Somatic cell mutagenicity in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages

With the intention of assessing the general performance, sensitivity and the underlying mechanisms of somatic cell mutagenicity assays in Drosophila, a study was undertaken to compare the effectiveness of 5 procarcinogens and 4 direct-acting agents in the white/white-coral eye mosaic assay (SMART) with their activity in early (premeiotic) male and female germ-cell stages, after exposure of Drosophila larvae. The outcome indicated a lack of agreement in the results from recessive lethal assays (SLRL) in comparison with the somatic mutation and recombination test (SMART). The procarcinogens 2-naphthylamine (NA), 3-methylcholanthrene (MC), 9,10-dimethylanthracene (DA) and 7,12-dimethylbenz[a]anthracene (DMBA), and the direct-acting mutagens bleomycin (BM), methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), were quite efficient in producing somatic recombination and mutations in white/white-coral larvae, as opposed to only weak effects in early germ-cell stages. 2-Acetylaminofluorene (2AAF) showed marginal effects in both germ cells and somatic tissue after exposure of female larvae, but was inactive in testis. The discrepancy in mutational response between somatic cells and premeiotic germ cells is most impressive for MMS and BM. There is sufficient evidence for attributing a good sized proportion of the encountered variation to efficient error-free DNA repair of premutational damage and to segregational elimination during meiosis of deleterious mutations: (1) The efficient point mutagen ENU was the but one agent producing high levels of viable genetic alterations in early germ cells and in somatic cells. A similar behaviour was previously described for diethylnitrosamine, which ethylates DNA in the same fashion as ENU. (2) In early germ-cell stages of mei-9L1 male larvae, MMS induced multiple mutations (putative clusters) at a low dose differing by a factor 20-40 from those needed to produce an equivalent response in repair-competent strains. This is consistent with the concept of an active excision repair in premeiotic cells. (3) In the case of EMS, next to DNA repair, germinal selection seems to restrict the realization of EMS-induced genetic damage in premeiotic cells. (4) Bleomycin-induced chromosome aberrations caused high mortality rates in males (hemizygous for an X-chromosome) but not in females. MMS and BM, agents known to show preference for chromosome aberration induction, produced 3-6-fold higher rates of somatic mutational events (SME) in female genotypes as compared with the other sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

A search for chromosome aberrations induced in mouse spermatogonia by chemical mutagens

Two established chemical mutagens—ethylmethanesulphonate (EMS) and triethylenemelamine (TEM)—were tested for the ability to induce chromosome aberrations in mouse spermatogonia. While not a single aberration was detected following the EMS treatment, a low frequency of translocations and fragments was found in the TEM groups. These findings are in agreement with the data obtained with the specific locus mutation test as applied to male mouse premeiotic germ cells but contrast with the effectiveness of these chemicals in breaking chromosomes in male mouse postmeiotic germ cells. A differential sensitivity of post- and premeiotic germ cells to any kind of genetic damage by these chemical mutagens is most likely to be the correct interpretation of all the data. However, it is also suggested that a high proportion of translocations induced in spermatogonia by chemical mutagens may not be detectable by present methods.     

Phenotypes of Drosophila homologs of human XPF and XPG to chemically-induced DNA modifications

DmXPF (mei9) and DmXPG (mus201) mutants are Drosophila homologs of the mammalian XPF and XPG genes, respectively. For Drosophila germ cells, causal correlations exist between the magnitude of a potentiating effect of a deficiency in these functions, measured as the M(NER-)/M(NER+) mutability ratio, and the type of DNA modification. M(NER-)/M(NER+) mutability ratios may vary with time interval between DNA adduct formation and repair, mutagen dose and depend also on the genetic endpoint measured. For forward mutations, there is no indication of any differential response of DmXPF compared to DmXPG.Subtle features appeared from a class-by-class comparison: (i) Methylating agents always produce higher M(NER-)/M(NER+) ratios than their ethylating analogs; (ii) M(NER-)/M(NER+) mutability ratios are significantly enhanced for cross-linking N-mustards, aziridine and di-epoxide compounds, but not for cross-linking nitrosoureas. The low hypermutability effects with bifunctional nitrogen mustards, aziridine and epoxide compounds are attributed to unrepaired mono-alkyl adducts; (iii) The efficient repair of mono-alkyl-adducts at ring nitrogens in wild-type germ cells is evident from the absence of a dose-response relationship for ethylene oxide, propylene imine and methyl methanesulfonate (MMS). These chemicals become powerful germline mutagens when the NER system is disrupted.Systematic studies of the type performed on germ cells are not available for somatic cells of Drosophila. The sparse data available show large differences in the response of germ cells and somatic cells. The bifunctional agent mechlorethamine (MEC) but not the monofunctional MMS or 2-chloroethylamine cause in NER(-) XXfemale symbol the highest potentiating effect on mitotic recombination. The causes of the discrepancy between the extraordinarily high activity of MEC in mus201 somatic cells and its low potentiating effect in germ cells is unknown at present.     

DNA single-strand breaks,double-strand breaks,and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. ¹³⁷Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methane sulfonate, ethyl methane sulfonate, ethyl nitrosurea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency.This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.Abbreviations DBCP dibromochlorpropane - DSB(s) DNA double-strand break(s) - EDB ethylene dibromide - EMS ethyl methane sulfonate - ENU ethyl nitrosurea - MC mitomycin C - MMS methyl methane sulfonate - SDS sodium dodecyl sulfate - SSB (s) DNA single-strand break(s) - TEM triethylene melamine - UDS unscheduled DNA synthesis     

The ratio of induced recessive lethals to ring-X loss has prognostic value in terms of functionality of chemical mutagens in Drosophila melanogaster

For 25 mutagens in Drosophila the ratio was determined between the induction of sex-linked recessive lethals (SLRL) and the induction of ring-X loss in male adults. For small monofunctional alkylating agents this ratio increases with decreasing s-value from 1.8 for methyl methanesulfonate (MMS) to 27 for ethylnitrosourea (ENU). For multifunctional cross-linking agents, however, the ratio varies within relatively narrow limits, ranging from 0.15 for cisplatin to 0.07 for tris-(1-aziridinyl)phosphineoxide (TEPA), while for most agents the ratio is around 0.12. The number of reactive groups seems to be of minor importance for compounds with more than one functionality as bi- and tri-functional agents show similar ratios. The systemic difference in the ratios between mono- and multi-functional agents suggests that different mechanisms are involved in the induction of SLRLs and ring-X loss. For ethyleneimine (EI) and ethyleneoxide (EO) low ratios of 0.32 and 0.60 respectively were observed which do not correlate with their s-values. An alternative chromosome-breaking mechanism may be responsible for this deviation, possibly alkylation of the phosphate backbone of DNA, followed by an intramolecular displacement of one of the deoxyribose groups by the beta-amino or the beta-hydroxy group. It is felt that the considerable difference between the ratios for monofunctional and multifunctional agents may be of prognostic value and can be used to obtain information on the mechanisms of mutagens with 'unknown' action, provided that structural features are taken into account. Hexamethylphosphoramide (HMPA), hexamethylmelamine (HEMEL), tetramethylurea (TMU) and dimethylpropyleneurea (DMPU) all show SLRL: ring-X loss ratios that match those of multifunctional agents, 0.08, 0.12, 0.08, and 0.16, respectively. The ratios for the pyrrolizidine alkaloids monocrotalin and seniciphilline, 0.053 and 0.24 respectively, also correspond with this group of mutagens. The low ratios for formaldehyde, 2-chloro-acetaldehyde and 2-chloroethyl methanesulfonate, 0.30, 0.052 and 0.36 respectively, are indicative that cross-linking may attribute considerably to their mutagenic action in Drosophila. On the other hand, not all mutagens containing 2 reactive groups act as cross-linking agents. The ratio for 1,2-dibromoethane, 2.7, indicates that it may act as a monofunctional agent. This is in accordance with the proposed activation mechanism by glutathione S-transferase, producing a monofunctional half-mustard derivative (Rannug, 1980; van Bladeren et al., 1981).     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Genetic damage by bifunctional agents in repair-active pre-meiotic stages of Drosophila males

Most of our understanding of germline mutagenesis in Drosophila is based on the DNA repair-inactive, haploid post-meiotic stages. The diploid, repair-active pre-meiotic stages are more relevant to the situation encountered in somatic cells. DNA mono-adducts induced by agents like methyl methanesulphonate (MMS) and ethylene oxide (EO) are well repaired in the pre-meiotic cell stages, and these agents show therefore, no or considerable lower mutagenic activity in these stages. In contrast, in this study the two bifunctional nitrogen mustards chlorambucil (CAB) and mechlorethamine (MEC) show significantly elevated mutant frequencies of both post- and pre-meiotic germ cells. Results were similar for the X-chromosomal and the autosomal (2nd) recessive lethal (RL) test. CAB and MEC were also active in stem cells, but in comparison with post-stem cell stages they seem to be better protected. The germ cell specific response in post- and pre-meiotic cell stages was for both nitrogen mustards comparable to mutagenic activity patterns observed in the specific locus test in the mouse.It was reported that for diepoxybutane (DEB), another cross-linking agent, the ratio of the RL frequency for the 2nd- and the X-chromosome was increased from 2.1 for post-meiotic stages to 9.5 for pre-meiotic stages. In own experiments aiming to confirm this observation, a high ratio was indeed found. The induction of large deletions by DEB could be the reason for this difference, since such lesions might include both a sex-linked lethal and a vital gene required for the development of spermatocytes into mature sperm. Similar differences were expected for CAB and MEC since they are also inducers of large deletions. But unexpectedly, no differences in 2nd/X RL ratio between post- and pre-meiotic cell stages were found for the nitrogen mustards. Possible causes such as distinct proportions of multi-locus deletions (MLDs), mitotic recombination and the formation of persistent lesions, are discussed.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

DNA-repair modulation using derivatives of 1,4-dihydroisonicotinic acid as an example

The influence of two derivatives of 1,4-dihydroisonicotinic acid on DNA-repair involved in chemical mutagenesis in Drosophila germ cells has been investigated. The compounds tested decreased the level of EMS-induced chromosome breakage and point mutations due to stimulation of maternal repair of DNA primary damage induced in spermatozoa as well as due to activation of DNA-repair in larvae and imago premeiotic stages of Drosophila males. Deficiency of DNA-repair systems leads to decrease in female and male germ-cell sensitivity to antimutagen action.     

The Drosophila wing test: a comparison of the sensitivity of different strains

A range of 5 chemical mutagens were tested in the Drosophila wing test using 2 different strains and carrying out the experiments in parallel under standardised conditions. The mutagens chosen for the study were the 2 alkylating agents MMS and ENU and the anti-cancer drugs methotrexate, cytosine-arabinoside and adriamycin. As a result of the different genetic backgrounds there was a marked variation in the response of the 2 strains to the mutagens.     

Biological effect of low doses of alkylating agents in drosophila studies

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Monitoring airborne genotoxicants in the rubber industry using genotoxicity tests and chemical analyses

This research was designed to examine the presence of mutagenic/carcinogenic compounds in airborne pollutants in the rubber industry using an integrated chemical/biological approach. Inhalable airborne particulate matter (PM-10: <10 μm) was collected in four rubber factories using a high-volume sampler equipped with a cascade impactor for particle fractionation. The organic extracts of two different fractions (0.5–10 μm and <0.5 μm) were examined for mutagenicity with the Ames test and for in vitro DNA-damaging activity in human leukocytes by single-cell microgel electrophoresis (Comet assay). The extracts were also studied by gas chromatography/mass spectrometry (GC/MS) for polycyclic aromatic hydrocarbon (PAH) content. Nitrosamines in ambient air were sampled on cartridges and analysed by GC with a thermal energy analyser (TEA) detector. Airborne volatile genotoxins were monitored in situ using a clastogenicity plant test (Tradescantia/micronuclei test). The results showed that airborne particulates were mainly very fine (<0.5 μm) and that trace amounts of genotoxic nitrosamines (N-nitrosodimethylamine: 0.10–0.98 μg/m³; N-nitrosomorpholine: 0.77–2.40 μg/m³) and PAH (total PAH: 0.34–11.35 μg/m³) were present in air samples. Some extracts, particularly those obtained from the finest fractions, were mutagenic with the Ames test and genotoxic with the Comet assay. In situ monitoring of volatile mutagens using the Tradescantia/micronuclei test gave positive results in two working environments. The results showed the applicability of this integrated chemical–biological approach for detecting volatile and non-volatile genotoxins and for monitoring genotoxic hazards in the rubber industry.     

Somatic cell mutagenesis in Drosophila: recovery of genetic damage in relation to the types of DNA lesions induced in mutationally unstable and stable X-chromosomes

This paper reports the results of a study on the genotoxic activities of 12 mutagens and clastogens of widely differing mode of action in somatic cells in vivo, i.e., in the eye primordia of Drosophila larvae. After emergence, adult flies were monitored for aberrantly colored sectors in the compound eyes of the following genotypes: UZ males and females (zeste) carrying a genetically unstable transposable element, SZ males and females (zeste) carrying a partial duplication of the w+ locus plus a transposon insert, white-coral/white (wco/w) females, w+/w females and w+ males. The UZ and SZ marker sets make it possible to monitor shifts from zeste to red (scored as mosaic red spots, RS) and for loss of the white locus (light spots, LS). wco/w+ females were scored for mosaic twin spots (TS) and LS, w+ genotypes for just LS. The genotoxins analyzed were methyl methanesulfonate (MMS), dimethyl sulfate (DMS) and ethylnitrosourea (ENU) (alkylating), adriamycin (AM) and daunomycin (DM) (intercalating), Trenimon, Thio-TEPA and cisplatin (DDP) (cross-linking), bleomycin (strand-breaking), 7,12-dimethylbenz[a]anthracene (DMBA) and 9,10-dimethylanthracene (DA) (bulky monoadducts) and cytosine arabinofuranoside (inhibition of DNA synthesis). The relative mutabilities with frequencies of mosaic light spots (LS) in w+/w female as the standard (relative mutability = 1) vs. genotypes UZ (RS in male) vs. SZ (RS in male) vs. w+ (LS in male) were 1:0.6:0.2:0.3 for MMS, 1:0.09:0.05:0.7 for DDP, and 1:1.6:0.2:1.0 for ENU, ENU showed exceptional behavior in that it was the only compound for which mutational response, measured by the induction of red spots, was highest with the UZ marker set. Occurrence of large light spots (LS) in male but not in female genotypes was negatively correlated with efficiency of agents for chromosomal damage, suggesting that in the hemizygous condition, as in males, selection of damaged cells and mitotic delay may have played a significant role. In general, the results indicate that there is no association between the ability of an agent to act as a clastogen and the recovery of aberrant (red spots) sectors in either the UZ or the SZ strain, and of single light spots (LS) in w+, UZ and SZ males. The possibility is considered that the process causing the genetic instability in the UZ strain is under genetic control, and that strong point mutagens such as ENU through efficient gene mutation induction can interfere with it.     

Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule.     

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.     

Influence of the yellow Locus on Sensitivity of Drosophila Germ Cells to Chemical Mutagens

The effect of theyellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the sex-linked recessive lethals (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin K; y; y ct v; and y mei-9 ^a) were treated by mutagenic agents and then crossed to Bascfemales. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that theyellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Bascmales were crossed to females of various genotypes including y and y mei-9 ^a ones. The crosses involving y females yielded F₁ progeny with high spontaneous lethality, whereas in F₂, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny F₁. The results obtained using the above two approaches suggest that theyellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.     

Comparative studies of the induction of somatic eye-color mutations in an unstable strain of Drosophila melanogaster by MMS and X-rays at different developmental stages

An unstable white locus in Drosophila melanogaster originally described by Rasmuson and Green (1974) and further by Rasmuson et al. (1978, 1980) contains an IS element. This constellation interacts with the zeste mutation and forms a mutationally unstable system that is sensitive to a variety of mutagens. Mutational shifts between zeste and wild-type eye color as well as deletions and transpositions of the white locus are frequently occurring in the unstable X-chromosome in germ line and in somatic tissue. Germinal mutations from zeste to wild-type eye color are associated with an insertion of a piece of DNA, proximal to the wsp site, and the shifts from red to zeste are caused by an excision of the same piece (Rasmuson, in preparation). Mutations to pigmentless phenotype are interpreted as deletions of the white locus, while they always are irreversible and show non-complementation with wsp. The somatic system can be used as a screening test for potential mutagens, described by Rasmuson et al. (1984). This survey is an attempt to correlate the size of the mutated area of the eyes with the age of the larvae at mutagen treatment. X-Rays and MMS were used to give an indication of the mechanism of the instability, according to the different kinds of DNA damage induced. The results show that the mean size of red spots decreased with increasing age of larvae at treatment, while the mutation frequencies were increased because of the multiplication of the cells in the eye anlage susceptible to the mutagens. This is contradictory to the hypothesis maintained by Fahmy and Fahmy (1980) that the somatic shifts are not mutagenic but epigenetic events, due to altered regulation of the gene expression. Red spots induced with MMS are smaller in size than X-ray-induced red spots, indicating a delay in the establishment of mutations from chemically-induced lesions compared to irradiation damage. White spots on the other hand were equally large in size, irrespective of inducing agent and about twice the size of the chemically-induced red spots, implying a faster and more direct action for fixation of deletions than for the production of MMS induced shifts in eye color from zeste to red.     

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.