Increase in mitotic recombination in diploid cells of Aspergillus nidulans in response to ethidium bromide

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 and 5.0 x 10 μM). Results indicate that the intercalating agent is potentially capable of inducing mitotic crossing-over in diploid A. nidulans cells.


Introduction
DNA topoisomerase II (topo II) is a ubiquitous enzyme that regulates DNA topologic interconversion during replication, transcription and genetic recombination decreasing torsional stress in DNA by introducing transient protein-bridged DNA breaks in both DNA strands.Through this function, topo II plays an essential role in the maintenance of genetic material integrity (Mo and Beck, 1999;Godard et al., 2002).Several topo II inhibitors, such as doxorrubicin, etoposide and ellipticine, have been extensively used as chemotherapeutic agents in anti-cancer treatments.Rather than inhibiting the catalytic activity of the topo II enzyme, they act by increasing levels of topo II mediated DNA cleavage.This mechanism converts essential enzyme into a potent cellular toxin, inducing apopitosis, cell cycle arrest and genotoxicity (Nitiss and Beck, 1996;Attia et al., 2002).In fact, long-term adverse effects of this class of chemotherapic items, such as infertility and increased incidence of secondary malignancies, have been shown (Tiburi et al., 2002).
In fact, mitotic crossing-over is not only a factor associated with proto-oncogenes activation by favoring gene amplification and transpositions, but also causes neoplasies progression through loss of heterozygosity at tumorsuppressor loci.Heterozygous cells bearing a normal and a defective tumor-suppressor allele may lose the remaining wild-type allele by somatic chromosomal exchange at mitosis (Zimmermann et al., 1966;Ramel et al., 1996;Weinberg, 1991).
In current in vivo study, the potential of ethidium bromide (EB), an intercalating inhibitor of topo II in inducing somatic recombination, was evaluated through Homozygotization Index (HI) in diploid Aspergillus nidulans cells (Pires and Zucchi, 1994;Baptista andCastro-Prado, 1997, Chiuchetta andCastro-Prado, 2002a andb).Loss of heterozygosity from nutritional markers was enhanced by the introduction of chromosome duplication DP (II-I) and uvsZ DNA repair mutation in tested cells (Baptista and Castro-Prado, 2001).The filamentous fungus Aspergillus nidulans has an extensive genetic homology to higher eukaryotes, which makes it a suitable model organism for monitoring genetic toxicity when the effects of chemical and physical agents are studied (Busso et al., 2001).

Strains
The A. nidulans strains used are described in Table 1.The Z1 mutant is derived from master strain UT448 after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).The mutant bears Dp(II,I) duplication: a segment

Culture Media
Minimum Medium (MM) was Czapek -Dox with 1.0% (w/v) glucose.Complete Medium (CM) has been previously described (Pontecorvo et al., 1953;Van de Vate and Jansen, 1978).Supplemented medium (SM) consisted of MM plus the nutriments required by each strain.Solid medium contained 1.5% agar.Incubation occurred at 37 °C.

Drug toxicity evaluation
Ethidium bromide (InvitrogenCo) was added to CM to obtain final 4.0 x 10 -3 and 5.0 x 10 -3 µM concentrations.Conidia of the Z1//A757 diploid strain were inoculated in plates containing CM (control) and CM + EB (treatment).Ten plates were used for each dose and for control.Colony diameters were measured after 24, 48, 72 and 96 h incubation.Colony diameters, with and without the drug, were compared by Student's t test.

Evaluation of recombinagenic potential
The Z1//A757 diploid strain conidia were inoculated in plates with MM + EB.Plates were incubated for 6 days at 37 °C.Treatment produced visible diploid sectors, identified by differentiated morphology of the original diploid.Sectors (D1-D4) were homozygous (+//+) or heterozygous (+//-or -//+) diploid segregants.They were never recessive homozygous (-//-) since they do not grow in MM.Diploid sectors were haploidized spontaneously in CM and then purified in MM.
After haploidization the haploid mitotic segregants from diploid D1 to D3 were purified in CM.Only stable mitotic segregants were selected for the recombinagenic test and determination of the Homozygotization Indexes (HI) (Pires and Zucchi, 1994;Franzoni et al., 1997).

Determination of the Homozygotization Indexes (HI)
Nutritional markers of the haploid segregants will segregate in the proportion (a) 4+:4-, if the drug fails to induce recombinagenesis or (b) 4+: 2-, if drug induces crossing-over (Figure 1).Thus Homozygotization Indexes (HI), or the ratio between prototrophic segregants and auxotrophic segregants, equal to or over 2.0 (4+/2-), show the recombinagenic effect of the compound under analysis (Figure 1) (Chiuchetta and Castro-Prado, 2002a and b).The HIs for markers from recessive homozygous diploids (-//-) cannot be calculated because they are not selected in MM.Results were compared by Yates correct Chi-Square test.

Results
Although the EB treatment of the Z1//A757 original diploid strain in MM does not allow the isolation of auxotrophic diploids (-//-), it is possible to obtain homozygous recessive diploids for conidia coloration markers (y and w ).The treatment of the Z1//A757 diploid strain with the intercalating agent allowed the isolation of prototrophic diploids with green (y+//y), yellow (y//y) and white (w //w) conidia (Figure 2).Phenotypic analyses of the D2 (yellow) and D4 (white) diploids showed they were recombinant for centromere-paba and w-centromere intervals, respectively.Treatment also produced homozygous and heterozygous diploid segregants (D1-D3) for nutritional markers and they were selected for HI determination.
Solid cultures of the Z1//A757 diploid strain in CM + EB (4.0 x 10 -3 µM or 5.0 x 10 -3 µM) exhibited reduction of the fungal growth after 48h incubation.However, after 72 to 96 h the mycelial growth of the diploid strain in the presence of EB was normal, when compared to the control (growth of diploid Z1 // A757 in CM) (Figure 3).
The untreated Z1 // A757 diploid strain exhibited increase in spontaneous mitotic exchange, and this result may be explained by the mitotic instability of the Z1 mutant.His > 2.0 were in fact obtained for markers from chromosomes I and IV of the Z1 // A757 diploid strain (Table 2, control).
The 5.0 x 10 -3 µM EB concentration produced D2 and D3 diploid strains.D2 diploid showed HIs > 2.0 only for ribo (chromosome I) and pyro (chromosome IV) markers, because it is a recombinant for the centromere-paba interval (chromosome I) and homozygous for the paba+, y and bi+ genes (Table 2, Figure 5).On the other hand, the D3 diploid strain showed HIs > 2.0 for all chromosome I markers (ribo, paba and bi genes) (Table 2).
Taken together, the HI values for diploids D1-D3 demonstrate that EB induces mitotic recombination in A. nidulans diploid cells.

Discussion
A. nidulans duplication strains are very unstable at mitosis, with slow growth caused by genetic imbalance.They occasionally produce faster-growing sectors originated by the spontaneous loss of part or all of either duplicated segment (Nga andRoper, 1968, 1969).
The strains' mitotic instability may be enhanced by the incorporation of a uvs mutation affecting DNA repair (Bur et al., 1982), since the spontaneous lesions that frequently occur in duplicated segments cannot be correctly repaired.It has also been shown that genome balance of heterozygous diploids for duplicated segments may be restored by deletion and mitotic crossing-over (Case and Roper, 1981).
The present study shows HIs > 2.0 for chromosomes I and IV markers of Z1//A757 strain (Table 2, control).Results indicate that mitotic crossing-over in this diploid strain is probably caused by the spontaneous unrepaired lesions in its DNA.The intrinsic factors that induce mitotic instability in the Z1//A757 genome transform the diploid into a sensitive system for the detection of recombinagenesis induced by DNA molecule-breaking agents.
Moreover, D1-D3 diploids, obtained after treatment with EB, showed significantly greater HI values than those of the control in both concentrations (Table 2).Further, only treated diploid strains produced homozygous diploids for conidial colour: D2 and D4 produced yellow and white conidia, respectively (Figure 2).Results indicate that the intercalating agent is potentially capable of inducing mitotic crossing-over in A. nidulans diploid cells heterozygous for an extra translocated segment.EB is a compound that intercalates reversibly between pair bases of DNA molecule affecting many DNA functions including DNA and RNA synthesis and mitotic activity (Heinen et al., 1976).The DNA-binding drug was classified as an inhibitor of Topoisomerase II (Snyder and Arnone, 2002), DNA polymerase alpha, delta and epsilon from Novikoff hepatoma cells (Fox et al., 1996) and the unwinding DNA and ATPase activities of the plant nuclear helicase PDH45 (Pham and Tuteja, 2002).
Covalent attachment of EB to DNA in nanomolar concentrations enhances topoisomerase II-mediated sin-gle-and double-strand DNA cleavage.Therefore, the conversion of the reversible EB-DNA complex into an irreversible adduct causes the transformation of the drug into a catalytic topoisomerase II inhibitor (Snyder and Arnone, 2002;Marx et al., 1997).
Stable DNA double-strand breaks generated by EB, through inhibition of DNA topoisomerase II, may become templates for recombination events and possibly explain the recombinagenic effect of the drug demonstrated in the present work.
DNA topoisomerase II inhibitors are the most successful anticancer drugs used to treat human malignancies.They increase the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme (Fortune and Osheroff, 2000).Nevertheless some of these antineoplastic agents have been associated with a distinct form of leukaemia characterised by chromosomal translocations as a treatment complication (Felix, 2001;Snyder, 2000).
Our research demonstrates that EB, a topoisomerase II inhibitor, induces mitotic crossing over in A. nidulans diploid cells heterozygous for duplicated segment Dp (II,I).Results suggest that DNA topoisomerase II inhibitors, such as EB, are potentially capable of inducing secondary malignancies mediated by mitotic crossing-over.

Figure 4 -
Figure 4 -Mitotic segregant with crinkled morphology produced by spontaneous haploidization of the D1 diploid strain.

Figure 5 -
Figure 5 -Schematic representation of chromosome I of the D2 diploid.Segregation of the parental (4) and the recombinant (2) chromatids towards the same mitotic pole results in the paba+, y, bi+ // paba+, y, bi+

Table 1 -
Genotype and origin of strains.