1,4-Dioxane: prediction of in vivo clastogenicity.

1,4-Dioxane was analyzed with the CASE program to determine the structural basis of its potential genotoxicity and carcinogenicity. These investigations led to the prediction that while 1,4-dioxane was not genotoxic in vitro, it was an inducer of micronuclei in the bone marrow of rats and a carcinogen for both rats and mice. If it is assumed that the induction of micronuclei is the result of DNA damage, then this potential and the previous report of the in vivo induction of DNA strand breaks in rat liver raise the possibility of a genotoxic action for 1,4-dioxane. However it is also conceivable that we have identified a structural feature which contributes to the induction of micronuclei by a non-genotoxic mechanism.     

Structural requirements for the induction of the SOS repair in bacteria by nitrated polycyclic aromatic hydrocarbons and related chemicals.

The CASE (computer-automated structure evaluation) methodology was used to investigate the structural basis of the SOS-inducing activity of 56 nitrated polycyclic aromatic hydrocarbons (nitroarenes, nPAH) and the unsubstituted parent PAH molecules. Based upon the presence and/or absence of structural features, CASE identified 5 activating (biophores) and 4 inactivating (biophobes) fragments responsible for the SOS-inducing activity. Based upon these fragments, CASE correctly calculated the genotoxicity of 94.6% of the molecules in the training set (sensitivity = 0.85, specificity = 1.0). Disregarding the questionable experimental results of the unexpected very weak direct-acting activity of the unsubstituted benzo[a]pyrene, dibenzo[a,h]anthracene and 7,12-dimethylbenz[a]anthracene, the concordance of the prediction was 100%, i.e., sensitivity = 1.0, specificity = 1.0. Additionally, the quantitative analysis of the SOS-inducing potency showed a good correlation between the experimental and predicted results. The present analyses indicate an identity in the structural determinants responsible for SOS induction in E. coli PQ37 (SOS chromotest) and mutagenicity in Salmonella typhimurium.     

Effects of hormone treatment on chromosomal radiosensitivity of somatic and germ cells of Snell's dwarf mice

The X-ray induction of micronuclei and structural chromosomal aberrations was studied in bone-marrow cells of normal and dwarf (dw) mice in combination with thyroxine and/or prolactin treatment or otherwise. Hormone treatment clearly increased micronuclei induction but not chromosome breakage, suggesting that indirect effects were involved. Since no clear differences in the timing of the final stage of erythropoiesis could be found, it is likely that the indirect effects are mediated via the formation-differentiation kinetics of erythroblasts. The induction of reciprocal translocations by X-rays in stem cell spermatogonia of dwarf mice was lower than in normals and treatment with prolactin, growth hormone and/or thyroxin, did not influence the chromosomal radiosensitivity of spermatogonial stem cells.     

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Structural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants

A set of 189 chemicals tested in the National Toxicology Program Cancer Bioassay was subjected to analysis by CASE, the Computer-Automated Structure Evaluation system. In the data set, 63% of the chemicals were carcinogens, approx. 40% of the carcinogens were non-genotoxic, i.e., they possessed neither "structural alerts" for DNA reactivity as defined by Ashby and Tennant, 1988, nor were they mutagenic for Salmonella. The data base can be characterized as a "combined rodent" compilation as chemicals were characterized as "carcinogenic" if they were carcinogenic in either rats or mice or both. CASE identified 23 fragments which accounted for the carcinogenicity, or lack thereof, of most of the chemicals. The sensitivity and specificity were unexpectedly high: 1.00 and 0.86, respectively. Based upon the identified biophores and biophobes, CASE performed exceedingly well in predicting the activity of chemicals not included among the 189 in the original set. CASE predicted correctly the carcinogenicity of non-genotoxic carcinogens thereby suggesting a structural commonality in the action of this group of carcinogens. As a matter of fact biophores restricted to non-genotoxic carcinogens were identified as were "non-electrophilic" biophores shared by genotoxic and non-genotoxic carcinogens. The findings suggest that the CASE program may help in the elucidation of the basis of the action of non-genotoxic carcinogens.     

The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: the Gene-Tox data base

The CASE structure-activity methodology has been applied to a Gene-Tox derived Salmonella mutagenicity data base consisting of 808 chemicals. Based upon qualitative structural features, CASE identified 29 activating and 3 inactivating structural determinants which correctly predicted the probability of carcinogenicity of 93.7% of the known mutagens and non-mutagens in the data base (sensitivity = 0.998, and specificity = 0.704). Additionally, based upon a qualitative structure-activity analysis, CASE's performance was even better, leading to a sensitivity of 0.981 and a specificity of 1.000. Using the structural determinants identified in this data base, CASE gave excellent predictions of the mutagenicity of chemicals not included in the data base. The identified biophores and biophobes can also be used to investigate the structural basis of the mutagenicity of various chemical classes.     

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites, benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F₁ mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites,benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F(1) mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in chinese hamster ovary cells

Arsenic is the first metal to be identified as a human carcinogen. Arsenite, one inorganic form of arsenic, has been found to induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety of in vitro systems. In this study of arsenite-induced genotoxicity represented as micronuclei production in Chinese hamster ovary cells (CHO-K1), we found that the calcium channel blocker, verapamil, can potentiate arsenite-induced micronuclei. And after arsenite treatment, the elevation of intracellular calcium was observed. When extracellular calcium was depleted during arsenite treatment, the arsenite-induced micronuclei formation was significantly suppressed. These data indicated that a calcium ion plays an essential role in arsenite-induced genotoxicity. Further, it was found that the cotreatment of arsenite and a calcium ionophore, A23187, can increase the micronuclei induction. In contrast, pretreatment of the intracellular calcium chelator, quin 2, significantly inhibited micronuclei production of arsenite administration. In addition, we measured the activity of calcium-and phospholipid-dependent protein kinase C (PKC) and found that arsenite can activate PKC activity in a dose-dependent manner. Subsequently, some PKC activators and inhibitors were applied to investigate the involvement of PKC on arsenite-induced micronuclei formation. It was found that H7, a PKC inhibitor, can depress but TPA, a PKC activator, can enhance arsenite-induced micronuclei significantly. These data indicated that arsenite exposure perturbs intracellular calcium homeostasis and activates PKC activity. As a result, the activation of PKC activity may play an important role in arsenite-induced genotoxicity. J. Cell. Biochem. 64:423–433. © 1997 Wiley-Liss, Inc.     

In vitro induction of micronuclei by monofunctional methanesulphonic acid esters: possible role of alkylation mechanisms

Six monofunctional alkylating methanesulphonates of widely varying structures were investigated in the in vitro micronucleus assay with Syrian hamster embryo fibroblast cells. The results were compared with the alkylating activities measured in the 4-(nitrobenzyl)pyridine test (NBP-test) and the N-methyl mercaptoimidazole (MMI-test) as measures for S(N)2 reactivity as well as in the triflouoroacetic acid (TFA) solvolysis and the hydrolysis reaction as measures for S(N)1 reactivity in order to provide insights into the role of alkylation mechanisms on induction of micronuclei. Moreover we compared the results of micronucleus assay with those of the Ames tests in strain TA 100 and TA1535 and with those of the SOS chromotest with the strains PQ37, PQ243, PM21 and GC 4798. The potency of methanesulphonates to induce micronuclei depended only to a certain degree, on the total alkylating activity (S(N)1 and S(N)2 reactivity). An inverse, significant correlation between the Ames test and the micronucleus assay was observed and an inverse correlation between the micronucleus assay and the SOS chromotest with the different strains. The results indicate that the primary mechanism leading to induction of micronuclei is not O-alkylation in DNA as it is the case in the Ames test with the hisG46 strains TA1535 and TA100 and not N-alkylation as with the SOS chromotest. There is evidence that protein alkylation, e.g. in the spindle apparatus in mitosis is decisive for induction of micronuclei by alkylating compounds. The structurally voluminous methanesulphonates 2-phenyl ethyl methanesulphonate and 1-phenyl-2-propyl methanesulphonate show a clear higher micronuclei inducing potency than the other tested though the bulky methanesulphonates possess a lower total alkylating activity than the others. This effect can be explained by a higher disturbance during mitosis after alkylation of the spindle apparatus with the structurally more bulky methanesulphonates.     

Correlation between cell survival and micronuclei formation in V79 cells treated with vindesine before exposure to different doses of gamma-radiation

Effect of 20 nM vindesine sulphate (VDS) treatment was studied on cell survival, growth kinetics and micronuclei induction in V79 cells exposed to 0-300 cGy of gamma-radiation at 16, 22 and 28 h post-irradiation. Treatment of V79 cells with VDS before exposure to different doses of gamma radiation resulted in a significant decline in cell survival and growth kinetic when compared with the concurrent PBS+irradiation group. The decline in cell survival and growth kinetics was dose related. Similarly, the cell proliferation indices also declined in a dose dependent manner in both PBS+irradiation and VDS+irradiation groups and this decline was higher in VDS+irradiation group in comparison with the PBS+irradiation group. In contrast, the frequency of micronuclei increased in a dose related manner in both PBS+irradiation and VDS+irradiation groups. However, the frequency of micronuclei was significantly greater in the VDS+irradiation group when compared to the PBS+irradiation group at all the post-irradiation time periods studied and the dose response for both groups was linear for all the scoring time periods. The biological response was determined by plotting surviving fraction and micronuclei frequencies on X- and Y-axes, respectively. The plot between surviving fraction and micronuclei induction showed a close correlation. The surviving fraction of V79 cells reduced with the increasing frequency of micronuclei in both groups and the relationship between micronuclei induction and cell survival could be fitted on a linear quadratic model.     

The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: the National Toxicology Program Data Base

A portion of the U.S. National Toxicology Program (NTP) Salmonella typhimurium mutagenicity data base was analyzed by CASE, an artificial intelligence SAR system. CASE identified 13 structural determinants which, with a high probability (p less than or equal to 0.05) predicted the likelihood of mutagenicity of the 243 chemicals in the data base (sensitivity = 0.989; specificity = 0.950) as well as of chemicals not included in the data base. CASE also identified an additional set of structures which were highly predictive of mutagenic potency (sensitivity = 0.949; specificity = 1.00). Even though there is little overlap among the chemicals included in the NTP and Gene-Tox Salmonella data bases, CASE found significant similarities between the structural determinants of the mutagenicity in the two data bases, thereby validating the analyses and indicating a commonality in the structural basis of mutagenicity.     

Structural basis of the mutagenicity of phenylazoaniline dyes

The CASE (Computer-Automated Structure Evaluation) methodology was used to gain an understanding of the basis of mutagenicity of phenylazoanilines. It was found that the activity of these molecules is dependent upon an intact moiety that spans the azo linkage, i.e., the azo bond must remain intact for mutagenicity. The study also addressed the effect of sulfonation on the activity of these azo dyes. It was revealed that sulfonation at only certain sites resulted in loss of mutagenicity. Sulfonation of the structural moiety responsible for the activity of phenylazoaniline dyes did not necessarily result in complete elimination of activity as this substitution could generate new structural moieties which contribute to the activity of the molecules.     

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate.

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEM.MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.     

DNA lesions sequestered in micronuclei induce a local defective-damage response

Micronuclei are good markers of chromosome instability and, among other disturbances, are closely related to double-strand break induction. The ability of DNA lesions sequestered in the micronuclear bodies to activate the complex damage-signalling network is highly controversial since some repair factors have not been consistently detected inside micronuclei. In order to better understand the efficiency of the response induced by micronuclear DNA damage, we have analyzed the presence of DNA damage-response factors and DNA degradation markers in these structures. Radiation-induced DNA double-strand breaks produce a modification of chromatin structural proteins, such as the H2AX histone, which is rapidly phosphorylated around the break site. Strikingly, we have been able to distinguish two different phosphoH2AX (γH2AX) labelling patterns in micronuclei: discrete foci, indicating DSB presence, and uniform labelling affecting the whole micronucleus, pointing to genomic DNA fragmentation. At early post-irradiation times we observed a high fraction of micronuclei displaying γH2AX foci. Co-localization experiments showed that only a small fraction of the DSBs in micronuclei were able to properly recruit the p53 binding protein 1 (53BP1) and the meiotic recombination 11 (MRE11). We suggest that trafficking defects through the micronuclear envelope compromise the recruitment of DNA damage-response factors. In contrast to micronuclei displaying γH2AX foci, we observed that micronuclei showing a γH2AX extensive-uniform labelling were more frequently observed at substantial post-irradiation times. By means of TUNEL assay, we proved that DNA degradation was carried out inside these micronuclei. Given this scenario, we propose that micronuclei carrying a non-repaired DSB are conduced to their elimination, thus favouring chromosome instability in terms of allele loss.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 micrograms/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 μg/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Biological responses in known bystander cells relative to known microbeam-irradiated cells

Normal human fibroblasts in plateau phase ( congruent with 95% G(1) phase) were stained with the vital nuclear dye Hoechst 33342 (blue fluorescence) or the vital cytoplasmic dye Cell Tracker Orange (orange fluorescence) and plated at a ratio of 1:1. Only the blue-fluorescing nuclei were microbeam-irradiated with a defined number of 90 keV/microm alpha particles. The orange-fluorescing cells were then "bystanders", i.e. not themselves hit but adjacent to cells that were. Hit cells showed a fluence-dependent induction of micronuclei as well as delays in progression from G(1) to S phase. Known bystander cells also showed enhanced frequencies of micronuclei (intermediate between those seen in irradiated and control cells) and transient cell cycle delays. However, the induction of micronuclei in bystander cells did not appear to be dependent on the fluence of the particles delivered to the neighboring hit cells. These are the first studies in which the bystander effect has been visualized directly rather than inferred. They indicate that the phenomenon has a quantitative basis and imply that the target for radiation effects cannot be considered to be the individual cell.