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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.

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.