Ekkehart W. Vogel

The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila.

The relationship in Drosophila males between chemical reaction pattern of mono-functional alkylating agents (AA), described in terms of primary alkylation pattern with DNA and proteins as well as the Swain--Scott s factor, and their biological effectiveness were investigated. The agents chosen for comparative analysis were the nitrosamides ENU and MNU, the methanesulfonic esters iPMS, EMS and MMS, the dialkylsulfate DMS, and the nitrosamines DEN and DMN. Parameters of their biological activity were mortality (LC50) of treated adult males, induction in post-meiotic stages of X-chromosomal recessive lethal mutations and 2--3 translocations after either adult feeding or injection. Induced frequencies of recessive lethals, determined for each AA with a range of concentrations, served as biological dosimeter for interaction with target DNA in the germ line. The results are interpreted as indicating for these AA a causal connection between the pattern of primary alkylation of DNA and the quality of genetic damage observed. 1. The agent with the lowest s value, ENU, and its pendant DEN, failed to produce translocations at mutation frequencies that reached 44% for ENU. The highest chromosome-breaking activity was attributed to AA with high s, MMS and DMS. For MMS, the proportions of translocations (T) to mutations (M) approximately reached a 1 : 1 ratio in stored spermatozoa, at a recessive-lethal frequency of 14%. Ability to break chromosomes, as indicated by the T : M ratios, decreased in the sequence MMS greater than or equal to DMS, MNU greater than DMN greater than EMS greater than iPMS greater than ENU = DEN. 2. Nearly the reversed sequence in relative mutagenci effectivenss was obtained when the (directly acting) AA were arranged on the basis of their CM4/LC50 ratios (CM4, the exposure condition producing 4% recessive lethals after injection): ENU greater than EMS greater than iPMS, MNU greater than MMS = DMS. 3. Among the AA, EMS had a somewhat unique position, in that it was slightly less effective in the translocation test, and also less cytotoxic but more mutagenic in the recessive-lethal test than one would expect from its s value. This is taken as an indication of the influence on biological effectiveness of factors other than the s value, e.g. methylation versus ethylation and the lipid/water partition ratio. An example of the latter was also provided by DMS which, although having the same s as MMS, with its 5-fold higher lipid/water partition ratio, was more toxic than MMS. 4. For those AA that were clearly active in the translocation tests--MMS, DMS, MNU, DMN and EMS--delayed formation of exchanges was observed. Only in 17 out of 555 translocation tests with positive response translocations were already found in progeny from unstored spermatozoa. Consequently, it was concluded that performance of storage experiments in Drosophila is an absolute necessity for the detection of this type of rearrangement by AA. 5...

Relationship between cell killing, chromosomal aberrations, sister-chromatid exchanges and point mutations induced by monofunctional alkylating agents in Chinese hamster cells a correlation with different ethylation products in DNA

Several monofunctional alkylating agents (AA) were compared for their ability to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCE) and point mutations in Chinese hamster cells (CHO and V79 cells). The AAs chosen varied in their reaction kinetics as well as their affinity to nucleophilic sites (different s values). AAs with low s values were more mutagenic in comparison to those with high s values, whereas the reverse was true for induction of cytotoxic effects. Neither SCEs nor chromosomal aberrations correlated with the induction of point mutations, indicating that different primary DNA lesions and repair pathways are involved in these biological processes. Molecular dosimetric studies indicate that O6 alkylation of guanine is the most probable cause of lesions in DNA leading to point mutations following treatment with ethyl methanesulphonate and ethyl nitrosourea.

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 somatic and germ cells in vivo

Alkylation-induced germ cell mutagenesis in the mouse versus Drosophila is compared based on data from forward mutation assays (specific-locus tests in the mouse and in Drosophila and multiple-locus assays in the latter species) but not including assays for structural chromosome aberrations. To facilitate comparisons between mouse and Drosophila, forward mutation test results have been grouped into three categories. Representatives of the first category are MMS (methyl methanesulfonate) and EO (ethylene oxide), alkylating agents with a high s value which predominantly react with ring nitrogens in DNA. ENU (N-ethyl-N-nitrosourea), MNU (N-methyl-N-nitrosourea), PRC (procarbazine), DEN (N-nitrosodiethylamine), and DMN (N-nitrosodimethylamine) belong to the second category. These agents have in common a considerable ability for modification at oxygens in DNA. Cross-linking agents (melphalan, chlorambucil, hexamethylphosphoramide) form the third category. The most unexpected, but encouraging outcome of this study is the identification of common features for three vastly different experimental indicators of genotoxicity: hereditary damage in Drosophila males, genetic damage in male mice, and tumors (TD50 estimates) in rodents. Based on the above three category classification scheme the following tentative conclusions are drawn. Monofunctional agents belonging to category 1, typified by MMS and EO, display genotoxic effects in male germ cell stages that have passed meiotic division. This phenomenon seems to be the consequence of a repair deficiency during spermiogenesis for a period of 3-4 days in Drosophila and 14 days in the mouse. We suggest that the reason for the high resistance of premeiotic stages, and the generally high TD50 estimates observed for this class in rodents, is the efficient error-free repair of N-alkylation damage. If we accept this hypothesis, then the increased carcinogenic potential in rodents, seen when comparing category 2 (ENU-type mutagens) to category 1 (MMS-type mutagens), along with the ability of category 2 genotoxins to induce genetic damage in premeiotic stages, must presumably be due to their enhanced ability for alkylations at oxygens in DNA; it is this property that actually distinguishes the two groups from each other. In contrast to category 1, examination of class 2 genotoxins (ENU and DEN) in premeiotic cells of Drosophila gave no indication for a significant role of germinal selection, and also removal by DNA repair was less dramatic compared to MMS. Thus category 2 mutagens are expected to display activity in a wide range of both post- and premeiotic germ cell stages. A number of these agents have been demonstrated to be among the most potent carcinogens in rodents.(ABSTRACT TRUNCATED AT 400 WORDS)

The occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in Drosophila melanogaster microsomes, and the importance of this metabolizing capacity for the screening of carcinogenic and mutagenic properties of foreign compounds.

Abstract The presence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in microsomes prepared from three strains of Drosophila melanogaster was investigated. The first microsomal preparations, for which mortar pounding was used to homogenize the insects, indicated the occurrence of cytochrome P-450, as concluded from carbon monoxide difference spectra, exhibiting an absorbance maximum at 452 nm. As the reproducibility of the mortar procedure was irregular, a new homogenization procedure was developed, using glass marbles. This method led to a better reproducibility and an increase in the quality of the carbon monoxide difference spectra. An empirical correction by a computer was used for analysis of the spectra. Aryl hydrocarbon hydroxylase activity, one of the expressions of the mixed function oxidase system mediated by cytochrome P-450, was studied by using benzo(a)pyrene as substrate and measuring enzymically produced 3-hydroxy-benzo(a)pyrene. The observed activity was NADPH dependent and was absent (i) when NADH was given as cofactor, (ii) when the microsomes were destroyed by boiling, and (iii) when they were deteriorated by treatment with deoxycholate. These findings provide the basis for further investigations, by which a better understanding of the metabolizing capacity of Drosophila can make this species even more meaningful for the screening of pre-carcinogens and indirectly acting mutagens.

Concentration-effect studies with MMS, TEB, 2,4,6-triCl-PDMT, and Den on the induction of dominant and recessive lethals, chromosome loss and translocations in drosophila sperm

Comparative tests were made with four mutagens, treating male germ cells, particularly mature sperm, of Drosophila melanogaster. Dominant lethals, sex-linked recessive lethals, sex-chromosome loss and partial loss, and in one test translocations were used as genetic and points. The four mutagens, methanesulphonate (MMS), 2,3,5,6-tetraethyleneimino-I,4-benzoquinone (TEB), I(2,4,6-trichlorophenyl)-3,3-dimethyltriazene (2,4,6-triC1-PDMT), and diethyl nitrosamine (DEN) are known to differ in their chemical properties and mode of mutagenic action. An apparent relationship between dominant lethal induction and other genetic damage was found only with TEB. All four mutagens are efficient inducers of sex-linked recessive lethals. At low concentrations there was no direct concentration-frequency relationships. The two direct mutagens, MMS and TEB were effective in the chromosome loss tests. DEN does not induce translocations or any of the other types of damage studied which can be attributed to chromosome breakage. It is concluded that the sex-linked recessive lethal test is a simple and efficient way of preliminary screening chemical mutagens with Drosophila melanogaster.

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

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems: IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)

Vinyl chloride mutagenesis in Drosophila melanogaster

In inhalation experiments, Drosophila males were exposed to vinyl chloride at concentrations of 200, 850, 10,000 30,000 or 50,000 ppm for 2 days, and to 30 or 850 ppm for 17 days. VCM was mutagenic in the recessive-lethal test both after short-term and long-term exposures. The lowest effective concentration (LEC) was 850 ppm after 2 day exposure, and this value could be lowered to 30 ppm by prolonging the exposure time to 17 days. With the concentration levels tested, the mutation frequency increased with concentrations and reached a plateau at 10,000 ppm. This indicates a substrate saturation effect. In contrast with the recessive lethal assay, negative results were obtained when tests on dominant lethals, translocations, entire and partial sex-chromosome loss were carried out with VCM at 30,000 ppm for 2 days. This finding of a false negative seems a logical consequence of the observed saturation effect, and strengthens the concept that there exist two effective concentrations for point mutations vs the induction of chromosome breakage events. Vinyl chloride monomer provides another example to support our view that chromosome breakage is not a reliable measure of mutagenic activity.

Genetical relationship between resistance to insecticides and procarcinogens in two Drosophila populations

Two Drosophila populations, one resistant and the other susceptible to several insecticides, were examined for response to several alkaryltriazenes (2,4,6-Cl3-PDMT; 3-PyDMT; 3-PyDET) and to DMN, DEN and AM. Hikone R (HR) males developed relative resistance to both mutation induction and cell killing by the triazenes, whereas Berlin K (BK) males showed increased tolerance to DMN, DEN, and AM. When F1 hybrid males from the two strains were treated with 2,4,6-Cl3-PDMT, the yield of recessive lethal mutations in the heterozygotes was nearly half of that found in homozygotes from the susceptible strain Berlin K. These findings indicate that in Drosophila metabolism of procarcinogens is controlled by multiple-gene systems located on several chromosomes. An extremely divergent response of the two genotypes was found following treatment with 3-PyDET; this triazene acts as a potent mutagen in BK males but is hardly mutagenic when tested in strain HR.