The MR P-type transposable elements and the genetic activities of mutagens and carcinogens in Drosophila melanogaster. I. N,N-Dimethylnitrosamine (DMN).

A technique was developed for the assay of the genetic activities of carcinogens in both the soma and germ line in the course of early larval development and to assess the extent of their modification through the introduction into the genome of the MR IInd autosome P-type transposable elements. The influence of MR on genotoxicity for a given treatment was indicated by the relative frequencies of non-MR (Cy) to MR-carrying sibs emerging within the same cultures. The viable genetic changes in the soma were classified as recombinational or mutational events on the basis of the comparative yields of mosaic sectors in females heterozygous for the markers y w sn carried in standard order (XS) or multiply inverted (XIn) X-chromosomes. The results with this technique are here described for the carcinogen DMN. In the absence of MR, the topical application of DMN induced no larval lethality up to the highest tested doses (20 mM), but raised the yields of the somatic sectors for all the test markers in the emerging females in accordance with a linear dose fit. Comparison of the XS and XIn data indicated that somatic mutagenesis by DMN entailed the induction of recombinational and mutational events in roughly equal proportions. The introduction of MR into the genome, whether patro- or matroclinously, resulted in dramatic and proportionately equivalent enhancements in the activities of DMN, both with respect to the induction of larval lethality and somatic sectoring. These activities increased exponentially with dose, following a 4th-degree polynomial course up to 10 mM, when larval lethality approached 100%. At lower dose levels, the yields of all sector types in the viable females also followed comparable polynomial curves at different heights, except for y sn in the XIn series, where sector recovery remained at the control level throughout the examined dose range. Analysis of the sector size distribution for eye and bristle mosaicism in the XS control and DMN-treated series gave statistically comparable mean values, irrespective of the presence or absence of MR, indicating that DMN alone, or in conjunction with the P elements, did not alter the timing of aberrant clone initiation. In contrast, estimates of the genetic induction events in the somatic primordia with 5 mM DMN indicated greatly increased response in the presence of MR, which was in excess of 20-fold for the mutational changes involving the sn locus.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic activity of the carcinogen N-nitrosodiethanolamine (NDELA) on unstable mutant alleles of the white locus in Drosophila melanogaster.

The genetic activities of the carcinogen N-nitrosodiethanolamine (NDELA) were assayed somatically and germinally on two stocks carrying unstable alleles of the white (w+) eye colour gene: one incorporating a TE (z TE w+; coded UZ), the other with a tandem duplication of part of the encoding w+ sequence (wi16). Treatment was applied topically on late embryonic and early larval stages over a wide dose range (0.01-2.0 M) and the general mutagenic levels actually achieved were measured in terms of the X-recessive mutations (lethals and visibles) recovered in Muller-5 tests on samples of the males scored for somatic sectoring. The carcinogen was germinally ineffective on the z TE w+ and wi16 loci even at the maximal tested dose (2.0 M), and was only weakly mutagenic with respect to the X-recessives (lethals + visibles) at massive doses (1.0-2.0 M). In contrast, it proved to be genetically effective somatically, inducing red eye sectors through regulatory effects involving the TE in the UZ stock and complete reversion to w+ in the case of wi16 at comparatively moderate doses (less than or equal to 0.26 M). The possible implications of the demonstration of the genetic activity of NDELA in the soma to the carcinogenic process, and to the wider problem of screening for environmental genotoxic compounds, are discussed.

Differential induction of altered gene expression by carcinogens at mutant alleles of a Drosophila locus with a transposable element.

Alterations in gene expression by carcinogens were analyzed on three unstable alleles of the white (w+) locus of Drosophilia melanogaster: white-crimson (wc); white-ivory 16 (wi16); and white-unstable 11 (wu11). Two of these alleles (wi16 and wu11) were spontaneous mutant derivatives of wc, which is known to harbor a transposable element. The compounds studied were dimethylnitrosamine, 7,12-dimethylbenz(a)anthracene, and aflatoxin B1. These carcinogens were topically applied on the early larval stages, and the genetic effects assayed were the alterations in eye color either to wild-type (w+) or to other w mutants, initiated both somatically and germinally, as well as the simultaneously induced X-chromosome recessive mutations. The tested compounds influenced the different unstable w alleles in a highly selective manner, both as a function of the inducing agent and the organization of the genome in the target cells. The same treatments raised the somatic reversions to w+ above the corresponding controls for wc and wi16, but not for wu11, whereas the simultaneous induction of other w mutant phenotypes occurred appreciably only with wc. Furthermore, these treatments gave high and variable somatic reversions to w+ with wi16, whereas the simultaneously induced germinal events were uniformly very low. The frequencies of altered expression at the unstable test loci, whether in the soma or germ line, were quantitatively uncorrelated with the mutagenic effects of the treatments in terms of the yield of X-chromosome recessive mutations assayed in the progeny of males emerging from the same treated larvae. There was also an association between the time of the induction of these alterations by the tested carcinogens in the soma and the cellular stage in genomic differentiation. Reversions to w+ were induced preferentially after the onset of genetic determination, whereas changes to the w mutant phenotypes occurred predominantly during the predetermination phases. The genetic properties of transposable elements and the manner of their response to carcinogens supported the hypothesis that nonviral cancer might arise from molecular processes similar to those involved in the evolution of retroviruses.

Misregulation versus mutation in the alteration of gene expression by carcinogens through interactions with transposable elements in Drosophila melanogaster.

Carcinogens from different chemical series were tested on the wild-type allele of the complex white (w+) locus, which comprised some 5 recombinational subunits, the proximal part (w+4.5) exhibiting regulatory interactions with the neighbouring gene zeste (z). Three w+ loci were used, with different proximal regulatory sequences, including an unstable locus with a TE. Altered expression with each z w+ complex was assayed on the basis of the induction of aberrantly pigmented eye sectors known to be diagnostic of the interaction between z and the dosage of the functionally active w+4.5 subunits. All the tested carcinogens (DMN, DMBA and AFB1) were poorly active in the induction of the putative somatic deletions causing white (w-) eye sectors. In contrast, they were highly effective on the regulatory w+4.5 sequences in all test loci, as indicated by the significantly higher yield of red eye sectors (w-4.5) above the controls. However, this effect varied as a function of the chemical structure of the test compound and the genetic organisation of the regulatory targets. Germinal mutagenicity of the test compounds was assayed on X chromosomes carrying stable and unstable w+ loci, after the injection of adults and topical application on newly hatched larvae. Both techniques revealed that there was no association between the induction of somatic alterations in gene expression and the germinally induced mutations, including the TE w+4.5 deletions. Furthermore, the somatic events, unlike mutations, showed an association with the time of genetic determination during eye-disc cell differentiation. The present results were compatible with the concept of somatic gene misregulation by carcinogens.

Genetic activities of 4-chloromethylbiphenyl, the 4-hydroxy derivative and benzyl chloride in the soma and germ line of Drosophila melanogaster.

The genetic activities of 4CMB, 4HMB and BC were assayed as regards the induction of somatic alterations in gene expression on an unstable w+ locus with an intragenic TE and all the simultaneously induced germinal mutations on the X-chromosome carrying this locus. The compounds were applied topically in solution at equimolar doses on late embryos and newly hatched larvae. The somatic events were scored as aberrantly pigmented eye sectors in the emerging adult males and the germinal mutations in their F2 progeny, according to the Muller-5 technique. The somatic events were expressed as red or while mosaic eye sectors; the former could be an outcome of the repression or deletion of the zeste-regulatory proximal subunits of w+ locus, and the latter generally attributable to deletions (w-) within its structural part. All 3 compounds were effective in the induction of red sectors at the higher tested doses (0.5-2.0 mM) and the level of this activity was virtually the same for 4CMB and 4HMB, but was 2-fold higher for BC. In contrast, the frequencies of the simultaneously scored white sectors were not raised significantly above the controls with 4CMB, but showed decisive increases above this level with both 4HMB and BC. The germinal X-chromosome mutations (recessive lethals and visibles) were only induced at the highest tested dose (2.0 mM), and their frequencies were virtually the same for all 3 compounds reaching a common level of about 0.6%, which is some 3-fold the normal control level for the test system. Specific-locus mutability at the TE w+ was suggestively positive only with BC.

Mutagenicity of hair dye components relative to the carcinogen benzidine in Drosophila melanogaster.

A comparative assay was undertaken in Drosophila melanogaster for the assessment of the mutagenic efficiency of the hair dye components m-toluene-diamine (m-TD) and 4-nitro-o-phenylenediamine (4-NOPD) relative to the aromatic amine human carcinogen benzidine (Bzd). The compounds were injected at equimolar dose ranges (5-20 mM) around the testes of adult males and their mutagenicities were measured separately on the various stages of spermatogenesis. Genetic activity was simultaneously assayed with respect to the overall induction of the X-chromosome recessives (lethals and visibles) relative to the specific effects on rDNA (expressed as bobbed mutations). All compounds exerted decisive mutagenicity both on the X-chromosome and the RNA genes, although their activities on the different genic sites varied between compounds and as a function of cell stage, but not in response to changes in dose, within the investigated molarity range. The mutagenicities and selectivities of the test compounds for rDNA gradually decreased in the order Bzd greater than m-TD greater than 4-NOPD, which correlated with the evidence-so far-about their carcinogenicities.

Mutagenicity of N-alpha-acetoxyethyl-N-ethylnitrosamine and N,N-diethylnitrosamine in relation to the mechanisms of metabolic activation of dialkylnitrosamines.

The mutagenicities of N-alpha-acetoxyethyl-N-ethylnitrosamine and N,N-diethylnitrosamine were compared in Drosophila for an assessment of the role of alpha-carbon ethyl oxidation in the toxicological activation of the amine. The relative genetic potencies of the two compounds were deduced from regression studies of the mutation frequencies on molar dose for the whole testicular tissue with respect to the induction of the overall X-chromosome recessives (lethals and visibles), representatives of the RNA genes (especially ribosomal DNA), and six specific euchromatic loci. Genetic activity per unit molar dose was invariably higher for the acetoxy compound than for the parent amine, but the two agents produced comparable mosaicism among corresponding mutational classes. This indicated that the same mutagenic reactive species were generated from the two agents, but more readily from the acetoxy derivative, which accorded with the hypothesis of nitrosamine metabolic activation through alpha-carbon alkyl hydroxylation. The differentials for the higher activity of the acetoxy compound varied between mutational classes: 20-fold for the X-chromosome recessives collectively or per locus, but only 5-fold for the specific ribosomal DNA deletions. These relationships indicated that the mutagenicities of these compounds were not entirely an outcome of DNA ethylation but were readily explicable on the basis of the additional generation of difunctional nitrosaldehydic metabolites, which were largely responsible for the induction of the ribosomal DNA deletions through DNA protein cross-link-age. The higher mutagenicity of the acetoxy compound relative to the parent amine on ribosomal DNA was in part countered by its much lower selectivity index for these genes. The possible relevance of these genetic features to the carcinogenicities of these compounds was considered.

Mutagenic selectivity of carcinogenic nitroso compounds: III. N,alpha-acetoxymethyl-N-methylnitrosamine.

A comparative genetic study was undertaken on the testicular tissue of Drosophila with N-alpha-acetoxymethol-N-METHYLNITROSAMINE (AcODMN) and its unsubstituted parent N,N-dimethylnitrosamine (DMN), to assess the role of intracellular metabolism on their mutagenicities. The relative genetic potencies of the two compounds were deduced from regression studies of the dose effect on the metabolically inert sperm and the metabolizing early germ cells (spermatocytes and spermatogonia) with respect to the induction of the non-specific X-chromosome recessives (lethals and visibles) and the specific effects on representatives of the RNA genes, especially rDNA. Genetic activity per unit molar dose was invariably higher for the acetoxy derivative as compared to the parent amine, but the differential in this respect varied significantly for various mutational classes and as a function of the metabolic level in the target cells. The induction of point-mutations (X-recessives) increased with the level of intracellular metabolism with bothe compounds and this was more pronounced with the parent amine, which was in accordance with the DNA methylation mechanism. In contrast, the yield of the specific rDNA deletions was not markedly enhanced with the increased metabolic activity in the early germ cells, especially with the acetoxy derivative. The induction of these deletions could not, therefore, be explained on the basis of DNA methylation, but was reconcilable with the posible generation of a nitroso-aldehydic metabolite, which could effect DNA-protein cross-linkage within the genic nucleoproteins. The two test compounds gave comparable frequencies of mosaicism among corresponding mutations and the same rDNA selectivity index, which indicated identical molecular mechanisms of mutagenesis. The higher genetic potency of the acetoxy derivative as compared to the parent amine would thus be indicative of its greater yield of the same mutagenic metabolites. Carcinogenicity studies with the two compounds paralleled the mutagenicity results, which whould suggest that the same molecular mechanisms could well be responsible for the initiation of both phenomena.

Genetic properties of N-alpha-acetoxymethyl-N-methylnitrosamine in relation to the metabolic activation of N,N-dimethylnitrosamine.

A genetic study was undertaken in Drosophila with N-alpha-acetoxymethyl-N-methylnitrosamine, a precursor of the alpha-hydroxymethyl derivative of N,N-dimethylnitrosamine, to assess the role of alpha-carbon oxidation in toxicological activation. Genetic activity was measured for the whole testicular tissue with respect to the general response of the X chromosome (recessive lethals and visibles), as well as certain specific genic sites, including representatives of the RNA genes. The biological activity of the acetoxy compound proved to be considerably higher than that of the parent amine with respect to both cytotoxicity and mutagenicity. At low and equitoxic molarities of the 2 agents (0.1 to 1.0 and 1.0 to 10.0 mM, respectively), dose-dependence for all the investigated genetic functions followed identical patterns, which were best described by quadratic dose curves. However, the regression coefficients for the acetoxy derivative were at least 1 order of magnitude higher than the corresponding values for the amine, indicating a consistent level of mutagenic activation as a result of the alpha-acetoxymethyl substitution. At mutagenically equivalent doses, the 2 compounds gave statistically comparable frequencies of mosaicism among corresponding mutational classes and equal ribosomal DNA selectivity indices, indicating identical molecular mechanisms of mutagenesis. The higher mutagenicity of N-alpha-acetoxymethyl-N-methylnitrosamine compared to N,N-dimethylnitrosamine was paralleled by its higher carcinogenicity which would suggest that the same effective metabolites might be involved in both processes.

Mutagenic selectivity of carcinogenic nitroso compounds. II. N,N-dimethylnitrosamine.

The genetic properties in the hepatocarcinogen N,N-dimethylnitrosamine (DMN) were examined in Drosophila for the assessment of the role of dose, cellular metabolism and genic target in its mutagenicity. Genetic activity was assayed with respect to the induction of the non-specific X-chromosome recessives (lethals and visibles) relative to the effects on specific genic sites, especially rDNA, which yields bobbed (bb) mutations. Dosses and germ cell types, which indicated that DMN induced at least some multiple-hit mutagenic events. The genetic activity of DMN was favoured by cellular metabolism for all mutational classes, as was indicated by the progressive increase in mutational classes, as was indicated by the progressive increase in mutation yield during spermatogenesis--from the metabolically inert mature sperm to the actively metabolizing spermatocyte and spermatogonia. The role of DNA methylation in the mutagenicity of DMN was deduced from quantitative assays for its genetic activity relative to the methylating nitrosamide--N-methyl-N-nitrosourethane (MNUr)--over the same dose range (1-10 mM) and on identical cell types and genic targets. In the metabolically inert cells (mature sperm), the two compounds were equally active with respect to the non-specific effects (X-recessives), but MNUr, but the two compounds were equally effective on rDNA. These results could not be entirely interpreted by the methylation hypothesis and indicated that a DMN aldehydic metabolite, structurally analogous to MNUr, might be responsible for the induction of the rDNA mutations. The rDNA selectivity index of DMN was significantly lower than for MNUr, which paralleled their relative carcinogenic verstilities. However, DMN was comparatively more effective on the tRNA genes, a feature which might be associated with its oncogenic specificity.

Mutagenic selectivity of carcinogenic nitroso compounds 2. n-methyl-N-nitrosourethane.

The mutagenicity of the carcinogen methylnitrosourethane (MNUr) was examined in Drosophila with a view to the determination of its activity on heterochromatic loci (especially rDNA) relative to those in the euchromatin. Assays were made of the yield of rDNA mutations (bobbed: bb) relative to other X-chromosome recessive lethals and visibles [X(l + v)] in the same male germ cells after treatment with different doses (1-10 mM) and at various stages in spermatogenesis. Dose dependence followed the same pattern for all genic loci and germ cell stages. In all instances, the regression of mutation frequency on injected molar dose was approximately linear, but could better be described by a quadratic dose curve. In contrast, the mutagenicity pattern during spermatogenesis varied according to the target genes. The response of the euchromatic loci reached a peak among the earlier germ cells (probably the spermatocytes), whereas that for the heterochromatic sites (including rDNA) was maximal in mature sperm. Mutagenic selectivity for rDNA with MNUr, as indicated by the percentage bb/X-mutations, was among the highest for the intrinsically reactive carcinogens (alkylating and arylating agents). This correlates with the strong carcinogenicity of MNUr and adds further support to the concept that rDNA mutations might well be a crucial step in cancer initiation.