Validation of the human T-lymphocyte cloning assay--ring test report from the EU concerted action on HPRT mutation (EUCAHM).

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P = 0.0004) and lnMF (P = 0.03), but there was no significant laboratory effect on the lnCE (P = 0.38) or lnMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide.

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

Gene-mutation assays in lambda lacZ transgenic mice: comparison of lacZ with endogenous genes in splenocytes and small intestinal epithelium.

Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used lambda lacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen cells, and (b) lacZ and Dlb-I in small intestine from lambda lacZ+/0/Dlb-Ia/b mice. Induction of mutations by ethyl- and methylnitrosourea (ENU, MNU) and ethyl methanesulphonate (EMS) was investigated at 7 weeks after a single i.p. dose of each of these chemicals. In the small intestine, treatment with various dosages of ENU (10-150 mg/kg) resulted in a linear dose-response in both lacZ and Dlb-I. MNU (30 mg/kg) was also mutagenic in lacZ and Dlb-I, while EMS (250 mg/kg) did not significantly induce mutations in either gene. In spleen, ENU gave a linear dose-related response in both lacZ and hprt, MNU induced mutation sin both lacZ and hprt, and EMS was only positive for lacZ. No differences in response were observed between single and split-dose treatment with ENU (1 x 50 or 5 x 10 mg/kg with a 1- or 7-day interval), both in spleen and small intestine, except for lacZ in small intestine, where the single high dose gave a significantly higher induction than the split dose with the 7-day interval. The overall results suggest that mutagenic effects of fractionated doses are generally additive. In most cases, the induction factor (ratio treated over controls) for mutations in lacZ was lower than that for hprt and Dlb-I, presumably due to a higher background in lacZ and/or a lower mutability of lacZ. The general concordance between the data for lacZ and the endogenous genes indicates that lambda lacZ transgenic mice are a suitable model to study induction of gene mutations in vivo.

Comparison of induction of hprt mutations by 1,3-butadiene and/or its metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane in lymphocytes from spleen of adult male mice and rats in vivo.

Induction of hprt mutations by 1,3-butadiene (BD) and its metabolites 1,2-epoxybutene (EB) and 1,2,3,4-diepoxybutane (DEB) was studied in lymphocytes from spleens of 6- to 14-week-old mice and 10- to 11-week-old rats. For unknown reasons, results from experiments with mice that received inhalation exposure to BD were quite variable. In the first experiment, mice were exposed for 5 days to 200, 500 or 1300 ppm and this resulted in a statistically significant, dose-dependent, induction of mutations. When the experiment was repeated and an extra expression time for mutations was included, it was not possible to detect induction of mutations. In a third experiment, a 6-day exposure to 500 ppm was mutagenic when mice with zero mutants were not excluded from the statistical analysis of the data. The monofunctional metabolite EB appeared to be mutagenic in mice (3 x 33 and 3 x 100 mg/kg), but not in rats (3 x 33 and 100 mg/kg or 30 days drinking water with 0.1, 0.3, or 1.0 mM EB). Contrary to expectations, there was no induction of mutations in mice and rats exposed to the bifunctional metabolite DEB (mice, 3 x 7, 21, 3 x 14, or 42 mg/kg; rats, 20 or 40 mg/kg or 30 days drinking water with 0.3 or 1 mM DEB), although in our earlier studies with mice and rats, DEB treatment significantly enhanced frequencies of micronuclei in splenocytes and in early spermatids of mice and rats. Some of these results differ from findings reported by other investigators. It is now becoming evident that these differences are, to a large extent, due to differences in age of the animals at the time of treatment. For example, the mutagenic potency of BD, EB and DEB was stronger in preweanling mice or 4-week-old mice than in 8- to 12-week-old adult mice.

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.

Development of a cloning assay with high cloning efficiency to detect induction of 6-thioguanine-resistant lymphocytes in spleen of adult mice following in vivo inhalation exposure to 1,3-butadiene.

A cloning assay with high cloning efficiency has been developed to detect spontaneous and induced 6-thioguanine-resistant T-lymphocytes (HPRT mutants) from the spleen of adult mice. The mean cloning efficiency in untreated male mice of 20-22 weeks old was 34.5 +/- 11.2% (SD) and the corresponding mutant frequency 0.7 +/- 0.8 (SD) x 10(-6). The cloning efficiencies obtained in this study are substantially higher than those reported previously by other investigators. Using this assay, it could be demonstrated that inhalation exposure of mice to 200, 500 or 1300 ppm of 1,3-butadiene for 6 h/day on 5 consecutive days caused a statistically significant induction of 6-thioguanine-resistant mutations in T-lymphocytes from spleens of adult mice exposed to 1300 ppm. The exposure to 1300 ppm resulted in a three-fold increase of the spontaneous mutant frequency. The mutant frequency after exposure to 500 ppm was higher than the control but the increase was not significant.

Frequencies of HPRT mutants and micronuclei in lymphocytes of cancer patients under chemotherapy: a prospective study.

Fifteen cancer patients, including 10 testicular carcinoma patients, were treated with several types of combination chemotherapy. Blood samples were collected before, during and after chemotherapy. Subsequently, lymphocytes were analyzed for frequencies of HPRT mutants (MF) and micronuclei (MNF). Significantly elevated MFs were detected in eight patients. Mean expression time (+/- SD) for mutations was 98 +/- 54 days (range: 42-172 days). In some patients, enhanced MFs persisted for a period of 430-490 days after cessation of chemotherapy. In five patients MNFs were increased 2-6-fold and the enhancement was fairly persistent. Ifosfamide and cyclophosphamide appeared to be the most mutagenic and clastogenic constituents of the chemotherapy, while evidence for adverse effects of adriamycin, 4-epi-adriamycin and bleomycin was equivocal. Results indicate that the clinical use of mutagenic drugs must be weighed against the risks of persistent genetic damage and secondary malignancies in cured patients and their potential offspring. Further studies are necessary to determine the true risks and incidence of such abnormalities following chemotherapy for curable forms of cancer.

Enhanced hprt mutant frequency but no significant difference in mutation spectrum between a smoking and a non-smoking human population.

Recently, we have observed a small (36%), but significant, enhancement of the frequency of 6-thioguanine (6-TG)-resistant T-lymphocytes in blood from smokers. The molecular nature of 43 hypoxanthine-guanine phosphoribosyltransferase (hprt) mutant T-lymphocyte clones from nine smoking individuals was determined to investigate whether the increase in hprt mutant frequency would lead to a changed mutation spectrum. The types and distribution of hprt mutations in smokers was compared with those found in 55 6-TGr T-lymphocyte clones from 12 members of a control group of non-smokers. From this control group 25 hprt mutants were novel, whereas 31 have been described previously. Among smokers and non-smokers, a similar proportion of base substitutions (approximately 35%), mutations causing aberrant splicing (approximately 37%), frameshifts (approximately 16%) and deletions (approximately 9%) was found. In both groups, GC----AT base pair changes were found to be predominant among transitions. However, whereas all types of transversions were about equally represented in non-smokers, GC----TA transversions were not recovered among smokers. Investigation of the distribution of base substitutions over the hprt coding region showed no differences between the two groups. These data provide no clues on the nature of DNA adducts induced by smoking, which are thought to be responsible for the increased mutation frequency at the hprt locus in T-lymphocytes from smokers.

Use of a T-lymphocyte clonal assay for determining HPRT mutant frequencies in individual rats.

Conditions for detection and isolation of HPRT- mutants in cloned rat T-lymphocytes from individual adult Lewis rats were determined. Similar to cloning of human T-cells, best results were obtained with lectin (PHA)-primed T-lymphocytes of rats. High cloning efficiencies, occasionally exceeding 50%, could be obtained when the target cells employed were isolated from cervical lymph nodes. Feeder cells used were splenocytes, irradiated with 40 Gy of X-rays after priming with Con A. Human interleukin-2, present in LAK supernatant, proved to be capable of inducing proliferative activity of rat T-lymphocytes and could replace conditioned medium from primed rat splenocytes. Under the conditions described in this paper, the frequency of mutants in the HPRT gene of T-lymphocytes in Lewis rats was about 80% lower than that found in human T-lymphocytes from adults. The inverse relationship between mutant frequency and cloning efficiency, clearly demonstrated for human data, could not be established for rats. Treatment of rats with N-ethyl-N-nitrosourea, a potent alkylating agent, resulted in a time- and dose-dependent induction of HPRT- mutants, demonstrating the usefulness of this system to study in vivo mutagenesis.

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.

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.

Radionuclide angiography with technetium-99m in vivo labeled erythrocytes does not lead to induction of mutations in the HPRT gene of human T-lymphocytes.

Mutant frequencies were measured in T-lymphocytes of patients undergoing radionuclide angiography with erythrocytes labeled in vivo with technetium-99m. Blood from 13 patients was sampled before and after (8-120 days) an injection with 750 MBq technetium-99m. Frequencies of HPRT- mutants were measured with the T-cell cloning method. Results indicated that the mean frequency of mutants after treatment was significantly below that measured before exposure. Thus, in contrast to published data, our results do not support the conclusion that radionuclide angiography with technetium-99m induces HPRT- mutations. Further analysis of our data indicated that the decrease in mutant frequency after exposure can be accounted for by an effect of cloning efficiency.