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Abstract Background Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. Results We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 106 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 106 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 106 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. Conclusions All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Background: Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. Results: We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 106 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 106 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 106 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. Conclusions: All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation. [ABSTRACT FROM AUTHOR]

In this study, the concentrations of 16 Polycyclic Aromatic Hydrocarbon (PAH), considered priority by US EPA (US Environmental Protection Agency), in fine particulate matter (PM2.5), were determined in a forest reserve and in an urban area in the city of Rio de Janeiro. The PM2.5 samples were collected in the Tijuca Forest (TF) and on the Maracanã campus of the State University of Rio de Janeiro (UERJ), using PM2.5 high-volume air samplers, from November 2015 to April 2016. The organic matter was extracted, separated by liquid chromatography, and analyzed by gas chromatography-mass spectrometry (GC-MS). The mean total PAH (excluding naphthalene, acenaphthene, and acenaphthylene) concentrations were 0.46 ± 0.61 ng m−3 and 1.12 ± 0.71 ng m−3 in PM2.5 collected at TF and UERJ, respectively. The diagnostic ratios suggested vehicular sources for both sites with no clear distinction between light and heavy vehicular sources. Cluster and principal component analyses were also used to clarify the possible PAH sources. Simulations of air mass trajectories confirmed the transport of pollutants from the city to the forest. Mutagenicity tests revealed that the PM collected in the UERJ presented mutagenic positive activity, likely for nitro-PAH and amino-PAH, which may be related to vehicular emissions. For the TF, although the forest was impacted by the pollutants, no positive activity was detected. Correlation and cluster analyses showed different PAH distributions for the TF and UERJ sites, which indicates that the TF receives the air masses from the city but is also impacted by local emissions. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) is a potentially useful technology to achieve a more precise evaluation of chemical mutagenicity. To establish NGS-based mutagenicity assays, which enable the direct detection of chemically induced mutations in a whole genome manner, the selection of appropriate biological resources and their precise genome sequences are essential. Here, we performed genome re-sequencing analyses of Salmonella typhimurium LT-2 strains TA98 and TA100, which have been frequently used in mutagenicity assays. We identified several strain-specific mutations including those that were relevant to their known phenotypes ( his, Δ uvrB and rfa). The details of rfa mutations were first clarified in this study, which was a frameshift variant in rfaF and a missense variant in rfaC in TA98 and TA100, respectively. The uvrB deletion in TA98 was larger than that in TA100, which suggested differences in defects of lipopolysaccharide synthesis between these strains. The re-sequenced genome data of TA98 and TA100 will help us establish NGS-based bacterial mutagenicity assays and understand the biological events seen in them. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Benzothiazole-piperazine derivatives prepared previously, 1h and 1j are potential anticancer agents. Since the mutagenic and genotoxic properties of anticancer drugs compose an essential issue to be researched, this study is focused on the analysis of the mutagenicity and genotoxicity of these molecules. The mutagenicity of 1h and 1j were determined by Ames test performed on Salmonella TA98 and TA100 strains. Sample 1j was mutagenic on TA98 bacterial strain. However, compound 1h was not mutagenic in bacterial strains TA98 and TA100 with and without S9 activation. The genotoxicity of 1h was evaluated by the chromosomal aberration assay on human lymphocytes. Compound 1h was also not genotoxic in human lymphocytes in vitro. All results revealed that, 1h was not mutagenic in the two Salmonella strains tested and was not genotoxic in chromosomal aberration assay. Therefore results demonstrate that the described molecule is promising as a new anticancer drug without mutagenicity. Also, after performing Ames test with other recommended bacterial strains and in vivo experiments can be used safely for the development of new structures exhibiting anticancer activities. [ABSTRACT FROM AUTHOR]

ALC67 is an N-acylated thiazolidine compound with promising anticancer activity that led to the recent discovery of a series of 3-propionyl thiazolidine-4-carboxylic acid ethyl esters as a family of novel antiproliferative agents. Since the mutagenic and genotoxic properties of marketed anticancer molecules constitute a main issue to be addressed, this study focused on the analysis of the mutagenicity, antimutagenecity, and genotoxicity of this molecule. The mutagenicity and antimutagenicity of ALC67 were evaluated by Ames test performed on Salmonella TA98 and TA100 strains. The genotoxicity of this molecule was investigated in the chromosomal aberration assay on human lymphocytes. All results revealed that the analyzed structure is not mutagenic in the two Salmonella strains tested and was not genotoxic in human lymphocytes in vitro. On the other hand, it showed a weak antimutagenic effect in these two bacterial strains. The above results indicate that after performing some more mutagenicity assays using the other recommended strains, this compound can be safely used for the development of new structures exhibiting anticancer activities. [ABSTRACT FROM AUTHOR]

Highlights: [•] Derivatives of 2-ethyl-2-methylsuccinic acid showed no mutagenic effect. [•] Most of the 2-ethyl-2-methylsuccinic acid derivatives showed antimutagenic effect. [•] 2,2-Diphenylsuccinic acid derivatives mutagenicity dependent on Vibrio harveyi strain. [•] There is no structure–mutagenicity relationship among the tested compounds. [•] V. harveyi test can be applied for preliminary assessment of mutagenicity. [Copyright &y& Elsevier]

Abstract: The role of the –N(CH2CH2OH)2 group in producing a mutagenic response from 4-((3-(2-hydroxyethoxy)4-amino)phenylazo)-N,N-bis(2-hydroxyethyl)aniline has been investigated. To accomplish this goal, a group of substituted 4,4′-diaminoazobenzene dyes was synthesized, and their structures were confirmed using 1H NMR, TOF-LC-ESI mass spectrometry, and combustion analysis. Mutagenicity was determined using the standard Ames test in Salmonella strains TA98, TA100, and TA1538 with and without S9 enzyme activation. The results of this study provide evidence that the mutagenicity of the parent dye arises from the metabolic cleavage of N-hydroxyethyl groups to give the corresponding –NHCH2CH2OH and –NH2 substituted monoazo dyes as direct-acting mutagens. All 5 of the dyes studied were mutagenic at various levels with and without S9 enzyme activation in TA1538. In addition, the results show that removing one N-hydroxyethyl group and capping both –OH groups in the parent dye did not affect mutagenicity, whereas removing both N-hydroxyethyl groups produced a strong direct-acting mutagen in all three bacterial strains. Increasing the length of the N-alkyl chain from two to three carbon atoms eliminated mutagenicity in TA98 without S9 activation. [Copyright &y& Elsevier]

Abstract: Detection of mutagenic pollution in natural environment is difficult as there are thousands of known chemical mutagens, and they have mutagenic effects usually at very low concentrations. Plants are able to accumulate various substances, including mutagens, in their tissues. Here, we demonstrate that rapid detection of mutagenic activities of compounds accumulated in plant tissues is possible using a recently developed microbiological mutagenicity assay, based on induction of bioluminescence of a dim mutant of a marine bacterium Vibrio harveyi. Using this assay, it was possible to detect mutagenic activity in extracts from relatively small amounts of tissues (1.5g) collected from plants, which were cultured in vitro for 30 days in the presence of nano-molar concentrations of various chemical mutagens. Moreover, contrary to control samples, cultured in vitro without any mutagens, significant mutagenicity was detected in several extracts of plant tissues collected from natural environment. The whole procedure was as short as 4h or less. [Copyright &y& Elsevier]

A novel microbiological mutagenicity assay, based on bioluminescence of a marine bacterium Vibrio harveyi mutant strain, potentially suitable for monitoring and assessment of mutagenic pollution of marine environment, has been described recently. Here, we tested the use of this assay, in combination with either mussels ( Mytilus sp.) or semipermeable membrane devices (SPMDs), in assessment of accumulation of mutagens in marine water (samples of Baltic Sea water were tested). Either similar results were obtained in both systems or higher signals in the SPMD-based system were detected, depending on the tested water samples. We conclude that the use of both mussels and SPMDs in combination with the V. harveyi bioluminescence mutagenicity assay is a method suitable for monitoring and assessment of accumulation of mutagenic pollutants in marine environment, but in some cases the SPMD-based system may provide a more sensitive test. [ABSTRACT FROM AUTHOR]

Abstract: Powdered coal samples were extracted by a hot water extraction (HWE) process, and the determination of total organic carbon (TOC) in the HWE eluent was performed by a TOC analyzer. The degree of TOC in the eluent greatly varied with kind of coals, and it tended to increase as the O/C value of coal increased. By use of heat-treated coals, which had somewhat smaller specific surface areas compared to the raw coals, it was shown that the degree of TOC in the eluent was also affected by specific surface area of coal. Almost no leaching was observed for hazardous heavy metals. The environmental impacts of the extract from the HWE eluent were evaluated by the Ames Salmonella mutagenicity assay and the estrogen receptor binding assay. The extracts from the HWE eluent did not show any notable mutagenicity. However, the extract from the HWE eluent for a lignite provided an appreciable affinity to estrogen receptor. [Copyright &y& Elsevier]

The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the β-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity. [Copyright &y& Elsevier]

Existing mutagenicity tests for metazoans lack the direct observation of enhanced germline mutation rates after exposure to anthropogenic substances, therefore being inefficient. Cadmium (Cd) is a metal described as a mutagen in mammalian cells and listed as a group 1 carcinogenic and mutagenic substance. But Cd mutagenesis mechanism is not yet clear. Therefore, in the present study, we propose a method coupling short-term mutation accumulation (MA) lines with subsequent whole genome sequencing (WGS) and a dedicated data analysis pipeline to investigate if chronic Cd exposure on Chironomus riparius can alter the rate at which de novo point mutations appear. Results show that Cd exposure did not affect the basal germline mutation rate nor the mutational spectrum in C. riparius, thereby arguing that exposed organisms might experience a range of other toxic effects before any mutagenic effect may occur. We show that it is possible to establish a practical and easily implemented pipeline to rapidly detect germ cell mutagens in a metazoan test organism. Furthermore, our data implicate that it is questionable to transfer mutagenicity assessments based on in vitro methods to complex metazoans. Image 1 • Chronic exposure to Cd does not exert mutagenicity in germ cells in C. riparius. • It was established a pipeline to detect germ cell mutagens in metazoans. • The pipeline has the potential to fill the existing gap of mutagenicity assays for multicellular organisms. [ABSTRACT FROM AUTHOR]