Effects of ionizing radiation and caffeine treatment on cyclin dependent kinase complexes in V79 hamster cells.

Exponentially growing V79 Chinese hamster lung fibroblasts irradiated with 7 Gy X-rays undergo cell cycle arrest in the S and G2 phases. These arrests are released, probably on completion of DNA repair. A premature release occurs after treatment of irradiated cells with caffeine. This release is accompanied by increased activity of the p34cdc2 serine/threonine protein kinase complex [Hain et al. (1993) Cancer Res. 53, 1507-1510]. We have investigated in V79 cells whether the association of p34cdc2 with its regulatory subunits cyclin A and B is affected by irradiation and subsequent caffeine treatment and found that this was not the case. The phosphorylation of p34cdc2 as assayed by mobility shift on SDS polyacrylamide gels was increased as early as 0.5 h after irradiation and decreased after subsequent caffeine treatment. A novel protein p40, detected with anti-PSTAIRE antibodies, appeared several fold more abundant than p34cdc2. Its phosphorylation state also changed after irradiation and after subsequent caffeine treatment.

Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes.

PURPOSE: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. METHODS AND MATERIALS: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. RESULTS: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r = 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r = 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen-ebb. Antigen-ebb was clearly observed in both cell types, and correlated significantly with cytotoxicity. A trend was observed between radiosensitivity and donor age, but there was no correlation for gender. Blood from a 4-year-old girl presenting with ICF demonstrated compromised radiation-induced cytotoxicity in her CD4 T-lymphocytes, and an 11-year-old boy presenting with AT demonstrated compromised radiation-induced cytotoxicity in both his CD4 and CD8 T-lymphocytes. CONCLUSION: We conclude that the assay provides a rapid means of determining radiosensitivity, can discriminate differences in radiation-induced cytotoxicity between individuals, and can be used as a rapid screen for genetically hypersensitive patients. Antigen-ebb offers interesting possibilities for molecular biological investigations, permitting characterization and isolation of abnormal but vital cells in the absence of clastogenic agents.

Heterologous expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae. Study of the valpromide-carbamazepine epoxide interaction.

A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity. The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected. Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide. A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate. For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes. Our results demonstrate the potential of heterologous gene expression in S. cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.

Predicting mammalian mutagenesis by submammalian assays: an application of database GEN.

A database containing qualitative information on the genotoxic activity of about 3000 chemicals is described. The initial aim for the construction of the database was to develop an instrument for comparing the performance of different genotoxicity assay systems. One application of the database is the prediction of expected results in any genotoxicity assay for chemicals that were tested in a small number of genotoxicity assays. The Bayesian prediction is calculated based on the sensitivities and specificities between any predictive test and the target test for which the prediction is to be determined. The predictivity of the system for in vivo mammalian assays is at present (with the exception of the micronucleus assay and the in vivo sister chromatid exchanges) limited, in particular because of the limited number of chemicals tested in the expensive in vivo assays and, in addition, due to the lack of sufficient information on negative compounds. A continued updating of the database will possibly help to overcome some of the present difficulties.

Functional expression of fused enzymes between human cytochrome P4501A1 and human NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae.

The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein. Anchoring of the fusion constructs in internal membranes either by the amino terminus of hOR or by the yeast peptide resulted in functional hybrid proteins, which were present in similar amounts as the authentic CYP1A1 in microsomal fractions of recombinant cells. Saccharomyces cerevisiae cells transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-CYP1A immunoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cpr1-) and wild-type (CPR1+) cells containing the fused enzymes exhibited CYP1A1-specific 7-ethoxyresorufin-O-deethylase activities. Reduced CO-difference spectra of microsomal fractions containing the fused enzymes indicated a proper incorporation of protoheme into the CYP1A1 domains. These results show that the chimeric proteins represent catalytically self-sufficient monooxygenase systems. The hOR domains of the hybrid proteins were also functional as cytochrome c reductases and able to activate the yeast P450 enzyme lanosterol-14 alpha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.

The somatic white-ivory eye spot test does not detect the same spectrum of genotoxic events as the wing somatic mutation and recombination test in Drosophila melanogaster.

A groups of six chemical compounds was tested in parallel in two different somatic genotoxicity assays in Drosophila melanogaster, the wing somatic mutation and recombination test (SMART) and the white-ivory eye spot test. The wing spot test makes use of the wing cell markers multiple wing hairs (mwh) and flare (flr) and detects both mitotic recombination and various types of mutational events. The white-ivory eye spot test makes use of the white-ivory (wi) quadruplication and detects the somatic reversion of the recessive eye color mutation wi to the wild-type (w+). Three- or two-day-old larvae were fed chronically with the compounds ethylnitrosourea (ENU), N-nitrosopyrrolidine (NNP), caffeine (CAF), chromium (VI) oxide (CRO), potassium chromate (POC), and 2,4-dichlorophenoxyacetic acid (2,4-D). All six compounds are genotoxic to various degrees in the wing spot test. The percentage of the genotoxic activity that is due to mitotic recombination was between 84% and 91% for the hexavalent chromium compounds CRO and POC and about 68% for 2,4-D. In contrast, ENU and NNP showed only 46% and 25% recombinagenic activity, respectively. In the white-ivory eye spot test, the three compounds (CRO, POC, and 2,4-D) with high recombinagenic activity and CAF were clearly nongenotoxic, whereas only ENU and NNP gave a positive response. From these results, it is concluded that the spectrum of genotoxic events detected by the two assays is different. In particular, the white-ivory eye spot test appears not to detect mitotic recombination the way the wing spot test does.

Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters.

Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M. This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII. The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment. The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9). Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin. Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments. In the case of the positive experiment, DES also induced putative recombinants. Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane). From our experiments it can be concluded that the hypothesis put forward by Parry et al. [Nature; 294:263-265], according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense. In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay.

We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems. Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM). Diethylstilboestrol (DES) showed solvent-dependent effects. DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM). Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM). Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM). Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols. The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix. The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol. Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process. The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively. Mercaptoethanol showed no effects up to 50 mM.

Reciprocal mitotic recombination is the predominant mechanism for the loss of a heterozygous gene in Saccharomyces cerevisiae.

The loss of a functional copy of a heterozygous tumor suppressor gene represents an important step during neoplastic transformation. In order to learn more about the genetic events that lead to spontaneous and drug-induced loss of heterozygosity, a diploid Saccharomyces cerevisiae strain was constructed that allows the detection of the loss of a heterozygous gene by means of direct selection. The strain contains a single functional URA3 gene copy inserted at the ADE2 locus located on the right arm of chromosome 15. In addition, the chromosome contains two other phenotypic marker genes, HIS3 which is located distal from URA3, and PHO80 which is closely linked to the centromere. The homologous chromosome lacks all three marker genes. Loss of the heterozygous copy of URA3 can easily be detected by 5-fluoro-orotic acid resistance of the resulting clones. Simple phenotypic tests of the resistant clones further allows one to distinguish whether the loss of the URA3 gene copy occurred by crossing over, chromosomal loss, or point mutation and gene conversion. Loss of heterozygosity was found to be induced in a dose-dependent fashion by UV radiation and by several chemical agents. All the tested mutagens induced loss of heterozygosity predominantly by crossing over.

Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae.

The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr3) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strain at higher levels than in the insecticide-sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH-cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B1 to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7,12-dimethylbenz[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), were metabolized in the transformed strain to cytotoxic products.

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Effects of chemical and physical agents on recombination events in cells of the germ line of male and female Drosophila melanogaster.

Genotoxic agents can induce mutations as well as recombination in the genetic material. The fruit fly Drosophila melanogaster was one of the first assay systems to test physical and chemical agents for recombinogenic effects. Such effects can be observed in cells of the germ line as well as in somatic cells. At present information is available on 54 agents, among them 48 chemicals that have been tested in cells of the germ line of males and/or females. Effects on meiotic recombination in female germ cells cannot simply be classified as positive or negative since for a number of agents, depending on the chromosome region studied, recombination frequencies may be increased, unaffected or decreased. The male germ line of D. melanogaster represents a unique situation because meiotic recombination does not occur. Among 25 agents tested in male germ cells 24 did induce male recombination, among them alkylating, intercalating and cross-linking agents, direct-acting ones as well as compounds needing metabolic activation. With several compounds the frequency of induced recombination is highest in the heterochromatic regions near the centromeres. In brood pattern analyses, e.g., after exposure of adult males to ionizing radiation, the first appearance of crossover progeny is indicative of the sampling of exposed spermatocytes. In premeiotic cells of the male and the female germ line mitotic recombination can occur. Upon clonal expansion of the recombinant cells, clusters of identical crossovers can be observed.

Statistical methods to decide whether mutagenicity test data from Drosophila assays indicate a positive, negative, or inconclusive result.

Two alternative hypotheses are used to distinguish among the possibilities of a positive, inconclusive, or negative result in Drosophila mutagenicity tests. In the null hypothesis one assumes that there is no difference in the mutation frequency between control and treated series. The alternative hypothesis postulates a priori that the treatment results in an increased mutation frequency that is m times the spontaneous frequency. To test against the hypotheses, the conditional binomial test according to Kastenbaum and Bowman or the chi 2 test for proportions may be applied. These 2 methods are in principle equivalent. An alternative method which is based on determining confidence limits of observed mutation frequencies also leads to the same conclusions. The practical calculations are formulated and an application is shown with a test example demonstrating the genotoxicity of the pyrrolizidine alkaloid 7-acetylintermedine in the somatic wing mosaic test. In the Appendix, the calculus for the 3 testing methods is explained with a numerical example.

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 flr3 (flare3) 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').

The vicinal chloroalcohols 1,3-dichloro-2-propanol (DC2P), 3-chloro-1,2-propanediol (3CPD) and 2-chloro-1,3-propanediol (2CPD) are not genotoxic in vivo in the wing spot test of Drosophila melanogaster.

In this study, the vicinal chloroalcohols 1,3-dichloro-2-propanol (DC2P), 3-chloro-1,2-propanediol (3CPD) and 2-chloro-1,3-propanediol (2CPD) were investigated for genotoxicity in the wing spot test of Drosophila. DC2P is an important starting material in many processes of synthesis in chemical industry. 3CPD as well as some related glycerol chlorohydrins were identified in protein hydrolysates industrially used for the production of food items such as seasonings, sauces and soups. The wing spot test is a somatic mutation and recombination test (SMART) and is a sensitive in vivo assay for the detection of mutagens and promutagens. The test was applied here in its standard version with normal bioactivation and in a variant with increased cytochrome P450-dependent bioactivation capacity. All three compounds were clearly non-genotoxic in these in vivo assays. The results are in agreement with recent findings which strongly suggest that positive genotoxicity results in in vitro testing of vicinal chloroalcohols such as DC2P are due to directly acting genotoxic intermediates arising from a chemical reaction with the culture medium rather than from enzymatic biotransformation.

Knowledge-based battery design of short-term tests based on dose information.

A construction of batteries of short-term tests (STTs) is described which is based on a classification of 73 chemicals in regard to their carcinogenicity. The 73 chemicals were studied within the U.S. National Toxicology Program (Ashby and Tennant, 1988). The batteries are validated using the classification of 35 additional chemicals. They are defined by logically structured combinations of rules. The single rules are defined by the z-scores of the logarithmic values of the limiting doses obtained from the 4 in vitro STTs used in the study by Ashby and Tennant. The limiting dose is defined as the lowest effective dose or the highest ineffective dose (Waters et al., 1987). The batteries are constructed by minimizing the number of disagreements with the classification by Ashby and Tennant. Compared with the results obtained from single STTs, 2 batteries of 3 STTs have higher concordances with the carcinogenicity data, namely 70% for the NTP data and 74-77% for the independent test data. In addition, a theoretical result shows that the proposed battery design, for a large enough learning set of chemicals, leads to results which are replicated with high probability on a large enough validation set. Based on the first results obtained with a limited number of chemicals it is concluded that the knowledge-based battery design is worth further development.

A genetic assay to detect chromosome gain and/or loss in somatic cells of Drosophila melanogaster.

A genetic short-term test is described that allows (i) detection and (ii) quantitative evaluation of aneuploidy induced in somatic cells of Drosophila melanogaster. In this somatic aneuploidy test (SAT) larvae of the genotype z w-/w+J Y are exposed to the test compound. Gain and/or loss of the w+J Y chromosome leads to the formation of aneuploid daughter cells: z w-/w/J Y/w+J Y and z w-/O, respectively. These cells are fully viable, proliferate and, when they are part of an eye primordium, form a yellow//white twin spot on the otherwise red background after metamorphosis. The number of eyes screened, the size and number of spots allow for a quantitative estimate of the frequency of induced aneuploidy. Induced aneuploidy was detected after exposure of larvae to X-rays and to vincristine. The somatic aneuploidy test seems to be a simple, sensitive and fast method to screen environmental chemicals for their ability to induce aneuploidy.

Genotoxicity testing with the somatic white-ivory system in the eye of Drosophila melanogaster.

The white-ivory test in Drosophila melanogaster is designed to detect chemically induced reversions of the sex-linked, recessive unstable eye-color mutation white-ivory to the wild-type form. After exposure of larvae reversions are detectable as clones of red facets in the eye of newly enclosed adult flies. Tester strains containing a quadruplication of the white-ivory gene on the X-chromosome(s) were used. In a strain with males carrying 4 copies of the gene and females carrying 8 copies of the gene, spontaneous reversions occurred proportional to the gene copy number. In contrast to this, chemically induced reversions occurred only 1.36 times more frequently in females (carrying 8 copies of the gene) than in males (carrying 4 copies). Since chemicals inducing different lesions in DNA (bleomycin, cyclophosphamide, daunomycin, diethyl sulfate and 7,12-dimethylbenz[a]anthracene) did induce statistically significant frequencies of reversions the test appears to be capable of detecting a wide variety of genotoxic chemicals with different modes of action. The recombinogen strychnine did not induce reversions.

Optimal experimental design and sample size for the statistical evaluation of data from somatic mutation and recombination tests (SMART) in Drosophila.

In genetic toxicology it is important to know whether chemicals should be regarded as clearly hazardous or whether they can be considered sufficiently safe, which latter would be the case from the genotoxicologist's view if their genotoxic effects are nil or at least significantly below a predefined minimal effect level. A previously presented statistical decision procedure which allows one to make precisely this distinction is now extended to the question of how optimal experimental sample size can be determined in advance for genotoxicity experiments using the somatic mutation and recombination tests (SMART) of Drosophila. Optimally, the statistical tests should have high power to minimise the chance for statistically inconclusive results. Based on the normal test, the statistical principles are explained, and in an application to the wing spot assay, it is shown how the practitioner can proceed to optimise sample size to achieve numerically satisfactory conditions for statistical testing. The somatic genotoxicity assays of Drosophila are in principle based on somatic spots (mutant clones) that are recovered in variable numbers on individual flies. The underlying frequency distributions are expected to be of the Poisson type. However, some care seems indicated with respect to this latter assumption, because pooling of data over individuals, sexes, and experiments, for sample, can (but need not) lead to data which are overdispersed, i.e., the data may show more variability than theoretically expected. It is an undesired effect of overdispersion that in comparisons of pooled totals it can lead to statistical testing which is too liberal, because overall it yields too many seemingly significant results. If individual variability considered alone is not in contradiction with Poisson expectation, however, experimental planning can help to minimise the undesired effects of overdispersion on statistical testing of pooled totals. The rule for the practice is to avoid disproportionate sampling. It is recalled that for optimal power in statistical testing, it is preferable to use equal total numbers of flies in the control and treated series. Statistical tests which are based on Poisson expectations are too liberal if there is overdispersion in the data due to excess individual variability. In this case we propose to use the U test as a non-parametric two-sample test and to adjust the estimated optimal sample size according to (i) the overdispersion observed in a large historical control and (ii) the relative efficiency of the U test in comparison to the t test and related parametric tests.

Drosophila wing-spot test: improved detectability of genotoxicity of polycyclic aromatic hydrocarbons.

The somatic mutation and recombination tests (SMART) using eyes or wings in the fruit fly Drosophila melanogaster are flexible and sensitive systems for the detection of genotoxicity of individual chemical compounds and complex mixtures. It is of special interest that adults and larvae possess cytochrome P-450-dependent activation systems able to metabolize most promutagens, e.g., nitrosamines, aflatoxins, pyrrolizidine alkaloids, safrole, etc. The polycyclic aromatic hydrocarbons (PAHs) represent a class of promutagens poorly detectable in Drosophila genotoxicity systems. Therefore, new tester strains for the wing-spot test were constructed by introducing chromosomes 1 and 2 from a wild-type strain with increased cytochrome P-450-dependent metabolism linked to a gene on chromosome 2. Previous investigations with the new strains showed their increased detection capability for diethylnitrosamine. Comparative tests with the 3 PAHs benzo[a]pyrene, benz[a]anthracene and 7,12-dimethylbenz[a]anthracene demonstrate, in a reproducible way, that with the new strains all 3 can be detected as active genotoxic compounds. The dose-response curves for all compounds show a plateau with higher exposures. This is interpreted as indicative of a saturation of the cytochrome P-450-dependent activation systems.