Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

The potential of 1,1'-hexamethylenebis [3-(3,5-dichloro-4-pyridyl)] urea to modify genotoxic actions of chemical mutagens in various test-systems

The main tasks of this investigation were to investigate the potential genotoxic effect of 1,1'-hexamethylenebis [3-(3,5-dichloro-4-pyridyl)] urea and to analyze its capacity to induce adaptive response (AR) against chemical mutagens in various test-systems. Microbiological, cytogenetical and biochemical end-points were used. The sensitivity of test systems can be arranged as followed: human lymphocyte cultures > Chlamydomonas reinhardtii > Hordeum vulgare. It was obtained that HMPU can induce oxidative stress in Chlamydomonas reinhardtii cells (7.5 x 10(-4) mol/l) and at appropriate experimental conditions can trigger an AR against chemical mutagens in Hordeum vulgare (7.5 x 10(-3) mol/l) and human lymphocytes (10(-5), 10(-6) mol/l). The extent of the AR induction was closely connected with the increase of the inter-treatment time between the conditioning concentration of HMPU and the challenge concentration of chemical mutagens.     

Vitamin C for DNA damage prevention

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels>50μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels>50μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels>50μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.     

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.     

Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.     

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome.

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48 h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.     

The use of rec-bacteria for testing of carcinogenic substances.

A series of rec-Escherichia coli strains were tested for their sensitivity to four known carcinogenic compounds by examination of a zone of inhibited bacterial growth around a central well containing the test chemical. The mutants recA-, recB-, recC-, and recA- recB- recC- were all more sensitive to the mutagens than the parental strain AB1157. The recB- recC- strain was examined with a larger series of compounds and was found to respond to many of the substances in a similar way as the Salmonella typhimurium strains of Ames but with some notable exceptions. Nitrosamines, with rat liver microsomal activation, could be detected at lower levels and a group of aromatic amino compounds failed to react with these rec-E. coli. An unusual feature of these rec-mutants is their sensitivity to mixtures of nitrosamines and 2-acetyl amino-fluorene in the absence of microsomal activation.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.     

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.     

Forward mutation assay for screening carcinogens by alkaline phosphatase constitutive mutations in Escherichia coli K-12

A new forward mutation assay was developed with Escherichia coli using alkaline phosphatase (APase) constitutive mutations as a genetic marker. Mutation in any one of the three regulator genes (phoR, T and S) is known to make the cell constitutive for APase synthesis and enable the mutants to form larger colonies on beta-glycerophosphate plate under the condition of excess inorganic phosphate. This property was used for qualitative and quantitative assay of chemical mutagens. Attempts were made to construct suitable strains for this assay by introduction of various genetic traits that might increase the sensitivity of mutation. Three known chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), and 4-nitroquinoline-1-oxide (NQNO)) were employed as reference compounds in the quantitative assay. Among the strains constructed, a tester strain with genetic markers tif-1, uvrA and pKM101 was the most sensitive to these compounds, judging from tests on concentration-dependent mutagenic activity. The merits and limitations of the present system are discussed.     

Evaluation of 2 different genetic markers for the detection of frameshift and missense mutagens in A. nidulans

21 chemicals, known to induce missense and/or frameshift mutations directly, were assayed for their ability to forward mutate a haploid strain of A. nidulans. 2 genetic markers for forward mutations were used, namely 8-azaguanine resistance and induction of meth A1 suppressors. Missense mutagens were usually active when tested with the plate-incorporation technique, whereas frameshift agents were ineffective; some of these, on the other hand, turned out to be positive when tested with a liquid-test procedure. The 2 genetic markers used showed a similar sensitivity (with only 2 exceptions) in detecting the chemical mutagens assayed.     

The effects of “Cell age” upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

Summary SummaryYeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52° C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C.Exponential phase cells showed maximum resistance to UV light and minimum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD ₅₀ gene.Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth.     

The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity

Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used for large, multi-site, and/or time series studies, for bioassay-directed fractionation studies, for identifying the presence of specific classes of mutagens, and for doing site- or source-comparisons for relative levels of airborne mutagens. Early research recognized that although carcinogenic PAHs were present in air samples they could not account for the majority of the mutagenic activity detected. The mutagenicity of airborne particulate organics is due to at least 500 identified compounds from varying chemical classes. Bioassay-directed fractionation studies for identifying toxicants are difficult to compare because they do not identify all of the mutagens present, and both the analytical and bioassay protocols vary from study to study. However, these studies show that the majority of mutagenicity is usually associated with moderately polar/highly polar classes of compounds that tend to contain nitroaromatic compounds, aromatic amines, and aromatic ketones. Smog chamber studies have shown that mutagenic aliphatic and aromatic nitrogen-containing compounds are produced in the atmosphere when organic compounds (even non-mutagenic compounds) are exposed to nitrogen oxides and sunlight. Reactions that occur in the atmosphere, therefore, can have a profound effect on the genotoxic burden of ambient air. This review illustrates that the mutagenesis protocol and tester strains should be selected based on the design and purpose of the study and that the correlation with animal cancer bioassay results depends upon chemical class. Future emphasis needs to be placed on volatile and semi-volatile genotoxicants, and on multi-national studies that identify, quantify, and apportion mutagenicity. Initial efforts at replacing the Salmonella assay for ambient air studies with some emerging technology should be initiated.     

The effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate

The genetic effect of some factors is generally evaluated as an average response of all individuals, without taking into account their potential differences. The presence of individual sensitivity in separate Drosophila organisms to the mutagenic influence of ethyl methanesulfonate was shown when analysing recessive sex-linked lethal mutations in germ cells of males. Different sensitivity of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains on a large scale. It is advisable to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution.     

Effect of chemical mutagens on steroidal sapogenins in Trigonella species

A two- to four-fold increase in the steroidal sapogenins (diosgenin and tigogenin) was observed in the plants and seeds obtained from seeds of T. corniculata and T. foenum-graecum treated with low concentrations of chemical mutagens (EMS, MMS and NaN₃). However, a decrease in their levels was recorded at high concentrations of the mutagens.     

X-ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.     

Effects of caffeine on sister-chromatid exchanges (SCE) in vivo.

Chinese hamsters were twice treated with caffeine via stomach tube. The single doses were either 20, 100, 200 or 400 mg per kg body weight. A dose-dependent increase was observed in the frequencies of SCE induced in vivo in bone-marrow cells. Two intraperitoneal injections of the chemical mutagens, cyclophosphamide or benzo[a]pyrene, led to a pronounced increase of the frequency of SCE. Simultaneous applications of the chemical mutagens and caffeine decreased the rate of SCE. The effect of caffeine per se to induce SCE, and the mechanisms by which caffeine reduces the level of SCE induced by chemical mutagens are discussed.     

Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.