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.

Expression of a Tardigrade Dsup Gene Enhances Genome Protection in Plants.

DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.

Construction of Gossypium barbadense Mutant Library Provides Genetic Resources for Cotton Germplasm Improvement.

Allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense) are cultivated worldwide for its white fiber. For centuries, conventional breeding approaches increase cotton yield at the cost of extensive erosion of natural genetic variability. Sea Island cotton (G. barbadense) is known for its superior fiber quality, but show poor adaptability as compared to Upland cotton. Here, in this study, we use ethylmethanesulfonate (EMS) as a mutagenic agent to induce genome-wide point mutations to improve the current germplasm resources of Sea Island cotton and develop diverse breeding lines with improved adaptability and excellent economic traits. We determined the optimal EMS experimental procedure suitable for construction of cotton mutant library. At M6 generation, mutant library comprised of lines with distinguished phenotypes of the plant architecture, leaf, flower, boll, and fiber. Genome-wide analysis of SNP distribution and density in yellow leaf mutant reflected the better quality of mutant library. Reduced photosynthetic efficiency and transmission electron microscopy of yellow leaf mutants revealed the effect of induced mutations at physiological and cellular level. Our mutant collection will serve as the valuable resource for basic research on cotton functional genomics, as well as cotton breeding.

Assessment of Phenotypic Variations and Correlation among Seed Composition Traits in Mutagenized Soybean Populations.

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous mutations. In mutation breeding, genetic variations from induced mutations provide abundant sources of alterations in important soybean traits; this facilitated the development of soybean germplasm with modified seed composition traits to meet the different needs of end users. In this study, a total of 2366 'Forrest'-derived M2 families were developed for both forward and reverse genetic studies. A subset of 881 M3 families was forward genetically screened to measure the contents of protein, oil, carbohydrates, and fatty acids. A total of 14 mutants were identified to have stable seed composition phenotypes observed in both M3 and M4 generations. Correlation analyses have been conducted among ten seed composition traits and compared to a collection of 103 soybean germplasms. Mainly, ethyl methanesulfonate (EMS) mutagenesis had a strong impact on the seed-composition correlation that was observed among the 103 soybean germplasms, which offers multiple benefits for the soybean farmers and industry to breed for desired multiple seed phenotypes.

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints.

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days -1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBCCD59- ) or reticulocytes (RETCD59- ) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Proteomics Analysis to Identify Proteins and Pathways Associated with the Novel Lesion Mimic Mutant E40 in Rice Using iTRAQ-Based Strategy.

A novel rice lesion mimic mutant (LMM) was isolated from the mutant population of Japonica rice cultivar Hitomebore generated by ethyl methane sulfonate (EMS) treatment. Compared with the wild-type (WT), the mutant, tentatively designated E40, developed necrotic lesions over the whole growth period along with detectable changes in several important agronomic traits including lower height, fewer tillers, lower yield, and premature death. To understand the molecular mechanism of mutation-induced phenotypic differences in E40, a proteomics-based approach was used to identify differentially accumulated proteins between E40 and WT. Proteomic data from isobaric tags for relative and absolute quantitation (iTRAQ) showed that 233 proteins were significantly up- or down-regulated in E40 compared with WT. These proteins are involved in diverse biological processes, but phenylpropanoid biosynthesis was the only up-regulated pathway. Differential expression of the genes encoding some candidate proteins with significant up- or down-regulation in E40 were further verified by qPCR. Consistent with the proteomic results, substance and energy flow in E40 shifted from basic metabolism to secondary metabolism, mainly phenylpropanoid biosynthesis, which is likely involved in the formation of leaf spots.

Generation of Loss-of-Function Mutants for Wheat Rust Disease Resistance Gene Cloning.

One of the most important tools to identify and validate rust resistance gene function is by producing loss-of-function mutants. Mutants can be produced using irradiation, chemicals, and insertions. Among all the mutagens, ethyl methanesulfonate (EMS) and sodium azide are most favored because of the ease of use and generation of random point mutations in the genome. The mutants so produced facilitate the isolation, identification and cloning of rust resistance genes. In this chapter we describe a protocol for seed mutagenesis of wheat with EMS and sodium azide.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.

In-utero exposure to nelfinavir-ethyl methyl sulfone.

Ethyl methyl sulfone contained in nelfinavir between 2007 and 2008 accidentally exposed embryos and fetuses to a powerful mutagen. We report data for 101 HIV-uninfected children exposed in utero included in the French prospective national cohort. The incidence of malformation was similar to that in the cohort as a whole with different drug exposures; no children had developed cancer after 9 years of follow-up.

Fine mapping of a dominant gene conferring chlorophyll-deficiency in Brassica napus.

Leaf colour regulation is important in photosynthesis and dry material production. Most of the reported chlorophyll-deficient loci are recessive. The dominant locus is rarely reported, although it may be more important than the recessive locus in the regulation of photosynthesis efficiency. During the present study, we mapped a chlorophyll-deficient dominant locus (CDE1) from the ethyl methanesulfonate-mutagenized Brassica napus line NJ7982. Using an F2 population derived from the chlorophyll-deficient mutant (cde1) and the canola variety 'zhongshuang11', a high-density linkage map was constructed, consisting of 19 linkage groups with 2,878 bins containing 13,347 SNP markers, with a total linkage map length of 1,968.6 cM. Next, the CDE1 locus was mapped in a 0.9-cM interval of chromosome C08 of B. napus, co-segregating with nine SNP markers. In the following fine-mapping of the gene using the inherited F2:3 populations of 620 individuals, the locus was identified in an interval with a length of 311 kb. A bioinformatics analysis revealed that the mapping interval contained 22 genes. These results produced a good foundation for continued research on the dominant locus involved in chlorophyll content regulation.

Ethyl methanesulphonate in a parenteral formulation of BMS-214662 mesylate, a selective farnesyltransferase inhibitor: formation and rate of hydrolysis.

The objectives of the present study were to investigate the formation and rate of hydrolysis of ethyl methanesulphonate (EMS) in BMS-214662 mesylate drug substance and parenteral formulation by a gas chromatographic/mass spectrometric (GC/MS) method. EMS levels in the drug substance ranged between 0.3 microg/g and 0.8 microg/g. The parenteral formulation contains ethanol and the reaction between residual free methane sulphonic acid and ethanol may lead to the formation of EMS. Given that EMS is a potent mutagen, it is therefore of vital importance to eliminate or reduce the risk of human exposure. Data indicate no significant increase in the levels of EMS following storage of the drug product for 18 weeks at 25 degrees C or six weeks at 60 degrees C indicating that the potential reaction between ethanol and free methane sulphonic acid may not occur in the BMS-214662 formulation under the storage conditions evaluated and therefore causes no plausible safety concerns of EMS exposure in humans. Kinetic studies were conducted by spiking 200 ppb of EMS into water and the diluted and undiluted parenteral formulation. The rates of hydrolysis of EMS at 25 degrees C followed pseudo-first order kinetics and were determined to be 2.35 x 10(-4)min(-1), 67.4 x 10(-4)min(-1), and 1.32 x 10(-4)min(-1) in water, undiluted, and diluted drug product, respectively.

EMS in Viracept--the course of events in 2007 and 2008 from the non-clinical safety point of view.

Viracept (nelfinavir) is an HIV protease inhibitor supplied by Roche outside the US, Canada and Japan. Viracept was first introduced by Roche in 1998. Although newer protease inhibitors have become available for the treatment of HIV, it is viewed as a useful medicine for patients who are intolerant to ritonavir (since it does not require ritonavir boosting), pregnant women, and patients in resource-limited settings, since the formulation is heat-stable and does not require refrigeration. The relatively high prevalence of HIV in some of the third world countries means that it was also a product of choice for young women of childbearing age, pregnant and nursing women and young children. On 18 May 2007 F. Hoffmann-La Roche received first reports of a "bad smell" of blisterpacked Viracept tablets and one adverse drug report of nausea and vomiting from patients in Spain. Subsequently, ethyl methanesulfonate (EMS), an established mutagen, carcinogen and teratogen was identified as the potential source of the bad smell. On 6 June 2007, Viracept was globally recalled as the extent of the contamination exceeded the guidances for permissible levels set by regulatory authorities by more than 1000-fold and hence human risk was not readily assessable. In the following, a compilation of the course of events from a non-clinical point of view is presented. This compilation only partially reflects the complexity of the case and the interactions between all parties between May/June 2007 and September 2008 and hence necessarily remains partly a subjective compilation of the authors of this article. This compilation serves also as an introduction into this Special Issue of Toxicology Letters. The data on the cause and levels of contamination, likely duration of intake and affected patient population can be found in the subsequent contributions. Most importantly, we share in other parts of this Special Issue with the scientific community the data and risk assessment arguments that supported the conclusion by the company and regulatory authorities that the levels of contamination with EMS posed no health risk to affected patients.

Considerations regarding a permitted daily exposure calculation for ethyl methanesulfonate.

Specification of human exposure limits to compounds with toxicities based on modes of action that allow considerations of a threshold and the safe estimation of a no-observed-effect level (NOEL) is normally based on acceptable daily intake (ADI) or permitted daily exposure (PDE) calculations using appropriate safety factors to account for differences between species, populations, length of observation and severity of lesions. In view of the reliable experimental evidence for a thresholded dose response of the genotoxicity of ethyl methanesulfonate (EMS) as reported in this special issue of Toxicology Letters such an acceptable daily intake proposal is made using the approach for setting permitted daily exposure limits outlined in appendix 3 of the ICH Q3C consensus guideline on residual solvents in pharmaceuticals (ICH, 2005). Up to now the specification of EMS exposure limits was based on the generic threshold of toxicological concern (TTC)-derived limit of 1.5mug/person/day as advocated by the CHMP [CHMP, 2006. Guideline on the limits of genotoxic impurities. www.emea.europa.eu/pdfs/human/swp/519902en.pdf (June 28, 2006) with Q&A www.emea.europa.eu/pdfs/human/swp/43199407en.pdf (June 25, 2008)] or on as low as technically achievable criteria. Such limits have been based on conservative linear dose-effect extrapolations corresponding to an excess cancer risk of 1 in 100,000. We now present an EMS-specific PDE based on the reliable demonstration of a NOEL for induction of mutations in vivo of 25mg/kg/day. Using the most conservative safety factors described in ICH Q3C we derive a PDE of approximately 100 microg/person/day using product safety factors still amounting to 12,000.

The Viracept (nelfinavir)--ethyl methanesulfonate case: a threshold risk assessment for human exposure to a genotoxic drug contamination?

In May 2007, the F. Hoffmann-La Roche Company became aware of a contamination of Viracept (nelfinavir) tablets by the mutagenic DNA-ethylating agent ethyl methanesulfonate (EMS) as a result of a production incident. HIV-patients could have been exposed for 3 months to daily doses of up to 2.75 mg EMS, i.e., about 50 microg/kg per day. In this special issue, 12 manuscripts have been assembled to provide comprehensive insight in what happened and how the incident was managed by Roche and handled by the regulatory agencies. In the first four papers, the course of events and the toxicological information available at the outset are summarized and a traditional cancer risk assessment on the basis of a linear default dose-response is made. Three articles then report on the experiments performed for an improved risk assessment. A standard 4-week toxicity study with EMS in the rat indicated an NOAEL of 20mg/kg per day. Extensive studies on the genotoxicity showed threshold-like dose responses for both chromosome damage (bone marrow micronucleus test) and gene mutation (lacZ transgenic MutaMouse test) in various organs of mice treated for up to 4 weeks, whereas ethylation of hemoglobin at the N-terminal valine increased linearly with dose. The difference between adduct formation in DNA and protein was interpreted by repair of DNA adducts that becomes saturated above a threshold concentration of EMS, regarded as the metrics for the rate of DNA ethylation. Elaborate toxicokinetic investigations in various animal species, coupled to appropriate modeling, were performed in order to extrapolate the animal data to humans. Using a threshold risk assessment based on estimated c(max) of EMS, a safety factor of 370 was derived for maximum doses ingested by Viracept patients. A number of critical points are addressed in this editorial, concerning (i) definitions and types of "thresholds", (ii) estimation of a confidence limit for a slope below the threshold dose, interpreted as an increment within background variation, (iii) implementation for other mutagens and for human risk assessment.

The mutagenic potentiator effect of chlorophyllin by the HGPRT assay.

Chlorophyllin, a sodium-copper salt derivative of chlorophyll-a and -b, was evaluated for antimutagenic activity against ethyl methane sulfonate by the hypoxanthin-guanine-phosphoribosyl transferase (HGPRT) assay. The results obtained suggest that this chlorophyllin can potentiate the mutagenicity of an alkylating agent which induces DNA damage.

Efficacy of vitamin-C (L-ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate.

The genotoxic effects of ethyl methane sulphonate (EMS) have been assessed in a fish, Oreochromis mossambicus with endpoints including chromosome aberrations, abnormal red blood cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen and kidney. EMS caused chromosomal aberrations, nuclear anomalies in red blood cells, abnormal sperm morphology, and alteration of protein synthesis in various tissues. Some of the EMS toxicity appeared to be modulated and ameliorated in this fish by vitamin-C treatment.

Induction of micronuclei in human cell lines and primary cells by combination treatment with gamma-radiation and ethyl methanesulfonate.

While testing for genotoxicity is usually performed on single chemicals, exposure of humans often involves combinations of agents. Previous results from this laboratory showed supra-additivity for the induction of micronuclei in p53-mutated mouse lymphoma L5178Y cells after combined treatment with gamma-radiation from a 137Cs source and ethyl methanesulfonate (EMS). The question now was whether supra-additivity was a general phenomenon for the genotoxicity of this combination of a physical and a chemical DNA-damaging agent or whether the result was species- and cell type-specific. The same combination of agents was investigated in two human lymphoblastoid cell lines, TK6 (wild-type p53) and WTK1 (mutated p53), and primary fibroblasts from a fetal human lung. Doses were in the linear dose-effect range, resulting in a 1.5- to 3-fold increase in micronuclei above control. Radiation doses were between 125 and 350 mGy, while the EMS concentrations were 20-50 microg/ml for the cell lines and 250-350 microg/ml for the primary cells. In none of the human test systems was supra-additivity observed. With the WTK1 cells, which are most similar to the mouse cells regarding p53 status, there was even a tendency for a sub-additive combination effect. Possible explanations for the difference to the mouse cells could be related to species-specific aspects, different consequences of the p53 mutations or the presence of additional mutations. It is concluded that caution is advised in the interpretation and extrapolation of experimental results of mixture toxicity data because the outcome could be highly specific for the given selection of agents, doses and assays.

Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells.

Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis. Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer. In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs. deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae. Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively. The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells. UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells. When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen. Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells. In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break. Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions. These results are discussed with respect to mechanisms of carcinogenesis.

Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4.

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Influence of anoxia and respiratory deficiency on the genotoxicity of some direct-acting alkylating agents in yeast.

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory-deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.