High Bioactivation Cross Research Articles

Recombinagenic activity of integerrimine, a pyrrolizidine alkaloid from Senecio brasiliensis, in somatic cells of Drosophila melanogaster

Integerrimine (ITR), a pyrrolizidine alkaloid from Senecio brasiliensis, was tested for genotoxicity using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. The compound was administered by chronic feeding (48 hours) of 3-day-old larvae. Two different crosses involving the markers flare (flr) and multiple wing hairs (mwh) were used, that is, the standard (ST) cross and the high bioactivation (HB) cross, which has a high cytochrome P450-dependent bioactivation capacity. In both crosses, the wings of two types of progeny were analyzed, that is, inversion-free marker heterozygotes and balancer heterozygotes carrying multiple inversions. ITR was found to be equally potent in inducing spots in a dose-related manner in the marker heterozygotes of both crosses. This indicates that the bioactivation capacity present in larvae of the ST cross is sufficient to reveal the genotoxic activity of ITR. In the balancer heterozygotes of both crosses, where all recombinational events are eliminated due to the inversions, the frequencies of induced spots were considerably reduced which documents the recombinagenic activity of ITR. Linear regression analysis of the dose response relationships for both genotypes shows that 85% to 90% of the wing spots are due to mitotic recombination. Environ. Mol. Mutagen 29:91–97, 1997 © 1997 Wiley-Liss, Inc.

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of the wing spot test in Drosophila melanogaster

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of the wing spot test in Drosophila melanogaster

Protection by coffee against somatic genotoxicity in drosophila: Role of bioactivation capacity

The protective effects of coffee against somatic mutation and mitotic recombination induced by cyclophosphamide (CPH), mitomycin C (MMC) and urethane (URE) were evaluated in the standard (ST) and high bioactivation (HB) crosses of the wing spot test in Drosophila melanogaster. These two crosses are characterized by different constitutive levels of cytochrome P-450-dependent enzyme activities. 3-day-old larvae transheterozygous for the wing cell markers mwh (multiple wing hairs) and flr 3 (flare 3) were fed until pupation on medium containing a genotoxin alone or its combination with different concentrations of instant coffee. Subsequently, the wings of the resulting adult flies were analysed for detecting single spots ( mwh or flr 3) originating from mutational or recombinational events as well as twin spots ( mwh and flr 3) originating exclusively from recombination. The results showed high sensitivity of the HB cross to URE. Co-administration of instant coffee was effective in exerting significant dose-related inhibitory effects on the genotoxicity of URE in the ST and the genetically susceptible HB cross. Similarly, coffee showed significant dose-related inhibitory effects on the genotoxicity of MMC in both crosses. The same protective effect was also observed with one concentration of coffee in combination with CPH. Pretreatment of 2-day-old HB larvae with coffee for 24 hr followed by treatment with URE was also effective in significantly reducing the induction of mutation and recombination. The magnitude of the protective effects of coffee against these three genotoxins was independent of the genotype of the larvae used for treatment, that is it was independent of the bioactivation capacity of these larvae. The study demonstrates the suitability of this assay for obtaining qualitative and quantitative data on the result of interactions among a genotoxin, an inhibitor of genotoxicity and bioactivation capacity of the host.

Genotoxic activity of environmentally important polycyclic aromatic hydrocarbons and their nitro derivatives in the wing spot test of Drosophila melanogaster

The genotoxicity of three polycyclic aromatic hydrocarbons (PAHs) and of three of their nitro derivatives was evaluated in the wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster. Two crosses were used, i.e. the standard cross (ST) and the improved high bioactivation cross (HB) which is characterised by an increased sensitivity to the genotoxic effects of promutagens and procarcinogens. Larvae trans-heterozygous for the two recessive wing cell markers multiple wing hairs ( mwh) and flare ( flr 3 ) were fed with the test compounds for 48 h. The wings of the surviving flies were analysed for the occurrence of single and twin spots. Naphthalene, 1-nitronaphthalene and 1,5-dinitronaphthalene proved to be more genotoxic in the HB cross than in the ST cross. Anthracene showed a clear genotoxic activity only in the HB cross whereas it was negative in the ST cross. 9-Nitroanthracene gave inconsistent results in both crosses. Phenanthrene was negative in the ST cross, but weakly positive in the HB cross. These results demonstrate that the genotoxic activity of these PAHs and their nitro derivatives can be detected with the somatic cells of the wing imaginal discs of larvae with high bioactivation capacity.

Tannic acid is not mutagenic in germ cells but weakly genotoxic in somatic cells of Drosophila melanogaster

Tannic acid (TA) was tested for genotoxic activity in three different assays (1-3) in Drosophila melanogaster by feeding of larvae or adult flies. TA did not induce sex-linked recessive lethals (1) nor sex-chromosome loss, mosaicism or non-disjunction (2) in male germ cells. In the wing somatic mutation and recombination test (SMART) (3) TA was found to be toxic for larvae of the high bioactivation cross and produced a weak positive response. These results suggest that this compound, when administered orally to larvae or adults of D. melanogaster, is not mutagenic and clastogenic in male germ cells, but weakly genotoxic in somatic cells of the wing imaginal disk.

Improved high bioactivation cross for the wing somatic mutation and recombination test in Drosophila melanogaster

The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Frölich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibit a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross ( ORR; flr 3 females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethyl-benz[ a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.

Yeast recombination assays for genotoxicity testing

The present study evaluated the mutagenic and recombinogenic effects of two commonly used anesthetic agents, ketamine and rocuronium bromide, in medicine using the wing somatic mutation and recombination test (SMART) in Drosophila. The standard (ST) cross and the high-bioactivation (HB) cross with high sensitivity to procarcinogens and promutagens were used. The SMART test is based on the loss of heterozygosity, which occurs via various mechanisms, such as chromosome loss and deletion, half-translocation, mitotic recombination, mutation, and non-disjunction. Genetic alterations occurring in the somatic cells of the wing's imaginal discs result in mutant clones in the wing blade. Three-day-old trans-heterozygous larvae with two recessive markers, multiple wing hairs (mwh) and flare (flr3), were treated with ketamine and rocuronium bromide. Analysis of the ST cross indicated that ketamine exhibited genotoxicity activity and that this activity was particularly dependent on homologous mitotic recombination at concentrations of 250 μg/ml and above. Rocuronium bromide did not exert mutagenic and/or recombinogenic effects. In the HB cross, ketamine at a concentration of 1000 μg/ml and rocuronium bromide at all concentrations, with the exception of 250 μg/ml (inconclusive), exerted genotoxic effects, which could also be associated with the increase in mitotic recombination.

New tester strains with improved bioactivation capacity for the Drosophila wing-spot test

In Drosophila melanogaster new tester strains for the somatic mutation and recombination test (SMART) in the wing were constructed with the aim of increasing the metabolic capacity to activate promutagens. Some aspects of the genetic control of the xenobiotics metabolism in Drosophila are already known. In the DDT-resistant strain Oregon R(R) the RI gene at position 65.0 on chromosome 2 is responsible for the high constitutive expression of cytochrome P-450-dependent activities typical for this strain. Therefore, chromosomes 1 and 2 in the original mwh (multiple wing hairs) and flr 3 (flare 3) tester strains were substituted for chromosomes 1 and 2 from the Oregon R(R) strain. In assays with the model promutagen diethylnitrosamine an increased sensitivity of about 2.5-fold was found for this new set of ‘HB’ strains (‘High Bioactivation (HB) cross’).

3175. Teratogenicity of ethylene thiourea and related compounds: Ruddick J. A., Newsome W. H. & Nash L. (1976). Correlation of teratogenicity and molecular structure: ethylenethiourea and related compounds. Teratology13, 263

The wing spot test in Drosophila melanogaster is a suitable system for the analysis of genotoxic activity of compounds that need metabolic transformation to render them active. We have analysed the genotoxicity of three fungicides for which it was reported that the metabolic processes taking place in vivo may determine their activity. The compounds analysed are captan, maneb, zineb and ethylenethiourea (ETU) (a metabolic derivative of ethylenebisdithiocarbamates like maneb and zineb). We have also evaluated the ability of ETU to form genotoxic derivatives in vivo analysing this compound in combined treatments with sodium nitrite. Both standard and high bioactivation NORR strains have been used. Captan, usually considered a mutagen in vitro but a non-mutagen in vivo, gave negative results in the wing spot test with both crosses. Positive results were obtained for maneb in the standard cross and for ETU in both the standard and the high bioactivation cross. The genotoxicities of maneb and ETU were higher when treatments were made on media in which nitrosation is favoured. A low absorption of the fungicide and an inefficient availability of the compound in the target may explain negative results obtained with zineb in both crosses. The results obtained in this study with the wing spot test demonstrate once again the suitability of this in vivo assay, in which absorption, distribution and metabolism processes take place, for the evaluation of genotoxicity of compounds to which humans are exposed.

Comprehensive evaluation for mutagenic activity of dieldrin

Among the great variety of genotoxicity assays available, the wing spot test in Drosophila melanogaster has some characteristics that make it very suited for the screening of genotoxic activity, i.e., it is an easy and inexpensive assay using a eukaryotic organism in vivo. One of the most interesting characteristics of the assay is its capacity to detect genotoxic activity of promutagens without the necessity of an exogenous metabolic activation system. In this paper we present results obtained with a recently developed high bioactivation cross of the wing spot test (NORR cross). The positive results obtained with the five well-known procarcinogens 7,12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, p-dimethylaminoazobenzene, diethylnitrosamine and urethane clearly show that the NORR strains are similar to the other high bioactivation strains previously described, but they lack their methodological disadvantages. We have tested six insecticides, which are characterised by having contradictory results in other genotoxicity tests, using both the standard and the high bioactivation (NORR) cross. The six insecticides analysed are the pyrethroid allethrin, the methylenedioxyphenolic compound piperonyl butoxide, the chlorinated hydrocarbons dieldrin and endrin, and the organophosphates dimethoate and malathion. We obtained negative results for all six compounds. Our results show the suitability of the wing spot test for the evaluation of compounds at the first level of genotoxicity testing.