Madeleine J.M. Nivard

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

SummaryThe vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr+) and excision-repair-deficient (exr−) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr+ strain and 24 from the exr− strain, were characterized by sequence analysis. In two mutants obtained from the exr+ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr+ strain, 22 (76%) were GC→AT transitions, 3 (10%) AT→TA transversions, 2 (6%) GC→TA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GC→AT transitions. Of the mutations in an exr− background, 12 (48%) were GC→AT transitions, 7 (28%) AT→TA transversions, 5 (20%) GC→TA transversions and 1 (4%) was a AT→GC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.

DNA damage, and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints.

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

The response of germ cells to ethylene oxide, propylene oxide, propylene imine and methyl methanesulfonate is a matter of cell stage-related DNA repair

We describe the consequences of a defect for nucleotide excision repair (NER) in oocytes for alkylation‐induced mutagenesis in different germ‐cell stages of Drosophila males. Mutant frequencies induced in NER+ condition (cross NER+ × NER+) were compared with those fixed in a NER‐ background (cross NER‐ × NER+), using the X‐linked recessive lethal assay (SLRL) for the measurement of forward mutations in 700 loci. In successive male germ‐cell stages exposed to a low dose of 2.4 mMh methyl methanesulfonate, efficient repair of premutational damage in spermatogonia and by the maternal repair system after fertilization was observed. Ethylene oxide (EO) and propylene oxide (PO) did not induce high mutant frequencies in postmeiotic germ cells when mutagenized males were mated with NER+ females: a 32‐fold increase in dose from 750 ppmh to 24,000 ppmh EO (≡LD50) led to no more than a 3‐fold enhancement in mutant frequency. However, up to a 17‐fold increase in mutant frequencies were obtained with NER‐ females. In matings with NER+ females, PO was about 10 times less mutagenic than EO. Suppression of the maternal NER system causeda hypermutability, which, on the average, was 2.4‐fold lower than for EO. This indicates that the 2‐hydroxyethyl adduct generated by EO is more efficiently repaired than the 2‐hydroxyípropyl adduct caused by PO. The low SLRL frequencies (0.2–0.9%) estimated for propylene imine (PI) in NER+ genotypes showed no relation to dose in the range from 1,500 to 48,000 ppmh. In the absence of NER, mutant frequencies were increased up to 29‐fold, and a dose‐dependent increase in mutations was observed for PI over the entire dose range. This study shows mutation induction by EO in postmeiotic Drosophila germ cells at exposure doses that are 800‐fold below those applied previously in the mouse specific‐locus test on spermatogonia [with negative response; Russell et al. (1984): Mutat Res 129:381–388] and 11‐fold below the EO dose for which increased dominant‐lethal responses and heritable translocations were documented in mice spermatozoa and spermatids [Generoso et al. (1990): Environ Mol Mutagen 16:126–131]. Environ. Mol. Mutagen. 29:124–135, 1997.

Mutagenic activity of ethylene oxide and propylene oxide under XPG proficient and deficient conditions in relation to N-7-(2-hydroxyalkyl)guanine levels in Drosophila

Ethylene oxide (EO) and propylene oxide (PO) are direct acting mutagens with high Swain-Scott s-values, which indicate that they react preferentially with ring nitrogens in the DNA. We have previously described that in the X-linked recessive lethal (RL) assay in Drosophila postmeiotic male germ cells EO is, per unit exposure dose, 5-10 times more mutagenic than PO. Furthermore, at the higher dose range of EO tested, 62.5-1000 ppm, up to 20-fold enhanced mutation rates were measured in the absence of maternal nucleotide excision repair (NER) compared to repair proficient conditions. The lower dose range of EO tested, 2-7.8 ppm, still produced a small increased mutation rate but without a significant elevated effect when the NER system is being suppressed. The lowest dose of PO tested, 15.6 ppm, produced only in NER- condition an increased mutation rate. The aim of the present study was to compare the mutagenic effect of EO and PO in the RL assay under XPG proficient and deficient conditions with the formation of N-7-(2-hydroxyethyl)guanine (7-HEG) and N-7-(2-hydroxypropyl)guanine (7-HPG), respectively, the major DNA adducts formed. The formation of 7-HEG and 7-HPG was investigated in Drosophila males exposed to EO and PO as a measure of internal dose for exposures ranging from 2 to 1000 or 2000 ppm, respectively, for 24h. Analysis of 7-HEG and 7-HPG, using a highly sensitive 32P-postlabelling assay, showed a linear increase of adduct levels over the entire dose range. The non-linear dose-response relationship for mutations could therefore not be explained by a reduced inhalation or increased detoxification at higher exposure levels. In analogy with the four times higher reactivity of EO the level of N-7-guanine alkylation per ppm was for EO 3.5-fold higher than that for PO. Per unit N-7-guanine alkylation EO was found to be slightly more mutagenic than PO, whereas PO was the more potent clastogenic agent. While this research has not identified the DNA lesions that cause the increase in repair deficient flies, it supports the hypothesis that efficient error-free repair of some N-alkylation products can explain why these agents tend to be weakly genotoxic or even inactive in repair-competent (premeiotic) germ cells of the mouse and the Drosophila fly.

Genotoxic effects of inhaled ethylene oxide, propylene oxide and butylene oxide on germ cells : sensitivity of genetic endpoints in relation to dose and repair status

We report here results on forward mutation induction (recessive lethal mutations, RL) in Drosophila spermatozoa and spermatids by the three 1,2-alkyl-epoxides ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO), at doses ranging from 47 to 24,000 ppm h for EO, 375 to 48,000 ppm h for PO, and 24,000 to 91,200 ppm h for BO. The results indicate for EO mutation induction at doses 500-fold below the LD50. In crosses of mutagenized NER+ males with NER+ females, the 500-fold increase in EO dose from 47 ppm h to 24,000 ppm h resulted in no more than a 17-fold enhanced mutant frequency in spermatozoa. This flat dose-response relationship is primarily the result of efficient repair of EO-induced DNA adducts in the fertilized egg, as was evident from the up to 40-fold or 240-fold increased mutant frequencies above NER- or NER+ background levels, respectively, in crosses with NER- females. With decreasing dose, MNER-/MNER+ ratios decreased from 9 to 14 at high doses down to approximately 1 at the two lowest doses, indicating that a small fraction of premutagenic lesions induced by EO cannot be repaired by the NER system of Drosophila. Linear extrapolation from high to low EO exposure led to an underestimation of the mutation frequency actually observed at low doses. The pattern of EO-induced ring chromosome loss (CL) differed in two respects from that observed for forward mutations: (a) an increase in CL frequencies was observed only at the two highest EO exposure levels, and (b) inactivation of the NER pathway by the mus201 mutant had no measurable effect on the occurrence of CL. The absence of a potentiating effect of mus201 on EO-induced clastogenicity suggests the formation of clastogenic DNA lesions not causing point mutations, and which are not repaired by NER. Consistent with an inversed correlation of reactivities towards N7-guanine and chain length of 1,2-alkyl-epoxides, the relative mutagenic efficiencies of EO:PO:BO are 100:7.2:1.8 for the NER+ groups, and 100:20:0.7 in the absence of NER. Although in Drosophila germ cells EO is also more effective as a clastogen than PO, the difference (EO:PO=100:58) is much smaller than for recessive mutations. These results provide another argument that DNA lesions generating base substitutions as opposed to those causing clastogenic damage may not be the same for these agents.

Characterization of the genotoxic action of three structurally related 1,2-dihaloalkanes in Drosophilia melanogaster

The genetic activity profiles of three structurally related dihaloalkanes, 1,2-dibromoethane (DBE), 1,2-dichloroethane (DCE) and 1-bromo-2-chloroethane (BCE), were compared in germ cells and somatic tissue of Drosophila melanogaster. The two genotoxicity indices estimated after mutagen exposure of male germ cells were (i) the hypermutability index fexr-/fexr+, measured by the increased frequency of induced recessive lethals (RL) in a strain defective in DNA excision repair (exr-), as compared to the wild type (exr+); (ii) the relative clastogenicity index CL/RL, expressed by the ratio of chromosomal aberrations (CL; ring-X loss) to RL determined in exr+ strains. The fexr-/fexr+ index for DBE was 4-5 times higher than those for DCE and BCE, suggesting a difference in the types of premutagenic lesions produced by DBE in comparison to DCE and BCE. The relative clastogenicity indices for BCE (CL/RL = 0.29) and DCE (0.41) are similar to the value of 0.37 estimated for DBE in an earlier study, all indicating that the three compounds or their metabolites are incapable of forming DNA crosslinks. In somatic cells, after inhalation treatment of female larvae, the effectiveness for the induction of interchromosomal recombination decreased in the order BCE > or = DBE > DCE. It is concluded that in accordance with other studies also in Drosophila the glutathione-mediated pathway is the major cause of genotoxicity caused by DBE, DCE and BCE.

DNA base sequence changes induced by diethyl sulfate in postmeiotic male germ cells of Drosophila melanogaster

SummaryThe DNA base sequence changes induced by diethyl sulfate (DES) were analyzed in postmeiotic male germ cells of Drosophila melanogaster. 31 transmissible vermilion mutants were recovered in F1 and F2 generations, with a frequency of 2.6 × 10−4 for the F1, and of 1.8−13 × 10−4 for the F2. The results show that DES induces both base pair substitutions (93%) and deletions (7%). In accord with its relatively high ability to alkylate oxygens in DNA, the most frequent type of sequence alteration among the basepair changes are GC-AT transitions, accounting for 73% of mutations, followed by transversions AT-TA (10%). DES also induced AT-GC transitions and AT-CG transversions. Both induced deletions were intralocus deletions, not occurring between basepair repeats. No influence of neighboring bases on the mutation position was found.

A novel method for the parallel monitoring of mitotic recombination and clastogenicity in somatic cells in vivo

Both homologous mitotic recombination (HMR), causing loss of heterozygosity (LOH) of the wild-type allele, and structural chromosome aberrations (CA) involve the formation of double-strand breaks in DNA. Whether the induction of CAs is always accompanied by HMR, or whether there exist DNA lesions specifically forming only one of the two end-points is unknown. Answering this fundamental question requires a system for the parallel detection of CAs and HMR, because only then is their analysis under strictly identical condition (dose, repair, genetic background) possible. We describe here a novel system for the parallel detection of HMR and loss of a whole chromosome as a measure of CA, utilizing somatic cells of Drosophila. In haploid germ cells of Drosophila, loss of a ring-shaped X-chromosome (rX) constitutes a frequent event providing an efficient method for measuring clastogenicity. For somatic cells, however, it was unclear whether the development of such a system would be feasible. The generally accepted notion has been that in XX female genotypes, loss of an entire X-chromosome acts as a cell lethal when generated at or shortly after blastoderm stage. However, here we show that rX-loss, if induced in pre-ommatidia cells of 3rd instar larvae, generates viable clones visible as small white patches in the red compound eye. To set up optimal conditions for the detection and quantification of rX-loss compared to HMR, several protocols were developed and tested against model carcinogens (methyl methanesulfonate, cisplatin and 7,12-dimethylbenz[a]anthracene). Generally, we find striking differences in the efficiency of these carcinogens for recombination when compared with clastogenicity. The cross-linking agent cisplatin is 4- to 6-fold more clastogenic than recombinagenic. 7,12-Dimethylbenz[a]-anthracene, on the contrary, produced less than a doubling effect for rX-loss but was highly active (20-times the background) for HMR. It appears therefore that both processes can be separated from each other. To the best of our knowledge, this is the first report suggesting, in terms of DNA adducts involved, qualitative differences between homologous recombination and clastogenic effects. Application of our system for studies on DNA repair may therefore provide new insight into the linkage of repair pathways in either of the two mechanisms.

Development of a Test System for Mutagenicity of Photosensitizers Using Drosophila melanogaster

In the past few years, there has been an increase in the application of photosensitizers for medical purposes. A good standardized test system for the evaluation of the mutagenic potentials of photosensitizers is therefore an indispensable device. In the standard Ames test, white light itself was proven to be mutagenic and the result influenced by the light source. Lack of a reliable positive control is another problem in many genotoxicity test systems used for the evaluation of mutagenicity of photosensitizers. Based on the validated somatic mutation and recombination test, known as SMART, and using Drosophila melanogaster, we developed the Photo‐SMART and demonstrated that methylene blue, known to induce photomutagenicity, can act as a positive control in the presented test system. The SMART scores for the loss of heterozygosity caused predominantly by homologous mitotic recombination. The Photo‐SMART can be used to detect photogenotoxicity caused by short‐lived photoproducts or by stable photoproducts or both. We demonstrated the Photo‐SMART to be a good standardized test system for the evaluation of mutagenic potentials of the photosensitizer 5,10,15‐tris(4‐methylpyridinium)‐20‐phenyl‐[21H,23H]‐porphine trichloride (TPP). We demonstrated that TPP was mutagenic using the Photo‐SMART. For hematoporphyrin, the results of the Photo‐SMART indicate the absence of mutagenicity.