Biotransformation of several structurally related 2B compounds to reactive metabolites in the somatic w/w+ assay of Drosophila melanogaster.

Biotransformation of several structurally related 2B compounds to reactive metabolites was evaluated in the somatic w/w+ assay of Drosophila melanogaster. Chemicals tested were the dichlorinated alkanes dichloromethane (DCM), 1,2-dichloroethane (DCE), and 1,3-dichloropropane (DCP); the thiouracil derivatives 5-methyl, 2-thiouracil (5M2TU), 6-methyl, 2-thiouracil (6M2TU), and 5-propyl, 2-thiouracil (5P2TU); and the plastic monomer styrene (STY) and its metabolite styrene 7,8-oxide (SO). The tester strains used consisted of one wild-type insecticide-susceptible (IS) laboratory strain (Leiden-S, ST), and two insecticide-resistant (IR) strains (Hikone-R, HK, and Haag-R, HG). The latter have high cytochrome P450-dependent bioactivation capacities. Drosophila larvae heterozygous for the wild-type report gene w+ were exposed chronically to at least three different exposure doses of each compound. A total of 53,694 eyes were analyzed. A positive genotoxic activity was obtained for DCM and for 6M2TU at all exposure doses and genotypes analyzed, and for SO in the IR strains HK-R and HG-R. An overall weakly recombinagenic response was shown by DCE and 5M2TU. The chemicals DCP, 5P2TU, and STY proved to be overall negative in IR as well as in IS strains, and SO was negative in the standard stock. Biotransformation mediated by cytochrome P450 monoxigenases to reactive metabolites is discussed.

Activity of aromatic amines in the eye: w/w+ somatic assay of Drosophila melanogaster.

Aromatic amines represent a category of classical environmental hazards which require biotransformation in order to exert their mutagenic and carcinogenic effects. The mutagenic activity of several aromatic amines was tested by means of the w/w+ somatic assay of Drosophila melanogaster employing a wild-type strain Leiden Standard (LS) and an insecticide-resistant stock Hikone-R (HK-R). Four monocyclicortho-anisidine (O-AN), ortho-toluidine (O-TO), 2,4-diaminoanisole (2,4-DAA) and 4-nitro-o-phenylenediamine (4-N-o-PDA), and one bicyclic- 4,4'-oxidianiline (4,4'-ODA) aromatic amines were tested by chronic exposure. Two separate experiments were run, and concurrent controls were treated with the solvent mixture. Results show that the ortho-anilines (O-AN and O-TO) were active in the assay, while the diamines containing a methoxy (2,4-DAA) or a nitro group (4-N-o-PDA) were inactive. The bicyclic aromatic amine, 4,4'-ODA, was the most potent. The genotoxic responsiveness of both strains was very similar.

Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster.

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds.

Genotoxic activation of hydrazine, two dialkylhydrazines, thiourea and ethylene thiourea in the somatic w/w + assay of Drosophila melanogaster.

Genotoxic activation of hydrazine (HZ), two symmetrical dialkylhydrazines, namely, 1,2dimethylhydrazine and 1,2-diethylhydrazine (SDMH and SDEH), thiourea (TU) and ethylene thiourea (ETU) has been evaluated by means of the w/w + somatic assay of Drosophila. Both low bioactivation insecticide-susceptible (IS) and high biotransformation insecticide-resistant (IR) strains were used. The combined application of insecticide-susceptible and insecticide-resistant strains should, in principle, detect somatic cell recombinagens in the Drosophila melanogaster in vivo w/w + assay. The IS strain was more susceptible to toxicity induced by the test chemicals than the IR stocks. Its performance in the biotransformation of the chemicals tested was rather poor. TU was inactive in all strains. With the active compounds, spot frequencies increased approximately linearly with dose for each spot type. SDEH gave a strong positive result in all three female genotypes exposed. HZ, ETU and SDMH were overall weakly positive in the IR strain Haag-79 (HG-R). Interestingly, ETU was clearly positive in the IR Hikone-R (HK-R) strain. A comparison of the recombinagenic potencies between the active and the weakly positive compounds, and among strains, showed pronounced genotype-dependent differences between the low and the high bioactivation strains. The ability of Drosophila to express several procarcinogens in relation to insecticide-resistance after activation catalyzed by CYP450 enzymes is discussed.

Mutagenicity of nickel sulphate in Drosophila melanogaster.

Nickel sulphate was injected into Drosophila melanogaster males at different concentrations in order to test the chemical for the induction of SLRL and SCL in germ cells. Nickel sulphate induced SLRL at concentrations tested, with the peak of activity at premeiotic and postmeiotic stages. It failed to produce SCL except at the highest concentration tested, where induction of XO males was significant for the pooled data.

Metabolic activation of four drugs in the eye mosaic assay measuring principally mitotic recombination in Drosophila melanogaster: differences in strain susceptibility and route of exposure.

One mycotoxin and three therapeutic drugs widely used in developing countries were examined for genotoxic activity by means of the w/w + somatic recombination assay. Streptozotocin (SZ), an antibiotic antineoplastic agent, gave a frequency of light spots almost one order of magnitude higher than those obtained with the carcinogen mycotoxin sterigmatocystin (STC), the antiprotozoal and antimicrobial metronidazole (MNZ), and the antifungal griseofulvin (GF). Thus the order of response was SZ > STC > MNZ > GF. Chronic treatment turned out to be the better route of exposure for these genotoxins when compared with surface treatment. The performance of the insecticide-resistant strain Hikone-R was better than that of the wild genotype LS (Leiden Standard). The positive test results obtained with all four chemicals showed that the P450 system of Drosophila is capable of metabolizing these genotoxins into electrophilic intermediates.

Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells.

The mutagenic potential of furfural was evaluated by means of the chromosome loss test in germ cells and the wing spot test in somatic cells of Drosophila melanogaster. The chromosome loss test was carried out employing repair-proficient as well as repair-deficient females. Males carried the compound Y chromosome, BSYy+. Two routes of administration were used: injection and feeding of adult males. Genetic damage was demonstrable after matings of treated males with females carrying the excision repair-deficient mutant mei-9a. The somatic mutation and recombination test was carried out treating 72-h transheterozygous mwh+/+flr3 larvae. Acute treatment of larvae was chosen as the method of exposure. Evidence indicates that furfural induces somatic damage as measured in the wing spot test.

Analysis of mitotic recombination induced by several mono- and bifunctional alkylating agents in the Drosophila wing-spot test.

Mitotic recombination induced by six alkylating agents has been studied in the wing-spot test of Drosophila melanogaster. The model mutagens chosen have different models of action at the DNA level. These are: the direct-acting small alkylating agent methylmethanesulfonate (MMS), the small promutagens N-dimethylnitrosamine (DMN) and N-diethylnitrosamine (DEN), the bifunctional cross-linking alkylating agents mitomycin C (MMC), chlorambucil (CLA) and monocrotaline (MCT). Flies of the standard cross (flr3 / TM3, Bds females and mwh males) were used to produce the larvae to be treated. Three-day old Drosophila larvae were exposed by chronic feeding for 48 h to three different concentrations of all six alkylating agents. Acute feeding for only 2 h was used in addition with DEN and MMC. Wings of the marker-heterozygous (mwh+ / + flr3) as well as of the balancer-heterozygous (mwh+ / TM3, Bds) progeny were analysed. The ranking of the compounds with respect to their genotoxic potency, based on mwh clone formation frequency in marker-heterozygous wings was: MMS > MNC > DMN > CLA approximately MCT > DEN. The ranking with respect to the induction of twin spots, which are produced by mitotic recombination exclusively, was: MMS > DMN > MMC > MCT > CLA > DEN. The quantitative determination of recombinagenic activity, based on mwh clone formation frequencies obtained in both types of wings, gave the following values: MMS, 93%; MCT, 87%; CLA, 80%; MMC, 73%; DMN, 67%; DEN, 22%. A clear relationship exists between the extent of N-alkylation of DNA and the efficiency of the monofunctional agents MMS and DMN as well of the bifunctional agents MCT, CLA and MMC to induce mitotic recombination. This contrasts with the ethylation of base oxygen atoms and the resulting lower efficiency of DEN to produce mitotic recombination.

Evidence for the absence of mutagenic activity of furfuryl alcohol in tests of germ cells in Drosophila melanogaster.

Furfuryl alcohol was evaluated for mutagenic activity in D. melanogaster by means of the sex-linked recessive lethal test and the sex-chromosome loss test. Brooding was employed in order to test different stages of spermatogenesis. No evidence was found of a mutagenic effect after adult injection and larval feeding.

Interactions of sulforaphane and dimethyl sulfoxide with methyl methanesulfonate, urethane, 4-nitroquinoline-1-oxide and hydrogen peroxide in the Drosophila melanogaster wing spot test.

Sulforaphane (SF) is an isothiocyanate present in Brassicaceae, vegetables that induce the detoxification of electrophiles and reactive oxygen species. SF has been correlated with chemoprevention mechanisms against degenerative diseases. We tested if the SF had an effect against methyl methanesulfonate (MMS), urethane (URE), 4-NQO and H(2)O(2). SF (>95% purity, 0.14, 0.28, 0.56 mM) was diluted in a DMSO/Tw80/EtOH mixture (DTE) corresponding to 25, 50, 100% of lyophilized broccoli. The SF treatment (0.14 mM) was positive for small spots in the ST cross and negative in the HB cross. In the HB cross, SF (0.28 mM) was genotoxic. In the ST cross, the SF treatments showed a tendency to reduce the genotoxic damage caused by MMS, which could be explained by the radical scavenging action of the DTE mixture. In the ST cross, the frequency of small spots in the SF 0.14 mM/URE treatment was similar to that of Water/URE, which can be explained by a DTE and SF scavenger action. In both crosses, the results for the direct oxidants, 4-NQO and H(2)O(2), were different and must be related to differential modulation of CYPs expression and the SF and DTE scavenger properties.

Strong intra-species variability in the metabolic conversion of six procarcinogens to somatic cell recombinagens in Drosophila.

Genetic heterogeneity in response to genotoxic carcinogens requiring metabolic conversion has been studied in Drosophila, using seven different genotypes in combination with the w/w+ eye mosaic assay for mitotic recombination. The set of tester strains examined consisted of four wild-type laboratory strains (Berlin-K, BK; Oregon-K, OK; Leiden-S, LS; and 91-C), and three DDT-resistant strains (91-R; Hikone-R, HR; and Haag-79, HG). Drosophila larvae heterozygous for the wild-type report gene w+ were exposed to benz[a]anthracene (BA), benzo[a]pyrene (BP), 9,10-dimethyl-anthracene (DA), monocrotaline (MC), N-nitrosodimethylamine (DMN) or vinyl bromide (VBr). The primary conclusion regarding this study is the up to 60-fold variation between different genotypes in mosaic spot frequencies induced by those procarcinogens and the non-existence of just one genotype to function as a 'super-strain' in the activation of different classes of procarcinogens. By contrast, the seven Drosophila strains show a similar response to the direct-acting agent methyl methanesulphonate. Among the six procarcinogens selected for this study, DMN and MC are readily detectable in all the distinct genotypes. Less satisfactory in terms of test performance are the either negative or weak test responses with BA in all four-wild type strains (LS, BK, OK and 91-C), and the weakly positive results for vinyl bromide in LS, BK and OK. The best overall test response was provided by strains HG and HR. It is concluded that the combined application of strains HG and HR safely and reliably should detect somatic cell recombinagens in the Drosophila w/w+ system.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450 gene clusters in Drosophila melanogaster.

Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.