Mutagenic Interactions Research Articles

Abstract 633: Thiopurines and mismatch repair deficiency cooperate to fuel TP53 mutagenesis and ALL relapse

Abstract Chemotherapy is curative for most children with acute lymphoblastic leukemia (ALL). Here we provide direct evidence that thiopurine chemotherapeutics can also directly induce drug resistance mutations leading to relapse. Using a large relapsed ALL cohort assembled from Chinese, US and German patients, we found that TP53 R248Q mutations were highly enriched at relapse compared to diagnosis. Relapse-specific TP53 R248Q was associated with the acquisition of MMR deficiency mutations in MSH2, MSH6, or PMS2 and a novel relapse-specific mutational signature. Using isogenic MCF10A cells with or without engineered MSH2 knockout, and the Nalm6 ALL cell line which has native MMR deficiency, we found that this novel signature was caused by a synergistic mutagenic interaction between thiopurine treatment and mismatch repair (MMR) deficiency (called the thio-dMMR signature) that contributes to a hypermutator phenotype and acquisition of TP53 R248Q in residual ALL during remission. Treatment-induced TP53-mutant clones then expand due to broad chemoresistance, leading to eventual relapse. Indeed, thiopurines preferentially induced C>T mutations at the center of NCG trinucleotides, which can lead to TP53 R248Q, and the thiopurine mutation rate was accelerated 2- to 10-fold in MMR-deficient ALL and cell lines. Thiopurine treatment induced C>T mutations preferentially on the transcribed strand, rather than the untranscribed strand, of mRNAs, which further increased the likelihood of TP53 R248Q induction. Further, experimental thiopurine treatment was able to directly induce TP53 R248Q variants in MMR-deficient cultured cells, including Nalm6 and MCF10A MSH2-/-, by activating the thio-dMMR mutational signature, while MMR-proficient MCF10A cells did not experience R248Q induction. The sequential acquisition of MMR deficiency mutations, followed by TP53 mutations, during post-diagnosis ALL evolution was supported by clonal evolution analysis of serial patient samples. p53 R248Q promoted resistance to multiple ALL chemotherapeutic agents, and was associated with on-treatment relapse and poor relapse-treatment response. Our findings indicate that the enrichment of TP53 R248Q in relapsed ALL is due to synergistic mutagenesis from thiopurine treatment and MMR deficiency, followed by selection for TP53 R248Q's chemoresistance phenotype. This suggests that cancer drug resistance mutations may not always pre-exist subclonally at diagnosis, but may be therapy-induced in some patients. Additionally, the qualitative and quantitative mutational signature output of a mutagen (e.g., thiopurines) can vary based on the genetic background. Finally, our findings suggest potential therapeutic strategies, including avoiding thiopurine treatment in MMR-deficient relapses, and therapeutic p53 mutant reactivation, to deal with this genetically-unstable, chemoresistant disease. Citation Format: Fan Yang, Samuel W. Brady, Huiying Sun, Chao Tang, Lijuan Du, Malwine Barz, Xiaotu Ma, Yao Chen, Houshun Fang, Xiaomeng Li, Pandurang Kolekar, Omkar Pathak, Jiaoyang Cai, Lixia Ding, Tianyi Wang, Arend von Stackelberg, Shuhong Shen, Caiwen Duan, Cornelia Eckert, Hongzhuan Chen, Yu Liu, Jeffery M. Klco, Hui Li, Benshang Li, Jinghui Zhang, Renate Kirschner-Schwabe, Bin-Bing S. Zhou. Thiopurines and mismatch repair deficiency cooperate to fuel TP53 mutagenesis and ALL relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 633.

Antioxidant, antibacterial, and antimutagenic activity of Piper nigrum seeds extracts.

Piper nigrum is a widely used plant in traditional remedies and known for its numerous biological properties. However, fraction-based antioxidant activity and their antimutagenic potential are not yet fully investigated. Different extracts of the seeds P. nigrum were obtained by sequential extraction in different solvents. All extracts were evaluated for antibacterial and antioxidant activities using different methods. The most active fraction was analyzed for antimutagenic activity using the Ames Salmonella test. The antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was found to be more prominent compared to ESβL producing Klebsiella pneumoniae isolates. The MIC values were found to be lower against MRSA than K. pneumoniae. The extract showing highest antioxidant activity (methanol extract) was further tested for antimutagenic activity both against direct and indirect-acting mutagens. A varying level of antimutagenic activity was shown by methanol extract at highest tested concentration (200 µg/plate). Alkaloids, phenols, and flavonoids were detected as major class of compounds in methanol extract. Gas chromatography-mass spectrometry (GC–MS) analysis showed the presence of various phytocompounds. Based on molecular docking of two major active phytocompounds (piperine and copaene), they were found to interact at the minor groove of DNA. Molecular dynamics (MD) simulation revealed that both the ligands were quite stable with DNA under physiological conditions. The ability of phytocompounds to interact with DNA might be reducing the interaction of mutagens and could be one of the possible mechanism of anti-mutagenic activity of P. nigrum extract. This study highlights the antioxidant and antimutagenic potential of Piper nigrum. The role of phytocompounds present in the bioactive extract is needed to be explored further for herbal drug research.

Soil mutagenic activity in hazardous waste site of Kalush City (Western Ukraine)

<p>Frequency and spectrum of cytogenetic disorders in the cells of root meristem sprouts of <em>Triticum aestivum L.,</em> induced by soil contamination with hexachlorobenzene in the territory of toxic waste ground and Dombrovskyi potassium ore mine (Kalush city, Ivano-Frankivsk oblast), were studied. The concentration of hexachlorobenzene exceeded MPC (maximum permissible concentration) by 1233.3–18350 times. It has been established that chemical soil contamination causes the increase of the frequency of cytogenetic aberrations by 1.8-3.8 times. Its highest indicators were found under the most intensive soil contamination with hexachlorobenzene in the areas of toxic waste ground which were not recultivated. Frequency of cytogenetic disorders exceeded the control level by 3.4-3.8 times. The maintenance of high mutagenic activity of hexachlorobenzene remains in the soil of the recultivated area of the ground is confirmed by statistically reliable increase of the number of aberrant cells by 2.6-3 times. It has been shown that soil contamination of Dombrovskyi mine with the complex of natural-mineral compounds of mining-chemical raw materials causes the increase of mutagenic activity level of low concentrations of chemical gene-toxic factors of techno-genic origin, which is seen in the induction of higher level of cytogenic disorders compared with spontaneous indicators by 1.8-2.4 times. Spectrum of chromosome aberrations, which mostly included acentric fragments, expanded due to induced bridges, ring chromosomes and micronuclei. The share increase of the cells with dicentrics, caused by the effect of soil contamination with hexachlorobenzene in the territory of toxic waste ground, confirms the radiomimetic effect, resulted from the effect of chemical mutagen in high concentrations. Chemical pollutants of the soil of all the studied areas show the ability to cause mitosis disorder, connected with the damage of mitotic apparatus. A significant increase of disorder frequency of chromosome segregation can be the result of the interaction of mutagens not only with microtube proteins of division spindle but also with heterochromatin of near-center areas of chromosomes, which is typical for the mechanism of the emergence of cytogenetic aberrations under the effect of chemical supermutagens in high concentrations. The increase of the cell number with multiple aberrations under the effect of soil contamination with hexachlorobenzene in different concentrations proves high gene-toxicity of the chemical compound and the threat of severe genetic outcomes when it gets into the environment.</p>

Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells.

Abstract 836: Gene-environment interaction of genome-wide association study-identified susceptibility loci and meat-cooking mutagens in renal cell carcinoma etiology

Abstract Background: Meat-cooking mutagens, including heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs), are formed as a result of meat cooking, preparation, and level of doneness and may increase the risk of renal cell carcinoma (RCC). We utilized data from an ongoing case-control study to evaluate the association between meat-cooking mutagens and the risk of RCC. We assessed the modification of these associations by known and suspected RCC risk factors such as smoking, obesity, alcohol use, physical activity and hypertension as well as established genetic variants identified through genome-wide association studies (GWAS). Methods: We investigated dietary intake of four meat-cooking mutagens among 659 incidence RCC cases recruited from the University of Texas MD Anderson Cancer Center in Houston, Texas and 699 healthy controls recruited via random digit dialing. We evaluate the association of PAH/HCA intake on RCC risk, and examined whether associations varied by known and suspected risk factors for RCC and genetic susceptibility variants previously identified from GWAS. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using tertiles of dietary PAH/HCA intake adjusting for total energy intake, fruit and vegetable intake, age, gender as well as known or suspected RCC risk factors. Results: Dietary intake of the mutagenic compounds 2-amino-3,8-dimethylimidazo-[4,5-f] quinoxaline (MeIQx) and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were significantly associated with an increased risk of RCC [(OR and 95%CI across tertiles:1.00 (ref), 1.28 (0.94-1.74), 1.95 (1.43-2.66); P-trend <0.001 and 1.00 (ref), 1.41 (1.04-1.90), 1.54 (1.14-2.07); P-trend = 0.02) respectively]. We observed evidence for interactions between PhIP and variants in two RCC susceptibility genes, ITPR2 (rs718314 multiplicative Pinteraction = 0.03, additive Pinteraction = 0.002) and EPAS1 (rs7579899 additive Pinteraction = 0.06), on modifying RCC risk. Conclusions: Intake of meat may increase the risk of RCC through mechanisms related to the cooking compounds MeIQx and PhIP. These associations may be modified by genetic susceptibility variants related to RCC. Further research is necessary to understand the biological mechanisms underlying these interactions. Citation Format: Stephanie C. Melkonian, Carrie R. Daniel, Yuanqing Ye, Nizar M. Tannir, Jose A. Karam, Surena F. Matin, Christopher G. Wood, Xifeng Wu. Gene-environment interaction of genome-wide association study-identified susceptibility loci and meat-cooking mutagens in renal cell carcinoma etiology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 836. doi:10.1158/1538-7445.AM2015-836

Clinical implications of neuropharmacogenetics

IntroductionPharmacogenetics aims to identify the underlying genetic factors participating in the variability of drug response. Indeed, genetic variability at the DNA or RNA levels can directly or indirectly modify the pharmacokinetic or the pharmacodynamic parameters of a drug. The ultimate aim of pharmacogenetics is to move towards a personalised medicine by predicting responders and non-responders, adjusting the dose of the treatment, and identifying individuals at risk of adverse drug effects. MethodsA literature research was performed in which we reviewed all pharmacogenetic studies in neurological disorders including neurodegenerative diseases, multiple sclerosis, stroke and epilepsy. ResultsSeveral pharmacogenetic studies have been performed in neurology, bringing insights into the inter-individual drug response variability and in the pathophysiology of neurological diseases. The principal implications of these studies for the management of patients in clinical practice are discussed. Conclusion/discussionAlthough several genetic factors have been identified in the modification of drug response in neurological disorders, most of them have a marginal predictive effect at the single gene level, suggesting mutagenic interactions as well as other factors related to drug interaction and disease subtypes. Most pharmacogenetic studies deserve further replication in independent populations and, ideally, in pharmacogenetic clinical trials to demonstrate their relevance in clinical practice.

Toxicity and Mutagenicity of Gulf of Mexico Waters During and After the Deepwater Horizon Oil Spill

The Deepwater Horizon oil spill is unparalleled among environmental hydrocarbon releases, because of the tremendous volume of oil, the additional contamination by dispersant, and the oceanic depth at which this release occurred. Here, we present data on general toxicity and mutagenicity of upper water column waters and, to a lesser degree, sediment porewater of the Northeastern Gulf of Mexico (NEGOM) and west Florida shelf (WFS) at the time of the Deepwater Horizon oil spill in 2010 and thereafter. During a research cruise in August 2010, analysis of water collected in the NEGOM indicated that samples of 3 of 14 (21%) stations were toxic to bacteria based on the Microtox assay, 4 of 13 (34%) were toxic to phytoplankton via the QwikLite assay, and 6 of 14 (43%) showed DNA damaging activity using the λ-Microscreen Prophage induction assay. The Microtox and Microscreen assays indicated that the degree of toxicity was correlated to total petroleum hydrocarbon concentration. Long-term monitoring of stations on the NEGOM and the WFS was undertaken by 8 and 6 cruises to these areas, respectively. Microtox toxicity was nearly totally absent by December 2010 in the Northeastern Gulf of Mexico (3 of 8 cruises with one positive station). In contrast, QwikLite toxicity assay yielded positives at each cruise, often at multiple stations or depths, indicating the greater sensitivity of the QwikLite assay to environmental factors. The Microscreen mutagenicity assays indicated that certain water column samples overlying the WFS were mutagenic at least 1.5 years after capping the Macondo well. Similarly, sediment porewater samples taken from 1000, 1200, and 1400 m from the slope off the WFS in June 2011 were also highly genotoxic. Our observations are consistent with a portion of the dispersed oil from the Macondo well area advecting to the southeast and upwelling onto the WFS, although other explanations exist. Organisms in contact with these waters might experience DNA damage that could lead to mutation and heritable alterations to the community pangenome. Such mutagenic interactions might not become apparent in higher organisms for years.

Interaction of mutagens in binary mixtures using the alkaline comet assay in human lymphocytes

Mutagens contained in complex mixtures can present synergistic interactions, either additive or antagonistic. Therefore, development of experimental approaches is necessary to elucidate which is the responsible agent for the effect in the mixtures. An experimental design was developed that allowed an understanding of the processes between the compounds of complex mixtures. Human lymphocytes were exposed to binary mixtures of the mutagens B[a]P, DMBA, Trp-P-1 and MX for 1 hour with or without S9. Viability was assessed with trypan blue dye and the genotoxicity by the comet assay. All of the hydrocarbon showed an effect with furanone. With and without S9, the most toxic interactions were observed between hydrocarbons. Synergistic interaction was observed without S9 between B [a] P and Trp-P-1 and between DMBA and Trp-P-1 with metabolic activity. Without S9 antagonistic interaction was observed only between Trp-P-1+DMBA, and with S9 between Trp-P-1+MX and MX+DMBA. It observed an increase dose dependent in tail length. Half the cultures showed genotoxic damage and increased cell damage. For each mixture, minimum concentrations were determined at which adverse effects are observed; for some only the maximum concentration was determined at which no adverse effects are observed. The processes between mutagens present in a mixture have become better understood, and the results validated an analytical model that determined which component had an effect on another. The results also showed that the type of compounds in the mixture determined whether or not a risk threshold was present.

Lethal and mutagenic interactions between γ-rays, cisplatin and etoposide at the cellular and molecular levels

Purpose: We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments.Materials and methods: The yeast Saccharomyces cerevisiae SC7K(lys2–3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry.Results: All three agents induced significant mutant frequency. The γ +Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ + Pt and γ + E combinations. Meanwhile, Pt + E combination showed lethal additivity and very low mutagenic synergism. Pt + E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ + Pt, γ + E and γ + Pt + E combinations.Conclusions: Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt + E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.

Lethal and mutagenic interactions between γ-rays, cisplatin and etoposide at the cellular and molecular levels

Lethal and mutagenic interactions between γ-rays, cisplatin and etoposide at the cellular and molecular levels

The influence of oral hygiene on salivary quality in the Ames Test, as a marker for genotoxic effects

Squamous cell carcinomas of the upper aerodigestive tract are a global health-political challenge. Accordingly, current research efforts are aimed towards the opportunities for early recognition of risk patients, and at the recognition of risk factors related to carcinogenesis. We determined the revertant number of the variety Salmonella typhimurium TA 98 and TA 100 after incubation, with saliva samples from 100 probands as a measure of a genotoxic environment within the oral cavity, depending on the dental status as measure of oral health. Beside chronic alcohol (p=0.032) and tobacco consumption (p<0.001), the dental status displayed in partial aspects (high plaque index, high number of decayed teeth, prosthetically not rehabilitated status, p <or= 0.050) a significant connection with the genotoxic salivary activity. The periodontal status and the type of restorative filling material, as well as dental prostheses, however, had no important influence on the revertant number (p >or= 0.104). Therefore, it can be concluded that the polymicrobial supragingival plaque, as a possible independent factor, possesses a relevant mutagenic interaction with saliva, and that individual oral health is a co-factor in the development of carcinomas in the oral cavity.

化学物質によるｉｎ ｖｉｔｒｏ及びｉｎ ｖｉｖｏにおける突然変異の定量的解析に関する研究

The mutagenicities of methyl, ethyl, isopropyl, and butyl methanesulfonates and the corresponding alkylderivatives of N-nitrosoureas were examined using E. coli Hs30R strain deficient in the excision repair system. Their mutagenic capacities were standardized by converting the experimentally obtained mutation frequencies into those per unit of the concentration-time integrated dose with various dimensions, μM×h, mM×h, and M×h. The mutation frequencies under various conceptual exposure conditions were computed by using those at unit integrated dose, on the assumption that the dose-response relation is linear on a log-log scale.An attempt was made to establish a procedure for kinetic formulation of the mutagenic chemical modification of the mutational target(s). Using the proposed kinetic formulation, a quantitative analysis was made of the temperature dependence of mutation frequency in terms of the activation energies for mutagenic modification induced in the cell by mutagens. E. coli Hs30R strain was treated with alkyl methanesulfonates and N-alkyl-N-nitrosoureas at 37, 25, and 15°C for 1 hour and the induced mutation frequencies were normalized in terms of the concentration-time integrated dose. The plot of the normalized mutation frequency versus the reciprocal of the reaction temperature gave a straight line which corresponds to the Arrehnius plot, enabling us to estimate the apparent activation energy of the initial chemical event leading to mutation.In order to study the mutagenic interaction between two chemicals, an analytical method for the classification of the mutagenic interactions as “equivalent” or “independent” is proposed. The mutation frequencies induced by simultaneous (combined) treatments of Salmonella typhimurium TA100 with two simple alkylating agents were compared with those induced by separate treatments with the two mutagens. Furthermore, the comparison was made of the frequencies of micronucleated reticulocytes (MNRETs) produced in ddY mice treated with model chemicals such as alkylating agents, spindle poisons, and an oxidizing agent. The results indicate that the analytical method proposed here is appropriate to evaluate the combined effects of chemicals both in bacterial mutagenesis and micronucleus induction.

Non-Linear QSAR Treatment of Genotoxicity

The nonlinear QSAR approach using the Chebyshev polynomial expansion and neural networks has been applied for the prediction of genotoxicity of compounds. The mutagenic toxicity of heteroaromatic and aromatic amines, measured by the Ames test, was correlated with the molecular descriptors calculated from the molecular structures using quantum-chemical methods. The quantitative models obtained were compared with the results of the linear QSAR treatment. The descriptors appearing in the models reveal the importance of mutagenic interactions of heteroaromatic amines via hydrogen bonding, of effects induced by the solvent, and of the size of compound. The dependence of molecular descriptors on environmental effects and on molecular conformations was analysed.

A Comprehensive QSAR Treatment of the Genotoxicity of Heteroaromatic and Aromatic Amines

A quantitative structure-activity relationship withR2=0.8344(R=0.9135)has been derived from a set of 95 heteroaromatic and aromatic amines to correlate and predict their mutagenic activity. It consists of six descriptors calculated from the molecular structures with quantum-chemical methods. The descriptors in the model reveal the importance in mutagenic interactions of heteroaromatic amines of hydrogen bonding, of effects induced by the solvent, and of the size of compound. The model also suggests that the amino group is critical for the reactive site.

Mutagenic interactions of model chemical mixtures

Although current methodology for human health risk assessment assumes additive interactions among the contaminants of a complex mixture, chemical interactions may occur which produce synergistic or antagonistic effects. In this study, the mutagenic response of three model compounds, benzo(a)pyrene (B(a)P), pentachlorophenol (PCP) and 2,4,6-trinitrotoluene (TNT), were tested individually and in binary and tertiary solutions, using the Salmonella/microsome assay with each of three bacterial tester strains (TA97a, TA98, and TA100). For all strains, B(a)P was mutagenic with metabolic activation (Arochlor 1254-induced Sprague-Dawley rat liver S9 fraction), TNT was mutagenic without metabolic activation, and pentachlorophenol was inactive both with and without metabolic activation. In binary and tertiary solutions, pentachlorophenol had no effect on the mutagenicity of B(a)P or TNT, independent of metabolic activation. For strain TA97a, the mutagenicity of B(a)P with metabolic activation was slightly decreased in the presence of TNT; the mutagenicity of TNT without metabolic activation was slightly decreased in the presence of B(a)P and PCP; and the mutagenicity of the tertiary solution (496 revertants/10 ug) with metabolic activation was lower than the mutagenicity of B(a)P alone (729 revertants/10 ug). The mutagenicity of B(a)P in strain TA98 with activation was inhibited by the addition of TNT. Studies conducted using several concentrations of TNT or B(a)P indicate that the inhibition of B(a)P mutagenicity was increased as the concentration of TNT increased. Assays performed using four concentrations of S9 indicated the inhibition of B(a)P mutagenicity was relatively unaffected by the level of S9. The data suggest that an interaction in the presence of TNT limits the concentration of B(a)P that is capable of reaching or binding with bacterial DNA.

Mutagenic interactions between X-rays and two promutagens, o-phenylenediamine and N-nitrosodimethylamine, in the stamen hairs of Tradescantia clone BNL 4430

Mutagenic interactions between X-rays and two promutagens, o-phenylenediamine (PDA) and N-nitrosodimethylamine (DMN), were studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots of this clone cultivated in a nutrient solution circulating growth chamber were used as tester plants. After determining dose–response curves for X-rays. PDA and DMN, combined treatments with PDA or DMN and X-rays were conducted, exposing acutely to X-rays 20 h before starting, at the midpoint of, or 20 h after completing the PDA or DMN treatments for 4 h. Clear synergistic effects in inducing somatic pink mutations were detected when X-rays were irradiated before the PDA or DMN treatments, resembling those confirmed earlier between maleic hydrazide (MH) and X-rays. On the contrary, clear antagonistic effects were observed when X-rays were given after the PDA or DMN treatments, also resembling those between MH and X-rays. When X-rayed at the midpoint of the PDA or DMN treatments, merely additive and synergistic effects were observed, respectively, differing from the antagonistic effects between MH and X-rays. The mutagenic synergisms detected were considered to be the results of interactions between DNA strand breaks (and the resultant chromosome breaks) induced by X-rays and those by PDA or DMN, whereas the mutagenic antagonisms observed were presumed to be due to X-ray-caused inhibition of the activation of PDA and DMN in the stamen–hair cells. The time periods required for penetrations into floral tissues and/or activations into mutagens seem different among PDA, DMN and MH.

Mutagenic synergism detected between 1,2-dibromoethane and X rays in the stamen hairs of Tradescantia clone BNL 4430.

Mutagenic interaction between 1,2-dibromoethane (EDB) and X rays was studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots of this clone cultivated in a nutrient solution circulating growth chamber were used as the tester plants. EDB is a promutagen and also a bifunctional alkylating agent with a high Swain-Scott substrate constant, but is thought to react probably via SN1 mechanism. After confirming the dose-dependent mutagenicities of aqueous solutions of EDB for the first time in Tradescantia stamen hairs, a combined treatment with EDB and X rays was conducted, exposing acutely to 578 mGy X rays at the midpoint of 66.5 mM EDB treatment for 4 h. The induced somatic mutation frequency determined after the combined treatment was significantly higher (at 0.1% level) than that expected from the additive effects of EDB and X rays, showing that EDB and X rays acted obviously synergistically. The confirmation of the mutagenic synergism between EDB and X rays is reported here for the first time, although a likelihood of synergistic effects of EDB with 3H beta rays has been suggested earlier.

Antagonistic effects of ethyl methanesulfonate and maleic hydrazide in inducing somatic mutations in the stamen hairs of Tradescantia clone BNL 4430.

Mutagenic interaction between ethyl methanesulfonate (EMS; a monofunctional alkylating agent) and maleic hydrazide (MH; a promutagen activated into a mutagen in plants highly likely by peroxidase) was studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. Since EMS has been shown to act synergistically with X rays in inducing mutations, and mutagenic synergisms have also been observed between X rays and MH by exposing to X rays before MH treatments, EMS and MH were expected to act synergistically at least by exposing to EMS before MH treatment. Three different combined treatments were conducted, i.e., by exposing for 4 h to 18.8 mM EMS 44 or 20 h before starting or 20 h after completing 1 mM MH treatments for 4 h. Unexpectedly, however, clear antagonistic effects in inducing somatic pink mutations were detected after all these combined treatments. Especially, the induced mutation frequency by exposing to EMS 44 h before the MH treatment was significantly lower than that induced by MH alone. The clear mutagenic antagonisms observed were thought to have resulted from EMS-caused inhibition of activation of MH by peroxidase, EMS ethylating and thus inactivating this enzyme or its precursors. Decreased peroxidase activities than those after treatments with MH alone were measured after two combined treatments, i.e., 12 h after the one exposing to EMS 44 h before MH and 24 h after the other exposing to EMS 20 h after MH, but the decreases were not large enough or their fluctuations were too large to judge them to be statistically significant. Comparisons of mutation frequencies induced by the combined treatments exposing to EMS before MH with those by MH alone suggest that there are some mechanisms (other than ethylation of peroxidase or its precursors) by which EMS suppresses the activation of MH.

Interactive Lethal and Mutagenic Effects of Ultraviolet Light and Bleomycin in Yeast: Synergism or Antagonism?

The mutagenic interactions of ultraviolet light and bleomycin in haploid populations of Saccharomyces cerevisiae were analyzed. Survival and mutation frequency as a function of different bleomycin concentrations after one conditioning dose of UV radiation were determined. Furthermore, corresponding interaction functions and sensitization factors were calculated. A synergistic interaction between UV light and bleomycin was shown for both lethal and mutagenic events when the cells were in nutrient broth during the treatments. Conversely, the interaction between UV light and bleomycin was antagonistic when the cells were in deionized water during the treatment. The magnitude of lethal and mutagenic interactions depends on dose, and thus presumably on the number of lesions. The observed interactions between UV light and bleomycin suggest that the mechanism that is most likely involved is the induction of repair systems with different error probabilities during the delay of cell division.

Synergistic effects of N-ethyl-N-nitrosourea (an alkylating agent with a low Swain-Scott substrate constant) and X-rays in the stamen hairs of tradescantia clone BNL 4430

The mutagenic interaction between N-ethyl-N-nitrosourea (ENU) and X-rays was tested in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. ENU, a monofunctional alkylating agent with a low Swain-Scott substrate constant (s) of 0.26, exhibited a strong cytotoxicity. ENU-induced somatic pink mutation frequency per 10(4) hair-cell divisions increased with increasing ENU dose, with a slope of 1.243 on a log-log graph, the slope value being similar to that for X-ray-induced mutation frequency. Three out of five combined treatments with ENU and X-rays produced mutation frequencies significantly higher than those expected from the additive effects of the two mutagens. Clear synergistic effects were detected when relatively higher X-ray doses were applied, resembling those confirmed earlier between methyl methanesulfonate (MMS) and X-rays, although the s value for ENU is very much smaller than that (0.88) for MMS. It is therefore concluded that mutagenic interactions between alkylating agents and X-rays do not have any clear relationship with the s values.