Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.     

Genetic change may be caused by interference with protein-protein interactions

Several aprotic polar solvents were shown to induce mitotic aneuploidy in yeast: diethyl ketone, γ-valerolactone, pyridine, pivalinic acid nitrile, phenylacetonitrile and fumaric acid dinitrile. Only fumaric acid dinitrile also strongly induced other types of genetic effects including mitotic crossing-over, mitotic gene conversion and point mutation. The other substances only induced aneuploidy and this only over a very narrow dose range.

The treatment protocol used suggested that these chemicals acted via interference with tubulin assembly and disassembly causing a malfunctioning of spindle fiber microtubules. This hypothesis was tested using twice recycled porcine brain tubulin. Diethyl ketone, γ-valerolactone, pyridine and phenylacetonitrile inhibited GTP-promoted assembly of porcine brain tubulin in vitro in the concentration range needed for the induction of mitotic aneuploidy in yeast. Pivalinic acid nitrile accelerated tubulin aggregation whereas fumaric acid dinitrile had no effect even at concentrations 18 times higher than the lowest tested concentration effective in yeast.

The in vitro experiments with porcine brain tubulin further suggest that genetic change can result from interference with specific protein-protein interactions. Fumaric acid dinitrile was the only exception since it did induce aneuploidy but had no effects on the assembly of porcine brain tubulin. This could be caused either by interference with protein-protein interactions other than between molecules during assembly and disassembly of microtubules or species-specific differences in susceptibility between yeast spindle and porcine brain tubulin.     

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae.

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induced mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.     

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induced mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.     

Genetic effects of 5-azacytidine in Saccharomyces cerevisiae

The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.     

Phenobarbital induces aneuploidy in Saccharomyces cerevisiae and stimulates the assembly of porcine brain tubulin

Phenobarbital (PB) specifically induces mitotic chromosomal malsegregation in the diploid Saccharomyces cerevisiae strain D61.M but no other genetic events such as mitotic recombination or point mutations. In accordance with the hypothesis that PB exerts its genotoxic activity by an interaction with tubulin, it stimulates the GTP-promoted assembly of porcine brain tubulin in vitro. This process is reversible thus excluding an unspecific denaturation of the tubulin protein by PB.     

The detection of mitotic and meiotic chromosome gain in the yeast Saccharomyces cerevisiae: effects of methyl benzimidazol-2-yl carbamate, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfoxide, propionitrile and cyclophosphamide monohydrate

The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents. The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel. 1988; Whittaker et al., 1988). Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain. Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain. Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C. MBC is postulated to act by binding directly to tubulin. The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics. The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation. Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.     

Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).     

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.     

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.     

Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

Aneuploidy-inducing chemicals in yeast evaluated by the micronucleus test

Chinese hamsters were exposed to acetone, methyl ethyl ketone, ethyl acetate and 2-methoxy ethyl acetate, known to be strong inducers of aneuploidy in the yeast Saccharomyces cerevisiae. All solvents yielded negative results in the micronucleus test, whereas the vinca alkaloid vindesine--used as a positive control substance--proved to act as a spindle poison in mammals in vivo.     

Genetic and anti-tubulin effects induced by pyridine derivatives

A series of pyridine derivatives, 2-methyl-, 2-chloro-, 2-acetyl-, 3-acetyl-, 4-acetyl, 2-phenyl-, 2,4-dimethyl-, 2,6-dimethyl- and 2-methyl-5-ethyl-pyridine, were shown to induce mitotic aneuploidy in strain D61.M of Saccharomyces cerevisiae. Induction of mitotic recombination was also observed with 3- and 4-acetylpyridine and 2-phenylpyridine in strain D61.M. 4-Acetylpyridine and 2-phenylpyridine were found to induce mitotic gene conversion and 2-phenyl-pyridine also induced reverse mutation in strain D7 of Saccharomyces cerevisiae. These two agents also inhibited the GTP-mediated assembly of porcine brain tubulin in vitro.     

Induction of aneuploidy by oncodazole (nocodazole), an anti-tubulin agent, and acetone

Oncodazole (nocodazole) is a compound which interacts with yeast and bovine tubulin. We have shown that it induces aneuploidy in the yeast Saccharomyces cerevisiae at very low concentrations. In the course of a search for an appropriate solvent for oncodazole we observed that acetone also induces mitotic aneuploidy in yeast. This effect of acetone was greatly enhanced when the treatment of growing cells at 28 degrees C was interrupted by a period of holding at ice-bath temperature.     

2（4′噻唑）苯丙咪唑对猪脑微管蛋白体外聚合的影响

本研究利用猪脑中分离纯化的微管蛋白聚合和解聚反应,分析了具有争议的非整倍体诱发剂2（4′噻唑）苯丙咪唑（thiabendazole,TBZ）对微管蛋白聚合状态的影响。秋水仙素(colchicine)为本研究的阳性对照物。结果发现2（4′噻唑）苯丙咪唑能显著抑制体外微管蛋白的聚合,并呈明显的剂量效应关系。研究表明,TBZ可能通过抑制微管蛋白聚合来影响染色体正常分离,诱发非整倍体。     

Malsegregation of chromosomes is not a major source of somatic mosaicism in Drosophila melanogaster

Two genotypes were constructed to determine whether some of the mosaic spots, on which the SMART (somatic mutation and recombination test) procedures are based may arise through malsegregation of the chromosomes. Both arms of the metacentric third chromosomes were labelled with marker mutations, and in this way one- and two-arm events (the former representing rearrangements or point mutations, the latter representing malsegregation) could be recorded separately. Although several hundred clones were identified following exposure of larvae to X-rays, colchicine or vinchicine or vincristine (all known inducers of malsegregation), none arose as a consequence of two-arm events. This suggests that malsegregation of the chromosomes plays little, if any role in the formation of mosaic spots. Instead, the clones develop due to mitotic recombinations, deletions or point mutations.     

Detection of chemically induced aneuploidy with plant test systems

The utility of plant test systems for detecting chemically induced aneuploidy was evaluated by using papers published in peer-reviewed journals. A total of 147 papers were provided to the group by the Environmental Mutagen Information Center. Based on the criteria established by the Gene-Tox Committee (Waters and Auletta, 1981), 22 papers were selected for in-depth review. Only those papers listing additional, missing, or lagging chromosomes in the meiotic or mitotic cells were included in this review. Although most plant test systems may be developed to utilize either mitotic or meiotic cells for cytogenetic analysis, only a few have been employed for this purpose. In this review, Allium cepa was found to be the most commonly used test system. Other species used less frequently were Vicia faba, Hordeum vulgare, Sorgham vulgare, and Pennisetum americanum. None of the plant test systems have been sufficiently utilized to warrant judgment for its sensitivity and specificity for detecting induced aneuploidy. A suggested protocol for detecting chromosomal malsegregation in meiotic or mitotic cells is presented. Further development and utilization of plant tissue culture techniques and morphological markers identifiable in the seedling stages is recommended for detecting chemically induced aneuploidy.     

Genetic effects of chlorinated ethylenes in the yeast Saccharomyces cerevisiae

The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae. From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates. Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not. The 2 ways of metabolic activation were compared and were found to cause approximately the same effect. In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M. The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss. In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM.