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Topoisomerase II (topo II) inhibitors are commonly used as chemotherapy to treat multiple types of cancer, though their use is also associated with the development of therapy related acute leukemias. While the chromosome-damaging effects of etoposide, a topo II poison, have been proposed to act through a threshold mechanism, little is known about the chromosome damaging effects and dose responses for the catalytic inhibitors of the enzyme. The current study was designed to further investigate the potencies and concentration-response relationships of several topoisomerase II inhibitors, including the topoisomerase II poison etoposide, as well as catalytic inhibitors aclarubicin, merbarone, ICRF-154 and ICRF-187 using both a traditional in vitro micronucleus assay as well as a flow-cytometry based version of the assay. Benchmark dose (BMD) analysis was used to identify models that best fit the data and estimate a BMD, in this case the concentration at which a one standard deviation increase above the control frequency would be expected. All of the agents tested were potent in inducing micronuclei in human lymphoblastoid TK6 cells, with significant increases seen at low micromolar, and in the cases of aclarubicin and etoposide, at low nanomolar concentrations. Use of the anti-kinetochore CREST antibody with the microscopy-based assay demonstrated that the vast majority of the micronuclei originated from chromosome breakage. In comparing the two versions of the micronucleus assay, significant increases in micronucleated cells were observed at similar or lower concentrations using the traditional microscopy-based assay. BMD modeling of the data exhibited several advantages and proved to be a valuable alternative for concentration-response analysis, producing points of departure comparable to those derived using traditional no-observed or lowest-observed genotoxic effect level (NOGEL or LOGEL) approaches., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Elevated frequencies of chromosomal aberrations have been observed in the lymphocytes of benzene-exposed workers. Similar changes occurring in the bone marrow may play an important role in the development of leukemia. The objective of this research has been to characterize chromosomal alterations induced by benzene in mice and humans and to investigate the potential role of inhibition of topoisomerase II in the myelotoxic effects of benzene. The research is presented in three sections corresponding to the specific aims of the project: genotoxicity studies in the mouse, topoisomerase II studies, and initial studies using a new fluorescence in situ hybridization (FISH) approach to detect chromosome alterations in benzene-exposed workers. The results of the mouse experiments indicate that both chromosome breakage and aneuploidy are induced in the bone marrow of B6C3F1 mice following benzene administration. Chromosome breakage is the predominant effect, and this occurs primarily in the mouse euchromatin. Significant breakage within the mouse heterochromatin was also observed, as was aneuploidy. Breakage in the mouse bone marrow erythrocytes increased as a function of both dose and duration of benzene administration. The aneuploidy resulting from benzene exposure in mice was a relatively infrequent event, with increases of both chromosome loss and hyperdiploidy being observed. In the topoisomerase studies, benzene or its metabolites were shown to inhibit topoisomerase II enzyme activity in an isolated enzyme system, in a human bone marrow-derived leukemia cell line, and in vivo in the bone marrow of treated mice. The decreased activity was probably due to the rapid degradation of the topoisomerase II protein in the treated cells. In the human biomonitoring studies, the feasibility of using FISH with tandem DNA probes to detect chromosome alterations in interphase granulocytes and lymphocytes of benzene-exposed workers was demonstrated. The results from the two worker studies were somewhat inconsistent, however. In the study of Estonian workers, characterized by lower exposures and a smaller sample size, the benzene-exposed workers exhibited elevated frequencies of breakage in the lq12 region as compared with those seen in controls. A suggestive trend toward increased hyperdiploidy was also seen, although the frequencies in the exposed workers were low and within the range of our laboratory's historical control frequencies. In the larger study of more highly exposed Chinese workers, no increase in breakage affecting the 1q12 region was seen among the exposed workers. A trend toward increased hyperdiploidy of chromosome 1 was seen in the exposed workers when the concentration of urinary benzene metabolites was used in conjunction with the frequency of hyperdiploidy observed in the lymphocytes of the individual workers. The results of these studies indicate that benzene exposure is characterized by chromosome breakage, primarily within the euchromatin, and modest increases in aneuploidy. These findings also provide the first direct evidence that benzene is capable of inhibiting the enzymatic activity of topoisomerase II in vivo, providing additional support for the hypothesis that inhibition of topoisomerase II contributes to benzene-induced toxicity and leukemogenesis.

Current models suggest that genomic instability is crucial in the accumulation of the multiple alterations required for tumorigenesis. However, the nature of the initial damage responsible for the origin of genomic instability remains poorly understood. In this investigation we demonstrate that the nucleotide analog 2,6-diaminopurine (DAP) can be used to induce highly focused damage to the large blocks of paracentromeric heterochromatin on chromosomes 1, 9 and 16. A large fraction of cells exposed to DAP exhibit undercondensation of alpha and classical heterochromatin which persists into metaphase. Subsequent chromosome breakage was observed for one of the target chromosomes by preferential exclusion of chromosome 16 fragments into micronuclei (P < 0.0001). The specificity of DAP-induced chromosomal breakage enabled us to utilize it as a reagent to demonstrate that paracentromeric heterochromatin is a sensitive target for the induction of persistent genomic instability. We observed a 100-fold increase in mutagenesis affecting a chromosome 16 marker (APRT) compared with marker loci on chromosomes 17 (TK) or X (HPRT). We previously reported that APRT- mutants were recovered at a high rate upon selection in DAP in a process involving recombinationally mediated loss of heterozygosity that extends from the telomere to the boundary region of the paracentromeric heterochromatin. Karyotypic analysis of DAP-resistant APRT- mutant clones demonstrated extensive genomic instability, particularly evidence of multiple and sequential events affecting chromosome 16. These data suggest that the heterochromatic breakage observed cytogenetically immediately following DAP exposure is also responsible for the initiation of persistent genomic instability.

Diethylstilbestrol (DES) and 17beta-estradiol (E2) are known inducers of aneuploidy and polyploidy in vivo and in vitro. Isolated human lymphocytes were treated with the stilbene estrogen DES (0.05-50 microM) and the steroid estrogen E2 (0.05-75 microM) in culture. Multicolor fluorescence in situ hybridization (FISH) with DNA probes for the centromere and adjacent heterochromatin regions of chromosomes 1, 9, and 16 was used to detect hyperdiploidy, polyploidy, and chromosomal breakage affecting these chromosomes. Using this FISH technique, significant nonlinear increases in hyperdiploidy were observed with both compounds, whereas no induction of chromosomal breakage affecting the pericentric heterochromatin regions of chromosomes 1, 9, and 16 could be detected. DES induced a maximum of approximately 13% hyperdiploid cells at 30 microM, whereas E2 showed its highest induction at 75 microM with 7% hyperdiploid cells. To distinguish hyperdiploidy from polyploidy, a FISH labeling strategy to detect multiple chromosomes simultaneously was established. Using this approach, we could show that most of the cells showing multiple hybridization regions after treatment with both chemicals were most likely the result of polyploidy rather than true hyperdiploidy. These results indicate that the induction of hyperdiploidy/polyploidy with DES and E2 show sublinear dose-response relationships with likely threshold concentrations in human lymphocytes and that FISH with multiple probes targeting different chromosomes can be used to estimate hyperdiploidy and polyploidy frequencies.

The in vitro micronucleus assay in conjunction with CREST-staining and fluorescence in situ hybridization (FISH) with centromere-specific DNA probes is being increasingly utilized for the detection of clastogenic and aneuploidy-inducing agents. Although potentially powerful techniques, both methods have unique characteristics that can influence sample processing and the interpretation of results. In this article, the use of the CREST and the FISH modifications of the in vitro micronucleus assay have been used to characterize the origin of the micronuclei induced by cyclophosphamide, 4,4'-methylene-bis(2-chloroaniline), 4-nitroquinoline N-oxide and ionizing radiation in metabolically competent MCL-5 cells or a derived cell line lacking metabolic activation. Using these results and our previous experiences with these techniques, a detailed comparison including the strengths and limitations of each technique as well as potential problems in performing each assay and in analyzing the data is discussed. In spite of their limitations, our results to date indicate that CREST-staining as well as FISH with centromere-specific DNA probes can be used to accurately distinguish micronuclei formed from chromosome loss from those originating from chromosome breakage and that these techniques can be valuable complements to the in vitro micronucleus assay.

A multicolour tandem labelling fluorescence in situ hybridization (FISH) procedure was used to compare the frequencies of radiation-induced chromosome breakage and hyperdiploidy of chromosome 1 occurring in non-cultured granulocytes and Go lymphocytes with those observed in cultured metaphase and interphase lymphocytes. Whole blood, obtained from healthy male donors, was exposed in vitro to 0, 100, 200, 300 and 400 cGy of ionizing radiation from a 137Cs source. Aliquots containing granulocytes and Go lymphocytes from each dose were treated immediately with hypotonic KCI on ice and harvested. Cells were hybridized with alpha- and classical satellite probes to the 1cen-q12 region of chromosome 1 and the frequencies of hyperdiploidy and breakage affecting this region were determined. Elevated dose-related frequencies of breakage were detectable in both lymphocytes and granulocytes immediately following radiation and decreased rapidly over the first 0.25-2 h. In a second series of experiments, the frequencies of hyperdiploidy and breakage for uncultured granulocytes and Go lymphocytes were compared with interphase and metaphase cells following 48-51 h of culture. Similar and significant dose-related increases in breakage were seen for the granulocytes, Go lymphocytes, 48 h cultured interphase and metaphase lymphocytes. A minor increase in hyperdiploidy was seen in the irradiated cultured cells, whereas no hyperdiploid cells were detected in the non-cultured cells. These results indicate that, in general, granulocytes and lymphocytes show similar sensitivity to radiation-induced damage and that cell culture is not required for chromosome breakage to be observed microscopically using this FISH procedure.

This article describes the induction of micronuclei, hyperdiploidy and chromosome breakage in human amniotic cells in vitro by amosite, chrysotile and crocidolite asbestos, and ceramic fibers. The response of human (amniotic fluid cells) and rodent (Syrian hamster embryo fibroblasts, SHE) cells to fiber treatment was compared using the micronucleus assay. The data of the rodent studies were taken from a previous investigation (Dopp, E. et al. (1995) Environ. Health Perspect., 103, 268-271). All types of mineral fibers caused a significant increase of micronucleated cells. The kinetochore analysis revealed that all three types of asbestos and ceramic fibers yielded similar effects. Approximately 50% of the induced micronuclei were kinetochore-negative indicating formation through clastogenic events. Human amniotic cells were much less susceptible than SHE cells to the induction of micronuclei by mineral fibers. This again demonstrates that SHE cells are more susceptible to chromosomal changes than human amniotic fluid cells. The application of fluorescence in situ hybridization (FISH) with tandem DNA probes yielded more detailed information about specific structural chromosome aberrations in the 1 (cen-q12) and 9 (cen-q12) regions and about abnormal numbers of chromosomes in interphase human amniotic fluid cells. Using this FISH approach we found a statistically significant increase of chromosomal breakage in the pericentric heterochromatin regions of chromosomes 1 and 9 in interphase human amniotic cells after exposure to asbestos and ceramic fibers compared to control cells. The number of hyperdiploid cells was also significantly increased. Our results show that asbestos fibers as well as ceramic fibers are inducers of structural and numerical chromosomal aberrations in human amniotic fluid cells.

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is a technique which has recently become widely used for the analysis of chromosome alterations in interphase and metaphase cells. In this report, a polymerase chain reaction (PCR)-based method is described for simultaneously amplifying and labelling probes targeting the alpha- and classical satellite regions of chromosome 9 using either plasmid or genomic DNA. Chromosome-specific probes were generated using readily obtainable plasmid DNA and genomic DNA from a hybrid cell line containing human chromosome 9 in a hamster cell background. The utility of these probes to detect and quantify structural and numerical aberrations in interphase cells was demonstrated using a new multicolor FISH strategy by comparing the frequencies of hyperdiploidy and chromosome breakage affecting the regions targeted by the probes in interphase and metaphase human lymphocytes irradiated during culture. The irradiated cells exhibited a significantly higher frequency of tetrasomy and breakage effecting the centromeric/pericentric region of chromosome 9 as compared with non-exposed cells. In general, similar frequencies of breakage and hyperdiploidy were observed in the interphase and metaphase preparations. These results show that DNA probes for the repetitive sequences in human chromosomes can be easily generated from genomic DNA and that these probes can be effectively used to detect chromosome breakage and aneuploidy in interphase and metaphase lymphocytes in vitro.

The hematopoietic and carcinogenic effects of benzene may result from an interaction of various benzene metabolites. Following the co-administration of phenol and hydroquinone, a synergistic increase in myelotoxicity and genotoxicity has been observed in the bone marrow of mice. To understand the mechanisms underlying these synergistic genotoxic effects we have studied the origin of micronuclei (MN) formed in bone marrow erythrocytes following the co-administration of these two metabolites. Phenol and hydroquinone were administered to male CD-1 mice by i.p. injection three times at 24 h intervals. The frequency of MN was evaluated in bone marrow cells harvested 24 h following the final dose. A marked increase in MN was observed in mice co-administered phenol and hydroquinone, which was significantly greater than that observed with the individual metabolites. Labeling with the CREST antibody and multicolor fluorescence in situ hybridization with the mouse major and minor satellite probes indicated that both chromosomal loss and breakage and occurred. The major increase in MN induced by the phenol and hydroquinone combination originated from breakage in the euchromatic region of the mouse chromosomes. The origin of MN in mice co-administered phenol and hydroquinone differed substantially from that induced by hydroquinone alone, but was almost identical to that seen in MN from benzene-treated mice. These results strongly support the hypothesis that interactive effects among benzene metabolites play an important role in the genotoxic and carcinogenic effects of benzene.

A novel multicolor fluorescence in situ hybridization approach, using an alpha satellite probe which labels the centromeric region on chromosome 1 and a classical satellite probe which targets an adjacent breakage-prone region (1q12), has been used to detect both hyperdiploidy and chromosome breakage in interphase human cells. With the use of this technique significant increases in chromosomal breakage were observed in interphase and metaphase lymphocytes irradiated in vitro. Metaphase analysis indicated that a significant proportion of these breakage events represented potentially stable aberrations such as translocations and inversions. A comparison of frequencies using a single classical satellite probe and the adjacent alpha and classical satellite probes indicated that this tandem label procedure allowed chromosomal breakage to be detected and distinguished from hyperdiploidy in untreated interphase lymphocytes, indicating the potential of this procedure for human biomonitoring. To determine whether this hybridization approach could detect alterations in humans, peripheral blood lymphocytes were obtained from a group of pesticide applicators and mixers and compared with a nonexposed control group. Significant increases in both hyperdiploidy and chromosomal breakage affecting the labeled region on chromosome 1 were observed in the pesticide-exposed group. These results indicate that this hybridization strategy allows hyperdiploidy and chromosomal breakage to be detected rapidly in interphase human cells and may facilitate the detection of chromosomal alterations in human populations exposed to carcinogenic and genotoxic agents using tissues which have not been previously amenable for cytogenetic analysis.

Benzene is a widely recognized human and animal carcinogen. In spite of considerable research, relatively little is known about the genotoxic events that accompany benzene exposure in vivo. To gain insights into the mechanisms underlying the genotoxic effects of benzene, we have characterized the origin of the micronuclei that are formed in bone marrow erythrocytes and splenic lymphocytes of benzene-treated mice using two molecular cytogenetic approaches: (a) fluorescence in situ hybridization with a centromeric DNA probe; and (b) staining with the calcinosis-Raynaud's phenomenon-esophageal dismobility-sclerodactyly-telangiectasia syndrome of scleroderma (CREST) antibody, an antibody recognizing a centromeric protein. Following the p.o. administration of benzene (220 or 440 mg/kg) to male CD-1 mice, a significant increase in micronuclei was observed in the bone marrow erythrocytes. In situ hybridization with a centromeric DNA probe and immunofluorescent staining with the CREST antibody indicated that the micronuclei in bone marrow erythrocytes were formed from both chromosome loss and breakage. The majority of the micronuclei originated from chromosome breakage. A dose-related increase in micronucleated cells was also observed in splenocyte cultures established from these benzene-treated animals. In contrast to the bone marrow erythrocyte results, the majority of benzene-induced micronuclei in the cytokinesis-blocked splenocytes labeled with the CREST antibody indicating that these micronuclei were the result of whole chromosome loss. These data demonstrate that both aneuploidy and chromosomal breakage are early genotoxic events induced by benzene or its metabolites in vivo and also indicate that the nature of the chromosomal alterations may vary depending on the target organ or cell type.

Increased frequencies of structural and numerical chromosomal aberrations have been observed in the lymphocytes of benzene-exposed workers. Similar aberrations occurring in bone-marrow cells may contribute to the increased incidence of leukemia seen in these populations. Fluorescence in situ hybridization with chromosome-specific DNA probes is a relatively new technique which shows promise for the identification of aneuploidy-inducing agents. In these studies, fluorescence in situ hybridization with several chromosome-specific DNA probes was used to investigate the ability of the benzene metabolite hydroquinone to induce hyperdiploidy in interphase human lymphocytes. Using a classical satellite probe specific for human chromosome 9, a significant dose-related increase in the frequency of cells containing 3 or more hybridization regions was observed following the in vitro exposure of lymphocytes to hydroquinone at concentrations from 75 to 150 microM. At the 100-microM concentration of hydroquinone, the frequency of nuclei containing 3 or more hybridization regions was determined using probes for chromosomes 1, 7 and 9. Significantly higher frequencies of affected nuclei were observed using the chromosome 1 and 9 probes when compared to the chromosome 7 probe. To establish whether this difference was due to the nonrandom involvement of these chromosomes in hydroquinone-induced hyperdiploidy or to chromosomal breakage within the chromosomal region targeted by these probes, a multicolor fluorescence in situ hybridization approach was developed using probes to two adjacent regions on chromosome 1. Using this tandem-labeling approach, the frequency of nuclei with multiple hybridization regions and the origin of the regions was determined by scoring slides labeled simultaneously with the chromosome 7 alpha satellite probe and the adjacent alpha and classical satellite probes for chromosome 1. The results of these studies confirmed that hydroquinone exposure resulted in a significant increase in hyperdiploid nuclei, but indicated that the different frequency of nuclei containing 3 or more hybridization regions observed using the chromosome 1 and 7 probes, was due to breakage within the chromosomal region targeted by the chromosome 1 classical satellite probe. These results indicate that hydroquinone may contribute significantly to the numerical and structural aberrations observed in benzene-exposed workers. In addition, the multicolor fluorescence in situ hybridization approach utilized in these studies promises to be a powerful technique for the detection of chromosomal breakage occurring in interphase human cells.

The mechanisms by which two quinone-forming compounds, hydroquinone (HQ) and tert-butyl-hydroquinone (tBHQ), induce chromosomal loss and breakage in a prostaglandin H synthase-containing V79 cell line have been investigated using the cytokinesis-block micronucleus assay with CREST antibody staining. Increased frequencies of CREST-positive micronuclei (indicating chromosome loss) and CREST-negative micronuclei (indicating chromosome breakage) were observed following exposure of cells to HQ and tBHQ. The formation of micronuclei by HQ, but not tBHQ, was dependent on arachidonic acid supplementation, indicating activation by prostaglandin H synthase. Since the oxidation of hydroquinones can result in the generation of oxygen radicals, the contribution of oxygen radicals to the formation of chromosomal alterations induced by HQ and tBHQ was investigated. In the presence of a superoxide-generating system consisting of hypoxanthine and xanthine oxidase, a significant increase in micronucleated cells was observed. These induced micronuclei consisted exclusively of CREST-negative micronuclei and their formation was completely inhibited by pretreatment with catalase. Catalase also significantly inhibited the CREST-negative micronuclei induced by HQ and tBHQ. In addition, glutathione treatment inhibited both CREST-positive and negative micronuclei induced by these phenolic compounds. These results indicate that both chromosome loss and breakage are induced by these two quinone-forming agents. Reactive oxygen species contribute to the chromosomal breakage induced by HQ and tBHQ but the observed chromosomal loss appears to result from other mechanisms such as an interference of quinone metabolites with spindle formation.

Benzene is an established human leukemogen. Workers occupationally exposed to benzene exhibit increased frequencies of both structural and numerical chromosomal aberrations in their peripheral blood lymphocytes. The metabolite(s) responsible for these chromosomal aberrations has not yet been identified. Using a modified micronucleus assay, we have examined the ability of the metabolites of benzene to induce chromosomal damage in human lymphocytes. An antikinetochore antibody was used to distinguish micronuclei that have a high probability of containing a whole chromosome (kinetochore positive) from those containing acentric fragments (kinetochore negative). In vitro treatments with the benzene metabolites hydroquinone, 1,4-benzoquinone, phenol, and catechol resulted in significant increases in micronuclei formation. Phenol, catechol, and 1,4-benzoquinone treatments resulted in moderate (2- to 5-fold) increases in micronuclei, whereas hydroquinone treatments resulted in a larger (11-fold) increase in micronuclei. Significant dose-related increases in kinetochore-positive micronucleated cells were not observed following 1,4-benzoquinone treatment but were observed following treatment with phenol, catechol, and hydroquinone. The higher efficacy of hydroquinone in inducing both total micronuclei and kinetochore-positive micronucleated cells when compared with catechol, phenol, and 1,4-benzoquinone suggests that hydroquinone is a major contributor to the clastogenicity and aneuploidy observed in the lymphocytes of benzene-exposed workers. Other metabolites may also contribute, however, to the genotoxic effects of benzene. Since consistent chromosomal aberrations are often observed in human leukemias, the ability of the phenolic metabolites of benzene to induce chromosomal damage in human cells also implicates them in benzene-induced leukemia.