Jonathan B. Ward

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.

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.

The identification and characterization of a urinary mutagen resulting from cigarette smoke

The urine of a cigarette smoker who excretes exceptionally mutagenic urine was analyzed for several factors affecting mutagenicity. S. typhimurium strain TA98 was always more sensitive to XAD-2 urine concentrates than TA100. With TA98, as high as 85 revertants per ml of urine were produced. It was observed that incubation with beta-glucuronidase was not required for expression of mutagenicity but that a complete S9 mix was needed to convert the material in the concentrate to the ultimate mutagenic species. TLC and HPLC separation of the XAD-2 urine concentrate resulted in the identification of trace amounts of the bladder carcinogen, 2-aminonaphthalene (beta-naphthylamine) and a considerable amount of a possible metabolite of 2-aminonaphthalene, 2-amino-7-naphthol. The identity of the compounds was confirmed by mass spectral analysis, and 2-amino-7-naphthol was shown to be a mutagen for TA100 and TA98 when activated by rat-liver S9.

Genetic monitoring of aluminum workers exposed to coal tar pitch volatiles

A group of 50 workers exposed to coal tar pitch volatiles (CTPV) in an aluminum reduction plant and a group of 50 non-exposed workers were selected to evaluate the genotoxic effects of CTPV exposure. A battery of tests was performed on 3 different body fluids; urine, blood and semen. Urine samples were evaluated for mutagenic constituents using the Ames/Salmonella assay. Cultured lymphocytes from blood samples were used to perform cytogenetic analysis. Semen samples were used to measure sperm count, percent abnormal sperm morphology and frequency of sperm carrying double fluorescent bodies (2-F). 14 of 28 (50%) exposed workers and 7 of 36 (19.4%) non-exposed workers had mutagenic urine. This difference was significant (p less than 0.01). Among the non-smokers a significantly higher percentage of workers who were exposed had positive urine (36%) compared to the non-exposed workers (5%) (p less than 0.05). Among the exposed group, more mechanics had mutagenic urine than did other types of workers. Overall chromosome aberration rates were similar in both exposed and non-exposed workers. Among exposed workers a significant inverse correlation (p less than 0.05) between age and chromatid aberration rate was observed. Results of semen analysis failed to detect differences between exposed and non-exposed workers. Results of these tests lend support to a battery approach to genetic monitoring and suggest a link between exposure to CTPV and genotoxic effects. Detection of exposure to mutagens at an early time offers an opportunity for disease prevention by the reduction of exposure.

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.

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.

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carcinogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1-10 parts per million for man and 0.04-0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and in characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risks associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal.(ABSTRACT TRUNCATED AT 400 WORDS)

Absence of mutagenicity in the urine of autopsy service workers exposed to formaldehyde: factors influencing mutagenicity testing of urine

SummaryHospital autopsy service workers and a matched control group were studied using a battery of genetic monitoring tests performed on samples of blood, semen and urine. The results of the analysis of urine for mutagens are described in this report. The participants in the study were matched with the controls for sex, age and their use of alcohol, tobacco and marijuana. Information was collected on general health, usage of medications and any exposure which might affect the outcome of the study. Individuals were sampled three times at approximately two month intervals. Time weighed average exposures to formaldehyde in the work areas were estimated at 0.61 to 1.32 ppm. Additionally, studies were carried out which examined various parameters affecting the testing of human urine samples for mutagenicity.No increase in mutagenicity was seen in the autopsy workers as compared to the control group. One individual who was receiving metronidazole and one control who smoked two packs of cigarettes per day had significantly mutagenic urine. A large proportion of the exposed individuals had toxic urine while only two of the control individuals had similar toxic urine. The material responsible for the toxicity has been isolated and purified but does not appear to be related to the formaldehyde exposure.Studies on the parameters affecting mutagenicity testing of urine with Salmonella typhimurium suggest that in the plate incorportion assay, TA100, but not TA98, can be affected by exogenous histidine. Furthermore, with the conditions employed in this study, 3 to 4% of labeled histidine added to urine samples was retained by the XAD-2 and subsequently eluted in the urine concentrate. Urinary histidine levels of unconcentrated samples ranged from 112 to 2614 nmol per ml (mean 994 nmol per ml) and the amount of histidine present correlated with the corresponding increases in histidine revertants with strain TA100.

Development of a rodent lung macrophage chromosome aberration assay

Lung macrophages are the first line of defense against inhaled xenobiotics. They are able to accumulate airborne particulates as well as having metabolic capability. They may thus be sensitive indicator cells for detecting inhalation exposure to environmental mutagens. Their usefulness as a short-term in vivo genotoxic assay has not, however, been adequately explored. We have systematically investigated the feasibility of developing a lung macrophage chromosome-aberration assay. It was found that with different types of spindle-binding chemicals (vinblastine and vincristine), and with improved harvesting procedures, an adequate number of metaphase cells can be collected from mice and Chinese hamsters. The chromosome aberration frequencies in macrophages from control mice and Chinese hamsters were found to be 1.2 +/- 2.3 and 0.75 +/- 2.2 per 100 cells respectively. These frequencies are within normal ranges for other somatic cells. After inhalation exposure to an occupational-exposure level of benzene (0, 0.1 and 1 ppm), significant dose-dependent induction of aberrations (1.2 +/- 2.3, 5.7 +/- 6.3 and 6.8 +/- 6.2 chromatid deletions per 100 cells resp.) were observed in the macrophages. Thus, these cells can be used as one of a battery of in vivo assays for inhalation exposure studies.