Effects of ENU dosage on mouse strains

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F₁ hybrids to ENU. The results show that most F₁ hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU. Received: 11 February 2000 / Accepted: 11 February 2000     

Estimating the number of coding mutations in genotypic and phenotypic driven <Emphasis Type="Italic">N</Emphasis>-ethyl-<Emphasis Type="Italic">N</Emphasis>-nitrosourea (ENU) screens: revisited

We recently described methods for estimating the number of N-ethyl-N-nitrosourea (ENU)-induced coding mutations in phenotypic and genotypic screens. In this article we revisit these methods, clarifying their application. In particular, we focus on the difference between unconditional and conditional probabilities. We also introduce a website to assist investigators in the application of these equations ().     

Partial desiccation augments plant regeneration from irradiated embryogenic cultures of sugarcane

Partial desiccation treatment was applied to improve plant regeneration response in irradiated in vitro cultures. Embryogenic callus cultures of sugarcane cv. Co-671 were exposed to different doses of gamma radiation (0–80 Gy) and radiation effect was evaluated in terms of post-irradiation callus recovery, growth and regeneration of plants. Proliferative capacity of cultures was inversely correlated with radiation dose as the percentage surviving cultures or white proliferating clumps (WPC) decreased as the radiation dose increased up to 80 Gy. LD50 was found to be around 20–30 Gy and at higher doses, poor regeneration frequency was observed after 4–6 weeks of post-irradiation culture. To stimulate regeneration response, irradiated cultures were subjected to partial desiccation for 6 h and the treatment resulted in enhanced plant regeneration response. The study suggests that partial desiccation treatment can be useful in stimulating regeneration response of irradiated in vitro cultures.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus

Summary Four antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 M APM or 10 M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 M), however, at a higher concentration (5 M) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations.     

Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Müll. Arg.)

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl₂ concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl₂ before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl₂ concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl₂ concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl₂ concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

A novel <Emphasis Type="Italic">Phex</Emphasis> mutation with defective glycosylation causes hypophosphatemia and rickets in mice

N-ethyl-N-nitrosourea (ENU) mutagenesis is a phenotype-driven approach with potential to assign function to every locus in the mouse genome. In this article, we describe a new mutation, Pug, as a mouse model for X-linked hypophosphatemic rickets (XLH) in human. Mice carrying the Pug mutation exhibit abnormal phenotypes including growth retardation, hypophosphatemia and decreased bone mineral density (BMD). The new mutation was mapped to X-chromosome between 65.4 cM and 66.6 cM, where Phex gene resides. Sequence analysis revealed a unique T-to-C transition mutation resulting in Phe-to-Ser substitution at amino acid 80 of PHEX protein. In vitro studies of Pug mutation demonstrated that PHEX^pug was incompletely glycosylated and sequestrated in the endoplasmic reticulum region of cell, whereas wild-type PHEX could be fully glycosylated and transported to the plasma membrane to exert its function as an endopeptidase. Taken together, the Pug mutant directly confirms the role of Phex in phosphate homeostasis and normal skeletal development and may serves as a new disease model of human hypophosphatemic rickets.     

Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

Biochemical characterization of a variant form of cytosolic epoxide hydrolase induced by parental exposure to N-ethyl-N-nitrosourea

1. ENU4 mice express a protein variant originally detected in a CBF1 mouse sired by a C57BL/6 mouse exposed to N-ethyl-N-nitrosourea. It appears to be an isolelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains.2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBF1 and ENU4 mice were more sensitive to the induction of palmitoyl CoA oxidase activity.3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar.     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.     

Flow cytometric analysis of the cell-cycle distribution of spleen lymphocytes isolated from Fischer 344 rats exposed to ethyl nitrosourea

Current studies in our laboratory are designed to determine the frequency of genotoxic responses induced in lymphocytes isolated from Fischer 344 rats. To evaluate the effect of a model compound, N-ethyl-N-nitrosourea (ENU), on the cell-cycle distribution of spleen lymphocytes, 8-week old, female Fischer 344 rats were injected i.p. with ENU and sacrificed 1, 2, 4, and 6 weeks afterexposure. Four replicate cultures per dose per exposure period were established and cells were cultured for 66 hr. Colcernid, an agent which blocks cells in mitosis and induces an accumulation of cells in the G₂ + M peak, was added to two of the four cultures as a positive control. After a 3 hr incubation, the cells were harvested, the nuclei stained with propidium iodide, and the DNA content of the individual nuclei was quantified by flow cytometry. As expected, exposure to Colcemid resulted in an accumulation of cells in the G₂ + M phase of the cell cycle, which was accompanied by a decrease in the Go + GI population. The increase in the G₂ + M population was significant (p < 0.05) in cultures of lymphocytes assayed at 4 and 6 weeks after exposure. The eflect of increasing ENU concentratiorl was an increase in the percentage of Sphase cells (p =0.05) and a decrease (p < 0.02) in the percentage of G₀ + G₁ cells. This finding was observed only in those lymphocytes isolated 1 week after exposure. These findings indicate that flow cytometric analysis of the distribution of cells within the cell-cycle may provide insight into the eflects of toxicant exposure on mamnzalian cells.Abbreviations BRdU bromodeoxyuridine - ENU N-ethyl-N-nitrosourea - FCM flow cytometry - PHA phytohemagglutinin - PI propidium iodide     

A comparative study of cytotoxic effects of N-ethyl-N-nitrosourea,adriamycin, and mono-(2-ethylhexyl)phthalate on mouse primordial germ cells

Several strategies for the assessment of reproductive toxicity of chemical compounds has have been proposed. In the present work, we devised experimental in vitro assays to test the effect of potential toxicants on proliferating primordial germ cells (PGCs) in vitro using recently developed methods for isolation and culture of mouse PGCs. Primordial germ cells are the embryonic precursors of gametes of the adult that carry the genome from generation to generation. Any damage or mutations caused to these cells by potential toxicants might impair normal reproduction and be transmitted to next generation. Three representative compounds, N-ethyl-N-nitrosourea (ENU), adriamycin (ADR), and mono-(2-ethylhexyl)phthalate (MEHP), toxic to different targets and known to affect germ cell development and impair fertility, were tested on PGCs in culture using three different experimental protocols. Survival and growth of PGCs and their ability to adhere to cell monolayers, were taken as endpoints for drug effects. For each compound, sublethal and acute toxicity doses were determined. In addition, information about the mechanisms of action of these compounds on PGCs was obtained. Whereas the effects of ENU and ADR on PGCs were attributable to growth inhibition and apoptosis induction, MEHP affected PGC adhesion to somatic cells without significantly altering their growth and survival. The results of our in vitro tests were not always exactly predictive of the effects of the tested compounds on PGCs in vivo, determined in parallel experiments in which pregnant mice were exposed to the same compounds. Nevertheless, they can provide information on the sensitivity of PGCs to the direct action of drugs or the mechanisms of action of such agents. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of 12C+5 ion beam irradiation on cell viability and plant regeneration in callus,protoplasts and cell suspensions of Lavatera thuringiaca

Summary The biological effects of irradiation with¹²C⁺⁵ ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with¹²C⁺⁵ ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the¹²C⁺⁵ ion beam in asymmetric protoplast fusion experiments is discussed.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

A Gain-of-Function Mutation in Adenylate Cyclase 3 Protects Mice from Diet-Induced Obesity

In a screen for genes that affect the metabolic response to high-fat diet (HFD), we selected one line of N-ethyl-N-nitrosourea (ENU)-mutagenized mice, Jll, with dominantly inherited resistance to diet-induced obesity (DIO). Mutant animals had dramatically reduced body weight and fat mass, and low basal insulin and glucose levels relative to unaffected controls. Both white adipose tissue (WAT) and brown adipose tissue (BAT) depots were smaller in mutant animals. Mutant animals fed a HFD gained only slightly more weight than animals fed regular chow, and were protected from hepatic lipid accumulation. The phenotype was genetically linked to a 5.7-Mb interval on chromosome 12, and sequencing of the entire interval identified a single coding mutation, predicted to cause a methionine-to-isoleucine substitution at position 279 of the Adcy3 protein (Adcy3^M279I, henceforth referred to as Adcy3^Jll). The mutant protein is hyperactive, possibly constitutively so, producing elevated levels of cyclic AMP in a cell-based assay. These mice demonstrate that increased Adcy3 activity robustly protect animals from diet-induced metabolic derangements.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

A gene-driven ENU-based approach to generating an allelic series in any gene

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F₁ ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.