Mutagenic selectivity of carcinogenic nitroso compounds I. N-methyl-N-nitrosourethane

The mutagenicity of the carcinogen methylnitrosourethane (MNUr) was examined in Drosophila with a view to the determination of its activity on heterochromatic loci (especially rDNA) relative to those in the euchromatin. Assays were made of the yield of rDNA mutations (bobbed: bb) relative to other X-chromosome recessive lethals and visibles [X(l + v)] in the same male germ cells after treatment with different doses (1–10 mM) and at various stages in spermatogenesis.Dose dependence followed the same pattern for all genic loci and germ cell stages. In all instances, the regression of mutation frequency on injected molar dose was approximately linear, but could better be described by a quadratic dose curve. In contrast, the mutagenicity pattern during spermatogenesis varied according to the target genes. The response of the euchromatic loci reached a peak among the earlier germ cells (probably the spermatocytes), whereas that for the heterochromatic sites (including rDNA) was maximal in mature sperm.Mutagenic selectivity for rDNA with MNUr, as indicated by the percentage bb/X-mutations, was among the highest for the intrinsically reactive carcinogens (alkylating and arylating agents). This correlates with the strong carcinogenicity of MNUr and adds further support to the concept that rDNA mutations might well be a crucial step in cancer initiation.     

Mutagenicity of a series of hexacoordinate rhodium(III) compounds

19 rhodium(III) compounds have been tested for genetic damaging capabilities using an Escherichia coli differential repair assay and for mutagenicity in the strains of Salmonella typhimurium. 10 of these were active in both assays. Presence of the plasmid pKM101 was required for mutagenicity in Salmonella. Both the composition of the ligands and the three-dimensional structures of the coordination complexes profoundly affect genetic activity. In general, the structure—activity relationships appear to favor complexes with (1) a +1 charge, (2) 2 relatively labile leaving groups with 4 more strongly bonded amine ligands, and (3) a relatively slow rate of exchange of the ligands which is characteristic of substitutionally inert coordination complexes.     

Differential effects of acetone or aroclor 1254 pretreatment on the microsomal activation of dimethylnitrosamine to a mutagen

Pretreatment of mice with acetone enhances the microsomal N-demethylation of dimethylnitrosamine (DMN) at low substrate concentrations (<5 mM), while pretreatment with Aroclor 1254 represses this activity at low, but enhances it at high (> 35 mM) DMN concentrations. To relate the activity of DMN demethylase with the mutagenicity of DMN, liver microsomes were isolated aseptically from mice 18 h after acetone (3 ml/kg, ip), 5 days after Aroclor 1254 treatment (500 mg/kg, ip), or after treatment with the appropriate injection vehicles, and incubated with S. typhimurium (TA 1535), NADPH and DMN (1, 3 or 70 mM) for 5 to 60 min. After a 48-h incubation on minimal media, revertants per plate were determined. Microsomes from acetone pretreated mice bioactivated DMN to a mutagen at significantly higher (p < 0.001) levels when incubations were performed at 1 mM DMN. Aroclor-1254 microsomes exhibited a decreased ability to convert DMN to a mutagen at both 1 and 3 mM DMN (p < 0.05) and a significantly higher (p < 0.05) ability at 70 mM DMN. These data and published reports suggest multiple microsomal enzymes for DMN bioactivation and that acetone may enhance the enzyme that operates at environmentally important levels of DMN.     

Mutagenicity of dimethylnitrosamine in the metabolic process by rat liver microsomes

The mutagenicity of dimethylnitrosamine (DMN) for bacteria was investigated by means of the metabolic activation process of the compound with rat liver microsomes.Three strains of streptomycin (SM)-dependent Escherichia coli having tetracycline (TC)-resistance factor (Sd-E. coli(TC)) were derived for this study. The reverse mutation in these strains from SM dependence to non-dependence was used as the marker for mutagenicity. The drug resistance factor (R factor) which was transferred to these strains was used in order to get around the bacterial contamination throughout the experiments. The study of the mutagenicity of DMN metabolites has been made by incubating DMN with rat liver microsomes and cofactor system in the presence of indicator bacterial cells.The reverse mutation was markedly induced for all of three strains in the complete incubation mixture but it was not observed when the cofactor system was omitted or the liver microsomal suspension was replaced by the kidney cell sap. When the indicator bacterial cells were added to the mixture in which DMN was previously incubated with the microsomes and cofactor system, the mutagenicity was extremely decreased.     

Comparative mutagenesis by aminofluorene derivatives A possible effect of DNA configuration

The mutagenicity of N-acetoxy-N-2-acetylaminofluorene-(N-acetoxy-2AAF) for Salmonella typhimuricum TA98 is greatly reduced when compared to that of N-hydroxy-2-aminofluorene. This decrease in mutagenic response is accompanied by the formation of a deoxyguanosine-2-acetylaminofluorene adduct. The deoxyguanosine-2-aminofluorene adduct, characteristic of cells exposed to N-hydroxy-2-aminofluorene, was not detected in N-acetoxy-2AAF-treated cells. Enzymic deacetylation of N-acetoxy-2AAF results in restoration of potent mutagenicity. N-Acetoxy-2-acetylamino-7-iodofluorene is also more mutagenic than N-acetoxy-2AAF. Because the acetylated and unacetylated guanine induce greatly different configurational changes, the results may be indicative that the introduction of the syn configuration and a possible shift to the Z-conformation at the mutational hot spot of Salmonella typhimurium TA98 [(dG-dC)₈] results in reduced mutagenic potency.     

Relative effectiveness of neutrons and X-rays in induction of crossing over in drosophila males

Relative biological effectiveness of neutrons vs. X-rays in inducing crossing-over in males of D. melanogaster was investigated using 812 and 834 rad of neutrons and the same dose of X-rays. Crossing-over was induced in spermatocytes and spermatogonia of adults and pupae. Neutrons were 4 times more effective in spermatocytes of adults and their effectiveness in pupal spermatocytes was even more. Neutrons also induced more exchanges in spermatogonial cells including predefinitive spermatogonia. Higher effectiveness of neutrons can be attributed to their high linear energy transfer.     

DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis.We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes.DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles.Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides.These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.     

Analysis of mutagen specificity Drosophila melanogaster

The previously reported difference between the mutational spectra of hydrazine (HZ) and hydroxylamine (HA) was confirmed for one selected locus (miniature) at which hydrazine produces no mutations in treated late larval spermatogonia or premeiotic spermatocytes sampled by 3 days' progeny. The genetically effective dose was measured in most experiments by the production of v mutants, and in a few by the production of sex-linked lethals. In a total of over 37 000 X-chromosomes (16 000 from previous, and over 21 000 from present experiments) treatment with HZ yielded no m mutation, but 90 v mutations. After treatment with genetically equivalent doses of HA, m and v mutations were about equally frequent. The ratio of visible mutations at the v locus to lethals on the X-chromosomes was exceptionally high after either treatment. So was the ratio of m mutations to lethals after treatment with HA.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6: SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF REACTION STEPS*

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH₃CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (^Dk_app ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO₂H and CH₃CO₂H, respectively. In neither case was a lag observed, which was unexpected considering the k_cat and K_m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).     

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle

The vasa gene is a reliable germline marker to study the origin and development of germ cells and gonads, although the gene product (mRNA or protein) varies between different species. However, there has been little study on vasa genes in holothuroids to date. Here we determined the expression characteristics of the Apostichopus japonicus vasa gene (Aj-vasa) during gametogenesis in the ovary and testis using in situ hybridization and immunohistochemistry. During oogenesis, the expression pattern of Aj-vasa coincided at the mRNA and protein levels. Intensive signals in oogonia decreased gradually with the development of oocytes. Interestingly, the pattern was different during spermatogenesis. The Aj-vasa mRNA level was the highest in spermatogonia, reduced in spermatocytes, low in spermatids and absent in spermatozoa, but the Aj-VASA protein was restricted to spermatogonia and early spermatocytes. These expression characteristics of Aj-vasa persisted in both male and female gonads throughout the reproductive cycle. Our findings show that Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells; moreover, Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. Our findings indicate that Aj-vasa is vital in the development and differentiation of germ cells.     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

Recombinational and mutational analyses of subspecific differentiation in Oryza sativa L.

Recombinational and mutational analyses were performed in an attempt to elucidate the nature of the genic differences and the ancestral relationship between the indica and japonica subspecies of Oryza sativa L. by taking representative varieties from each group. The absence of cytological aberrations at meiosis in indica×japonica hybrids suggest that genetic differences are predominantly genic rather than chromosomal. The pattern of variation induced in the key characters, and the frequency and spectra of mutations obtained in each group, failed to support the hypothesis that indica has given rise to japonica, and suggested that the two subspecies have originated from a common ancestor.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine,dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O⁶-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.