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.

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.

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.

Induced mutagenic effects in the nucleotide excision repair deficient Drosophila mutant mus201(D1), expressing a truncated XPG protein.

Defects in nucleotide excision repair (NER) as defined by the UV sensitivity of xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) patients has lead to the identification of most of the genes involved: XPA through XPG, CSA and CSB. Whereas XP patients often show an increased risk for skin cancer after exposure to sunlight, this is not the case for patients with CS and TTD. Several CS patients have been shown to carry a defect in the XPG gene. The XPG, a structure specific endonuclease makes the incision 3' of damage and is also involved in the subsequent 5'incision during the NER process. In addition, XPG plays a role in the removal of oxidative DNA damage. The Drosophila XPG gene was isolated and based on the molecular defect of a spontaneous (insertion) and an EMS induced mutant, it was shown that a mutated XPG is responsible for the Drosophila mutagen-sensitive mutants mus201. One of these mutants, mus201(D1) has been used extensively in studies of the effects and mechanisms of many chemical mutagens as well as X-rays. The results of these studies are discussed in the light of the finding that mus201p is the Drosophila homologue of XPG.

Parallel monitoring of mitotic recombination, clastogenicity and teratogenic effects in eye tissue of Drosophila.

Loss of heterozygosity (LOH) of the wild-type allele by structural chromosome aberrations (SCAs), homologous mitotic recombination (HMR) or intra-chromosomal (deletion/amplification) recombination (ICR) plays a crucial role in multistage carcinogenesis. We describe here an in vivo system, enabling the detection of all three chromosome breakage-related events in the same genetic experiment, with eye tissue of Drosophila as targets. This modification of the white/white(+) system enables to measure, simultaneously, HMR and ICR on the X-chromosome, and loss of a ring-shaped X-chromosome, utilizing the eye color gene white. Optimal conditions for the detection and quantification of SCAs (ring-X loss) compared to HMR are discussed in detail. Emerging new techniques comprise the parallel detection of HMR on chromosomes X and 3, using the tumor suppressor gene warts in addition to the X-linked marker white. Another modification of the white/white(+) system measures, again in parallel, HMR and chromosome duplication (non-disjunction).

The in vivo genetic activity profile of the monofunctional nitrogen mustard 2-chloroethylamine differs drastically from its bifunctional counterpart mechlorethamine.

The property of forming crosslinks within DNA is seen as the major cause of the high carcinogenic, genotoxic and anti-neoplastic potency of bifunctional nitrogen mustards. To further investigate the importance for genotoxicity of a second reactive group in a molecule, the genetic activity profiles of the bifunctional nitrogen mustard mechlorethamine (MEC) and its monofunctional counterpart 2-chloroethylamine (CEA) were compared, using several in vivo end points in Drosophila. When post-meiotic male germ cells were alkylated by CEA and then transferred to nucleotide excision repair (NER)-proficient oocytes, no more than up to 4-fold increased forward mutation frequencies were induced. With oocytes deficient for XPG (DmXPG), frequencies were enhanced up to 50 times. For MEC mutation frequencies increased up to 40 times the background, whereas only a low hypermutability was observed when DmXPG were used instead of wild-type females, indicating that nitrogen mustard-induced monoadducts, in contrast to crosslinks, are efficiently repaired by the NER system. Specific locus mutations generated in the vermilion gene by CEA under NER(-) conditions were almost exclusively base pair substitutions (93%). The high proportion of mutations at guanine positions indicates a strong contribution of N7-alkylguanine to the mutational spectrum. MEC induced 64% deletions and other DNA rearrangements in crosses of males with DmXPG females. The small portion of point mutations (36%) was further reduced to approximately 20% with NER(+) females. Inactivation of NER had no potentiating effect on clastogenic events (chromosome loss) induced by CEA, which is in sharp contrast to the strongly enhanced forward mutation frequencies measured with DmXPG females. The weak genotoxic effectiveness of CEA under NER(+) conditions is clearly due to efficient error-free repair of monoalkyl adducts. These results further support the concept that bifunctional nitrogen mustards exert their mutagenic activity through formation of DNA crosslinks and that DNA monoadducts make only a minor contribution to their genotoxic activity.

Genetic effects of exocyclic DNA adducts in vivo: heritable genetic damage in comparison with loss of heterozygosity in somatic cells.

Etheno adducts are promutagenic lesions which generate point mutations, deletions, homologous recombination and gross structural DNA aberrations. High ratios of chromosome loss to forward mutations characterize vinyl bromide, vinyl chloride, ethyl carbamate, vinyl carbamate and its epoxide as effective clastogens in postmeiotic germ cells of Drosophila melanogaster. Of the mutants induced by vinyl carbamate at the vermilion gene, 68% were intra-locus or multi-locus deletions. In view of the far-reaching concordance between mutation spectra in mice and Drosophila observed in specific-locus tests with genotoxic agents, etheno bases are expected to generate mainly deletions in male mammals in the postmeiotic germ-cell stages. Twenty-two of 23 base substitutions induced in the vermilion gene after treatment of postmeiotic stages with vinyl carbamate or vinyl bromide fall into four categories of mutations expected from etheno bases: GC-->AT, AT-->GC, GC-->TA and AT-->TA base-pair changes. These types of point mutations occurred in mutated proto-oncogenes of tumours induced in rodents by vinyl chloride, ethyl carbamate or their metabolites. Of interest is the ability of vinyl carbamate to produce persistent lesions in otherwise highly repair-active premeiotic cells of Drosophila, leading to mutations of yet unknown nature. Etheno bases are also potent pro-clastogenic lesions in somatic cells in vivo. Strongly positive responses were reported for ethyl carbamate and vinyl carbamate in assays for micronucleus formation in mouse bone marrow and in the Drosophila white+/white eye mosaic test. Loss of heterozygosity in somatic cells of Drosophila was due primarily to ring-X chromosome loss, followed by homologous mitotic recombination. Particularly striking is the near failure of ethyl carbamate and vinyl carbamate to generate significant frequencies of intrachromosomal recombination. The overall genetic activity profiles of etheno adduct-forming chemicals in mice and in Drosophila support the hypothesis that vinyl carbamate is the proximate mutagen of ethyl carbamate, and vinyl carbamate epoxide is the ultimate electrophilic mutagen and carcinogen.

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system.

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.

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.

Germ cell mutagenesis in Drosophila: multiple endpoint analysis.

Genotoxic carcinogens, able to damage DNA by alkylation reactions, represent a very diverse class of agents which are capable of producing a wide range of DNA modifications. The mechanisms leading to genetic changes as a result of exposure to alkylating agents (AAs) have been studied in male germ cells of Drosophila using a structure-activity relationship approach (SAR). The analytical tools available concern both genetic and molecular assays. The genetic tests enable to quantify excision repair and clastogenic potency of the AA after treatment of post-meiotic male germ cells and to determine the degree of germ-cell specificity, i.e., the mutagenic effectiveness in post- versus premeiotic cell stages. For a selected group of alkylating agents the molecular spectra have been studied in post-meiotic cell stages. On the basis of these descriptors clear SAR's between genotoxic activity in germ cells and physico-chemical parameters (s-values and O6/N7-alkylguanine adducts) and carcinogenic potency in rodents became apparent, resulting in five distinct classes of alkylating agents so far. These classes are: 1) SN2-type monofunctional AAs, 2) SN1-type monofunctional AAs, 3) polyfunctional AAs, 4) agents able to form etheno-DNA adducts, and 5) aflatoxin B1 (AFB1) a bulky-adduct forming agent. The recent finding that the molecular data obtained with Drosophila and data of the specific locus tests in male mice show remarkable similarities for most genotoxic agents supports the view that Drosophila is a useful model system for the study of transgenerational damage.

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.

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 close to or at 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.

Strand-specific mutation induction by 1,2-dibromomethane at the hypoxanthine-guanine phosphoribosyltransferase locus of Chinese hamster ovary cells.

The nature of mutations induced by 1,2-dibromoethane (DBE) at the hprt (hypoxanthine-guanine phosphoribosyl-transferase) gene was analysed in Chinese hamster ovary (CHO-9) cells. Molecular characterization of 36 hprt mutants at the cDNA level yielded 19 GC-->AT transitions, two AT-->CG transversions, three frameshift mutations, two identical small deletions and 10 exon deletions. Further analysis of the deletion mutants by amplification of specific exons from genomic DNA showed two more GC-->AT transitions at splice sites and an approximately 70 bp deletion. Assuming that the S-[2-(N7-guanyl)ethyl]glutathione adduct is responsible for the GC-->AT transitions, 90% of the affected guanines were located in the non-transcribed strand of the hprt gene, suggesting a strand bias in repair of this adduct. Nearest neighbour analysis of induced GC-->AT transitions indicates a preference for a 5'-PyPuG DNA sequence, i.e. 15/21 mutated guanines were located in either a TGG or a CAG DNA sequence. These molecular data on DBE-induced mutations showed similar features as data from a study by Graves et al. (Mutagenesis, 11, 229-233, 1996) in which they analyzed 13 hprt mutants induced by DBE in CHO-K1 cells. Six of the seven GC-->AT mutations were on positions mutated more than once among the 36 hprt mutants in the present study. The combined findings suggest that some positions seem to be hot spots for DBE-induced mutations. Concerning the relevance of these in vitro studies for germ cell mutagenesis the conclusion may be that these data lend further support to the view that mutation spectra derived from in vitro systems have little predictive value for the nature of mutations induced in post-meiotic germ cells in vivo, as demonstrated for other alkylating agents in both Drosophila and mice.

Preferential formation of deletions following in vivo exposure of postmeiotic Drosophila germ cells to the DNA etheno-adduct-forming carcinogen vinyl carbamate.

DNA sequence changes induced in the vermilion gene of Drosophila following in vivo treatment of postmeiotic male germ cells with vinyl carbamate (VCA), an etheno-adduct-forming carcinogen, are primarily deletions. With VCA, 65% (13/20) of the vermilion mutants isolated from crosses of NER+ (nucleotide excision repair) males with NER+ females and 40% (6/15) obtained from matings with NER- females were intra- or multi-locus deletions. Due to the insufficiently low mutagenic activity in NER+ genotypes of vinyl bromide (VB), another epsilon-adduct-forming carcinogen, vermilion mutants could only be isolated from crosses of VB-treated males with NER- females. Of 14 vermilion mutants induced by VB, three carried large deletions. Twenty-two of 23 base substitutions derived from either VCA or VB experiments fell into one of the four categories expected from epsilon-adducts: three vermilion mutants had GC-->AT transitions, five had AT-->GC transitions, 7 carried GC-->TA transversions, and 7 were AT-->TA transversions. In view of the similarities in the response of mouse and Drosophila germ lines to several classes of alkylating agents, a high incidence of deletions is predicted to occur as well in postmeiotic germ cells of mice exposed to these types of agents.

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+female female x NER+male) were compared with those fixed in a NER- background (cross NER-female female x NER+male), 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 mM x h 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 ppm x h to 24,000 ppm x h EO (approximately 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 caused a 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-hydroxypropyl 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 ppm x h. 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].

Mutational spectra induced under distinct excision repair conditions by the 3 methylating agents N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and N-nitrosodimethylamine in postmeiotic male germ cells of Drosophila.

This paper describes the analysis of mutations induced at the vermilion locus in postmeiotic male germ cell stages of Drosophila exposed to 3 different N-methyl-N-nitroso compounds: N-methyl-N-nitrosourea (MNU); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); and N-nitrosodimethylamine (DMN). With MNU and DMN, the impact of DNA nucleotide excision repair (NER) on the spectra of mutations was studied. Mutants were isolated from F1 (mutations fixed before the first mitotic replication after fertilization) and F2 (mutations fixed following one or more mitotic replications; mosaics in F1) generations. The vermilion system enables the analysis of both intra- and inter-locus DNA changes for which several techniques have been adapted: (1) amplification of the vermilion gene by PCR, cloning of the fragment and sequence analysis of ssDNA; (2) Southern blot hybridization; and (3) cytological analysis of polytene chromosomes. In total, 49 MNU (26 from the exr+ genotype and 23 from the exr- genotype), 47 DMN (28 from the exr+ genotype and 19 from the exr- genotype) and 16 MNNG-induced mutations were characterized. The F1 spectra of all 3 agents contained base-pair changes and deletions (intra- and multi-locus) in a ratio of roughly 1 to 1, indicating a significant contribution of nitrogen DNA adducts to the spectra. In all F2 spectra the levels of base-pair changes were significantly higher compared to those in the F1 spectra, a finding also made for methyl methanesulfonate-induced mutations in earlier studies. There is an increase of mutations of, especially, the transversion types of mutations under exr- conditions in comparison to the exr+ situation. The induced transversions, clearly present in all spectra (exr+ and exr-), are presumably caused by N-methyl DNA adducts, which upon release from the DNA backbone lead to apurinic sites in a time-related process. Regarding the occurrence of transitions, it turned out for all 3 mutagens that the AT-->GC type strongly dominated the GC-->AT transitions. This suggest that O6-methylguanine is efficiently repaired, in contrast to O4-methylthymine. Based on the data obtained in the vermilion system with ENU, we propose, in addition, that the Drosophila alkyltransferase system repairs O6-methylguanine more efficiently than O6-ethylguanine.

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

Characterization by two-endpoint comparisons of the genetic toxicity profiles of vinyl chloride and related etheno-adduct forming carcinogens in Drosophila.

The genetic toxicity profiles of vinyl chloride (VCl), vinyl bromide (VBr), ethyl carbamate (EC), vinyl carbamate (VC) and some structurally related chemicals were investigated in both somatic and germ cells of Drosophila melanogaster. In the white/white+ eye mosaic assay, a screening system measuring predominantly homologous recombination in somatic cells, only marginal genotoxic activities were observed for acetyl chloride (ACl), glycolaldehyde (GCA), 2,2'-dichlorodiethyl ether (DDE) and methyl carbamate (MC), whereas VCl, 2-chloroacetaldehyde (CAA), VBr, 2-bromoacetaldehyde (BAA) and EC were clearly recombinogenic in the assay. Those chemicals proven to be recombinogenic in somatic cells were investigated further in postmeiotic male germ cells, utilizing as descriptors of their genotoxicity I(CL/RL) and M(exr-)/M(exr+) indices. The I(CL/RL) index is the rate of induced chromosome loss (CL), a clastogenic event, divided by the forward mutation rate, measured as recessive lethal (RL) mutations in 700 loci of the X-chromosome. The M(exr-)/M(exr+) mutation enhancement ratio is obtained by determining RL under excision repair deficient versus repair proficient conditions. With I(CL/RL) values (2.7-6.9) similar to those obtained for cross-linking agents, vinyl chloride, vinyl bromide, ethyl carbamate and vinyl carbamate are all efficient clastogenic agents in Drosophila germ cells. In the absence of excision repair, however, neither CEO nor CAA gave a hypermutability response (M(exr-)/M(exr+) approximately 1). By contrast, VCl, VBr, EC and VC showed clearly enhanced M(exr-)/M(exr+) ratios, suggesting that these compounds produce some repairable DNA modification(s) that are not generated by their epoxides. This unexpected finding points to the formation of other, yet unknown, metabolites of vinyl chloride, vinyl bromide, ethyl carbamate and vinyl carbamate. Our results support the concept that the epoxides chloroethylene oxide (CEO), bromoethylene oxide (BEO) and vinyl carbamate epoxide (VCO) are the most essential mutagenic intermediates. Compared to chloroethylene oxide (CEO), 2-chloroacetaldehyde (CAA) was approximately 50 times less effective in the induction of RL, whereas BAA was inactive as a mutagen. These findings are consistent with the general view that CAA and BAA play no major role in the genotoxic action of vinyl halides.

Hexamethylmelamine is a potent inducer of deletions in male germ cells of Drosophila melanogaster.

Chemotherapy-related second tumors constitute a matter of concern in cancer treatment. Therefore, it is of great interest to elucidate the mechanisms by which cytostatic drugs exert their mutagenic and/or carcinogenic activity besides the anticancer effect and the possible relationship among them. A useful and informative approach to this problem is the analysis of the mutation spectra induced by these drugs in eukaryotic organisms. Sequence analysis of the mutations induced by hexamethylmelamine, a crosslinking agent extensively used in the treatment of ovarian cancer, in male germ cells of Drosophila was conducted using the v locus as reporter gene. Both intra-locus and multi-locus deletions were induced whereas based changes were almost absent. Thus, it is proposed that deletions are likely to be involved in the generation of second malignancies in hexamethylmelamine-treated patients. It has to be stressed that systems, such as v, capable of efficiently recovering mutations caused by big losses of DNA, should be used for the study of mutational spectra induced by cross-linking agents.

The cross-linking agent hexamethylphosphoramide predominantly induces intra-locus and multi-locus deletions in postmeiotic germ cells of Drosophila.

The nature of DNA sequence changes induced by the cross-linking agent hexamethylphosphoramide (HMPA) within and in the vicinity of the vermilion locus of Drosophila melanogaster that produce a vermilion mutant phenotype was analyzed after exposure of postmeiotic male germ cells. Mutagenized males were mated to either females wild-type (exr+) for nucleotide excision repair (NER) or to females having a deficiency (exr-) for NER. Rearrangements, mostly deletions, represented by far the most frequent type of mutational events induced by HMPA that are detected as vermilion mutations. In the exr+ group, all but one (a double substitution) of 21 mutants characterized were large sequence changes: we found 5 intra-locus deletions, 3 intra-locus deletions associated with insertions and 12 multi-locus deletions. When taken together, deletions and deletion/insertion mutations represent 96% of the HMPA-induced DNA modifications obtained under proficient repair conditions. Of the 10 mutants obtained from crosses with exr- females, 6 intra-locus and 2 multi-locus deletions were found, as opposed to just 1 point mutation and 1 double substitution. The "hypomutability effect" observed with exr- genotypes in relation to the wild type seems to be caused by a decrease in the frequency of multi-locus deletions in the former group. The results suggest that the NER system is involved in the generation of multi-locus deletions, whereas intra-locus deletions appear to be formed through a postreplication slipped-misrepair pathway. It is concluded that an eukaryotic in vivo system with no limitations for the recovery of multi-locus deletions, such as vermilion, should be used for the analysis of DNA damage induced by cross-linking agents.