Cytogenetic damage and occupational exposure

ENVIRONMENTAL RESEARCH56, 25--30 (1991)

Cytogenetic Damage and Occupational Exposure I. Exposure to Stone Dust

R. C. SOBTI AND D. K. BHARDWAJ Environment and Cancer Research Unit, Department of Zoology, Panjab University, Chandigarh-160014, India Received October 18, 1990 Cytogenetic investigations were carried out on 50 workers exposed to stone dust in a stone crusher industry and on 25 control subjects never exposed to such dust. The frequency of chromosomal aberrations and sister chromatid exchanges in exposed individuals was significantly higher than that in controls (P < 0.01). The cytogenetic indices demonstrated a clear dependence on the working environment. The effect of smoking and/or alcoholic habits coupled with exposure to stone dust has also been investigated. The results indicate that the mutagenic risk in the working environment is probably associated with silica dust in the area. © 1991AcademicPress, Inc,

INTRODUCTION

Hazards of certain working environments have been the matter of great concern for many years. For centuries stone has been used primarily for building material and roads; thus, a fraction of people working in the stone industries engaged in drilling, cutting, sieving, road and building material, and crushing of stones are exposed to the stone dust occupationally. According to Guenel et al. (1989), stone crushing has the highest exposure median index of 2.6 as compared to 1.0 for drilling, 0.9 for sieving, 0.5 for cutting, and 2.1 for road and building material industry. Occupational exposure to dust has been linked with excess mortality from stomach cancer (Wright et al., 1988; McDowall, 1984), lung diseases such as bronchial carcinoma and mesothelioma (Chamberlain et al., 1982), and pneumoconiosis (Glover et al., 1980). All these indicate the carcinogenic risk of the occupation, but no conclusions have been drawn because of lack of studies using carcinogenic and/or mutagenic assays. Although these assays have been applied on the persons exposed to various other environments such as wood dust (Jones and Smith, 1986; Chebotarev, 1984), fertilizers (Mandal, 1990), and petroleum exhaust (Simeonova et al., 1989), the studies on persons exposed to stone dust are lacking. It is because of this that the present studies were undertaken. MATERIALS AND METHODS

Fifty male workers from the stone crushing units near Chandigarh (India) constituted the exposed group for the present study. Sand stone was being crushed in these units for the building material. The composition of the sand stone as revealed after the analysis done at the Center for Advance Studies in Geology, P.U. Chandigarh, was: 50-60% silica (SiO2) , 14-16% aluminum oxide ( A L 2 0 3 ) , 4-6% 25 0013-9351/91 $3.00 Copyright© 1991by AcademicPress, Inc. All fightsof reproductionin any formreserved.

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iron oxide, 4-5% CaO, 2-4% MgO, 2-3% K20, 1-2% Na20, and 0.7-0.8% TiO 2. A major component of the stone crushed in those units was thus silica (SiO2). Heparinized blood samples of these 50 workers were transferred to the laboratory in an ice box. Twenty-five age-matched controls who were never exposed to such dust were obtained from the teachers and students of the Panjab University, Chandigarh. The mean age of the exposed group was 30.92 years, and that of controls was 30.36 years. The subjects of both the exposed and control group were further categorized according to their smoking and/or alcoholic habits. The subject was considered a smoker if he smoked more than 10 cigarettes a day and an alcoholic if he took an average of 200 ml of alcohol per day. Culture. Blood samples of controls were kept in a freezer at 4°C for the time equivalent to the transportation time of exposed group samples. Whole blood cultures were set up in duplicate using 4 ml of RPMI medium (Gibco, U.S.A.) supplemented with 20% fetal calf serum (HiSera, UK), 0.1 ml of phytohemagglutinin M (Borroughs Wellcome, UK) and 25 ~g/ml of BrdU(Sigma, U.S.A.). The cultures were maintained at 37°C for 72 hr. After 69 hr of incubation, colchicine treatment was given to arrest the metaphases. Hypotonic treatment was given with 0.075 M KC1 solution and fixation was done with 3:1 methanol:acetic acid treatment. Flame-dried slides were made.

Sister Chromatid Differential Staining Slides were processed for SCEs according to the method of Perry and Wolf (1974) with slight modifications. After staining with Hoechst 33258, the slides were kept in the warm 2 x SSC solution in the sun for 3 to 4 hr.

Scoring of Slides Slides of both the exposed and control groups were coded blindly and studied under microscope for sister chromatid exchanges and chromosomal aberrations. One hundred metaphases per individual were studied for chromosome aberrations and 25 metaphases with differential staining were studied for sister chromatid exchange analysis. Slides were decoded after the complete data were obtained. The data thus obtained were subjected to statistical evaluation using test of variance (one way ANOVA). RESULTS

The incidence of chromosomal aberrations in the exposed group was 2.72% as compared to 1.28% in the control group (Table 1). The mean value of SCEs was 7.50 per cell in the exposed group as against 5.16 per cell in controls (Table 2). These variations in the values of chromosomal aberrations and SCEs were highly significant (P < 0.01). After comparison of the frequency of chromatid gaps and breaks with that of controls, highly significant variation (P < 0.01) in the frequency of chromatid gaps and significant variation (P < 0.05) in chromatid breaks of exposed group were apparent (Table 1).

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CYTOGENETIC DAMAGE, I TABLE 1 FREQUENCY OF CHROMOSOMAL ABERRATIONS (CAs) IN EXPOSED AND CONTROL GROUPS E x p o s e d group

Total aberrations C h r o m a t i d gaps C h r o m a t i d breaks C A s in alcoholics C A s in nonalcoholics C A s in s m o k e r s C A s in n o n s m o k e r s

Control group

n

Mean ~ SEM

n

50 50 50 32 18 34 16

2.72** 1.76" 0.68 2.78* 2.61" 2.88** 2.37

25 25 25 13 12 11 14

-+ 0.25 -+ 0.23 -+ 0.15 -+ 0.3 -+ 0.42 -+ 0.29 + 0.44

M e a n +-- S E M 1.28 1.04 0.24 1.53 1.00 1.27 1.28

+ 0.25 + 0.23 + 0.13 -+ 0.40 -+ 0.30 +-- 0.33 --- 0.33

F-value 13.77 4.04 3.29 4.06 7.89 8.28 3.61

Note. C A s , c h r o m o s o m a l aberrations; n, n u m b e r of subjects; SEM, standard error of mean. * Significant variation at P < 0.05 level. ** Significant variation at P < 0.01 level.

Impact of Alcohol When chromosomal aberrations were compared separately for alcoholics of exposed and control groups, a significant increase was observed in the former: 2.72 aberrations per individual compared to 1.53 in control (Table 1). After comparison of the SCEs frequency of alcoholics and nonalcoholics of exposed group with their respective controls, the increase in the SCE frequency of both alcoholics and nonalcoholics of exposed group was found to be highly significant (P < 0.01) and the values were 7.65 and 7.27 SCEs per cell, respectively, for alcoholics and nonalcoholics of exposed group and 5.29 and 5.01 SCEs per cell for their respective controls (Table 2). When the frequency of SCEs and chromosome aberrations was compared in alcoholic and nonalcoholic subjects of the exposed group itself, no marked variation in the chromosome aberrations or the frequency of SCEs in alcoholics compared to nonalcoholics was observed.

Impact of Smoking When comparison was made in chromosome aberrations of smokers of exposed and control groups, significant variations were noticed (P < 0.01). TABLE 2 FREQUENCY OF SISTER CHROMATID EXCHANGES (SEEs) IN EXPOSED AND CONTROL GROUPS E x p o s e d group

Total S C E s Alcoholics Nonalcoholics Smokers Nonsmokers S m o k e r s and alcoholics

Control group

n

M e a n -+ S E M

n

50 32 18 34 16 22

7.51"* 7.65** 7.27** 7.80** 6.92** 7.88**

25 13 12 11 14 8

Note. n, n u m b e r of subjects. ** Significant variation at P < 0.01 level.

--- 0.17 -+ 0.22 -+ 0.24 +- 0.18 +- 0.32 +_ 0.24

M e a n -+ S E M 5.16 5.29 5.01 5.45 4.92 5.57

--+ 0.10 -+ 0.17 +- 0.11 +-- 0.17 +- 0.10 +- 0.22

F-value 89.54 36.76 56.18 48.10 33.76 31.36

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.After comparison of the frequency of SCEs in smokers and nonsmokers of exposed group with their respective controls, the increase in the mean SCEs was found to be highly significant (P < 0.01), values being 7.80 and 6.92 SCEs per cell for smokers and nonsmokers of exposed group and 5.45 and 4.92 SCEs per cell for controls, respectively (Table 2). During comparison of the frequency of SCEs and chromosome aberrations in smokers and nonsmokers of the exposed group itself, a significant variation (P < 0.01) in the SCEs of smokers became apparent, whereas there was no significant difference between chromosome aberrations of smokers versus those of nonsmokers. A slightly significant increase in the frequency of SCEs was encountered in subjects who were both alcoholics and smokers as compared to alcoholics only. There was, however, no significant variation when compared to smokers only (Table 2). The SCEs and chromosome aberrations increased constantly with the increase in the duration of exposure (Table 3). The values for SCEs were 7.24, 7.84, 7.72, and 7.84 SCEs per cell for the exposure of 0-5, 6-10, 11-20, and 21-30 years, respectively. The values of chromosome aberrations were also duration dependent; these were 2, 3.42, 3.55, and 4% for 0-5, 6-10, 11-20, and 21-30 years of exposure respectively.

DISCUSSION The chromatid gaps and not the chromatid breaks were much more abundant in the exposed group. No significant difference was found between the increase in chromosome aberrations of smokers versus those of nonsmokers or in those of alcoholics versus those of nonalcoholics of the exposed group. This indicated that smoking and/or alcoholism were not responsible for increased chromosome aberrations observed in the exposed group. But when the frequency of chromosomal aberrations was compared to the duration of exposure of the subjects, a highly positive correlation was apparent between CAs and the increasing exposure duration. Sister chromatid exchange analysis showed that the variation in mean SCE frequency of exposed and control groups was very high. It was clearly evident TABLE 3 FREQUENCY OF S f E s AND CAs WITHIN THE EXPOSED GROUP WITH DIFFERENT DURATIONS OF EXPOSURE TO THE STONE DUST

E x p o s u r e in years

n

0--5 6--10 !1-20 21-30

26 14 9 1

SCEs Mean -+ SEM 7.24 7.84 7.72 7.84

± 0.26 -+ 0.31 -+ 0.32 ± 0.00

CAs Mean ± SEM 2.00 3.42 3.50 4.00

± ±

0.35 0.38 0.55 0.00

Note. SCEs, Sister chromatid exchanges; CAs, chromosomal aberrations; SEM, standard error of mean.

CYTOGENETIC DAMAGE, I

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that with the increasing duration of exposure, the number of SCEs per cell also increased. When the frequency of SCEs of smokers and nonsmokers, and that of alcoholics and nonatcoholics of the exposed group was compared, a highly significant variation was observed in the SCE frequency of smokers as compared to that of nonsmokers, but there was no variation in SCE frequency of alcoholics and nonalcoholics of the exposed group. It has been reported that there is no positive correlation between SCE frequency and chromosomal aberrations, and our results agree with these contentions. We have found a correlation between the total number of SCEs and aberrations and with that of increasing exposure duration, but the rest of the parameters did not show any positive correlation between SCE frequency and chromosomal aberrations. There was no positive correlation between the habit of smoking and taking alcohol coupled with exposure to the dust and chromosomal aberrations, whereas in the case of SCEs, a highly significant correlation was seen with smoking and stone dust exposure. There are reports that smoking increases the risk of cancer such as the cancer of buccopharyngeal cavity, larynx, lung, and bladder in people occupationally exposed to the dust (Bross et al., 1978). Since stone dust has been reported to be a risk factor in various cancers (McDowall, 1984; Chamberlain et al., 1982), it is possible that smoking and stone dust may have additive effects in this regard. There was a slightly significant variation in the frequency of SCEs in alcoholics compared to that of nonalcoholics in the exposed group. Smoking alone coupled with dust exposure induced SCEs as much as 7.80 per cell, the value equal to 7,88 per cell in subjects who were alcoholics and smokers, thus making smoking a factor much more likely than alcohol drinking to induce SCEs. Airborne particles act as sites for the condensation of chemical carcinogens in cigarette smoke and in the urban environment (Natusch and Wallace, I974). The present results indicate more genetic damage in the form of SCEs in smokers occupationally exposed to stone dust (mainly silica), thus setting them at a higher risk of developing a disease such as cancer. In addition to the opinion that removal of personnel from harmful environments significantly reduces the chromosomal damage in the due course of time (Sram et at., 1983), it is felt that a regular cytogenetic checkup of these workers should be conducted and the personnel with a higher incidence of chromosome breakage and/or SCEs should be removed from that work environment and provided a job elsewhere. This is the first report on cytogenetic monitoring of stone crushing workers.

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SOBTI AND BHARDWAJ McDermott, M., and Oldharn, P. D. (1980). Effects of exposure to slate dust in North Wales. Br. J. Ind. Med. 37, 152-162.

Guenel, P., Breum, N. O., and Lynge, E. (1989). Exposure to silica dust in the Danish stone industry. Scand. J. Work Environ. Health 15(2), 147-153. Jones, P. A., and Smith, L. C. (1986). Personal exposures to wood dust of wood workshops in the furniture industry in the High Wycombe area. A statistical comparison of 1983 and 1976/1977 Survey results. J1. Occup. Hyg. 30(2), 171-184. Mandal, P. K. (1990). Monitoring of cytogenetic effects in occupational workers of a fertilizer factor. In "XV Annual Conference of Indian Society of Human Genetics and National Symposium on Chromosome in Health and Disease." McDowall, M. E. (1984). A mortality study of cement workers. Br. J. Ind. Med. 41, 17%182. Natusch, D. S., and Wallace, J. R. (1974). Urban aerosol toxicity: The influence of particle size. Science 186, 695--699. Perry, P., and Wolf, S. (1974). New Giemsa method for differential staining of sister chromatids. Nature (London) 125, 156-158. Simeonova, M., Georgieva, V., and Alexiev, C. (1989). Cytogenetic investigations of human subjects occupationally exposed to chemicals from the petrolium processing industry. Environ. Res. 48, 145-153. Sram, R. J., Dobias, L., Pastorkova, A., Rossner, P., and Janca, L. (1983). Effect of ascorbic acid prophylaxis on the frequency of chromosome aberrations in the peripheral lymphocytes of coaltar workers. Mutat. Res. 120, 181-186. Wright, W. E., Bernstien, L., Peters, J. M., Garbrant, D. H., and Mack, T. M. (1988). Adenocarcinoma of the stomach and exposure to occupational dust. Am. J. Epidemiol. 128, 64-73.