In vitro micronucleus screening of pharmaceutical candidates by flow cytometry in Chinese hamster V79 cells

We previously reported a high concordance of in vitro micronucleus (MNvit) results obtained by flow cytometry to the known cytogenetic activity often commercially available compounds mentioned as validation compounds in an early draft of the OECD MNvit TG487 [Bryce et al., 2010; Organization for Economic Co-operation and Development(OECD), 2007]. The current study investigated this method in Chinese hamster V79 cells with pharmaceutical compounds of unknown genotoxic potential. Twenty-five compounds from several therapeutic areas such as oncology, neuroscience and immunological research were tested in the flow cytometry assay, and for comparison using the cytokinesis-block microscopy assay. Five of these 25 compounds were considered positive for micronucleus induction by the microscopy assessment. In all cases, the results from the flow cytometry assess ment matched the results of the microscopy assay. Thus, flow cytometry is a viable method for assessing the aneugenic/clastogenic potential of pharmaceutical drug candidates. The flow method offered several advantages over traditional microscopy. For instance, the ratio of micronuclei (MN) to 10,000 nuclei was evaluated in less than 2 min vs.15 min to manually assess 600 binucleate cells. Evaluation by flow cytometry can be automated,freeing resources and eliminating scorer fatigue.The assay may also provide for mechanistic understanding of MN formation based on size and the ratio of nuclei with sub-2N DNA content, allowing for discrimination between aneugenic and clastogenic compounds.     

Evaluation of the mutagenicity of beta-myrcene in mammalian cells in vitro

The genotoxicity of the terpene beta-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 micrograms/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt-locus in V79-cells. Tests with and without S9-mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE-inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.     

Novel statistical approach for evaluating flow cytometric in vitro micronucleus data

Statistical methods currently recommended for analysis of in vitro micronucleus data are based on small sample sizes. The tests are designed to evaluate linear trends and differences between treated and control samples. When using flow cytometric analysis, >5 times the number of cells are easily evaluated, and the variance estimates from these large samples are small. Application of these recommended tests to large samples resulted in statistically significant outcomes which were not considered to be biologically meaningful. Alternative statistical methods for testing trends and differences among treatments that were either widely used, or sample‐size independent, were investigated. Using data from 95 experiments (from 2011–2013) where 19% of the experiments were considered positive, results for the various statistical methods were compared. When using either the recommended or alternate methods, 42–68% of the experiments resulted in statistically significant results (p < 0.05). A new concept was then tested using the same data sets: the “z′ factor”, designed to identify ‘hits’ during high throughput screening. Using this simple‐to‐compute statistic the number of significant calls was reduced to 27%. Then, when combined with a biological criterion based on historical vehicle control data, there was restoration of the original positive frequency (19%). Given the larger sample sizes evaluated using flow cytometry, we have demonstrated that traditional statistical tests may be overly sensitive to small changes in micronucleus induction, and that a simple‐to‐compute index of separation (z′) may be a better tool for analysis, provided that the response is first determined to be biologically meaningful. Environ. Mol. Mutagen. 57:589–604, 2016. © 2016 Wiley Periodicals, Inc.     

Effects of spermine on formation of HGPRT− mutants induced by ethylmethanesulfonate,methylmethanesulfonate, and mitomycin C in V79 chinese hamster cells

Spermine is a polyamine found in bacteria, animal, and plant tissues. It is involved in a variety of biological processes, and its interaction with DMA stabilizes the secondary structure of the double helix. Spermine is one of the first reported antimutagens, reducing the mutation rate in several prokaryotic test systems, while in eukaryotic organisms conflicting results have been obtained. In light of the significant antimutagenic effect of spermine, it is important to evaluate its activity in mammalian cells in culture. The present study was undertaken to evaluate the ability of spermine to suppress the level of HGPRT mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C. Spermine reduced the mutation frequency induced by ethylmethanesulfonate and methylmethanesulfonate but did not affect survival; with mitomycin C survival was reduced but mutation rate was not influenced.     

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluation of flow cytometric immunophenotyping and DNA analysis for detection of malignant cells in serosal cavity fluids

The serosal cavities are frequent sites of tumor metastasis. The distinction between carcinoma cells, inflammatory cells, and reactive or malignant mesothelial cells can be difficult in cytology. Multicolor flow cytometry (FCM) provides the opportunity to evaluate multiple antigens simultaneously, making it possible to characterize various cell populations. In this study, we aimed to assess the diagnostic accuracy of FCM immunophenotyping and DNA in comparison with serum tumor markers and classic cytology for detection of malignant cells in pleural and ascitic fluids. One hundred and nineteen samples of body cavity fluids were analyzed. Immunophenotyping was performed by four‐color immunofluorescent staining using monoclonal antibodies against Ber‐EP4, cytokeratin, CD3, and CD45. The DNA analysis by FCM was also performed. In addition, serum CA19‐9, CEA, AFP, and CA125 were analyzed. Ber‐EP4 marker had the highest sensitivity (73%) and specificity (95.5%) in the detection of carcinoma cells in serous fluid and correlated with cytology in most of cases (73%). The mean of DI differed statistically in patients with malignant effusions than in benign one. DI showed no difference in fluids due to infiltration of malignant epithelial cells or hematopoitic malignancy or due to hepatocellular carcinoma developing in cirrhotic liver. Thus, flow cytometry appears to aid not only in the detection of malignant cells but also in the characterization of cell type. On the other hand, although DNA ploidy examination had better sensitivity; it had no advantage over conventional cytopathological examination in identification of malignant cells. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Overexpression of heme oxygenase-1 (HO-1) in V79 cells results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Heme oxygenase (HO) catalyzes the rate-limiting step in the oxidative degradation of heme to biliverdin. The isoform HO-1 is inducible by a variety of agents causing oxidative stress and has been suggested to play an important role in cellular protection against oxidant-mediated cell damage. Using treatment of cell cultures with hyperbaric oxygen (HBO) as a model for oxidative stress, we have shown an induction of HO-1 in isolated human lymphocytes after a single HBO exposure and protection of these cells against DNA damage by subsequent oxidative stress. In contrast, V79 Chinese hamster cells showed neither a comparable adaptive protection nor an induction of HO-1 after HBO exposure, which makes this cell line an attractive model system for a further characterization of HO-1-mediated protection. In the present study, we investigated whether overexpression of HO-1 renders V79 cells more resistant to DNA damage induced by HBO. Transient transfection of V79 cells with a full-length human HO-1 cDNA resulted in a 2-3-fold increase in HO-1 protein levels. Comet assay experiments with and without FPG posttreatment for the determination of oxidative DNA base damage showed that HO-1 overexpressing V79 cells were significantly protected against oxidative DNA damage induced by a single HBO exposure. Furthermore, HO-1-transfected cells exhibited a clearly reduced induction of micronuclei after HBO treatment. Since the observed protective effects were abolished by cotreatment with the HO-1 inhibitor tin-mesoporphyrin, our study suggests that a low-level overexpression of HO-1 provides protection against oxidative DNA damage induced by HBO.     

Flow cytometric analysis of micronucleated reticulocytes: Time- and dose-dependent response of known mutagens in mice, using multiple blood sampling

According to the current Organization of Economic Cooperation and Development (OECD) and International Committee on Harmonization (ICH) guidelines for the mammalian erythrocyte micronucleus (MN) test, analysis of peripheral blood reticulocytes (RETs) for the presence of micronuclei can be performed using flow cytometry. The MicroFlow PLUS method (Litron Laboratories, Rochester, NY) for MN analysis by flow cytometry is based on the binding of FITC-labeled antibodies to the CD71 transferrin receptor of immature RETs, on parallel RNA degradation, and on propidium iodide staining of DNA present as micronuclei. The objective of this study was to assess the sensitivity of this flow cytometry method to detect time- and dose-dependent induction of micronuclei in mouse peripheral blood RETs after treatment with nine chemical agents. Five known clastogens, two known aneugens, and two compounds previously reported to be inactive in the mouse bone marrow MN test were evaluated at three dose levels. Multiple blood sampling of the same animal before and at two time points after treatment was conducted. All known mutagens produced a dose-dependent increase in micronucleated reticulocytes (MN-RETs); the compounds previously shown to be inactive in the in vivo MN test were also negative using the present methodology. The highest frequency of MN-RETs was observed at 48 hr after treatment, except for 5-fluorouracil, which had its peak response at 72 hr. The results indicate that micronuclei can be measured by multiple blood sampling of the same animal before and after treatment without altering the sensitivity of the assay. The results confirm that the flow cytometric assessment of MN-RETs in mouse peripheral blood using MicroFlow PLUS is a sensitive method with high analysis throughput, and robust quality control.     

A flow cytometric method for the detection of adenosine deaminase in mononuclear cells

The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells. Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked. This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde. The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments. The fluorescence profile of the cells was then analyzed by flow cytometry. Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines. In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations. Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence. Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells.     

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis

Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.     

Using CTLL-2 and CTLL-2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

In vitro assays for chromosome aberrations (i.e., in vitro micronucleus and in vitro metaphase analysis tests) frequently produce false-positive or exaggerated-positive results. Our previous work suggested that apoptosis interferes with these tests, producing misleading results. These previous studies were conducted by performing the in vitro micronucleus test in CTLL-2 cells and a CTLL-2 cell derivative stably transfected with the apoptosis inhibitor gene bcl2. In the present study, these previous observations were extended by examining micronucleus induction with a larger number of compounds in both CTLL-2 and CTLL-2 bcl2 cells and measuring apoptosis with annexin V-FITC. Both cell lines were treated with different classes of compounds that were anticipated to be exclusively apoptosis inducers, or compounds known to be clastogens or aneugens, some of which were anticipated to be both genotoxic and apoptotic. We were able to confirm that compounds that are only apoptogenic induced micronuclei in CTLL-2 but not CTLL-2 bcl2 cells, indicating that the positive responses are due to apoptosis in CTLL-2 cells. Some genotoxins (clastogens and aneugens) did not produce apoptosis by the annexin V assay and gave similar responses in CTLL-2 and CTLL-2 bcl2 cells. Finally, higher responses were induced in CTLL-2 cells compared to CTLL-2 bcl2 cells that were treated with aneugens or clastogens that were also apoptosis inducers, suggesting that the greater response in CTLL-2 cells is a consequence of both genotoxicity and apoptosis. Finally, it was demonstrated that just eliminating CTLL-2 cells having three or more micronuclei from scoring was not adequate for correctly evaluating agents that only produce apoptosis. The results indicate that coupling the in vitro micronucleus test in both CTLL-2 and CTLL-2 bcl2 cells with the measurement of apoptosis is able to distinguish the genotoxic effects of a test compound from its apoptotic potential and is able to avoid interference from apoptosis in the in vitro micronucleus test. These observations may provide the basis for a useful genotoxicity assay.     

Induction of micronuclei in human sperm-hamster egg hybrids at the two-cell stage after in vitro gamma-irradiation of human spermatozoa

The efficiency of the micronucleus test to assess radiation-induced chromosomal damage in human spermatozoa has been investigated. Micronuclei were scored in human sperm-hamster egg hybrids at the two-cell stage, after exposure of human spermatozoa to in vitro gamma-rays at doses of 0.00, 0.10, 0.25, 0.50, 1.00, 2.00, and 4.00 Gy. The relationship between the yield of micronuclei per two-cell stage as well as the percentage of two-cell stages with micronuclei and the different doses of irradiation were fitted to linear equations. To evaluate whether scoring micronuclei is useful for the quantification of chromosomal damage occurring in human spermatozoa, induced micronuclei at the different doses of sperm irradiation were compared to the induction of breaks and fragments in sperm-derived chromosomes. After interspecific fertilization of zona-free hamster oocytes by irradiated spermatozoa, a total of 699 fertilized eggs at the two-cell stage and a total of 387 sperm-derived complements were analyzed. The incidence of fertilized eggs with micronuclei at the two-cell stage coincided well with the incidence of sperm-derived chromosome breaks and fragments (e.g., 8.9% vs. 6.7% in the 0.25 Gy group and 52.8% vs. 58.6% in the 4.00 Gy group). A similar correlation was found between the number of micronuclei per two-cell stage and the number of breaks and fragments per sperm complement (0.09 vs. 0.07 in the 0.25 Gy group and 0.71 vs. 0.81 in the 4.00 Gy group). The results show that this test system can be used for the quantification of spontaneous or induced chromosomal damage in human spermatozoa. © 1995 Wiley-Liss, Inc.     

Flow cytometric 96‐well microplate‐based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96‐well plate (Bryce SM et al. [2010]: Mutat Res 703:191‐199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non‐genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5‐ to 2‐cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN‐fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non‐genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide‐positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96‐well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluation of the micronucleus test in vitro using chinese hamster cells: Results of four chemicals weakly positive in the in vivo micronucleus test

A rapid and simple procedure for the micronucleus test (MNT) in vitro using Chinese hamster ovary (CHO) cells was established in our laboratory. The assay is intended to quickly screen chromosomal aberrations in vitro within the framework of industrial genotoxicity studies. To test the sensitivity of the assay in the experiments described here, four substances, classified as noncarcinogens but reported as weak inducers of micronuclei (MN) in bone-marrow cells of mice, were evaluated in the MNT in vitro. Of the four compounds, ascorbic acid, phenol, and 2,6-diaminotoluene proved to be genotoxic in the MNT in vitro. Titanium dioxide, which could not be dissolved in the culture medium, did not induce MN. The MNT in vitro proved to be quick and relatively simple and to yield highly reproducible results when testing the four chemicals. © 1995 Wiley-Liss, Inc.     

Comparison of the TUNEL,lamin B and annexin V methods for the detection of apoptosis by flow cytometry

The present study was performed in order to compare the sensitivity of 3 apoptosis-detection methods using a model cell culture after induction of apoptosis. Evaluation was performed by means of flow cytometry and light microscopy. It appeared that both TUNEL and annexin V methods are sensitive and specific and produced similar data in all measurements. The immunocytochemical detection of lamin B was less reliable than the other methods.     

Efficacy of low dose temozolomide in combination with bortezomib in U87 glioma cells: a flow cytometric analysis

Introduction

Maximizing responses of malignant gliomas is hampered by resistance to temozolomide (TMZ). Increasing efficacy but not toxicity is a key issue when testing drug combinations. The antimyeloma agent bortezomib (BZ) has shown promising results in vitro and is currently being tested in glioblastoma (GBM) patients. In this study we investigate whether reduction of TMZ dosage is feasible without compromising the antitumor effect of TMZ-BZ combination.

Material and methods

U87 GBM cells were treated with increasing doses of TMZ (1, 10, 100, 1000 µM), BZ (0.001, 0.01, 0.1, 1) and the combination during a 48-hour period, and apoptotic or/and necrotic cell death was evaluated by flow cytometry.

Results

Bortezomib alone at a dose as low as 0.001 µM markedly induced cell death, particularly late apoptosis, to a level which was comparable with high TMZ dosage. For combination treatments, the dose of 0.1 µM BZ, which was more potent than the maximal dose of TMZ (1000 µM), was chosen to be added to increasing TMZ concentrations. The combination of 0.1 BZ µM BZ with low doses of TMZ (1, 10 µM) further increased the cell death rate in an additive manner, at levels higher than those induced by high doses of TMZ monotherapy (100, 1000 µM).

Conclusions

Efficacy of TMZ-BZ combination is feasible with low doses of TMZ in vitro.     

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids offer different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DMA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating sper-matogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage. © 1994 Wiley-Liss, Inc.     

Decemtione (Imidan)-induced single-strand breaks to human DNA, mutations at the hgprt locus of V79 cells, and morphological transformations of embryo cells.

To study the genotoxic activity of Decemtione (Imidan), this substance was subjected to a series of tests. After preliminary cytotoxicity testing, the capacity of Decemtione to damage human DNA was determined by alkaline elution of DNA and DNA unwinding. Both tests gave positive results, suggesting that Decemtione was able to induce single-strand breaks in DNA. This capacity was higher in the absence and lower in the presence of the S9 fraction. The potential mutagenicity of Decemtione was followed on the basis of its ability to induce resistance to 6-thioguanine in V79 hamster cells. Unlike the induction of single-strand breaks, Decemtione showed, in the absence of the metabolic activation system, a very weak mutagenic effect, which was, however, significantly higher in the presence of the S9 fraction. The ability of the substance to transform diploid cells under in vitro conditions was followed on the basis of morphological transformation of Syrian hamster embryo cells. The results showed that Decemtione, like positive carcinogenes, induced a significant elevation in morphologically transformed colonies of embryo cells. The results suggest a carcinogenic potential of this organophosphate insecticide.