Elbert B. Whorton

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.

Chromosome aberrations and response to γ-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.

Elevated frequencies of hprt mutant lymphocytes in cigarette-smoking mothers and their newborns.

Maternal cigarette smoking during pregnancy has been associated with increased perinatal mortality and low birth weight. Several epidemiological studies have demonstrated an association between smoking during pregnancy and an elevated risk of hematopoietic cancer in the child, but other studies have failed to confirm this association. We have used an assay for somatic cell mutation to evaluate the in utero effects of exposure to maternal cigarette smoking. Cord blood samples were obtained from 10 newborns whose mothers smoked cigarettes during pregnancy and 10 newborns of non-smoking mothers. Blood samples were also obtained from 5 of the smoking and 5 of the non-smoking mothers. Smoking status was confirmed in all samples by testing the blood plasma for cotinine. The frequency of lymphocytes containing mutations at the hypoxanthine phosphoribosyltransferase (hprt) locus was determined with an autoradiographic assay using cells that had been cryopreserved. The mothers who were smokers had a mean frequency (+/- SE) of 3.08 (+/- 0.55) variant (mutant) cells per 10(6) evaluatable lymphocytes. The frequency (Vf) in non-smokers was 1.07 (+/- 0.17) x 10(-6). The Vf of newborns of smokers was 2.17 (+/- 0.24) x 10(-6), and newborns of non-smokers had a Vf of 0.77 (+/- 0.13) x 10(-6). In both mothers and newborns the difference in Vf between smokers and non-smokers was statistically significant (p < 0.05). Maternal and newborn Vfs were significantly correlated (r = 0.88; p < 0.004), and there was a positive association (r = 0.86; p < 0.001) between the reported number of cigarettes smoked per day and the Vfs. This study provides further evidence that maternal smoking may be hazardous to the future health of children exposed in utero to mutagenic agents in cigarette smoke.

The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice

Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.

Frequencies of hprt mutant lymphocytes in marijuana-smoking mothers and their newborns

Reports of increases in the prevalence of marijuana smoking, especially among young people, have led to concerns about possible genotoxic effects from marijuana use due to exposure to the mutagenic and carcinogenic agents present in marijuana smoke. Prior studies of the adverse health consequences of marijuana smoking, using disease outcomes, have sometimes been confounded by the fact that most marijuana smokers also smoke tobacco. In the present study, the potential mutagenic effects of marijuana smoking were investigated with a somatic cell mutation assay that detects mutations occurring in vivo in the hprt gene. Subjects were volunteers recruited from a prenatal clinic that performs urine drug screens on all consenting patients. Blood samples were collected from 17 subjects whose drug screens indicated marijuana use, but who did not smoke tobacco or use cocaine or opiates, and 17 non-smokers with negative drug screens. Absence of tobacco use was confirmed by plasma cotinine tests. Cord blood samples were collected from newborns of 5 of the marijuana smokers and 5 non-smokers. Lymphocytes were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The frequency of variant (mutant) lymphocytes (Vf) in the 17 non-smokers (+/- standard error) was 1.93 (+/- 0.17) per million evaluatable cells. The Vf of 17 marijuana smokers was more than three-fold higher, 6.48 (+/- 0.48) x 10(-6), a significant difference, p < 0.001. Cord blood lymphocytes from 5 newborns of non-smokers had a Vf of 0.85 (+/- 0.23) x 10(-6), compared to 2.55 (+/- 0.60) x 10(-6) for 5 newborns of marijuana smokers, significantly higher, p < 0.05. Because of the known association between increases in somatic mutations and the development of malignancies, this study indicates that marijuana smokers may have an elevated risk of cancer. For pregnant marijuana smokers, there is also concern for the possibility of genotoxic effects on the fetus, resulting in heightened risk of birth defects or childhood cancer.

Sperm count, morphology and fluorescent body frequency in autopsy service workers exposed to formaldehyde

A battery of monitoring tests that could indicate genetic damage was used to investigate occupational formaldehyde exposure in a population of a hospital autopsy service workers. 11 exposed individuals and 11 matched controls were evaluated for sperm count, abnormal sperm morphology and 2F-body frequency. Subjects were matched for sex, age and customary use of alcohol, tobacco and marijuana. Additional information was collected on health, medications and other exposures to toxins. 10 subjects were employed for 4.3 months (range 1-11 months) prior to the first sample and 1 was employed for several years. Formaldehyde exposures were episodic but with a time weighed average between 0.61 and 1.32 ppm (weekly exposure range 3-40 ppm X h). Exposed and control subjects were sampled 3 times at 2-3 month intervals. Sperm morphology was also evaluated in B6C3F1 mice after 5 daily oral doses of 100 mg/kg formalin. No increase in abnormal morphology was detected in the treated animals. In humans, no statistically significant differences were observed between the exposed and control groups for the observed variables. Reduced sperm count correlated with increased abnormal morphology and 2F-body frequency in the exposed group but not in the control group. Evaluation of the impact of incidental exposures suggests a reduced count with marijuana use and increased abnormal morphology with medications used by controls. No effects on sperm were seen from formaldehyde or its metabolites in this population after occupational exposure, nor in mice following a high acute exposure. It is possible that minor effects might have occurred. The lack of an effect in this study may be due to a lack of statistical power to detect effects at this exposure level.

Variations in the proportion of abnormal cells and required sample sizes for human cytogenetic studies

Abstract A study of cytogenetic aberration rates in human beings was conducted (1) to determine the magnitude of variation in one index of aberration, that of the proportion of abnormal cells, and (2) to estimate the number of subjects and the number of cells per subject needed to detect significant increases of predetermined size over a control value with a type I error of α = 0.05 and typeII errors of β = 0.20 or 0.10. The index of interest, the proportion of abnormal cells, was defined as the ratio of cells with at least one aberration to all cells scored. The study subjects were 239 persons seen for preemployment evaluation at a division of a large U.S. chemical company. 200 cells were scored on each subject. The proportion of abnormal cells for this groups was 0.022. The variance among subjects was found to be significantly larger than the variance with subjects ( p Several combinations of sample size requirements were calculated for the purpose of determining the most reasonable number of subjects per group and cells per subject. The detection of relatively small increases over a control value requires either more subjects or more cells per subject, and from the point of view of the costs associated with the scoring of cells, greater savings will be accomplished in most cases by increasing the number of subjects, rather than increasing only the number of cells per subject.

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.

Dominant lethal effects of n-butyl glycidyl ether in mice.

Using the dominant lethal assay, the ability of n-butyl glycidyl ether to induce mutations in male mice was investigated. No significant dose-related changes either in pregnancy rates or in average number of implants per pregnant female were found. However, while the results were not altogether conclusive, there was evidence of a significant increase in fetal death rates by the end of the first week after the highest dosage was administered.

The influence of multiple scorers on cytogenetics study results

Cytogenetics data resulting from one laboratory in a multiple-laboratory study were analyzed to determine if 5 well-trained scorers produced significantly different results using metaphase scoring procedures. Although the scorers reached the same general conclusion, results show that scorer differences exists (p less than 0.01). Consequently, all participating scorers in a laboratory should be used equally in all treatment groups and the results should be analyzed accordingly to account for scorer variations. This is easily accomplished in controlled prospective experiments; however, it is often difficult in retrospective studies using data which exists. In such studies, every effort should be made to analyze and interpret the data so that scorer differences are taken into account. For severely damaged cells not only were there scorer differences but the difference were greater at higher doses. This phenomenon may be related to the operational definition of a severely damaged cell, since scorers who identify more damage than other scorers would logically tend to classify more cells as severely damaged both overall and at lower doses.