The results of assays in Drosophila as indicators of exposure to carcinogens.

Drosophila has fulfilled a dual function in the field of genetic toxicology: for use in short-term tests for identifying carcinogens and in a model for studies of the mechanisms of mutagenesis by chemicals. Until the mid-1980s, use of Drosophila in short-term tests was restricted to assays for genetic damage in germ cells, mostly in males. The largest database, on 700-750 chemicals, is available for the test for sex-linked recessive lethal (SLRL) forward mutation. The database for assays of the consequences of chromosomal breakage--reciprocal translocations and chromosome loss--is smaller, with about 100 chemicals tested. Comparative studies conducted within the US National Toxicology Program showed that SLRL is a better end-point than reciprocal translocation: of 66 chemicals (68 entries) that induced SLRL, only 28 (41%) induced reciprocal translocation. The major weakness of the SLRL assay is its low sensitivity (0.27-0.79) for mammalian genotoxins. A strength of the SLRL mutation test is its high specificity, which is close to 1. Thus, whereas a negative response in Drosophila provides little evidence for genotoxicity, a positive response (SLRL frequency > or = five times the control level) provides good evidence that a chemical is a trans-species mutagen and probably also carcinogenic to mammals. The poor performance of the SLRL test revealed in several collaborative studies led to the development of assays for recombination in somatic cells of Drosophila. Two of these tests have been evaluated for all known classes of genotoxic chemical: the mwh/flr wing spot test on more than 400 chemicals and the white/white+ eye spot test on about 220 chemicals. Of 24 carcinogens that gave negative or inconclusive test results in the SLRL assay, 22 gave positive results in one or both of the somatic systems. Their better performance in comparison with the germ-line assays is primarily the result of their low cost (5-10% of that needed for an SLRL assay), allowing use of multiple doses and protocols and the use of distinct tester strains with heterogeneity for activation of procarcinogens. For qualitative and quantitative studies on structure-activity and activity-activity relationship, only germ-line system have been used. In general, clear relationships between physico-chemical parameters (s values, O6/N7-alkylguanine ratios), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (from mice and Drosophila) became apparent when the following descriptors were used: (1) estimates of TD50 (lifetime doses expressed in milligrams per kilogram body weight or millimoles per kilogram body weight) from bioassays for cancer in rodents; (2) the degree of germ-cell specificity, i.e. the ability of a genotoxic agent to induce mutations at practically any stage of development of Drosophila and mouse spermatogenesis, as opposed to a more specific response in postmeiotic stages of both species; (3) the M(NER-)/M(NER+) hypermutability ratio, determined in a repair assay in Drosophila germ cells; (4) the ratio of chromosomal aberrations to SLRL in postmeiotic germ cells of Drosophila, i.e. the comparative efficiency of a carcinogen to induce these two end-points; (5) mutational spectra induced at single loci, i.e. the seven loci used in the specific-locus test in mice and the vermilion, white and rosy genes of Drosophila; and (6) the doubling doses in milligrams or millimoles per kilogram for specific locus induction in mice. On the basis of these parameters, alkylating agents were classified into three categories in terms of germ-cell specificity, which is primarily due to stage-related differences in DNA repair, clastogenic efficiency, type of mutation spectra and carcinogenic potency in rodents. The three categories allow predictions of the genotoxicity of alkylating agents but not yet for other categories of genotoxic carcinogens.

Influence of nucleotide excision repair and of dose on the types of vermilion mutations induced by diethyl sulfate in postmeiotic male germ cells of Drosophila.

The role of a defect for nucleotide excision repair (NER) in oocytes on the repair of DNA ethyl adducts induced by diethyl sulfate (DES) in male germ cells of Drosophila was analysed. Frequencies of mutations at multiple loci (recessive lethal mutations) and at the vermilion gene induced in NER+ conditions (cross NER+ x NER+) were compared with those fixed in a NER- background (NER- x NER+). The M(NER-)/M(NER+) mutability ratios for two DES concentrations, 10 mM and 15 mM, were 2.21 and 1.49, respectively, indicating that NER repairs part of the DES-induced damage. The majority of 28 fertile vermilion mutations produced by DES in NER- are transitions, both GC-AT (46.4%) and AT-GC (21.4%) transitions are found, the consequences of O6-ethylguanine and O4-ethylthymine, respectively. Transversions (21.5%), one +1 frameshift mutation (3.6%) and two deletions (7.1%) are most likely the result of N-alkylation damage. Furthermore, the DES-induced mutation spectra show interesting differences in relation to the exposure dose. All 10 mutants isolated in this and a previous [L.M. Sierra, A. Pastink, M.J.M. Nivard, E.W. Vogel, DNA base sequence changes induced by DES in postmeiotic male germ cells of Drosophila melanogaster, Mol. Gen. Genet. 237 (1993) 370-374] study from experiments with low DES-effectiveness are exclusively transitions, independent whether the females were of the NER+ or NER-genotype. This indicates that at lower DES effectiveness only O-alkylation damage is relevant, and that N-alkylation damage is repaired. In experiments revealing high DES-effectiveness, vermilion mutations representing N-alkylation damage reached 43% (9/21) with NER- and 26% (7/27) with NER+ females, suggesting (i) that NER becomes involved at high adduct levels because then the base excision repair (BER) may be saturated, and (ii) that this involvement of NER causes the relative decrease from 43% to 26% N-alkylation mediated sequence changes.