Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations.

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

A K homology (KH) domain protein identified by a forward genetic screen affects bundle sheath anatomy in Arabidopsis thaliana.

Because of their photosynthetic capacity, leaves function as solar panels providing the basis for the growth of the entire plant. Although the molecular mechanisms of leaf development have been well studied in model dicot and monocot species, a lot of information is still needed about the interplay of the genes that regulate cell division and differentiation and thereby affect the photosynthetic performance of the leaf. We were specifically interested in understanding the differentiation of mesophyll and bundle sheath cells in Arabidopsis thaliana and aimed to identify genes that are involved in determining bundle sheath anatomy. To this end, we established a forward genetic screen by using ethyl methanesulfonate (EMS) for mutagenizing a reporter line expressing a chloroplast-targeted green fluorescent protein (sGFP) under the control of a bundle sheath-specific promoter. Based on the GFP fluorescence phenotype, numerous mutants were produced, and by pursuing a mapping-by-sequencing approach, the genomic segments containing mutated candidate genes were identified. One of the lines with an enhanced GFP fluorescence phenotype (named ELEVATED BUNDLE SHEATH CELLS SIGNAL 1 [ebss1]) was selected for further study, and the responsible gene was verified by CRISPR/Cas9-based mutagenesis of candidate genes located in the mapped genomic segment. The verified gene, At2g25970, encodes a K homology (KH) domain-containing protein.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Effect of ethyl methanesulfonate mediated mutation for enhancing morpho-physio-biochemical and yield contributing traits of fragrant rice.

Background: Chemical mutagenesis has been successfully used for increasing genetic diversity in crop plants. More than 800 novel mutant types of rice (Oryza sativa L.) have been developed through the successful application of numerous mutagenic agents. Among a wide variety of chemical mutagens, ethyl-methane-sulfonate (EMS) is the alkylating agent that is most commonly employed in crop plants because it frequently induces nucleotide substitutions as detected in numerous genomes. Methods: In this study, seeds of the widely consumed Basmati rice variety (Super Basmati, Oryza sativa L.) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1.0%, and 1.25% to broaden its narrow genetic base. Results: Sensitivity to a chemical mutagen such as ethyl methanesulfonate (EMS) was determined in the M1 generation. Results in M1 generation revealed that as the levels of applied EMS increased, there was a significant reduction in the germination percent, root length, shoot length, plant height, productive tillers, panicle length, sterile spikelet, total spikelet, and fertility percent as compared to the control under field conditions. All the aforementioned parameters decreased but there was an increase in EMS mutagens in an approximately linear fashion. Furthermore, there was no germination at 1.25% of EMS treatment for seed germination. A 50% germination was recorded between 0.50% and 0.75% EMS treatments. After germination, the subsequent parameters, viz. root length and shoot length had LD50 between 05.0% and 0.75% EMS dose levels. Significant variation was noticed in the photosynthetic and water related attributes of fragrant rice. The linear increase in the enzymatic attributes was noticed by the EMS mediated treatments. After the establishment of the plants in the M1 generation in the field, it was observed that LD50 for fertility percentage was at EMS 1.0% level, for the rice variety. Conclusion: Hence, it is concluded that for creating genetic variability in the rice variety (Super Basmati), EMS doses from 0.5% to 0.75% are the most efficient, and effective.

Enhancing the Antibiotic Production by Thermophilic Bacteria Isolated from Hot Spring Waters via Ethyl Methanesulfonate Mutagenesis.

Antibiotic-resistant bacteria represent a serious public health threat. For that reason, the development of new and effective antibiotics to control pathogens has become necessary. The current study aims to search for new microorganisms expressing antibiotic production capacity. Fifteen sites covering a wide range of harsh environmental conditions in Egypt were investigated. Two hundred and eighty bacterial isolates were obtained and then tested against pathogenic bacteria using the agar disk diffusion technique. Fifty-two (18.6% of the total) of the isolates exhibited antagonistic properties, which affected one or more of the tested pathogens. The isolate 113 was identified as Bacillus licheniformis and isolate 10 was identified as Brevibacillus borstelensis using the 16S rRNA technique. The B. licheniformis strain was stronger in antibiotic production against S. typhi, M. luteus, and P. ariginosa, whereas the strain Br. borstelensis was more efficient against B. cereus, E. coli, and Klebs. sp. The sensitivity of the strains to commercial antibiotics showed that B. licheniformis was highly sensitive to seven commercial antibiotics, whereas Br. borstelensis was sensitive to nine antibiotics. The two strains were subjected to ethyl methanesulfonate (EMS) mutagenesis to obtain mutants with a higher antibiotic production. The total bacterial count was measured after treatment with EMS mutagen and showed a significant gradual increase in the antimicrobial activity, which was achieved via shaking in the presence of EMS for 60 min. High antimicrobial activities were noted with 17 and 14 mutants from the B. licheniformis and Br. borstelensis strains, respectively. The mutant B. licheniformis (M15/Amo) was more active than the parent strain against S. aureus (212.5%), while the mutant Br. borstelensis (B7/Neo) was more effective against S. typhi (83.3%). The present study demonstrates the possibility of obtaining potent antibiotic-producing bacteria in hot spring waters and further improving the indigenous bacterial capacity to produce antibiotics by using EMS mutagenesis.

The effects of mutations on gene expression and alternative splicing.

Understanding the relationship between mutations and their genomic and phenotypic consequences has been a longstanding goal of evolutionary biology. However, few studies have investigated the impact of mutations on gene expression and alternative splicing on the genome-wide scale. In this study, we aim to bridge this knowledge gap by utilizing whole-genome sequencing data and RNA sequencing data from 16 obligately parthenogenetic Daphnia mutant lines to investigate the effects of ethyl methanesulfonate-induced mutations on gene expression and alternative splicing. Using rigorous analyses of mutations, expression changes and alternative splicing, we show that trans-effects are the major contributor to the variance in gene expression and alternative splicing between the wild-type and mutant lines, whereas cis mutations only affected a limited number of genes and do not always alter gene expression. Moreover, we show that there is a significant association between differentially expressed genes and exonic mutations, indicating that exonic mutations are an important driver of altered gene expression.

Variations in Total Protein and Amino Acids in the Sequenced Sorghum Mutant Library.

Sorghum (Sorghum bicolor) is the fifth most important cereal crop worldwide; however, its utilization in food products can be limited due to reduced nutritional quality related to amino acid composition and protein digestibility in cooked products. Low essential amino acid levels and digestibility are influenced by the composition of the sorghum seed storage proteins, kafirins. In this study, we report a core collection of 206 sorghum mutant lines with altered seed storage proteins. Wet lab chemistry analysis was conducted to evaluate the total protein content and 23 amino acids, including 19 protein-bound and 4 non-protein amino acids. We identified mutant lines with diverse compositions of essential and non-essential amino acids. The highest total protein content in these lines was almost double that of the wild-type (BTx623). The mutants identified in this study can be used as a genetic resource to improve the sorghum grain quality and determine the molecular mechanisms underlying the biosynthesis of storage protein and starch in sorghum seeds.

Investigation of mutation load and rate in androgenic mutant lines of rapeseed in early generations evaluated by high-density SNP genotyping.

Oilseed rape (Brassica napus) is an important oil crop distributed worldwide with a broad adaptation to different climate zones. The cultivation of rapeseed is one of the most commercially viable areas in crop production. Altogether 269,093 ha of rapeseed are cultivated in Kazakhstan. However, all rapeseed cultivars and lines cultivated in Kazakhstan on an industrial scale predominantly belong to the foreign breeding system. Therefore, the formation of a diverse genetic pool for breeding new, highly productive cultivars adopted to the environmental conditions of Kazakhstan is the most important goal in country selection programs. In this work, we have developed ethyl methanesulfonate (EMS) doubled haploid mutant lines from plant material of cultivars 'Galant' and 'Kris' to broad diversity of rapeseed in Kazakhstan. The development of mutant lines was performed via embryo callusogenesis or embryo secondary callusogenesis. Mutants were investigated by Brassica90k SNP array, and we were able to locate 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. The most frequent combination of mutations according to reference controls was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%). We revealed 5606.27 markers for 'Kris' and 4893.01 markers for 'Galant' by mutation occurrence. Most mutation occurrences were occupied by double mutations where progenitors and offspring were homozygous by different alleles, enabling the selection of appropriate genotypes in a short period of time. Regarding the biological impact of mutations, 861 variants were reported as having a low predicted impact, with 1042 as moderate and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Protein encoding genes, such as wall-associated receptor kinase-like protein 5, TAO1-like disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein, contained more than two variable positions, with an impact on their biological activities. Nevertheless, the obtained mutant lines were able to survive and reproduce. Mutant lines, which include moderate and high impact mutations in encoding genes, are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions. For the first time, a collection of mutant lines was developed in our country to improve the selection of local rapeseed cultivars.

Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population.

Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

Optimized methods for random and targeted mutagenesis in field pea (Pisum sativum L.).

Field pea is an important pulse crop for its dense nutritional profile and contribution to sustainable agricultural practices. Recently, it has received extensive attention as a potential leading source of plant-based proteins. However, the adoption of peas as a mainstream source of proteins is affected by a relatively moderate protein content, anti-nutritional factors and high levels of off-flavor components that reduce protein quality. Availability of genetic variation for desirable seed quality traits is the foundation for the sustainable development of pea varieties with improved protein content and quality. Mutagenesis has been an important tool in gene functional characterization studies and creating genetic variability for crop breeding. Large-scale mutagenesis of a crop using physical and chemical agents requires diligent selection of the mutagen and optimization of its dose to increase the frequency of mutations. In this study, we present detailed optimized protocols for physical and chemical mutagenesis of pea using gamma irradiation and ethyl methanesulfonate (EMS), respectively. Gamma radiation and EMS titration kill curves were established to identify optimal doses of the two mutagenic agents. Based on germination, survival rate and growth phenotypes, a gamma radiation dose of 225 Gy and EMS concentration of 5 mm were selected as optimal dosages for mutagenesis in field pea. The presented protocol has been modified from previously established mutagenesis protocols in other crop plants. Our results indicate that the optimal mutagen dosage is genotype dependent. CRISPR/Cas-based gene editing provides a precise and rapid method for targeted genetic manipulation in plants. With the recent success of gene editing in pea using CRISPR/Cas, this innovative technology is expected to become an integral component of the gene discovery and crop improvement toolkit in pea. Here, we describe an optimized methods for targeted mutagenesis of pea protoplasts, including mesophyll protoplast extraction, PEG-mediated transformation and gene editing of a LOX gene using CRISPR/Cas system. The general strategies and methods of mutagenesis described here provide an essential resource for mutation breeding and functional genomics studies in pea. These methods also provide a foundation for similar studies in other crops.

Measuring the frequency and distribution of meiotic crossovers in homozygous barley inbred lines.

We report a novel approach for establishing the number and position of CO events in individual homozygous inbred plants by combining low level EMS mutagenesis, speed breeding, whole genome shotgun sequencing and sliding window analysis of the induced molecular variant data. We demonstrate the approach by exploring CO frequency and distribution in self-fertilised progeny of the inbred barley cultivar Bowman and compare these observations to similar data obtained from a Bowman nearly isogenic line (BW230 Hvmlh3) containing a mutation in the DNA mismatch repair gene HvMLH3. We have previously shown that Hvmlh3 decreases both plant fertility and recombination by ~50%. We compare our results to those from previously published traditional genetic analysis of F3 families derived from multiple F2 lines containing WT or mutant alleles of HvMLH3, revealing a high level of correspondence between analyses. We discuss possible applications of the approach in streamlining the assessment of recombination in plant meiosis research.

Development of Ethyl Methanesulfonate Mutant Edamame Soybean (Glycine max (L.) Merr.) Populations and Forward and Reverse Genetic Screening for Early-Flowering Mutants.

Induced mutation is a viable breeding strategy that is widely utilized in the development of elite plant varieties. We aimed to improve a variety of edamame by constructing novel mutant populations using the ethyl methanesulfonate in soybeans (Glycine max (L.) Merr.). In the M2 population, the flowering stage showed a considerable standard deviation compared to the wild type, confirming that the mutant populations had the expected DNA mutations. To identify the DNA mutations in the mutant populations, we used the targeting induced local lesions in genomes (TILLING) method, which is a reverse genetic method, to search for soybean flowering-related gene mutants. A total of 30 mutants from E1, E3, E4, and PhyA1 genes, which are known to be highly effective genes, or their homologous gene for flowering and maturation found in soybean quantitative trait locus analyses were isolated from our TILLING screening. Among these mutants, there were eleven nonsynonymous substitution mutants, one nonsense mutant, and two single nucleotide deletion mutants that could be expected to reduce or eliminate gene function. The e1, e3, and e4 mutants obtained in this study flowered considerably earlier than the wild type. In particular, the e1 mutant with a nonsynonymous substitution flowered approximately 1 month after sowing regardless of the sowing date, and its harvest date was approximately 1 month earlier than that of the wild type. Mutations identified using the TILLING method could not only be used as gel-based DNA markers with the same manipulation method, but the mutations could also be detected as DNA markers by the high-resolution melting method. These results indicate that mutations achieved without chromosome modification by crossbreeding are effective for early and practical improvement of superior varieties and that efficient selection of mutants by reverse genetics is an effective method for the identification of genetic modifications. The edamame mutant populations developed in this study are believed to possess various useful alleles which may be applicable in the search for mutations that lead to improved edamame yield and eating quality beyond the flowering stage.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Dissecting Enhanced Carbohydrate and Pigment Productivity in Mutants of Nannochloropsis oculata Using Metabolomics and Lipidomics.

Random mutagenesis is an effective strategy for enhancing cellular traits. In this study, we used the mutagen ethyl methanesulfonate to create fast-growing Nannochloropsis oculata mutants. When cultivated in a photobioreactor with a diel cycle, two mutants exhibited 2.2-fold higher carbohydrate productivity and 3.5-4.0-fold higher pigment productivity than the wild type, while one of them also showed 2.5-fold higher lipid productivity. A comprehensive physiological, metabolomic, and lipidomic study showed that the mutants had high levels of glucose-, galactose-, and xylose-based carbohydrates. Their high growth rate was attributed to increased chlorophyll a content, improved nitrogen assimilation, storage, and recycling, and low monogalactosyldiacyl glycerol/digalactosyldiacyl glycerol ratio, which was responsible for higher biomass productivity. The investigation revealed upregulation of lipid precursors, shedding light on high lipid accumulation. The derived algae strains are capable of increasing the biosynthesis of value-added storage molecules without impairing growth, rendering them promising candidates for commercial development in future biorefineries.

Improving microalgae for biotechnology - From genetics to synthetic biology - Moving forward but not there yet.

Microalgae are a diverse group of photosynthetic organisms that can be exploited for the production of different compounds, ranging from crude biomass and biofuels to high value-added biochemicals and synthetic proteins. Traditionally, algal biotechnology relies on bioprospecting to identify new highly productive strains and more recently, on forward genetics to further enhance productivity. However, it has become clear that further improvements in algal productivity for biotechnology is impossible without combining traditional tools with the arising molecular genetics toolkit. We review recent advantages in developing high throughput screening methods, preparing genome-wide mutant libraries, and establishing genome editing techniques. We discuss how algae can be improved in terms of photosynthetic efficiency, biofuel and high value-added compound production. Finally, we critically evaluate developments over recent years and explore future potential in the field.

Corrigendum: A Novel Banana Mutant "RF 1" (Musa spp. ABB, Pisang Awak Subgroup) for Improved Agronomic Traits and Enhanced Cold Tolerance and Disease Resistance.

[This corrects the article DOI: 10.3389/fpls.2021.730718.].

A Novel Banana Mutant "RF 1" (Musa spp. ABB, Pisang Awak Subgroup) for Improved Agronomic Traits and Enhanced Cold Tolerance and Disease Resistance.

Banana is a major fruit crop grown in tropical and subtropical regions worldwide. Among cultivars, "FenJiao, FJ" (Musa spp. ABB, Pisang Awak subgroup) is a popular variety of bananas, due to its better sugar-acid blend and relatively small fruit shape. However, because the traditional FJ variety grows relatively high in height, it is vulnerable to lodging and unsuitable for harvesting. In this study, we sought desirable banana mutants by carrying out ethyl methanesulfonate (EMS) mutagenesis with the FJ cultivar. After the FJ shoot tips had been treated with 0.8% (v/v) EMS for 4 h, we obtained a stably inherited mutant, here called "ReFen 1" (RF1), and also observed a semi-dwarfing phenotype. Compared with the wild type (FJ), this RF1 mutant featured consistently improved agronomic traits during 5-year field experiments conducted in three distinct locations in China. Notably, the RF1 plants showed significantly enhanced cold tolerance and Sigatoka disease resistance, mainly due to a substantially increased soluble content of sugar and greater starch accumulation along with reduced cellulose deposition. Therefore, this study not only demonstrated how a powerful genetic strategy can be used in fruit crop breeding but also provided insight into the identification of novel genes for agronomic trait improvement in bananas and beyond.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells.

BACKGROUND: We previously showed that hormetic responses can be established in cell activity tests using human and murine adherent cells. This time, we examined whether hormetic responses can be established in cell proliferation tests using suspended human and murine lymphoid cells. METHODS: Human lymphoblastoid cells (TK6) and mouse lymphoma cells (L5178Y) were cultured in multi-well culture plates and treated with mitomycin C, ethyl methansulfonate, hygromycin B, aclarubicin or colchicine at various dose levels and the number of cells was measured at varied times using a flow cytometer. RESULTS: When the ratio of the number of cells treated with a test chemical to those in the negative control was plotted, the dose-response relationship typically showed a reverse U-shaped curve, indicating the occurrence of hormesis and existence of thresholds in cell toxicity. The hormetic responses depended largely on the test chemical, dose level and exposure time. When examining responses over the course of time, a J-shaped or fallen S-shaped curve was also observed. CONCLUSIONS: The dose-response relationship showed a reverse U-shaped curve, a hallmark of hormesis, at least some time points for all chemicals tested here, indicating that chemical hormesis can be established in in vitro cell proliferation tests.

Highly Efficient and Comprehensive Identification of Ethyl Methanesulfonate-Induced Mutations in Nicotiana tabacum L. by Whole-Genome and Whole-Exome Sequencing.

Tobacco (Nicotiana tabacum L.) is a complex allotetraploid species with a large 4.5-Gb genome that carries duplicated gene copies. In this study, we describe the development of a whole-exome sequencing (WES) procedure in tobacco and its application to characterize a test population of ethyl methanesulfonate (EMS)-induced mutations. A probe set covering 50.3-Mb protein coding regions was designed from a reference tobacco genome. The EMS-induced mutations in 19 individual M2 lines were analyzed using our mutation analysis pipeline optimized to minimize false positives/negatives. In the target regions, the on-target rate of WES was approximately 75%, and 61,146 mutations were detected in the 19 M2 lines. Most of the mutations (98.8%) were single nucleotide variants, and 95.6% of them were C/G to T/A transitions. The number of mutations detected in the target coding sequences by WES was 93.5% of the mutations detected by whole-genome sequencing (WGS). The amount of sequencing data necessary for efficient mutation detection was significantly lower in WES (11.2 Gb), which is only 6.2% of the required amount in WGS (180 Gb). Thus, WES was almost comparable to WGS in performance but is more cost effective. Therefore, the developed target exome sequencing, which could become a fundamental tool in high-throughput mutation identification, renders the genome-wide analysis of tobacco highly efficient.

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing.

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 µg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.