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

Mutation Research 419 Ž1998. 145–154

Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1,3-butadiene-exposed workers a ˇ ´ a,) , P. Rossner R.J. Sram , K. Peltonen b, K. Podrazilova´ a , G. Mrackova ¨ ˇ ´ a, N.A. Demopoulos c , G. Stephanou c , D. Vlachodimitropoulos c , F. Darroudi d , A.D. Tates d a

Laboratory of Genetic Ecotoxicology, c r o Regional Institute of Hygiene of Central Bohemia and Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Vıdenska ´ ˇ ´ 1083, 142 20, Prague, Czech Republic b Finnish Institute of Occupational Health, Helsinki, Finland c Department of Biology, UniÕersity of Patras, Patras, Greece d MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden UniÕersity, Leiden, The Netherlands Received 17 July 1998; revised 21 August 1998; accepted 16 September 1998

Abstract The association of occupational exposure to 1,3-butadiene ŽBD. 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 ŽCA., sister chromatid exchanges ŽSCE., cells with a high frequency of SCE ŽHFC., micronuclei, comet assay parameters like tail length ŽTL. and percentage of DNA in tail w T Ž%.x and polymorphisms of GSTM1 and GSTT1 genotypes. BD exposure with a median value of 0.53 mgrm3 Žrange: 0.024–23.0. significantly increased Ža. the percentage of cells with chromosomal aberrations in exposed vs. control groups Ž3.11% vs. 2.03%, P - 0.01., Žb. the frequency of SCE per cell Ž6.96 vs. 4.87, P - 0.001., and Žc. the percentage of HFC Ž19.9% 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. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 1,3-Butadiene; Cytogenetic biomarker; GSTM1 and GSTT1 genotype; Effect of smoking; Human lymphocyte

Abbreviations: BD: 1,3-butadiene; CA: chromosomal aberrations; GC: gas chromatography; GSTM1: glutathione-S-transferase M1; GSTT1: glutathione-S-transferase T1; HFC: cells with high frequency of SCE; MN: micronuclei; SCE: sister chromatid exchanges; WBC: total white blood cells ) Corresponding author. Tel.: q420-2-475-2596; Fax: q420-2-475-2785; E-mail: [email protected] 1383-5718r98 r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 3 5 - 1

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1. Introduction

1,3-butadiene ŽBD, CAS No. 106-99-0. is an important product of the petrochemical industry; its annual production in the world is estimated to exceed 5 million tons w1x. It is mainly used as a monomer in the manufacture of various synthetic rubber and plastic polymers and copolymers, the largest single use being styrene–butadiene rubber for tires and tire products. Interest in the risk assessment of butadiene is related to its occupational as well as environmental exposure. Environmental exposure to BD mostly stems from motor vehicles. Burning of organic material produces emissions containing minor amounts of BD. Also, a tobacco smoke contains small amount of BD; one cigarette produces 0.4 mg BD as sidestream smoke w2x. Exposure to BD in ambient air is lower than 2–22 mgrm3 Žconversion factor: mgrm3 s 2.21 = ppm., while in smoking indoor air, it is lower than 10–20 mgrm3 w3x. BD is emitted in automobile exhaust at 5.6–6.1 mgrkm, being the second most important toxicant from automobile engines according to the United States Environmental Protection Agency ŽUS EPA. w4x. Butadiene was shown to be genotoxic in vitro and in vivo and carcinogenic in rodents, with mice being more sensitive than rats w5x. Epidemiological studies in the BD industries have resulted in a significant association between occupational exposure to BD and increased risk of hematopoietic cancers. The International Agency for Research on Cancer ŽIARC. w3x placed BD in the category 2A—Probably carcinogenic to humans. A survey of several epidemiological studies w6–10x indicated an elevated SMR Žstandardized mortality ratio. for all lymphopoietic cancers, especially leukemia and non-Hodgkin lymphoma. The increased risk of cancer is probably related to the high exposure levels of BD in the early years of plant operations. For example, the excess of leukemia is believed to be found in workers who were employed for 10 or more years, who have 20 or more years of latency and who were first employed in the 1950s. Ward et al. w10x interpreted the results of their mortality study as providing evidence for the carcinogenic action of BD in humans. Koppikar w11x con-

cluded that the leukemia mortality increased with increasing exposure to BD. Given these recent epidemiological findings, human studies using non- or pre-disease endpoints as indicators for genotoxicity became increasingly important. The possibility of using biomarkers, as alternatives to diseases, for determining health hazards associated with human toxic exposures received a high priority w12x. Sorsa et al. w13x reported results from the monitoring of BD-exposed workers in Finland, Portugal, and the Czech Republic with several biomarkers. They did not find evidence for an exposure-related increase in frequencies of chromosomal aberrations, sister-chromatid exchanges ŽSCE., or micronuclei ŽMN. in peripheral lymphocytes. Glutathione-S-transferase polymorphism for the T1 ŽGSTT1. gene has recently been defined as a possible biomarker of susceptibility to BD and its metabolites w14x. BD-exposed workers who were of the GSTT1 null genotype showed significantly higher frequencies of chromosomal aberrations than the appropriate controls w15x. In the same group of workers, hemoglobin adduct levels of butadiene monoepoxide ŽBMO. were increased w16x. Ward et al. w17x, using the autoradiographic assay for HPRT mutations in lymphocytes, recently reported an increase of the mutant frequency in exposed workers in the US. Tates et al. w18x, however, could not detect an increase of the mutant frequency when they used the clonal assay to study mutation induction in lymphocytes from BD-exposed workers in the Czech Republic. In the present paper, we will describe the results of cytogenetic biomarkers in the study among BDexposed workers in the Czech Republic.

2. Materials and methods 2.1. Study population Blood samples were collected from 19 exposed and 19 control subjects from a petrochemical company in the Czech Republic. Exposed subjects came from a BD monomer production unit. The mean age of the male workers was 43.1 " 9.5 Ž"SD., and the

ˇ ´ et al.r Mutation Research 419 (1998) 145–154 R.J. Sram

mean employment time was 15.3 " 10.5 years. Matched control subjects were from the heat production unit from the same company and had a mean age of 40.5 " 10.4. Blood samples were collected in November 1994. One shift before the blood sampling, personal and stationary monitoring of air levels of butadiene in the course of 8 h was conducted. Informed consent was obtained from each subject prior to the beginning of this study. A questionnaire was administered to each subject to determine individual life style Že.g., smoking, diet, and alcohol consumption.. Any persons with medical treatment, radiography, or vaccination within the previous three months were not included in the study. 2.2. Air monitoring of BD The air samples were analyzed in Helsinki using a gas chromatograph equipped with FID detector ŽHP 5890, Hewlett Packard, CA, USA.. For personal and ambient monitoring, passive monitors of 3M-type 3520 with a back-up section Ž3M, St. Paul, MN. were used. A diffusion rate of 42.8 lrmin was used in calculations. Acetonitrile was used for the desorption of BD and Chrompack PLOTrKCL columns for the analysis of BD by GC w19x. 2.3. Blood samples and cell cultiÕation Cells to be analyzed for the presence of chromosomal aberrations and SCE were cultivated and processed within 24 h after blood collection in Prague. Venous blood was taken from each subject using heparinized vacutainer tubes. Duplicate lymphocyte cultures were set-up by adding 0.5 ml of whole blood to 5 ml of RPMI medium ŽSEVAC Praha, Czech Republic., supplemented with 20% inactivated fetal calf serum, antibiotics, and glutamine. Lymphocytes were stimulated by 1% phytohaemagglutinin ŽWellcome PHA 15.. For the evaluation of chromosomal aberrations, the cultures were incubated for 48 h at 378C. A final concentration of 2 = 10 y 7 M of Colcemid was added 2 h before fixation. Slide preparation and staining of chromosomes were carried out by our standard cytogenetic method w13x. For the evaluation of frequencies of SCE, lymphocytes were incubated in the presence of 5-

147

bromodeoxyuridine ŽBrdU. at the final concentration of 15 mgrml in the dark for 68 h at 378C. The cells were harvested according to the standard procedure. Processing of lymphocytes and staining of slides were performed according to the FPG method of Perry and Wolff w20x. The processing of cells for the analysis of micronuclei in cytochalasin B-blocked binucleate lymphocytes was performed in Leiden. One milliliter of fresh blood was used to set up two cultures for measuring micronuclei in binucleated lymphocytes. After a culture time of 44 h, cytochalasin B ŽSigma. Žfinal concentration of 6 mgrml. was added to the cultures. Cultures were incubated at 378C for a total culture time of 72 h. The methodology was published previously w21x. The Comet assay procedure on isolated lymphocytes was performed in Prague according to Singh et al. w22x, with times for lysis, unwinding, and electrophoresis of 60, 60, and 40 min, respectively w23x. 2.4. Cytogenetic analysis For the analysis of chromosomal aberrations, 100 metaphases per subject were analyzed in Prague Žfrom two slides., and 100 in Patras Žfrom another two slides.. Four categories of chromosomal aberrations were evaluated, i.e., chromatid ŽBX . and chromosome breaks ŽBY ., and chromatid ŽEX . and chromosome exchanges ŽEY .. Gaps were not scored as aberrations. All cells carrying breaks or exchanges were counted as aberrant cells ŽAB. C... For the scoring of SCE, 50 second division metaphases were analyzed according to the method of Perry and Wolff w20x. The threshold value to define HFC was calculated according to Carrano and Moore w24x. The cells as being HFC were identified if the minimum number of SCEsrcell was 10. For the micronucleus frequency, 1000 binucleate cells per subject were analyzed for the presence of micronuclei. It was also recorded whether the micronucleated binucleates carried one, two or more micronuclei per cell. Furthermore, an analysis was made of the percentage of mono-, bi-, tri- or tetranucleated cells. The Comet assay samples were analyzed using an image analyzer system ŽKomet, version 2.3; Kinetic Imaging, UK.. The following comet parameters were

NS NS NS S S NS NS NS S NS S S S S NS NS NS NS S

S NS NS S S NS S NS S S NS NS NS NS NS S NS S S

Smoking

q y y y q y q y y y q q y y q y y y q

q q q q q q q q q y y y q y q y q q q

GSTM1

q q q q q y q q y q y q q q q q q q y

q q q q q q y q q q q y q q q q q q q

GSTT1

1 2 2 1 3 4.5 1 3.5 1.5 2 2.5 1.5 1.5 1.5 1 2 1 2.5 3.5

4 2.5 3 4 1.5 4 4 3 6 2 2.5 1 4 2 5.5 1.5 3.5 2.5 2.5

AB. C. Ž% .

X

1 1.5 0 0.5 2.5 1.5 0 1.5 1 1.5 2 1.5 1 1 1 1.5 1 1.5 2.5

3 1.5 1.5 1.5 1 2 4 2.5 3.5 1.5 2 0.5 2.5 2 3 1 3.5 2 1.5

B

0 0 0 0 0 0 0 0 0.5 0 0 0 0 0 0 0 0 0 0

0 0 0 1.5 0 0.5 0 0 0.5 0 0 0 0 0 0 0 0 0 0

X

E

0 0.5 2 0.5 0.5 2.5 1 2 0 0.5 1 0 0.5 0.5 0 0.5 0 1 0.5

1.5 0.5 0 0 0.5 1.5 0 0.5 1.5 0.5 0.5 0.5 1 0 2.5 0.5 0 0 0.5

Y

B

0 0 1 0 0 0.5 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0.5 1.5 0.5 0 0 0 0.5 0.5 0 0 0.5 0.5 0 0 0 0 0.5 0.5

Y

E

5.88 6.52 4.80 4.60 5.02 5.20 5.60 4.06 3.88 2.36 6.88

3.38 5.24 4.28 4.40 5.60

5.10

4.88 3.56 4.28 8.36 7.34 6.98 8.78 6.52 8.50 7.70 6.02 7.08 8.02 6.78 8.16 9.10 6.98 7.38 5.82

SCEr Cell

4 – 0 4 2 0 8 – 12 14 6 4 0 4 6 2 0 0 4

6 0 0 28 24 12 36 14 46 24 12 24 22 16 28 44 16 24 2

HFC Ž% .

29 19 5 22 5 29 13 25 8 14 19 2 3 6 12 9 17 20 21

20 15 7 22 17 28 20 18 26 16 11 12 13 12 17 12 14 20 9

MN

7.39 7.65 7.44 5.32 4.72 6.43 4.77 4.75 6.21 5.98 5.67 5.97 9.01 5.47 6.04 7.64 5.49 5.85 6.93

5.24 4.90 5.84 6.30 7.92 10.11 6.27 6.79 6.56 6.92 5.75 5.80 3.96 4.67 5.77 5.55 4.61 4.56 4.96

T Ž% .

6.31 7.02 6.67 5.61 5.96 5.61 6.31 6.91 6.31 6.31 4.91 5.61 8.42 5.61 6.31 7.02 4.91 5.61 5.26

5.61 5.61 5.61 6.31 12.28 13.68 7.02 7.02 7.72 7.72 5.61 6.31 5.61 4.91 4.21 5.26 4.91 5.61 4.91

TL Žm m .

X Y S: Smoker, NS: nonsmoker, GSTM1 q : positive, GSTM1 y : null, GSTT1 q : positive, GSTT1 y : null genotype, AB. C. Ž% .: aberrant cells carrying chromosomal aberrations, B : chromatid break, B : chromosome X Y break, E : chromatid exchange, E : chromosome exchange Žnumber per 100 metaphases., SCErcell: number of sister chromatid exchanges per cell, HFC Ž% .: cells with high frequency of SCE, MN: number of micronuclei per 1000 binucleated cells, T Ž% .: percentage DNA in tail, TL: tail length.

- 0.005 - 0.005 0.03 0.028 0.027 - 0.005 - 0.005 - 0.006 - 0.006 - 0.006 0.048 0.036 0.038 0.038 - 0.005 - 0.005 - 0.006 - 0.006 0.15

Control group 251 31 252 51 253 54 254 52 255 24 256 59 257 54 258 56 259 39 260 55 261 47 262 36 263 20 264 31 265 44 266 39 267 49 268 51 269 50

0.118 0.07 8.7 1.15 5.88 - 0.011 0.77 2.96 0.97 - 0.011

BD Žmgrm 3 .

17.0 3.08 0.30 0.34 0.53 23.05 0.20 0.05

Age Žyears.

Exposed group 211 57 212 48 213 45 214 41 221 50 222 48 223 28 224 38 226 59 227 23 231 44 232 43 233 44 234 43 241 57 242 49 243 31 244 49 245 24

Number

Table 1 Individual data on each subject—exposure to 1,3-butadiene, smoking, genotypes GSTM1 and GSTT1, cytogenetic parameters

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ˇ ´ et al.r Mutation Research 419 (1998) 145–154 R.J. Sram

evaluated for 100 images per sample: percentage of DNA in tail w T Ž%.x, tail length wTL Žmm.x, and tail moment ŽTM s percentage T = TL.. Medians of these parameters for 100 images were used for the characterization of each individual subject. The slides for each cytogenetic endpoint were coded, so that the analyst was not aware of the exposure status. 2.5. Genotyping GSTM1 polymorphism was determined by the polymerase chain reaction ŽPCR. described by Zhong et al. w25x with a slight modification Žusing DNA isolated from WBC.. Two of three primers used could also anneal to another class M gene ŽGSTM4., while the third was specific for the GSTM1 gene. The GSTM1 null genotype was identified on the basis of the absence of the GSTM1-specific fragment. The consistent presence of the other fragment was used as an internal standard to detect a failure of the amplification reaction. GSTT1 genotype was determined according to Pemble et al. w26x. 2.6. Statistical analysis Statistical analysis was performed using the STATGRAPHICS Plus 7.0 package ŽMagnuistics, Rockville, MD.. Nonparametric methods were chosen for a group-wide evaluation of the individual data that did not follow a normal distribution. The Mann–Whitney rank sum U-test as well as t-test were used for comparison of two samples. Correlations were performed by the Spearman rank correlation test. The multifactor analysis of variance procedure was used to analyze the association between cytogenetic biomarkers and independent variables while controlling for the potential influence of the other variables.

3. Results

days as the blood samples. In all samples, the mean concentration of BD was lower than 1 mgrm3. Using personal monitoring with 3M passive monitors, 58% of the workers were found to be exposed to a BD concentration lower than 1 mgrm3 and 21% to a concentrations higher than 5 mgrm3. Controls values varied between 0.009 and 0.27 mgrm3. Individual data for exposure, smoking and all cytogenetic biomarkers are given in Table 1. The results concerning the effect of BD exposure on the frequency of chromosomal aberrations are presented in Table 2. As the results of analysis between Prague and Patras did not differ, they were pooled together. Occupational exposure to a median BD concentration of 0.53 mgrm3 resulted in a significant increase of the percentage of cells with one or more chromosomal aberrations Ž P - 0.01.. There was no difference in the percentage of aberrant cells between exposed smokers and nonsmokers, even though the nonsmokers received a three-fold higher BD exposure Ž1.73 mgrm3 in nonsmokers vs. 0.53 mgrm3 in smokers.. Also, in the controls, there was no difference in aberration frequencies for smokers and nonsmokers. The effect of BD exposure on the frequency of SCErcell is shown in Table 3. The mean frequency of SCErcell for the workers exposed to BD was 6.96 SCErcell and 4.87 SCErcell for controls. The difference is highly significant Ž P - 0.001.. There is

Table 2 Chromosomal aberrations, effect of exposure to 1,3-butadiene Group

N Exposure Žmgrm3 . AB. C. Ž%.

Exposed Controls

19 0.53 a Ž0.024–23.0. b 3.11"1.33 c ) Ž1.0–6.0. b 19 0.013 Ž0.009–0.27. 2.03"1.01 Ž1.0–4.5.

Exposed Smokers 9 0.53 Ž0.024–5.89. Nonsmokers 10 1.73 Ž0.024–23.0.

3.11"1.50 Ž1.5–6.0. 3.10"1.24 Ž1.0–5.5.

Controls Smokers 9 0.038 Ž0.012–0.27. 2.00"0.89 Ž1.0–3.5. Nonsmokers 10 0.012 Ž0.009–0.03. 2.05"1.13 Ž1.0–4.5. a

Median. Range. c Mean"SD. ) P - 0.01. AB. C. Ž%.: aberrant cells carrying chromosomal aberrations. b

Ambient air samples were collected in several places of the monomer production unit on the same

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150

Table 3 SCE—effect of exposure to 1,3-butadiene Group

N

SCErcell

HFC Ž%.

mean

SD

mean

SD

Exposed Controls

19 17

6.96) 4.87

1.51 1.11

19.9) 4.1

13.4 4.2

Exposed Smokers Nonsmokers

9 10

7.54 6.44

1.40 1.48

26.0 14.4

15.1 9.2

Controls Smokers Nonsmokers

8 9

5.24 4.54

0.82 1.29

4.5 3.8

3.8 4.8

) P - 0.001. HFC: cells with high frequency of SCE.

also a significant difference between the exposed and unexposed groups with respect to the frequency of HFC Ž P - 0.001.. A significant effect of smoking on the frequency of SCErcell was not observed within both groups. Higher percentage of HFC in exposed smokers as compared to exposed nonsmokers was on the borderline of significance Ž P s 0.056.. Micronucleus data for exposed and control groups are presented in Table 4. Exposure to BD did not result in an increased frequency of micronuclei in binucleate cells. When the group of exposed and unexposed subjects were each subdivided into smokers and nonsmokers, a significant difference was observed between smokers and nonsmokers in both

the exposed and the control groups. The effect of smoking in exposed subjects was significant Ž P 0.05.. In controls, the effect of smoking went, unexpectedly, in the opposite direction Ž P - 0.05.. In the controls, 89% of the micronucleated binucleates carried just one micronucleus and 11% had two or more micronuclei. In exposed subjects, the corresponding figures were 92% and 8%. In control subjects, 52% of the cells were mononucleated, 44% were binucleated and 4% were tri- or tetranucleated. The percentages for exposed subjects were 33%, 60%, and 7%, respectively. With respect to comet assay parameters, there was not any significant difference between exposed and non-exposed subjects ŽTable 4.. Nevertheless, when subjects were subdivided into smokers and nonsmokers, some significant differences between subgroups could be detected. The tail length was significantly longer in exposed smokers than in exposed nonsmokers Ž P - 0.05., but the tail length was significantly shorter in unexposed smokers than in unexposed nonsmokers Ž P - 0.05.. For the parameter T Ž%., a significant difference in the unexpected direction was found between exposed and unexposed nonsmokers Ž P - 0.05.. The GSTM1 and GSTT1 genotypes Žnull, positive. among exposed and control subjects are presented in Table 5. GSTM1 genotype affected the frequency of chromosomal aberrations only in exposed group, null genotype having lower frequency

Table 4 Comet assay and micronuclei by cytochalasin B—effect of exposure to 1,3-butadiene Group

N

T Ž%.

TL Žmm.

TM

MN

Exposed Controls

19 19

5.65a Ž3.81–10.11. b 5.97 Ž4.71–9.01.

5.61 Ž4.21–13.68. 5.96 Ž4.91–8.42.

0.31 Ž0.20–1.13. 0.33 Ž0.24–0.68.

16.26 " 5.53 c 14.63 " 8.59

Exposed Smokers Nonsmokers

9 10

6.27 Ž4.56–7.86. 5.60 Ž3.81–10.11.

6.32) Ž4.91–12.28. 5.61 Ž4.21–13.68.

0.42 Ž0.23–0.85. 0.29 Ž0.20–1.13.

18.00 " 5.17) 14.70 " 5.62

Controls Smokers Nonsmokers

9 10

5.71) Ž4.72–9.01. 6.01 Ž4.71–7.60.

5.71) Ž4.91–8.42. 6.32 Ž4.91–7.02.

0.31 Ž0.24–0.68. 0.35 Ž0.26–0.51.

10.75 " 8.45) 17.45 " 7.88

a

Median. Range. c Mean " SD. ) P - 0.05. Comet assay parameters. T Ž%.: percentage DNA in tail, TL: tail length, TM: tail moment, MN: number of micronuclei per 1000 binucleated cells. b

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Table 5 Effect of GSTM1 and GSTT1 genotypes on chromosomal aberrations—SCE and HFC in subject exposed to 1,3-butadiene Group

Genotype

N

AB. C. Ž%.

N

SCErcell

HFC Ž%.

Exposed

GSTM1 positive null GSTT1 positive null

14 5 17 2

3.57 " 1.21a )) 1.80 " 0.57 3.18 " 1.29 2.50 " 2.12

14 5 17 2

6.83 " 1.64 7.34 " 1.16 6.85 " 1.53 7.93 " 1.20

18.4 " 13.8 24.0 " 12.2 18.7 " 13.5 30.0 " 8.5

Controls

GSTM1 positive null GSTT1 positive null

7 12 15 4

1.93 " 1.06 2.08 " 1.02 1.76 " 0.78) 3.0 " 1.29

7 10 13 4

5.27 " 0.86 4.59 " 1.23 4.68 " 1.09 5.49 " 1.12

4.6 " 1.1 3.6 " 2.6 3.7 " 4.0 5.0 " 5.3

a

Mean " SD. ) P - 0.05. )) P - 0.01. AB. C. Ž%.: aberrant cells carrying chromosomal aberrations, SCE: sister chromatid exchanges, HFC: cells with high frequency of SCE.

Ž P - 0.01.. GSTT1 null genotype increased chromosomal aberrations in controls Ž P - 0.05.. No significant effect of both genotypes was observed on SCE or HFC. Using Spearman rank correlation analysis, individual exposure to BD correlated with chromosomal aberrations Ž r s 0.353, P - 0.05., SCE Ž r s 0.534, P - 0.005.; HFC Ž r s 0.504, P - 0.005.. The correlations in the subgroups were as follows: CA for nonsmokers Ž r s 0.340, P s 0.138., for smokers Ž r s 0.472, P s 0.058.; SCE for nonsmokers Ž r s 0.479, P - 0.05., for smokers Ž r s 0.592, P - 0.05.; HFC for nonsmokers Ž r s 0.505, P - 0.05., for smokers Ž r s 0.553, P - 0.05.. In the multifactorial analysis of variance, after accounting for BD exposure, smoking, GSTM1, GSTT1, and age, BD exposure affected significantly the frequency of chromosomal aberrations Ž P - 0.05. and HFC Ž P - 0.001.. HFC was also affected by smoking Ž P - 0.05.. The effect of GSTM1 genotype Table 6 Effects of 1,3-butadiene exposure, smoking, GSTM1 and GSTT1 genotypes and age on chromosomal aberrations, SCE and HFC by multifactor analysis of variance procedure Factors

CA

SCE

HFC

BD exposure Smoking GSTM1 GSTT1 Age

P s 0.0365 P s 0.1048 P s 0.0682 P s 0.0918 P s 0.4559

P s 0.2380 P s 0.2410 P s 0.3404 P s 0.6279 P s 0.4813

P s 0.0099 P s 0.0455 P s 0.6639 P s 0.1651 P s 0.2007

on chromosomal aberrations was on the borderline of significance Ž P s 0.068. ŽTable 6.. 4. Discussion The results in this paper represent the first report indicating a significant increase of frequencies of chromosomal aberrations and SCE in a group of workers occupationally exposed to BD. The group of workers from the same petrochemical company was sampled already in 1992 and 1993, but no effect of exposure on induction of CA and SCE could be detected w13x. The samplings in 1992 and 1993 occurred at the beginning of October and the sampling reported in this paper took place at the end of November 1994 with another group. The exposed subjects of the 1992 group consisted of workers in the monomer production and polymerization units, whereas the 1993 and 1994 samples were collected exclusively in the monomer production unit. It may be, that the results for the samples collected at the beginning of October 1992 and 1993 were affected by the summer vacation in a sense that there was less accumulation of damage than for the 1994 samples collected late November 1994. During the years 1992–1994, there were no changes in the technology that could have resulted in a decreaserincrease of the exposure dose. Thus, apart from the ‘holiday’ aspect, there were no other known factors that could explain why there was no effect of exposure on frequencies of CA and SCE in samples collected in

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1992 and 1993. The exposure evaluation in 1992 cannot be compared with that performed in 1994 because half of the exposed group in 1992 was from the polymerization unit w13x. The significant increase of the percentage of cells with chromosomal aberrations in exposed workers of the 1994 study group could not have been affected by GSTT1 genotype because only two GSTT1 null carriers were found in the exposed group as compared to just four carriers in the controls. When we reanalyzed SCE samples from the 1993 batch, 6.62 SCErcell were measured in the exposed group vs. 5.44 SCErcell in the controls Ž P - 0.001.. The same data were also analyzed for the occurrence of HFC, and it was found that 16.9% of the cells in exposed subjects were HFC as opposed to 6.4% in the controls. This difference was statistically significant Ž P - 0.01.. Multifactorial analysis showed that chromosomal aberrations and HFC were predominantly affected by BD exposure. Evaluating the effect of smoking in exposed workers, we found that the exposure dose for BD was about three times higher in nonsmokers than in smokers. Therefore, no effect of smoking on the percentage of cells with chromosomal aberrations was observed. There was also no significant effect of smoking on SCE in exposed group as well as controls. The difference between smokers and nonsmokers observed in exposed group was on the borderline of significance Ž P s 0.056.. Multifactorial analysis showed that the effect of smoking was pronounced only in HFC. Peculiar but concordant results were observed with the comet and micronucleus assays. For the parameters measured with these assays, there were no significant differences between exposed and control groups. However, when each of these two groups was subdivided into smokers and nonsmokers, DNA damage in exposed smokers was stronger than in exposed nonsmokers. DNA damage in the opposite direction was found comparing unexposed smokers and nonsmokers. The biological significance of these unexpected results is not known. Our results on smoking and BD exposure, determining HFC, comet parameters and micronuclei are also different from the postulate of Oesch et al. w27x that cigarette smoking protects against some additional genotoxic insults. Our results rather seem to

indicate that smoking increased the genotoxicity of BD. Recently, Sasiadek and Paprocka-Borowicz w28x published a paper on an adaptive response to monoepoxybutene in in vitro experiments on SCE in human lymphocytes. From these results, it may be hypothesized that low concentrations of BD probably induce detoxification pathways, which later diminish the effect of larger exposures to BD. If this idea is correct, then the low total BD dose during the work shift is more important for determining the relationship between biomarkers and exposure, than the impact of occupational peak exposures. If the cytogenetic parameters used to study the effects of BD exposure were ranked in order of decreasing sensitivity, then the sequence would be: HFC ) SCE ) CA ) MN ; comet assay parameters. SCE and especially HFC, may be judged as being the most sensitive biomarker for detection of BD exposure. Waters and Nolan summarizing the ECrUS Workshop on the genetic risk of BD and the parallelogram approach concluded ‘‘ . . . the biomonitoring studies conducted so far have not revealed convincing evidence that sister chromatid exchanges, chromosomal aberrations or micronuclei are induced in blood lymphocytes of humans exposed at the generally low level Ž- 1 ppm, i.e., - 2.2 mgrm3 . in ambient air in the BD manufacturing industry’’ w29x. The results of our study may be an indication that cytogenetic biomarkers are positive also at low exposure level to BD. Our data on exposure dose were interpreted as representing the occupational exposure during the last 8 h before the blood sampling. Considering the relationship between exposure and cytogenetic endpoints, it may be advisable to follow each subject for several shifts, to obtain more accurate information about the real exposure to BD. We observed an increase of genetic damage in smokers in the BD-exposed workers for HFC, MN, and comet assay parameters, too. It seems that smoking could be an extra risk factor for workers that are occupationally exposed to BD. This hypothesis should be, however, carefully analyzed in the future. The results presented in this study have shown that although exposure levels in modern plants were substantially reduced, a significant induction of cytogenetic damage could still be demonstrated.

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Acknowledgements w13x

We are grateful to Dr. Blanka Binkova´ for the statistical analysis. The authors would like to thank Prof. H.G. Neumann, University of Wurzburg, for ¨ his enthusiastic coordination of EC grant. The research was supported by the EC grant ERBCIPACT93-0228 and the grant from the Regional Institute of Hygiene of Central Bohemia. The authors wish to express their gratitude towards the workers taking part in the study as well as persons responsible for excellent arrangements.

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