We use these probabilities in the estimation of genome saturation and the probability that individual proteins have been adequately tested for function in specific genetic screens. We estimate the proportion of essential autosomal genes in Mus musculus (C57BL/6J) and show that viable mutations in essential genes are more likely to induce phenotype than mutations in non-essential genes.

There is growing interest in quantitative analysis of in vivo genetic toxicity dose-response data, and use of point-of-departure (PoD) metrics such as the benchmark dose (BMD) for human health risk assessment (HHRA). Currently, multiple transgenic rodent (TGR) assay variants, employing different rodent strains and reporter transgenes, are used for the assessment of chemically-induced genotoxic effects in vivo. However, regulatory issues arise when different PoD values (e.g., lower BMD confidence intervals or BMDLs) are obtained for the same compound across different TGR assay variants. This study therefore employed the BMD approach to examine the ability of different TGR variants to yield comparable genotoxic potency estimates. Review of over 2000 dose-response datasets identified suitably-matched dose-response data for three compounds (ethyl methanesulfonate or EMS, N-ethyl-N-nitrosourea or ENU, and dimethylnitrosamine or DMN) across four commonly-used murine TGR variants (Muta™Mouse lacZ, Muta™Mouse cII, gpt delta and BigBlue® lacI). Dose-response analyses provided no conclusive evidence that TGR variant choice significantly influences the derived genotoxic potency estimate. This conclusion was reliant upon taking into account the importance of comparing BMD confidence intervals as opposed to directly comparing PoD values (e.g., comparing BMDLs). Comparisons with earlier works suggested that with respect to potency determination, tissue choice is potentially more important than choice of TGR assay variant. Scoring multiple tissues selected on the basis of supporting toxicokinetic information is therefore recommended. Finally, we used typical within-group variances to estimate preliminary endpoint-specific benchmark response (BMR) values across several TGR variants/tissues. We discuss why such values are required for routine use of genetic toxicity PoDs for HHRA. Environ. Mol. Mutagen. 58:632-643, 2017. © 2017 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc., (© 2017 Her Majesty the Queen in Right of Canada 2017. Reproduced with permission of the Minister of Health, Canada. Environmental and Molecular 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.

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

A cancer bioassay on hexavalent chromium Cr(VI) in drinking water reported increased incidences of duodenal tumors in B6C3F1 mice at exposures of 30-180ppm, and oral cavity tumors in F344 rats at 180ppm. A subsequent transgenic rodent (TGR) in vivo mutation assay in Big Blue® TgF344 rats found that exposure to 180ppm Cr(VI) in drinking water for 28days did not increase cII transgene mutant frequency (MF) in the oral cavity (Thompson et al., 2015). Herein, we extend our analysis to the duodenum of these same TgF344 rats. At study termination, duodenum chromium levels were below either the limit of detection or quantification in control rats, but were 24.6±3.8μg/g in Cr(VI)-treated rats. The MF in control (23.2×10 -6 ) and Cr(VI)-treated rats (22.7×10 -6 ) were nearly identical. In contrast, the MF in the duodenum of rats exposed to 1-ethyl-1-nitrosourea for six days (study days 1, 2, 3, 12, 19, 26) increased 24-fold to 557×10 -6 . These findings indicate that mutagenicity is unlikely an early initiating event in Cr(VI)-induced intestinal carcinogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Camurati-Engelmann disease (CED) is a rare sclerosing bone disorder in humans with autosomal dominant inheritance. Mutations in the gene (TGFB1) that encodes transforming growth factor-β1 (TGF-β1) are causative for CED. TGF-β1 signaling is enhanced by the CED-causing mutations. In this study, we performed Tgfb1 mutation screening in an ENU-mutagenized mouse genomic DNA library. We identified a missense mutation in which cysteine was substituted by serine at position 225 (p.C225S), that corresponded to the CED-causing mutation (p.C225R). TGF-β1 mutant protein carrying p.C225S was secreted normally into the extracellular space. Reporter gene assays showed that the p.C225S mutants enhanced TGF-β signaling at the same level as p.C225R mutants. We generated p.C225S homozygous mice and confirmed that the mature TGF-β1 levels in the culture supernatants of the calvarial cells from the homozygotes were significantly higher than those from wild-type mice. Although the skull and femur are sclerotic in CED, these phenotypes were not observed in p.C225S homozygous mice. These results suggest that human and mouse bone tissue react differently to TGF-β1. These findings are useful to pharmacological studies using mouse models in developing drugs that will target TGF-β signaling.

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n-ethyl-n-nitrosourea (ENU), 6-mercaptopurine (6-MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration-dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration-related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6-MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real-time PCR results showed that the mRNA level expression of p53 increased and bcl-2 decreased significantly with the increasing ENU, 6-MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6-MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6-MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99-107, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Leukemia is often accompanied by enhanced susceptibility to infection due to compromised B cell and T cell functions. The alterations in macrophage functions in leukemia remain less investigated. Herein, we examined macrophages for their functions and responsiveness to IL-3 and GM-CSF in ENU-induced leukemia in BALB/c mice. We observed that the macrophages from the leukemic mice had less phagocytic activity, reduced chemotactic activity in response to monocyte chemoattractant protein-1(MCP-1), and decreased expression of iNOS, GM-CSF, TLR2, TLR4, IFN-γ and TNF-α. These impaired macrophage functions in leukemic mice were significantly corrected by IL-3 and GM-CSF treatment indicating the therapeutic benefit of these two cytokines in leukemia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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, 75 mg/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.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis., Competing Interests: The authors have declared that no competing interests exist.

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.

The rodent Pig-a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol-anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig-a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti-CD59 antibodies and flow cytometry. The Pig-a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28-day general toxicology studies. However, as recommended in the Organisation for Economic Co-operation and Development Test Guidelines for genotoxicity testing, laboratories are expected to demonstrate their proficiency. While this has historically involved the extensive use of animals, here we describe an alternative approach based on a series of blood dilutions covering a range of mutant frequencies. The experiments described herein utilized either non-fluorescent anti-CD59 antibodies to provide elevated numbers of mutant-like cells, or a low volume blood sample from a single N-ethyl-N-nitrosourea treated animal. Results from these so-called reconstruction experiments from four independent laboratories showed good overall precision (correlation coefficients: 0.9979-0.9999) and accuracy (estimated slope: 0.71-1.09) of mutant cell scoring, which was further confirmed by Bland-Altman analysis. These data strongly support the use of reconstruction experiments for training purposes and demonstrating laboratory proficiency with very few animals, an ideal situation given the typically conflicting goals of demonstrating laboratory proficiency and reducing the use of animals. Environ. Mol. Mutagen. 57:678-686, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p < 0.02) in erythrocytes as compared to non-obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1 (MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1 (MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1 (MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the "bystander effect". This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1 (MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.