Genotoxic activity of 2-amino-N-hydroxylaminopurine ( AHA ) in Aspergillus

In Aspergillus nidulans, as well as in other eukaryotic cells, not all base analogs are mutagenic. For example, 2-aminopurine (2-AP) is non-mutagenic or weakly mutagenic for eukaryotes while it is mutagenic for bacteria. Because of their potential use in genetical research, an effort has been made to find base analogs mutagenic for eukaryotic cells. Work in this field has been successful: in fact, 6hydroxylamino-purine (HAP) and 2-amino-N-hydroxylaminopurine (AHA) have been found mutagenic for yeast as well as for other eukaryotic cells. (Pavlov et al. 1991, Mut. Res. 253:33-46). In particular, Brockman et al. (Mut. Res. 177:61-75, 1987) tested the mutagenic activity of HAP and AHA in Neurospora crassa and found that AHA is about equally mutagenic as HAP at low doses but more mutagenic at high doses. In this paper we report the genotoxic activity of AHA in A. nidulans. In this mold, we have tested AHA-induced lethality and mutagenic and recombinogenic effects Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol42/iss1/2 Genotoxic activity of 2-amino-N-hydroxylaminopurine (AHA) in Aspergillus nidulans N. Babudri, G. Morpurgo, C. Checchi, S. Pimpinelli and A. Marini Istituto di Anatomia Comparata, Università dei Perugia, Italy In Aspergillus nidulans, as well as in other eukaryotic cells, not all base analogs are mutagenic. For example, 2-aminopurine (2-AP) is non-mutagenic or weakly mutagenic for eukaryotes while it is mutagenic for bacteria. Because of their potential use in genetical research, an effort has been made to find base analogs mutagenic for eukaryotic cells. Work in this field has been successful: in fact, 6hydroxylaminopurine (HAP) and 2-amino-N-hydroxylaminopurine (AHA) have been found mutagenic for yeast as well as for other eukaryotic cells. (Pavlov et al. 1991, Mut. Res. 253:33-46). In particular, Brockman et al. (Mut. Res. 177:61-75, 1987) tested the mutagenic activity of HAP and AHA in Neurospora crassa and found that AHA is about equally mutagenic as HAP at low doses but more mutagenic at high doses. In this paper we report the genotoxic activity of AHA in A. nidulans. In this mold, we have tested AHA-induced lethality and mutagenic and recombinogenic effects. We only summarize briefly the technical approach followed in our experiments; for more detailed information see Babudri et al. 1993, Mut. Res. 321:19-26. 1. Germinating conidia (6.5 h of incubation) were treated with AHA for 1.5 h in soft agar medium (0.45% agar in liquid minimal medium). 2. The mutagenic activity was tested by selecting p-fluorophenylalanine resistant (FPAR) mutants on minimal medium plus FPA. 3. Only colonies that grew and conidiated well were regarded as true FPAR mutants. FPA resistance may be due to mutations arising at several loci: therefore, to estimate the frequency of mutation at one locus (fpaA) we tested resistance of FPAR colonies on minimal medium (pH 4.5) plus 3-amino-L-tyrosine and phenyl-anthranilic acid. On this medium, only fpaA mutants are viable (Calvori and Morpurgo, 1966, Mut. Res. 230:187-195). 4. Recombinational activity as tested by selecting fpaA/fpaA colonies (resistant to FPA) arising from +/fpaA diploids (sensitive to FPA). Mutagenic activity of AHA was tested in 4 experiments with AHA doses ranging from 1 ug/ml to 10 ug/ml. In Table 1 we report the mutagenic potency of AHA evaluated as fpaA mutants per viable cell and the percentage of survival with respect to the controls. A clear dose-effect relationship was not evident in the range of doses tested: for example, note in the Table the frequency obtained at 10 ug/ml and the similar values obtained at 1 and 2.5 ug/ml. Published by New Prairie Press, 2017 As for recombinogenic acitvity of AHA, no enhancement was observed in AHA-treated conidia, compared with controls (Table 2). On the basis of these results, we can conclude that AHA is mutagenic (but not lethal) for A. nidulans germinating conidia. When we compare the data with AHA to those obtained with HAP in Aspergillus (Babudri et al. 1993, Mut. Res. 321:19-26) this main conclusion can be drawn: AHA is a less potent mutagen than HAP in A. nidulans in the dose range tested, a result which is opposite to that obtained in Neurospora (Brockman et al. 1987, Mut. Res. 177:61-75) but is in agreement with that obtained by Pavlov et al. (Mut. Res. 1991, 253:33-46) with the yeast Saccharomyces cerevisiae. It must be noted that we have compared the mutagenic potency of HAP and AHA at doses where the mutation frequency has already reached a plateau, notwithstanding the surviving fraction was not affected at all; in fact, statistical analysis of data obtained with AHA shows that the peak in mutation frequency at 5 ug/ml is not statistically significant. We wish to express our gratitude to Dr. Y. Pavlov who provided us with AHA. This work has been supported by the Ministero della Ricerca scientifica e tecnologica. Table 1. Surviving fraction and frequency of AHA-induced fpaA mutants in a haploid strain of Aspergillus nidulans. The mean values of four experiments and the standard errors of the means are reported. AHA dose fpaA mutants survivors (ug/ml) per viable cell (% of controls) 0 0.16 x 10(-5)(+/0.18) 1 4.80 x 10(-5)(+/0.75) 95 2.5 5.40 x 10(-5)(+/1.21) 102 5 15.50 x 10(-5)(+/5.1) 93 10 3.00 x 10(-5)(+/0.54) 89 Table 2. Frequency of AHA-induced FPA resistant recombinants selected at the fpaA locus in a diploid strain fpaA/+ (mean values from two experiments) AHA dose (ug/ml) fpaA/fpaA recombinants per viable cell 0 1.2 x 10(-4) 1 1.5 x 10(-4) 2 1.3 x 10(-4) http://newprairiepress.org/fgr/vol42/iss1/2 DOI: 10.4148/1941-4765.1332


Genotoxic activity of 2-amino-N-hydroxylaminopurine (AHA) in Aspergillus nidulans
In Aspergillus nidulans, as well as in other eukaryotic cells, not all base analogs are mutagenic.For example, 2-aminopurine (2-AP) is non-mutagenic or weakly mutagenic for eukaryotes while it is mutagenic for bacteria.
Because of their potential use in genetical research, an effort has been made to find base analogs mutagenic for eukaryotic cells.Work in this field has been successful: in fact, 6-hydroxylaminopurine (HAP) and 2-amino-N-hydroxylaminopurine (AHA) have been found mutagenic for yeast as well as for other eukaryotic cells.(Pavlov et al. 1991, Mut. Res. 253:33-46).In particular, Brockman et al. (Mut. Res. 177:61-75, 1987) tested the mutagenic activity of HAP and AHA in Neurospora crassa and found that AHA is about equally mutagenic as HAP at low doses but more mutagenic at high doses.
In this paper we report the genotoxic activity of AHA in A. nidulans.In this mold, we have tested AHA-induced lethality and mutagenic and recombinogenic effects.
We only summarize briefly the technical approach followed in our experiments; for more detailed information see Babudri et al. 1993, Mut. Res. 321:19-26.1. Germinating conidia (6.5 h of incubation) were treated with AHA for 1.5 h in soft agar medium (0.45% agar in liquid minimal medium).
2. The mutagenic activity was tested by selecting p-fluorophenylalanine resistant (FPAR) mutants on minimal medium plus FPA.
3. Only colonies that grew and conidiated well were regarded as true FPAR mutants.FPA resistance may be due to mutations arising at several loci: therefore, to estimate the frequency of mutation at one locus (fpaA) we tested resistance of FPAR colonies on minimal medium (pH 4.5) plus 3-amino-L-tyrosine and phenyl-anthranilic acid.On this medium, only fpaA mutants are viable (Calvori and Morpurgo, 1966, Mut. Res. 230:187-195).
4. Recombinational activity as tested by selecting fpaA/fpaA colonies (resistant to FPA) arising from +/fpaA diploids (sensitive to FPA).Mutagenic activity of AHA was tested in 4 experiments with AHA doses ranging from 1 ug/ml to 10 ug/ml.
In Table 1 we report the mutagenic potency of AHA evaluated as fpaA mutants per viable cell and the percentage of survival with respect to the controls.A clear dose-effect relationship was not evident in the range of doses tested: for example, note in the Table the frequency obtained at 10 ug/ml and the similar values obtained at 1 and 2.5 ug/ml.
As for recombinogenic acitvity of AHA, no enhancement was observed in AHA-treated conidia, compared with controls (Table 2).
On the basis of these results, we can conclude that AHA is mutagenic (but not lethal) for A. nidulans germinating conidia.
When we compare the data with AHA to those obtained with HAP in Aspergillus (Babudri et al. 1993, Mut. Res. 321:19-26) this main conclusion can be drawn: AHA is a less potent mutagen than HAP in A. nidulans in the dose range tested, a result which is opposite to that obtained in Neurospora (Brockman et al. 1987, Mut. Res. 177:61-75) but is in agreement with that obtained by Pavlov et al. (Mut. Res. 1991, 253:33-46) with the yeast Saccharomyces cerevisiae.It must be noted that we have compared the mutagenic potency of HAP and AHA at doses where the mutation frequency has already reached a plateau, notwithstanding the surviving fraction was not affected at all; in fact, statistical analysis of data obtained with AHA shows that the peak in mutation frequency at 5 ug/ml is not statistically significant.
We wish to express our gratitude to Dr. Y. Pavlov who provided us with AHA.This work has been supported by the Ministero della Ricerca scientifica e tecnologica.

Table 1 .
Surviving fraction and frequency of AHA-induced fpaA mutants in a haploid strain of Aspergillus nidulans.The mean values of four experiments and the standard errors of the means are reported.