Marinel M. Ammenheuser

Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study.

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).

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.

Single nucleotide polymorphisms of the DNA repair gene XPD/ERCC2 alter mRNA expression

Objectives Epidemiological studies documented associations between single nucleotide polymorphisms (SNPs) in the nucleotide excision repair gene XPD/ERCC2 and cancer risk. Little is known, however, about the underlying mechanisms for these associations. We explored a novel mechanism that could further explain the reported risk-modifying effect of these SNPs on disease susceptibility. Methods Using quantitative real-time polymerase chain reaction, we examined the relationship between three SNPs in the XPD gene (R156R in exon 6, D312N in exon 10 and K751Q in exon 23) and mRNA levels as a potential mechanism by which these SNPs could alter DNA repair capacity and affect disease risk. To further investigate the mechanism(s) by which these SNPs alter mRNA transcription levels, we performed a localized Mfold structure analysis on the mRNA sequence surrounding the studied SNPs. Results All three SNPs studied, alone and in combination, significantly decreased constitutive XPD mRNA levels (P<0.003) in lymphocytes of healthy subjects. The decrease in mRNA levels was significantly greater in smokers and was exacerbated by smoking duration and intensity. The decrease was more pronounced in older than in younger subjects. The R156R and the K751Q polymorphisms were predicted to alter mRNA secondary structure, indicating that these SNPs potentially affect local folding and mRNA stability. Conclusions Our results provide novel mechanistic explanations for epidemiological studies linking these SNPs to elevated cancer risk and emphasize the importance of comprehensively investigating the effect of both synonymous and nonsynonymous SNPs as risk modifiers by considering their potential effects on gene expression, protein translation and functions.

Genotoxicity in workers exposed to methyl bromide

To address the genotoxicity of in vivo methyl bromide (CAS 74-83-9) exposure in humans, we collected blood and oropharyngeal cells as part of a cross-sectional morbidity study of methyl bromide-exposed fumigation workers and their referents. Micronuclei were measured in lymphocytes and oropharyngeal cells, and hypoxanthine-guanine phosphoribosyl transferase gene (hprt) mutations were measured in lymphocytes. A total of 32 workers and 28 referents provided specimens. Among current non-smokers, mean hprt variant frequencies (Vfs) were found to be elevated among workers compared to referents (geometric mean: workers=4.49x10(-6), referents=2.96x10-(6); two-sided p=0.22); this difference was more pronounced among workers with 4 h or more of recent methyl bromide exposure compared to referents (geometric mean: workers=6.56x10(-6), referents=2.96x10(-6); two-sided p=0.06). Mean oropharyngeal cell micronuclei were higher among workers compared to referents (mean: workers=2.00, referents=1.31; two-sided p=0.08); the results were similar when workers with 4 h or more of recent methyl bromide exposure were compared to referents (mean: workers=2.07, referents=1.31; two-sided p=0.13). No consistent differences between workers and referents were observed for frequencies of kinetochore-negative lymphocyte micronuclei, or kinetochore-positive lymphocyte micronuclei. The study was limited by a sample size sufficient only for detecting relatively large differences, absence of a reliable method to measure the intensity of workplace methyl bromide exposures, and relatively infrequent methyl bromide exposure (e.g., the median length of exposure to methyl bromide during the 2 weeks preceding the survey was 4 h). In conclusion, our findings provide some evidence that methyl bromide exposure may be associated with genotoxic effects in lymphocytes and oropharyngeal cells. Further study on the genotoxicity of methyl bromide exposure in humans is warranted.

Frequencies of hprt mutant lymphocytes in smokers, non-smokers, and former smokers

Previous work with the autoradiographic mutant lymphocyte assay has provided information about the time‐course of development of hprt mutations and the persistence of detectable mutant cells in human subjects following therapeutic exposures to genotoxic agents. These early studies also revealed elevations in frequencies of mutant cells in pretreatment blood samples from patients who were current tobacco smokers, but no information was available on former smokers. In the present study, blood samples were obtained from 21 healthy former tobacco smokers who had quit smoking at least 1 year before sampling, 42 subjects who had never smoked, and 23 tobacco smokers. Plasma from all samples was tested for cotinine, a metabolite of nicotine. Current smokers were categorized as heavy smokers (≥10 cigarettes per day, cotinine ≥90 ng/ml plasma) and light smokers (<10/day, cotinine < 90 ng/ml). Lymphocytes from the blood samples were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The 21 former tobacco smokers had a mean variant (mutant) frequency (Vf ± standard error) of 1.97 (±0.13) per million evaluatable cells. The Vf of 42 subjects who had never smoked was 1.74 (±0.13) × 10‐6, not significantly different from the former smokers. The smokers had Vfs of 8.09 (±0.78) × 10‐6 for 18 heavy smokers and 5.22 (±1.02) × 10‐6 for five light smokers. The two categories of smokers had frequencies of mutant cells significantly different from each other, and each was significantly higher than non‐smokers and former smokers (P < 0.05). Vfs were significantly correlated with both cotinine concentrations and the number of cigarettes smoked per day, P < 0.001. This study demonstrates the sensitivity of the autoradiographic hprt assay for detecting mutagenic effects related to chronic low‐level exposures to genotoxins, and indicates that this assay is more likely to detect the effects of recent rather than past exposures. Environ. Mol. Mutagen. 30:131–138, 1997.

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.

Assessment of butadiene exposure in synthetic rubber manufacturing workers in Texas using frequencies of hprt mutant lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to manufacture synthetic rubber, is a mutagen and carcinogen. Because past occupational exposures have been associated with an increased risk of leukemia, there has been a dramatic reduction in workplace exposure standards. The health benefits of these reduced levels of occupational exposure to BD will be difficult to evaluate using relatively insensitive traditional epidemiological studies; however, biomarkers can be used to determine whether there are genotoxic effects associated with recent exposures to BD. In past studies of BD-exposed workers in Southeast Texas, we observed an increase in the frequency of lymphocytes with mutations in a reporter gene, hprt. Frequencies of hprt mutant cells correlated with air levels of BD and with the concentration of a BD metabolite in urine. Average exposures to 1-3 parts per million (p.p.m.) of BD were associated with a threefold increase in hprt variant (mutant) frequencies (Vfs). We now report results from a follow-up study of workers in a synthetic rubber plant in Southeast Texas. Thirty-seven workers were evaluated on three occasions over a 2-week period for exposure to BD by the use of personal organic vapor monitors and by determining the concentration of a BD metabolite in urine. The frequency of hprt mutants was determined, by autoradiography, with lymphocyte samples collected 2 weeks after the final exposure measurement. Based on their work locations, the study participants were assigned to high-exposure (N=22) or low-exposure (N=15) groups. The BD exposure, +/-standard error, of the workers in the high-exposure group (1.65+/-0.52 p.p.m.) was significantly greater than the low-exposure group (0.07+/-0.03 p.p.m.; P<0.01). The frequency of hprt mutant lymphocytes was also significantly different in the two groups (high, 10.67+/-1.5 x 10(-6); low, 3.54+/-0.6 x 10(-6); P<0.001). The concentration of the urine metabolite was greater in the high-exposure group, but the difference was not significant. The correlation coefficient between hprt Vf and BD exposure levels was r=0.44 (CI(95), 0.11-0.69; P=0.011). This study reproduced the findings from a previous study at this plant. Although studies of butadiene-exposed workers in other countries have not detected an effect of exposure on frequencies of hprt mutant lymphocytes, we have repeatedly observed this result in our studies in Texas.

The L84F polymorphism in the O6-Methylguanine-DNA-Methyltransferase (MGMT) gene is associated with increased hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes of tobacco smokers.

Objectives O6-methylguanine-DNA-methyltransferase (MGMT) is a crucial DNA repair protein that removes DNA adducts formed by alkylating mutagens. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. Their biological significance, however, is not known. Methods We used a newly modified cloning HPRT mutant lymphocyte assay to test the hypothesis that inheritance of the L84F and I143V coding single nucleotide polymorphism in the MGMT gene is associated with increases in HPRT mutant frequency in lymphocytes of individuals exposed to alkylating agents. In addition, we expanded and sequenced 109 mutant clones to test the hypothesis that the mutation spectrum would shift to a larger percentage of base substitutions and G→A transition mutations in cells with L84F and I143 V coding single nucleotide polymorphisms. Results We observed no significant effect for the I143 V coding single nucleotide polymorphism on mutant frequency. In contrast, we observed a significant increase in mutant frequency (P<0.01) in lymphocytes from smokers with the 84F coding single nucleotide polymorphism compared with smokers homozygous for the referent L84 wild-type allele. A multiple regression analysis indicated that the mutant frequency increased significantly as a function of the 84F coding single nucleotide polymorphism and smoking, according to the model; mutant frequency (×10−5)=0.90+0.618 (84F polymorphism)+0.46 (smoking) with R2=0.22. Mutation spectra analysis revealed an apparent increase, which was short of statistical significance (P=0.08), in base substitutions in cells with the 84F polymorphism. Conclusions These new data suggest that the 84F coding single nucleotide polymorphism may alter the phenotype of the MGMT protein, resulting in suboptimal repair of O6-methylguanine lesions after exposure to alkylating agents.