Diploid Strain Of Aspergillus Nidulans Research Articles

Gene homozygosis and mitotic recombination induced by camptothecin and irinotecan in Aspergillus nidulans diploid cells.

Mitotic recombination is a process involved in carcinogenesis which can lead to genetic loss through the loss of heterozygosity. The recombinogenic potentials of two anticancer drugs topoisomerase I inhibitors, camptothecin (CPT) and irinotecan (CPT-11), were evaluated in the present study. The homozygotization assay, which assess the induction of mitotic recombination and gene homozygosis, as well as the heterozygous A757//UT448 diploid strain of Aspergillus nidulans were employed. The three non-cytotoxic concentrations of CPT (3.5 ng mL-1, 10.5 ng mL-1 and 17.4 ng mL-1) were found to induce both mitotic recombination and gene homozygosis. CPT treatment produced three diploids homozygous, for nutritional and conidia color genes, and Homozygotization Indices (HI) significantly different from negative control. On the other hand, only the highest CPT-11 concentration tested (18 µg mL-1), corresponding to the maximal single chemotherapeutic dose, produced HI values higher than 2.0 and significantly different from negative control HI values. The recombinogenic effects of both topoisomerase I blockers were associated with the recombinational repair of DNA strand breaks induced by CPT and CPT-11. The anticancer drugs CPT and CPT-11 may be characterized as secondary malignancies promoters in cancer patients after chemotherapy treatment.

Metformin's performance in in vitro and in vivo genetic toxicology studies

Metformin is a hypoglycemiant drug prescribed for the treatment and control of the type 2 diabetes mellitus. Recently, the potential efficacy of this antidiabetic drug as an anticancer agent has been demonstrated in various mammalian cancer cells. This report evaluates the mutagenic as well as the recombinogenic potentials of the metformin drug in therapeutically relevant plasma concentrations (12.5 µM, 25.0 µM or 50.0 µM). Since the loss of heterozygosity is a process associated with carcinogenesis, the recombinogenic potential of such a drug was evaluated by the homozygotization assay using a heterozygous diploid strain of Aspergillus nidulans. The homozigotization indices (HI) for the genetic markers from the metformin-treated diploids were not statistically different from the negative control (non-treated diploids). For the first time, this indicated a lack of recombinogenic activity of the antidiabetic drug. The mutagenic potential of the metformin drug was evaluated by the chromosome aberrations and the micronuclei tests in human lymphocytes cultures. The metformin drug did not show any significant increase either in the numerical or in the structural chromosome aberrations and did not affect significantly the mitotic index when compared to the negative control. In the in vitro micronucleus test, the drug did not increase the number of micronuclei or nuclear buds when compared with the negative control. The data in this study suggest that the metformin drug is not a secondary cancer inducer, since it has neither showed recombinogenic nor mutagenic activities when used in pharmacological concentrations.

Mitotic crossing-over induced by two commercial herbicides in diploid strains of the fungus Aspergillus nidulans

Some herbicides are suspected of promoting teratogenic, carcinogenic and mutagenic events. Detection of induced mitotic crossing-over has proven to be an indirect way of testing the carcinogenic properties of suspicious substances, because mitotic crossing-over is involved in the multistep process of carcinogenesis. We examined mitotic crossing-over induced by two commercial herbicides (diuron and trifluralin) in diploid strains of Aspergillus nidulans based on the homozygotization index. Low doses (2.5 microg/mL) of diuron were sufficient to increase the mean homozygotization index in 2.1 and 11.3 times for UT448//UT196 and Dp II-I//UT196, respectively, whereas the same dose of trifluralin increased this mean only 1.2 (UT448//UT196) and 3.5 (Dp II-I//UT196) times, respectively. The lower homozygotization index value found for trifluralin could be due to its interference with mitotic crossing-over in eukaryotic cells. We concluded that the diploid Dp II-I//UT196 of A. nidulans is more sensitive to organic compounds than UT448//UT196; these compounds cause recombinational events at a greater frequency in the latter diploid. This system holds promise as an initial test for carcinogenicity of organic compounds, including herbicides.

Recombinogenic activity of fluoxetine in Aspergillus nidulans

The recombinogenic potential of fluoxetine, an antidepressant widely prescribed in the treatment of depressive disorders in cancer patients, was investigated in this study. A heterozygous diploid strain of Aspergillus nidulans was utilized. Fluoxetine at 7.5, 15, and 30μM concentrations induced homozygosity of several nutritional genetic markers and significantly increased their homozygotization index values. Since mitotic recombination is a mechanism leading to malignant growth through the loss of a functional copy of a heterozygous tumor-suppressor gene, fluoxetine may be characterized as an inducer of secondary malignances in cancer patients after antidepressant treatment.

Genotoxic Potentials of Natural Products Detected by a Short-Term Test Using Diploid Strains of Aspergillus nidulans

Main aim is to evaluate the genotoxic potentials of three classes of natural products, a sesquiterpene lactone (cynaropicrin), a saponin (stigmast-7en-3� -ol-glucopyranoside) and a steroid (stigmasterol), at low doses, using the Ho- mozygotization Index test (HI). This short-term test, deals with the determination of alterations in mitotic crossing-over frequencies eventually involving heterozygous auxotrophic genes in diploid strains of Aspergillus nidulans. In this way, treatments of UT448//UT184 and Dp II-I//UT184 diploid strains with the natural products showed that saponin and lac- tone significantly increased the homozygotization index (HI) of both strains, especially for the diploid Dp II-I//UT184. No effect was observed with the steroid. On this account it presents remarkable potentials to detect the geno- toxic/carcinogenic effects of bioactive natural products. Furthermore, this is the first time the low doses effects of bioac- tive natural products were detected using A. nidulans.

Genotoxicity of Achillea millefolium essential oil in diploid cells of Aspergillus nidulans

The essential oil of Achillea millefolium is commonly used in folk medicine for the treatment of several diseases and has been demonstrated previously to exert an in vitro antimicrobial activity against human pathogens. Current study investigates the genotoxic activity of A. millefolium oil. The oil's major constituents are: chamazulene (42.15%), sabinene (19.72%), terpin-4-ol (5.22%), beta-caryophyllene (4.44%) and eucalyptol (3.10%), comprising 74.63% of the total. The oil's genotoxic evaluation was performed at concentrations of 0.13 microL/mL, 0.19 microL/mL and 0.25 microL/mL with a heterozygous diploid strain of Aspergillus nidulans, named A757//UT448, with green conidia. A statistically significant increasing number of yellow and white mitotic recombinants, per colony, of the diploid strain was reported after oil treatment with 0.19 microL/mL and 0.25 microL/mL concentrations. The genotoxicity of the oil was associated with the induction of mitotic non-disjunction or crossing-over by oil.

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.

Loss of heterozygosity by mitotic recombination in diploid strain of Aspergillus nidulans in response to castor oil plant detergent

Somatic recombination in heterozygous diploid cells may be a promotional agent of neoplasms by inducing homozygosity of defective genes. Tumor suppressor genes may in this way be completely suppressed in recombinant cells. In this work, the genotoxic effects of detergent derived from the castor oil plant (Ricinus communis) in heterozygous diploid cells of Aspergillus nidulans are evaluated. Previous studies have evaluated the application of this substance in endodontic treatments as an irrigating solution. The recombinogenic potential of the compound has been studied through the production of homozygous cells for nutritional markers riboA1, pabaA124, biA1, methA17, and pyroA4. Detergent was diluted to 1:10, 1:20, and 1:40, and morphologic alterations, delay in conidiophore development, and mitotic recombination occurrence were reported for the three dilutions. Although past studies have demonstrated the antimicrobial action of the detergent under analysis, our results revealed its cytotoxic effects and recombinogenic potential.

Cremophor EL stimulates mitotic recombination in uvsH//uvsH diploid strain of Aspergillus nidulans.

Cremophor EL is a solubilizer and emulsifier agent used in the pharmaceutical and foodstuff industries. The solvent is the principal constituent of paclitaxel's clinical formulation vehicle. Since mitotic recombination plays a crucial role in multistep carcinogenesis, the study of the recombinagenic potential of chemical compounds is of the utmost importance. In our research genotoxicity of cremophor EL has been studied by using an uvsH//uvsH diploid strain of Aspergillus nidulans. Since it spends a great part of its cell cycle in the G2period, this fungus is a special screening system for the study of mitotic recombination induced by chemical substances. Homozygotization Indexes (HI) for paba and bi markers from heterozygous B211//A837 diploid strain were determined for the evaluation of the recombinagenic effect of cremophor EL. It has been shown that cremophor EL induces increase in mitotic crossing-over events at nontoxic concentrations (0.05 and 0.075% v/v).

Recombinagenic effect of cryptolepine in uvsH+//uvsH+ and uvsH//uvsH diploid strains of Aspergillus nidulans.

Recombinagenic potential of the alkaloid cryptolepine was evaluated in two diploid strains of Aspergillus nidulans--a wild type strain (uvsH+//uvsH+) and a DNA-repair-deficient one (uvsH//uvsH). Treatment of both strains with cryptolepine failed to alter colony growth in culture; its recombinagenic potential was determined by the homozygotization index (in which events of mitotic exchange may cause expression of genes previously masked by the dominant allele). Mitotic crossing-overs were induced by 7 and 14 mg/L doses of cryptolepine in both diploid A. nidulans strains.

UV light induced accumulation of variability in a diploid strain of Aspergillus nidulans

The accumulated variability in asexual species was evaluated in Aspergillus nidulans diploid cells after repeated cycles of UV irradiation. The results show that diploid cells can accumulate a very high genetic variability in the heterozygous condition as previously shown with the base analog 6-N-hydroxylaminopurine (HAP).

Gene Inactivation in the Parasexual Cycle of Duplication Strains of Aspergillus nidulans.

The aim of this work is to demonstrate occurrence of GIS (Gene Inactivation System) during the mitotic haploidisation of a diploid strain of Aspergillus nidulans, heterozygous for a duplicated segment. The DWM strain bears Dp (II, I) duplication and has already been submitted to GIS. This strain was used to make the DWM-Aza/PabaA diploid strain after 5-Azacytidine treatment. Dp (II, I) is a II → I insertional duplication including Acr, w and meth + genes. Mitotic segre-gants were obtained from DWM-Aza//PabaA diploid strain which showed inactivation of duplicated genes only These results were confirmed by the gene reactivation achieved by demethylating treatment of the mitotic segregants with 5-Azacytidine. GIS effect was also observed during the sexual cycle of the mitotic segregants, showing that the demethylating treatment makes the duplicated genes very sensitive to GIS. The reduction of the ascospore viability per cleistothecium has also been demonstrated and it has been related to GIS action during the sexual cycle of the duplication strains of A. nidulans.

6-N-hydroxylaminopurine (HAP)-induced accumulation of variability in haploid and diploid strains of Aspergillus nidulans.

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.

On the Recombinogenic Activity of Norfloxacin in a Diploid Strain of Aspergillus nidulans.

The recombinogenic effect of norfloxacin was studied in a heterozygous diploid Aspergillus nidulans strain. Both 0.8 and 1.5, μg/ml of norfloxacin produced homozygosity of recessive genes in chromosomes I and II. The same concentrations also induced crossing-over in mitotic segregants, but did not produce morphological changes or differences in the growth curve.

Effects of sterol biosynthesis inhibitors on mitosis

The sterol biosynthesis inhibitors (SBIs) fenarimol, triadimenol, bitertanol, miconazole, clotrimazole, and fenpropimorph increase sectoring in a heterozygous diploid strain of Aspergillus nidulans, but only at concentrations highly inhibitory to growth. Analysis of color segregants suggest that SBIs cause chromosome nondisjunction. That fenarimol and clotrimazole disturb the mitotic process was also shown by cytological studies. In treated A. nidulans germ tubes it was impossible to observe the normal stages of mitotic division with the light microscope. At the electron microscope level, an abnormal nuclear division was observed, with a characteristic cross membrane dividing the nucleus into two unequal parts, with dissimilar chromatin content. In Chinese hamster cells, the two inhibitors increased the mitotic index and caused mitotic abnormalities such as chromosome clumping and chromatin fragmentation to more than two nuclear fragments. The mechanism of the genetic activity of SBIs is discussed and interference with the regulatory action of sterols is suggested.

Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans

The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on their resting or germinating conidia. Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the diploid strain as well as mitotic recombination frequencies. We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.

Genetic effects of phthalimide fungicides on diploid Aspergillus nidulans

AbstractA few specimens of ‘Ridomil 25 WP’, a commercial formulation of metalaxyl, showed high genetic activity on a heterozygous diploid strain of Aspergillus nidulans. This activity was due to an impurity which was identified by mass and n.m.r. spectroscopy as captafol. This and the related fungicides captan, dichlofluanid, and tolyfluanid greatly increased the yield of euploid colour segregants from the diploid strain. At low captafol concentrations, most of the induced segregants resulted from mitotic crossing over but chromosome non‐disjunction or loss was also caused at high concentrations. Mitotic crossing over could be induced also by vapour‐phase action of captafol and captan. The mechanism by which these phthalimides may exert their genetic effects is discussed.

Estimation of relative DNA content in nuclei of races of Phytophthora megasperma f. sp. glycinea by quantitative fluorescence microscopy

The fluorochrome 4,6-diamidino-2-phenylindole was used as a stain for nuclear DNA in the fungus Phytophthora megasperma f. sp. glycinea. Measurements of DNA contents were made cytofluorometrically using known haploid and diploid strains of Aspergillus nidulans as standards. No significant differences between DNA levels in nine races of Phytophthora megasperma f. sp. glycinea were found. The results are consistent with genetical data that all nine races are diploid in the vegetative state.Key words: Phytophthora, ploidy, fungi, DNA content, fluorescence.

Disruptive effects of ethyl alcohol on mitotic chromosome segregation in diploid and haploid strains of Aspergillus nidulans

To identify, with certainty, the primary genotoxic effects of ethanol, condidia from diploid strains of Aspergillus nidulans were treated during early germination with ethyl alcohol, and all the resulting segregants from large samples were analysed in detail. Results were identical whether technical grade (95%), or highly purified ‘absolute’, alcohol was diluted to obtain the effective low levels of ethanol (3–6%). This makes it unlikely that trace contaminants, rather than ethanol itself, caused the observed induced segregation. At the most effective concentrations survival was about 50%, but over half of the colonies were abnormal and showed sectoring phenotypes. Higher concentrations were too inhibitory for growth. In practically all cases, when ‘abnormals’ were replated, aneuploids of various types were recovered. Most aneuploids were hyperdiploid, including a fraction of simple trisomics, and some were even polyploid types. All showed chromosomal-type segregation in diploid sectors, often segregating for genetic markers of many different chromosomes. Mitotic crossing-over was slightly increased, but probably not induced, since an equally high spontaneous frequency was observed among replated aneuploid types. To eliminate conclusively the possibility that chromosome breakage was the primary effect of ethanol, which might indirectly produce aneuploid-like types, haploid conidia were also treated. Up to 8% abnormals, mainly hyperhaploids, were obtained (at about 20–50% survival). When diploid and haploid strains were treated identically with ethanol in liquid media after a few hours of pregermination, frequencies of abnormals were similar for short treatments, but higher in diploid strains for longer ones (10–20% aneuploids). The abnormal colonies from the haploid strain were replated and visually identified: about 2 3 were typical n + 1 hyperhaploids, and most others were n + 2 or 3 or more, including a few 2 n + 1 trisomics. It is concluded that as a primary effect, alcohol interferes with, and probably arrests, mitotic segregation, and causes chromosome missegregation and nondisjunction. In most cases, the resulting nuclei contain increased numbers of chromosomes and show high frequencies of chromosome loss.

Determination of nuclear DNA content in fungi using mithramycin: vegetative diploidy in Armillaria mellea confirmed

Armillaria mellea, a phytopathogenic fungus, is the only member of the Agaricales (Basidiomycetes) whose fertile vegetative phase in nature is thought to be diploid, rather than dikaryotic. To examine the vegetative ploidy of A. mellea, we used the DNA-binding antibiotic, mithramycin, for fluorometry of in situ nuclear DNA. The measurements of nuclear DNA content indicated that strains derived from single basidiospores of A. mellea were haploid and that strains derived from matings of isolates of single spores were diploid. These data confirm the results of earlier genetic experiments, which show haploidy and diploidy in unmated and mated strains, respectively. Nuclear DNA measurements in known haploid and diploid strains of Aspergillus nidulans confirmed the validity of our protocol.