Mutation Research, 210 (1989) 345-351 345 Elsevier

MTR 04705

Detection of 6-thioguanine resistance in human peripheral blood lymphocytes (PBL) of industrial workers and lung cancer patients

Anna Tompa and Eva Sapi National Institute of Occupational Health, Nagyvarad ter 2., Budapest (Hungary)

(Received 6 June 1988) (Revision received 23 August 1988)

(Accepted 24 August 1988)

Keywords: 6-Thioguanine resistance; Peripheral blood lymphocytes, human; Lung cancer; Occupational health

Summary

Human peripheral blood lymphocytes (PBL) were selected for 6-thioguanine (6-TG) resistance in short-term (42-h) cultures in 110 high-cancer-risk industrial workers, 131 primary lung cancer patients and 96 low-risk controls. The lymphocytes were cultured and stimulated by phytohemagglutinin (PHA). A labeling index (LI) was scored using light microscope autoradiography, based on the lymphocyte's ability to incorporate tritiated thymidine with or without selective agent 6-TG. The number of 6-TG-resistant cells increased in the high-occupational-cancer-risk group of vinyl chloride- and mixed organic industrial dust (MOID)-exposed workers as well as in the primary lung cancer patients. The results were compared with the low-occupational-cancer-risk groups and with samples taken from the 70 healthy individuals and 26 hospitalized, non-cancerous controls. In both risk-exposed groups the frequency of 6-TG-resistant lymphocytes was significantly higher (p < 0.01) than in the controls. These results suggest that the original Strauss and Albertini (1977, 1979) method can be used to study qualitative risk assessment in carcinogen- or mutagen-exposed occupational groups.

Screening of the mutagenic effect of different environmental and industrial chemicals in humans is a current, unsolved problem in cancer preven- tion and occupational health. One of the newer approaches is to detect alterations in human lymphocytes by determining their acquired resis- tance to 6-thioguanine (6-TG). Resistant lympho- cytes can be quantified directly by using autora- diographic methods to measure their ability to

Correspondence: Dr. Anna Tompa, National Institute of Oc- cupational Health, P.O. Box 22, Nagyvarad ter 2., Budapest H-1450 (Hungary).

incorporate [3H]thymidine in culture. The re- sistance to 6-TG is caused by a deficiency of hypoxanthine-guanine-phosphoribosyl-trans- ferase (HPRT) activity coded on the X-chro- mosome (Albertini et al., 1985; DeMars et al., 1981). Viable 6-TG-resistant, HPRT-deficient somatic cells exhibit phenotypic changes, and they are able to respond to the stimulating agent phy- tohemagglutinin (PHA) with blastogenic transfor- mation (Everson et al., 1985). This method makes it possible to evaluate the additional genetic damage in somatic cells induced by different ex- posure factors as previously described by Strauss and Albertini (1977, 1979) and since repeated by

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

346

others (Albertini, 1985; Amneus and Erikson, 1986; Cox and Masson, 1978; Dempsey and Mor- ley, 1983; Evans and Vijayalaxmi, 1981; Lange and Prantner, 1982; Morley et al., 1983; Sander- son et al., 1984; Tompa et al., 1983; Vijayalaxmi and Evans, 1984).

Our study was performed to determine the induction of 6-TG-resistant cells in workers who had been exposed to mutagens or carcinogens in their working place. We measured the number of TG-resistant lymphocytes in 4 groups of industrial workers exposed to mixed organic industrial dust (MOID), vinyl chloride, asbestos and quartz. The results were compared with those obtained from non-exposed healthy controls and with primary lung cancer patients.

Material and methods

Whole blood was obtained by venipuncture into heparinized (0.01 ml hepar in/10 ml blood) syringes. The blood was then diluted with Hanks' balanced solution to a ratio of 3:1. The lympho- cytes were isolated by Ficol l -Hypaque density gradient centrifugation. At first, donor sera were heat inactivated and filtered prior to use in self- culture at a concentration of 30%. Parallel to the self-cultured samples we used fetal calf serum (Gibco) to optimise culture conditions. In later stages of the experiments self-sera appeared to be unnecessary and were subsequently omitted. To stimulate blastogenesis of T lymphocytes phyto- hemagglutinin-M (PHA) was used in each culture (0.03 ml /10 ml RPMI-1640 medium (Gibco)), and no antibiotics were added. The lymphocyte sus- pension was adjusted to one million cel ls /ml of medium. Cultures from each sample were divided into 4 subgroups: (1) Controls, 3 tubes containing RPMI-1640 medium supplemented with 30% fetal calf serum and no PHA; (2) PHA-stimulated group, cells cultured in the presence of PHA dis- solved in RPMI-1640 medium; (3) 6-TG group, cultures supplemented with 6-thioguanine (Sigma) in 10 - 4 M concentration; (4) pH control for 6-TG, containing only 10 - 4 M 6-TG in the control medium.

The cells were incubated for 42 h at 37 °C in humidified CO 2 (7.5% in air) using a LABOR- MIM incubator (Hungary). In the final 6-12 h of

incubation [3H]thymidine (Amersham), diluted to a final concentration of 1 #Ci /ml , was added to each culture. The ability of the cells to incorporate tritiated thymidine was determined using autora- diographic methods. To this end PHA-stimulated cell nuclei were fixed in a solution of methanol- acetic acid and formaldehyde fixative in the ratios: 7.5 : 2.0 : 0.5, respectively. Then a 0.01-ml volume of this solution was dropped onto coverslips and stained with aceto-orcein dye or methyl-green py- ronine. The air-dried slides were then dipped in Ilford G5 emulsion, and the slides were exposed under refrigeration at 4 ° C for 3 days. The slides were then developed in Kodak D19 developer. Using a light microscope, 2500 cells were counted from the PHA-stimulated group in order to calcu- late the labeling index (LI) for PHA. In the 6-TG group the total number of cells placed on the coverslips was scored and counted in a Buerker chamber before preparing smears. In these cells the LI was calculated by the mathematical for- mula:

LI(PHA + T G ) / L I ( P H A )

to give the variant frequency of VF (Strauss and Albertini, 1977). The significance of elevation in VF values and of the LI(PHA) was statistically analyzed using the non-parametric Mann-Whit- ney test applied to the ranked measurements (Zar, 1984). The groups of different occupationally ex- posed donors were selected randomly and did not consider age, sex or smoking habits although the questionnaires contained data concerning life style, smoking habits, drug and alcohol consumption, diseases, oral contraceptives, housing and social status.

Results (see Table 1)

Blood samples from 110 industrial workers were cultured in vitro for blastogenesis of T lympho- cytes following PHA stimulation. The expected level of VF values was between 5 x 10 -4 and 4 × 10 -6, as based on our healthy and hospital- ized (but non-cancerous) control group data.

The sample was divided into 4 main groups according to the nature of chemical exposure in the workers' environment. The first exposure group

TABLE 1

Groups Number of Mean age % of Signif. LI(PHA) cases in years elevated level a (%)

(range) VF values

347

Controls 70 34.4 (19-60) 1.4 23.3 + 8 Hospitalized controls 26 60.0 (23-83) 0 - 16.6 + 9 M O l D 35 53.8 (42-60) 58.0 p < 0.01 12.2 + 6 Vinyl chloride 21 41.0 (32-48) 47.3 p < 0.01 16.4 + 7 Asbestos 41 53.0 (38-58) 21.9 p < 0.1 24.0 + 9 Quartz 13 45.2 (37-62) 15.3 p < 0.1 18.6 + 7 Primary lung cancer 131 65.6 (34-79) 64.8 p < 0.01 14.5 + 9

a Using M a n n - W h i t n e y test. Positive V F = the value is above 4 × 10 -4 level.

comprised those workers who had been exposed for 20 or more years to MOID, containing differ- ent polycyclic aromatic hydrocarbons, mineral oil

mists, tars, etc. The mean age of workers in this group was 53.8 years (range, 42-60) at the time of the study. The PHA stimulation rate was lower

TABLE 2

F R E Q U E N C Y OF 6-TG-RESISTANT LYMPHOCYTES IN CONTROLS A N D IN I N D U S T R I A L W O R K E R S

VF

10-2

10-3

10-4

10-5

10 -6

mmmm

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Normal Hospital M O l D Vinyl Asbestos Quartz Lung control control chloride cancer

348

(LI(PHA) = 12.2%) than in the healthy control group (23.3%). The rate was also similar to the average value obtained from the hospitalized, non-cancer patients (16.6%), all of whom suffered from obstructive respiratory diseases and whose mean age was 60 years (range, 23-83). The VF values of the MOID-exposed group were elevated; 58% of 35 samples showed significant ( p < 0.01) increases as compared to non-exposed controls where only 1 sample (1.4%) in 70 cases showed an elevated VF (see point diagram: Table 2). In hospitalized controls no elevated VFs were ob- served among the 26 donors.

The group of 21 workers who had been exposed to vinyl chloride for 10-15 years had a mean age of 41 years (range, 32-48). In these the PHA stimulation rate was also decreased, to 16.4%, whereas the VF values were above the expected level in 47.6% of the 21 cases. This elevation of VF value was significantly higher ( p < 0.01) in com- parison to both healthy and hospitalized controls.

Asbestos workers ( N = 41) with typical clinical symptoms of asbestosis, were examined in the third group of industrial workers. In this group the mean age was 53 years (range, 38-58), The PHA stimulation rate of 24% was similar to that of the healthy controls. The VF values of these patients were not significantly increased ( p < 0.1) in comparison to the controls; only 21.9% of the donors had an elevated VF.

In addition to these 3 occupationally high- cancer-risk groups we studied a group of in- dustrial workers who had been exposed to a non- mutagenic industrial dust, DQ-quartz, and who had contracted silicosis. The work records of these patients were carefully examined in order to ex- clude any previous long-term exposure to carcino- genic materials in their working environment. However, their smoking habits were similar to those in the other groups; 45-50% of them were smokers. In the healthy control group smoking itself caused no significant differences in the VF values. Therefore, the effect of smoking on the other occupationally exposed groups was not indi- cated. The PHA stimulation rate, compared with healthy individuals, was found to be somewhat decreased (18.6%) and the VF values did not increase significantly ( p <0.1) . In the low- cancer-risk group of industrial workers only 2 cases showed higher VF values (15.3%).

The incidence of mutan t 6-TG-res is tant lymphocytes among high- and low-cancer-risk groups of occupationally exposed people was com- pared with 131 primary lung cancer patients. Their average age was 65.6 years (range, 34-79) which matched that of the hospitalized control group. The PHA stimulation rate was also similar to the hospitalized controls (14.5%). In addition, the mu- tation frequency was increased significantly (p < 0.01), and 64.8% of the 131 patients showed elevated VF rates. The samples from cancer pa- tients were obtained before chemotherapy or X-ray treatment. The distribution of variant frequencies is shown on the point diagram (Table 2). In the control groups the VF values varied over the range of 5 x 10 6-4 x 10 -4. In the MOID-exposed group it was elevated up to 5 x 1 0 2 and in the vinyl chloride exposed group it varied between 2 x 1 0 5 and 4 x 1 0 ~. In asbestos workers VF values increased up to 3 x 10 ~, though most fell below the VF limit of 4 x 10 4. In the positive control group of lung cancer patients the majority (64.8%) of the samples showed VFs above the VF limit though the observed range was between 10 ~' and 8 × 10 2.

Discussion

Several methods for detecting occupational risks and for monitoring groups exposed to mutagenic and carcinogenic chemicals are available (Pero and Norden, 1981; Sorsa et al., 1982; Strauss, 1982; Tompa, 1980; Zetterberg, 1984). However, it is clear that these methods are not applicable for forecasting of individual cancer risks in practice. In all human monitoring studies we have to con- sider the heterogeneity of different working groups resulting from individual sensitivities to the sus- pect chemicals which in turn influences the stan- dardized values of obtained data. Controls, for example, have a key importance in the interpre- tation of changes in PHA stimulation rate or in the VF values. In our studies the number of 6-TG-res is tant lymphocytes in healthy and hospitalized non-cancer patients was below the expected level. At the same time, in 65% of all patients with pr imary lung carcinoma, the best known environmentally induced cancer in humans (Blot et al., 1982; Damme, 1982; Goldsmith, 1980), the spontaneous rate of HPRT-deficient lympho-

cytes showed a significantly elevated rate (p < 0.01).

Accordingly, we have used this group as a positive control to estimate the significance of HPRT-deficient lymphocytes determined in 4 groups of workers with different exposures to industrial carcinogens. We sampled 35 workers exposed to MOIDs. Some 58% of these workers had a higher incidence of 6-TG-resistant T lymphocytes. This was found to be a significant elevation of the variant frequency in comparison to the controls (p < 0.01). The lymphocytes in vinyl chloride-exposed workers (N = 21) also had increased mutation rates, and they showed higher VF values in 47.3% of all cases. In asbestos workers (N = 41) with diagnosed asbestosis in their clinical trials, only 21.9% showed an elevated VF. This is most probably due to the fact that asbestos is not mutagenic itself. The carcinogenicity of asbestos probably derives from epigenetic factors, the exact nature of which is still far from clear. The quartz- exposed, silicotic group comprises workers ex- posed to a non-mutagenic chemical. They were included in the study in order to determine how pneumoconiosis itself may influence the 6-TG-re- sistance level of PBL cells. The lowest VF values were found in this group; only 15.35% of silicotic workers showed VFs elevated above the healthy and hospitalized controls.

These data indicate that the original Strauss- Albertini method may be used for demonstrating genotoxic effects of certain occupational and en- vironmental factors -- if they are real genotoxi- cants. However, the method as it is often applied has one serious limitation. We can interpret only group data using statistics. At the moment, geno- toxicity of occupational factors to the individual can only be tested using self-controls, i.e. compari- son of donor HPRT deficiency before entering the working place and, subsequently, during and after work under exposed conditions (Tompa et al., 1983).

One should take into consideration other possi- ble limiting factors of the method used in this study. Since its introduction by Strauss and A1- bertini (1977) as a human point-mutation assay, and as modified by them in 1979, numerous stud- ies have shown its value in analyzing changes in the number of 6-TG-resistant lymphocytes in dif-

349

ferent X-irradiated (Albertini, 1982; Cox and Masson, 1978; Evans and Vijayalaxmi, 1981; Sanderson et al., 1984; Vijayalaxmi and Evans, 1984) or chemotherapy-treated humans (Lange and Prantner, 1982; Morley et al., 1985). More re- cently, Albertini et al. (1988) have demonstrated the applicability of both short-term autoradio- graphic and long-term cell-cloning techniques as valid in vivo indicators of human somatic cell mutagenicity. They found that smoking itself caused no significant elevation in mutation rate when they used clonal assays to detect 6-TG-re- sistant lymphocytes. In our study we found similar results when we analyzed the effect of smoking in the healthy control group. Therefore, we did not further consider the effects of smoking in any of the occupationally exposed groups. Nevertheless, their study showed no elevation in 6-TG-resistant lymphocytes in nurses who routinely handled chemotherapeutic agents.

Since many chemical carcinogens also prove to be mutagenic in experimental systems (Kopelo- vich, 1982; Morley et al., 1985; Pero and Norden, 1981), a mutagenic theory of cancer development in humans must be considered. This suggests that a mutational event in somatic cells may be a prerequisite for the malignant transformation. Therefore, it seems evident that the mutation rate in human PBL cells has a potential predictive value in cancer development (Morley et al., 1983; Zetterberg, 1984).

When assaying for point-mutations in humans we can only detect mispairings of complementary bases indirectly caused by their alkylation. It is, of course, this mispairing which subsequently alters DNA expression (Morley et al., 1985; Pero and Norden, 1981). In using the original method of Strauss and Albertini (1979), the question arises whether it is capable of detecting real point-muta- tions. It is known that the [3H]thymidine-labeled, so-called "mutant", lymphocytes are not sensitive to the 6-TG treatment during their blastogenesis. Most investigators attempt to overcome these problems by using modifications to the original method. For example, an approach which used cloning techniques (Albertini et al., 1982; Amneus and Erikson, 1986; Featherstone et al., 1987; Strauss, 1982) permitted the study of mutation frequency changes. Though less sensitive, this

350

method is thought to be more specific than the original autoradiographic method. Using cloning techniques, O'Neill et al. (1987) showed that cryopreservation itself caused no differences in the occurrence of 6-TG-resistant cells, but that it did decrease the viability and blastogenesis of T lymphocytes. In our study we did not use cryopreservation, and thus avoided the extremely low PHA stimulation rate typically found in the occupationally exposed groups (Table 1).

Recent, careful investigations by Featherstone et al. (1987) suggested that both the original and cloning method may show phenotypical rather than genotypical characteristics of the 6-TG resis- tance of T lymphocytes. However, these authors maintained that it was still possible to use this method to study qualitative parameters of human chemical mutagenesis. In our work the significant differences between the various exposed groups and their controls show that the method is clearly useful for practical purposes. We need to consider the possibility of improving the accuracy of this method and, perhaps, to combine it with chro- mosome aberration analysis and DNA-repair capacity measurements. Such studies could serve to elucidate more precisely the nature of the spontaneous development of 6-TG-resistant lymphocytes during in vitro blastogenesis.

Acknowledgements

We are grateful to Professor J.R. Haight for reading an initial draft of this manuscript and to M. Fiil6p and I. Rrthhti for skilful technical assis- tance.

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