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Chromosomal aberrations are associated with cancer, birth defects, and pregnancy loss. Previous studies using banding techniques have revealed that chromosomal alterations induced in human peripheral lymphocytes by many genotoxic agents occur nonrandomly throughout the genome. One of the regions prone to breakage is the centromeric heterochromatin of chromosome 1. We have developed a fluorescence in situ hybridization (FISH) procedure using tandem DNA probes to distinguish hyperdiploidy from breakage occurring in this region. Interphase nuclei exhibiting breakage or exchanges affecting the 1cen-1q12 region can readily be distinguished from nuclei hyperdiploid for this chromosome by identifying the number and location of the hybridization signals. This hybridization approach was tested using cultured human lymphocytes treated with a series of known aneuploidy-inducing agents (colchicine, diethylstilbestrol, and vincristine sulfate), several potent clastogens (ionizing radiation, mitomycin C, and etoposide), as well as so-dium arsenite and hydroquinone, agents that have been reported to have relatively weak aneuploidy-inducing and clastogenic activity. Significant increases in chromosomal alterations were seen with all agents tested and the results were generally consistent with those previously seen using standard cytogenetic techniques. Treatment with colchicine, diethylstilbestrol, and vincristine sulfate resulted in high frequencies of primarily hyperdiploid nuclei, and cells exposed to radiation, mitomycin C, and etoposide exhibited elevated frequencies of breakage affecting the 1cen-1q12 region. Sodium arsenite and hydroquinone induced relatively minor but significant increases in both hyperdiploidy and breakage. These results indicate that this tandem labeling approach can be used to distinguish aneuploidy-inducing agents from those causing breakage in interphase human cells and may be a valuable procedure for monitoring human populations exposed to genotoxic agents. Environ. Mol. Mutagen. 29:161–167, 1997. © 1997 Wiley-Liss, Inc.

Chromosomal aberrations are associated with cancer, birth defects, and pregnancy loss. Previous studies using banding techniques have revealed that chromosomal alterations induced in human peripheral lymphocytes by many genotoxic agents occur nonrandomly throughout the genome. One of the regions prone to breakage is the centromeric heterochromatin of chromosome 1. We have developed a fluorescence in situ hybridization (FISH) procedure using tandem DNA probes to distinguish hyperdiploidy from breakage occurring in this region. Interphase nuclei exhibiting breakage or exchanges affecting the 1cen‐1q12 region can readily be distinguished from nuclei hyperdiploid for this chromosome by identifying the number and location of the hybridization signals. This hybridization approach was tested using cultured human lymphocytes treated with a series of known aneuploidy‐inducing agents (colchicine, diethylstilbestrol, and vincristine sulfate), several potent clastogens (ionizing radiation, mitomycin C, and etoposide), as well as so‐dium arsenite and hydroquinone, agents that have been reported to have relatively weak aneuploidy‐inducing and clastogenic activity. Significant increases in chromosomal alterations were seen with all agents tested and the results were generally consistent with those previously seen using standard cytogenetic techniques. Treatment with colchicine, diethylstilbestrol, and vincristine sulfate resulted in high frequencies of primarily hyperdiploid nuclei, and cells exposed to radiation, mitomycin C, and etoposide exhibited elevated frequencies of breakage affecting the 1cen‐1q12 region. Sodium arsenite and hydroquinone induced relatively minor but significant increases in both hyperdiploidy and breakage. These results indicate that this tandem labeling approach can be used to distinguish aneuploidy‐inducing agents from those causing breakage in interphase human cells and may be a valuable procedure for monitoring human populations exposed to genotoxic agents. Environ. Mol. Mutagen. 29:161–167, 1997. © 1997 Wiley‐Liss, Inc.

Diethylstilbestrol (DES) and 17β‐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 μM) and the steroid estrogen E2(0.05–75 μM) 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 μM, whereas E2showed its highest induction at 75 μM 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 E2show 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. Environ. Mol. Mutagen. 31:263–273, 1998 © 1998 Wiley‐Liss, Inc.

The oxidation of phenol catalyzed by human myeloperoxidase and horseradish peroxidase resulted in extensive binding of phenol-derived metabolites to boiled rat liver protein. This binding paralleled closely the removal of phenol from the incubations and was inhibited from 83 to 99% by the addition of the antioxidants, ascorbate and glutathione, suggesting that metabolism and binding were occurring via a one-electron oxidation pathway. Metabolic studies employing both human myeloperoxidase and horseradish peroxidase resulted in the identification of 4,4'-biphenol and diphenoquinone as the principal identifiable metabolites. The addition of reduced glutathione to incubations containing horseradish peroxidase resulted in the formation of two conjugate species. These conjugate species were identified by fast atom bombardment mass spectrometry to be glutathione conjugates of diphenoquinone. The major gluthathione conjugate was identified as 3-(glutathion-S-yl)-4,4'-biphenol by NMR spectroscopy. These results suggest that the formation of highly reactive species through the peroxidase-mediated metabolism of phenol and other phenolic compounds could play an important role in the hematopoietic toxicity observed during chronic benzene exposure.

Epidemiological studies have shown that a high incidence of oral cancers is associated with chewing betel quid. Since chromosomal aberrations are involved in many types of cancers, we investigated whether increased frequencies of chromosomal alterations could be detected in the oral mucosa cells of betel quid chewers as compared to non-chewers. Due to the difficulty in culturing these epithelial cells, we used multicolor FISH with adjacent DNA probes to detect hyperdiploidy and breakage/exchanges affecting the 1cen-q12 region in interphase cells. Buccal mucosa cells from 19 male betel quid chewers and 23 non-chewers were hybridized and 1000 cells per donor were evaluated. A highly significant increase in the frequency of breakage affecting 1cen-1q12 region was observed in the mucosa cells of the chewers as compared to the non-chewers. A good correlation was also seen between breakage and duration of chewing. A modest increase in hyperdiploidy for chromosome 1 was also observed among chewers who had used betel quid for many years. These results indicate that this FISH approach can be useful for human biomonitoring, particularly for detecting alterations in non-dividing cells.

The induction of loss of heterozygosity (LOH) by the environmental carcinogen N-nitrosodimethylamine (NDMA), and the factors that influence the recovery of LOH mutations were studied in two directly related human lymphoblastoid cell lines, AHH-1 (h2E1.v2) and MCL-5. Initially, the NDMA-induced mutation frequency at the heterozygous tk locus in AHH-1 cells was observed to be 5-fold higher in AHH-1 compared with MCL-5. Molecular analysis of NDMA-induced TK- mutants indicated that the induced mutant fraction attributable to small intragenic mutations was similar in both cell lines. However, the induced mutant fraction, because of LOH, was 18-fold greater in AHH-1. In addition, LOH mutations were more extensive among TK- mutants derived from AHH-1 cells. We hypothesized that the increased recovery of large LOH mutations in AHH-1 cells could be attributable to reduced apoptotic capacity, as it has been reported that AHH-1 cells carry a heterozygous mutation in the p53 locus, whereas MCL-5 cells are homozygous wild-type. Analysis of the kinetics of apoptosis showed that the apoptotic response of the AHH-1 cell line was diminished and delayed compared with MCL-5. Based on the analyses presented here, and several recent reports, it is suggested that the recovery of LOH mutations in p53 deficient cell lines is affected not only by abnormalities in cellular apoptotic response, but also involves a number of p53-mediated responses to DNA damage.

Nitrosodimethylamine (NDMA) is a potent mutagen and animal carcinogen to which many people are exposed through the consumption of contaminated food and the use of tobacco products. Although the mutational specificity of NDMA has been studied in bacteria, little is known about the specific types of mutations induced by NDMA in the human genome. Knowledge of the mutational spectrum of NDMA in human genes may help to substantiate the role of NDMA in the etiology of human cancers. In the current study, the mutational spectrum of NDMA was characterized at the tk and hprt loci, in human lymphoblastoid cells capable of metabolically activating NDMA. A number of patterns were observed among NDMA-induced mutations. At both marker loci, G;C→A:T transitions dominated the mutational spectrum of NDMA, which were indicative of the mutagenicity of the O⁶meG lesion. In addition, the majority of G:C→A:T mutations occurred at guanines 3' to another guanine. Almost all of these mutations originated on the non-transcribed strand, which suggests that transcription-coupled repair influenced the distribution of G:C→A:T transitions at the tk and hprt loci. Furthermore, the observation of hotspots for G:C→A:T mutations, within both loci, suggests that differential repair kinetics may exist, and consequently affect the distribution of mutations. Finally, a comparison of the site specificity of G:C→A:T mutations at the tk and hprt loci, indicated that the gene used for mutational analysis influenced the site specificity of NDMA-induced mutations, and possibly reflects the number of 5'-GG-3' sites in the tk and hprt loci that when mutated would yield a mutant phenotype.

Diethylstilbestrol (DES) and 17β-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 μM) and the steroid estrogen E2 (0.05–75 μM) 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 μM, whereas E2 showed its highest induction at 75 μM 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. Environ. Mol. Mutagen. 31:263–273, 1998 © 1998 Wiley-Liss, Inc.

o-Phenylphenol (OPP) and its sodium salt (SOPP) are broad spectrum fungicides and antibacterials to which humans are frequently exposed. Both OPP and SOPP have been found to cause cancer in the urinary bladder of male F344 rats at high doses, and the metabolite phenylhydroquinone (PHQ) is believed to play a key role in the carcinogenicity of these compounds. Tumor formation in the treated animals has also been shown to be significantly influenced by urinary pH. To provide additional insights into the mechanisms of OPP carcinogenesis, we have investigated the autoxidation of PHQ over the pH range commonly found in the urine of OPP- and SOPP-treated rats. Over the pH range studied (6.3−7.6), a curvilinear relationship between rate of PHQ oxidation and pH was observed. Phenylbenzoquinone (PBQ) was formed during the autoxidation of PHQ, with a formation yield of 0.92 ± 0.02. In addition, the effects of PBQ and oxygen concentrations on PHQ autoxidation and the nonenzymatic conversion of PBQ to PHQ were also studied. Our data indicate that the production of reactive metabolites from PHQ involves a pH-independent (i.e., oxygen-dependent) and a pH-dependent pathway and that the rate of pH-dependent PHQ autoxidation was found to be enhanced by the presence of PBQ. A reaction mechanism has been formulated to explain the experimental data observed, with ionization of PHQ semiquinone being identified as a key step in reactive species production for the pH-dependent pathway. By combining data from OPP animal carcinogenicity studies with the proposed reaction pathway, a good correlation between the proposed formation of reactive species and bladder lesions was observed. These results indicate that the pH-dependent autoxidation of free PHQ metabolite in the urine may potentially be responsible for the tumorigenic effects of OPP and SOPP observed in the rat bladder.

N-Nitrosodimethylamine (NDMA) is a potent mutagen and animal carcinogen to which many people are exposed through the consumption of contaminated food and the use of tobacco products. Although the mutational specificity of NDMA has been studied in bacteria, little is known about the specific types of mutations induced by NDMA in the human genome. Knowledge of the mutational spectrum of NDMA in human genes may help to substantiate the role of NDMA in the etiology of human cancers. In the current study, the mutational spectrum of NDMA was characterized at the tk and hprt loci, in human lymphoblastoid cells capable of metabolically activating NDMA. A number of patterns were observed among NDMA-induced mutations. At both marker loci, G:C-->A:T transitions dominated the mutational spectrum of NDMA, which were indicative of the mutagenicity of the O6meG lesion. In addition, the majority of G:C-->A:T mutations occurred at guanines 3' to another guanine. Almost all of these mutations originated on the non-transcribed strand, which suggests that transcription-coupled repair influenced the distribution of G:C-->A:T transitions at the tk and hprt loci. Furthermore, the observation of hotspots for G:C-->A:T mutations, within both loci, suggests that differential repair kinetics may exist, and consequently affect the distribution of mutations. Finally, a comparison of the site specificity of G:C-->A:T mutations at the tk and hprt loci, indicated that the gene used for mutational analysis influenced the site specificity of NDMA-induced mutations, and possibly reflects the number of 5'-GG-3' sites in the tk and hprt loci that when mutated would yield a mutant phenotype.