Time- and concentration-dependent genomic responses of the rat airway to inhaled nickel sulfate.

While insoluble nickel subsulfide (Ni3 S2 ) was carcinogenic in the lung in a 2-year rat bioassay, soluble nickel sulfate hexahydrate (NiSO4* 6H2 O) was not. To investigate whether differences in the cellular responses to these two nickel compounds could underlie their differential activities, we conducted parallel studies to determine the gene expression changes in micro-dissected lung distal airway cells from Fischer 344 rats following inhalation of the two compounds for one and four weeks (6 hr per day, 5 days per week). The results of the Ni3 S2 study have been reported previously; this paper reports the results for NiSO4 and provides a comparative analysis. The cellular responses to NiSO4 were highly similar to those previously reported for Ni3 S2 , and a set of genes was identified whose expression could be used as biomarkers for comparing cellular nickel effects from in vitro or in vivo studies with soluble NiSO4 and particulate Ni3 S2 . Evaluation of the genomic concentration-responses for the two compounds suggests that the highest inhaled concentration in the tumor bioassay for NiSO4 , which was limited by toxicity, may not have achieved the Ni concentrations at which tumors were observed in the Ni3 S2 bioassay. However, several key differences in the immune responses to NiSO4 and Ni3 S2 were identified that may result from the differential intracellular disposition of Ni from NiSO4 entering the cell as an ion rather than as a slowly soluble Ni3 S2 particle. These differences may also contribute to the observation of tumors in the bioassay for Ni3 S2 but not NiSO4 . Environ. Mol. Mutagen. 58:607-618, 2017. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Time- and concentration-dependent genomic responses of the rat airway to inhaled nickel subsulfide.

OBJECTIVE: To provide insights into the mode of action for Ni3S2 lung carcinogenicity by examining gene expression changes in target cells after inhalation exposure. METHODS: Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer 344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m(3) (0.03, 0.06, 0.11, and 0.44 mgNi/m(3)) for one and four weeks (6h/day, 5 days/week). RESULTS: Broncho-alveolar lavage fluid evaluation and lung histopathology provided evidence of inflammation only at the two highest concentrations, which were similar to those tested in the 2-year bioassay. The number of statistically significant up- and down-regulated genes decreased markedly from one to four weeks of exposure, suggesting adaptation. Cell signal pathway enrichment at both time-points primarily reflected responses to toxicity, including inflammatory and proliferative signaling. While proliferative signaling was up-regulated at both time points, some inflammatory signaling reversed from down-regulation at 1 week to up-regulation at 4 weeks. CONCLUSIONS: These results support a mode of action for Ni3S2 carcinogenicity driven by chronic toxicity, inflammation and proliferation, leading to mis-replication, rather than by direct genotoxicity. Benchmark dose (BMD) analysis identified the lowest pathway transcriptional BMD exposure concentration as 0.026 mgNi/m(3), for apoptosis/survival signaling. When conducted on the basis of lung Ni concentration the lowest pathway BMD was 0.64 µgNi/g lung, for immune/inflammatory signaling. IMPLICATIONS: These highly conservative BMDs could be used to derive a point of departure in a nonlinear risk assessment for Ni3S2 toxicity and carcinogenicity.

Evaluation of the human nickel retention model with workplace data.

A previously developed dosimetry model of inhaled Ni compounds in the human lung has been expanded by incorporating three additional factors: (1) inhalability, (2) mixed breathing mode, and (3) clearance rate coefficient of a Ni compounds' mixture. Predicted lung burdens of Ni compounds from the expanded model for workers at different departments in a Ni refinery plant compared favorably with the reported data. Although many uncertainties remain, the present model represents a first step toward developing a more meaningful risk assessment tool that will allow for more accurate extrapolation of animal data to humans.

Carcinogenicity assessment of selected nickel compounds.

The early epidemiological data indicated different carcinogenic risks from inhalation of different nickel compounds, but it was not clear what characteristics governed the intrinsic carcinogenic hazard of the various nickel compounds. Based on the earlier results, all soluble and insoluble nickel compounds were assumed to have the same carcinogenic mechanism albeit different potencies. Recent in vivo and in vitro studies challenged this assumption. In this paper an attempt is made to integrate the most relevant human, animal, and in vitro data into a general model that can help understand the different carcinogenic potentials of the various nickel compounds. In this perspective, it is recognized that there are two main components that could contribute to the development of lung cancer via exposure to certain nickel compounds. The first component corresponds to the heritable changes (genetic or epigenetic) derived from the direct or indirect actions of nickel compounds. The second component may be the promotion of cell proliferation elicited by certain nickel compounds. The different contributions of three nickel compounds to these two components are presented. This paper emphasizes the importance of recognizing the individuality of the different nickel species in reaching regulatory decisions and the fact that different risk assessment considerations may apply for compounds that appear to produce immortality and cancer by genetic/epigenetic mechanisms (like nickel subsulfide), compounds that may present a threshold for the induction of tumors in rats (like high-temperature nickel oxide), or compounds that may only have an enhancing effect on carcinogenicity (like nickel sulfate).

Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele.

We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the alpha subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rifr, lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by approximately 3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A.T-->T.A are affected most strongly (approximately 6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A.T-->G.C base-pair substitution hotspot in the lac operator is also reduced (approximately 5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.

The Escherichia coli galK2 papillation assay: its specificity and application to seven newly isolated mutator strains.

The Escherichia coli dnaE and dnaQ genes encode, respectively, the alpha (polymerase) and epsilon (proofreading) subunits of DNA polymerase III. Mutations in these genes resulting in mutator or antimutator phenotypes provide important tools to understand the mechanisms by which mutations occur. One way to isolate such strains is the use of papillation assays. We used one such assay based on the reversion of the galK2 allele in cells grown on MacConkey-Gal plates. Here, we describe the identification of the galK2 mutation and its possible reversion pathways, and the characterization of 7 mutators isolated using this system. 1 mutator resided in dnaE and 6 in dnaQ. Sequencing of the galK2 allele revealed a G.C-->T.A transversion at base pair 571 that changed a glu codon (GAA) to a stop codon (TAA). The analysis of 319 revertants showed that a Gal+ phenotype can be achieved by A.T-->G.C transition, A.T-->T.A transversion and A.T-->C.G transversion. We characterized the mutator phenotypes of the newly isolated mutators by determining (i) their mutation frequencies to resistance to rifampicin and nalidixic acid in both wild-type and mutL backgrounds, (ii) their temperature sensitivity and medium dependence and (iii) their mutational specificity (by analyzing the nature of galK revertants). Based on the genomic locations of their mutations, specificity of reversion pathways and magnitude of mutator effects, the mutators can be grouped into 3 classes. These classes may represent different mutational mechanisms that include defective base insertion, defective proofreading and interference with the postreplicative mismatch-repair system.

Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene.

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)

Transcription-repair coupling determines the strandedness of ultraviolet mutagenesis in Escherichia coli.

We have analyzed the spectra of UV-induced mutations in the lacI gene of a wild-type and an mfd strain of Escherichia coli. mfd strains have been recently proposed to be deficient in a factor coupling DNA repair and transcription. Analysis of UV-induced mutations occurring at adjacent pyrimidines showed that mutations in the wild-type strain arose largely from the nontranscribed strand but arose predominantly from the transcribed strand in the mfd strain. The overall strand switch was 14-fold. One mutation, G.C-->A.T in the lacI initiation codon, showed a > 300-fold shift. No effect was observed for mutations at non-pyrimidine-pyrimidine sequences. These results provide in vivo evidence for a key role of the mfd gene in controlling the strandedness of mutagenesis and support the proposed role of the mfd gene product in directing DNA excision repair to the transcribed strand of a damaged gene.

Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species.

Previous work has described the novel ability to modulate in vitro the activity of restriction endonuclease NaeI from Nocardia aerocoligenes by using cleavable DNA and spermidine [Conrad & Topal (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. In this paper we report the results of a study of 49 type II restriction enzymes from a variety of bacterial species. On the basis of the rates of cleavage observed, we found that in addition to expected cleavable sites a number of enzymes had slow and resistant cognate recognition sites. Resistant sites were identified for BspMI, NaeI, and NarI; slow sites were identified for HpaII, NaeI, and SacII. Cleavage of these sites was found to be significantly enhanced by the addition of cleavable DNA or spermidine. We demonstrate that for BspMI, as for NaeI, activator DNAs increased Vmax without altering Km, whereas for HpaII, NarI, and SacII activator DNAs decreased Km without changing Vmax. Comparison among the Kms for NaeI cleavage of several different substrates demonstrated that distant DNA sequences can affect DNA recognition by the activated enzyme. Our observations extend DNA activation of the Nocardia NaeI endonuclease to restriction endonucleases from Nocardia argentinensis (NarI), Bacillus species M (BspMI), Haemophilus parainfluenza (HpaII), and Streptomyces achromogenes (SacII). In addition, activation has now been found to affect slow as well as resistant recognition sites.

Growth of mammalian cells at high oxygen concentrations.

Oxygen, although essential to the growth of mammalian cells in vitro and in vivo, has been widely reported to be toxic at concentrations at or above the oxygen concentration in culture medium equilibrated with air (approximately 200 microM). We were therefore surprised to note that a diploid human B-cell line (TK6) was able to proliferate normally while exposed to 380 microM-oxygen. This observation was extended to Vero (African Green monkey kidney) cells, and Sp2/0-derived murine transfectomas producing antibody. Using an experimental system with a high capacity for oxygen transfer, we determined the growth rates of the three cell lines at controlled oxygen concentrations ranging from 80 microM to 910 microM. Each of these cell types was able to grow normally at oxygen concentrations up to 360-380 microM. At oxygen concentrations above 380 microM, a significant increase in the apparent doubling times of the cells was observed. No adverse effect of oxygen on TK6 cell survival was seen for concentrations ranging from 60 microM to 410 microM. We conclude that exponential growth at nearly maximum growth rates was observed for Sp2/0-derived cells, TK6, and Vero cells at constant oxygen concentrations up to 400 microM (twice air saturation). These findings have substantial implications for process control in the production of cells and cell-derived materials. The intrinsic oxygen transfer rate of any cell culture vessel can be increased severalfold by raising the oxygen gas concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A statistical model to estimate variance in long term-low dose mutation assays: testing of the model in a human lymphoblastoid mutation assay.

Long term-low dose mutation assays offer a means to study the genetic effects of environmental mutagens at concentrations relevant to human exposure. These assays involve continuous induction of mutants, serial dilution of cultures and sampling to determine the mutant fraction as a function of time and mutagen concentration. An arithmetic model for the expected variance among identically treated cultures is presented. This model provides means to calculate a predicted variance of the mutant fractions and mutation rates in typical long term-low dose experiments. We have calculated the expected variances of the mutant fraction with this model and compared them to the observed variances among 4 independent experiments in which human lymphoblastoid cells were treated for 5, 10, 15 and 20 days with a non-toxic concentration of the mutagen 4-aminobiphenyl. Mutations at the HPRT locus were measured by determining the 6-thioguanine-resistant mutant fraction. The expected and observed variances of the mutant fractions are in close agreement. This model is adequate to predict the variance of the mutant fraction and should be useful in experimental design and objective evaluation of long term-low dose mutation assays.