10 H. AKSOY ET AL. JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 2006; 26: 10–15 Published online 12 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1098 Genotoxicity study in lymphocytes of offset printing workers Hüseyin Aksoy,1 Serkan Yılmaz,1 Mustafa Çelik,2 Deniz Yüzba6ıoglu1* and Fatma Ünal1 1 2 Gazi Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, 06500 Teknikokullar, Ankara, Turkey Sütçü Imam Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Kahramanmara6, Turkey Received 18 April 2004; Revised 20 June 2005; Accepted 20 June 2005 ABSTRACT: The potential cytogenetic damage in offset printing workers was evaluated using sister chromatid exchanges (SCEs), chromosome aberrations (CAs) and micronuclei (MN) as biomarkers in peripheral lymphocytes of 26 volunteers (14 workers, 12 controls). The CA, SCE and MN frequency of offset printing workers was significantly higher than in their controls. The replication index (RI) was not affected while the mitotic index (MI) was affected most in the workers. It can be concluded from this study that chronic occupational exposure to printing dyes and thinner may lead to a slightly increased risk of genetic damage among offset printing workers. Copyright © 2005 John Wiley & Sons, Ltd. KEY WORDS: offset printing workers; occupational exposure; sister chromatid exchanges (SCEs); chromosome aberrations (CAs); micronuclei (MN) Introduction In recent years, there has been an increased problem of human exposure to potentially toxic chemicals in the work place. A large volume of epidemiological data deals with potential cancer risks in printing processes. Svensson et al. (1990) reported that a significant increase in cancers of the respiratory tract was observed among rotogravure printers. Aerosols from high speed rotary printing machines consist of ink mists, which are believed to induce lung cancer (Leon et al., 1994). In addition some studies reported that kidney cancer, urinary bladder cancer and leukemia were shown among printing workers (IARC, 1996). Offset printing workers are exposed to offset printing dyes and thinner. Offset printing dyes contain cobalt and hydroquinone. Cobalt is a heavy metallic chemical element. It is a member of group VIII of the periodic table. It is used for cancer therapy and in industry for detecting flaws in metal parts. The overall genetic evaluation was that cobalt and its compounds are possibly carcinogenic to humans (group 2B) (IARC, 1991). In the same evaluation, cobalt (II) compounds were reported to induce DNA damage, DNA protein cross links, gene mutations, sister chromatid exchanges and aneuploidy in in vitro studies on animal and human cells. There was some evidence that cobalt (II) compounds could also induce aneuploidy in vivo in Syrian hamster bone marrow and testes (IARC, 1991). Another chemical used in printing, * Correspondence to: Deniz Yuzbasıoglu, Gazi Üniversitesi, Fen-Edebiyat Fakultesi, Biyologi Bölümü, 06500 Teknikokullar, Ankara, Turkey. E-mail: deniz@gazi.edu.tr Copyright © 2005 John Wiley & Sons, Ltd. hydroquinone, is extensively used as a reducing agent and as a photographic developer. It is a skin-lightening agent and is used in cosmetics, hair dyes and medical preparations (WHO, 1996). Knadle (1985) reported that hydroquinone induced sister chromatid exchanges (SCEs) at relatively high concentrations in human lymphocytes. Stillman et al. (1999) reported that hydroquinone induced hypoploidy in a human lymphoblastoid cell line GM09948. Offset printing workers also use widely an industrial thinner containing toluene (about 65%) for the cleaning of the offset machines. Bauchinger et al. (1982) and Schmid et al. (1985) reported an increased incidence of SCEs in individuals occupationally exposed to toluene. A high incidence of chromosomal aberrations (CA) and sister chromatid exchanges in the lymphocytes of rotogravure printing workers has been reported. The CA and SCE frequencies were higher in workers than in the control groups (Pelclova et al., 1990; Hammer et al., 1998). However, some studies in the literature showed that there was no relationship between chronic occupational exposure to toluene and increased incidence of chromosomal aberration (Haglung et al., 1980; Maki-Paakkanen et al., 1980; Richer et al., 1993). Genotoxicity biomarkers have received considerable interest as tools for detecting human genotoxic exposure and effects. The largest databases are available for CA; a high CA level has been associated with an increased cancer risk while the frequencies of SCEs and micronucleus (MN) were not associated with cancer risk (Hagmar et al., 1998a,b; Bonassi et al., 2000). The induction of SCEs has been described as a rapid and sensitive end-point for testing mutagenicity. An increase of SCE frequency can be an indicator of persistent DNA J. Appl. Toxicol. 2006; 26: 10–15 GENOTOXICITY STUDY IN OFFSET PRINTING WORKERS 11 damage (Palitti et al., 1982; Cardoso et al., 2001). The cytokinesis-blocked micronucleus assay is used to detect acentric chromosome fragments or whole chromosomes left upon nucleus division and visible as small additional nuclei in the cytoplasm. Hence, the appearance of micronuclei should point to clastogenic and/or aneugenic effects (Giri et al., 2002). This paper analysed cytogenetic damage in peripheral blood lymphocytes of offset printing workers by using CAs, SCEs and micronucleus assay (MN). Changes in cell proliferation kinetics through the replication index (RI) and cytotoxic effect by means of the mitotic index (MI) were also evaluated. dried at room temperature and mounted with depex. For the SCE study, the slides were stained with Giemsa according to the method of Speit and Houpter (1985) with some modifications. The mitotic index (MI) was determined by scoring 1000 cells from each donor. Chromosomal abnormalities were scored from 100 well-spread metaphases per donor. For the occurrence of the number of SCEs, a total of 25 cells from each donor under second metaphases was scored. In addition, 100 cells from each donor were scored for the determination of the replication index (RI). The RI, calculated according to the following formula [1 × M1] + [2 × M2] + [3 × M3]/N Material and Methods Twenty six volunteers (14 exposed, 12 controls) were studied in this work. The volunteers were selected according to their ages and smoking habit. Three age groups were selected: 16–25, 26–35 and 36–45. None of the volunteers had any problems relating to health, alcohol and drug consumption. Six of the exposed workers were smokers, the others were nonsmokers. Their ages were in the range 18–45 years and averaged 27.64 years. The mean duration of employment was 10.36 years (min. 1 year, max. 30 years). The working period was 45 h per week. A total of 12 male blood donors from the Gazi University of Ankara were used as a control group. This group consisted of six smokers and six nonsmokers, aged 22–38 years (average 28.70 years). The preparations of cells were made by the following procedures. SCE and CA Analysis Human peripheral blood cells were used as the test systems. Heparinized blood (1/10: 2 ml venous blood containing 0.2 ml heparin) was collected and stored at +4 °C for 30 min. The whole blood was added to 2.5 ml chromosome medium B (Biochrom) supplemented with 10 µg ml−1 bromodeoxyuridine. The cultures were incubated at 37 °C for 72 h. 0.06 µg ml−1 colchicine was added 2 h prior to the harvesting of the culture. Then, the cells were harvested by centrifugation (1200 rpm, 10 min), and the pellet was resuspended in a hypotonic solution of 0.075 M KCl for 30 min at 37 °C. Following this process, the cells were centrifuged again and fixed in a cold methanol:acetic acid (3:1) mixture for 35 min at +4 °C. At the end of this procedure, the cells were treated with fixative two times. Then slides were made by dropping and air drying. For chromosome aberrations, slides were stained with 5% Giemsa (pH = 6.8) prepared in Sorensen buffer solution, for 20–25 min, and then washed in distilled water, Copyright © 2005 John Wiley & Sons, Ltd. where N represents the number of observed cells, M1, M2 and M3 represent the number of cells undergoing first, second and third mitosis (Lin et al., 1987). MN Analysis In this analysis the blood samples were added to 2.5 ml chromosome medium B (Biochrom). The cultures were incubated at 37 °C for 72 h. At 44 h after beginning the culture, cytocalasin-B (5.2 µg ml−1) was added to each culture. Then, the cells were harvested by centrifugation (1000 rpm, 10 min), and the pellets were resuspended in a hypotonic solution of 0.075 M KCl for 5 min at +4 °C. The cells were again centrifuged and fixed in the cold methanol:acetic acid (3:1) mixture for 15 min. The fixation procedure was applied three times. Into the last fixative, 1% formaldehyde was added to preserve the cytoplasm. The slides were made by dropping and air drying. For the MN analysis, the slides were stained with 5% Giemsa (pH = 6.8), prepared in Sorensen buffer solution, for 20–25 min, washed in distilled water, dried at room temperature and mounted with depex. Micronuclei were scored from 1000 binucleated cells per donor. Statistical Analysis The significance between the percentage of abnormal cells, CA/cell, RI, MI and MN in cultures and their controls were determined using the z-test. The significance between mean SCE in treated cultures and their controls were determined using the t-test. Results In this study, 26 volunteers were studied. Of these, 14 were offset printing workers and 12 were controls. As shown in the Fig. 1 and Table 1, the abnormal cell ratio J. Appl. Toxicol. 2006; 26: 10–15 12 H. AKSOY ET AL. Table 1. Types and distribution of chromosome aberrations in offset printing workers according to smoking habit and age Group Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers Age 16–25 16–25 26–35 26–35 36–45 36–45 Smoking habit Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker No. of subjects 2 3 2 3 2 1 2 3 2 2 2 2 No. of cells scored 200 300 200 300 200 100 200 300 200 200 200 200 Aberrations Abnormal cell ± SE (%) B′ B″ SU DC ER F P CE 6 23 — 29 8 10 1 27 6 13 2 15 — 6 — 3 — 1 — 3 — 6 — 3 — 16 3 12 — 2 3 3 — 17 1 2 — 9 — 15 — 1 — 4 2 9 1 2 — 1 — 2 — 1 — — — — — — — 1 — 1 — — — 4 — 4 1 2 — — — — — — — 1 — 4 — — — — — 1 2 — — — — — — — 3.00 16.00 1.50 18.33 2.50 13.33 2.00 14.00 2.00 26.00 2.50 11.00 ± ± ± ± ± ± ± ± ± ± ± ± 1.21 2.12a 0.86 2.24a 1.10 3.40a 0.98 2.00a 0.99 3.10a 1.00 2.21a CA/cell ± SE 0.030 0.190 0.015 0.210 0.050 0.150 0.020 0.140 0.040 0.270 0.025 0.120 ± ± ± ± ± ± ± ± ± ± ± ± 0.01 0.02a 0.01 0.02a 0.01 0.04a 0.01 0.06a 0.01 0.03a 0.01 0.02a B′, chromatid break; B″, chromosome break; SU, sister chromatid union; DC, dicentric chromosome; ER, endoreduplication; F, fragment; P, polyploidy; CE, chromatid exchange. a Significant from the control P < 0.001 (z-test). Figure 1. Chromosome aberrations in peripheral blood lymphocytes of smoker and nonsmoker offset printing workers and their controls was significantly increased compared with their controls. In the smoker workers the increase of abnormal cells was higher than in the nonsmokers. The CA/cell ratio significantly increased in exposed groups when compared with the controls. Also the increase of CA/cell was higher in the smoker groups than in the nonsmoker groups in the 26–35 and 36–45 age groups (Table 1). The eight types of abnormalities recorded were chromatid and chromosome breaks, sister chromatid union, dicentric chromosomes, endoreduplication, fragment, polyploidy and chromatid exchange. Chromatid breaks and sister chromatid union were observed as the most common aberrations (Fig. 2). The replication index (RI) was not affected in the worker groups but the mitotic index was decreased significantly in all the age groups of nonsmokers (Table 2). The SCE frequency was significantly increased in exposed groups when compared with their controls (Table 2, Fig. 3). In all exposed groups, the SCE frequency was higher in the smoker workers than in the nonsmoker workers. The micronuclei frequency was significantly increased in all exposed groups except in the smoker workers Copyright © 2005 John Wiley & Sons, Ltd. Figure 2. Most common aberrations observed in offset printing workers, (a) chromatid break (b) sister chromatid union of the 16–25 age group (Table 3). The frequency of micronuclei was significantly higher in exposed groups compared with their controls in total (Fig. 4). In all J. Appl. Toxicol. 2006; 26: 10–15 GENOTOXICITY STUDY IN OFFSET PRINTING WORKERS 13 Table 2. Distribution of SCE, RI and MI in peripheral blood lymphocytes of offset printing workers according to smoking habit and age Groups Ages Smoking habit No. of subjects No. of cells scored (SCE) Min-max SCE Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers 16–25 Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker 2 3 2 3 2 1 2 3 2 2 2 2 50 75 50 75 50 25 50 75 50 50 50 50 2–12 5–20 2–9 5–15 1–14 5–20 1–19 4–17 5–23 5–20 2–8 6–15 a b c d Significant Significant Significant Significant 16–25 26–35 26–35 36–45 36–45 from from from from the the the the control control control control P P P P < < < < SCE/cell ± SE 6.28 9.97 4.68 8.75 5.68 9.48 4.70 8.51 8.20 9.80 5.12 9.20 ± ± ± ± ± ± ± ± ± ± ± ± 0.41 0.35b 0.23 0.29b 0.45 0.74b 0.46 0.29b 0.61 0.47a 0.29 0.32b M1 M2 M3 38 48 40 46 10 16 42 54 30 69 44 29 50 108 57 93 50 25 81 105 52 66 70 62 112 144 103 161 140 59 77 141 118 65 86 109 RI ± SE 2.37 2.32 2.32 2.38 2.75 2.43 2.18 2.29 2.44 1.98 2.21 2.40 ± ± ± ± ± ± ± ± ± ± ± ± 0.05 0.04 0.05 0.04 0.04 0.08 0.05 0.04 0.05 0.06 0.06 0.06 MI ± SE (%) 3.20 3.90 5.50 3.83 4.20 4.60 7.60 3.57 4.50 3.85 6.25 4.05 ± ± ± ± ± ± ± ± ± ± ± ± 0.39 0.35 0.51 0.35c 0.44 0.66 0.59 0.34d 0.46 0.43 0.54 0.44c 0.05 (t-test) 0.01 (t-test) 0.01 (z-test) 0.001 (z-test) Table 3. Distribution of MN in peripheral blood lymphocytes of offset printing workers according to smoking habit and age Groups Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers Controls Workers a b Ages 16–25 16–25 26–35 26–35 36–45 36–45 Smoking habit Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker Smoker Smoker Nonsmoker Nonsmoker No. of subjects BN cells scored 2 3 2 3 2 1 2 3 2 2 2 2 2000 3000 2000 3000 2000 1000 2000 3000 2000 2000 2000 2000 Distribution of BN cells according to the no. of MN (1) (2) (3) (4) 8 16 4 15 10 11 2 20 4 19 8 18 0 2 0 1 0 1 0 4 0 2 2 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MN/cell (%) 0.40 0.67 0.20 0.67 0.50 1.30 0.10 0.93 0.20 1.15 0.60 1.00 ± ± ± ± ± ± ± ± ± ± ± ± 0.138 0.148 0.094 0.148a 0.154 0.357b 0.063 0.173b 0.094 0.236b 0.170 0.221a Significant from the control P < 0.05 (z-test) Significant from the control P < 0.001 (z-test) Figure 3. Sister chromatid exchanges in peripheral blood of offset printing workers according to smoking habit and their controls Figure 4. MN in peripheral blood of offset printing workers according to smoking habit and their controls exposed groups (except the 16–25 age group), the MN frequency was higher in the smoker workers than in the nonsmoker workers but this result was not statistically significant. Discussion Copyright © 2005 John Wiley & Sons, Ltd. In this study the CA, SCE and MN (except smokers of the 16–25 age group) frequency of exposed workers J. Appl. Toxicol. 2006; 26: 10–15 14 H. AKSOY ET AL. significantly increased in all the age groups compared with their controls. The MI value was significantly decreased only in the nonsmoker groups. However, the RI value was not affected in workers when compared with the controls. In this investigation, eight types of chromosomal aberrations were observed and it was found that the most common aberrations were chromatid breaks and sister chromatid union. Pelclova et al. (1990) reported that chromatid and chromosome breaks, gaps, chromatid and chromosome exchanges were observed in rotogravure printing workers. Genotoxicity of toluene was investigated in rotogravure printing plant workers and a significant increase was observed only in chromatid breaks (Pelclova et al., 2000). Printing dyes used by offset printing workers contain cobalt and hydroquinone. Some researchers have shown that cobalt was possibly carcinogenic to humans (IARC, 1991) and that hydroquinone was positive for cytogenetic effects, which included induction of micronuclei (Yager et al., 1990; Robertson et al., 1991; Vian et al., 1995) and sister chromatid exchanges (Erexson et al., 1985; Knadle, 1985). However, Roza et al. (2003) reported that hydroquinone was cytotoxic, but did not induce chromosomal aberrations in human lymphocytes culture in vitro. Offset printing workers also use thinner for machine cleaning processes. Thinner contains a high amount of toluene and some researchers have reported that occupational exposure to toluene increased the incidence of SCEs (Bauchinger et al., 1982; Schmid et al., 1985; Pelclova et al., 2000). In contrast, some studies showed that there was no relationship between chronic occupational exposure to toluene and increased incidence of chromosomal aberration and sister chromatid exchanges (Haglung et al., 1980; Maki-Paakkanen et al., 1980; Richer et al., 1993). Pelclova et al. (1990) reported that CA frequency was increased in rotogravure printing workers compared with controls. In another study Hammer et al. (1998) reported that the SCE frequency was significant at a high confidence level in rotogravure printing workers. The authors pointed out a strong relationship between individual toluene burden and the genotoxic risk of the exposed workers. Many studies have been carried out to evaluate the cytogenetic effects of occupational exposure to various health hazard chemicals in different field workers. Sardas et al. (1994) reported that the frequency of SCE was higher in workers employed in car-painting workshops. The genotoxicity study carried out by Topaktas et al. (2002) in workers of an iron and steel factory showed that the frequency of CA was higher, while the frequency of the SCE was not in all the smoker-nonsmoker workers, than in the smoker-nonsmoker control groups. In addition, the authors observed that there was no significant decrease in the RI, but the MI was significantly lower in exposed groups than in the controls. Bogadi-Sare Copyright © 2005 John Wiley & Sons, Ltd. et al. (1997) reported that exposure to benzene and toluene increased the CA frequency in the lymphocytes of workers. The genotoxicity study of Swedish paint industry workers showed that there was no significant increase in the frequency of CA and SCE (Haglung et al., 1980). Pastor et al. (2001) reported that a comparison between workers and controls did not reveal any statistical significant difference in the MN frequency for either lymphocytes or buccal cells in Greek farmers. Genotoxicity research in wooden furniture workers has shown that micronuclei and SCE frequency in exposed groups was higher than in the controls (Elavarasi et al., 2002). Iravathy Goud et al. (2004) reported that there is a significant increase in the frequency of MN in buccal epithelial cells and peripheral blood lymphocytes as well as chromosomal aberrations in individuals working with photocopying machines. A significant increase in the formation of SCE, CA and MN frequencies were reported in Pb-Zn miners exposed to heavy metals (Bilban, 1998). In the literature, several studies demonstrated that CA, SCE and MN frequencies generally increased in workers occupationally exposed to the environmental contaminants discussed above. Some of these investigations revealed that there is a relationship between age, smoking and increase in abnormalities (Maki-Paakkanen et al., 1980; Topaktas et al., 2002). Whereas, some investigators reported that there was no relationship between age, smoking and increasing abnormalities (Khalil et al., 1994; Surrales et al., 1997). Data obtained in this study showed that the CA, SCE and MN frequency of the exposed workers were higher than the controls. The CA frequency and CA/cell was almost equal in smoker and nonsmoker workers in the 16–25 and 26–35 age groups but higher only in smokers of the 36– 45 age group than in the nonsmokers. A strong relationship between CA and smoking habit, age and exposure to pollutants was observed. In all exposed groups, the SCE and MN (except the 16–25 age group) frequency was higher in smokers than in nonsmokers. However, the increase of SCE and MN frequency was not associated with age. As a result, the present study showed that the CA analysis was more sensitive to environmental contaminants than other cytogenetic end-points. It can be concluded from this study that chronic occupational exposure to printing dyes and toluene leads to an increased risk of genetic damage among offset printing workers. The genotoxic and potential carcinogenic risks of printing dyes and toluene should be taken into account in the process of offset printing. References Bauchinger M, Schmid E, Dresp J, Kolin-Gerresheim J, Hauf R, Suhr E. 1982. Chromosome changes in lymphocytes after occupational exposure to toluene. Mutat. Res. 102: 439– 445. J. Appl. Toxicol. 2006; 26: 10–15 GENOTOXICITY STUDY IN OFFSET PRINTING WORKERS 15 Bilban M. 1998. 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