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

The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster was employed to determine the recombinagenic and mutagenic activity of four chemicals in an in vivo eukaryotic system. Two different crosses involving the wing cell markers mwh and flr(3) were used: the standard cross and a high bioactivation cross. The high bioactivation cross is characterized by a high constitutive level of cytochromes P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Three-day-old larvae derived from both crosses were treated chronically with the oxidizing agent potassium chromate and with the three procarcinogens cyclophosphamide, p-dimethylaminoazobenzene and 9,10-dimethylanthracene. From both crosses two types of progeny were obtained: marker-heterozygous and balancer-heterozygous. The wings of both genotypes were analysed for the occurrence of single and twin spots expressing the mwh and/or flr(3) mutant phenotypes. In the marker-heterozygous genotype the spots can be due either to mitotic recombination or to mutation. In contrast, in the balancer-heterozygous genotype only mutational events lead to spot formation, all recombination events being eliminated. The oxidizing agent potassium chromate was equally and highly genotoxic in both crosses. Surprisingly, the promutagen cyclophosphamide also showed equal genotoxicity in both crosses, whereas p-dimethylaminoazobenzene was negative in the standard cross, but clearly genotoxic in the high bioactivation cross. 9,10-Dimethylanthracene showed a rather weak genotoxicity in the high bioactivation cross. Analyses of the dose-response relationships for mwh clones recorded in the two wing genotypes demonstrated that all four compounds are recombinagenic. The fraction of all genotoxic events which are due to mitotic recombination ranged from 83% (9,10-dimethylanthracene) to 99% (p-dimethylaminoazobenzene). These results demonstrate that the wing spot test in Drosophila is most suited to the detection of recombinagenic activity of genotoxic chemicals.

Genotoxicity testing of biotechnology-derived products. Report of a GUM task force. Gesellschaft für Umweltmutationsforschung.

Various aspects of genotoxicity testing of biotechnology-derived products are discussed based on information gathered from a questionnaire which was sent to about 30 predominantly European companies. Feedback was received from 13 companies on 78 compounds, mostly recombinant proteins but also on a number of nonrecombinant proteins, which had been assessed for genotoxicity in a total of 177 tests. Four of the 78 compounds appeared to elicit reproducible genotoxic effects. For one of these compounds, the activity could be related to a nonpeptidic linker molecule. No scientifically convincing rationale for the other three compounds could be established, although, at least for two compounds, their activity may be connected with the enzymatic/hormonal activity. In addition to the survey, published reports on genotoxicity testing of biotechnology products were reviewed. The data are discussed relative to whether genotoxicity testing is a valuable exercise when assessing potentially toxic liabilities of biotechnology-derived compounds. It is concluded that genotoxicity testing is generally inappropriate and unnecessary, a position which is in accordance with the available guidelines addressing this area. For the 'average' protein, electrophilic reactions are difficult to envision. Indirect reactions via DNA metabolism and growth regulation seem possible for only very specific proteins such as nucleases, growth factors, cytokines. No information on testing of different types of biotechnology-derived products (e.g., ribozymes, antisense-oligonucleotides, DNA vaccines) has been received in the questionnaires. Discussion of their potential to cause genotoxic changes was based on literature reports. Even for those products for which concerns of genotoxic/tumourigenic potential cannot be completely ruled out, e.g., because of their interaction with DNA metabolism or proliferation control, the performance of standard genotoxicity assays generally appears to be of little value. All information, including also information on the occurrence of genotoxic impurities, has been utilized to formulate a decision tree approach for the genotoxicity testing of biotechnology-derived products.

Antigenotoxicity studies in Drosophila melanogaster.

The fruit fly Drosophila melangaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests (SMART) have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs (mwh,3-0.3) and flare (flr,3-38.8). Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compound(s) or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, specific types of translocation, etc.). The analysis of two different genotypes (one with structurally normal chromosomes, one with a multiply inverted balancer chromosome) allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies presented: (1) instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and (2) ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite.

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

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.

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells.

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the beta-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9 x 10(-4), whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8 x 10(-4). The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.

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.

A plasmid rescue to investigate mutagenesis in transgenic D. melanogaster.

We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo. Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis. The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors. The resulting circular plasmids are then transformed back into E. coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal. The number of inactivated versus intact lacZ genes directly indicates the mutation frequency. By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely. Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4). Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively.

Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells.

The interactions in vitro of heavy metals Cd(II), Co(II), Cu(II), Ni(II), Pb(II), and Zn(II) with cytochrome P4501A (CYP1A) induction response and enzyme activity were studied in fish hepatoma cells PLHC-1. Cells were simultaneously exposed to heavy metals and to 3-methylcholanthrene (3-MC), an inducer of CYP1A. Heavy metals were added to the cells in different concentrations. Cytotoxicity were measured in the neutral red (NR) assay, relative CYP1A protein contents in an enzyme-linked immunosorbent assay (ELISA), and CYP1A activities in the ethoxyresorufin-O-deethylase (EROD) assay. All metals had a more pronounced effect on EROD activity than on CYP1A protein content and cytotoxicity. For the most active metal Cd(II), a 50% inhibition of EROD activity was observed at significantly lower concentrations (2.2 x 10(-5) M) than a 50% reduction of CYP1A protein (5.3 x 10(-5) M), and a 50% cytotoxicity (1.4 x 10(-4) M). The inhibitory potency of the metals had the following order: Cd(II) > Ni(II) > Cu(II) > Co(II) = Zn(II) > Pb(II). In a second set of experiments, lysates of 3-MC-induced cells were exposed to heavy metals. Cd(II) and Cu(II) caused a 50% inhibition of EROD activity at significantly lower concentrations than in the experiments with living cells, at 8.2 x 10(-6) M and 1.3 x 10(-5) M, respectively, whereas the effect by Co(II) occurred at a significantly higher concentration (8.2 x 10(-4) M). The results indicate that Cd(II) and Cu(II) in particular may affect the CYP1A system of the liver of fish at low concentrations through direct inhibition of the CYP1A enzyme activity. CYP1A induction response in fish liver is increasingly being used in biomonitoring programs. In the environment, interactions of CYP1A-inducing and CYP1A-inhibiting components (such as heavy metals) can be expected and must be taken into consideration.

Induction of somatic mutation and recombination by four inhibitors of eukaryotic topoisomerases assayed in the wing spot test of Drosophila melanogaster.

Four inhibitors of eukaryotic topoisomerases were investigated for genotoxic effects in the wing spot test of Drosophila melanogaster. As a somatic mutation and recombination test (SMART) this assay assesses mitotic recombination and mutational events of various kinds. We studied camptothecin as a topoisomerase I inhibitor, as well as ellipticine as an intercalating inhibitor and teniposide and etoposide as two non-intercalating inhibitors of topoisomerase II. Wing spots were induced in flies trans-heterozygous for the recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) as well as in flies heterozygous for mwh and the multiply inverted TM3 balancer chromosome. All four compounds proved significantly genotoxic in this test. The spot induction frequencies formally standardized to the millimolar unit of exposure dose decreased in the order camptothecin > teniposide > ellipticine greater, similar etoposide in the mwh/flr3 inversion-free genotype. In the mwh/TM3 genotype, in which mitotic crossing over is suppressed because of the inversion-heterozygosity, the observed spot frequencies were considerably reduced, but to different extents. In this genotype, spot induction by ellipticine was not statistically significant, and it was determined that >99% of the spots are due to mitotic recombination in mwh/flr3 flies. For the other compounds, spot induction in the inversion-heterozygous genotype was significant. The relative contribution of recombination to total spot induction in the inversion-free genotype was 88% for camptothecin. It was significantly lower for the two epipodophyllotoxins teniposide (71%) and etoposide (59%). Only suggestions can be proffered at present as to how these proportions could be related to the primary damage produced by the respective compounds on the chromosomes.

Detection of genotoxic activity in native hospital waste water by the umuC test.

The genotoxic potential of the waste water of a hospital was evaluated by the umuC test. Within 2 years over 800 native waste water samples were analysed. Genotoxic activity was found in 13% of the samples. The highest genotoxic activity occurred in the morning hours, but genotoxic samples were detected also during the day and at night. 96% of the genotoxic waste water samples revealed a genotoxic potential without growth inhibition of test bacteria monitored as OD600, in the same way as antineoplastic drugs like mitomycin C or cisplatin. 4% of the genotoxic waste water samples showed combined cytotoxic and genotoxic activities as seen in control experiments using glutaraldehyde containing disinfectants and certain antibiotics.

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.

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.

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae.

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequences encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-delta CYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-delta CYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14 alpha-demethylase.

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.

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.

Distinct area distribution differences of micronuclei induced by clastogenic and aneuploidogenic chemicals in the bone marrow of the CD-1 mouse.

To distinguish between aneuploidogenic and clastogenic effects of test chemicals, area distributions of micronuclei (MN) in polychromatic erythrocytes (PE) from the mouse bone marrow were measured using an image analysis system. Triethylenemelamine (TEM), cytosine-beta-D-arabinofuranoside (ara-C), urethane (URT), cyclosphamide (CP), mitomycin C (MMC), colcemid (COL) and tubulazole C (TUB) were investigated for the induction of micronucleus area distributions. The area distribution of micronuclei of untreated mice was also determined. Reproducible small differences between the clastogens and the aneuploidogens were observed after measuring 1100-1200 micronuclei. A common feature of the distribution curves was a shoulder region in the same area range for all clastogens. The aneuploidogens COL and TUB showed a plateau (= wide peak) in this clastogenic shoulder region. For all clastogens, the integrated area of shoulder over a fitted function (shoulder strength) was evaluated. MMC and CP, thought to have some aneuploidogenic potential, showed an increased shoulder strength compared to TEM, ara-C and URT. The control area distribution had no similarities to the area distribution of either clastogens or aneuploidogens. In a further experiment, we attempted to correlate the size of micronuclei determined after treatment with the aneuploidogenic chemicals to the size of whole chromosomes. Micronuclei found by image analysis which bear chromosome-like structures (judged by light microscopy) were manually identified. This selection of micronuclei was area-distributed to determine the mean size of these micronuclei. None of the peaks and plateaus in the area distributions obtained with the aneuploidogenic chemicals could be attributed to the size of a chromosome.

DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes.

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily.

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).

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.