Co-carcinogenic effect of beta-carotene.
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3T3 Cells↗
Biomedical subjects
M S Legator
Publications and source records attributed to M S Legator.
Cytogenetic effects from exposure to mixed pesticides and the influence from genetic susceptibility.
Exposure to pesticides remains a major environmental health problem. Health risk from such exposure needs to be more precisely understood. We conducted three different cytogenetic assays to elucidate the biological effects of exposure to mixed pesticides in 20 Costa Rica farmers (all nonsmokers) compared with 20 matched controls. The farmers were also exposed to dibromochloropropane during the early employment years, and most of them experienced sterility/fertility problems. Our data show that the farmers had consistently higher frequencies of chromosome aberrations, as determined by the standard chromosome aberration assay, and significantly abnormal DNA repair responses (p < 0.05), as determined by the challenge assay, but no statistically significant differences in the tandem-probe fluorescence in situ hybridization (FISH) assay (p > 0.05). Genotype analysis indicates that farmers with certain "unfavorable" versions of polymorphic metabolizing genes (cytochrome P4502E1, the glutathione S-transferases mu and theta, and the paraoxonase genes) had significantly more biological effects, as determined by all three cytogenetic assays, than both the farmers with the "favorable" alleles and the matched controls. A unique observation is that, in individuals who had inherited any of the mentioned "unfavorable" alleles, farmers were consistently underrepresented. In conclusion, the Costa Rican farmers were exposed to genotoxic agents, most likely pesticides, which expressed the induction of biological and adverse health effects. The farmers who had inherited "unfavorable" metabolizing alleles were more susceptible to genotoxic effects than those with "favorable" alleles. Our genotype data suggest that the well-recognized "healthy worker effect" may be influenced by unrecognized occupational selection pressure against genetically susceptible individuals.
Adult↗
Underestimating risk for three important human carcinogens: vinyl chloride, benzene, and butadiene.
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Animals↗
Abnormal DNA repair activities in lymphocytes of workers exposed to 1,3-butadiene.
Exposure to high concentrations of butadiene has been shown to cause cancer among exposed workers. We have conducted a biomarker study to elucidate whether current butadiene exposure conditions are hazardous to workers. Twenty-four workers exposed consistently to butadiene were matched with 19 co-workers who had much less contact with butadiene and who served as our controls. In the standard cytogenetic assay, there was no difference in chromosome aberration frequencies between the exposed and control groups. In the challenge assay, the exposed group shows a consistent, but non-significant, increase in chromosome aberrations indicating some abnormality in DNA repair response. The observed dicentric frequency in the challenge assay (indicative of abnormal repair of damaged chromosomes) is significantly correlated with a butadiene metabolite, 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, in urine (r = 0.52; p = 0.04). Furthermore, cigarette smokers had consistently abnormal repair response compared with non-smokers for both the control and exposed groups. A small subset of the studied workers were evaluated for toxicant-induced DNA repair deficiency using an independent cat-host cell reactivation (CAT-HCR) assay. When cigarette smokers and non-smokers were combined in our analysis, we observed that the exposed group (n = 9) had a significant reduction of DNA repair activities (p = 0.009) compared with the control group (n = 6). Cigarette smoking contributed significantly to the effect as exposed smokers (n = 4) had a significant reduction in DNA repair activities (p = 0.04) compared with exposed non-smokers. The results from the two independently conducted assays support each other and confirm the previously reported abnormal DNA repair response in another group of butadiene workers. In conclusion, our data indicates that exposure to environmental toxicants, such as butadiene, can cause DNA repair defects. Therefore, the current butadiene exposure conditions are still hazardous to workers. However, our data indicates that butadiene is not a potent genotoxic agent. Furthermore, the butadiene-induced effect is significantly enhanced by the cigarette smoking habit.
Adult↗
Two-color ratio-coding of chromosome targets in fluorescence in situ hybridization: quantitative analysis and reproducibility.
Ratio coding in fluorescence in situ hybridizations has the potential to identify more DNA and RNA targets simultaneously using fewer fluorescent labels than other multi-color techniques. Ratio coding uses hybridization probes containing different proportions of two or more distinguishable labels to stain each target. In order to better define the limits of ratio coding applied to chromosome detection, we have 1) examined methods of processing electronic images of ratio-coded hybridizations to increase our ability to visually and quantitatively distinguish different stained targets and 2) examined the reproducibility of target identification. A number of hybridizations were performed using eight ratios of two fluorescent labels to identify whole chromosomes in metaphase spreads, and using five ratios of two labels on repetitive sequence probes to identify chromosomes in metaphase spreads and interphase nuclei. Greater visual discrimination was afforded by color composite images which expanded the color range across the entire visual spectrum. Quantitative ratio measurements on 25 metaphase spreads predicted that eight different chromosomes can be identified simultaneously with 96% accuracy using whole chromosome probes. Analysis of a smaller data set predicted that six different chromosomes can be identified with 97% accuracy using repetitive sequence probes.
Chromosome Mapping↗
Biological monitoring for mutagenic effects of occupational exposure to butadiene.
The use of biological markers in the evaluation of human exposure to hazardous agents has increased rapidly in recent years. Because 1,3-butadiene is a mutagenic carcinogen, existing occupational levels of exposure may be appropriately evaluated using somatic cell mutation as a biomarker. Previously, we have described a biomarker study of workers in a butadiene monomer plant (Ward et al., 1994). We now report results from a second study of the same group of workers, conducted after plant modernization, and present preliminary results from a study of exposures in a styrene butadiene rubber (SBR) plant. Air levels of butadiene were determined using either charcoal tubes with air pumps or passive badge dosimeters. The quantity of a butadiene metabolite in the urine was used as a biomarker of exposure and the mutagenic effects of exposure were measured using the autoradiographic hprt mutant lymphocyte assay. In all three studies, the frequencies of hprt mutants were significantly elevated in workers from the areas of highest exposure when compared to workers from lower exposure areas or non-exposed subjects. The concentration of the urinary metabolite was significantly increased in high-exposed workers in the first study of monomer plant workers but not in the second. In the first monomer plant study, historical air concentrations of butadiene were higher in the production units than in the central control unit. While concurrent determined air concentrations were not elevated in the second monomer plant study, they were elevated in high exposure areas in the SBR plant study. Mutant frequencies in the lower-exposure and the non-exposed groups were consistent with historical values for non-smoking individuals who were not exposed to known mutagens. The use of biomarkers, including the hprt mutant lymphocyte assay, may be of great value in determining an appropriate occupational exposure limit for butadiene.
Butadienes↗
Chromosome damage and DNA repair response in lymphocytes of women who had children with neural tube defects.
Mothers who resided in Brownsville, Texas and who had children with neural tube defects (NTD) were studied to determine whether exposure to environmental mutagens may be a cause of abnormal reproductive outcomes. Peripheral blood lymphocytes from 19 of the mothers who had children with NTD and from 14 matched mothers who had normal children and who resided in Corpus Christ. Texas were investigated using the standard cytogenetic assay and a challenge assay to determine the existence of chromosome aberrations and abnormal DNA repair response. No differences were observed when the spontaneous and the challenged chromosome aberration frequencies were compared between the core and the control groups. Our data suggests that the core group was not exposed to mutagens at levels to cause significant increases of chromosome aberrations or to cause abnormal DNA repair response as determined by our assays. However, exposure to non-mutagenic environmental teratogens cannot be ruled out.
Adult↗
Monitoring populations for DNA repair deficiency and for cancer susceptibility.
The induction of a mutator phenotype has been hypothesized to cause the accumulation of multiple mutations in the development of cancer. Recent evidence suggests that the mutator phenotype is associated with DNA repair deficiencies. We have been using a challenge assay to study exposed populations to test our hypothesis that exposure to environmental toxicants induce DNA repair deficiency in somatic cells. In this assay, lymphocytes were irradiated in vitro to challenge cells to repair the radiation-induction DNA strand breaks. An increase of chromosome aberrations in the challenged cells from toxicant-exposed populations compared to nonexposed populations is used to indicate abnormal DNA repair response. From studies of cigarette smokers, butadiene-exposed workers, and uranium-exposed residents, the assay showed that these exposed populations had mutagen-induced abnormal DNA repair response. The phenomenon was also demonstrated using experimental animals. Mice were exposed in vivo to two different doses of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) and their lymphocytes were challenged with one dose of a radiomimetic chemical, bleomycin, in vitro. These challenged lymphocytes showed an MNNG dose-dependent increase of abnormal DNA repair response. In a population that was potentially exposed to teratogens--mothers having children with neural tube defects--lymphocytes from these mothers did not have the abnormal response in our assay. In studies with patients, we reported that lymphocytes from Down's syndrome patients have the abnormal DNA repair response. Lymphocytes from skin cancer-prone patients (epidermodysplasia verruciformis) have normal response to gamma-ray challenge but abnormal response to UV-light challenge. These patient studies also indicate that the challenge assay is useful in documenting the radiosensitivity of Down's syndrome and the UV sensitivity in EV patients. In most cases, the challenge assay is more sensitive in detecting biological effects than the standard chromosome aberration assay. Our series of studies indicates that the challenge assay can be used to document biological effects from exposure to mutagens and that the effect is an abnormal DNA repair response. This abnormality can increase the risk for development of cancer. The repair deficiency is currently being validated using a plasmid transfection (host-reactivation) assay. The need to integrate chromosome aberration and the challenge assays with other relevant assays for better documentation of biological effects and for more precise prediction of health risk will be presented. Our experience in using genetic polymorphism and host-reactivation assays will be discussed.
Animals↗
Chromosome aberrations and response to gamma-ray challenge in lymphocytes of workers exposed to 1,3-butadiene.
An integrated population monitoring study was initiated to investigate whether occupational exposure to current low levels of butadiene is mutagenic to workers. Ten exposed workers (mean production area concentration of 3.5 ppm) and 10 matched plant controls (mean exposure to 0.03 ppm) were selected and blood samples were collected for our study. The standard cytogenetic assay was used to determine chromosome aberration frequencies. In addition, a challenge assay was used to determine response to gamma-rays as an indication of DNA repair deficiencies. In the latter assay, cells were exposed to gamma-rays at the G1 phase of the cell cycle in vitro and the frequencies of chromosome aberrations in the first post-irradiation metaphase cells were quantitated. Based on results of the cytogenetic assay, the exposed group had a higher frequency of cells with chromosome aberrations and higher chromatid breaks per 100 cells compared with the control. However, the difference was not significant (p > 0.1). With the challenge assay, the exposed group had a higher frequency of aberrant cells (p < 0.04), chromatid breaks (p < 0.05), deletions (p < 0.07), and dicentrics (p < 0.02) than the controls. In addition, the dicentric frequencies from workers were significantly correlated with the presence of a butadiene metabolite [1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane] in urine with a correlation of coefficient of 0.6 (p < 0.01). Two outliers were identified and our interpretation of their responses will be discussed. This study indicates that the workers had exposure-induced mutagenic effects. Together with the observation of gene mutation in a subset of the present population, this study indicates that the current occupational exposure to butadiene may not be safe to workers.
Adult↗
Biomarker monitoring of a population residing near uranium mining activities.
We investigated whether residents residing near uranium mining operations (target population), who are potentially exposed to toxicants from mining waste, have increased genotoxic effects compared with people residing elsewhere (reference population). Population surveys were conducted, and 24 target and 24 reference residents were selected. The selected subjects and controls were matched on age and gender and they were nonsmokers. Blood samples were collected for laboratory studies. The standard cytogenetic assay was used to determine chromosome aberration frequencies, and the challenge assay was used to investigate DNA repair responses. We found that individuals who resided near uranium mining operations had a higher mean frequency of cells with chromosome aberrations and higher deletion frequency but lower dicentric frequency than the reference group, although the difference was not statistically significant. After cells were challenged by exposure to gamma-rays, the target population had a significantly higher frequency of cells with chromosome aberrations and deletion frequency than the reference group. The latter observation is indicative of abnormal DNA repair response in the target population.
Biomarkers↗
Fluorescence in situ hybridization: powerful molecular tool for cancer prognosis.
We review several aspects of fluorescence in situ hybridization (FISH) technology that demonstrate its breadth and power in detecting and monitoring genetic abnormalities associated with cancers. The clinical utility of FISH in disease management is demonstrated in several examples, including trisomy 8 detection with high specificity and sensitivity in patients with myeloid leukemias; trisomy 12 detection with higher efficiency than conventional cytogenetics in patients with chronic lymphocytic leukemia; assessment of engraftment success, chimerism, and relapse in opposite sex bone marrow transplantation; and correlation of trisomy 7 with survival time in patients with prostate tumors. Advances in FISH technology include multicolor analyses, which permit the simultaneous detection of several genetic abnormalities by using cohybridization of probes labeled with several fluorescent labels or label combinations, and comparative genomic hybridization, a relatively new method whereby a single hybridization can reveal aberrations across the entire genome.
Chromosome Aberrations↗
Genetic toxicology: lessons from the past, directions for the future.
Twenty-five years after the founding of the Environmental Mutagen Society, the need to detect germinal cell mutagens remains an unfulfilled goal. Instead we embarked upon a journey to characterize carcinogens by detecting mutations in nonmammalian or in vitro assays. Genetic toxicologists, in an attempt to devise a quick efficient strategy for detecting chemical carcinogens, committed two basic errors. The first error set us off on a 25-year quest for the definitive short-term assay for characterizing carcinogens; the second error, derived from the first, was to focus our efforts mostly toward determining effects in somatic cells rather than also looking for chemically induced effects in germinal cells. The time is now at hand to reestablish the credibility of the field of genetic toxicology and achieve the goals of the initial founders of the society. Having realized the shortcomings of nonmammalian studies, we should devote our efforts to developing suitable germinal cell assays in animals and where possible in humans. In addition to identifying germinal cell mutagens, we should also expand our efforts to evaluate chronic low-level exposures of human populations to potential cancer causing agents, using the rapidly developing methods of genetic biomonitoring.
Animals↗
Application of integrated genetic monitoring: the optimal approach for detecting environmental carcinogens.
Short-term in vitro genetic toxicity assays have not fulfilled their anticipated role in predicting the carcinogenicity of environmental agents reliably and economically. A reduction in emphasis from nonanimal systems to relevant animal assays and population monitoring will help to reestablish the credibility of this field. An analysis of the various steps in the carcinogenic process indicates the biological responses occurring during these stages can be utilized for early detection of environmental carcinogens. Emphasis should be placed on using the earliest significant response that indicates genetic damage (e.g., gene mutations and chromosome alterations). Assays that detect pregenomic damage (e.g., adduct formation), without evidence of subsequent heritable genetic alterations, may produce misleading results for risk assessment and should not be considered as stand-alone monitoring procedures. Late biological responses may occur in tissues or organs where genetic damage may be difficult to measure, and the opportunity for intervention diminishes as we approach the clinical outcome. For example, analyzing localized cells that contain activated protooncogenes and inactivated tumor suppressor genes, although they further document adverse response from exposure to carcinogens, may be of greater value for indicating clinical outcome than for genetic monitoring. With few notable exceptions, the window of opportunity for genetic monitoring is the period after exposure where genetic damage is evident and where circulating lymphocytes can faithfully record this damage. An ongoing study of butadiene-exposed workers illustrates an optimum protocol, where multiple assays can be carried out and correlated with both external and internal measurements of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Butadienes↗
hprt mutant lymphocyte frequencies in workers at a 1,3-butadiene production plant.
1,3-Butadiene is a major industrial chemical that has been shown to be a carcinogen at multiple sites in mice and rats at concentrations as low as 6.25 ppm. Occupational exposures have been reduced in response to these findings, but it may not be possible to determine by using traditional epidemiological methods, whether current exposure levels are adequate for protection of worker health. However, it is possible to evaluate the biological significance of exposure to genotoxic chemicals at the time of exposure by measuring levels of genetic damage in exposed populations. We have conducted a pilot study to evaluate the effects of butadiene exposure on the frequencies of lymphocytes containing mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in workers in a butadiene production plant. At the same time, urine specimens from the same individuals were collected and evaluated for the presence of butadiene-specific metabolites. Eight workers from areas of the plant where the highest exposures to butadiene occur were compared to five workers from plant areas where butadiene exposures were low. In addition, six subjects with no occupational exposure to butadiene were also studied as outside controls. All of the subjects were nonsmokers. An air sampling survey conducted for 6 months, and ending about 3 months before the study, indicated that average butadiene levels in the air of the high-exposure areas were about 3.5 +/- 7.5 ppm. They were 0.03 +/- 0.03 ppm in the low-exposure areas. Peripheral blood lymphocytes from the subjects were assayed using an autoradiographic test for hprt mutations.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Public health policies regarding hazardous waste sites and cigarette smoking: an argument by analogy.
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Environmental Exposure↗
Evaluation of the clastogenic effects of 2-methoxyethanol in mice.
2-Methoxyethanol (ethylene glycol monomethyl ether; EGME) is present in many industrial and consumer products, therefore, many individuals in the population are exposed to EGME. Although the toxicity of this compound is well documented its genotoxicity has not been adequately investigated using updated cytogenetic procedures. We have conducted studies to determine the clastogenic effects of EGME and its metabolite, methoxyacetaldehyde (MALD), in bone-marrow cells of B6C3F1 mice after their acute and subchronic exposure to the chemicals by the oral route. In addition, the effects after acute intravenous treatment with EGME were investigated. Mice treated with cyclophosphamide (CP) under similar experimental conditions were used as positive controls. Mice treated acutely with EGME or MALD were also implanted with bromodeoxyuridine tablets to label cells so that only cells at their first post-treatment mitoses were selected for chromosome analyses. Our data show that none of the concentrations of EGME (35-2500 mg/kg body weight) nor MALD (20-1000 mg/kg) caused any induction of chromosome aberrations even though cytotoxic doses were used. On the other hand, CP caused significant increases in chromosome damage. The data suggest that EGME and MALD are either non-clastogenic in vivo or that our mice are able to detoxify the two chemicals. In order to clarify these possibilities, pharmacokinetic and metabolic studies need to be conducted.
Acetaldehyde↗
Use of haemoglobin adducts for biomonitoring exposure to 1,3-butadiene.
Measurement of specific adducts to haemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in man. Adducts of 1,2-epoxybutene with the N-terminal valine in haemoglobin were determined in male B6C3F1 mice and Sprague-Dawley rats following exposure by inhalation to low concentrations of 1,3-butadiene (0, 2, 10 or 100 ppm, 6 h/day, 5 days/week, 4 weeks; animals killed within 1 h after the last exposure). The adduct level increased linearly with butadiene concentration in the mice, whereas a deviation from linearity was observed in the rats. After exposure to 100 ppm butadiene, the adduct levels were four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Adduct levels of about 1-3 pmol/g globin were recorded in human subjects (nonsmokers) who worked in a production area where butadiene levels of about 1 ppm had been recorded in a survey conducted three to nine months prior to this study. Increased adduct levels were also observed in cigarette smokers (two subjects) who were not exposed occupationally to butadiene. Although preliminary, the data suggest that the adduct levels, and consequently the doses of epoxybutene per parts per million-hour of butadiene are lower in humans than in mice and rats. The adduct levels are much lower than those seen after occupational exposures to corresponding air levels of ethylene oxide and are lower than those seen after exposure to ethylene.
Adult↗
Elevated somatic cell mutant frequencies and altered DNA repair responses in nonsmoking workers exposed to 1,3-butadiene.
Epidemiological evidence indicates that 1,3-butadiene is a human carcinogen; however, the epidemiological studies reflect past levels of exposures in the work place, which in all likelihood were higher than present-day levels. Studies of the metabolism of butadiene suggest that there may be qualitative differences among species in this respect. The question therefore arises as to the mutagenic and carcinogenic effects of butadiene in humans. The biomonitoring study reported here was designed to determine whether current exposure to this chemical in the work place is sufficient to induce mutations and/or to alter DNA repair functions, and to determine further whether levels of urinary metabolites correlate with the effects of exposure to butadiene. Nonsmoking workers in a butadiene production plant, who were exposed to levels of 1-3 ppm butadiene, were evaluated for hprt mutant frequencies, cytogenetic effects, including a challenge test to detect DNA repair deficiencies, and formation of protein adducts. We report here the results of the initial study on hprt mutant frequencies and of the challenge assay and the correlation between the results of these assays and levels of butadiene metabolite in the urine. A single metabolite of butadiene, 1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane, was detected in the urine of all subjects. The concentration of the metabolite in the highly exposed group of workers was significantly higher than that in the group exposed to low concentrations or that in outside control groups. The correlation between the level of the metabolite in urine and the frequency of hprt mutants was r = 0.85 (p < 0.0003). Initial observations from a newly developed cytogenetic assay intended to measure altered DNA repair capability (the challenge assay) indicate a statistically significant increase in the high-exposure group relative to the low-exposure group (p < 0.01). This study thus reinforces and extends the results of the epidemiological studies and indicates that present exposure levels may not be sufficiently low to protect workers. It also provides evidence that levels of the urinary metabolite that was considered to be indicative of insensitivity correlate with the frequencies of somatic mutations and exposure to butadiene.
Butadienes↗