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D A Eastmond

Publications and source records attributed to D A Eastmond.

At least 19 recordsLinked to original sources
PubMed2006

Micronuclei and cell proliferation as early biological markers of ortho-phenylphenol-induced changes in the bladder of male F344 rats.

ortho-Phenylphenol (OPP) and its sodium salt, sodium ortho-phenylphenate (SOPP), are widely used fungicides and antibacterial agents known to cause tumors in the bladders of male F344 rats. Previous studies in our laboratory have shown that micronuclei and cell proliferation were induced in the bladders of treated rats by a high dose of OPP. In our present studies, we investigated the relationship in dose response between these two biomarkers and previously reported tumor formation in the bladders of male F344 rats. Significant non-linear increases in micronuclei (MN) and BrdU-labeling were seen in the bladder cells of rats treated with the 8000 and 12,500 ppm doses of OPP and at 20,000 ppm SOPP. CREST anti-kinetochore staining showed that the micronuclei originated from both chromosomal loss and breakage. In addition, increases in MN were detected in the bladder but not in the bone marrow, underscoring the value of assessing genotoxicity in the target organ. In summary, these studies clearly show that at high doses, OPP and SOPP are genotoxic to the rat bladder. These results also indicate that micronucleus formation and cell proliferation can detect early OPP-induced changes in the rat bladder and may be useful as biomarkers for bladder carcinogens.

Agrochemicals↗
PubMed2005

Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-dioxane in the bone marrow and liver of young CD-1 mice.

1,4-Dioxane, a widely used industrial chemical and rodent hepatocarcinogen, has produced mixed, largely negative results in the mouse erythrocyte micronucleus assay. In contrast, a recent report has indicated that 1,4-dioxane induces micronuclei in mouse hepatocytes following in vivo treatment. The objective of this study was to confirm these earlier results and identify the origin of the induced micronuclei. Following an initial range-finding study, mice were administered 1,4-dioxane by gavage at doses ranging from 1500 to 3500 mg/kg. The test animals were also implanted with BrdU-releasing osmotic pumps to allow cell proliferation to be measured in the liver and to increase the sensitivity of the hepatocyte assay. Upon sacrifice, the frequency of micronuclei in the bone marrow erythrocytes and in the proliferating BrdU-labeled hepatocytes was determined. Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80-90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed. Based on these results, we conclude that at high doses: (i) dioxane exerts genotoxic effects in both the mouse bone marrow and liver; (ii) the induced micronuclei are formed primarily from chromosomal breakage; and (iii) dioxane can interfere with cell proliferation in both the liver and bone marrow.

Animals↗
PubMed2005

Chromosomal malsegregation and micronucleus induction in vitro by the DNA topoisomerase II inhibitor fisetin.

The plant flavonol fisetin is a common dietary component that has a variety of established biological effects, one of which is the inhibition of the enzyme DNA topoisomerase II (topo II). Compounds that inhibit topo II can exert genotoxic effects such as DNA double strand breaks, which can lead to the induction of kinetochore- or CREST-negative micronuclei. Despite reports that fisetin is an effective topoisomerase II inhibitor, its genotoxic effects have not yet been well characterized. Genotoxicity testing of fisetin was conducted in TK6 and HL60 cell lines and the cells were analyzed for malsegregating chromosomes as well as for the induction of micronuclei. Using the cytokinesis-blocked CREST micronucleus assay to discriminate between micronuclei formed from chromosomal breakage (CREST-negative) and chromosomal loss (CREST-positive), a statistically significant increase in CREST-positive micronuclei was seen for all doses tested in both cell lines. CREST-negative micronuclei, however, were significantly increased at the higher test concentrations in the TK6 cell line. These data indicate that at low concentrations fisetin is primarily exerting its genotoxic effects through chromosomal loss and that the induction of DNA breaks is a secondary effect occurring at higher doses. To confirm these results, the ability of fisetin to inhibit human topoisomerase II-alpha was verified in an isolated enzyme system as was its ability to interfere with chromosome segregation during the anaphase and telophase periods of the cell cycle. Fisetin was confirmed to be an effective topo II inhibitor. In addition, significant increases in the number of mis-segregating chromosomes were observed in fisetin-treated cells from both cell lines. We conclude that fisetin is an aneugen at low concentrations capable of interfering with proper chromosomal segregation and that it is also an effective topo II inhibitor, which exerts clastogenic effects at higher concentrations.

Chromosomes, Human↗
PubMed2003

Evaluation of hyperdiploidy in the bladder epithelial cells of male F344 rats treated with ortho-phenylphenol.

Ortho-phenylphenol (OPP) is a broad-spectrum fungicide and anti-bacterial agent that has been shown to cause bladder cancer in male F344 rats. An earlier study to investigate the potential role of aneuploidy in OPP-induced bladder carcinogenicity, failed to detect increases in frequencies of hyperdiploidy/polyploidy in treated animals, presumably due to the presence of polyploid cells in the bladder. To overcome this problem, we utilized a novel approach to determine increases in numerical alterations in the slowly dividing replicating cells of the rat bladder following treatment with OPP. Collagenase digestion of the bladder was used to enrich for actively-dividing cells and FISH in conjunction with BrdU was employed to detect hyperdiploidy in the replicating interphase cells. Initial studies were performed using FISH with a chromosome 4 probe. Follow-up studies were conducted with OPP and a positive control, vinblastine sulfate using probes for chromosomes 4 and 19. No significant increases in hyperdiploidy/polyploidy were seen in the replicating bladder cells of the OPP-treated rats using FISH with either the chromosome 4 or 19 probes. As expected, no significant increases in hyperdiploidy were seen in the non-replicating cells. In contrast, highly significant increases in hyperdiploidy/polyploidy, as detected using FISH with probes for either chromosome 4 or 19, were seen in the replicating cells from rats treated with a combination of OPP and vinblastine. The inability to detect increases in hyperdiploidy/polyploidy in the bladder of OPP-treated rats indicates that chromosome gain is unlikely to play a major role in the early genotoxic effects of OPP. However, the increase in hyperdiploidy/polyploidy induced by vinblastine sulfate in OPP-treated rats, clearly demonstrates that this approach using FISH in combination with BrdU is capable of detecting changes in chromosome number even in slowly-dividing tissues, such as the urinary bladder.

Animals↗
PubMed2003

Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points.

The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9-120 micro g/ml) in the presence of either cytochalasin B (CYB) (3 micro g/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 micro M). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific alpha-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents.

Aneugens↗
PubMed2002

Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker.

Aneuploidy is associated with spontaneous abortions, birth defects, and many types of human cancers. Currently there are few assays developed for the efficient detection of aneuploidy in vivo. However, with the recent availability of chromosome-specific DNA probes for the rat, fluorescence in situ hybridization (FISH) techniques could be used for the rapid and sensitive detection of aneuploidy in different tissue and cell types. In order to develop a system that can detect alterations in chromosome number in rat cells in vitro, we treated cultured rat lymphocytes with three aneugens-noscapine hydrochloride (0-150 microM) and vincristine and vinblastine sulfate (0-0.06 microM). 5-Bromo-2-deoxyuridine (BrdU; 1 microM) was added to the culture medium to allow proliferating and non-proliferating cells to be distinguished. To test this assay under in vivo conditions, 21-day-old male Sprague-Dawley rats were subcutaneously implanted with osmotic pumps that delivered BrdU (approximately 12 mg/kg per day) continuously. These rats were administered vinblastine sulfate (0, 0.5 and 1mg/kg) by intraperitoneal injection. The rat lymphocytes and hepatocytes incorporating BrdU were detected by immuno-fluorescent labeling, and FISH with a rat chromosome 4 probe was performed on the labeled and unlabeled cells. Highly significant increases in hyperdiploidy were seen in the replicating rat lymphocytes treated with noscapine, vincristine or vinblastine in vitro and in the rat hepatocytes treated with vinblastine in vivo. In contrast, no significant increase in hyperdiploidy was observed in the non-replicating cells. These results demonstrate that this BrdU-enhanced FISH assay with chromosome-specific rat probes can be used to efficiently detect numerical chromosomal aberrations in vitro and in vivo in slowly or moderately replicating rat tissues. The combination of BrdU-labeling and FISH allows the scoring of hyperdiploidy to be focused on the actively replicating cells, thereby increasing the sensitivity of the FISH technique.

Aneuploidy↗
PubMed2002

Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o-phenylphenol.

o-Phenylphenol (OPP), a widely used fungicide and antibacterial agent, has been considered to be among the top 10 home and garden pesticides used in the USA. Earlier studies have consistently shown that the sodium salt of OPP (SOPP) causes bladder cancer in male Fischer 344 (F344) rats, whereas OPP has produced variable results. This difference has been attributed to the presence of the sodium salt. To determine cellular and genetic alterations in the rat bladder and the influence of the sodium salt, F344 rats were administered 2% OPP, 2% NaCl and 2% NaCl + 2% OPP in their diet for 14 days. Twenty-four hours before being killed the animals were administered 5-bromo-2'-deoxyuridine (BrdU) by i.p. injection. Bladder cells were isolated, stained with DAPI and scored for the presence of micronuclei and incorporation of BrdU into replicating cells. To determine changes in chromosome number, we used fluorescence in situ hybridization (FISH) with a DNA probe for rat chromosome 4. Significant increases in the frequency of micronuclei and BrdU incorporation were seen in bladder cells of rats from all treatment groups. In contrast, the frequency of hyperdiploidy/polyploidy in treated animals was not increased over that seen in controls. A high control frequency of cells with three or more hybridization signals was seen, probably due to the presence of polyploid cells in the bladder. The presence of polyploid cells combined with cytotoxicity and compensatory cell proliferation makes it difficult to determine whether OPP is capable of inducing aneuploidy in the rat urothelium. In summary, these studies show that OPP can cause cellular and chromosomal alterations in rat bladder cells in the absence of the sodium salt. These results also indicate that at high concentrations the sodium salt can enhance chromosomal damage in the rat urothelium.

Aneuploidy↗
PubMed2001

Characterization and mechanisms of chromosomal alterations induced by benzene in mice and humans.

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.

Adult↗
PubMed2000

The genotoxicity of 3-nitrobenzanthrone and the nitropyrene lactones in human lymphoblasts.

Polycyclic aromatic hydrocarbons (PAH) and nitrated polycyclic aromatic compounds (nitro-PAC) have been found to be mutagenic in bacterial and human cells as well as carcinogenic in rodents. In this investigation, the genotoxic effects of 3-nitrobenzanthrone (3NB) and a mixture of nitropyrene lactones (NPLs) were determined using forward mutation assays performed in two human B-lymphoblastoid cell lines, MCL-5 and h1A1v2, which are responsive to the nitro-PAC class of compounds. Mutagenicity of the compounds was determined at the heterozygous tk locus and the hemizygous hprt locus, thus, identifying both large-scale loss of heterozygosity (LOH) events as well as intragenic mutagenic events. Genotoxicity was also determined using the CREST modified micronucleus assay, which detects chromosomal loss and breakage events. Results indicate 3NB is an effective human cell mutagen, significantly inducing mutations at the tk and hprt loci in both cell lines, and inducing micronuclei in the h1A1v2 cell line. The NPL isomers are also mutagenic, inducing mutations at the two loci as well as micronuclei in both cell lines. Because of their mutagenic potencies and their presence in ambient air, further assessments should be made of human exposures to these nitro-PAC and the potential health risks involved.

B-Lymphocytes↗
PubMed1999

Persistence of chromosomal alterations affecting the 1cen-q12 region in a human lymphoblastoid cell line exposed to diepoxybutane and mitomycin C.

Multicolor fluorescence in situ hybridization (FISH) with tandem-labeling probes for the 1cen-q12 region is a potential biomarker for the detection of structural chromosomal aberrations (CAs) in human cells. To determine the suitability of this technique for biomonitoring humans exposed to 1,3-butadiene (BD) and to characterize the alterations induced as well as their stability over time, the human lymphoblastoid cell line AZH-1 was treated with 5 microM diepoxybutane (DEB) or the positive control mitomycin C (MMC; 0.1 microM) for 24 h. Following the removal of the test chemicals, cell cultures were grown for an additional 19 days in the absence of the test compound. Using the tandem FISH technique, aliquots from the main cultures were examined for the induction of CAs affecting the 1cen-q12 region at various intervals. A significant increase in chromosomal breakage/exchanges affecting the 1cen-q12 region was seen in both the DEB- and MMC-treated interphase and metaphase cells. The damage peaked at approximately 48 h following the addition of the test compound and declined with time. However, at day 20, the frequency of aberrant cells was still significantly higher than the control levels. For comparison, the frequency of micronuclei (MN) formed and their origin was determined using the cytochalasin B-modified MN assay and FISH with a pancentromeric probe. Showing a similar pattern, the frequency of centronere-negative MN peaked at 48 h, but however was not significantly elevated above control levels at 20 days. At early time points, aberrations detected using the FISH assay consisted of nearly equal proportions of unstable- and stable-type aberrations, while at the later time points, translocations were the predominant aberration type. In addition, the use of tandem-label FISH in combination with BrdU-immunfluorescence staining, showed that almost identical frequencies of structural aberrations could be seen in actively replicating and non-replicating cell populations. These studies indicate that a small but significant proportion of the alterations detected using this FISH technique persists over time and that this technique may be valuable for biomonitoring chromosomal alterations in BD-exposed populations.

Bromodeoxyuridine↗
PubMed1999

Evidence for oxidative metabolism in the genotoxicity of the atmospheric reaction product 2-nitronaphthalene in human lymphoblastoid cell lines.

2-Nitronaphthalene (2NN) has been identified as a mutagenic atmospheric reaction product of naphthalene in the Ames bacterial reversion assay. Recent experiments have shown this nitroarene to be genotoxic in a human lymphoblastoid cell line (MCL-5) transfected with plasmids encoding epoxide hydrolase and four cytochrome P450 monooxygenase activities. The present study investigated the genotoxicity of 2NN in two related human B-lymphoblastoid cell lines, h1A1v2 containing a single P450 isozyme (cytochrome P450 1A1) and L3 cells which are isogenic with MCL-5 cells and are distinguished only by the absence of transfected plasmids. The results indicate that 2NN-induced mutagenesis at the heterozygous thymidine kinase (tk) locus was dependent on metabolic activities provided by the transfected plasmids in MCL-5; no significant induction of mutants was observed in L3 cells studied in parallel. A similar induction of mutation was observed in h1A1v2 and MCL-5 cell lines at the tk locus and no induction was observed at the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus. The induction of mutations in h1A1v2 cells suggests that cytochrome P450 1A1 alone can activate 2NN to a mutagenic species, however, this interpretation may be confounded by differences between the h1A1v2 and MCL-5 cell lines. The observed genotoxic activity induced by 2NN prompted testing of the amino analogue, beta-naphthylamine (betaNA), to investigate potential similarities in the metabolic activation pathways of the two compounds. The negative response of betaNA in all cell lines suggests that 2NN and betaNA are not activated in these human cells by similar metabolic pathways.

2-Naphthylamine↗
PubMed1999

Dose-dependent binding of ortho-phenylphenol to protein but not DNA in the urinary bladder of male F344 rats.

ortho-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that is also known to be highly effective in inducing bladder tumors in male F344 rats. At present, neither the role of the urinary bladder in the bioactivation of OPP metabolites nor the nature of the molecular target is understood. To address these issues, we investigated the relationship between OPP dosage and macromolecular adduct formation in the urinary bladder of male F344 rats. Male F344 rats were treated with 0, 15, 50, 125, 250, 500, 1000 mg/kg of OPP and its radiocarbon analogue via oral gavage. The dosed rats were euthanized after 24 h, and the proteins were extracted from the liver, kidney, and bladder. The amount of radioactivity associated with the extracted protein was quantified using highly sensitive accelerator mass spectrometry. Protein binding in liver and kidney exhibited a linear or modest curvilinear relationship over the dose range studied. In the urinary bladder, however, a pronounced nonlinear relationship between protein adduct levels and administered dose was observed. The measured protein adduct levels were in agreement with the predicted concentrations of phenylbenzoquinone based on a proposed mechanism involving free phenylhydroquinone autoxidation in the urine. Unlike protein binding, DNA adducts measured from the same bladder samples did not show a significant difference from the control group. These data are consistent with the hypothesis that OPP is an indirect acting carcinogen, and that regenerative hyperplasia due to OPP-metabolite cytotoxicity and/or binding of OPP metabolites to protein targets may play an important role in OPP-induced bladder carcinogenesis.

Animals↗
PubMed1999

Noscapine hydrochloride disrupts the mitotic spindle in mammalian cells and induces aneuploidy as well as polyploidy in cultured human lymphocytes.

Noscapine hydrochloride is a centrally acting antitussive opium derivative widely used in cough suppressants. Recent studies have reported that noscapine is a potent inducer of polyploidy but not of aneuploidy in vitro. To obtain more comprehensive information about the cytogenetic effects of this compound, we treated cultured human lymphocytes (HPL) and Chinese hamster ovary (CHO) cells with various concentrations of noscapine hydrochloride. Using a differential staining technique noscapine was shown to disrupt the mitotic spindle at concentrations < 5 micrograms/ml in both cell types. The use of multicolor fluorescence in situ hybridization (FISH) on noscapine-treated human lymphocytes showed a dose-dependent induction of hyperdiploidy of chromosome 1 but not of chromosomal breakage in the 1cen-q12 region under in vitro conditions, indicating that noscapine is specifically inducing numerical chromosomal aberrations. FISH with probes targeting different chromosomes revealed that noscapine is capable of inducing both polyploidy and true hyperdiploidy. Our results show that noscapine, by disrupting the function of the mitotic spindle, has the ability to induce aneuploidy and not uniquely polyploidy as previously reported. By using these types of molecular cytogenetic techniques, it should be possible to evaluate the ability of noscapine to induce aneuploidy in the upper intestinal tract in vivo.

Aneuploidy↗
PubMed1999

Evaluation of micronuclei and chromosomal breakage in the 1cen-q12 region by the butadiene metabolites epoxybutene and diepoxybutane in cultured human lymphocytes.

1,3-Butadiene is a widely used industrial chemical and common environmental pollutant that has been associated with increased risks of leukemias and lymphomas. Butadiene and its metabolites, 1, 2-epoxybutene (EB) and diepoxybutane (DEB), have been shown to be genotoxic in a wide variety of test organisms. The objective of this research was to evaluate techniques for the rapid detection of chromosomal alterations occurring in humans exposed to butadiene. We have used a multicolored fluorescence in situ hybridization (FISH) method and the CREST-modified micronucleus assay to detect chromosomal breakage induced by EB (10-300 microM) and DEB (0.5-10 microM) in cultured human lymphocytes. A significant dose-related increase in the formation of micronuclei was seen in lymphocytes treated with DEB at concentrations as low as 2.5 microM, but not with EB over the dose range tested. Over 80% of the micronuclei induced by DEB were CREST-negative, indicating their origin from chromosomal breakage. Multicolor FISH using two adjacent chromosome-specific probes showed a significant increase in chromosomal breakage in the 1cen-q12 region induced by DEB at concentrations as low as 2.5 microM, but not by EB. Since DEB is likely to be one of the metabolites contributing to the genotoxic effects of butadiene, the sensitivity of the tandem FISH approach to detect breakage induced by diepoxybutane indicates that this technique may be useful for monitoring chromosomal alterations in butadiene-exposed workers.

Butadienes↗
PubMed1999

Evaluation of the potential health effects of the atmospheric reaction products of polycyclic aromatic hydrocarbons.

The genotoxic risks from exposure to polycyclic aromatic hydrocarbons (PAHs) have long been recognized. Less well understood are the potential genotoxic risks of the atmospheric reaction products of this class of compounds. In this investigation, we have utilized several human cell assays to evaluate the genotoxicity of naphthalene, phenanthrene, and their atmospheric reaction products 1-nitronaphthalene, 2-nitronaphthalene (2NN), 1-hydroxy-2NN, 2-hydroxy-1-nitronaphthalene, 1,4-naphthoquinone, and 2-nitrodibenzopyranone (2NDBP). In addition, simulated atmospheric reaction products of naphthalene were generated in a 6,700 liter (L) Teflon environmental chamber, collected on a solid adsorbent, extracted, and fractionated by normal-phase high-performance liquid chromatography (HPLC). Individual fractions were then analyzed using gas chromatography/mass spectrometry (GC/MS), and tested for genotoxic effects. Genotoxicity was primarily determined using the human B-lymphoblastoid cell line, MCL-5, which expresses several transfected P450 and epoxide hydrolase genes. Mutagenicity was evaluated at both the heterozygous thymidine kinase (tk) locus and the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus, permitting detection of both intragenic and chromosomal scale mutational events. Test compounds were also screened using the CREST modified micronucleus assay. The results indicate that 2NN and 2NDBP possess greater mutagenic potency than their parent compounds, and, interestingly, both compounds induced significant increases in mutation frequency at the tk but not the hprt locus. These findings suggest a mechanistic difference in human cell response to 2NN and 2NDBP as compared to bacteria, where both compounds were previously shown to induce point mutations in the Salmonella typhimurium reversion assay. The genotoxicity of 2NN and 2NDBP in human cells, together with their high concentrations in ambient air relative to nitro-PAHs directly emitted from combustion sources, emphasizes the need to consider atmospheric reaction products of PAHs in assessments of the genotoxicity of air pollutants. We also investigated whether transfected cytochrome P450 monooxygenase activities were required to activate 2NN and 2NDBP to genotoxic species, and whether a single enzyme could be sufficient for metabolic activation. Three directly related cell lines with multiple (MCL-5), single (AHH-1 1A1), or no (L3) transfected cytochrome P450 genes were used. AHH-1 is additionally distinguished by elevated mutagenic response at the tk locus, a heterozygous mutation in p53, and apoptosis capacity. The effect of these metabolic and genetic differences on genotoxicity of 2NN, 2NDBP, and beta-naphthylamine (beta NA) was also investigated. The results indicated that 2NN and 2NDBP were not activated to genotoxic species through nitroreduction pathways. Mutagenicity induced at the tk locus was dependent on oxidative metabolism, provided by transfected cytochrome P450 enzymes in MCL-5 and AHH-1 1A1. Mutagenicity was not observed in the L3 cell line, which does not carry transfected cytochrome P450 activities. The negative response of beta NA in all cell lines indicates that, contrary to previous hypotheses, 2NN and beta NA are not activated by similar metabolic pathways in these human cell lines. Taken as a whole, these results suggest that the genotoxicity of nitro-PAHs in human cells requires oxidative metabolism.

Air Pollutants↗
PubMed1998

Dose-response studies of the induction of hyperdiploidy and polyploidy by diethylstilbestrol and 17beta-estradiol in cultured human lymphocytes using multicolor fluorescence in situ hybridization.

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.

Benzimidazoles↗
PubMed1998

Sequence specific mutations induced by N-nitrosodimethylamine at two marker loci in metabolically competent human lymphoblastoid cells.

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.

B-Lymphocytes↗