Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential. Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion. BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain. However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential. Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative. Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy. Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU. Hydroxyurea actually decreased toxicity of the BCNU. Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug. Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.     

‘Petite’ mutagenesis and mitotic crossing-over in yeast by DNA-targeted alkylating agents

Although the biological properties (cytotoxicity, mutagenicity and carcinogenicity) of alkylating agents result from their bonding interactions with DNA, such compounds generally do not show any special binding affinity for DNA. A series of acridine-linked aniline mustards of widely-varying alkylator reactivity have been designed as DNA-directed alkylating agents. We have considered whether such DNA targeting has an effect on mutagenic properties by evaluating this series of drugs in comparison with their untargeted counterparts for toxic, recombinogenic and mutagenic properties in Saccharomyces cerevisae strain D5. The simple untargeted aniline mustards are effective inducers of mitotic crossing-over in this strain, but resemble other reported alkylators in being rather inefficient inducers of the “petite” or mitochondrial mutation in yeast. However, the majority of the DNA-targeted mustards were very efficient petite, mutagens, while showing little evidence of mitotic crossing-over or other nuclear events. The 100% conversion of cells into petites and the lack of a differential between growing and non-growing cells are similar to the effects of the well characterised mitochondrial mutagen ethidium bromide. These data suggest very different modes of action between the DNA-targeted alkylators and their non-targeted counterparts.     

Mutagenic, recombinogenic and antimitochondrial effects of nitracrine analogues in Saccharomyces cerevisiae

The mutagenic and recombinogenic potential of 9-[(3-dimethylaminopropyl)amino]acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied using 3 different strains of Saccharomyces cerevisiae. The parent compound slightly enhanced the frequency of total aberrant colonies in diploid strains D5 and D7, but showed no evidence of recombinogenic effects. Each of the nitroacridines enhanced the frequency of total aberrant colonies in strains D5 and D7, but only the 1- and 4-nitro compounds significantly enhanced mitotic crossing-over (measured as twin spotted colonies in strains D5 and D7) or gene conversion in D7. The 3- and 4-nitro derivatives were effective mitochondrial mutagens, substantially increasing the frequency of 'petite' mutants in strains D5 and 5178B.     

Different test systems in Aspergillus nidulans for the evaluation of mitotic gene conversion,crossing-over and non-disjunction

The wide variety of genetic alterations produced by environmental mutagens has increased the necessity of using experimental microorganisms to reveal the induction of such genetic events with short-term tests. Aspergillus nidulans, because of its well-developed genetic system and the availability of morphological markers easy to score, can be profitably used in mutagen testing.The constitution of particular diploid strains of A. nidulans able to detect the induction of mitotic gene conversion, mitotic crossing-over and mitotic non-disjunction with selective procedures are described and validated with standard mutagens: methyl methanesulphonate and UV radiation (lacking a specific genetic activity), benomyl and p-fluorophenylalanine (with a specific genetic activity).The possibility of using mammalian metabolic activation of promutagens in A. nidulans in vitro was tested with cyclophosphamide, with positive results in all the tested genetic systems. A method that increases the sensitivity of conidia to mutagenic treatments is described; its application appeared to be particularly useful in experiments on crossing-over and non-disjunction.     

The genetic toxicology of acridines

Acridine and its derivatives are planar polycyclic aromatic molecules which bind tightly but reversibly to DNA by intercalation, but do not usually covalently interact with it. Acridines have a broad spectrum of biological activities, and a number of derivatives are widely used as antibacterial, antiprotozoal and anticancer drugs. Simple acridines show activity as frameshift mutagens, especially in bacteriophage and bacterial assays, by virtue of their intercalative DNA-binding ability. Acridines bearing additional fused aromatic rings (benzacridines) show little activity as frameshift mutagens, but interact covalently with DNA following metabolic activation (forming predominantly base-pair substitution mutations). Compounds where the acridine acts as a carrier to target alkylating agents to DNA (e.g. the ICR compounds) cause predominantly frameshift as well as base-pair substitution mutations in both bacterial and mammalian cells. Nitroacridines may act as simple acridines or (following nitro group reduction) as alkylating agents, depending upon the position of the nitro group. Acridine-based topoisomerase II inhibitors, although frameshift mutagens in bacteria and bacteriophage systems, are primarily chromosomal mutagens in mammalian cells. These mutagenic activities are important, since the compounds have considerable potential as clinical antitumour drugs. Although evidence suggests that simple acridines are not animal or human carcinogens, a number of the derived compounds are highly active in this capacity.     

Environmental mutagens may be implicated in the emergence of drug-resistant microorganisms

The emergence of drug-resistant microorganisms is an important medical and social problem. Drug-resistant microorganisms are thought to grow selectively in the presence of antibiotics. Most clinically isolated drug-resistant microorganisms have mutations in the target genes for the drugs. While any of the many mutagens in the environment may cause such genetic mutations, no reports have yet described whether these mutagens can confer drug resistance to clinically important microorganisms. We investigated how environmental mutagens might be implicated in acquired resistance to antibiotics in clinically important microorganisms, which causes human diseases. We selected mutagens found in the environment, in cigarette smoke, or in drugs, and then exposed Pseudomonas aeruginosa to them. After exposure, the incidence of rifampicin- and ciprofloxacin-resistant P. aeruginosa strains markedly increased, and we found mutations in genes for the antibiotic-target molecule. These mutations were similar to those found in drug-resistant microorganisms isolated from clinical samples. Our findings show that environmental mutagens, and an anticancer drug, are capable of inducing drug-resistant P. aeruginosa similar to strains found in clinical settings.     

Relationships between structure, toxicity and genetic effects in Salmonella typhimurium and Saccharomyces cerevisiae for substituted aniline mustards

A series of 4-substituted aniline mustards of widely varying reactivities have been evaluated for their mutagenic effects in Salmonella typhimurium strains of varying uvrB gene and plasmid status, and for their ability to cause mitotic crossing-over in Saccharomyces cerevisiae. The 4-methyl aniline mustard N,N-bis(2-chloroethyl)-4-methylaniline and its corresponding half-mustard N-(2-chloroethyl)-4-methylaniline showed widely different effects in the various bacterial strains, with the half-mustard being much less toxic than the full mustard in the uvrB- strain TA100. However, in the uvrB+ strain TA1978+, possessing an intact excision repair system, both compounds were equally toxic and the full mustard was the more mutagenic. Both compounds were equally effective in promoting mitotic crossing-over in yeast. For a series of 4-substituted full mustards, the toxicity in S. typhimurium strain TA100 correlated with substituent electronic parameters in the same way as does mammalian cell toxicity, supporting the view that the primary mode of toxicity is via DNA cross-linking, even for unreactive analogues. However, there were no obvious correlations between substituent physiochemical properties and mutagenic potential in bacteria, suggesting that mutagenic events are subject to a variety of influences other than the reactivity of the mustard group. In contrast, the most chemically reactive compounds were the most toxic and most recombinogenic in yeast.     

Genotoxic, mutagenic and recombinogenic effects of rauwolfia alkaloids

In the last decade, the possible correlation between the use of reserpine and rauwolfia drugs as antihypertensive agents and breast cancer incidence has been investigated. For the purpose of evaluating the mutagenic and genotoxic effects of these drugs, reserpine and ajmalicine were studied using the SOS Chromotest and the induction of gene conversion, crossing-over and reverse mutation in the yeast diploid strain XS2316. The results indicated a lack of genotoxic, mutagenic and recombinogenic effects.     

Genetic effects of N-nitroso-N-methylurea on Saccharomyces cerevisiae yeasts. I. The influence of radiosensitivity mutations on lethal and recombinogenic effects]

Effect of mutations rad2 and rad54 in homozygous state on survival, mitotic segregation and crossing-over induced by NMU in yeast was studied. Mutation rad2 did not influence on these effects of NMU. The mutation rad54 increased sensitivity to the lethal effect, the frequencies of NMU-induced segregation and crossing-over were decreased in the strain rad54 rad54. The recombinogenic effect of NMU on yeast was lower than under the action of UV and gamma rays.     

Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans.

Thirteen pesticides, aminotriazole, benomyl, captafol, captan, dalapon-Na, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, neburon, picloram and tordon were tested for ability to induce (1) point mutations to 8-azaguanine resistance, (2) mitotic crossing-over, and (3) mitotic non-disjunction and haploidization in Aspergillus nidulans. Tests were performed at three different pHs, i.e. 4.5, 7, 8.2. Three of the pesticides, captan , captafol and dichlorvos induced point mutations; dichlorvos also induced a high frequency of mitotic crossing-over and non-disjunction; benomyl induced a very high frequency of non-disjunction whereas aminotriazole induced weakly both types of somatic segregation.     

Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans

Thirteen pesticides, aminotriazole, benomyl, captafol, captan, dalapon-Na, dichlorvos, dinobuton, dodine, ioxynil, mecoprop, neburon, picloram and tordon were tested for ability to induce (1) point mutations to 8-azaguanine resistance, (2) mitotic crossing-over, and (3) mitotic non-disjunction and haploidization in Aspergillus nidulans. Tests were performed at three different pHs, i.e. 4.5, 7, 8.2. Three of the pesticides, captan, captafol and dichlorvos induced point mutations; dichlorvos also induced a high frequency of mitotic crossing-over and non-disjunction; benomyl induced a very high frequency of non-disjunction whereas aminotriazole induced weakly both types of somatic segregation.     

Induction of mitotic crossing-over in diploid strains of Aspergillus nidulans using low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individually analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.     

Specific cellular defects in patients with Fanconi anemia

Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N-methyl-N1-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.     

Microtubule-targeting agents are clinically successful due to both mitotic and interphase impairment of microtubule function

Microtubules undergo continual dynamic changes in mitotic cells as the mitotic spindle forms and is broken down and in interphase cells where they play a central role in intracellular trafficking, cell signaling, cell migration, and angiogenesis. Compounds that target the microtubule have been hugely successful in the clinic as chemotherapeutics, and this success is likely due to their ability to target cells regardless of their cell cycle stage. Additionally, new generation antibody-conjugated microtubule-targeting agents are improving the targeting of these drugs to tumors. Microtubule-targeting agents have been shown to have anti-angiogenic and vascular-disrupting properties as well as effects on cellular migration, intracellular trafficking, and cell secretion. There are a number of these compounds in development that target the vasculature, and different formulations of clinically used drugs are being developed to take advantage of these anti-angiogenic properties. Microtubule-targeting agents have also been shown to have the potential to treat neurodegenerative diseases, such as Alzheimer’s disease. Thus, drugs that target the microtubule will continue to have a major impact in oncology not only as anti-mitotics but also as potent inhibitors of interphase functions, and in future may also prove to be effective in reducing the consequences of neurodegenerative disease.     

The mutagenic effects of diacridines and diquinolines in microbial systems

Two series of difunctional DNA-intercalating agents (diacridines and diquinolines) were tested for mutagenic properties in Salmonella typhimurium strain TA1537, and for 'petite' mutagenesis activity in Saccharomyces cerevisiae, and also compared in terms of their structural, lipophilic and DNA-binding properties. Diacridines with only a short chain length were monointercalators, while those with an alkyl linker chain longer than C6 were bisintercalators. Although the bisintercalators especially bound very tightly to DNA, none of these compounds was as effective a frameshift mutagen in TA1537 as the parent chromophore 9-aminoacridine. However, the two (monointercalating) diacridines of shortest chain length were still able to cause frameshifts, and this ability returned (albeit weakly) in the bisintercalators of longest chain length. Although 9-aminoacridine showed no ability for 'petite' mutagenesis, the diacridines of longer chain length were very effective in causing this mitochondrial event. In the quinoline series, both the parent chromophore (4-aminoquinoline) and all the diquinolines were weak monointercalators. None of these compounds showed any ability for frameshift mutagenesis, although some were very weak mitochondrial mutagens. It is concluded that linking two acridines produces compounds whose mutagenic properties might have been predicted from our current knowledge of the parent molecules. However, despite a similar ability to intercalate DNA, the diquinolines show no resemblance to acridines in their mutagenic properties.     

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Parallel RNAi and compound screens identify the PDK1 pathway as a target for tamoxifen sensitization

Tamoxifen is the most commonly used drug to treat breast cancer and acts by blocking ERalpha (oestrogen receptor alpha) signalling. Although highly effective, its usefulness is limited by the development of resistance. Given this, strategies that limit resistance by sensitizing cells to tamoxifen may be of use in the clinic. To gain insight into how this might be achieved, we used chemical and genetic screens to identify targets and small-molecule inhibitors that cause tamoxifen sensitization. A high-throughput genetic screen, using an RNA interference library targeting 779 kinases and related proteins, identified the PDK1 (phosphoinositide-dependent kinase 1) signalling pathway as a strong determinant of sensitivity to multiple ERalpha antagonists, including tamoxifen. A chemical screen using existing drugs and known kinase inhibitors also identified inhibitors of the PDK1 pathway, including triciribine and tetrandrine. Aside from identifying novel agents and targets for tamoxifen sensitization, this approach also provides evidence that performing chemical and genetic screens in parallel may be useful.     

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program

The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.     

Recombinogenic and mutagenic effects of the antitumor antibiotic bleomycin in Aspergillus nidulans

Bleomycin, an antibiotic and antineoplastic drug that inhibits DNA synthesis and causes several types of chromosomal aberration, was found to increase mitotic recombination in Aspergillus nidulans. Heterozygous prototrophic diploid strains grown on media containing bleomycin produced significant increases of yellow and white sectors compared with controls. Further, the increased colour segregants were due to mitotic crossing-over, whereas the non-dis junctional segregants remained at the control level. Bleomycin also induced point mutations in the methionine-suppressor system of the methGl biAl strain of Aspergillus nidulans. Conidia treated in suspension with various concentrations of bleomycin increased the methionine-independent mutants 30-fold and more.