A.D. Tates

Ctrogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes

A rat-liver microsomal system in vitro has been used to activate two indirectly acting carcinogens, DMN and DEN. On activation, both compounds were extremely potent in inducing chromosomal aberrations as well as sister chromatid exchanges in Chinese hamster cells. The implications of these findings and the potential utility of this technique to detect mutagens/carcinogens are discussed.

A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo

A micronucleus assay in vivo has been developed that utilizes freshly isolated hepatocytes from livers of hepatectomized rats. In a small validation study, increased frequencies of micronuclei were detected in rats exposed to DEN and DMN before or after hepatectomy. The method is suitable for the detection of clastogenic effects of compounds or their metabolites that are too short-lived to reach the classical target cells used in cytogenetic studies. Because the non-hepatectomized liver shows very low levels of mitosis, the liver-micronucleus assay can also be used for the study of storage effects and the clastogenic effects of chronic exposures to mutagens/carcinogens.

Biological and chemical monitoring of occupational exposure to ethylene oxide.

Studies were carried out on two populations occupationally exposed to ethylene oxide (EtO) using different physical and biological parameters. Blood samples were collected from 9 hospital workers (EI) and 15 factory workers (EII) engaged in sterilization of medical equipment with EtO and from matched controls (CI and CII). Average exposure levels during 4 months (the lifespan of erythrocytes) prior to blood sampling were estimated from levels of N-(2-hydroxyethyl)valine adducts in hemoglobin. They were significantly enhanced in EI and EII and corresponded to a 40-h time-weighted average of 0.025 ppm in EI and 5 ppm in EII. Exposures were usually received in bursts with EtO concentrations in air ranging from 22 to 72 ppm in EI and 14 to 400 ppm in EII. All samples were analyzed for HPRT mutants (MFs), chromosomal aberrations (CAs), micronuclei (MN) and SCEs. MFs were significantly enhanced by 60% in EII but not in EI. These results are the first demonstration of mutation induction in man by ethylene oxide. CAs were significantly enhanced in EI and EII by 130% and 260% respectively. MN were not enhanced in EI but significantly in EII(217%). The mean frequency of SCEs was significantly elevated by 20% in EI and by almost 100% in EII. SCE was the only parameter that allowed distinction between daily and occasionally exposed workers in EII. An interesting finding in exposed workers was the large increase of the percentage of cells with high frequencies of SCE (3-4 times in EI and 17-fold in EII). The relative sensitivity of endpoints for detection of EtO exposure in the present investigation was in the following order: HOEtVal adducts greater than SCEs greater than chromosomal aberrations greater than micronuclei greater than HPRT mutants.

An analysis of in vivo hprt mutant frequency in circulating T-lymphocytes in the normal human population: a comparison of four datasets☆

In this paper, we have compared mutant frequency data at the hprt locus in circulating T-lymphocytes from four large datasets obtained in the UK (Sussex), the USA (Vermont), France (Paris) and The Netherlands (Leiden). In total, data from > 500 non-exposed individuals ranging in age from newborns (cord blood samples) to > 80 years old have been included in the analysis. Based on raw data provided by the four laboratories, a model is presented for the analysis of mutant frequency estimations for population monitoring. For three of the laboratories, a considerable body of data was provided on replicate estimates of mutant frequency from single blood samples, as well as estimates from repeat blood samples obtained over a period of time from many of the individual subjects. This enabled us to analyse the sources of variation in the estimation of mutant frequency. Although some variation was apparent in the results from the four laboratories, overall the data were in general agreement. Thus, in all laboratories, cellular cloning efficiency of T-cells was generally high (> 30%), although in each laboratory considerable variation between experiments and subjects was seen. Mutant frequency per clonable T-cell was in general found to be inversely related to cloning efficiency. With the exception of a few outliers (which are to be expected), mutant frequencies at this locus were in the same range in each dataset; no effect of subject gender was found, but an overall clear age effect was apparent. When log mutant frequency was analysed vs log (age + 0.5) a consistent trend from birth to old age was seen. In contrast, the effect of the smoking habit did differ between the laboratories, there being an association of smoking with a significant increase in mutant frequency in the Sussex and Leiden datasets, but not in those from the Vermont or Paris datasets. Possible reasons for this are discussed. One of the objectives of population monitoring is an ability to detect the effect of accidental or environmental exposure to mutagens and carcinogens among exposed persons. The large body of data from non-exposed subjects we have analysed in this paper has enabled us to estimate the size of an effect that could be detected, and the number of individuals required to detect a significant effect, taking known sources of variation into account.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations

The clonal assay was used to measure frequencies of 6-thioguanine-resistant (HPRT) T-lymphocytes in 111 donors from the following 5 control populations: 55 adult healthy volunteers; 20 untreated cancer patients; 8 healthy hospital workers serving as controls for 9 hospital workers sterilizing equipment with ethylene oxide; 15 factory workers serving as controls for 15 workers occupationally exposed to high doses of ethylene oxide; 13 pretreatment samples from donors undergoing a diagnostic test with Technetium-99m for an analysis of heart function. With respect to mutant frequency (MF), cloning efficiency (CE) and age distribution, the first 4 populations were identical. The Technetium group had significantly higher MFs and lower CEs but this can be attributed to the higher mean age of this group. Using the total data base, we calculated the following relationships between MF, CE, age and smoking: (1) ln MF = 4.23-0.63 x ln CE indicating that a doubling of the CE has the effect of decreasing the MF by 37%, (2) ln MF = 0.71 + 0.03 x age meaning that the MF increases by 3% from one year to the next, (3) ln CE = 4.87-0.04 X age indicating that the CE decreases by 0.98% from one year to the next, (4) ln MF = 3.25-0.52 x ln CE + 0.02 X age being the equation quantifying the interrelationship between MF, CE and age, (5) ln MF = 3.32-0.56 x ln CE + 0.01 x age + 0.31 s (where s = 1 for smokers and s = 0 for nonsmokers). Using the latter equation, which allows for effects of CE and age on the MF, a statistically significant effect of smoking could be established. For any combination of CE and age smoking has the effect of increasing the MF by 36%. The above conclusions and calculations remain essentially the same when donors with cloning efficiencies lower than 10 or 20% are excluded from the data base.

Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1,3-butadiene-exposed workers

The association of occupational exposure to 1,3-butadiene (BD) and induction of cytogenetic damage in peripheral lymphocytes was studied in 19 male workers from a monomer production unit and 19 control subjects from a heat production unit. The exposure to BD was measured by passive personal monitors. The following biomarkers were used: chromosomal aberrations (CA), sister chromatid exchanges (SCE), cells with a high frequency of SCE (HFC), micronuclei, comet assay parameters like tail length (TL) and percentage of DNA in tail [T (%)] and polymorphisms of GSTM1 and GSTT1 genotypes. BD exposure with a median value of 0.53 mg/m3 (range: 0.024-23.0) significantly increased (a) the percentage of cells with chromosomal aberrations in exposed vs. control groups (3.11% vs. 2.03%, P<0.01), (b) the frequency of SCE per cell (6.96 vs. 4.87, P<0.001), and (c) the percentage of HFC (19.9% vs. 4.1%, P<0.001). BD exposure had no significant effects on formation of micronuclei and on comet assay parameters. Effect of smoking was observed only for HFC in BD-exposed group. GSTM1 genotype affected chromosomal aberrations in exposed group, while GSTT1 genotype affected chromosomal aberrations in controls. No effect of GSTM1 or GSTT1 genotypes was observed on any other biomarkers used.

Biological effect monitoring in industrial workers from the Czech Republic exposed to low levels of butadiene

Blood samples were collected twice (in 1993 and 1994) from 19 workers exposed to 1,3-butadiene and 19 matched controls. Three exposed and three control subjects were the same in 1993 and 1994. Personal passive dosimetry was performed in 1993 and twice in 1994 on the day preceding blood sampling. Mean exposure level in 1994 was 1.76 +/- 4.20 ppm (S.D.) and individual exposure levels ranged between 0.012 ppm (detection limit) and 19.77 ppm. Using the clonal assay, geometric mean of hprt mutant frequencies adjusted for cloning efficiency, age and smoking were, respectively, 7.85 (+/- 7.09) x 10(-6) and 10.14 (+/- 9.16) x 10(-6) in pooled (1993 plus 1994) exposed and control subjects. The difference was not statistically significant indicating that 1,3-butadiene did not induce a detectable increase in mutations at the hprt locus. A similar result was obtained for the 1994 subjects alone. There was no difference between adjusted geometric mean mutant frequencies of exposed and unexposed non-smokers or between exposed and unexposed smokers. Analysis of chromosomal aberrations in lymphocytes from 1994 subjects indicated that the percentage of aberrant cells was significantly enhanced in exposed subjects. In 1993 (data not shown), it was impossible to demonstrate a significant increase of aberrant cells in subjects exposed to 1,3-butadiene. Frequencies of micronuclei in cytochalasin-B blocked binucleate lymphocytes in exposed and unexposed 1994 subjects were not significantly different. This was also the case for earlier samples analyzed in the same plant. Using the comet assay for 1994 subjects, no statistically significant difference was found between the whole group of exposed and unexposed subjects. This was true for both the comet tail length and the percentage of DNA in the tail. In exposed smokers, however, the comet tail length was significantly longer than in unexposed smokers. Unexpectedly, in unexposed smokers the tail length was significantly shorter than in unexposed non-smokers. It was also unexpected that the percentage of DNA in the comet tail was significantly lower in exposed non-smokers than in unexposed non-smokers.

Measurement of frequencies of HPRT mutants, chromosomal aberrations, micronuclei, sister-chromatid exchanges and cells with high frequencies of SCEs in styrene/dichloromethane-exposed workers

Frequencies of HPRT mutants (MFs), chromosomal aberrations with or without gaps (CA+; CA-), aberrant cells (AC), micronuclei (MN), sister-chromatid exchanges (SCEs) and cells with high frequencies of SCEs (HFCs) were measured in lymphocytes collected from 46 workers occupationally exposed to styrene and dichloromethane (DCM = methylene chloride). These parameters were also determined in 23 controls. Time-weighted average (TWA) values for styrene and DCM exposure during an 8-h working day were respectively 70 mg/m3 (range: 0-598) and 108 mg/m3 (range: 0-742). These values correspond to TWA values of 17 ppm styrene and 31 ppm DCM. In exposed workers, all cytogenetic parameters were significantly enhanced (P < 0.0001; one-sided), but, due to the lack of appropriate control data, no definite conclusions could be drawn concerning the mutagenicity of styrene/DCM exposure. Duration of exposure was not correlated with genetic effects analyzed. The TWA value for styrene was not correlated with the extent of genetic damage detected, but the TWA value for DCM was positively correlated with the frequencies of chromosome aberrations (with gaps) and aberrant cells. These observations make it difficult to decide whether styrene or DCM, or both chemicals, induced the cytogenetic effects observed in exposed workers. Using the present styrene/DCM data, earlier ethylene oxide data and unpublished epichlorohydrin data, the relative sensitivity of the genetic endpoints to detect genotoxic exposure was: HFC > CA- > CA+ > SCE > MN > HPRT.

A micronucleus method for detection of meiotic micronuclei in male germ cells of mammals

A new rapid micronucleus method is presented for the detection of chromosomal damage induced in spermatocyte stages of mammals. Analysis of micronuclei is done in early spermatids that have been isolated from testis tubules in a special testis isolation medium supplemented with enzymes (collagenase, trypsin and DNAase).

Biomonitoring of occupational exposure to styrene in a plastics lamination plant

A comprehensive approach to biological monitoring of 44 workers occupationally exposed to styrene in a hand lamination plant was performed by using several end-points: styrene in workplace air, styrene in exhaled air, styrene in blood, DNA strand breaks (SBs) and oxidised bases in mononuclear leukocytes, chromosomal aberrations in lymphocytes, immune parameters and genotyping of polymorphic genes of some xenobiotic-metabolizing enzymes (CYP 1A1, EPHX, GSTM1 and GSTP1). We found a significantly higher number of DNA SBs, measured by a modified comet assay, in mononuclear leukocytes of the styrene-exposed workers compared with results from 19 unexposed controls (P<0.001). A fairly strong correlation was observed between SBs and years of exposure (P<0.001, r=0.545). The styrene-exposed workers also showed a significantly increased frequency of chromosomal aberrations (P<0.0001 for highly exposed group, P<0.004 for medium-exposed group, and P=0.0001 for low-exposed group). The proliferative response of T-lymphocytes stimulated with concanavalin A was significantly suppressed in people exposed to styrene (P<0.05). We recorded a significant increase of the percentage of monocytes in differential white blood cell counts in the exposed group (P<0.05). Using flow cytometry, we found an increased expression of adhesion molecules CD62L, CD18, CD11a, CD11b, CD49d and CD54 in the exposed workers as compared with the control group (P<0.05).