L. María Sierra

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.

The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds.

The somatic mutation and recombination w/w+ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity raised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w+ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.

The hypermutability conferred by the mus308 mutation of Drosophila is not specific for cross-linking agents

The hypersensitivity of the mus308 mutant of D. melanogaster to cross-linking agents has been suggested to be the consequence of a possible defect of this mutant in DNA cross-link repair. Moreover, the mus308 mutation has been proposed as an animal model for the study of Fanconis anemia. In order to obtain more information about the function controlled by this locus, we have measured the mutability of the mus308 mutant to several mutagens with different modes of action using the sex-linked recessive lethal test. We show that this mutation confers hypermutability not only to the cross-linking agents tested, hexamethylphosphoramide and hexamethylmelamine, but to the point mutagen N-ethyl-N-nitrosourea as well, whereas the response to methyl methanesulfonate was normal. The results suggest that the mus308 locus is not defective in a repair pathway specific for cross-links but is rather involved in a step of a more general post-replication repair process responsible for the removal of non-excised adducts.

Drosophila comet assay: insights, uses, and future perspectives

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type.

Methodological aspects of the white-ivory assay of Drosophila melanogaster

The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f/Dp(1:1:1:1)wi,y2, although the use of strain FM6,l(1)66a/Dp(1:1:1:1)wi,y2;st/st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU > HMPA > MMS approximately EMS.

Initial studies on quantitative DNA induced oxidation by gel electrophoresis (GE)-ICP-MS

One of the most important consequences of oxidative stress is the induction of oxidation in DNA molecules. Permanent modification of genetic material resulting from “oxidative damage” incidents represents the first step involved in mutagenesis, carcinogenesis, and aging. Therefore, there is an urgent need for monitoring DNA oxidative damage in a quantitative way. For this purpose, the present work evaluates the use of gel electrophoresis (GE) on-line coupled to inductively coupled plasma-mass spectrometry (ICP-MS) to address induced oxidative events in plasmid DNA pBlueScript SK (2961 bp). Oxidative stress is induced in the samples by addition of Fe2+ and H2O2 through the Fenton reaction. After optimization of the set-up for large DNA fragments, the GE separation of the different oxidation products (as induced by the Fenton reaction) were followed by 31P+ monitoring with ICP-MS and compared with conventional slab gels. The main advantage with the proposed set-up is that the quantification of each resulting oxidation product could be performed using just an inorganic phosphate as internal standard.

Influence of nucleotide excision repair and of dose on the types of vermilion mutations induced by diethyl sulfate in postmeiotic male germ cells of Drosophila

The role of a defect for nucleotide excision repair (NER) in oocytes on the repair of DNA ethyl adducts induced by diethyl sulfate (DES) in male germ cells of Drosophila was analysed. Frequencies of mutations at multiple loci (recessive lethal mutations) and at the vermilion gene induced in NER+ conditions (cross NER+ x NER+) were compared with those fixed in a NER- background (NER- x NER+). The M(NER-)/M(NER+) mutability ratios for two DES concentrations, 10 mM and 15 mM, were 2.21 and 1.49, respectively, indicating that NER repairs part of the DES-induced damage. The majority of 28 fertile vermilion mutations produced by DES in NER- are transitions, both GC-AT (46.4%) and AT-GC (21.4%) transitions are found, the consequences of O6-ethylguanine and O4-ethylthymine, respectively. Transversions (21.5%), one +1 frameshift mutation (3.6%) and two deletions (7.1%) are most likely the result of N-alkylation damage. Furthermore, the DES-induced mutation spectra show interesting differences in relation to the exposure dose. All 10 mutants isolated in this and a previous [L.M. Sierra, A. Pastink, M.J.M. Nivard, E.W. Vogel, DNA base sequence changes induced by DES in postmeiotic male germ cells of Drosophila melanogaster, Mol. Gen. Genet. 237 (1993) 370-374] study from experiments with low DES-effectiveness are exclusively transitions, independent whether the females were of the NER+ or NER-genotype. This indicates that at lower DES effectiveness only O-alkylation damage is relevant, and that N-alkylation damage is repaired. In experiments revealing high DES-effectiveness, vermilion mutations representing N-alkylation damage reached 43% (9/21) with NER- and 26% (7/27) with NER+ females, suggesting (i) that NER becomes involved at high adduct levels because then the base excision repair (BER) may be saturated, and (ii) that this involvement of NER causes the relative decrease from 43% to 26% N-alkylation mediated sequence changes.

'Non-genotoxic' carcinogens evaluated using the white-ivory assay of Drosophila melanogaster

Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster. These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination. Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative. These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D. melanogaster.

Genotoxicity and DNA Repair

The Salmonella typhimurium /mammalian microsome assay is the most widely used short-term test to identify genetic damage. This is used to assess the mutagenic and antimutagenic potential of compounds and mixtures. This assay uses histidine-dependent strains to detect mutations, e.g., substitutions, additions, or deletions of one or several DNA nucleotides reverting originally changed gene sequence of the tester strains. The addition of a mutagenic chemical agent to a plate of cultured cells results in the growth of mutant colonies; the number of such colonies is an indicator of the mutagenic potency of the agent. The Ames test has many advantages, it is a very versatile assay, its different modifi cations have been developed to determine mutagenic potencies, and it is recommended by several regulatory agencies. This chapter provides a detailed description of how the standard plate incorporation method should be performed, including the experimental design and interpretation of results.

The importance of distinct metabolites of N-nitrosodiethylamine for its in vivo mutagenic specificity.

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.