Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose-response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.     

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Release of nitric oxide together with carbon-centered radicals from N-nitrosamines by ultraviolet light irradiation

Solutions of N-nitrosamines, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomorpholine and N-nitrosopyrrolidine in phosphate buffer (pH 7.4) were irradiated by ultraviolet (UV) light at room temperature. The N-nitrosamines were extensively degraded due to irradiation for 120 min in a time-dependent fashion as monitored by UV-absorption or high performance liquid chromatographic analysis. Carbon-centered radicals were generated from four N-nitrosamines during the short time irradiation of 10–60 s as monitored by electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-α-phenylnitrone as spin traps. Nitric oxide (NO) was generated during the short time irradiation as monitored by ESR technique using cysteine-Fe(II) complex, N-methyl-d-glucamine dithiocarbamate and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Significant amounts of nitrite (4–16%) from four N-nitrosamines and also a significant amount of nitrate (4%) was produced from N-nitrosodimethylamine during the irradiation time of 120 min. Released NO from the N-nitrosamines must be converted into nitrite through intermediary reactive nitrogen oxide species including nitrogen dioxide and dinitrogen trioxide in contact with dissolved oxygen.     

DNA-repair genes and vitamin E in the prevention of <Emphasis Type="Italic">N</Emphasis>-nitrosodiethylamine mutagenicity

Nitrosamines are stable compounds, biologically and chemically inert unless activated. In biological systems, N-nitrosodiethylamine (NDEA) can be activated by a variety of enzymes, leading to aldehydes and/or intermediates which are themselves alkylating agents. Additionally, it has been shown that NDEA causes reactive oxygen species (ROS) production and induces mutagenicity. The cell defense seeks to neutralize ROS that escape the primary defense mechanisms (antioxidants) by DNA-repair mechanisms. NDEA is present at low concentrations in major dietary sources, like cured meats, salami, millet flour, and dried cuttlefish, where NDEA mutagenicity has been detected. These facts lead us to evaluate vitamin E as a ROS scavenger, in Escherichia coli mutants system, against genotoxicity induced by NDEA at low concentrations under exogenous metabolic activation. Statistical analysis were performed in order to compare the effects of NDEA-induced genotoxicity (a) between the mutants and the wild-type strains, at the same metabolic activation conditions and, (b) between the same strains in the presence or in the absence of vitamin E (150 μM). The indirect evaluation of ROS production by NDEA metabolizing shows that vitamin E protects E. coli cells proficient or deficient in the DNA-repair genes from cytotoxic effects. Our results underscore the role of scavenger molecules such as vitamin E in the diet, avoiding lesions induced by NDEA at low concentrations, via ROS, that could be repaired by nucleotide excision repair and base excision repair proteins.     

Random mutagenesis and use of 2-deoxy-D-glucose as an antimetabolite for selection of α-amylase-overproducing mutants of Aspergillus oryzae

By using 2-deoxy-D-glucose, selection of different mutants of Aspergillus oryzae PTCC 5164, which were produced by random mutagenesis by u.v. radiation, nitrous acid and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), was studied. 2-Deoxy-D-glucose, a well-known antimetabolite, was used to isolate derepressed mutants. The mutational and lethal effects of these mutagens on conidia of A. oryzae were compared and the frequency distribution of isolated mutants, in the presence of 2-deoxy-D-glucose, was determined. Potent mutants, which produced higher dextrinizing and saccharogenic activities, were isolated. The best strain was a result of mutagenesis by nitrous acid, which produced 6.73 times more dextrinizing and 5.13 times more saccharogenic activity than the parent strain. In general, the mutants obtained by nitrous acid and u.v. were more potent than those obtained by MNNG.     

Inheritance of susceptibility to induction of nephroblastomas in the Noble rat

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.     

Argininosuccinate synthetase as a marker of transformed Syrian hamster epidermal cells

Thirteen continuous lines of Syrian hamster epidermal cells were isolated following direct treatment of epidermal cells with N-methyl-N′-nitro-N-nitrosoguanidine, or from epidermal cells cultured from animals treated transplacentally with 7,12-dimethylbenz(a)anthracene or dimethylnitrosamine. The epidermal origin of the lines was evidenced by the presence of desmosomes with radiating tonofilaments, keratin fibers, and the ability of the cells to use citrulline in place of arginine corresponding with their high argininosuccinate synthetase activity.     

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells

Summary The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.     

Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine

Chemopreventive effect of S-allylcysteine (constituent of garlic) on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis was evaluated in Wistar rats. Significantly decreased lipid peroxidation products (thiobarbituric acid reactive substances-TBARS and lipid hydroperoxides) with increased level of reduced glutathione, increased activities of glutathione S-transferase, and glutathione peroxidase were observed in liver of NDEA-treated rats when compared with control rats. The activities of superoxide dismutase and catalase were significantly decreased in tumor tissue when compared with control. Administration of S-allylcysteine (SAC) showed the inhibition of tumor incidence, modulated the lipid peroxidation, and increased the reduced glutathione, glutathione-dependent enzymes, superoxide dismutase, and catalase in NDEA-induced carcinogenesis. From our results, we speculate that S-allylcysteine mediates its chemopreventive effects by modulating lipid peroxidation, GST stimulation, and by increasing the antioxidants. Hence SAC prevents cells from loss of oxidative capacity in NDEA-induced hepatocarcinogenesis.     

Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis

Nannochloropsis oculata was subjected to N-methyl-N-nitrosourea-induced mutagenesis under the selection pressure of quizalofop, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity with the objective of generating genetically tractable mutants with altered fatty acid metabolism. Two mutants, QUIZ1 and QUIZ2, with stable resistance to quizalofop were isolated and partially characterized. The growth properties and morphology of the mutants appeared identical with the parent strain. However thermo-tolerance was observed in the mutants. Enhanced resistance to quizalofop suggested the presence of herbicide resistant isoforms of ACCase. In vitro assays for ACCase activity showed that ACCase in the wild strains was much more sensitive to quizalofop than the mutant strains. Gas chromatographic analysis of fatty acids revealed that the mutant strains were rich in polyunsaturated fatty acids (n– 3PUFAs), as well as total fatty acid contents; this was accompanied by a concomitant increase in triacylglycerol (TAG) followed by linoleic acid (18:2), arachidonic acid (20:4 n– 6) and EPA (20:5 n– 3). These results suggest that an increased substrate pool (malonyl-CoA) (due to increased specific activity of ACCase) in the mutant strains in vivo and in vitro may have led to the increased TAG accumulation. Random mutagenesis was shown to be a good tool to manipulate PUFAs and EPA in Nannochloropsis. The strains developed will be useful in understanding fatty acid metabolism using genetic and biochemical approaches and also for their direct use in mariculture.     

Evaluation of two alternative metabolic pathways for creatinine degradation in microorganisms

The microbial decomposition of creatinine was found to proceed mainly via N-methylhydantoin or creatine as the first degradation product. Either N-methylhydantoin or urea or both were detected as metabolites derived from creatinine in various microorganisms, and creatinine deiminase and creatinine amidohydrolase activities were detected concomitantly. N-Methylhydantoin hydrolase and N-carbamoylsarcosine amidohydrolase were found to be formed inducibly in the presence of creatinine or N-methylhydantoin. Three microorganisms which decompose creatinine in different ways were screened from soil. Pseudomonas putida 77 rapidly metabolized creatinine solely via N-methylhydantoin. Degradation of creatinine proceeded with both creatine and N-methylhydantoin as the first degradation products at the same time in Pseudomonas sp. H21. Pseudomonas sp. 0114 was found to metabolize creatinine mainly via creatine and to also metabolize N-methylhydantoin. Changes in the metabolites of creatinine during a cultivation or enzyme reaction were found to be closely related to the enzyme activities of interest which are regulated by creatinine or N-methylhydantoin in different ways depending on the microbial strain.     

Brain Stem GABA Receptor Functional Regulation During Rat Liver Cell Proliferation

GABAergic alterations in brain stem during compensatory hyperplasia after partial hepatectomy (PH), lead nitrate (LN)-induced direct hyperplasia, and N-nitrosodiethylamine (NDEA)-induced neoplasia in liver were investigated. GABA content decreased in brain stem of PH- and NDEA-treated rats while it increased in LN-treated rats. GABA_A receptor number and affinity in brain stem membrane preparations of rats showed a significant decrease in PH- and NDEA-treated rats. The GABA_B receptor number increased in PH- and NDEA-treated rats with an increase in affinity. The results of the present study indicate that liver cell proliferation is influencing the brain stem GABAergic neurotransmission and these changes regulate the hepatic proliferation through the sympathetic stimulation.     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Interaction ofN-acetyl-4-, 7-, 8- or 9-deoxyneuraminic acids andN-acetyl-4-, 7- or 8-mono-epi- and-7,8-di-epineuraminic acids withN-acetylneuraminate lyase

Various deoxy- and epi-derivatives ofN-acetylneuraminic acid were synthesized and tested for their substrate properties withN-acetylneuraminate lyase fromClostridium perfringens.N-Acetyl-9-deoxyneuraminic acid is a good substrate,N-acetylneuraminic acid derivatives with epimeric configuration at C-7, C-8 or both are cleaved slowly, whileN-acetyl-4-epi-,N-acetyl-4-deoxy-,N-acetyl-7-deoxy-andN-acetyl-8-deoxyneuraminic acid are resistant to enzyme action.N-Acetyl-4-deoxyneuraminic acid andN-acetyl-4-epineuraminic acid competitively inhibit the enzyme. These studies give further insight into a mechanism proposed for the reversible cleavage of sialic acids byN-acetylneuraminate lyase.     

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Improvement in Inulinase Production by Simultaneous Action of Physical and Chemical Mutagenesis in Penicillium purpurogenum

Summary An extracellular inulinase (fructanase)-producing strain of Penicillium purpurogenum was isolated from the rhizosphere soil of chicory. Conidia of this selected strain were subjected to simultaneous treatment with NTG–UV (N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet radiation) and EtBr–UV (Ethidium bromide–ultraviolet radiation). After mutagenesis, colonies were screened and among them a few were selected to carry out the inulinase study, which showed a significantly higher inulinase activity with higher I/S (inulin/sucrose) ratio in all the selected colonies, indicating enhancement of inulinase production after mutagenic treatments in all the selected mutants.