A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

Transformation of cultured epithelial cells by ethylnitrosourea: Altered expression of type I procollagen chains

Cultured epithelial rodent cells were transformed in vitro using ethylnitrosourea as a carcinogen either alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The frequency of transformation in the absence of TPA was 5·10^?4 at 10 μg/ml ethylnitrosourea. Growth of ethylnitrosourea-treated cells in TPA-substituted medium increased the transformation frequency 8-fold. Colonies of transformed cells were isolated from soft agar and analyzed for the production of pericellular matrix glycoproteins. The ethylnitrosourea-transformed cells retained pericellular matrix structures, typical of the nontransformed control cells. Parent cells produced into their culture media fibronectin and procollagen types I and III as their major pericellular glycoproteins. The ethylnitrosourea-transformed cells synthesized and secreted altered procollagen polypeptides. The procollagen of ethylnitrosourea-transformed cells apparently consisted mainly of homotrimeric proα1 molecules, with smaller amounts of basement membrane procollagen-like chains. Fibronectin synthesis or secretion was not affected by ethylnitrosourea-induced transformation, but the production of fibronectin was enhanced in the transformed cultures treated with TPA. Also, the deposition of procollagen and fibronectin into the pericellular matrix was not affected by ethylnitrosourea-transformation. Very similar changes had previously been observed in murine sarcoma virus-transformed cells. The change of procollagen type I thus appears to be a correlate of malignant transformation of cultured epithelial cells. The results indicate that ethylnitrosourea can induce malignant transformation of epithelial cells in culture and modify production and deposition of pericellular glycoproteins.     

Biochemical Defect of the hph-1 Mouse Mutant Is a Deficiency in GTP-Cyclohydrolase Activity

A hyperphenylalaninemic mouse mutant, hph-1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid-dihydropteridine reductase activity is also normal. GTP-cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP-cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4-dihydroxyphenylalanine (DOPA) and serotonin.     

Effect of elongation factor Tu on the conformation of phenylalanyl-tRNAPhe

Structural features of the tRNAPhe molecule upon ternary complex formation with the bacterial elongation factor Tu were investigated. Phosphodiester bonds at positions 18 and 34 were found to be labilized in bound tRNA. Conversely, a higher stability of the phosphodiester links at positions 20, 21 and 36 was detected. Using ethylnitrosourea as a chemical probe a conformational change occurring at phosphate position 53 was observed in complexed tRNA. These results are interpreted by a structural rearrangement of the nucleic acid induced by complex formation.     

Neutral Glycosphingolipids and Ceramide Composition of Ethylnitrosourea-Induced Rat Neural Tumors: Accumulation of Ceramide in Tumors

Abstract: Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20 mg/kg body weight, in the tail vein of the mother. The neutral glycosphingolipid, sulfatide, and ceramide composition of the tumors and the normal tissues from which the tumors originated is described. The content of nonhydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) containing ceramide in all the neural tumors so far examined was significantly increased compared with the corresponding normal neural tissue. Some 8 to 18 mol% of total neutral glycolipids was as ceramide in neurinomas, oligodendrogliomas, and menin-giomas. Lactosylceramide in normal neural tissues was about 1 mol% of the total neutral glycosphingolipids. In various neural tumors lactosylceramide increased up to 8 mol%. NFA- and HFA-containing cerebrosides constitute 94–100% of the neutral glycosphingolipids in normal neural tissues. In various neural tumors the mol percent of cerebrosides was significantly reduced. A high performance liquid chromatographic method was modified to analyze simultaneously ceramides, cerebrosides, and higher neutral glycosphingolipids.     

Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea

Experiments were performed to characterize the age-related patterns of appearance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hprt) mutant T lymphocytes in thymus and spleen following exposure of preweanling (12-day-old), weanling (22-day-old), and young adult (8-week-old) male B6C3F1 mice to ethylnitrosourea (ENU). Mice were given single i.p. injections of 0 or 40 mg ENU/kg and then groups of animals were necropsied from 2 h to 116 days after treatment to examine the relationships between exposure, cell loss and proliferation, and the frequency of Hprt mutant T cells in thymus and spleen. Hprt mutant frequency (Mf) data for thymus of ENU-exposed (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) male C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of spleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen. 9 (1987) 317–329.], were compared to results in B6C3F1 mice. Isolated T cells were cultured in the presence of mitogen, growth factor, and 6-thioguanine to detect Hprt mutants. The time required to achieve maximum Mfs in thymus was uniformly found at 2 weeks after ENU treatment, while the times needed to reach peak values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cells in spleen are largely defined by the physiologically based, age-dependent trafficking of mutant cells from or through the thymus. Three modes of handling the resulting Hprt Mf data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed, and (iii) comparing the change in Mfs over time (or the mutant T cell ‘manifestation’ curves in treated vs. control mice) in each age group post-exposure. Measuring the Mfs in spleen at multiple time points after cessation of exposure and integrating the frequency of mutants as a function of time appeared to be the superior method for comparing mutagenic responses in different age groups. Some of the underlying assumptions of this approach, as well as its strengths and weaknesses, are discussed.     

Isolation of developmental mutants of the ascidian Ciona savignyi

Ascidians have been used extensively as model animals for experimental embryology. We report here the results of a pilot study with the aim of developing genetic methods for the ascidian Ciona savignyi. The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to induce point mutations. F1 animals, produced by using sperm from ENU-treated animals to fertilize untreated eggs, were grown to reproductive age. Sperm and eggs collected from the hermaphrodite F1 adults were used to generate self-fertilized F2 broods, which were then screened for recessive, zygotically acting mutations. Animals carrying potential mutations were outcrossed to wild type to test for the heritability of the phenotypes. We report on a number of mutants isolated using this method, including several with abnormalities in tail and notochord development. Received: 15 March 1999 / Accepted: 6 May 1999     

Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea

Experiments were performed to characterize the age-related patterns of appearance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hprt) mutant T lymphocytes in thymus and spleen following exposure of preweanling (12-day-old), weanling (22-day-old), and young adult (8-week-old) male B6C3F1 mice to ethylnitrosourea (ENU). Mice were given single i.p. injections of 0 or 40 mg ENU/kg and then groups of animals were necropsied from 2 h to 116 days after treatment to examine the relationships between exposure, cell loss and proliferation, and the frequency of Hprt mutant T cells in thymus and spleen. Hprt mutant frequency (Mf) data for thymus of ENU-exposed (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) male C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of spleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen. 9 (1987) 317–329.], were compared to results in B6C3F1 mice. Isolated T cells were cultured in the presence of mitogen, growth factor, and 6-thioguanine to detect Hprt mutants. The time required to achieve maximum Mfs in thymus was uniformly found at 2 weeks after ENU treatment, while the times needed to reach peak values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cells in spleen are largely defined by the physiologically based, age-dependent trafficking of mutant cells from or through the thymus. Three modes of handling the resulting Hprt Mf data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed, and (iii) comparing the change in Mfs over time (or the mutant T cell ‘manifestation’ curves in treated vs. control mice) in each age group post-exposure. Measuring the Mfs in spleen at multiple time points after cessation of exposure and integrating the frequency of mutants as a function of time appeared to be the superior method for comparing mutagenic responses in different age groups. Some of the underlying assumptions of this approach, as well as its strengths and weaknesses, are discussed.     

Alkylation damage, DNA repair and mutagenesis in human cells

17 human cell lines that differ significantly in level of O⁶-alkylguanine-DNA alkyltransferase (AGT) activity were identified by comparing their sensitivity to the cytotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and determining the level of AGT activity in cell extracts from the various lines by measuring the decrease in radiolabeled O⁶-methylguanine from DNA, using high-performance liquid chromatography. 9 lines exhibited high levels of AGT activity, 2 showed an intermediate level (25–50% of the mean of those with the higher levels), and 6 exhibited very low or virtually undetectable levels of AGT. Included were several lines that are very deficient in capacity for nucleotide excision repair. When representatives from the 3 categories of cell lines defined by the level of AGT activity were compared for sensitivity to the cytotoxic and mutagenic effect of MNNG, they showed an inverse correlation between the degree of cell killing and frequency of mutants induced and the level of AGT activity. The cells' capacity for nucleotide excision repair did not affect these results. Exposure of cells with a high level of AGT activity to O⁶-methylguanine in the medium reduced the AGT activity 60–80%. These pre-treated cells exhibited a significantly higher frequency of MNNG-induced mutants than did cells that were not pre-treated, suggesting that the O⁶-methylguanine lesion in DNA is responsible for a significant proportion of the mutations induced. Cell strains containing substrates for assaying intrachromosomal homologous recombination were constructed using parental cell lines from each of the 3 categories of AGT activity. These strains showed an inverse correlation between the level of AGT activity and the frequency of MNNG-induced recombination. When various cell lines representing the 3 categories of AGT activity were compared for sensitivity to ethylnitrosourea, the results were consistent with AGT and nucleotide excision repair playing a role in preventing cell killing and mutation induction by this agent.