Exposure To N-ethyl-N-nitrosourea Research Articles

Mutant TRP53-R172H Has Gain-of-Function or Dominant-Negative Effects in Response to Different Hematopoietic Stressors in Mice

Introduction. Mutations in TP53 are common (~18%) in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia and often undergo loss of heterozygosity. Our understanding of the hematopoietic consequences of expressing mutant TP53-R175H, one of the most common mutations in MDS, is incomplete. In addition, whether TP53-R175H confers a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapy has not been fully explored. Methods. We used a constitutive knock-in mouse model expressing TRP53-R172H (G-to-A substitution at nucleotide 515), corresponding to human mutant TP53-R175H. We generated Trp53R172H/+ and Trp53R172H/R172H mice and compared them to wild type (WT), Trp53+/-, and Trp53-/- mice. Peripheral blood (PB) and bone marrow (BM) was analyzed in non-BM transplant conditions, following a non-competitive BM transplant, and following a competitive BM transplant with or without exposure to N-ethyl-N-nitrosourea (ENU) and 5-fluorouracil (5FU). Results. BM hematopoietic stem and progenitor cells (HSPC), including LSK-SLAM cells, were increased in Trp53+/- and Trp53-/-mice (n=4-8, 8-15 weeks old, P<0.01), but not Trp53R172H/+ and Trp53R172H/R172H mice. In order to study the hematopoietic cell-intrinsic properties of mutant TRP53, we transplanted whole BM into lethally irradiated congenic recipient mice and monitored survival. The median overall survival was dependent on the Trp53 genotype of donor cells: WT cells (100% survival at 1 year), Trp53R172H/+ (60% survival at 1 year), Trp53+/- (31 weeks), Trp53-/- (20 weeks), and Trp53R172H/R172H (18 weeks) (n=12-20, P<0.01 for all genotypes vs. WT). To test long-term HSC function of mutant cells, we performed a competitive BM transplant by injecting equal numbers of test and congenic competitor BM into lethally irradiated congenic recipient mice and monitored PB chimerism of recipient mice for 16 weeks. There was PB competitive advantage for all TRP53 mutant cells compared to WT competitor cells, with Trp53-/- cells having the most significant advantage compared to all other mutant genotypes (n=9-11, P<0.05). The results suggest that mutant TRP53-R172H has distinct properties compared to Trp53 deletions, and not consistent with loss-of-function. TP53 mutant cells can clonally expand in patients following cytotoxic chemotherapy. Therefore, we investigated the response of TRP53R172H/+ mutant cells to alkylator (ENU) exposure. We created mixed BM chimeric mice by transplanting test (WT, Trp53+/-, Trp53R172H/+ and Trp53-/-) and WT competitor BM in a 1:3 ratio, respectively. Following engraftment, chimeric mice received vehicle or ENU (2 doses of 100 mg/kg, 9 days apart). ENU-exposed Trp53R172H/+ cells have a robust PB multilineage competitive advantage relative to placebo (Fig. 1A, n = 4-5, 2-fold increase at 10 weeks post-ENU, P<0.001). This expansion was greater than the rise observed for ENU-treated Trp53+/- cells relative to vehicle treatment (1.67-fold relative to vehicle), and similar to the expansion of Trp53-/- cells, regardless of ENU. BM cells from Trp53R172H/+ mice were resistant to ENU-induced p21 expression and cell cycle arrest observed in WT and Trp53+/- mice (n = 4-5, P<0.001, Fig. 1B, C). The results suggest that mutant TRP53-R172H induces a dominant-negative effect following ENU exposure, similar to prior reports following irradiation. Next, we asked whether mutant TRP53-R172H has similar or different effects as Trp53 deletion following exposure to an alternative chemotherapy (5FU). We first treated mice with a single dose of 5FU (200 mg/kg) to deplete cells and monitored WBC count recovery for 4 weeks. Trp53R172H/+ mice had significantly higher recovery WBC counts compared to WT, Trp53+/-, andTrp53-/- mice (n = 5-15, P<0.05, Fig. 1D). Next, we exposed mutant mice to 4 doses of 5FU (150mg/kg x 1 dose, 90 mg/kg x 3 doses, once per week) and monitored survival. We observed that all Trp53-/- mice survived, while Trp53R172H/+ mice had a median survival of 21 days, and Trp53+/- and WT mice had the shortest median survival (13 and 14.5 days, respectively, Fig. 1E). The results suggest that mutant TRP53-R172H cells display a gain-of-function property following a single dose of 5FU. Collectively, the results indicate that mutant TRP53-R172H may induce a gain-of-function or a dominant-negative effect depending on the exposure to specific hematopoietic stresses. Disclosures No relevant conflicts of interest to declare.

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

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.

Abstract 4807: Braf mutations initiate the development of rat gliomas induced by postnatal exposure to N-ethyl-N-nitrosourea (ENU)

Abstract Purpose A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T -> A mutations in the transmembrane domain of the neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas was unknown. The objectives of this study were to identify driver mutations in ENU-induced rat gliomas. Methods We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. Results T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41-55% of all somatic single nucleotide mutations). T->A mutations were detected in all 5 rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, while peritumoral brain tissue of either strain (n=16) had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. Conclusions Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU. Citation Format: Kaishi Satomi, Qi Wang, Ji Eun Oh, Barbara Hutter, Benedikt Brors, Nicolle Diessl, Hai-Kun Liu, Stephan Wolf, Otmar Wiestler, Paul Kleihues, Bernd Koelsch, Andrea Kindler-Rohrborn, Hiroko Ohgaki. Braf mutations initiate the development of rat gliomas induced by postnatal exposure to N-ethyl-N-nitrosourea (ENU) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4807. doi:10.1158/1538-7445.AM2017-4807

Abstract 3144: Prediction of ALK mutations associated with acquired resistance to lorlatinib

Abstract Anaplastic lymphoma kinase (ALK) rearrangements are important therapeutic targets in non-small cell lung cancer. They are currently treated with the first-generation ALK inhibitor crizotnib followed by more potent, second-generation ALK inhibitors, such as ceritinib, alectinib, or brigatinib. We reported different spectrums of ALK resistance mutations in the biopsies from patients progressing on these drugs. G1202R mutation was found more frequently after treatment with second generation ALK inhibitors. In addition to these drugs, the third-generation ALK inhibitor lorlatinib is currently being evaluated in phase 2 clinical trial. Ba/F3 models indicated that all single ALK mutants are sensitive to lorlatinib and some compound ALK mutations are resistant to lorlatinib. In this study, we performed accelerated mutagenesis screen on Ba/F3 models to predict the resistance mutations which potentially emerge in the patients treated with lorlatinib. Briefly, Ba/F3 cells expressing wild type EML4-ALK or mutant EML4-ALK containing C1156Y, F1174C, L1196M, G1202R, or G1269A were exposed to N-ethyl-N-nitrosourea (ENU). After a 24-hour incubation in normal media, the cells were seeded in 96-well plates and incubated in lorlatinib for 4 weeks. ALK kinase domain was sequenced in clones growing in lorlatinib to identify possible new mutations. As a result, Ba/F3 cells harboring wild type EML4-ALK did not show any mutation on ALK kinase domain. Crizotinib was used as a control to validate the efficiency of mutagenesis. We identified eight different mutations in clones growing in crizotinib, and those were reflecting the spectrum of mutations in the crizotinib-treated patients. Ba/F3 cells with mutant EML4-ALK showed additional compound mutations after incubation with lorlatinib. Those mutations included L1196M + L1198F and G1202R + L1198F which showed high resistance to lorlatinib in Ba/F3 models. Ba/F3 cells with different mutant EML4-ALK showed a distinct spectrum and different frequency of additional mutations. In conclusion, this study predicted that no single mutation would emerge to confer resistance to lorlatinib. Thus, compound mutations and ALK-independent mechanisms become essential mechanisms for lorlatinib resistance. Citation Format: Satoshi Yoda, Leila Dardaei, Manrose Singh, Jeffrey A. Engelman, Alice T. Shaw, Aaron N. Hata. Prediction of ALK mutations associated with acquired resistance to lorlatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2017-3144

Differences in micronucleus induction in peripheral blood reticulocytes of mice exposed to <i>N</i>-ethyl-<i>N</i>-nitrosourea at light and dark dosing times

Mammals, including human beings, have a circadian clock system to regulate behavioral and physiological processes. In this study, we investigated the effect of dosing time on micronucleus induction in the bone marrow by evaluating the frequencies of micronucleated peripheral reticulocytes (MNRETs) in mice exposed to N-ethyl-N-nitrosourea (ENU) to assess any difference in genotoxic sensitivity to chemicals between light and dark periods (inactive phase for rodents and active phase for rodents). Male C3H/He mice were treated intraperitoneally with ENU (12.5 or 25 mg/kg body weight) at zeitgeber time (ZT) 3 in the light period or ZT15 in the dark period, and then the time courses of the frequencies of the MNRETs were determined. The frequencies of the MNRETs induced by ENU increased time-dependently and peaked at 48 hr after treatment for ZT3 and ZT15, and were obviously higher in the ZT15 treatment group than the ZT3 treatment group. The MNRETs were measured at 48 hr after treatment with ENU (25 mg/kg body weight) at various dosing times (ZT0, 3, 6, 12, 15 and 18). The frequencies of the MNRETs in mice treated at ZT0, 15 and 18 were significantly higher than those in mice treated at ZT3, 6 and 12. These results suggest that genotoxic sensitivity to chemicals in mouse bone marrow is different between light and dark periods maybe due to different biological responses (detoxification, cell cycle, DNA repair, etc.) related to circadian rhythms.

International Pig‐a gene mutation assay trial: Evaluation of transferability across 14 laboratories

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.

NG2-expressing glial precursor cells are a new potential oligodendroglioma cell initiating population in N -ethyl- N -nitrosourea-induced gliomagenesis

Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.

Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains.

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response-sequentially involving macrophages, T helper- and cytotoxic T lymphocytes-is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.

Differential mutation of transgenic and endogenous loci in vivo

Although chemicals usually induce very similar frequencies of mutations in transgenes and endogenous genes in vivo when given acutely, chronic exposure to N-ethyl- N-nitrosourea (ENU) produced a more complex pattern in which the endogenous locus was spared many mutations. Here, we demonstrate that the effect is neither ENU-specific nor locus-specific, and thus, may be important in the extrapolations of risk assessment and in understanding mutational mechanisms. During chronic mutagen exposure, mutations at the transgene accumulate linearly with time, i.e. in direct proportion to the dose received. In contrast, mutations at the endogenous gene are much less frequent than those of the transgene early in the exposure period and the accumulation is not linear with time, but rather accelerates as the exposure continues. Previous comparisons involved the endogenous Dlb-1 locus and the lacI transgene from the Big Blue™Mouse in the small intestine. These experiments involved the Dlb-1 locus and the lacZ transgene from the Muta™Mouse in the small intestine and the hprt locus and the lacZ transgene in splenocytes. Comparisons were made in both tissues after acute and chronic exposures to ENU, the original mutagen, and in the small intestine after exposures to benzo(a)pyrene. All comparisons showed that during chronic exposures mutations at the transgene accumulate linearly with the increasing duration of exposure, whereas induced mutations of the endogenous gene initially accumulate at a slower rate. Thus, the difference in mutational response observed during low chronic treatment is not unique to a particular transgene, endogenous gene, tissue, or mutagen used, but may be a general phenomenon of such genes.

Effects of extended chronic exposures on endogenous and transgenic loci: Implications for low-dose extrapolations

Although transgenic and endogenous loci generally respond alike to acute mutagenic exposures, those loci that have been tested respond differently to daily or continuous exposures. During chronic exposures, the transgenes accumulate mutations linearly, whereas the endogenous loci are less mutable initially but later accumulate mutations at an accelerating rate. The result is a reverse dose rate effect in which the same total dose is more mutagenic for the endogenous locus when spread over a longer time. This makes extrapolations to still lower chronic exposures uncertain. Here we report extension of a chronic exposure to N-ethyl-N-nitrosourea (ENU) in drinking water to longer times and to lower doses. The F(1) of MutaMouse males x SWR that were used permit detection of mutations at both lacZ and Dlb-1. Both of these mutations were found to be genetically neutral over this period. Extension of the exposure from 30 to 90 days at 94 microg/ml/d showed a continuation of the curves found previously for 10 to 30 days, namely, linear for mutations of the lacZ transgene and concave upward for the Dlb-1 endogenous gene. A simple model for these data is presented. Of the extended exposures, only the highest dose produced a significant increase in Dlb-1 mutant frequency, an increase consistent with the model. The time (at 2.8 microg/ml/d), concentration (after 480 days exposure), and dose (concentration x duration of exposure) response curves were not significantly different from linearity. The data for the transgene are not as convincing, due to the high spontaneous mutant frequency, obscuring the induced response.

Effects of extended chronic exposures on endogenous and transgenic loci: Implications for low‐dose extrapolations

Although transgenic and endogenous loci generally respond alike to acute mutagenic exposures, those loci that have been tested respond differently to daily or continuous exposures. During chronic exposures, the transgenes accumulate mutations linearly, whereas the endogenous loci are less mutable initially but later accumulate mutations at an accelerating rate. The result is a reverse dose rate effect in which the same total dose is more mutagenic for the endogenous locus when spread over a longer time. This makes extrapolations to still lower chronic exposures uncertain. Here we report extension of a chronic exposure to N-ethyl-N-nitrosourea (ENU) in drinking water to longer times and to lower doses. The F1 of Muta™Mouse males × SWR that were used permit detection of mutations at both lacZ and Dlb-1. Both of these mutations were found to be genetically neutral over this period. Extension of the exposure from 30 to 90 days at 94 μg/ml/d showed a continuation of the curves found previously for 10 to 30 days, namely, linear for mutations of the lacZ transgene and concave upward for the Dlb-1 endogenous gene. A simple model for these data is presented. Of the extended exposures, only the highest dose produced a significant increase in Dlb-1 mutant frequency, an increase consistent with the model. The time (at 2.8 μg/ml/d), concentration (after 480 days exposure), and dose (concentration × duration of exposure) response curves were not significantly different from linearity. The data for the transgene are not as convincing, due to the high spontaneous mutant frequency, obscuring the induced response. Environ. Mol. Mutagen. 34:208–215, 1999 © 1999 Wiley-Liss, Inc.

Studies on DNA repair defects in degenerative brain disease.

Using the alkaline filter elution technique we determined the induction and disappearance of single-strand breaks (SSB) in freshly isolated peripheral blood lymphocytes (PBL) from 43 Alzheimer disease (AD) patients and from 48 healthy, age- and sex-matched control subjects following in vitro exposure to N-ethyl-N-nitrosourea (ENU), methyl methanesulphonate (MMS), or gamma (gamma)-radiation. No differences in SSB disappearance between AD patients and controls were observed after treatment of PBL with MMS or gamma-rays. After treatment with ENU, however, the amount of SSB disappearance was significantly lower in PBL from familial, but not in PBL from sporadic AD patients. ENU repair in PBL from neurological controls was comparable to that found in normal age-matched controls, indicating that the lower amount of ENU repair in familial AD patients is not a consequence of neuronal degeneration. These tentative findings are discussed in relation to the aetiology of AD.

Rat model of the human “Triton” tumor: direct genetic evidence for the myogenic differentiation capacity of schwannoma cells using the mutant neu gene as a cell lineage marker

Abstract Spontaneous myogenic differentiation was observed in 2 out of 15 cases when cells from schwannomas induced in the offspring of BDIX rats by transplacental exposure to N-ethyl- N-nitrosourea (EtNU) were grown in monolayer culture following fluorescence-activated cell sorting with monoclonal antibody (Mab) 217c. Myotubes and numerous mononucleated cells no longer expressed the Schwann cell antigens 217c and S-100 protein, but rather revealed the presence of desmin, the α-sarcomeric form (α-sr) of actin, and the cell surface antigen specified by Mab RB21–7, a 250 kD glycoprotein sharing an epitope with the neural cell adhesion molecule (N-CAM). Subcutaneous reimplantation of such cells into syngeneic animals led to the appearance of tumors composed of both S-100 positive Schwann cells and desmin and α-sr-actin positive rhabdomyoblasts, thus closely resembling the human “Triton” tumor. With the use of the polymerase chain reaction and allele-specific oli-gonucleotide hybridization, DNA isolated from individual myotubes was analyzed for the presence of a T→A transversion mutation at nucleotide 2012 of the neu gene, which is diagnostic of EtNU-induced rat schwannomas. All of the amplified DNA isolates contained the mutant neu allele, thus providing direct genetic proof for the capacity of mammalian neuroectodermal cells for myogenic differentiation.

Decreased DNA repair capacity in familial, but not in sporadic Alzheimer's disease

Using the alkaline filter elution technique we determined the induction and disappearance of DNA single-strand breaks (SSB) in freshly isolated peripheral blood lymphocytes (PBL) from 43 Alzheimer's disease (AD) patients and 48 normal, healthy age- and sex-matched control subjects following in vitro exposure to N-ethyl- N-nitrosourea (ENU). The mean percentage SSB disappearance in PBL from control subjects at 1 h after ENU treatment was 41.4 ± 2.9%; this was not significantly different from that found in samples from AD patients which had no (n = 16) or one (n = 12) first-degree relative with dementia (42.5 ± 8.2% and 43.0 ± 4.4%, respectively; p>0.75). However, in PBL of 15 AD patients with at least two first-degree relatives with dementia the mean percentage SSB disappearance was 23.6 ± 5.8%, which was significantly lower than that found in controls ( p<0.01) or in the other AD patients ( p<0.02).

Induction and disappearance of DNA strand breaks and/or alkalilabile sites in human lymphocytes exposed to N-ethyl-N-nitrosourea

We investigated the induction and disappearance of DNA lesions that are detected as single-strand breaks (SSBs) with the alkaline filter elution technique, in human peripheral blood lymphocytes (PBLs) following exposure to N-ethyl-N-nitrosourea (ENU). In PBLs of the 15 individuals studied, 35 +/- 16% (mean +/- SD) of the SSBs present at the end of a 20 min treatment disappeared within 1 h; up to 24 h post-treatment no further disappearance was observed. Interindividual differences in SSB disappearance were considerable; in two cases, almost no SSBs disappeared over the 1 h period. In PBLs of the same 15 individuals 85 +/- 3% (mean +/- SD) of the SSBs induced by 4 Gy of gamma-rays were found to disappear within 1 h, which indicated no interindividual differences. Multiple blood sampling at 1 month intervals indicated that interindividual differences in ENU-induced SSB repair are constant and do not vary with time. The low rate of SSB disappearance appeared to correlate with low cell survival after ENU exposure, measured as a reduction in phytohemagglutinin-stimulated incorporation of [3H]-thymidine. The cytosine analog 1-beta-D-arabinofuranosyl-cytosine completely inhibited SSB disappearance, indicating that the removal of the repairable ENU-induced SSBs involves excision repair events.

High frequency of mosaic mutants produced by N-ethyl-N-nitrosourea exposure of mouse zygotes.

Mouse zygotes containing one multiple-recessive parental genome (a, b; p cch; d se; s) and the corresponding wild-type alleles in the other were exposed to N-ethyl-N-nitrosourea (ENU) at various stages in vivo. At weaning age, the resulting mice were examined for mutations at the marked loci as well as at others producing externally visible phenotypes. Because of viability problems in one of two reciprocal crosses, the bulk of the mutagenesis data are derived from the cross that detects recessive mutations in the maternal genome. The mutation rate was approximately 8 times higher in groups treated 2.5-3 hr postmating (sperm entry, completion of second meiotic division) than in those injected 5-6 hr postmating (pronuclear formation). In the former more sensitive zygote population, the mutation rate is about an order of magnitude greater than that induced by the same ENU exposure (50 mg/kg) to spermatogonial stem cells. Of 11 mutants recovered, 8 were mosaics. Progeny tests have demonstrated germ-line involvement for most of the mosaics, and the average fraction of the germ line carrying the mutation is close to 50%. The nature of the mutations indicates (i) that the mosaicism results not from misassortment at the first cleavage but from mutation affecting one DNA strand of the maternal chromosome, and (ii) that the mutations are intragenic lesions rather than multilocus deletions, thus resembling ENU-induced mutations in spermatogonia. The finding that mosaicism for presumed point mutations is readily inducible by ENU treatment of zygotes may provide a means of generating genetic materials that can be of use for developmental studies.

Effect of Nocardia rubra cell wall skeleton and/or cyclophosphamide on leukemogenesis induced by N-ethyl-N-nitrosourea in Donryu rats.

The effect of Nocardia rubra cell wall skeleton (N-CWS) and/or cyclophosphamide (CP) on chemical carcinogenesis was examined in female Donryu rats exposed to N-ethyl-N-nitrosourea (ENU) in the drinking water for 6 weeks. Five administrations of N-CWS following ENU treatment caused a slight prolongation of the average survival of rats but did not reduce the incidence of leukemia. CP given on two occasions after ENU treatment caused a moderate prolongation of average survival period and a moderate reduction of the incidence of leukemia, but significant differences from ENU-treated control group values were not observed after statistical analysis. Combined treatment with N-CWS and CP after ENU treatment caused prolongation of the average survival period of rats and a statistically significant reduction in the incidence of leukemia. The present experiment indicates that combined treatment with N-CWS and CP effectively reduces induction of leukemia by ENU in rats, although other types of tumors were not affected.

N-Ethyl-N-nitrosourea-Induced Spinal Tumors in an Inbred Strain of W Albino Rats2

Exposure to N-ethyl-N-nitrosourea (ENU) either transplacentally via the maternal bloodstream or postnatally by direct injection into the cerebellum or the cisterna magna resulted in a high incidence of spinal tumors in an inbred strain of W albino rats. After prenatal exposure to 60 mg ENU/kg maternal body weight, as many as 92% of the offspring developed 1 or more tumors in the spinal cord, whereas after postnatal exposure to 0.2 mg ENU/animal, 50% of the animals eventually developed spinal tumors. These tumors included relatively pure oligodendrogllomas, astrocytomas, and the usual mixed gllomas. Obvious clinical symptoms of paralysis of the limbs and weight loss accompanying the high incidence of tumors in the spinal cord make this system pertinent to the study of carcinogenesis in the central nervous system as well as to the study of related problems to the incidence of these tumors are discussed.