Sister-chromatid exchanges in lymphocytes from styrene-exposed boat builders

Mutation Research, 241 (1990) 215-221 Elsevier

215

MUTGEN 01554

Sister-chromatid exchanges in lymphocytes from styrene-exposed boat builders K a r l T. K e l s e y

1,2, T h o m a s

J. S m i t h 3, S. K a t h e r i n e H a m m o n d a n d J o h n B. Little ~

3, R i c h a r d L e t z 4

t Laboratory of Radiobiology and z Occupational Health Program, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, 3 Department of Family and Community Medicine, University of Massachusetts Medical Center, Worcester, MA 01655 and 4Dioision of Environmental Sciences, Mt. Sinai School of Medicine, New York, N Y 10029 ( U.S.A.)

(Received 9 January 1990) (Accepted 11 January 1990)

Keywords: Sister-chromatid exchange; Chromosomes; Human lymphocytes; Styrene; Boat builders

Summary Sister-chromatid exchanges (SCE) were measured in peripheral blood lymphocytes from 40 workers in the boat-building trade. Twenty of these workers were exposed to significant amounts of styrene. The mean air concentration of styrene in the breathing zone of the boat builders was 209 m g / m 3 in the 7 exposed current smokers and 230 m g / m 3 in the 13 exposed non-smokers. Urinary styrene metabolites were also measured and the mean mandelic acid/creatinine ratios in the exposed, smokers was 275 mg/g, and in the exposed, non-smokers 323 mg/g. The SCE frequency in lymphocytes from the styrene-exposed group did not differ from that in the controls, although smoking significantly induced SCE in these workers.

Styrene and its metabolic intermediate, styrene7,8-oxide, are both mutagenic and clastogenic (Vainio et al., 1984, 1985). Styrene also induces sister-chromatid exchanges (SCE) in human lymphocytes (Norppa et al., 1983). Exposure to styrene in the workplace has been associated with increased frequencies of SCE in the peripheral blood lymphocytes of workers chronically exposed to high doses of styrene (Anderson et al., 1980; Camurri et al., 1983). However, other studies have reported that chronic, low-level styrene exposure

Correspondence: Dr. ICT. Kelsey, Laboratory of Radiobiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 (U.S.A.).

does not induce cytogenetic changes (Maki-Paakkanen 1987; Hansteen et al., 1984; Watanabe et al., 1981, 1983; Meretoja et al.,+1978). Styrene is widely used in the manufacture of polyester resin board, synthetic rubber and fiberreinforced plastic boats. Boat builders may be exposed to particularly high concentrations of styrene. Since many fabrication operations are conducted on a small scale, there is also concern about potential wide-spread elevated styrene exposure in the poorly controlled environments sometimes seen in small industrial settings. In order to investigate further the level of styrene exposure which produces chromosomal changes in exposed workers, we have studied 20 individuals actively working in the boat-building fabrication

0165-1218/90/$03.50 © 1990 Elsevier Science Pubfishers B.V. (Biomedical Division)

216

process and 20 controls who also work in this industry. Materials and methods

Subject selection Volunteer workers in two small fiber-reinforced boat-building companies were studied. All workers in each company were invited to participate; two workers declined. Controls were volunteer administrative personal and workers who had no contact with the fabrication process at the time of our investigation but who were working with each company. Precise matching of controls to exposed workers was not possible owing to the small size of these companies. All of the volunteers completed an interviewer-administered questionnaire which included an occupational history, medical history, smoking status, history of medication use, coffee consumption, and drug and alcohol use. The subjects were all afebrile at the time of study and had no history of recent viral illness or vaccination.

Styrene exposure Exposure to styrene vapors was evaluated by 3 techniques: personal monitoring with charcoal tubes and small air pumps (Eller, 1984); exhaled breath sampling (Hammond, 1988); and urinalysis for styrene metabolites. Ambient styrene was collected by personal sampling through the full work shift on charcoal tubes (150 rag) with battery operated pumps (100 ml/min). These samples were desorbed in 1 ml of carbon disulfide and analyzed with by gas chromatography (GC) and flame ionization detection on a Shimadzu 6AM (glass column, 2 m × 3 ram, 10% OV275 on chromosorb WAW 100/120 mesh, initial temperature of 90°C held for 5 min and then increased at 20°C/min to 200°C). The overaU limit of detection was 0.1 ppm averaged over an 8-h exposure, and the reported coefficient of variation was 15%. Breath analyses were conducted in separate rooms removed from the styrene exposure. The breath collection system is designed to discard the first 500 ml of expired air (from the alveolar region) onto a Tenax tube. A vacuum pump draws the expired air through the tube while the subject exhales. The breath collection system has been

described in detail elsewhere (Hammond et al., 1988). The samples were stored on dry ice in the field, and at - 2 0 ° in the laboratory prior to analysis. The samples were desorbed with 1.5 ml heptane. Two methods, liquid chromatography and gas chromatography, were used to analyze the styrene. A Waters liquid chromatograph was used with a Vydeac No. 201 TP54 octadecylsilane colunto and a mobile phase of 50% acetonitrile at 1.5 ml/min; 10 /tl of sample was injected. Absorbance at 254 nm was observed. A Hewlett Packard model 5890 GC equipped with a mass selective detector (MSD) was also used to confirm identity and quantitate styrene in exhaled breath (30 m FB1 fused silica capillary column, initial temperature of 70°C held for 2 min and then increased at 20°C/min to 170°C; 3/~1 of sample was injected by an autosampler). The sensitivity of the mass selective detector was increased by quantitating at one specific mass, 117, which was the predominant one in the styrene mass spectrum. Excretion of styrene metabolites was measured by the collection of spot urine samples at the end of the work shift. Mandelic acid was measured colorimetrically by a commercial laboratory (Eller, 1984).

Sister-chromatid exchange determination Peripheral venous blood from each worker was drawn into 10-ml heparinized vacutainer tubes (Becton-Dickinson) and stored at room temperature in insulated boxes kindly provided by ESA Laboratories Inc. (Bedford, MA). The blood tubes were coded with individual study identification numbers at the time of venipuncture. Blood was drawn from all participating workers during the same week exhaled breath styrene analysis, breathing zone personal sampling for styrene, and urine collection for styrene metabolites were carried out. Whole blood cultures were initiated within 24 h of venipuncture. Whole blood (0.5 ml) was added to 5-ml volume of RPMI 1640 culture medium with L-glutamine containing 10% fetal calf serum (Reheis), 1% phytohemagglutinin-M @HA-M) (Difco), penicillin (100 units/ml), streptomycin (100 /~g/ml) and 18 /~g/ml of 5-bromodeoxyuridine was also added to the culture medium at the time of initiation of duplicate cultures. Cultures

217

were incubated for 72 h in complete darkness at 37°C in an atmosphere of 5% CO2/95% air. Colcemid (0.1 ttg/ml) was added to each culture 2 h before processing. The lymphocytes were harvested and treated with 10 ml of hypotonlc KC1 (75 mM) for 8 min. Cells were then fixed at ambient temperature in freshly prepared methanol:glacial acetic acid (3:1, v/v). The cell suspension was washed twice in fixative, and slides were prepared by an air-dry technique. The slides were stained for SCE analysis by a modification of the fluorescence-plus-giemsa technique of Perry and Wolff (1974). Each slide was stained for 10 min in Hoechst 33258 (5/,g/ml) in double distilled water and mounted in a phosphate buffer (pH 6.8). The slides were then exposed to black light from two 15-W tubes for 70 rain at a distance of 1.0 cm and stained with 5% giemsa in phosphate buffer (pH 7.0) for 3 min One reader blindly scored all of the slides. For

each mean SCE determination, only mitoses with 40-46 chromosomes were examined. Fifty cells were scored for each worker.

Statistical analysis Group mean SCE frequencies were compared using Student's t-test. Analysis of variance (ANOVA) was also used to measure the effect of additional parameters on SCE frequency. To further examine the effect of styrene on SCE, the outlier population in the distribution of SCE frequencies was examined separately, using a variation of previously described methods (Kelsey et al., 1988; Moore et al., 1984). Results The study population included 40 currently employed workers in the boat building trade. The population demographics are summarized in Ta-

TABLE i D E M O G R A P H I C CHARACTERISTICS OF STUDY POPULATION

Styrene exposed Value

Controls Range

Value

Range

20- 58

N = 20 37.5 + 13.4 19 males, I female

19-63

Gender

N=20 33.9 + 13.4 20 males

Smoking status Current Mean cigarette consumption Never

7 28.1 pk-yrs 6

1-106

Mean age + S.D.

Coffee consumption (current) Alcohol consumption (current)

N ffi 18

4.2 cups/day

Bookkeeper

1 - 20

N ffi 19

9.4 drinks/week

Currentjobs Laminator supervisors Laminators Glassmen Carpenters Maintenance Boatyard

8

6 9

2 1 1 1 0

Purchasing agent

0

Insurance agent

0

Truck driver Sailmaker Managerial

0 0 0

1-40

26.3 pk-yrs 7 N=16 3.0 cups/day N=17 8.0 drinks

3-69

1-12 2-30

218 ble 1. Twenty of the workers were actively involved in production of fiber-reinforced boats with significant styrene exposure. The remaining 20 had only minimal indirect measured styrene exposure or no styrene exposure, and were designated as unexposed controls. Nine control workers had no personal sampling for styrene, no exhaled breath analysis, and no measurement of styrene urinary metabolites done since they worked in areas physically separated from the production facility and had no known contact with styrene. For these workers, the exposure parameters were assumed to be zero, as a best estimate. All controls with potential indirect exposure to styrene were sampled with personal breathing zone measures, exhaled breath analysis, and with urinary metabolite determination. The styrene T W A for the controls who were sampled was 8.1 + 11 m g / m 3 in non-smokers and 25.7 m g / m 3 in the smokers. In order to estimate the effect of styrene exposure on SCE frequency, the exposed and control individuals were compared, by smoking status. As is seen in Table 2, no significant increase in SCE was associated with styrene exposure in either the smokers or non-smokers. The population had comparable ages, although the exposed workers had a significantly longer tenure in the trade. Measurements of styrene urinary metabolites (the mandelic acid/creatinine ratio) and the styrene time-weighted average (TWA) breathing-zone per-

sonal sampling showed styrene exposure to be comparable in the exposed non-smokers and smokers. The measured styrene T W A ranged from 7.5 m g / m 3 to 570.8 m g / m 3 in the exposed smokers and from 25.3 m g / m 3 to 564.1 m g / m 3 in the exposed non-smokers. It is important to note that these workers periodically used respiratory protection and 5 of the most heavily exposed laminators/glassmen wore respiratory protection on the day that the styrene breathing-zone measurements were made. Use of respirators reduced the exhaled breath concentrations of styrene immediately after high-exposure tasks to less than half the levels found in those with comparable exposures who did not use respirators ( H a m m o n d et al., 1988). An additional approach was used to examine the effect of measured styrene exposure (including both air T W A and urinary metabolites) on SCE frequency. Analysis of variance (ANOVA) showed, both overall and in each exposed group, that no increase in SCE frequency was associated with any measure of styrene exposure. Therefore, there was no effect of styrene dose on SCE frequency. Previous studies have shown that analysis of the outlier portion of the individual SCE frequency distribution is sometimes more sensitive to the effects of exposure to genotoxins (Tucker et al., 1988). Therefore, we examined the mean SCE frequency in the highest 10% of the individual SCE frequency distribution (high SCE frequency cells; H F C s ) for each worker, by exposure and

TABLE 2 SCE IN STYRENE-EXPOSED BOAT BUILDERS N

Age

Yrs. in trade a

Mandelicacid creatinine (mg/g)

Control Non-smokers Smokers

12 8

37.3 26.3

1 1

13 + 14 214- 20

Exposed Non-smokers Smokers

13 7

34.8 32.3

7.2 8.6

323 4-224 2754-241

TWA styrene a (mg/m3)

Air samplinga time (min)

SCE/ceU 4-S.D.

MeanSCE of HFCs b

3.3 + 7 10.1 4- 9

508.2 390.5

6.23 + 0.9 7.21 4-1.3

11.55 + 1.6 12.97 4-2.0

230.1 4-185 209.84-227

495.4 507.0

6.45 4-0.7 7.164-1.3

11.61 + 1.4 13.124-1.8

a A_rithlnelic mean value. b Mean of means for H F C in each worker studied. Mean of the highest 5 S C E / c e l l values was used to calculate individual me a n SCE frequency of H F C for each worker.

219 TABLE 3 ANALYSIS O F S T Y R E N E IN E X H A L E D B R E A T H (ppm) Pre-shift

(range)

Post-shift

(range)

Mean of differences a

(range)

Control Non-smoker Smoker

2.3+3.2 3.6 + 4.2

(0 - 6.0) b (0 -14.5)

2.3+ 4.1 5.6 + 7.3

(0 -18.1) (0 -22.1)

0.4 2.8

( - 2 . 7 - 2.8) b (0 - 7.6)

Exposed Non-smoker Smoker c

5.94-6.3 5.44-2.6

(1.3-17.4) b (1.3- 6.8)

12.44- 9.0 15.54-16.2

(4.0--35.8) (8.0-45.0)

6.9 12.4

(--5.6--30.5) b (-3.4-43.7)

a M e a n of [(after shift)-Coefore shift)] for each worker. b One pre~shift measure on one worker was not done. c One worker was not measured.

smoking status. As can be seen in Table 2, we again did not observe any elevation in mean SCE frequency in the pooled HFCs which was attributable to exposure. The effect of cigarette smoking on mean SCE frequency in the pooled HFCs is not significant within groups; however, when the exposed and control cohorts are combined, the difference attributable to smoking is significant (p < 0.05). To further test the effect of acute styrene exposure on SCE, we measured styrene in the exhaled breath of each worker before and after the same workshift that was also monitored for styrene breathing-zone exposure and styrene urinary metabolites. Table 3 shows the mean values for the workers previously studied with personal air sampling and urinary styrene metabolites. ANOVA again confirmed that there was no effect of exposure on SCE, showing no association of SCE frequency with styrene breath analysis measured before or after the shift or with the difference between the two, controlling for smoking, using either baseline SCE frequency or the mean SCE frequency of HFCs as the endpoint. Smoking elevated baseline SCE frequency in both the control and the exposed groups. Within groups, this difference does not reach statistical significance. However, as in the case of HFCs, when the exposed and control groups are combined, the smokers have a significantly higher mean SCE frequency than the non-smokers, with the difference approaching one SCE per cell. Age,

coffee and alcohol consumption were also tested for their effect on SCE frequency (both baseline and HFCs) within groups and in the entire cohort using an analysis of variance. Controlling for smoking and styrene exposure, no significant effect of these variables on SCE frequency was noted (data not shown). Discussion

Exposure to high levels of styrene in the workplace has been previously shown to induce SCE in peripheral blood lymphocytes (Anderson et al., 1980; Camurri et al., 1983). However, several investigators have also reported no elevation in SCE frequency in workers exposed to mean air concentrations of styrene below 200 / t g / m 3 (Maki-Paakkanen et al., 1987; Hansteen et al., 1984; Watanabe et ai., 1981, 1983; Meretoja et al., 1978). We have studied a group of boat builders exposed to a mean air concentration slightly above this level and also found no increase in SCE frequency in these workers. In addition, we have also examined other measures of styrene exposure, including urinary metabolites and exhaled breath concentrations, and noted no association between them and SCE frequency. Hence, our findings are consistent with those of Watanabe et al. (1981, 1983), Meretoja et al. (1978), Hensteen et al. (1984) and Maki-Paakkanen (1987), showing no induction of SCE in lymphocytes of workers chronically exposed to relatively low levels of styrene. We have also shown smoking to elevate SCE

220 frequency in these workers. The magnitude of this elevation is consistent with our observations in other occupationally exposed groups (Kelsey et al., 1986, 1988). Analysis of m e a n SCE frequencies in H F C s also reflected the difference in SCE attributable to smoking and showed no association between any measure of styrene exposure and HFCs. C o m p a r i s o n of the studies of the effect of styrene exposure on SCE shows that the positive studies included workers whose exposure clearly exceeded 400 m g / m 3, including exposures in excess of 1000 m g / m 3 (Andersson et al., 1980; Camurri et al., 1983). U r i n a r y mandelic a c i d / creatinine ratios also exceeded 1100 in some cases, with the m e a n in the study of Camurri et al. (1983) being 472 m g / m l . Hence, the present study m a y indicate that air levels of styrene above 400 m g / m 3 a n d / o r urinary mandelic a c i d / c r e a t i n i n e ratios in excess of 400 m g / g are necessary before the baseline SCE frequency in peripheral b l o o d lymphocytes will be appreciably affected by styrene exposure. However, it is i m p o r t a n t to note that the kinetics of styrene absorption, distribution and metabolism are complex (Engstrom et al., 1978). Consequently, the exposure parameters that we have used m a y not always represent those m o s t relevant for understanding the induction of chrom o s o m e d a m a g e in target tissues. Recently, Yager et al. reported that exposure to low doses of styrene induced SCE when exposure was measured b y a compilation of several measurements conducted over one year (Yager et al., 1989). In addition, the use of respirators b y co-workers in our study with higher exposures also p r o b a b l y reduces the dose of styrene. W e did not have access to previous industrial hygiene data f r o m the sites studied and d o not, therefore, have information regarding chronic exposure history or the history of peak exposures in these workplaces. Hence, we c a n n o t adequately assess the potentially i m p o r t a n t role of exposure over time, p e a k exposures, or dose rate in producing cytogenetic changes.

Acknowledgements The authors would like to thank Marilyn Hallock and Susan Woskie for field work, R i c h a r d

Childs, J o A n n e Shatkin and C o y l a Woskie for laboratory analysis, D e b r a Wright for technical assistance and Virginia Braga and K a r e n K a n e for manuscript preparation.

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