RESUMEN
Aspergillus nidulans is a fungal model organism extensively used in genetic approaches. It may reproduce sexually and asexually, with a well-defined parasexual cycle. The current paper demonstrates that the limitation of nitrogen source facilitates the production of A. nidulans's nonmeiotic recombinants directly from heterokaryons, without the recovery of the diploid phase. Heterokaryons formed between master strains were inoculated in sodium nitrate-low (basal medium [BM]) and sodium nitrate-rich media (minimal medium [MM]). All mitotic segregants produced by the heterokaryons were tested for their mitotic stability in the presence of benomyl, the haploidizing agent. Only mitotically stable haploid segregants were selected for subsequent analysis. Phenotypic analyses of such haploids favored the characterization of nonmeiotic recombinants. As the number of such recombinants was higher in BM than in MM, nitrogen limitation may have facilitated the isolation of nonmeiotic recombinants from heterokaryons by stimulating nuclear fusion still inside the heterokaryotic mycelium as a survival strategy.
Asunto(s)
Aspergillus nidulans/genética , Mitosis , Nitrógeno/química , Recombinación Genética , Benomilo/química , Medios de Cultivo/química , Diploidia , Haploidia , Nitratos/químicaRESUMEN
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.
Asunto(s)
Aspergillus nidulans/efectos de los fármacos , Camptotecina/análogos & derivados , Camptotecina/toxicidad , Recombinación Genética/efectos de los fármacos , Inhibidores de Topoisomerasa I/toxicidad , Aspergillus nidulans/genética , Diploidia , Homocigoto , Irinotecán , Mitosis/efectos de los fármacos , Mitosis/genética , Pruebas de MutagenicidadRESUMEN
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.
Asunto(s)
Metformina/toxicidad , Mutágenos/toxicidad , Adulto , Aspergillus nidulans/citología , Aspergillus nidulans/efectos de los fármacos , Cromátides/metabolismo , Aberraciones Cromosómicas/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Cromosomas de los Mamíferos/metabolismo , Diploidia , Femenino , Haploidia , Heterocigoto , Homocigoto , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Pruebas de Micronúcleos , Índice Mitótico , Adulto JovenRESUMEN
The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.
Asunto(s)
Colletotrichum/genética , Variación Genética/genética , Glycine max/microbiología , Mutación/genética , Brasil , Colletotrichum/clasificación , Colletotrichum/aislamiento & purificación , Genotipo , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
This report evaluates the potential of the antidepressant drug citalopram to induce homozygotization of genes previously present in a heterozygous condition, by homologous recombination. In order to address this question, a heterozygous diploid strain of the filamentous fungus Aspergillus nidulans and the homozygotization assay were utilized. Non-cytotoxic concentrations of citalopram (50, 75 and 100 µmol/L) showed a strong recombinogenic effect in A. nidulans, inducing homozygosis of the diploid strain's nutritional markers. The genetic markers exhibited homozygotization index (HI) rates higher than 2.0 and significantly different from HI control ones. Since citalopram has been previously characterized as a DNA synthesis inhibitor, the recombinogenic potential of this antidepressant in A. nidulans may be associated with the recombinational repair of citalopram-induced DNA strand breaks.
Asunto(s)
Antidepresivos de Segunda Generación/toxicidad , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Citalopram/toxicidad , Mutágenos/toxicidad , Recombinación Genética/efectos de los fármacos , Aspergillus nidulans/citología , Carcinógenos/toxicidad , Intercambio Genético/efectos de los fármacos , Daño del ADN , Reparación del ADN , Trastorno Depresivo/complicaciones , Trastorno Depresivo/tratamiento farmacológico , Diploidia , Genes Fúngicos , Marcadores Genéticos , Heterocigoto , Homocigoto , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/complicaciones , Neoplasias/etiologíaRESUMEN
Heterokaryosis is the initial step of the parasexual cycle, a process that provides genetic variability in filamentous fungi through the production of heterozygous diploid nuclei. To characterize the parasexual cycle in Colletotrichum lindemuthianum, we evaluated the presence of heterokaryosis, vegetative compatibility reactions, and diploid formation among isolates of Race 65 collected from different Brazilian states. Vegetative compatibility groups were identified among the isolates according to their ability to form heterokaryons. Two heterozygous diploids were selected from compatible heterokaryons, which were characterized by the segregation of the parental auxotrophic markers and by RAPD profiles.
Asunto(s)
Colletotrichum/crecimiento & desarrollo , Brasil , Colletotrichum/genética , Colletotrichum/aislamiento & purificación , Diploidia , ReproducciónRESUMEN
The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.
Asunto(s)
Colletotrichum/genética , Variación Genética/genética , Mutación/genética , Glycine max/microbiología , Brasil , Colletotrichum/clasificación , Colletotrichum/aislamiento & purificación , Genotipo , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
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 30 microM 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 malignancies in cancer patients after antidepressant treatment.
Asunto(s)
Antidepresivos de Segunda Generación/farmacología , Aspergillus nidulans/efectos de los fármacos , Fluoxetina/farmacología , Pérdida de Heterocigocidad/efectos de los fármacos , Mutación/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Antidepresivos de Segunda Generación/efectos adversos , Aspergillus nidulans/genética , Intercambio Genético , Diploidia , Relación Dosis-Respuesta a Droga , Fluoxetina/efectos adversos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Heterocigoto , Humanos , Pérdida de Heterocigocidad/genética , Pruebas de Sensibilidad Microbiana , Mutación/fisiologíaRESUMEN
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).
Asunto(s)
Aspergillus nidulans/efectos de los fármacos , Intercambio Genético/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas Fúngicas/efectos de los fármacos , Glicerol/análogos & derivados , Glicerol/toxicidad , Tensoactivos/toxicidad , Aspergillus nidulans/citología , Diploidia , Mitosis/efectos de los fármacos , Pruebas de Mutagenicidad/métodosRESUMEN
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 percent v/v).
Asunto(s)
Aspergillus nidulans , Intercambio Genético , Tensoactivos , Ácido 4-Aminobenzoico , Aspergillus nidulans , Diploidia , Mitosis , Pruebas de MutagenicidadRESUMEN
Ethidium bromide (EB) is an intercalating inhibitor of topoisomerase II and its activities are related to chemotherapeutic drugs used in anti-cancer treatments. EB promotes several genotoxic effects in exposed cells by stabilising the DNA-enzyme complex. The recombinagenic potential of EB was evaluated in our in vivo study by the loss of heterozygosity of nutritional markers in diploid Aspergillus nidulans cells through Homozygotization Index (HI). A DNA repair mutation, uvsZ and a chromosome duplication DP (II-I) were introduced in the genome of tested cells to obtain a sensitive system for the recombinagenesis detection. EB-treated diploid cells had HI values significantly greater than the control at both concentrations (4.0 x 10-3 and 5.0 x 10-3 mM). Results indicate that the intercalating agent is potentially capable of inducing mitotic crossing-over in diploid A. nidulans cells